Applications of magnetic and electromagnetic forces in micro-analytical systems.

Novel applications of magnetic fields in analytical chemistry have become a remarkable trend in the last two decades. Various magnetic forces have been employed for the migration, orientation, manipulation, and trapping of microparticles, and new analytical platforms for separating and detecting molecules have been proposed. Magnetic materials such as functional magnetic nanoparticles, magnetic nanocomposites, and specially designed magnetic solids and liquids have also been developed for analytical purposes. Numerous attractive applications of magnetic and electromagnetic forces on magnetic and non-magnetic materials have been studied, but fundamental studies to understand the working principles of magnetic forces have been challenging. These studies will form a new field of magneto-analytical science, which should be developed as an interdisciplinary field. In this review, essential pioneering works and recent attractive developments are presented.

Sequentially addressable dielectrophoretic array for high-throughput sorting of large-volume biological compartments.

Droplet microfluidics has become a powerful tool in precision medicine, green biotechnology, and cell therapy for single-cell analysis and selection by virtue of its ability to effectively confine cells. However, there remains a fundamental trade-off between droplet volume and sorting throughput, limiting the advantages of droplet microfluidics to small droplets (<10 pl) that are incompatible with long-term maintenance and growth of most cells. We present a sequentially addressable dielectrophoretic array (SADA) sorter to overcome this problem. The SADA sorter uses an on-chip array of electrodes activated and deactivated in a sequence synchronized to the speed and position of a passing target droplet to deliver an accumulated dielectrophoretic force and gently pull it in the direction of sorting in a high-speed flow. We use it to demonstrate large-droplet sorting with ~20-fold higher throughputs than conventional techniques and apply it to long-term single-cell analysis of Saccharomyces cerevisiae based on their growth rate.

Pretreatment of donor islets with the Na(+) /Ca(2+) exchanger inhibitor improves the efficiency of islet transplantation.

Pancreatic islet transplantation is an attractive therapy for the treatment of insulin-dependent diabetes mellitus. However, the low efficiency of this procedure necessitating sequential transplantations of islets with the use of 2-3 donors for a single recipient, mainly due to the early loss of transplanted islets, hampers its clinical application. Previously, we have shown in mice that a large amount of HMGB1 is released from islets soon after their transplantation and that this triggers innate immune rejection with activation of DC, NKT cells and neutrophils to produce IFN-γ, ultimately leading to the early loss of transplanted islets. Thus, HMGB1 release plays an initial pivotal role in this process; however, its mechanism remains unclear. Here we demonstrate that release of HMGB1 from transplanted islets is due to hypoxic damage resulting from Ca(2+) influx into ß cells through the Na(+) /Ca(2+) exchanger (NCX). Moreover, the hypoxia-induced ß cell damage was prevented by pretreatment with an NCX-specific inhibitor prior to transplantation, resulting in protection and long-term survival of transplanted mouse and human islets when grafted into mice. These findings suggest a novel strategy with potentially great impact to improve the efficiency of islet transplantation in clinical settings by targeting donor islets rather than recipients.

Less invasive technique for EC-IC bypass.

BACKGROUND: We introduce less invasive technique for superficial temporal to middle cerebral artery (STA-MCA) anastomosis, and also described an innovative technique to preoperatively identify the recipient artery using three dimensional CT angiography (3D CTA). Objective. In a period of 28 months between January 2004 and April 2006, 39 EC-IC bypass were performed for hemodynamic compromised patients (including 9 patients with Moyamoya disease) using less invasive technique. METHODS: Operative technique is as follows: 1) A parietal or frontal branch of STA and cortical arteries could be identified on the original images of 3D CTA. The most suitable segment of both the artery provided as donor and recipient arteries for EC-IC bypass. The distance between the afore-mentioned segment of donor artery (STA) and the superior border of the helix were calculated. 2) A 5 cm linear skin incision on the STA, the center of which was the point measured on preoperative 3D CTA, was made. The temporal muscle was divided in the same fashion, and a 3 cm small craniotomy was made. The recipient artery could be identified on the center of the craniotomy. End-to-side anastomosis was performed in the usual way. RESULTS: Operation times were 115-172 min (mean 154 min) and intraoperative blood loss was 20-60 ml (mean 38 ml). All bypasses were patent on the post-operative 3D CTA. CONCLUSIONS: This technique for EC-IC bypass was less invasive and cosmetically excellent. 3D CTA provides useful information for planning of the less invasive EC-IC bypass.

Treatment of cerebral aneurysms: surgical clipping and coil embolization.

SUMMARY: The present series provides a balanced overview of the treatment of aneurysms in surgical clipping and coil embolization. Between January 2004 and March 2006, 76 consecutive patients with cerebral aneurysms underwent endovascular embolization and/or surgical clipping. Of these, 42 patients suffered an aneurysmal subarachnoid hemorrhage (SAH), while the remaining 34 patients had nonruptured cerebral aneurysms. Of the 23 surgically treated patients, 17 (73.9%) achieved a favorable outcome. Of the 19 patients who underwent endovascular embolization, 12 (63.2%) achieved a favorable outcome. Three patients (15.8%) who underwent endovascular embolization needed to undergo re-treatments, while no re-treatment was needed in the surgically treated patients. Of the 34 nonruptured aneurysms, 12 (35.3%) were treated using surgical clipping, while 22 (64.7%) underwent endovascular embolization. The complication rates of the two treatment modalities demonstrated no significant difference. A combined microsurgical-endovascular team approach is thus considered to provide the most effective means to achieve favorable outcomes for patients with cerebral aneurysms.

Surface-enhanced Raman spectroscopy of dodecanethiol-bound silver nanoparticles at the liquid/liquid interface.

Adsorption and domain formation of dodecanethiol (DT)-bound silver nanoparticles (SNPs) at the cyclohexane/water interface were studied by means of total internal reflection (TIR) light scattering microscopy and TIR surface-enhanced Raman scattering (SERS). By the TIR light scattering microscopy, the extent of the interfacial adsorption and domain formation of SNP was observed, which was produced by the reaction between citrate-reduced SNPs in the aqueous phase and DT in the cyclohexane phase. The Raman spectra of DT on SNP showed that the relative intensity ratios of gauche to trans conformers in the nu(C-S) band region decreased with the increase of the initial concentration of DT, suggesting the change from the liquidlike structure to the solidlike structure of the DT. The residue of the negative charges on the SNPs at the interface was detected by the resonance SERS (SERRS) peaks of the adsorbed cationic porphyrin, 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphine (TMPyP). The efficiency of the interfacial SNPs domains as a SERS substrate for TMPyP strongly depended on the adsorption state of the DT.

Nitric oxide synthase expression is increased by occlusal force in rat periodontal ligament.

OBJECTIVES: The goal of this study was to investigate the role of nitric oxide synthase (NOS) in occlusal force-induced signal transduction in rat periodontal ligament (PDL). DESIGN: Rats were fitted with a bite plate and a metal cap to the maxillary and mandibular incisors, respectively, to eliminate the occlusal forces on rat molars. One group was sacrificed at 7 days (exclusion group), while the remaining rats had their appliances removed to reestablish molar occlusal contact (reload group) and were sacrificed 7 days thereafter. Another group of rats (normal group) were left completely untreated. Frozen cross sections of the upper first molars were stained with NADPH-diaphorase to quantify NOS activity. The distal sides of the disto-palatal roots of the upper first molars were examined, and the number and the area of stained cells in the PDL were measured. RESULTS: In the normal group, NOS expression was detected in blood vessels, monocyte-macrophages, fibroblastic cells and osteoclastic cells. NOS expression was lower in the exclusion group when compared with the normal group or the reload group (p < 0.05), and the exclusion group exhibited occluded blood vessels and a narrowing of PDL. In contrast, in the reload group the PDL and blood vessel structure had recovered and NOS expression was increased to the level of the controls. CONCLUSION: Occlusal force resulted in increased NOS expression. NO may mediate changes in PDL structure in response to occlusal force.

Effect of nitric oxide on the recovery of the hypofunctional periodontal ligament.

The relationship between occlusal stimuli and a hypofunctional periodontal ligament (PDL) structure has been reported, though changes in occlusal recovery conditions were still unclear. Nitric oxide (NO) produced by NO synthase (NOS) is considered a factor for vascular and immune system control, and it increases according to mechanical stimuli. The objective of this study was to examine the relationship between NOS and occlusal stimuli in PDL by comparing hypofunction with occlusal recovery. The study focused on the expression of endothelial NOS (eNOS) and inducible NOS (iNOS). Their expression significantly decreased in occlusal hypofunction compared with the control group and increased close to normal in an occlusal recovery group. The change in the immunopositive area was more dramatic than the immunopositive cell number. Moreover, the rate of iNOS increase was higher than that of eNOS. This study suggests that NO plays an important role in the recovery of the hypofunctional PDL.

Dielectrophoresis of microbioparticles in water with planar and capillary quadrupole electrodes.

Dielectrophoresis of single microbioparticles was measured in a planar quadrupole microelectrode (50 mum or 65 mum in working area radius) with a microscope. Carbon and polystyrene microparticles, yeast cells and DNA molecules (about 40 kbp) were adopted as a sample. Their dielectrophoretic mobilities were analysed quantitatively with their intrinsic and surface conductivity, their permittivities and their sizes as well as the conductivity and permittivity of aqueous media. Using the dielectrophoretic mobilities obtained with the planar quadrupole microelectrode, some instances of the separation performance between the microparticles were demonstrated with a fabricated capillary quadrupole microelectrode (82.5 mum in bore radius) under the field flow fractionation regime.

Magnetophoretic velocimetry of manganese(II) in a single emulsion droplet at the femtomole level.

We developed a new experimental technique named magnetophoretic velocimetry to determine a small amount of paramagnetic species in a single microdroplet. The magnetophoretic velocity of an aqueous droplet containing paramagnetic metal ion dispersed in an organic medium could response to a very small amount of the metal ion under an inhomogeneous magnetic field. The paramagnetic droplet (2 approximately 8 microm diam) used as a test sample in this study was the aqueous droplet of manganese(II) chloride dispersed in ethylbenzoate whose density was nearly equal to water. A pair of small Nd-Fe-B magnets placed with a gap of 400 microm generated an inhomogeneous magnetic field between the edges, at which the product of the magnetic flux density and the gradient, B(dB/dx), was as high as 410 T2 m(-1). When a silica capillary containing the emulsion was inserted into the gap between the magnets, the magnetophoretic migration of the droplets was observed with a video microscope. The magnetophoretic velocity divided by the squared radius of the droplet was proportional to the MnCl2 concentration in the droplet, as predicted by a theoretical calculation. The estimated detection limit in this simple method was lower than 10(-16) mol for manganese(II).

Migration mechanism of bases and nucleosides in oil-in-water microemulsion capillary electrophoresis.

The electrophoretic behaviors of five bases and corresponding nucleosides in the oil in water (o/w) microemulsion capillary electrophoresis, microemulsion electrokinetic chromatography (MEEKC), were examined in comparison with those in normal capillary zone electrophoresis (CZE). The microemulsion systems were composed of heptane, sodium dodecyl sulfate (SDS), 1-butanol and 10 mM phosphate buffer (pH 7.0) or toluene, SDS, 1-butanol and 5 mM carbonate buffer (pH 10.0). CZE was carried out in the range of pH 9.7-10.9, and the dissociation constants, pKa, of the bases and nucleosides and the electrophoretic mobilities of the anionic forms were determined. The electrophoretic behaviors of the solutes in the microemulsion systems were analyzed from their pKa, the electrophoretic mobilities of the anions determined by CZE, and the distribution constants, K(D), of the neutral forms between the microemulsion droplets and the outer aqueous phase. The importance of adsorption mechanism in MEEKC system was suggested from the correlation between log K(D) and log P.

Direct spectrophotometric measurements of acid-catalyzed complexation of palladium(II) with 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol at the heptane/water interface by a centrifugal liquid membrane method.

The centrifugal liquid membrane (CLM) method, designed for the rapid sample injection, was applied to the kinetic study of the complexation of palladium(II) with 2-(5-bromo-2-pyridylazo)-5-diethyaminophenol (5-Br-PADAP) in the heptane/water system. The formation rates of Pd(II)-5-Br-PADAP complex, which existed only at the heptane/water interface, could be directly measured by the CLM method combined with transmission spectrophotometry. We found that the formation rates of Pd(II)-5-Br-PADAP complex were accelerated by the protonation of 5-Br-PADAP at the diethylamino-group that did not coordinate to Pd(II) ion and that the rate constant for the reaction of protonated 5-Br-PADAP at the interface was close to that in the aqueous phase. The present study demonstrated that the CLM method was easily applicable for the measurements of relatively fast interfacial reactions.

Flow fractionation of microparticles under a dielectrophoretic field in a quadrupole electrode capillary.

A technique of fractionation for microparticles was proposed that utilized a unique combination of a dielectrophoretic (DEP) field generated by a quadrupole electrode and a laminar flow in a capillary of 82.5 microm in radius. The fabricated capillary possessed four platinum wires in its inside wall as a quadrupole electrode. In a nonuniform electric field generated by the quadrupole electrode, microparticles, such as polystyrene and carbon, in water experienced DEP forces in the radial direction. When a sample solution was pumped in, an ideal laminar flow perpendicular to the DEP force was formed inside the capillary. The microparticles dynamically migrated by the DEP force across the laminar flow while they were carried by the flow. A theoretical model taking the DEP force and the laminar flow pattern into account predicted the elution profiles of the single microparticles quantitatively. The elution times of the microparticles depended on the dielectric properties and the sizes of the microparticles, as well as the voltage and frequency of the applied alternating current.

Posttranslational modification of the glycosylation inhibiting factor (GIF) gene product generates bioactive GIF.

Glycosylation inhibiting factor (GIF) and macrophage migration inhibitory factor (MIF) share an identical structure gene. Here we unravel two steps of posttranslational modifications in GIF/MIF molecules in human suppressor T (Ts) cell hybridomas. Peptide mapping and MS analysis of the affinity-purified GIF from the Ts cells revealed that one modification is cysteinylation at Cys-60, and the other is phosphorylation at Ser-91. Cysteinylated GIF, but not the wild-type GIF/MIF, possessed immunosuppressive effects on the in vitro IgE antibody response and had high affinity for GIF receptors on the T helper hybridoma cells. In vitro treatment of wild-type recombinant human GIF/MIF with cystine resulted in preferential cysteinylation of Cys-60 in the molecules. The cysteinylated recombinant human GIF and the Ts hybridoma-derived cysteinylated GIF were comparable both in the affinity for the receptors and in the immunosuppressive activity. Polyclonal antibodies specific for a stretch of the amino acid sequence in alpha2-helix of GIF bound bioactive cysteinylated GIF but failed to bind wild-type GIF/MIF. These results strongly suggest that cysteinylation of Cys-60 and consequent conformational changes in the GIF/MIF molecules are responsible for the generation of GIF bioactivity.

Proteomic approach to the identification of cell membrane proteins.

The expression of plasma membrane proteins in human monocyte-derived U937 cells was examined by cell disruption and isolation of microsomal fractions. Two alternative procedures for cell disruption, Dounce homogenization and nitrogen cavitation, were compared. Cell homogenization and sequential centrifugation resulted in an approximately fivefold enrichment of plasma membrane proteins in the microsomal fraction. However, identification of 30 such apparently enriched proteins by two-dimensional (2-D) electrophoresis, proteolytic digestion, and mass spectrometry revealed that only eight were plasma membrane proteins, the remaining 22 being contaminants. In contrast, nitrogen cavitation followed by sequential centrifugation and solubilization of proteins with sodium dodecyl sulfate (SDS) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) detergent yielded subcellular fractions, including microsomes, that showed little overlap in constituent proteins as revealed by 2-D electrophoresis. These results highlight the importance of obtaining pure plasma membranes and complete solubilization of membrane proteins for proteomic analysis.

Immunosuppressive activities of recombinant glycosylation-inhibiting factor mutants.

We have shown previously that glycosylation-inhibiting factor (GIF) in culture supernatants of suppressor T cell (Ts) hybridomas had bioactivity, while the same cells contained a substantial quantity of inactive GIF in cytosol. Mass-spectrometric analysis of GIF in the culture supernatant and cytosol of a Ts hybridoma provided direct evidence that GIF protein was posttranslationally modified in the Ts cells, and that the GIF bioactivity is associated with the posttranslationally modified species. Assuming that conformational changes induced by the posttranslational modifications are responsible for generation of bioactivity, we constructed cysteine mutants of human rGIF (rhGIF) in which cysteine at position 57, 60, or 81 was replaced with Ala, and the mutants were expressed in Escherichia coli. Replacement of Cys57 or Cys60 with Ala resulted in generation of bioactivity, while replacement of Cys81 with Ala failed to do so. It was also found that replacement of Cys57 with Ala and carboxymethylation of a sulfhydryl group in Cys60 synergistically increased the GIF bioactivity of the GIF derivatives. A mutated GIF protein, in which Cys57 and Asn106 in the rhGIF were replaced with Ala and Ser, respectively, had immunosuppressive effects on the IgE and IgG1 Ab responses of BDF1 mice to DNP-OVA, while wild-type rhGIF did not. Evidence was obtained that the mutated GIF suppressed Ag priming of Th cells for the Ab responses and proliferative response.

EBI1/CCR7 is a new member of dendritic cell chemokine receptor that is up-regulated upon maturation.

Dendritic cells (DC) that are stimulated with inflammatory mediators can maturate and migrate from nonlymphoid tissues to lymphoid organs to initiate T cell-mediated immune responses. This migratory step is closely related to the maturation of the DC. In an attempt to identify chemokine receptors that might influence migration and are selectively expressed in mature DC, we have discovered that the chemokine receptor, EBI1/CCR7, is strikingly up-regulated upon maturation in three distinct culture systems: 1) mouse bone marrow-derived DC, 2) mouse epidermal Langerhans cells, and 3) human monocyte-derived DC. The EBI1/CCR7 expressed in mature DC is functional because ELC/MIP-3beta, recently identified as a ligand of EBI1/CCR7, induces a rise in intracellular free calcium concentrations and directional migration of human monocyte-derived mature DC (HLA-DRhigh, CD1a(low), CD14-, CD25+, CD83+, and CD86high) in a dose-dependent manner, but not of immature DC (HLA-DRlow, CD1a(high), CD14-, CD25-, CD83-, and CD86-). In contrast, macrophage inflammatory protein-1alpha (MIP-1alpha), monocyte chemotactic protein-3 (MCP-3), and RANTES are active on immature DC but not on mature DC. Thus, it seems likely that MIP-1alpha, MCP-3, and RANTES can mediate the migration of immature DC located in peripheral sites, whereas ELC/MIP-3beta can direct the migration of Ag-carrying DC from peripheral inflammatory sites, where DC are stimulated to up-regulate the expression of EBI1/CCR7, to lymphoid organs. It is postulated that different chemokines and chemokine receptors are involved in DC migration in vivo, depending on the maturation state of DC.

Polyamide microcapsules containing alginic acid: extractability of metal ions and surface characterization by XPS.

Polyamide microcapsules containing alginic acid as a water-soluble macromolecular ligand (Alg-MC) were prepared by the interfacial polycondensation of sebacoyldichloride with hexamethylenediamine in a w/o emulsion system. The mean diameter of the microcapsules was 1.2 microns. The extractabilities of Cu(II), Ni(II), Co(II) and Ag(I) into the Alg-MC were examined and the highest uptake was found for Cu(II). It was ascertained that not only the inner ligand solution but also the membrane can accumulate the metal ions. The surface composition of the microcapsules was characterized by X-ray photo-electron spectroscopy (XPS) and it was found that some functional groups of alginic acid were present at the surface penetrating the membrane.

Neutralization of biological activity and inhibition of receptor binding by antibodies against human thrombopoietin.

Thrombopoietin (TPO) is a recently isolated cytokine that primarily regulates megakaryocytopoiesis and thrombopoiesis. We recently reported the development of a variety of antibodies (Abs) to synthetic peptides of human (h)TPO and to recombinant human TPO (rhTPO). In this study, we characterized the Abs and mapped immunologically distinct areas of the molecule. Among the five different antipeptide polyclonal Abs, only one, raised against synthetic peptide D8 to Q28, neutralized the TPO-dependent growth of FDCP-2 cells expressing human Mpl (FDCP-hMpl5 cells). One out of seven anti-rhTPO monoclonal Abs, designated as TN1, also showed neutralizing activity. TN1 was found to be specifically reactive with two proteolytic fragments, residues S1 to R117 and A60 to K122 of hTPO, indicating that the epitope(s) of TN1 is localized in residues A60 to R117 of the molecule. These two neutralizing Abs inhibited the binding of biotinylated rhTPO to FDCP-hMpl5 cells. On the other hand, the other Abs, which reacted with five polypeptides of S47 to D62, L108 to A126, N172 to A190, S262 to T284, and P306 to G332 of hTPO, did not show either the neutralizing activity or the ability to inhibit the binding of biotinylated rhTPO to the cell surface hMpl. These findings indicate that two regions, residues D8 to Q28 and A60 to R117 of hTPO, may contain the domains associated with its receptor, C-Mpl. These Abs characterized here are valuable for studying the structural analysis and the biological function of hTPO mediated by its receptor.