In Vitro Assessment of Biocontrol Effects on Fusarium Head Blight and Deoxynivalenol (DON) Accumulation by DON-Degrading Bacteria.

Fusarium head blight (FHB) of cereals is a severe disease caused by the Fusarium graminearum species complex. It leads to the accumulation of the mycotoxin deoxynivalenol (DON) in grains and other plant tissues and causes substantial economic losses throughout the world. DON is one of the most troublesome mycotoxins because it is a virulence factor to host plants, including wheat, and exhibits toxicity to plants and animals. To control both FHB and DON accumulation, a biological control approach using DON-degrading bacteria (DDBs) is promising. Here, we performed a disease control assay using an in vitro petri dish test composed of germinated wheat seeds inoculated with F. graminearum (Fg) and DDBs. Determination of both grown leaf lengths and hyphal lesion lengths as a measure of disease severity showed that the inoculation of seeds with the DDBs Devosia sp. strain NKJ1 and Nocardioides spp. strains SS3 or SS4 were protective against the leaf growth inhibition caused by Fg. Furthermore, it was as effective against DON accumulation. The inoculation with strains SS3 or SS4 also reduced the inhibitory effect on leaves treated with 10 µg mL-1 DON solution (without Fg). These results indicate that the DDBs partially suppress the disease by degrading DON.

Draft Genome Sequence of Deoxynivalenol-Degrading Actinomycete Nocardioides sp. Strain LS1, Isolated from Wheat Leaves in Japan.

Actinomycete Nocardioides sp. strain LS1, isolated from wheat leaf, is a bacterium that degrades and assimilates the mycotoxin deoxynivalenol (DON) as the carbon source. This is the first study of the genome sequence of the DON-degrading genus Nocardioides, and it facilitates the study of genes encoding the DON-degrading pathway.

Evaluation of a surface plasmon resonance imaging-based multiplex O-antigen serogrouping for Escherichia coli using eleven major serotypes of Shiga -toxin-producing E. coli.

The early detection of Shiga toxin-producing Escherichia coli (STEC) is important for early diagnosis and preventing the spread of STEC. Although the confirmatory test for STEC should be based on the detection of Shiga toxin using molecular analysis, isolation permits additional characterization of STEC using a variety of methods, including O:H serotyping. The conventional slide agglutination O-antigen serogrouping used in many clinical laboratories is laborious and time-consuming. Surface plasmon resonance (SPR)-based immunosensors are commonly used to investigate a large variety of bio-interactions such as antibody/antigen, peptide/antibody, DNA/DNA, and antibody/bacteria interactions. SPR imaging (SPRi) is characterized by multiplexing capabilities for rapidly screening (approximately 100 to several hundred sensorgrams in parallel) molecules. SPRi-based O-antigen serogrouping method for STEC was recently developed by detecting the interactions between O-antigen-specific antibodies and bacterial cells themselves. The aim of this study was to evaluate its performance for E. coli serogrouping using clinical STEC isolates by comparing the results of slide agglutination tests. We tested a total of 188 isolates, including O26, O45, O91, O103, O111, O115, O121, O128, O145, O157, and O159. The overall sensitivity of SPRi-based O-antigen serogrouping was 98.9%. Only two O157 isolates were misidentified as nontypeable and O121. The detection limits of all serotypes were distributed between 1.1 × 106 and 17.6 × 106 CFU/ml. Pulsed-field gel electrophoresis (PFGE) revealed the heterogeneity of the examined isolates. In conclusion, SPRi is a useful method for the O-antigen serogrouping of STEC isolates, but the further evaluation of non-O157 minor serogroups is needed.

Development of a Surface Plasmon Resonance-Based Immunosensor for Detection of 10 Major O-Antigens on Shiga Toxin-Producing Escherichia coli, with a Gel Displacement Technique To Remove Bound Bacteria.

A surface plasmon resonance-based immunosensor (SPR-immunosensor) was developed for the detection of Shiga toxin-producing Escherichia coli (STEC) belonging to the O-antigen groups O26, O91, O103, O111, O115, O121, O128, O145, O157, and O159. The polyclonal antibodies (PoAbs) generated against each of the STEC O-antigen types in rabbits were purified and were immobilized on the sensor chip at 0.5 mg/mL. The limit of detection for STEC O157 by the SPR-immunosensor was found to be 6.3 × 10(4) cells for 75 s. Each of the examined 10 O-antigens on the STECs was detected by the corresponding PoAb with almost no reaction to the other PoAbs. The detected STECs were sufficiently removed from the PoAbs using gelatin or agarose gel without deactivation of the PoAbs, enabling repeatable use of the sensor chip. The developed SPR-immunosensor can be applied for the detection of multiple STEC O-antigens. Furthermore, the new antigen removal technique using the gel displacement approach can be utilized with various immunosensors to improve the detection of pathogens in clinical and public health settings.

Nano-analysis of DNA conformation changes induced by transcription factor complex binding using plasmonic nanodimers.

The plasmon resonant wavelength for a pair of gold nanoparticles, or gold nanodimer, increases inversely with the gap distance between the two nanoparticles. Taking advantage of this property, we performed nanoscale measurements of DNA conformation changes induced by transcription factor binding. Gold nanoparticles were bridged by double-stranded DC5 DNA that included binding sequences for the transcription factors SOX2 and PAX6, which interact on the DC5 enhancer sequence and activate transcription. The gold nanodimers bound by SOX2 shifted the plasmon resonant wavelength from 586.8 to 604.1 nm, indicating that SOX2 binding induces DNA bending. When the SOX2 formed a ternary complex with PAX6 on DC5, the plasmon resonant wavelength showed a further shift to 611.6 nm, indicating additional bending in the DC5 sequence. Furthermore, we investigated DNA conformation changes induced by SOX2 and PAX6 on the DC5-con sequence, which is a consensus sequence of DC5 for the PAX6 binding region that strengthens the PAX6 binding but at the same time disrupts SOX2-PAX6-dependent transcriptional activation. When the PAX6 binding sequence in DC5 was altered to DC5-con, the plasmon resonant wavelength shifted much less to 606.5 nm, which is more comparable to the 603.9 nm by SOX2 alone. These results demonstrate that SOX2-PAX6 cobinding induces a large conformation change in DC5 DNA.

Improved analysis for determining diffusion coefficients from short, single-molecule trajectories with photoblinking.

Two maximum likelihood estimation (MLE) methods were developed for optimizing the analysis of single-molecule trajectories that include phenomena such as experimental noise, photoblinking, photobleaching, and translation or rotation out of the collection plane. In particular, short, single-molecule trajectories with photoblinking were studied, and our method was compared to existing analytical techniques applied to simulated data. The optimal method for various experimental cases was established, and the optimized MLE method was applied to a real experimental system: single-molecule diffusion of fluorescent molecular machines known as nanocars.

[The effect of substituting latanoprost 0.005% for unoprostone 0.12%].

PURPOSE: To evaluate the effect of substituting latanoprost(LAT) 0.005% for unoprostone(UNO) 0.12% after a trial of unilateral treatment. METHODS: We treated 30 patients with primary open-angle glaucoma(n = 8), ocular hypertension (n = 1), or normal-tension glaucoma(n = 21) with UNO for 4 weeks in one eye and then substituted LAT for UNO. Four weeks later we measured the intraocular pressure(IOP) in the ipsilateral eye. RESULTS: The mean baseline IOP level was 18.6 +/- 3.8(mean +/- standard deviation) mmHg. The mean IOP levels(reduction rates) after UNO and LAT therapy were 16.7 +/- 3.1 mmHg (16.6%) and 14.1 +/- 3.2 mmHg (28.9%), respectively(p < 0.001). All patients who responded to UNO also responded to LAT; however, 55% of those who did not respond to UNO responded to LAT. CONCLUSIONS: If LAT is substituted for UNO, it can be predicted that 63.3% of the patients will respond.

Protective effect of nilvadipine against glutamate neurotoxicity in purified retinal ganglion cells.

We determined the effect of nilvadipine, a dihydropyridine-type calcium channel blocker, in preventing glutamate neurotoxicity in purified retinal ganglion cells (RGCs). RGCs were purified from dissociated rat retinal cells (postnatal days 6-8), using a modified two-step panning method, and cultured in serum-free medium containing neurotrophic factors and forskolin. RGC survival after exposure to glutamate (25 microM) with nilvadipine or other calcium channel blockers was measured by calcein-acetoxymethyl ester staining after 3 days in culture. Changes in the level of intracellular Ca(2+) ([Ca(2+)](i)) were measured with fura-2 fluorescence. Induction of apoptosis was evaluated using the TDT-dUTP terminal nick-end labeling technique. The neurotoxic effects of low doses of glutamate were blocked by a specific alpha-amino-3-dihydro-5-methylisoxazole-4-propionate-kainate receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (20 microM). Simultaneous application of nilvadipine (1-100 nM) with glutamate protected against glutamate neurotoxicity in a dose-dependent manner. Calcium-imaging experiments showed that the glutamate-evoked [Ca(2+)](i) increase was significantly blocked by nilvadipine (P<0.001), but not nifedipine and diltiazem, in about 50% of RGCs. In addition, the application of nilvadipine significantly reduced glutamate-induced apoptosis (P<0.001). These findings suggest that nilvadipine may partly inhibit glutamate-induced apoptotic cell death by blocking calcium influx via voltage-dependent calcium channels in purified RGCs.

ABCA4 gene mutations in Japanese patients with Stargardt disease and retinitis pigmentosa.

PURPOSE: To evaluate photoreceptor cell-specific adenosine triphosphate (ATP)-binding cassette transporter (ABCA4) gene mutations in Japanese patients with Stargardt disease (STGD) and the correlation of these mutations to clinical phenotypes. METHODS: Serum was obtained from 10 unrelated Japanese patients with STGD and 96 unrelated Japanese patients with autosomal recessive retinitis pigmentosa (arRP). All 50 ABCA4 gene exons of the patients with STGD were screened for mutations by a combination of single-strand conformation polymorphism analysis and polymerase chain reaction (PCR) direct-sequencing techniques. By restriction enzyme digestion, primer extension analysis, and PCR direct sequencing techniques, the patients with arRP were screened for three segregated, presumably null ABCA4 gene mutations observed in Japanese patients with STGD. RESULTS: Three novel, presumably null mutations of the ABCA4 gene, IVS7-45_952delinsTCTGACC, IVS12+2T-->G, and 1894delA, were identified. The Arg2149stop mutation that had been found in a white patient with STGD in a prior study was also found in a Japanese patient. Two arRP-affected siblings and two unrelated patients with STGD were found to be homozygous for the same IVS12+2T-->G mutation, and three other arRP-affected siblings were carriers of the IVS12+2T-->G mutation and/or the IVS7-45_952delinsTCTGACC mutation. These three siblings with arRP showed only atrophic degeneration in the macula early after the onset of the disease, and STGD had been diagnosed. CONCLUSIONS: Three novel ABCA4 gene mutations were identified in Japanese patients with STGD and arRP. Mutations in the ABCA4 gene can cause panretinal degeneration that changes its clinical appearance from STGD to arRP over time.