Development of a simultaneous electrorotation device with microwells for monitoring the rotation rates of multiple single cells upon chemical stimulation.

Here, we described a unique simultaneous electrorotation (ROT) device for monitoring the rotation rate of Jurkat cells via chemical stimulation without fluorescent labeling and an algorithm for estimating cell rotation rates. The device comprised two pairs of interdigitated array electrodes that were stacked orthogonally through a 20 µm-thick insulating layer with rectangular microwells. Four microelectrodes (two were patterned on the bottom of the microwells and the other two on the insulating layer) were arranged on each side of the rectangular microwells. The cells, which were trapped in the microwells, underwent ROT when AC voltages were applied to the four microelectrodes to generate a rotating electric field. These microwells maintained the cells even in fluid flows. Thereafter, the ROT rates of the trapped cells were estimated and monitored during the stimulation. We demonstrated the feasibility of estimating the chemical efficiency of cells by monitoring the ROT rates of the cells. After introducing a Jurkat cell suspension into the device, the cells were subjected to ROT by applying an AC signal. Further, the rotating cells were chemically stimulated by adding an ionomycin (a calcium ionophore)-containing aliquot. The ROT rate of the ionomycin-stimulated cells decreased gradually to 90% of the initial rate after 30 s. The ROT rate was reduced by an increase in membrane capacitance. Thus, our device enabled the simultaneous chemical stimulation-induced monitoring of the alterations in the membrane capacitances of many cells without fluorescent labeling.

Determination of membrane capacitance and cytoplasm conductivity by simultaneous electrorotation.

Membrane capacitances and cytoplasm conductivities of hematopoietic cells were investigated by simultaneous electrorotation (ROT) systems of multiple cells. Simultaneous ROT was achieved by the rotation of electric fields in grid arrays formed with three-dimensional interdigitated array (3D-IDA) electrodes that can be easily fabricated using two substrates with IDA electrodes. When AC signals were applied to four microband electrodes with a 90° phase difference to each electrode, cells dispersed randomly in the 3D-IDA device started to rotate and moved to the center of each grid. Multiple cells were simultaneously rotated at the center of grids without friction from contact with other cells and substrates. The averages and variance of ROT rates of cells at each frequency can be measured during a single operation of the device within 5 min, resulting in the acquisition of ROT spectra. Membrane capacitances and cytoplasm conductivities of hematopoietic cells (K562 cells, Jurkat cells, and THP-1 cells) were determined by fitting ROT spectra obtained experimentally to the curves calculated theoretically. The values determined by using the simultaneous ROT systems well coincided with the values reported previously. The membrane capacitances and cytoplasm conductivities of WEHI-231 cells were firstly determined to be 8.89 ± 0.25 mF m-2 and 0.28 ± 0.03 S m-1, respectively. Furthermore, the difference of the ROT rates based on the difference of the electric properties of cells was applied to discriminate the types of cells. The acquisition of rotation rates of multiple cells within a single operation makes the statistical analysis extremely profitable for determining the electrical properties of cells.

Effects of trans-2-hexenal and cis-3-hexenal on post-harvest strawberry.

Green leaf volatiles are emitted by green plants and induce defence responses. Those with antifungal activities in plants may replace chemicals as natural post-harvest treatments. We investigated the postharvest treatment of strawberry with trans-2-hexenal and cis-3-hexenal and observed a decrease in the mould infection rate. To determine the volatiles' functions, we conducted a component analysis of the volatiles released from trans-2-hexenal-treated strawberry and analysed gene expression. Several acetates, which were expected to be metabolites of trans-2-hexenal in fruit, were released from treated strawberry; however, these acetates did not inhibit fungal growth. The gene expression analysis suggested that postharvest strawberries were not protected by jasmonic acid-mediated signalling but by another stress-related protein. Harvested strawberries experience stress induced by harvest-related injuries and are unable to perform photosynthesis, which might result in different responses than in normal plants.

Identifying stress markers in skin gases by analysing gas collected from subjects undergoing the Trier social stress test and performing statistical analysis.

In recent years, there has been great interest in mental health disorders. It is very important for people with mental health disorders to protect themselves from the stressful conditions that can occur as part of daily life before their symptoms become worse. We attempted to identify stress markers in skin gases in order to study the stress condition non-invasively in real time. We investigated changes in the skin gas components relative to the normal skin gas components of the subjects when stress was induced in the subjects using the Trier social stress test method, and we attempted to identify stress markers from those changes. We used a statistical analysis method and identified six stress markers in skin gases released from the armpits of the subjects.

N-Cα Bond Cleavage of Zinc-Polyhistidine Complexes in Electron Transfer Dissociation Mediated by Zwitterion Formation: Experimental Evidence and Theoretical Analysis of the Utah-Washington Model.

Electron capture dissociation (ECD) and electron transfer dissociation (ETD) of gas-phase ions are widely used for peptide/protein sequencing by mass spectrometry. To understand the general mechanism of ECD/ETD of peptides, we focused on the ETD fragmentation of metal-peptide complexes in the absence of remote protons. Since Zn(2+) strongly binds to neutral histidine residues in peptides, Zn(2+)-polyhistidine complexation does not generate any remote protons. However, in the absence of remote protons, electron transfer to the Zn(2+)-polyhistidine complex induced the N-Cα bond cleavage. The formation pathway for the ETD products was investigated by density functional theory calculations. The calculations showed that the charge-reduced zinc-peptide radical, [M + Zn](•+), can exist in the low-energy zwitterionic amide π* states, which underwent homolytic N-Cα bond dissociation. The homolytic cleavage resulted in the donation of an electron from the N-Cα bond to the nitrogen atom, producing an iminoenol c' anion. The counterpart z(•) radical contained a radical site on the α-carbon atom. The iminoenol c' anion then abstracted a proton to presumably form the more stable amide c' fragment. The current experimental and computational joint study strongly suggested that the N-Cα bond cleavage occurred through the aminoketyl radical-anion formation for Zn(2+)-polyhistidine complexes in ETD.