Novel microarrays for simultaneous serodiagnosis of multiple antiviral antibodies.

We developed an automated diagnostic system for the detection of virus-specific immunoglobulin Gs (IgGs) that was based on a microarray platform. We compared efficacies of our automated system with conventional enzyme immunoassays (EIAs). Viruses were immobilized to microarrays using a radical cross-linking reaction that was induced by photo-irradiation. A new photoreactive polymer containing perfluorophenyl azide (PFPA) and poly(ethylene glycol) methacrylate was prepared and coated on plates. Inactivated measles, rubella, mumps, Varicella-Zoster and recombinant Epstein-Barr viruse antigen were added to coated plates, and irradiated with ultraviolet light to facilitate immobilization. Virus-specific IgGs in healthy human sera were assayed using these prepared microarrays and the results obtained compared with those from conventional EIAs. We observed high correlation (0.79-0.96) in the results between the automated microarray technique and EIAs. The microarray-based assay was more rapid, involved less reagents and sample, and was easier to conduct compared with conventional EIA techniques. The automated microarray system was further improved by introducing reagent storage reservoirs inside the chamber, thereby conserving the use of expensive reagents and antibodies. We considered the microarray format to be suitable for rapid and multiple serological diagnoses of viral diseases that could be developed further for clinical applications.

An automated multiplex specific IgE assay system using a photoimmobilized microarray.

An automated microarray diagnostic system for specific IgE using photoimmobilized allergen has been developed. Photoimmobilization is useful for preparing microarrays, where various types of biological components are covalently immobilized on a plate. Because the immobilization is based on a photo-induced radical cross-linking reaction, it does not require specific functional groups on the immobilized components. Here, an aqueous solution of a photoreactive poly(ethylene glycol)-based polymer was spin-coated on a plate, and an aqueous solution of each allergen was microspotted on the coated plate and allowed to dry in air. Finally, the plate was irradiated with an ultraviolet lamp for covalent immobilization. An automated machine using these plates was developed for the assay of antigen-specific IgE. Initially, the patient serum was added to the microarray plate, and after reaction of the microspotted allergen with IgE, the adsorbed IgE was detected by a peroxidase-conjugated anti-IgE-antibody. The chemical luminescence intensity of the substrate decomposed by the peroxidase was automatically detected using a sensitive charge-coupled device camera. All the allergens were immobilized stably using this method, which was used to screen for allergen-specific IgE. The results were comparable with those using conventional specific IgE. Using this system, six different allergen-specific IgE were assayed using 10 µL of serum within a period of 20 min.

Evaluation of prosthetic venous valves, fabricated by electrospinning, for percutaneous treatment of chronic venous insufficiency.

Chronic venous insufficiency (CVI) remains a major health problem worldwide. Direct venous valve surgical repair and venous segment transplantation are clinical options; however, they are highly invasive procedures. The objectives of this study were to fabricate prosthetic venous valves (PVVs) by electrospinning, for percutaneous treatment of CVI, and evaluate their hydrodynamic characteristics in vitro at the same locations and under the same flow conditions. The PVVs consisted of polyurethane fiber scaffolds attached to a cobalt-chromium stent. PVVs with two different valve-leaflet configurations were compared: biomimetic PVV (bPVV) and open PVV (oPVV). A balloon catheter was used to implant the devices in a poly(vinyl chloride) tube and the column outlet was set at a height of 100 cm above the test valve to simulate the elevation of the heart above a distal vein valve while standing; 50 wt% glycerin solution was used as the test fluid. The devices were evaluated for antegrade flow, effect of ankle flexion, and stagnation zones around the valve leaflets. During sudden hydrostatic backpressure, little leakage and constant peripheral pressure were observed for the devices; under forward pulsatile pressure of 0-4 mmHg, to simulate the effect of breathing, the oPVV had a higher flow rate than the bPVV. With regard to the effect of ankle flexion, the oPVV was functionless. Moreover, the stagnation zone around the oPVV valve leaflets was larger than that around the bPVV valve leaflets. These results suggest that the bPVV would be clinically suitable for percutaneous treatment of CVI.

Analysis of gene expression changes associated with long-lasting synaptic enhancement in hippocampal slice cultures after repetitive exposures to glutamate.

We have previously shown that repetitive exposures to glutamate (100 muM, 3 min, three times at 24-hr intervals) induced a long-lasting synaptic enhancement accompanied by synaptogenesis in rat hippocampal slice cultures, a phenomenon termed RISE (for repetitive LTP-induced synaptic enhancement). To investigate the molecular mechanisms underlying RISE, we first analyzed the time course of gene expression changes between 4 hr and 12 days after repetitive stimulation using an original oligonucleotide microarray: "synaptoarray." The results demonstrated that changes in the expression of synapse-related genes were induced in two time phases, an early phase of 24-96 hr and a late phase of 6-12 days after the third stimulation. Comprehensive screening at 48 hr after the third stimulation using commercially available high-density microarrays provided candidate genes responsible for RISE. From real-time PCR analysis of these and related genes, two categories of genes were identified, 1) genes previously reported to be induced by physiological as well as epileptic activity (bdnf, grm5, rgs2, syt4, ania4/carp/dclk) and 2) genes involved in cofilin-based regulation of actin filament dynamics (ywhaz, ssh1l, pak4, limk1, cfl). In the first category, synaptotagmin 4 showed a third stimulation-specific up-regulation also at the protein level. Five genes in the second category were coordinately up-regulated by the second stimulation, resulting in a decrease in cofilin phosphorylation and an enhancement of actin filament dynamics. In contrast, after the third stimulation, they were differentially regulated to increase cofilin phosphorylation and enhance actin polymerization, which may be a key step leading to the establishment of RISE.

miR-210 promotes osteoblastic differentiation through inhibition of AcvR1b.

Although microRNAs (miRNAs) are involved in many biological processes, the mechanisms whereby miRNAs regulate osteoblastic differentiation are poorly understood. Here, we found that BMP-4-induced osteoblastic differentiation of bone marrow-derived ST2 stromal cells was promoted and repressed after transfection of sense and antisense miR-210, respectively. A reporter assay demonstrated that the activin A receptor type 1B (AcvR1b) gene was a target for miR-210. Furthermore, inhibition of transforming growth factor-beta (TGF-beta)/activin signaling in ST2 cells with SB431542 promoted osteoblastic differentiation. We conclude that miR-210 acts as a positive regulator of osteoblastic differentiation by inhibiting the TGF-beta/activin signaling pathway through inhibition of AcvR1b.

Improvement and analysis of a micro Raman probe.

A micro Raman probe (MRP) with a 600 microm diameter, which we previously reported as the narrowest achieved to date, was further improved by introducing high-quality optical filters and a collecting lens at the tip. We fabricated the MRP with a high collection efficiency, a wider collection wavelength, and a high signal-to-noise ratio. We compared two types of probes: one with a lens-tipped end and one with a flat tip. We experimentally tested the performance of these MRPs to evaluate the detection properties defined by parameters such as the optical purity against inherent Raman background noise due to optical fibers, the sensitivity, and the viewing area. Finally, we demonstrated their effectiveness in measurements of standard Raman samples and applied them to measurements of plastic and human skin samples in situ.

miR-125b inhibits osteoblastic differentiation by down-regulation of cell proliferation.

Although various microRNAs regulate cell differentiation and proliferation, no miRNA has been reported so far to play an important role in the regulation of osteoblast differentiation. Here we describe the role of miR-125b in osteoblastic differentiation in mouse mesenchymal stem cells, ST2, by regulating cell proliferation. The expression of miR-125b was time-dependently increased in ST2 cells, and the increase in miR-125b expression was attenuated in osteoblastic-differentiated ST2 cells induced by BMP-4. The transfection of exogenous miR-125b inhibited proliferation of ST2 cells and caused inhibition of osteoblastic differentiation. In contrast, when the endogenous miR-125b was blocked by transfection of its antisense RNA molecule, alkaline phosphatase activity after BMP-4 treatment was elevated. These results strongly suggest that miR-125b is involved in osteoblastic differentiation through the regulation of cell proliferation.

Development and validation of microarray-based assay for epidemiological study of MRSA.

We have developed a microarray-based assay for the genotyping of Staphylococcus aureus strains. A DNA microarray consisting of 221 genes with 390 oligonucleotide probes was designed to identify characteristic genes or gene alleles of S. aureus. The 221 genes were chosen on the basis of the following criteria: (i) genes used as control for the microarray system, (ii) virulence genes, (iii) resistance genes and their regulators, and (iv) genes constituting genomic islands, e.g., SCCmec. The microarray system was established by determining the method to prepare targets by random-primer labeling with chromosomal DNA and the conditions for hybridization. We verified the system by using DNAs of seven strains, the genome of which has been fully sequenced. Furthermore, the presence of 32 genes and the types of SCCmec elements and coagulase genes carried by another 27 strains were examined and compared with the results of PCR. As a result, the presence or absence of 182 genes out of the 221 genes was verified. Our data showed the usefulness of the oligonucleotide microarray based assay in identifying important marker sets, such as toxin genes, resistance genes, SCCmec elements, and coagulase genes, for the molecular epidemiology of S. aureus.

Retrospective evaluation of treatment outcome in Japanese children with complete unilateral cleft lip and palate. Part 1: Five-year-olds' index for dental arch relationships.

OBJECTIVE: To evaluate the dental arch relationships of Japanese children with complete unilateral cleft lip and palate (UCLP) and to examine the 5-year-olds' index for its validity. DESIGN: Retrospective study and comparison with previous reports. SUBJECTS: One hundred thirty-six children with complete UCLP who received primary cheiloplasty and palatoplasty in the Kyushu University Hospital from 1966 to 1999. MATERIALS: Dental models taken from children 53 to 67 months of age and their cephalograms. METHODS: Study models were assessed using five scores; 1=excellent, 2=good, 3=fair, 4=poor, and 5=very poor, in accordance with the 5-year-olds' index and also evaluated using Huddart and Bodenham's numerical classification. Dental arch widths, three-dimensional maxillary dental arch form, and lateral cephalograms were traced and measured. The outcome by 5-year-olds' index was compared with Huddart and Bodenham's numerical classification, dental arch dimensions, and cephalometric measurements. RESULTS: Occlusal outcome evaluated by the 5-year-olds' index was rated 2.95, which was classified as fair. This index rating showed a significant relationship with numerical classification and dental arch length, but not with dental arch width. The index showed a relationship with mandibular form and position, but not with maxillary position. CONCLUSION: The occlusal outcome of the cases with UCLP was fair as evaluated using the 5-year-olds' index. The index evaluates the anteroposterior relationship of maxillary/mandibular dental arches but does not evaluate the collapse of maxillary segments.

In vivo Raman study of the living rat esophagus and stomach using a micro-Raman probe under an endoscope.

A small endoscope system equipped with a micro Raman probe is developed for in vivo Raman measurements in living rats. The measurements are done under anesthesia and artificial respiration to minimize the impact on the rats. Raman spectra of living rat esophagus and stomach are successfully measured. Our results suggest that the Raman spectra reflect subsurface tissue structure that cannot be distinguished in the endoscope image. After the experiments, rats recover without any aftereffects. It is verified that the Raman measurement using the present system is safe and noninvasive for rats.

Identification of synaptic activity-dependent genes by exposure of cultured cortical neurons to tetrodotoxin followed by its withdrawal.

Activity-dependent gene expression is one of the key mechanisms of synaptic plasticity that form the basis of higher order functions such as learning and memory. In the present study, we surveyed for activity-dependent genes by analyzing gene expression changes accompanying reversible inhibition of synaptic activity by tetrodotoxin (TTX) using two types of DNA microarrays; our focused oligo DNA microarray "Synaptoarray" and the commercially available high-density array. Cerebral cortical cells from E18 rat embryos were cultured for 14 days to ensure synaptogenesis, then treated with 1 muM TTX for 48 hr without detectable effect on cell viability. Synaptic density estimated by the amount of Synapsin I and Synaptotagmin I was decreased 21-24% by TTX treatment, but recovered to the control level 48 hr after TTX withdrawal. Comparison of gene expression profiles by competitive hybridization of fluorescently labeled cRNA from TTX-treated and control cells showed an overall downregulation of the genes on the Synaptoarray by TTX-treatment with different recovery rates after TTX withdrawal. With 16 representative genes, microarray data were validated by real-time PCR analysis. Genes most severely downregulated by TTX and upregulated above the control level at 5 hr after TTX withdrawal were munc13-1 (involved in docking and priming of synaptic vesicles) and Shank2 (involved in the postsynaptic scaffold). In addition, comprehensive screening at 5 hr after TTX withdrawal using high density arrays resulted in additional identification of Rgs2, a regulator of trimeric G-protein signaling, as an activity-dependent gene. These three genes are thus likely to be key factors in the regulation of synaptic plasticity. (c) 2007 Wiley-Liss, Inc.

Detection of cRNA hybridized on a DNA chip using a tetrakis-acridinyl peptide cassette, consisting of TAP and partially self-complementary oligonucleotide, d[A18(TA)51].

A tetrakis-acridinyl peptide (TAP) cassette, consisting of a double-stranded region of alternating AT sequence bound to TAP and a single stranded overhanging sequence of continuous dA, was prepared by mixing TAP with d[A18(TA)51]. A TAP cassette could be applied to the fluorometric detection of hybridized DNA on the DNA chip, which was prepared by stamping a 45-meric DNA probe onto a gold-coated plastic chip using a high-precision spotter developed at RIKEN. Spots on the DNA chip were imaged by a CCD camera after hybridization with 65-meric target single-stranded DNAs carrying a continuous dA20 sequence (dA tail) on the DNA chip after treatment with a TAP cassette. Their fluorescence intensity on the DNA chip showed a good linear correlation with the concentration of the target DNAs in the range from 10 pM to 1 nM. Fluorescence of their spots derived from the TAP cassette remaining on the surface of the DNA chip through the dA tail of the hybridized target DNA. Furthermore, the TAP cassette could be successfully applied to the quantitative detection of complementary RNAs (cRNAs) prepared from rat brain with reverse transcription and in vitro transcription.

Efficient characterization for protein crystals using confocal Raman spectroscopy.

Confocal Raman spectroscopy was applied to the characterization of various states emerging in the screening of protein crystallization. Four main characterized states, namely single crystals, microcrystals, precipitates, and clear drops without solid materials, appear in a droplet for crystallization; the first three states should be critically distinguished and characterized because of the limitations of visual observation under an optical microscope. Using lysozyme and other proteins, crystallization was performed by the hanging drop vapor diffusion technique and was monitored through an automated confocal Raman system. Prior to the spectroscopic analysis, an optical microscope with a charge-coupled device (CCD) camera and associated image processing software were used to rapidly identify the XY locations to be measured spectroscopically by focusing the laser beam on a test sample. Instead of the current image analysis by optical microscopy, confocal Raman spectroscopy with a high spatial resolution was used to identify the state of protein crystallization. Such real-time Raman monitoring also distinguished real protein crystals from pseudo-protein crystals emerging in a crystallization droplet.

Intravascular Raman spectroscopic catheter for molecular diagnosis of atherosclerotic coronary disease.

An intravascular catheter for Raman spectroscopic detection and analysis of coronary atherosclerotic disease has been developed. The catheter, having an outer diameter of 2 mm, consisted of a side-view-type micro-Raman probe, an imaging fiber bundle, a working channel (injection drain), and a balloon. By inflating the balloon, the probe was brought close to the inner wall of a modeled blood flow system and detected a phantom target buried in the wall. Results obtained demonstrate the possibility of using the spectroscopic catheter for molecular diagnosis of coronary lesions.

Fluorescence-suppressed Raman technique for quantitative analysis of protein solution using a micro-Raman probe, the shifted excitation method, and partial least squares regression analysis.

A practical Raman analyzing technique with suppression of the strong fluorescent background in order to obtain quantitative information is proposed in the present study. The technique is based on the shifted excitation method and partial least squares regression (PLSR) analysis. The Raman system consists of a single Raman spectrometer, a background-free electrically tunable Ti:Sapphire laser (BF-ETL), and a micro-Raman probe (MRP). The system allows one to obtain reliable shifted excitation Raman spectra with a simple operation. The PLSR analysis successfully provides quantitative information from the obtained spectra with the suppression of random noise including photon shot noise. The present study demonstrates that the technique is effective for extracting quantitative information concealed behind a fluorescent background that is more than 200 times stronger than the Raman signal.

Automatic continuous scanning and random-access switching of mid-infrared waves generated by difference-frequency mixing.

We report the rapid tuning of mid-infrared waves beyond 5 microm emitted in difference-frequency mixing with an electronically tuned dual-wavelength Ti:Al2O3 laser used as a pumping source. Simultaneous rapid tuning of the dual wavelengths, which satisfy phase matching in AgGaS2, allows rapid random access switching and continuous tuning of mid-infrared wavelengths. In random-access switching, the mid-infrared wavelength is tuned every pulse shot at a repetition rate of 1 kHz. Mid-infrared wavelengths continuously tuned from 5.2 to 7.2 microm, from 7.0 to 9.1 microm, and from 8.9 to 12.0 microm are achieved at phase-matched angles of 55 degrees, 50 degrees, and 45 degrees, respectively.

Flexural rigidity of individual microtubules measured by a buckling force with optical traps.

We used direct buckling force measurements with optical traps to determine the flexural rigidity of individual microtubules bound to polystyrene beads. To optimize the accuracy of the measurement, we used two optical traps and antibody-coated beads to manipulate each microtubule. We then applied a new analytical model assuming nonaxial buckling. Paclitaxel-stabilized microtubules were polymerized from purified tubulin, and the average microtubule rigidity was calculated as 2.0 x 10(-24) Nm2 using this novel microtubule buckling system. This value was not dependent on microtubule length. We also measured the rigidity of paclitaxel-free microtubules, and obtained the value of 7.9 x 10(-24) Nm2, which is nearly four times that measured for paclitaxel-stabilized microtubules.

Photo-cross-linked small-molecule microarrays as chemical genomic tools for dissecting protein-ligand interactions.

We have developed a unique photo-cross-linking approach for immobilizing a variety of small molecules in a functional-group-independent manner. Our approach depends on the reactivity of the carbene species generated from trifluoromethylaryldiazirine upon UV irradiation. It was demonstrated in model experiments that the photogenerated carbenes were able to react with every small molecule tested, and they produced multiple conjugates in most cases. It was also found in on-array immobilization experiments that various small molecules were immobilized, and the immobilized small molecules retained their ability to interact with their binding proteins. With this approach, photo-cross-linked microarrays of about 2000 natural products and drugs were constructed. This photo-cross-linked microarray format was found to be useful not merely for ligand screening but also to study the structure-activity relationship, that is, the relationship between the structural motif (or pharmacophore) found in small molecules and its binding affinity toward a protein, by taking advantage of the nonselective nature of the photo-cross-linking process.

Raman probe using a single hollow waveguide.

A simple Raman probe was realized using a single flexible hollow waveguide (HW). A HW coated with a silver film, which had reasonable transmission and little optical background noise, was used as a bidirectional transmission fiber for both the excitation and collection of Raman scattered light. The HW itself generated no Raman scattering or fluorescence noise during transmission. A complex filtering system at the end of the waveguide was thus unnecessary. In addition, the measured Raman spectra showed better signal-to-noise ratios than a conventional Raman fiber probe. The HW's suitability as a Raman fiber probe was also demonstrated.

Micro-optical fiber probe for use in an intravascular Raman endoscope.

We believe that we have developed the narrowest optical-fiber Raman probe ever reported, 600 microm in total diameter, that can be inserted into coronary arteries. The selection of suitable optical fibers, filters, and a processing method is discussed. Custom-made filters attached to the front end of a probe eliminate the background Raman signals of the optical fiber itself. The experimental evaluation of various optical fibers is carried out for the selection of suitable fibers. Measurement of the Raman spectra of an atherosclerotic lesion of a rabbit artery in vitro demonstrates the excellent performance of the micro-Raman probe.