Simple and sensitive bacterial quantification by a flow-based kinetic exclusion fluorescence immunoassay.

A flow-based immunoassay system utilizing secondary-antibody coated microbeads and Cy5-secondary antibody for signal production was successfully developed to quantitate target bacteria with a kinetic exclusion assay (KinExA 3000 Instrument). It directly measured the concentration of unliganded antibody separated from the equilibrated mixture of antibody and bacteria through a 0.2 microm polyethersulfone membrane, enabling it to quantify the concentration of bacteria. The novel method demonstrated the qualities of rapidness, sensitivity, high accuracy and reproducibility, and ease to perform. Detection of Pseudomonas aeruginosa and Staphylococcus aureus was accomplished with low detection limits of 4.10 x 10(6) and 5.20 x l0(4)cells/mL, respectively, with an assay time of less than 15 min. The working ranges for quantification were 4.10 x l0(6) to 1.64 x l0(10)cells/mL for P. aeruginosa, and 5.20 x l0(4) to 1.04 x l0(9)cells/mL for S. aureus. It yielded an assay with at least 10-fold greater sensitivity than ELISA and could correctly assess the concentration of predominant bacterium spiked in the mixture of P. aeruginosa and S. aureus. With this reliable platform, the average amount of antibody bound by one cell in the maximum capability could be further provided: (1.6-2.5) x l0(5) antibodies for one P. aeruginosa cell and (2.2-2.7) x l0(8) antibodies for one S. aureus cell. The KinExA system is flexible to determine different kinds of bacteria conveniently by using anti-mouse IgG as the same immobilizing agent. However, a higher specificity of the antibodies to the target bacteria will be required for the use of this system with higher detection sensitivity.

Case study of electrochemical metal removal from actual sediment, sludge, sewage and scallop organs and subsequent pH adjustment of sediment for agricultural use.

In this paper we propose a method for chemical-free removal of metal from lake sediment, and its subsequent pH adjustment, based on electrochemical migration and precipitation. Such a method would enable the utilization of sediment as composting material. Sediment was placed in the anode side of a dual-bath electrochemical reactor separated by a thimble-shape cellulose filter from the cathode side, which was filled with pure water. When voltage was applied, contaminant metals in the sediment on the anode side migrated toward the cathode side, and precipitated due to the alkaline conditions caused by the cathodic reaction. After 10 days of electrolysis with 400 mA of constant current of 150 g wet lake sediment, the removal ratios of 13 kinds of elements after the electrochemical treatment were measured. Cd and Zn, the elements for which agricultural standards apply, showed 98% and 86% removal, respectively. The type of metal removed changed over time, and the order of removal was roughly from light metals to heavy metals. The acidified lake sediment after electrolysis could be neutralized without significant recontamination with Zn and Cd by using the alkaline cathode solution collected during electrolysis under a condition of tap water overflow at a rate of 1.5 L/h. The electrochemical metal removal method was effective not only for lake sediment, but also for municipal sludge cake, human sewage, and contaminated scallop organs. Cathode overflow during electrolysis tended to increase metal removal and decrease required voltage.

Axitinib-induced reversible posterior leukoencephalopathy syndrome in a patient with metastatic renal cell carcinoma.

A 61-year-old woman with metastatic renal carcinoma was treated with axitinib as a second-line tyrosine kinase inhibitor. Thirteen days after the treatment, the patient developed reversible posterior leukoencephalopathy syndrome (RPLS). Her symptoms and imaging findings resolved after withdrawal of axitinib, blood pressure control, and administration of glycerin and levetiracetam. RPLS should be kept in mind as a possible rare adverse event after axitinib administration.

Symbiotic association in Chlorella culture.

Chlorella sorokiniana IAM C-212 has long been maintained in slant culture as a mixed strain, representing an associated natural microbial consortium. In this study, the consortium was separated and five nonalgal constituents, a fungal strain (CSSF-1), and four bacterial strains (CSSB-1, CSSB-2, CSSB-3, and CSSB-4) were isolated and identified. 16S rDNA sequence analysis revealed that strains CSSB-1, CSSB-2, CSSB-3, and CSSB-4 were close to Ralstonia pickettii (99.8% identity), Sphingomonas sp. DD38 (99.4% identity), Microbacterium trichotecenolyticum (98.6% identity), and Micrococcus luteus (98.6% identity) respectively. 18S rDNA sequence analysis revealed that strain CSSF-1 resembled Acremonium-like hyphomycete KR21-2 (98.8%). The fungal strain CSSF-1 and one of the bacterial strains, CSSB-3, were found to promote the growth of Chlorella while the presence of bacterial strains CSSB-1 and CSSB-2 had no effect. Strain CSSB-4 could not be subcultured so its role was not elucidated. These results show that the interaction between Chlorella and its symbionts under photoautotrophic conditions involved both mutualism and commensalisms. The chlorophyll content of mixed strain was stable in long-term cultivation (7 months) while the chlorophyll content of a pure culture showed a marked decline. Electron microscopic analysis showed the two bacterial strains CSSB-2 and CSSB-3 were harbored on the sheath excreted by Chlorella, while the fungal strain CSSF-1 and the bacterial strain CSSB-1 directly adhered to the Chlorella cell surface. This report is the first observation of a symbiotic relationship among fungus, bacteria, and Chlorella, and the first observation of direct adhesion of fungus and bacteria to Chlorella in a consortium.

Effect of power-frequency magnetic fields on genome-scale gene expression in Saccharomyces cerevisiae.

To estimate the effect of 50 Hz magnetic-field exposure on genome-wide gene expression, the yeast Saccharomyces cerevisiae was used as a model for eukaryotes. 2D PAGE (about 1,000 spots) for protein and cDNA microarray (about 5,900 genes) analysis for mRNA were performed. The cells were exposed to 50 Hz vertical magnetic fields at 10, 150 or 300 mT r.m.s. for 24 h. As positive controls, the cells were exposed to aerobic conditions, heat (40 degrees C) or minimal medium. The 2D PAGE and microarray analyses for the positive controls showed high-confidence differential expression of many genes including those for known or unknown proteins and mRNAs. For magnetic-field exposure, no high-confidence changes in expression were observed for proteins or genes that were related to heat-shock response, DNA repair, respiration, protein synthesis and the cell cycle. Principal component analysis showed no statistically significant difference in principal components, with only insignificant differences between the magnetic-field intensities studied. In contrast, the principal components for the positive controls were significantly different. The results indicate that a 50 Hz magnetic field below 300 mT did not act as a general stress factor like heat shock or DNA damage, as had been reported previously by others. This study failed to find a plausible differential gene expression that would point to a possible mechanism of an effect of magnetic fields. The findings provide no evidence that the magnetic-field exposure alters the fundamental mechanism of translation and transcription in eukaryotic cells.

Evaluation of a compact bench top immunoassay analyzer for automatic and near continuous monitoring of a sample for environmental contaminants.

A compact bench top immunoassay analyzer is evaluated and shown to possess sufficient automation to allow continuous unattended sampling and measuring while still achieving the theoretical (antibody affinity based) detection limit for analyte. The system is comprised of antigen coated particles in a disposable flow cell held at the focus of a filter fluorometer. Capture of fluorescently labeled antibody from the flow stream is inhibited by analyte in the sample, allowing analyte concentrations to be determined from the fluorescent intensity. The disposable cell was designed to allow easy end user changing of test specificity, e.g. for selection of any member of a panel of environmental contaminants. Standard curves are shown for six analytes of environmental interest, dioxin F114 (2,3,4,7,8-PeCDF), the pesticide Fenitrothion, three coplanar PCBs, including the most toxic, PCB 126, and estradiol. In each case the curves are constructed using antibody concentrations at or below the Kd of the antibody, assuring that the sensitivity shown is limited by the antibody itself rather than the analyzer. The dynamic range for the six analytes investigated ranged from a low of 5 to 340 pM for fenitrothion to a high of 0.8 to 59 nM for dioxin F114, and is correlated to the antibody Kd in every case. Data is also shown for 17 consecutive samples, including both high and low values, measured completely automatically over a period of hours. With further development and characterization, the bench top analyzer is expected to fill an important niche in environmental testing.

Analysis of gene expression in yeast protoplasts using DNA microarrays and their application for efficient production of invertase and alpha-glucosidase.

The global gene expression of cultured Saccharomyces cerevisiae protoplasts was compared with that of cells using DNA microarray. Quantitative and qualitative analyses revealed that after 6 h of cultivation, 416 gene transcript levels (about 7.1% in all) in the cultured protoplasts were different from those in the cells. Various characteristics and functions of the protoplasts were predicted from the analysis of the gene functions. The cultured protoplasts were more sensitive to oxidative stress than the cultured cells. Their cell cycles were arrested at the G1 phase and cell wall synthesis was promoted. Carbohydrate metabolism was activated in cultured protoplasts, while amino acid biosynthesis was inhibited. Furthermore, some genes associated with the secretory pathway of metabolites were activated, leading to active secretion of these metabolites into the broth. As an example of the application of DNA microarray analysis, we developed two novel methods for the production of useful enzymes based on the characteristics of protoplasts. One was the production of invertase based on the activated secretory pathway, while the other was the production of alpha-glucosidase based on the activated carbohydrate metabolism. The secretion of invertase and alpha-glucosidase was promoted in cultured protoplasts. The invertase and alpha-glucosidase productivities in the cultured protoplasts were 657 U and 218 U, respectively. On the other hand, only 227 U of invertase was produced, while alpha-glucosidase was not detected, in the cultured cells. The fragile protoplasts were immobilized in agarose gel to protect them from hydrodynamic stress. Four repeated-batch cultures with the immobilized protoplasts were performed, leading to the production of 1574 U of invertase and 739 U of alpha-glucosidase. The same productivities were obtained when this system was scaled up by 10-fold (invertase: 13304 U; alpha-glucosidase: 7688 U).

Kinetic rate constant for electron transfer between ferrous ions and novel Rusticyanin isoform in Acidithiobacillus ferrooxidans.

Here we report the kinetic rate constant for electron transfer from ferrous ions to a novel rusticyanin isoform in Acidithiobacillus ferrooxidans. The second order rate constant for this isoform is shown to be approximately one half that of the previously known type, 0.09 M(-1)s(-1) vs. 0.14 M(-1)s(-1).

Isolation and identification of a novel strain of the genus Ochrobactrum with phenol-degrading activity.

A bacterial strain AS1 belonging to the genus Ochrobactrum, was isolated from an enriched phenol-activated sludge in Egypt. This strain grew aerobically on phenol as the sole carbon source using the meta-cleavage pathway at high phenol-degrading rates compared with those in a previous report.

Delayed surgical treatment for a traumatic bilateral cervical facet joint dislocation using a posterior-anterior approach: a case report.

INTRODUCTION: There have been few reports of patients with bilateral cervical facet dislocations that remain untreated for eight weeks or more. We report the case of a 76-year-old man with an old bilateral cervical facet joint dislocation fracture that was treated by posterior-anterior reduction and fixation. CASE PRESENTATION: A 76-year-old Asian man was involved in a road traffic accident. He presented with neck pain and arm pain on his right side, but motor weakness and paralysis were not observed. He was treated conservatively; however, instability and spondylolisthesis at the C5 to C6 joint increased eight weeks after the injury. We performed a posterior-anterior reduction and fixation. After surgery, bony union was achieved, and his neck pain and arm pain disappeared. CONCLUSION: We recommend reduction and fixation surgery if a patient has an old bilateral facet joint dislocation fracture in the cervical spine.

A surgical treatment for adult muscular torticollis.

Adult presentation of neglected congenital muscular torticollis (CMT) is rare. Therefore, efficacy of surgical treatment for adult CMT is unclear. We experienced a case of neglected CMT in a 28-year-old male patient and report the surgical result here. We conducted unipolar resection at the distal end of the sternocleidomastoid muscle (SCM). After surgery, the range of neck movement and head tilt improved, and his appearance was cosmetically improved despite the long-standing nature of the deformity. We concluded that surgical management of adult patients with neglected congenital muscular torticollis may be a treatment option.

Sensitive detection of ochratoxin A in wine and cereals using fluorescence-based immunosensing.

Ochratoxin A (OTA) is a mycotoxin found in a wide range of food and feedstuffs. Intake of OTA-contaminated food causes health concern due to the harmful effects reported on humans and animals. Much effort is currently devoted to set up and optimise highly sensitive and accurate methods of OTA analysis. This work describes the comparison of fluorescence-based immunosensing strategies for the analysis of OTA. First, an indirect competitive fluoroimmunoassay was designed and optimised. The assay enabled the quantification of the toxin at the levels set by the European legislation. Then, a flow-immunoassay based on kinetic exclusion measurements was developed. It showed the theoretical lowest limit of detection enabled by the affinity of the anti-OTA antibody (IC(80)=12ngL(-1) in the assay solution). Wine and cereal samples were analysed using the optimised flow system. No significant matrix effects were observed after simple pre-treatment of wine and OTA extraction from corn-flakes samples. This simple and highly sensitive automated biosensing-system allows OTA quantification in food and beverages. It is envisaged as a powerful tool for rapid and reliable toxin screening.

High-sensitive flow-based kinetic exclusion assay for okadaic acid assessment in shellfish samples.

Episodes of shellfish contamination with okadaic acid (OA) are a human health threat that is causing increasing concern. As a way to overcome the shortcomings involved in the reference methods of analysis set by legislations, alternative procedures are envisaged. This paper describes the development of different immunosensors for the analysis of OA, focusing on the comparison of their sensitivity, precision, ease of use and sample matrix effects. Initially, a surface plasmon resonance (SPR)-based immunosensor was developed, which enabled the quantification of the toxin in mussel samples at concentrations in the range of the 160 microg kg(-1) European regulatory limit with good percentages of recovery. Nevertheless, calibration curves with spiked mussel samples showed that matrix effects could not be neglected. Alternatively, a flow-immunosensing system based on kinetic exclusion measurements was developed achieving the theoretical lowest limit of detection enabled by the affinity of the anti-OA antibody (IC(70)=0.03 microg L(-1) in the assay solution). This highly sensitive automated system allows rapid and reliable OA quantification, with no significant matrix effects for the analysis of spiked mussel and scallop samples. Performance features such as high sensitivity and precision, low limits of detection and simplicity of the analysis protocol, shows the biosensing-systems based on kinetic exclusion measurements for toxin detection in shellfish samples as highly performing tools for rapid and continuous screening.

Least detectable concentration and dynamic range of three immunoassay systems using the same antibody.

The least detectable concentration (LDC) and dynamic range (DR) of three immunoassay systems are compared using four distinct antibodies (all specific for estradiol but with different affinities) on each system. The systems evaluated include the industry standard, ELISA, and two biosensors, surface plasmon resonance and kinetic exclusion. In all cases, the measurements of inhibition curves (response vs estradiol concentration) were contracted to outside experts (the biosensor manufacturers themselves in the case of the biosensors), each of whom was supplied with the same blind samples. Each biosensor manufacturer also reported an estimate of the equilibrium dissociation constant (Kd) for each of the antibodies. The LDC and DR observed for the kinetic exclusion biosensor are consistent with an interpretation of Kd limited detection while that from the other biosensor and ELISA show limits of detection somewhat above those expected for Kd limited performance. The determined LDC and DR of each biosensor is self-consistent in the sense that none of the inhibition data contradicts theoretical limits associated with the Kd as measured on that system; however, some contradictions are apparent across platforms. The use of multiple antibodies of differing Kd improves confidence that the observed differences in performance are associated with the immunoassay system rather than the particular analyte.

Acidianus manzaensis sp. nov., a novel thermoacidophilic archaeon growing autotrophically by the oxidation of H2 with the reduction of Fe3+.

A novel thermoacidophilic iron-reducing Archaeon, strain NA-1, was isolated from a hot fumarole in Manza, Japan. Strain NA-1 could grow autotrophically using H2 or S0 as an electron donor and Fe3+ as an electron acceptor, and also could grow heterotrophically using some organic compounds. Fe3+ and O2 served as electron acceptors for growth. However, S0, NO3-, NO2-, SO4(2-), Mn4+, fumarate, and Fe2O3 did not serve as electron acceptors. The ranges of growth temperature and pH were 60-90 degrees C (optimum: 80 degrees C) and pH 1.0-5.0 (optimum: pH 1.2-1.5), respectively. Cells were nearly regular cocci with an envelope comprised of the cytoplasmic membrane and a single outer S-layer. The crenarchaeal-specific quinone (cardariellaquinone) was detected, and the genomic DNA G + C content was 29.9 mol%. From 16S rDNA analysis, it was determined that strain NA-1 is closely related to Acidianus ambivalens (93.1%) and Acidianus infernus (93.0%). However, differences revealed by phylogenetic and phenotypic analyses clearly show that strain NA-1 represents a new species, Acidianus manzaensis, sp. nov., making it the first identified thermoacidophilic iron-reducing microorganism (strain NA-1T = NBRC 100595 = ATCC BAA 1057).

Improving an immunoassay response to related polychlorinated biphenyl analytes by mixing antibodies.

Immunoassays for detection of a class of closely related antigens, e.g., PCBs, have often been too specific (responding strongly to some members of the class and missing others) and no general method for adjusting the response has been described. In this paper, the difference in the response of a model immunoassay to different Kanechlors (Japanese commercial mixtures of PCBs, analogous to Aroclors in the United States) is reduced from 20- or 50-fold (depending on which antibody is used) to 3-fold when the antibodies are mixed at the proper ratio. A mathematical model based on competitive binding of two antibodies for up to four antigens has been developed and used to describe the assay performance and to predict optimum mix ratios for the antibodies used. The model (based on separate measurement of each antibody's effective Kd for each Kanechlor) provides an excellent fit to the measured mixed antibody assay response. The model is also successful in identifying cases where mixing monoclonal antibodies will not improve the response. It is thought the method described will have applicability in a variety of cases where the analytical goal is semiquantitative screening based on the total quantity of an unknown mixture of related compounds.

Photosynthetic productivity of conical helical tubular photobioreactor incorporating Chlorella sorokiniana under field conditions.

The photosynthetic performance of a conical, helical tubular photobioreactor (HTP) incorporating Chlorella sorokiniana was investigated under conditions of high temperature and light intensity during midsummer in an outdoor environment. Although the culture medium temperature exceeded 40 degrees C for approximately 5 h each day, peaking at 47.5 degrees C under sunny conditions, a photosynthetic productivity of 30.0 g x m(-2) (installation area) x day(-1) and a photosynthetic efficiency of 8.66% [photosynthetically active radiation (PAR), 400-700 nm] were achieved. A maximum photosynthetic productivity of 33.2 g x m(-2) x day(-1) was achieved on a sunny day, when solar energy input was also maximal (11.5 MJ x m(-2) x day(-1) [PAR]). On the other hand, a maximum photosynthetic efficiency of 9.54% was obtained on a day that was rainy in the morning and cloudy in the afternoon, and there was relatively little solar energy input. The average daily photosynthetic efficiency over the two culture periods (August 4 to 7 and August 10 to 13, 1999) was 7.25%. Thus, a high level of photosynthetic performance was achieved in the conical HTP incorporating Chlorella sorokiniana despite the fact that culture medium temperature was not controlled. The use of Chlorella sorokiniana in the conical HTP should be a good choice to produce microalgal biomass during the summer under field conditions.

Electrochemical regeneration of Fe(III) to support growth on anaerobic iron respiration.

Here we describe artificial help for the respiratory electron flow supporting anaerobic growth of Thiobacillus ferrooxidans through exogenous electrolysis. Flux between H(2) and a anode through cells was accomplished with electrochemical regeneration of iron. The electrochemical help resulted in a 12-fold increase in yield compared with the yield observed in its absence.

Anaerobic respiration using Fe(3+), S(0), and H(2) in the chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans.

The chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans has been known as an aerobe that respires on iron and sulfur. Here we show that the bacterium could chemolithoautotrophically grow not only on H(2)/O(2) under aerobic conditions but also on H(2)/Fe(3+), H(2)/S(0), or S(0)/Fe(3+) under anaerobic conditions. Anaerobic respiration using Fe(3+) or S(0) as an electron acceptor and H(2) or S(0) as an electron donor serves as a primary energy source of the bacterium. Anaerobic respiration based on reduction of Fe(3+) induced the bacterium to synthesize significant amounts of a c-type cytochrome that was purified as an acid-stable and soluble 28-kDa monomer. The purified cytochrome in the oxidized form was reduced in the presence of the crude extract, and the reduced cytochrome was reoxidized by Fe(3+). Respiration based on reduction of Fe(3+) coupled to oxidation of a c-type cytochrome may be involved in the primary mechanism of energy production in the bacterium on anaerobic iron respiration.