Novel separation mode of HPLC based on phase-separation multiphase flow.

A novel separation mode for high-performance liquid chromatography (HPLC) is proposed based on phase-separation multiphase flow. A commercially available HPLC system was used with a packed-separation column of octadecyl-silica (ODS)-modified particles. Water/acetonitrile/ethyl acetate ternary mixed solutions, (a) 1:8:1, (b) 1:3:1, and (c) 16:3:1 (v/v/v), were delivered into the system as an eluent at 20 and 5 °C. The ternary mixed solution acted as a two-phase separation solution leading to phase-separation multiphase flow. The solution flowed in the column homogeneously and heterogeneously at 20 and 5 °C, respectively. 1-Naphthol (NA) and 2,6-naphthalenedisulfonic acid (NDS) were injected into the system as model analytes. At 20 °C, the analyte mixture did not separate in solutions (a) and (b) while it separated in solution (c) with the elution order of NDS followed by NA. At 5 °C, it did not separate in solution (a), while it separated in solution (b) with elution order of NA followed by NDS and solution (c) with elution order of NDS followed by NA more effectively than 20 °C. The separation behavior and elution order are possibly caused by the phase-separation multiphase flow.

Periodicity in ultrasonic atomization involving beads-fountain oscillations and mist generation: Effects of driving frequency.

Ultrasonic atomization induced by high driving frequency, generally on the order of 1 MHz or higher, could involve a liquid fountain in the form of a corrugated jet, or a chain of "beads" of submillimeter diameter in contact. This study concerns dynamics/instability of such beads fountain, observed under lower input power density (≤ 6 W/cm2) of the "flat" ultrasound transducer with a "regulating" nozzle equipped, exhibiting time-varying characteristics with certain periodicity. High-speed, high-resolution images are processed for quantitative elucidation: frequency analysis (fast Fourier transform) and time-frequency analysis (discrete wavelet transform) are employed, respectively, to evaluate dominant frequencies of beads-surface oscillations and to reveal factor(s) triggering mist emergence. The resulting time variation in the measured (or apparent) fountain structure, associated with the recurring-beads size scalable to the ultrasound wavelength, subsumes periodic nature predictable from simple physical modeling as well as principle. It is further found that such dynamics in (time-series data for) the fountain structure at given height(s) along a series of beads would signal "bursting" of liquid droplets emanating out of a highly deformed bead often followed by a cloud of tiny droplets, or mist. In particular, the bursting appears to be not a completely random phenomenon but should concur with the fountain periodicity with a limited extent of probability.

Microfluidic behavior of ternary mixed solutions of water/acetonitrile/ethyl acetate through experiments and computer simulations.

When ternary mixed solutions of water/acetonitrile/ethyl acetate are delivered into a microspace under laminar flow conditions, the solvent molecules show specific microfluidic flows, such as microfluidic inverted flow and tube radial distribution flow, which have been applied to novel analytical methods. In this paper, inverted flow was examined using various Y-type microchannels that had mixing angles of 0°, 90°, 180°, and 270°. Inverted flow was experimentally observed and the trigger phenomenon was also successfully expressed through computer simulations. Tube radial distribution flow, that is, annular flow, in a capillary tube is reported to cause exchange of the inner and outer phases based on the solvent composition of the ternary mixed solution. Tube radial distribution flow for an organic solvent-rich inner and a water-rich outer phases, as well as for a water-rich inner and an organic solvent-rich outer phases, could be well recreated by computer simulations for a ternary mixed solution. This highlights the effectiveness of computer simulations for such flow scenarios and will allow optimization of the operating conditions and design of microfluidic analytical devices.

Consecutive Sample Injection Analysis in Tube Radial Distribution Chromatography.

Tube radial distribution chromatography based on the tube radial distribution flow, or annular flow, in an open-tubular capillary has been reported, where the annular flow is created through phase-separation multiphase flow. We have proposed the first-ever procedure for consecutive sample injection analysis using chromatography. In basic terms, a commercially available HPLC system could be used with a sample injector (0.2 µL volume) and a fused-silica capillary tube (250 cm long) as a separation column instead of a normal packed one, while the built-in detection cell was replaced by improved on-capillary detection. A ternary mixed solution of water/acetonitrile/ethyl acetate (3:8:4 volume ratio) was delivered into the capillary tube as an eluent at a flow rate of 2.0 µL min-1. Model sample solutions of 1-naphthol and 2,6-naphthalenedisulfonic acid were consecutively analyzed by the present chromatography with a processing rate of 6 samples per hour. Simple and rapid consecutive analysis could be performed because washing and initialization of the separation tube was no longer necessary. The obtained results provide clues to developing new methodologies which combine features of both chromatography (separation) and the flow injection method (consecutive analysis).

Phase Separation and Collection of Annular Flow by Phase Transformation.

A polyethylene glycol/citrate mixed solution was fed into a single channel of a Y-type micro-channel on a microchip as an aqueous two-phase system. A phase separation multi-phase flow with a liquid-liquid interface was generated due to a phase transformation. An annular flow, one of the flow types in the phase separation multi-phase flow, was observed through bright-field microscopy. The flow consisted of citrate-rich inner and polyethylene glycol-rich outer phases. We attempted to separate and collect the two phases in the single channel into two separate Y-type channels. When the pressure losses for the separated channels were not very different, we observed symmetric flow in the Y-type channel. When the pressure losses were quite different, the polyethylene glycol-rich phase with higher viscosity was selectively distributed to the separated channel with lower pressure loss. Thus, the polyethylene glycol-rich phase was successfully and intentionally collected from the chosen Y-type channel via the creation of annular flow in the single channel.

Development of Tube Radial Distribution Chromatography Based on Phase-Separation Multiphase Flow Created via Pressure Loss.

A tube radial distribution chromatography (TRDC) method based on phase-separated multiphase flow created through phase transformation via temperature change has been developed. These systems typically required a temperature-controlling device containing a water bath and a stirrer. Herein, we proposed a novel TRDC system without a cooling device, where the phase transformation was achieved via pressure loss in a capillary tube of 50 µm inner diameter and 550 cm length. Model analytes were successfully separated with the developed TRDC system, which provided a simplified platform and helped to clarify the operating principle of TRDC based on phase transformation in a capillary tube.

Microfluidic Inverted Flow of Ternary Water/Hydrophilic/Hydrophobic Organic Solvent Solution in a Y-Type Microchannel and a Proposal of the Response Microfluidic Analysis through the Experiment.

Two solutions that are individually fed at the same flow rate into two separate microchannels of a microchip, combine to form a single channel (a Y-type microchannel). This flow is either parallel for immiscible solutions or initially parallel, but then becomes homogeneous through diffusion, for miscible solutions. However, a new type of microfluidic behavior in a Y-type microchannel that was neither parallel nor homogeneous flow has been observed using, for example, water/acetonitrile (3:4.5, v/v) and acetonitrile/ethyl acetate (3.5:4, v/v) mixed solutions. Each mixed solution was marked with distinctive dyes and delivered at the same flow rate into a Y-type microchannel under laminar flow conditions. In the single channel, the two phases were initially observed to flow in parallel, but then apparently swapped to flow on the opposite wall while retaining parallel flow with a slight change in the components of the two phases. We have named this type of laminar flow "microfluidic inverted flow" for ternary water/hydrophilic/hydrophobic organic solvent mixed solutions. The inverted flow of a ternary water/acetonitrile/ethyl acetate system was examined in detail under various flow conditions. We also proposed a concept of response microfluidic analysis based on such microfluidic inverted flow.

Ionic Specificity in Rapid Coagulation of Silica Nanoparticles.

The Smoluchowski theory has been widely accepted as the basic theory to estimate the rapid coagulation rate of colloidal particles in electrolyte solutions. However, because the size and specificity of molecules and ions are not taken into account, the theory is applicable only if the particle size is large enough to neglect the effects caused by the structured layers composed of water molecules, ions, and hydrated ions adsorbed on the colloidal surface. In the present study, the rapid coagulation rates of silica nanoparticles in concentrated chloride and potassium solutions were measured by using a low-angle light-scattering apparatus, and the dependence of the experimental value of rapid coagulation rate, KER, on the particle diameter, Dp, and also on the Gibbs free energy of hydration of ions, ΔGhyd, was investigated extensively. The following were found. (1) When the particle size was small enough, the value of KER reduced exponentially not only with the decreasing particle size but also with the increasing value of (-ΔGhyd) of cations (counterions) in the case of chloride solutions and with that of anions (coions) in the case of potassium solutions. (2) Silica nanoparticles of Dp ≲ 70 nm in 1 M KNO3 and KSCN solutions did not coagulate at all, although they coagulated at Dp ≳ 100 nm as in the other potassium solutions. These unexpected phenomena were explained by the proposed mechanisms.

Orders of Magnitude Reduction of Rapid Coagulation Rate with Decreasing Size of Silica Nanoparticles.

The modification of the classical Smoluchowski theory for the rapid coagulation rate of colloidal particles, which takes account of the effect of the squeezing flow between colliding particles, has been widely accepted because it predicts experimental results adequately. However, it is not clear whether the modified theory, in which the coagulation rate is independent of the particle size, is applicable even to nanoparticles in solutions. In the present study, the rapid coagulation rates of silica particles in various 2 M chloride and 1 M potassium solutions were measured by using a low-angle light-scattering apparatus, and the dependence of rapid coagulation rate on the particle diameter, Dp, was investigated extensively. It was clearly shown that the rapid coagulation rate of spherical silica particles reduces by the orders of magnitude with decreasing particle size at Dp ≤ 300 nm, whereas it coincides with the value predicted by the modified theory at Dp ≥ 300 nm. A possible mechanism is proposed, and an analytical equation, which predicts the dramatic reduction in the rapid coagulation rate with decreasing particle size, is derived.

Pro-apoptotic protein glyceraldehyde-3-phosphate dehydrogenase promotes the formation of Lewy body-like inclusions.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has long been recognized as a classical glycolytic protein; however, previous studies by our group and others have demonstrated that GAPDH is a general mediator initiating one or more apoptotic cascades. Our most recent findings have elucidated that an expression of a pro-apoptotic protein GAPDH is critically regulated at the promoter region of the gene. Apoptotic signals for its subsequent aggregate formation and nuclear translocation are controlled by the respective functional domains harboured within its cDNA component. In this study, coexpression of GAPDH with either wild-type or mutant (A53T) alpha-synuclein and less likely with beta-synuclein in transfected COS-7 cells was found to induce Lewy body-like cytoplasmic inclusions. Unlike its full-length construct, the deleted mutant GAPDH construct (C66) abolished these apoptotic signals, disfavouring the formation of inclusions. The generated inclusions were ubiquitin- and thioflavin S-positive appearing fibrils. Furthermore, GAPDH coimmunoprecipitated with wild-type alpha-synuclein in this paradigm. Importantly, immunohistochemical examinations of post mortem materials from patients with sporadic Parkinson's disease revealed the colocalized profiles immunoreactive against these two proteins in the peripheral zone of Lewy bodies from the affected brain regions (i.e. locus coeruleus). Moreover, a quantitative assessment showed that about 20% of Lewy bodies displayed both antigenicities. These results suggest that pro-apoptotic protein GAPDH may be involved in the Lewy body formation in vivo, probably associated with the apoptotic death pathway.

Disclosure of a pro-apoptotic glyceraldehyde-3-phosphate dehydrogenase promoter: anti-dementia drugs depress its activation in apoptosis.

Overexpression and subsequent nuclear accumulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is involved in neuronal apoptosis induced by several stimuli in which GAPDH antisense oligonucleotides specifically block the increment (2 approximately 3 fold) of GAPDH mRNA contents occurring prior to neuronal death. However, these agents do not affect the basal, constitutive mRNA contents. This suggests that there may be distinct gene regulations for GAPDH mRNA expression. Herein, we cloned two types of promoter regions upstream of this gene; viz., #104 (1.02-kb) and #302 (2.46-kb). These fragments were inserted into the pGL3 luciferase reporter system and transiently transfected into cultured cerebellar neurons undergoing cytosine arabinonucleoside-induced apoptosis. The functional analysis of these constructs revealed that #104, but not #302, increased luciferase activity in response to the apoptotic stimulus. Deletion and replacement mutation analysis of the #104 fragment disclosed the promoter core harbored between the 154-bp and 84-bp domains (3.5-fold activity of the control). Furthermore, anti-dementia drugs (such as Cognex and Aricept) markedly depress the expression of this pro-apoptotic GAPDH promoter activity. Interestingly, immunocytochemical examination of human post-mortem materials from patients with Alzheimer's disease revealed nuclear aggregated GAPDH in neurons of the affected brain regions, implying an association with apoptotic cell death. The current findings indicate that induction of the pro-apoptotic protein GAPDH is genetically regulated at the level of promoter activation, and this protein may be an important molecular target for developing anti-apoptotic therapeutic agents in certain neurological illnesses.

Proapoptotic protein glyceraldehyde-3-phosphate dehydrogenase: a possible site of action of antiapoptotic drugs.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has long been recognized as a classical glycolytic protein and has been used as a "housekeeping" gene in studies of genetic expression and regulation. However, recent advances reveal that GAPDH displays diverse nonglycolytic functions depending on its subcellular localization. Among those functions, one of the most intriguing is likely to be the induction of apoptosis. Previous works by our group and others have demonstrated that the overexpression of GAPDH and its subsequent nuclear translocation appear to be implicated in the initiation of one or more apoptotic cascades and also in the etiology of some neurological diseases. This review addresses new data demonstrating that a protein, referred to as proapoptotic protein GAPDH, with a quite mundane function in healthy cells behaves very differently during cell suicide, and summarizes emphatically the importance of this protein as a putative molecular target in developing antiapoptotic therapeutic agents for the treatment of certain neurodegenerative disorders.