Optimization of Growth Conditions for Test Fungus Cultures used in Direct Bioautographic TLC Detection. 3. Test Fungus: Candida albicans

JPC – Journal of Planar Chromatography – Modern TLC - Optimum conditions have been established for culture of the fungus Candida albicans (ATCC 90028) for microbial detection of zones...     

Optimization of Culture Conditions for Enhanced Lysine Production Using Engineered Escherichia coli

In this study, culture conditions, including dissolved oxygen (DO) content, presence of osmoprotectants, residual glucose concentration, and ammonium sulfate-feeding strategies, were investigated for decreasing the inhibition effects of acetic acid, ammonium, and osmotic stress on l-lysine fermentation by Escherichia coli. The results revealed that higher DO content and lower residual glucose concentration could decrease acetic acid accumulation, betaine supplementation could enhance osmotic stress tolerance, and variable speed ammonium sulfate-feeding strategy could decrease ammonium inhibition. Thus, with 25 % DO content, 0–5.0 g/L of residual glucose concentration, and 1.5 g/L of betaine supplementation, 134.9 g/L of l-lysine was obtained after 72 h of culture, with l-lysine yield and productivity of 45.4 % and 1.9 g/(L?·?h), respectively.     

Metabolic Engineering of Escherichia coli Cells for Ethanol Production under Aerobic Conditions

The ethanol production by recombinant Escherichia coli introducing of pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB) from Zymomonas mobilis was investigated under aerobic conditions. In aerobic culture (K_La = 1.5 min^-1), the cells expressing pdc and adhB produced 0.4 g l^-1 ethanol when cultured for 18 h. This value was improved in BW25113Δpta/pHfdh/pTadhB-pdc following 4 g l^-1 formate feeding at 0.8 g l^-1 ethanol. In higher oxygenation level (K_La = 6.1 min^-1), the production of ethanol was further enhanced at 1.79 g l^-1 ± 0.37 g l^-1 after 24 h cultivation. Formate was found not detectable at the end of culture, indicating complete degradation this organic acid to regenerate NADH from NAD⁺. The culture strategy was effective to inactivate lactate dehydrogenase, which is major competitor for ethanol production in utilizing NADH.     

铂纳米颗粒修饰电极对大肠杆菌的电化学快速检测

本文采用了电化学沉积法制备了铂纳米颗粒化学修饰电极（PtNP/GCE）,并将它应用于大肠杆菌的检测。原理是基于检测大肠杆菌溶液中酶与底物的反应产物,对氨基酚,实现了对大肠杆菌的快速检测。采用了铂纳米颗粒修饰电极,并对检测系统进行优化,提高大肠杆菌的检测灵敏度。大肠杆菌浓度在50—1.0×10⁵cfu/ml与响应电流成良好的线性关系,最低检测限为20 cfu/ml,检测时间在4个小时以内。与传统方法相比,该电化学方法能很好地满足食品安全、环境监控和临床医学等领域中快速检测的要求。     

Optimization of TLC Detection by Phosphomolybdic Acid Staining for Robust Quantification of Cholesterol and Bile Acids

JPC – Journal of Planar Chromatography – Modern TLC - In this paper we describe a robust and sensitive detection procedure for cholesterol and selected bile acids (cholic acid,...     

Contact-electro-catalysis for Direct Synthesis of H2O2 under Ambient Conditions

Hydrogen peroxide (H₂O₂) is an indispensable basic reagent in various industries, such as textile bleach, chemical synthesis, and environmental protection. However, it is challenging to prepare H₂O₂ in a green, safe, simple and efficient way under ambient conditions. Here, we found that H₂O₂ could be synthesized using a catalytic pathway only by contact charging a two-phase interface at room temperature and normal pressure. Particularly, under the action of mechanical force, electron transfer occurs during physical contact between polytetrafluoroethylene particles and deionized water/O₂ interfaces, inducing the generation of reactive free radicals (⋅OH and ⋅O₂⁻ ), and the free radicals could react to form H₂O₂, yielding as high as 313 μmol L⁻¹ h⁻¹. In addition, the new reaction device could show long-term stable H₂O₂ production. This work provides a novel method for the efficient preparation of H₂O₂, which may also stimulate further explorations on contact-electrification-induced chemistry process.     

Optimization of the Culture Medium Used for Direct TLC-Bioautography. Application to the Detection of Antimicrobial Compounds from Cordia gilletii De Wild (Boraginaceae)

JPC – Journal of Planar Chromatography – Modern TLC - TLC-bioautography, a convenient and simple mean of testing plant extracts and pure substances for their effects on pathogenic...     

Detection reagents used for on-plate identification of organic pesticides in biological samples with preliminary separation by TLC/HPTLC

Many specific and non-specific chromogenic spray reagents have been used to detect organic pesticides (organophosphorus, organochlorine, carbamates, and pyrethroids) on thin-layer chromatographic (TLC)/high-performance thin-layer chromatographic (HPTLC) plates. To realize high sensitivity and improved selectivity, several chromogenic reagents have been introduced. The physical properties of organic pesticides reported so far are also presented in tabular form. The colors produced on TLC plates due to reactions taking place between pesticides and spray reagents are illustrated in the form of chromatograms, and reaction mechanism is also presented.

     

EDX-Spectra Simulation in Electron Probe Microanalysis. Optimization of Excitation Conditions and Detection Limits

Standardless determination of absolutely calculated concentrations (P/B based) has been successfully applied for over a decade. The PUzaf-method [1, 2] works without normalization to 100 per cent and without any information in addition to the spectrum, apart from excitation conditions (E ₀, geometry) and detector parameters. The simulation of EPMA spectra is simply the inverse procedure of this real standardless spectra evaluation. The absolute calculation of P/B-ratios based on a fundamental parameter method and a reliable computation of bremsstrahlung over the entire distribution of energies are the basis for the simulation. The accuracy of the data base is crucial for the quality of a simulation [4]. The addition of detector effects and count-statistics are necessary to compute the artificial spectra close to a real EDX data acquisition.     

Electrophoretic Behavior Study of Bacteria of Pseudomonas aeruginosa, Edwardsiella tarda and Enteropathogenic Escherichia coli by Capillary Electrophoresis with UV and Fluorescence Detection

Capillary electrophoresis approaches have been utilized for the study of bacteria under specific experimental conditions. The main objective within our research work was to study electrophoretic behaviors of Pseudomonas aeruginosa by means of capillary electrophoresis with UV and fluorescence detection. Edwardsiella tarda and Enteropathogenic escherichia coli were also included in the study. The results showed that proper pretreatment (vortexing or sonication) for each bacterial sample before injection was necessary to disperse the clusters of cells, which is helpful to observe the single peaks and better peak shape of bacteria during electrophoresis. Apart from this, it was found that ionic strength of buffer affected mobilities of Pseudomonas aeruginosa as a result of increasing of buffer concentration from 25 mM to 150 mM. Moreover, sharp and single peaks were still observed without significant increase of noise in the concentration range. Eventually, mixtures of bacteria were well separated under optimized separation conditions with UV and fluorescence detection. In the mean time, comparison of concentration sensitivities for Pseudomonas aeruginosa by UV and fluorescence detection was made. Blue light emitting diode induced fluorescence detection was found to be more sensitive (8.5-fold higher) than UV detection with home-made fluorescence detection system. Generally, proposed CE methods for the analysis of bacteria could be potentially valuable for the monitoring of bacteria contamination in real life.     

An anti E. coli O157:H7 antibody-immobilized microcantilever for the detection of Escherichia coli (E. coli).

A silicon microcantilever sensor was developed for the detection of Escherichia coli O157:H7. The microcantilever was modified by anti-E. coli O157:H7 antibodies on the silicon surface of the cantilever. When the aquaria E. coli O157:H7 positive sample is injected into the fluid cell where the microcantilever is held, the microcantilever bends upon the recognition of the E. coli O157:H7 antigen by the antibodies on the surface of the microcantilever. A negative control sample that does not contain E. coli O157:H7 antigen did not cause any bending of the microcantilever. The detection limit of the sensor was 1 x 10(6) cfu/mL when the assay time was < 2 h.     

The gene-protein database of Escherichia coli: edition 5.

The gene-protein database of Escherichia coli is both an index relating a gene to its protein product on two-dimensional gels, and a catalog of information about the function, regulation, and genetics of individual proteins obtained from two-dimensional gel analysis or collated from the literature. Edition 5 has 102 new entries--a 15% increase in the number of annotated two-dimensional gel spots. The large increase in this edition was accomplished in part by the use of a new method for expression analysis of ordered segments of the E. coli genome, which has resulted in linking 50 gel spots to their genes (or open reading frames) and another 45 to specific regions of the chromosome awaiting the availability of DNA sequence information. Communication of information from the scientific community resulted in additional identifications and regulatory information. To increase accessibility of the database it has been placed in the repository at the National Center for Biotechnology Information (NCBI) at the National Library of Medicine under the name ECO2DBASE. It will be updated twice yearly. This edition of the gene-protein database is estimated to contain entries for one-sixth of the protein-encoding genes of E. coli.     

Disinfection of Escherichia Coli Gram Negative Bacteria Using Surface Modified TiO2: Optimization of Ag Metallization and Depiction of Charge Transfer Mechanism

The antibacterial activity of silver deposited TiO₂ (Ag‐TiO₂) against Gram negative Escherichia coli bacteria was investigated by varying the Ag metal content from 0.10 to 0.50% on the surface of TiO₂. Ag depositions by the photoreduction method were found to be stable. Surface silver metallization was confirmed by EDAX and XPS studies. Photoluminescence studies show that the charge carrier recombination is less for 0.1% Ag‐TiO₂ and this catalyst shows superior bactericidal activity under solar light irradiation compared to Sol gel TiO₂ (SG‐TiO₂) due to the surface plasmon effect. The energy levels of deposited Ag are dependent on the Ag content and it varies from ?4.64 eV to ?1.30 eV with respect to the vacuum energy level based on atomic silver to bulk silver deposits. The ability of electron transfer from Ag deposit to O₂ depends on the position of the energy levels. The 0.25% and 0.50% Ag depositions showed detrimental effect on bactericidal activity due to the mismatch of energy levels. The effect of the EROS (External generation of the Reactive Oxygen Species by 0.1% Ag‐TiO₂) and IROS (Interior generation of Reactive Oxygen Species within the bacteria) on the bactericidal inactivation is discussed in detail.     

The gene-protein database of Escherichia coli: edition 4.

The gene-protein database of Escherichia coli has as its core an index that links each of the protein spots from a two-dimensional polyacrylamide gel to the gene that encodes the protein. Additional information about each protein and its gene is generated from two-dimensional gel analysis or collated from the literature to form the database. Earlier editions of the database have provided periodic updates of information. The current edition does this, but also introduces a new reference gel image produced by an electrophoresis system recently adopted in this laboratory. The new gel system was chosen because it offers an improved opportunity for other investigations to produce close replicas of the reference gel pattern, thereby allowing easier access to the information of the database and encouraging independent contribution to the database. The new gel format also is larger and hence more compatible with computer assisted image analysis, which has become essential for a project of this magnitude. This edition continues the use of the former reference gel images, but adds a reference image of an equilibrium gel of E. coli strain W3110 produced by the new standardized gel system. At this time, 55% of the protein spots annotated on the previous equilibrium reference gel for this organism have been located on the new reference image, and these identifications are included in the tables of the database.     

Direct Analysis in Real Time-Mass Spectrometry for the Rapid Detection of Metabolites of Aconite Alkaloids in Intestinal Bacteria

In the present work, direct analysis of real time ionization combined with multi-stage tandem mass spectrometry (DART-MSⁿ) was used to investigate the metabolic profile of aconite alkaloids in rat intestinal bacteria. A total of 36 metabolites from three aconite alkaloids were identified by using DART-MSⁿ, and the feasibility of quantitative analysis of these analytes was examined. Key parameters of the DART ion source, such as helium gas temperature and pressure, the source-to-MS distance, and the speed of the autosampler, were optimized to achieve high sensitivity, enhance reproducibility, and reduce the occurrence of fragmentation. The instrument analysis time for one sample can be less than 10 s for this method. Compared with ESI-MS and UPLC-MS, the DART-MS is more efficient for directly detecting metabolic samples, and has the advantage of being a simple, high-speed, high-throughput method. Graphical Abstract

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Detection of Escherichia coli O157:H7 DNA using two fluorescence polarization methods

Using stx 2 gene in verotoxin-producing Escherichia coli O157:H7 as a target DNA, polymerase chain reaction (PCR) amplification has been combined with fluorescence polarization (FP) by two distinct combination protocols. The first approach (PCR-probe-FP) requires that fluorescence labeled specific probes are hybridized with the asymmetric PCR product. In the second protocol (PCR-primer-FP), the fluorescence labeled primer is used in PCR amplification. In both methods, the PCR products are detected using fluorescence polarization. Hybridization (in the PCR-probe-FP method) and conversion (in the PCR-primer-FP method) of 5′-fluorescence labeled oligodeoxynucleotide to the PCR product are monitored by an increase in the anisotropy ratio. The results demonstrate the importance of asymmetric PCR (in the first method) and the selection of dye-modified primer concentration (in the second method) for designing a polarization strategy for the detection of DNA sequence. It has been found that the methods can be used for the identification of infectious agents. This system has also been applied to the determination of Escherichia coli O157:H7 strains.     

Bead-Based Optical Immunoassay Using Quantum-Dot Labeling and Immunocomplex Dissociation for Detection of Escherichia coli O157:H7

An immunoassay for Escherichia coli O157:H7 using quantum-dot (QD) labeling and subsequent release of the QD labels from immunocomplexes has been developed. The assay principle is that anti-E. coli O157:H7 conjugated immunomagnetic beads are used to capture E. coli O157:H7; this is followed by the binding of QD labeled antibodies to form sandwich immunocomplexes (Bead-Cell-QD); a dissociation buffer then elutes QDs from immunocomplexes by denaturing antibody or lysing cell; the fluorescence signal of the eluted QDs is measured to quantify E. coli O157:H7. This proposed approach avoids the interference of bead autofluorescence in signal transduction and, thus, enhances the detection resolution, while keeping the fast magnetic separation and sandwich binding of two selective antibodies for high specificity.     

Reveal E. coli 2.0 method for detection of Escherichia coli O157:H7 in raw beef

Reveal E. coli 2.0 is a new lateral-flow immunodiagnostic test for detection of E. coli O157:H7 and O157:NM in raw beef trim and ground beef. Compared with the original Reveal E. coli O157:H7 assay, the new test utilizes a unique antibody combination resulting in improved test specificity. The device architecture and test procedure have also been modified, and a single enrichment protocol was developed which allows the test to be performed at any point during an enrichment period of 12 to 20 h. Results of inclusivity and exclusivity testing showed that the test is specific for E. coli serotypes O157:H7 and O157:NM, with the exception of two strains of O157:H38 and one strain of O157:H43 which produced positive reactions. In internal and independent laboratory trials comparing the Reveal 2.0 method to the U.S. Department of Agriculture-Food Safety and Inspection Service reference culture procedure for detection of E. coli O157:H7 in 65 and 375 g raw beef trim and ground beef samples, there were no statistically significant differences in method performance with the exception of a single internal trial with 375 g ground beef samples in which the Reveal method produced significantly more positive results. There were no unconfirmed positive results by the Reveal assay, for specificity of 100%. Results of ruggedness testing showed that the Reveal test produces accurate results even with substantial deviation in sample volume or device incubation time or temperature. However, addition of the promoter reagent to the test sample prior to introducing the test device is essential to proper test performance.     

Optimization of Growth Medium and Enzyme Assay Conditions for Crude Cellulases Produced by a Novel Thermophilic and Cellulolytic Bacterium, Anoxybacillus sp. 527

A newly isolated Anoxybacillus sp. 527 was found to grow on crystalline cellulose as sole carbon and energy sources. Cellulases secreted by strain 527 were better induced by cellobiose, followed by glucose, lactose, sucrose, and cellulose. Cellulase secretion was enhanced by an optimized medium. Cellulase activity was increased by the addition of Ca²⁺ and NH₄⁺ and achieved maximum as 7.0 FPU ml⁻¹ at 70 °C and pH 6.0. Even at 100 °C, the enzymes were still active, which implies their potential application in large-scale cellulose conversion process.     

Crosslinking studies on the Ca2+, Mg2+-activated ATPase of Escherichia coli.

Crosslinking of membrane proteins of Escherichia coli with dithiobis (succinimidyl propionate) (DSP) resulted in loss of several enzyme activities including the Ca2+, Mg2+-activated ATPase. This enzyme was crosslinked by DSP to the membrane and was not released by dialysis at low ionic strength in the absence of dithiothreitol which could cleave the crosslinking group. DSP inactivated both phosphohydrolase and coupling activities of the solubilized ATPase. Loss of hydrolytic activity could be correlated with the extent of reaction of the alpha and/or beta subunits of the enzyme. The loss of coupling activity appeared to be associated with modification of the gamma and/or delta subunits.