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Polyhydroxyalkanoate (PHA) is one of the biocompatible and biodegradable plastics that can be produced and accumulated as granules inside microorganisms. In this study, a new approach to rapidly quantify a short-chain-length PHA, polyhydroxybutyrate (PHB), produced from genetically engineered

The use of colloidal nanoparticles suffers from the drawbacks of potential color interference and substrate-induced aggregation. To overcome the limitations, a catalyst was developed by crosslinking Cu-doped carbon dots (Cu-CDs) with chitosan. Cu-CDs with high peroxidase activity were prepared by using a rapid microwave-assisted method. The Cu-CDs containing 6.88% of Cu had an average particle size of 2.25 nm and exhibited 9% of fluorescence quantum yield. The nanozyme/film composite was prepared by crosslinking between the amino groups of Cu-CDs and those of chitosan via a glutaraldehyde linker. A H2O2-mediated tetramethylbenzidine (TMB) oxidation reaction was use to evaluate the peroxidase activity of the film. Based on the TMB color changes, colorimetric assays were developed for the detection of H2O2 and glucose at an absorption wavelength 652 nm. Under the optimal conditions, the linear ranges for H2O2 and glucose were 0.625–40 µM and 1.9–125 µM, respectively, and the detection limits were 0.12 µM and 0.69 µM, respectively. The colorimetric assay was also applied to analyze diluted human serum samples spiked with glucose. Furthermore, this biodegradable, non-toxic, and easy-to-handle nanozyme composite could be stored for over 4 weeks without a significant decrease in activity. Graphical abstract

We present a sensitive and rapid screening method for the determination of β‐lactamase activity of antibiotic‐resistant bacteria, by designing a pH‐sensitive fluorescent dye‐doped mesoporous silica nanoparticle encapsulated with penicillin G as a substrate. When penicillin G was hydrolysed by β‐lactamase and converted into penicilloic acid, the acidic environment resulted in fluorescence quenching of the dye. The dye‐doped mesoporous nanoparticles not only enhanced the β‐lactamase‐catalyzed reaction rate but also stablized the substrate, penicillin G, which degrades into penicilloic acid in a water solution without β‐lactamase. Twentyfive clinical bacterial samples were tested and the antibiotic resistant and susceptible strains were identified. The proposed method may detect the presence of β ‐lactamases of clinically relevant samples in less than 1 hour. Moreover, the detection limit of β‐lactamase activity was as low as 7.8×10−4 U/mL, which was determined within two hours., A pH‐sensitive fluorescent dye‐doped mesoporous silica nanoparticle encapsulated with penicillin G as a substrate was used for a screening assay to determine the β‐lactamase activity of antibiotic‐resistant bacteria. Twentyfive clinical bacterial samples were tested and the antibiotic resistant and susceptible strains were successfully identified.

Ensuring food safety requires a rapid and reliable method for detecting food-borne pathogens. Mass spectrometry has been demonstrated as a powerful tool to classify pure bacterial species. However, matrix interference from food backgrounds may lead to false results because of the suppression of microbial signals. It is useful to develop a method for bacterial enrichment and marker identification in food samples. Magnetic zirconia nanoparticles were used to concentrate spiked microorganisms from apple juice/lettuce under specific conditions (pH 4.5). Bacterial identification was achieved using nanoLC–MS. Selected reaction monitoring of bacteria-related peptides was applied for the first time to identify bacteria including Staphylococcus aureus and Escherichia coli. This study presents an accurate means for bacterial identification in food matrixes using MS. The analysis time is less than 90 min and the minimum concentration of E. coli detected was 5 × 103 CFU/mL. The interaction between bacteria and the magnetic nanoparticles was electrostatic and nonspecific, in contrast to immunoassays which require specific antibodies. The targeted peptide analysis focuses on the bacterial markers, thus significantly simplifying the analysis and leading to an accurate identification of bacteria. Keywords: Food matrixes, Mass spectrometry, Magnetic nanoparticles, Pathogen, Selected reaction monitoring

Oxidative stress resulting from reactive oxygen species (ROS) is known to play a key role in numerous neurological disorders, including neuropathic pain. Morphine is one of the commonly used opioids for pain management. However, long-term administration of morphine results in morphine antinociceptive tolerance (MAT) through elevation of ROS and suppression of natural antioxidant defense mechanisms. Recently, mesoporous polydopamine (MPDA) nanoparticles (NPS) have been known to possess strong antioxidant properties. We speculated that morphine delivery through an antioxidant nanocarrier might be a reasonable strategy to alleviate MAT. MPDAs showed a high drug loading efficiency of &sim, 50%, which was much higher than conventional NPS. Spectral and in vitro studies suggest a superior ROS scavenging ability of NPS. Results from a rat neuropathic pain model demonstrate that MPDA-loaded morphine (MPDA@Mor) is efficient in minimizing MAT with prolonged analgesic effect and suppression of pro-inflammatory cytokines. Additionally, serum levels of liver enzymes and levels of endogenous antioxidants were measured in the liver. Treatment with free morphine resulted in elevated levels of liver enzymes and significantly lowered the activities of endogenous antioxidant enzymes in comparison with the control and MPDA@Mor-treated group. Histopathological examination of the liver revealed that MPDA@Mor can significantly reduce the hepatotoxic effects of morphine. Taken together, our current work will provide an important insight into the development of safe and effective nano-antioxidant platforms for neuropathic pain management.

A nucleic acid aptamer that specifically recognizes methicillin-resistant Staphylococcus aureus (MRSA) has been immobilized on magnetic nanoparticles to capture the target bacteria prior to mass spectrometry analysis. After the MRSA species were captured, they were further eluted from the nanoparticles and identified using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The combination of aptamer-based capture/enrichment and MS analysis of microorganisms took advantage of the selectivity of both techniques and should enhance the accuracy of MRSA identification. The capture and elution efficiencies for MRSA were optimized by examining factors such as incubation time, temperature, and elution solvents. The aptamer-modified magnetic nanoparticles showed a capture rate of more than 90% under the optimized condition, whereas the capture rates were less than 11% for non-target bacteria. The as-prepared nanoparticles exhibited only a 5% decrease in the capture rate and a 9% decrease in the elution rate after 10 successive cycles of utilization. Most importantly, the aptamer-modified nanoparticles revealed an excellent selectivity towards MRSA in bacterial mixtures. The capture of MRSA at a concentration of 102 CFU/mL remained at a good percentage of 82% even when the other two species were at 104 times higher concentration (106 CFU/mL). Further, the eluted MRSA bacteria were successfully identified using MALDI mass spectrometry.

Boron and nitrogen co-doped carbon dots (B, N-CDs) were fabricated through a simple, one-step hydrothermal reaction of citric acid, boric acid, and tris base. The obtained B, N-CDs exhibit excitation-dependent fluorescence, high quantum yield (QY), biocompatibility, photostability, and aqueous solubility. The QY was substantially increased to 57% by doping boron atoms. Furthermore, the fluorescence intensity of B, N-CDs was temperature-dependent and decreased linearly from 283 to 333 K. The prepared B, N-CDs were used as a fluorescence probe for the detection of para-nitrophenol (p-NP) and Fe (III) ions with low detection limits of 0.17 μM and 0.30 μM, respectively. Moreover, the presence of p-NP could be further confirmed by a colorimetric assay. The fluorescent probe has been applied to determine p-NP and Fe (III) in a spiked serum sample and spiked water samples (lake and tap water). Moreover, the as-prepared B, N-CDs were of low toxicity and capable of bioimaging.

The impact of microwave irradiation on the in-solution digestion processes and the detection limit of proteins are systematically studied. Kinetic processes of many peptides produced through the trypsin digestion of various proteins under microwave heating at 50°C were investigated with MALDI-MS. This study also examines the detection limits and digestion completeness of individual proteins under microwave heating at 50°C and at different time intervals (1, 5 and 30 min) using LC-MS. We conclude that, if the peptides without missed cleavage dictate the detection limit, conventional digestion will lead to a better detection limit. The detection limit may not differ between microwave and conventional heating if the peptides with missed cleavage sites and strong intensity are formed at the very early stage (i.e., less than 1 min) and are not further digested throughout the entire digestion process. The digestion of E. coli lysate was compared under conventional and short time (microwave) conditions. The number of proteins identified under conventional heating exceeded that obtained from microwave heating over heating periods less than 5 min. The overall results show that the microwave-assisted digestion is not complete. Although the sequence coverage might be better, the detection limit might be worse than that under conventional heating. This article is protected by copyright. All rights reserved

The effect of microwave heating on lipase-catalyzed reaction remains controversial. It is not clear whether the reaction rate enhancements are purely due to thermal/heating effects or to non-thermal effects. Therefore, quantitative mass spectrometry was used to conduct accurate kinetic analysis of lipase-catalyzed hydrolysis of triolein by microwave and conventional heating. Commercial lipases from Candida rugosa (CRL), Porcine Pancreas (PPL), and Burkholderia cepacia (BCL) were used. Hydrolysis reactions were performed at various temperatures and pH levels, along with various amounts of buffer and enzymes. Hydrolysis product yields at each time point using an internal-standard method showed no significant difference between microwave and conventional heating conditions when the reaction was carried out at the same temperature. CRL showed optimum catalytic activity at 37 °C, while PPL and BCL had better activities at 50 °C. The phosphate buffer was found to give a better hydrolysis yield than the Tris-HCl buffer. Overall results prove that a non-thermal effect does not exist in microwave-assisted lipase hydrolysis of triolein. Therefore, conventional heating at high temperatures (e.g., 50 °C) can be also used to accelerate hydrolysis reactions.

A simple label-free method was developed for the quantification of the herbicide-resistant gene-related protein 5-enolpyruvylshikimate-3-phosphate synthase using multiple reaction monitoring liquid chromatography–mass spectrometry. Sample pretreatment procedures including ion exchange chromatography and CaCl2 precipitation were used to purify the 5-enolpyruvylshikimate-3-phosphate synthase protein. Quantification of various percentages of genetically modified soya (0.5–100%) was performed by selecting suitable endogenous soybean peptides as internal standards. Results indicated that Gly P (QGDVFVVPR) and Lec P (LQLNK) are useful internal standards for the quantification of low and high percentages of genetically modified soya, respectively. Linear regression analysis of both calibration curves yielded good linearity with R2 of 0.99. This approach is a convenient and accurate quantification method for genetically modified soya at a level as low as 0.5% (less than the current EU threshold for labeling genetically modified soya).

This study addresses the issue of whether the magnetite Fe3O4 nanoparticle shows a true spin-glass like behavior or if this is an artifact of interparticle interaction. Transmission electron microscopy and X-ray diffraction pattern analysis confirms the formation of pseudospherical Fe3O4 nanoparticles with a grain size of 8(1) nm. The magnetic moment relaxation M(t) follows a stretched exponential and power law function, confirming the presence of multiple-magnetic anisotropic barriers (β = 0.51(1)) and weak-interparticle interaction (n = 0.60(4)). The observed field cooling (FC) memory effect occurs because of the presence of magnetic anisotropy arising from a broad size distribution and weak-interparticle interactions. The absence of the zero-field-cooled (ZFC) memory effect signals that the Fe3O4 nanoparticle system is not a true spin-glass system, and the observed decreasing behavior of magnetization in FC measurement is an artifact of the weak-interparticle interaction.

The development of nanotechnology in biology and medicine has raised the need for conjugation of nanoparticles (NPs) to biomolecules. In this study, magnetic and functionalized magnetic iron oxide nanoparticles were synthesized and used as affinity probes to capture Gram-positive/negative bacteria. The morphology and properties of the magnetic NPs were examined by transmission electron microscopy, Fourier transform infrared spectroscopy, and zeta potential measurements. Furthermore, this study investigated the interaction between functionalized magnetic nanoparticles and Gram positive/negative bacteria. The positively and negatively charged magnetic nanoparticles include functionalities of Fe3O4, SiO2, TiO2, ZrO2, poly ethyleneimine (PEI) and poly acrylic acid. Their capture efficiencies for bacteria were investigated based on factors such as zeta potential, concentration and pH value. PEI particles carry a positive charge over a range of pH values from 3 to 10, and the particles were found to be an excellent candidate for capturing bacteria over such pH range. Since the binding force is mainly electrostatic, the architecture and orientation of the functional groups on the NP surface are not critical. Finally the captured bacteria were analyzed using matrix-assisted laser desorption/ionization mass spectrometry. The minimum detection limit was 10(4) CFU/mL and the analysis time was reduced to be less than 1 hour. In addition, the detection limit could be reduced to an extremely low concentration of 50 CFU/mL when captured bacteria were cultivated.

The direct modulation of Antrodia cinnamomea (AC) on the prominent role of liver fibrosis-hepatic stellate cells (HSCs) in situ remains unclear. Firstly, the administration of A. cinnamomea mycelial extract (ACME) could improve liver morphology and histological changes including collagen formation and GPT activity in the liver of thioacetamide (TAA)-injured rats. The morphology and fatty acid restore of TAA-induced HSCs (THSCs) returned to the non-chemical induced HSCs (NHSCs) type as measured by immunofluorescence and Oil Red O staining. PPARγ was upregulated associated with the lowering of α-SMA protein in NHSC-ACME. ACME inhibited the MMP-2 activity in NHSCs by gelatin Zymography. After LC-MS/MS, the cytoskeleton (tubulin, lamin A) and heat shock protein 8 in NHSC-ACME, and guanylate kinase, brain-specific kinase, SG-II and p55 proteins were downregulated in THSC-ACME. Whereas MHC class II, SMC6 protein, and phospholipase D were upregulated in NHSC-ACME. Furthermore, PKG-1 was downregulated in NHSC-ACME and upregulated in THSC-ACME. SG-II and p55 proteins were downregulated in NHSC-ACME and THSC-ACME by Western blotting. Taken together, the beneficial effect of A. cinnamomea on the induction of HSC cellular proteins is potentially applied as an alternative and complementary medicine for the prevention and amelioration of a liver injury.

Viral replication depends on host proteins to supply energy and replication accessories for the sufficient production of viral progeny. In this study, we identified fructose-bisphosphate aldolase A as a binding partner of Japanese encephalitis virus (JEV) untranslated regions (UTRs) on the antigenome via RNA affinity capture and mass spectrometry. Direct interaction of aldolase A with JEV RNAs was confirmed by gel mobility shift assay and colocalization with active replication of double-stranded RNA in JEV-infected cells. Infection of JEV caused an increase in aldolase A expression of up to 33%. Knocking down aldolase A reduced viral translation, genome replication, and viral production significantly. Furthermore, JEV infection consumed 50% of cellular ATP, and the ATP level decreased by 70% in the aldolase A-knockdown cells. Overexpression of aldolase A in aldolase A-knockdown cells increased ATP levels significantly. Taken together, these results indicate that JEV replication requires aldolase A and consumes ATP. This is the first report of direct involvement of a host metabolic enzyme, aldolase A protein, in JEV replication.

Sample preparation methods used for genetically modified organisms (GMOs) analysis are often time consuming, require extensive manual manipulation, and result in limited amounts of purified protein, which may complicate the detection of low-abundance GM protein. A robust sample pretreatment method prior to mass spectrometry (MS) detection of the transgenic protein (5-enolpyruvylshikimate-3-phosphate synthase [CP4 EPSPS]) present in Roundup Ready soya is investigated. Liquid chromatography-multiple reaction monitoring tandem MS (nano LC-MS/MS-MRM) was used for the detection and quantification of CP4 EPSPS. Gold nanoparticles (AuNPs) and concanavalin A (Con A)-immobilized Sepharose 4B were used as selective probes for the separation of the major storage proteins in soybeans. AuNPs that enable the capture of cysteine-containing proteins were used to reduce the complexity of the crude extract of GM soya. Con A-sepharose was used for the affinity capture of β-conglycinin and other glycoproteins of soya prior to enzymatic digestion. The methods enabled the detection of unique peptides of CP4 EPSPS at a level as low as 0.5% of GM soya in MRM mode. Stable-isotope dimethyl labeling was further applied to the quantification of GM soya. Both probes exhibited high selectivity and efficiency for the affinity capture of storage proteins, leading to the quantitative detection at 0.5% GM soya, which is a level below the current European Union's threshold for food labeling. The square correlation coefficients were greater than 0.99. The approach for sample preparation is very simple without the need for time-consuming protein prefractionation or separation procedures and thus presents a significant improvement over existing methods for the analysis of the GM soya protein.

Microwave-assisted proteolytic digestion methods have evolved into a highly effective approach and serve as an alternative to conventional overnight digestion. This approach typically exploits the unique microwave properties to facilitate the digestion of proteins into their peptides within minutes. Conventional digestion is carried out at 37°C while microwave-assisted digestion requires much higher and sometimes inconsistent temperatures. Thus, this study aims to investigate whether the faster reaction rate is due to the microwave quantum effect or the thermal effect. Quantitative mass spectrometry was used to conduct kinetic analysis of tryptic digestion for several proteins by microwave and conventional heating. The percentages of digestion products relative to internal standards showed no significant difference between microwave and conventional heating conditions at the same digestion temperature. The optimum temperature for tryptic digestion was determined to be 50°C. Furthermore, this study compares the digestion completeness indicators of several proteins under microwave and conventional heating. Again, the values obtained from microwave and conventional heating were similar given identical temperatures. The overall results prove that a nonthermal effect does not exist in microwave-assisted tryptic digestion. Therefore, conventional heating at high temperatures (50°C) can be also used to accelerate digestion reactions.

Background Antrodia cinnamomea and its host Cinnamomum kanehirae are both endemic species unique to Taiwan. Many studies have confirmed that A. cinnamomea is rich in polysaccharides and triterpenoids that may carry medicinal effects in anti-cancer, anti-inflammation, anti-hypertension, and anti-oxidation. Therefore it is of interest to study the chemical variation of regular orange-red strains and white strains, which included naturally occurring and blue-light induced white A. cinnamomea. Results The chemical profiles of A. cinnamomea extracts at different growth stages were compared using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). The TLC and HPLC profiles indicated that specific triterpenoids varied between white and regular strains. Moreover, the compounds of blue-light induced white strain were similar to those of naturally occurring white strain but retained specific chemical characteristics in more polar region of the HPLC chromatogram of regular strain. Conclusions Blue-light radiation could change color of the regular A. cinnamomea from orange–red to white by changing its secondary metabolism and growth condition. Naturally occurring white strain did not show a significantly different composition of triterpenoid profiles up to eight weeks old when compared with the triterpenoid profiles of the regular strain at the same age. The ergostane-type triterpenoids were found existing in both young mycelia and old mycelia with fruiting body in artificial agar-plate medium culture, suggesting a more diversified biosynthetic pathway in artificial agar-plate culture rather than wild or submerged culture.

The impact of microwave irradiation on the in-solution digestion processes and the detection limit of proteins are systematically studied. Kinetic processes of many peptides produced through the trypsin digestion of various proteins under microwave heating at 50°C were investigated with MALDI-MS. This study also examines the detection limits and digestion completeness of individual proteins under microwave heating at 50°C and at different time intervals (1, 5 and 30 min) using LC-MS. We conclude that if the peptides without missed cleavage dictate the detection limit, conventional digestion will lead to a better detection limit. The detection limit may not differ between the microwave and conventional heating if the peptides with missed cleavage sites and strong intensity are formed at the very early stage (i.e., less than 1 min) and are not further digested throughout the entire digestion process. The digestion of Escherichia coli lysate was compared under conventional and short time (microwave) conditions. The number of proteins identified under conventional heating exceeded that obtained from microwave heating over heating periods less than 5 min. The overall results show that the microwave-assisted digestion is not complete. Although the sequence coverage might be better, the detection limit might be worse than that under conventional heating.

The analysis of bacteria in foods is a challenge because the food matrices might severely interfere with bacterial detection. Pathogens in milk/pudding/coffee were isolated with magnetized zirconium hydroxide, directly cultivated without colony isolation, and then analyzed using MALDI and LC–MS/MS. The analysis was less time-consuming than conventional biological methods because a colony isolation procedure was not required. Further, the MS analysis of bacteria was broad-spectrum detection that did not need prior knowledge of the target information such as DNA sequences, as required in PCR detection. The results showed that the bacteria stayed alive when attached to the synthesized magnetic metal hydroxide. We could concentrate and identify both Gram-positive ( Enterococcus faecalis and Staphylococcus aureus ) and Gram-negative ( Escherichia coli and Vibrio parahaemolyticus ) bacterial cells using the magnetized zirconium hydroxide immobilization approach. The detection limit of E. faecalis spiked into milk was down to a level as low as 32 CFU/mL when cultivation of the isolated bacteria was performed prior to MS analysis. The immobilization-based MS approach is simple and allows analysis of microorganisms in complex food samples.

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.

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 degrees 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.

Reaction kinetics of Reactive Blue C.I. 19 (RB 19) dye and sodium hypochlorite (NaOCl) were investigated. Decline of RB 19 was monitored continuously by an ultraviolet (UV)-Vis spectrophotometer to avoid sampling errors. The purpose of this study is to develop a kinetic model for reactor and process design to establish hypochlorite oxidation as a viable alternative to advanced oxidation processes for textile effluent decolorization. Results were in agreement with 0.5-order reaction kinetics with respect to the RB 19 concentration. The lumped rate constant of the pseudo-0.5-order reaction is proportional to the NaOCl concentration. The Arrhenius activation energy obtained from the slope of the best-fit line is 76.6 kJ/mol, with a corresponding preexponential factor of 1.388 × 1010. The reaction rate is strongly affected by the pH of the solution. During the oxidation decomposition process, color was rapidly removed by the NaOCl but chemical oxygen demand (COD) resisted the treatment. A molar ratio...

Mycoplasma hyopneumoniae colonizes at the porcine respiratory-ciliated epithelial cells and causes the enzootic pneumonia of swine. The adhesion step is crucial in the colonization process. A few adhesion molecules have been characterized, and the concurrent receptors from the porcine ciliated cells have also been suggested to recognize the adhesion molecules. In the present study, the interactions between M. hyopneumoniae and porcine tracheal ciliated cells were investigated by employing the Far-Western blotting method. The results indicate that aconitase, lamin A/C, and peroxiredoxin of the porcine tracheal ciliated cell may interact specifically with the mycoplasmal proteins. We speculate that these mycoplasmal proteins could be secreted cleavage products, and their relative small size may enable them to penetrate into ciliated cells interfering with important metabolic pathways and other critical cellular processes.

New macrocyclic Schiff base Pd(II) compounds were synthesized by treating N4 and N2O2 macrocycles with palladium chloride in a 1 : 1 ratio. The resulting macrocyclic compounds were characterized by elemental, IR, 1H-NMR, 13C-NMR, mass, molar conductance, magnetic susceptibility, electronic spectra, and thermal analysis. These compounds were used as catalysts in the development of an efficient catalytic method for reduction of organic substrates having nitro, olefinic, acetylenic, and aldehyde groups under mild reaction conditions. The biological activities of all the macrocycles and macrocyclic Pd(II) compounds have been tested against gram positive (Bacillus subtilis and Staphylococcus aureus) and gram negative (Escherichia coli and Klebsiella pneumonia) bacteria and found to be more active than commercially available antibacterial drugs like Streptomycin and Ampicillin.

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.

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.

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.

Mass spectrometry (MS) has become a powerful and popular method to analyze macromolecules from biological systems towards the application of clinical chemistry. Disease markers related to infections can be identified with MS analysis in combination with electrophoresis or chromatographic separations. Further, direct analysis of whole pathogenic bacterial cells (taken directly from a colony) by MS can reveal specific biomarkers that can be used for taxonomy. A brief introduction to the two advanced ionization techniques, electrospray ionization and matrix-assisted laser desorption/ionization, for MS is provided in this review. Sample preparation, separation and MS-related techniques for staphylococcal proteins analysis are summarized. The review is concluded with some current clinical applications of mass spectrometry in the area of biomarker research, vaccine development, diagnosis and strain typing of infectious Staphylococci.

CE-MS/MS analysis of proteolytic digests of bacterial cell extracts was combined with SEQUEST searching and a new scoring system to identify bacteria species in bacterial mixtures. Searches of MS/MS spectra against protein databases enabled the identification of bacterial species by the matching of the proteins associated with the corresponding species. An empirical scoring function was obtained by evaluating the SEQUEST search results of 38 samples that contained single bacterial species. The scoring by the empirical function helped move up the positive identification results from their original positions in the ranking based on Xcorr values alone. Therefore, the identification of bacteria in the samples that contained bacterial mixtures was improved. Bacterial species in 20 bacterial mixtures, including one real sample, were correctly identified by database searches and the new scoring function.

Sample preparation is the first and the most crucial step in the identification of microorganisms. In this chapter, an overview is presented on different sample preparation steps used in the context of rapid analysis of microorganisms by mass spectrometry (MS). Sample preparation methods that might eliminate the need for culturing of the target cells (the key rate-limiting step) to obtain detectable signals are important for the rapid analysis of microorganisms. Microbial cell and biomarker enrichment through various affinity techniques are useful to enhance the sensitivity and accuracy of microbial identification. Combining the methodologies for cell lysis and biomarker solubilization will facilitate the rapid identification of microorganisms. Efficient biomarker separation techniques such as liquid chromatography may guarantee a more accurate MS identification of drug-resistance-related biomolecules in microbial mixtures or complex biological samples. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is the method of choice for the rapid identification of microorganisms. This chapter concludes with a brief discussion of the selection of MALDI matrices and matrix solvents.

Analysis of microbial mixtures in complex systems, such as clinical samples, using mass spectrometry can be challenging because the specimens may contain mixtures of several pathogens or both pathogens and nonpathogens. We have successfully applied capillary electrophoresis-selective MS/MS of unique peptide marker ions to the identification of common pathogens in clinical diagnosis. We searched the CE-MS/MS spectra acquired from the proteolytic digests of pure bacterial cell extracts against protein databases. The identified peptides that matched a protein associated with a particular pathogen were selected as marker ions to identify that bacterium in clinical specimens. Thirty-four clinical specimens, obtained from pus, wound, sputum, and urine samples, were analyzed using both biochemical and selective MS/MS methods. The bacteria in these clinical samples were cultivated directly, without prior isolation of a pure colony, before performing the selective MS/MS analyses. The bacteria analyzed included both Gram-positive and -negative strains. The match with respect to the pathogens identified was good between the biochemical and the selective MS/MS methods; the matching rate was 91%. The rate was as high as 97% when not considering two specimens for which the bacteria were not grown successfully. Two of the specimens that we identified using the biochemical method as containing two bacterial species were confirmed also through selective tandem MS analysis.

This paper describes a method—using a combination of LC-MS/MS of selected bacteria-specific peptides and database search—for determining the species of bacteria present in a mixture. We identified the proteotypic peptides that were associated with specific bacteria by searching protein databases for the LC-MS/MS data. The retention time windows for specific peptide markers were used as an extra constraint so that the peptide markers of many bacterial species could be analyzed in a single LC-selective proteotypic-peptide analysis (SPA). We performed LC-MS/MS analyses on the proteolytic digest of cell extracts and monitored only the selected marker peptide ions at given elution time windows. The corresponding bacterial species could be characterized when the selected peptides that eluted at expected elution windows were identified correctly from the database. We managed to identify up to eight bacterial species simultaneously during a single LC-MS/MS analysis, as well as bacteria mixed in various abundances. Two marker ions having similar values of m/z, but obtained from two different bacterial samples, which would otherwise be selected as precursors within mass tolerance and would complicate the MS/MS data, were time-resolved using LC and then used to correctly identify their bacterial sources. The coupling of selective MS/MS monitoring with separation methods, such as LC, provides a highly selective and accurate analytical method for characterizing complex mixtures of bacterial species. Copyright © 2006 John Wiley & Sons, Ltd.

The work describes the accelerated enzymatic digestion of several proteins in various solvent systems under microwave irradiation. The tryptic fragments of the proteins were analyzed by matrix-assisted laser desorption/ionization mass spectrometry. Under the influence of rapid microwave heating, these enzymatic reactions can proceed in a solvent such as chloroform, which, under traditional digestion conditions, renders the enzyme inactive. The digestion efficiencies and sequence coverages were increased when the trypsin digestions occurred in acetonitrile-, methanol- and chloroform-containing solutions that were heated under microwave irradiation for 10 min using a commercial microwave applicator. The percentage of the protein digested under microwave irradiation increased with the relative acetonitrile content, but decreased as the methanol content was increased. These observations suggest that acetonitrile does not deactivate the enzyme during the irradiation period; in contrast, methanol does deactivate it. In all cases, the digestion efficiencies under microwave irradiation exceed those under conventional conditions.

Protein particles undergo Brownian motion and collisions in solution. The diffusive collisions may lead to aggregation. For proteins to fold successfully the process has to occur quickly and before significant collision takes place. The speed of protein folding was deduced by studying the correlation time of a lysozyme refolding process from autocorrelation function analysis of the mean collision time and aggregation/soluble ratio of protein. It is a measure of time before which an aggregate can be formed and also is the time measure for a protein to fold into a stable state. We report on the protein folding stabilizing time of a lysozyme system to be 25.5-27.5 micros (

In this paper, we propose a new strategy for identifying specific bacteria in bacterial mixtures by using CE-selective MS/MS of peptide marker ions associated with the bacteria of interest. We searched the CE-MS/MS spectra acquired from the proteolytic digests of pure bacterial cell extracts against protein databases. The identified peptides that match the protein associated with the corresponding species were selected as marker ions for bacterial identification. Specific peptide marker ions were obtained for each of the following three pathogens: Pseudomonas aeruginasa, Staphylococcus aureus, and Staphylococcus epidermidis. To identify a bacterial species in a sample, we performed CE-MS/MS analysis of the selected marker ions in the proteolytic digest of the cell extract and then performed protein database searches. The selected peptides that we identified correctly from Xcorr values ranking at the top of the search results allowed us to identify the corresponding bacterial species present in the sample. We have applied this method successfully to the identification of various mixtures of the three pathogens. Even minor bacterial species present at a concentration of 1% can be identified with great confidence. This method for CE-MS/MS analysis of bacteria-specific marker peptides provides excellent selectivity and high accuracy when identifying bacterial species in complex systems. In addition, we have used this approach to identify P. aeruginasa in a saliva sample spiked with E.coli and P. aeruginasa.

This work examines the interactions between a large polypeptide, oxidized insulin chain A (ICA), and several metal ions (Na + , K + , Mn 2+ , Fe 2+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ , and Pd 2+ ) by using tandem mass spectrometry. Fragmentation spectra of the anionic complexes show that each metal ion complex undergoes specific types of interactions, suggesting that different conformations are favored for each complex. The overall results are consistent with the data obtained from ion mobility measurements. Thus, the tandem mass spectrometry approach represents a complementary method for probing the conformations of metal–peptide complexes. The fragmentation spectra obtained from the complexes of the enzyme-digested ICA fragments with metal ions also reflect specific metal–peptide interactions parallel with those for corresponding metal–ICA complexes.

Collisionally ac ti vated dis so ci a tion (CAD) of glycerophosphoglycerol, glycerophosphocholine, gly cero phosphoethanolamine, and glycerophosphoserine spe cies was ex am ined us ing quadrupole ion trap mass spec trom e try (QITMS). Since the spec tral pat terns ob tained by QITMS may dif fer from those ob tained us ing sec tor or quadrupole mass spec trom e try, meth ods em ployed in the struc ture anal y sis of phospholipids on a sec tor or quadrupole mass spec trom e ter are not nec es sar ily use ful on an ion trap mass spec trom e t er. CAD (or MS/MS) of sodiated phospholipids was pro posed to struc tur ally an a lyze phospholipids. The CAD spec tra of such ions ob tained by QITMS re vealed in for ma tive ions. Prod uct ions that cor re sponded to the loss of po lar heads iden ti fied the lipid classes. Prod uct ions formed by the loss of fatty acyl sub stitu ents from sn-1 and sn-2 po si tions were also ob served. A pref er en tial loss of the sn-1 fatty acyl group over the loss of the sn-2 fatty acyl group was ob served among the phospholipid stan dards ex am ined. This fa cil i tated the de ter mi na tion of the iden ti ties and po si tions of fatty acyl sub stitu ents.

Metastable decay rates of two peptides, RPPGFSPF and PKPQQFFGLM, were determined from ions produced in an external matrix-assisted laser desorption/ionization source with a Fourier transform mass spectrometer. An isolation and subtraction method that gives difference spectra was employed to monitor the product formation and metastable decays. The dependence of metastable decay rates on laser fluences and matrixes was demonstrated.

Reactions of gold anions and cations generated by laser desorption/ionization were studied in the FTICR spectrometer. Au− associated with C6F6 to give the novel Au−(C6F6) complex, whose binding energy was estimated to be 24 ± 4 kcal mol−1 from analysis of the radiative association (RA) kinetics. Au+ associated with C6F5H to give Au+(C6F5H), with binding energy estimated to be 31 kcal mol−1. Au+ reacted with C6H6 to form the well known Au+(C6H6) and Au+(C6H6)2 complexes. The observation of rapid charge transfer from Au+(C6H6) to C6H6 was interpreted as showing that benzene binds more strongly to neutral Au than to Au+. The neutral Au–C6H6 bond is accordingly concluded to be stronger than about 70 kcal mol−1.

Association reactions of Cd+ with benzene and of Ag+ with acetone and several unsaturated hydrocarbons were observed in the Fourier-transform ion cyclotron resonance (FT-ICR) spectrometer. The reactions were presumed to occur by radiative association (RA) involving infrared photon emission, and the kinetics were analyzed to derive bond strengths for the ion−neutral complexes. To supply the structures, infrared frequencies, and infrared intensities required for this analysis, ab initio calculations at the Hartree−Fock (HF) and second-order Moeller−Plesset perturbation theory (MP2) levels were carried out for the reactants and the association complexes, and the results are reported. The RA kinetics analysis yielded values for the binding energies of 1.41 ± 0.2 eV for Cd+(benzene), 1.68 ± 0.2 eV for Ag+(benzene), 1.66 ± 0.2 eV for Ag+(acetone), 1.70 ± 0.2 eV for Ag+(isoprene), and 1.71 ± 0.2 eV for Ag+(2-pentene). The MP2-derived modeling gave higher (and more reliable) binding energies than the HF-derived m...

Hot ions of 9-cyanophenanthrene were produced by multiphoton ionization at 193 nm. The radiative cooling rate of the hot ions was measured thermometrically using time-resolved photodissociation (TRPD) as the internal energy probe. In this application of the TRPD technique two-photon dissociation of the ions at 355 nm was used to measure the rate for the C 2 H 2 loss channel as a function of cooling time following the ionization laser pulse. Since the TRPD rate depends on the ion internal energy the dissociation rate measurements gave a cooling curve for the initially hot ions. The radiative cooling time constant was measured as 330 ms. The cooling rate observed here is faster than the radiative cooling rates of comparable nonpolar ions. This enhancement of radiative cooling is attributed to high vibrational transition dipole moments associated with the polar CN group.

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 (?-BTX), a neurotoxin derived from Bungarus multicinctus with specific blockade of alpha7-nicotinic acetylcholine receptor (?7-nAChR). The electrostatic binding of cND??-BTX was mediated by the negative charge of the cND and the positive charge of the ?-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 ?-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 ?-BTX presented a yellow color at the same location, which indicated that ?-BTX proteins were conjugated with cND particles. Xenopus laevis's oocytes expressed the human ?7-nAChR proteins by microinjection with ?7-nAChR mRNA. The cND??-BTX complexes were bound to ?7-nAChR locating on the cell membrane of oocytes and human lung A549 cancer cells analyzed by laser scanning confocal microscopy. The choline-evoked ?7-nAChR-mediated inward currents of the oocytes were blocked by cND??-BTX complexes in a concentration-dependent manner using two-electrode voltage-clamp recording. Furthermore, the fluorescence intensity of cND??-BTX binding on A549 cells could be quantified by flow cytometry. These results indicate that cND-conjugated ?-BTX still preserves its biological activity in blocking the function of ?7-nAChR, and provide a visual system showing the binding of ?-BTX to ?7-nAChR.

Tri-tert-butylbenzene ions were heated by CO 2 laser irradiation to an average internal energy of 0.63 eV in the ion cyclotron resonance ion trap. Their subsequent cooling by collisional and radiative processes was measured thermometrically using time-resolved photodissociation (TRPD) at 615 nm. Master equation modeling of ion heating and cooling was carried out to calculate the internal energy distribution of the ions for deconvolution of the TRPD curves. Ion cooling in both low-pressure (radiative cooling) and high-pressure (collisional cooling) regimes was indistinguishable from simple exponential energy decay

Phosphopeptides have been isolated and concentrated by use of polyethyleneimine (PEI)-modified magnetic nanoparticles as an extremely specific affinity probe. The particles specifically captured phosphopeptides from a tryptic digest of a protein mixture that contained 0.07% (mole/mole) phosphoproteins, which is the highest specificity obtained to date. The time required for enrichment of the phosphopeptides was 1 min only. PEI-modified magnetic nanoparticles carry positive charges over a wide range of pH—between 3 and 11. This feature means the particles are effectively dispersed in solution during phosphopeptide capture. Mass spectrometric analysis revealed the very high efficiency of enrichment of phosphopeptides that contain both single and multiply-phosphorylated sites. The detection limit in the analysis of phosphopeptides obtained from both bovine α-casein and β-casein by matrix-assisted laser desorption/ionization mass spectrometry was 5 fmol. This approach was also used to enrich the phosphopeptides in a protein digest obtained from non-fat milk.