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The osmotic stress imposed on microorganisms by hypotonic conditions is perceived to regulate water and solute flux via cell membranes, which are crucial for survival. Some cells that fail to perceive osmotic stress die because this results in the rupture of the cell membrane. The flux through the membrane is characterized by the membrane permeability, which is measured using a stopped-flow apparatus in response to a millisecond-order osmolarity change. However, the obtained data are an ensemble average of each cell response. Additionally, the measurement of permeability, considering cellular viability, contributes to a more accurate evaluation of osmoadaptation. Here, we present a novel on-chip instantaneous extracellular solution exchange method using an air–liquid interface. The presented method provides a concurrent evaluation at the single-cell level in response to a millisecond-order osmotic shock, considering cellular viability by solution exchange. This method utilizes a liquid bridge with a locally formed droplet on the surface of a micropillar fabricated inside a microchannel. We evaluated a solution exchange time of 3.6 ms and applied this method to Synechocystis PCC 6803 under two different osmolarity conditions. The live/dead ratio of 1 M to 0.5 M osmotic down shock condition was 78.8/21.2% while that of 1 M to 0.25 M osmotic down shock condition was 40.0/60.0%. We evaluated the water permeability of two groups: cells that were still live before and after osmotic shock (hereafter named cell type 1), and cells that were live before but were dead 10 minutes after osmotic shock (hereafter named cell type 2). The results indicated that the water permeability of cell type 2 was higher than that of cell type 1. The results obtained using the presented methods confirmed that the effect of osmotic stress can be accurately evaluated using single-cell analysis. [ABSTRACT FROM AUTHOR]

The integration of liquid exchange and microfluidic chips plays a critical role in the biomedical and biophysical fields as it enables the control of the extracellular environment and allows for the simultaneous stimulation and detection of single cells. In this study, we present a novel approach for measuring the transient response of single cells using a system integrated with a microfluidic chip and a probe with a dual pump. The system was composed of a probe with a dual pump system, a microfluidic chip, optical tweezers, an external manipulator, an external piezo actuator, etc. Particularly, we incorporated the probe with the dual pump to allow for high-speed liquid change, and the localized flow control enabled a low disturbance contact force detection of single cells on the chip. Using this system, we measured the transient response of the cell swelling against the osmotic shock with a very fine time resolution. To demonstrate the concept, we first designed the double-barreled pipette, which was assembled with two piezo pumps to achieve a probe with the dual pump system, allowing for simultaneous liquid injection and suction. The microfluidic chip with on-chip probes was fabricated, and the integrated force sensor was calibrated. Second, we characterized the performance of the probe with the dual pump system, and the effect of the analysis position and area of the liquid exchange time was investigated. In addition, we optimized the applied injection voltage to achieve a complete concentration change, and the average liquid exchange time was achieved at approximately 3.33 ms. Finally, we demonstrated that the force sensor was only subjected to minor disturbances during the liquid exchange. This system was utilized to measure the deformation and the reactive force of Synechocystis sp. strain PCC 6803 in osmotic shock, with an average response time of approximately 16.33 ms. This system reveals the transient response of compressed single cells under millisecond osmotic shock which has the potential to characterize the accurate physiological function of ion channels. [ABSTRACT FROM AUTHOR]

Prevention and treatment of Alzheimer disease are urgent problems for elderly people in developed countries. We previously reported that nobiletin, a poly-methoxylated flavone from the citrus peel, improved the symptoms in various types of animal models of memory loss and activated the cAMP responsive element (CRE)-dependent transcription in PC12 cells. Nobiletin activated the cAMP/PKA/MEK/Erk/MAPK signaling pathway without using the TrkA signaling activated by nerve growth factor (NGF). Here, we examined the effect of combination of nobiletin and NGF on the CRE-dependent transcription in PC12 cells. Although NGF alone had little effect on the CRE-dependent transcription, NGF markedly enhanced the CRE-dependent transcription induced by nobiletin. The NGF-induced enhancement was neutralized by a TrkA antagonist, K252a. This effect of NGF was effective on the early signaling event elicited by nobiletin. These results suggested that there was crosstalk between NGF and nobiletin signaling in activating the CRE-dependent transcription in PC12 cells. Key words: Alzheimer disease, CRE-dependent transcription, flavone, nerve growth factor, neuron, nobiletin, PC12 cells. La prevention et le traitement de la maladie d'Alzheimer sont des problemes urgents pour les personnes agees des pays developpes. Anterieurement, nous avons rapporte que la nobiletine, une flavone poly-methoxylee que l'on trouve dans la pelure d'agrumes, permet d'attenuer les symptomes dans divers types de modeles de perte de memoire chez l'animal et d'activer la transcription dependante des elements de reponse a l'AMPc (CRE) dans les cellules PC12. En fait, la nobiletine activait la voie de signalisation AMPc/PKA/MEK/Erk/MAPK sans utiliser la signalisation de la TrkA activee par le facteur de croissance neuronal (NGF). Dans les presents travaux, nous avons etudie les effets de l'association de la nobiletine avec le NGF sur la transcription dependante des CRE dans les cellules PC12. Nous avons observe que l'administration du NGF seul avait peu d'effet sur la transcription dependante des CRE, mais qu'elle amplifiait de facon marquee la transcription dependante des CRE induite par la nobiletine. En fait, l'amplification engendree par le NGF etait neutralisee par le K252a, un antagoniste de la TrkA. En definitive, le NGF agissait des le debut du processus de signalisation engendre par la nobiletine. Ces resultats donnent a penser qu'il y aurait un couplage entre le NGF et la signalisation par la nobiletine pour l'activation de la transcription dependante des CRE dans les cellules PC12. [Traduit par la Redaction] Mots-cles: maladie d'Alzheimer, transcription dependante des CRE, flavone, facteur de croissance neuronal, neurone, nobiletine, cellules PC12., Introduction Alzheimer disease (AD) is a neurodegenerative disorder characterized by impaired cognition and memory loss. The progression of AD takes more than 10 years and the illness results in a [...]

The [Na.sup.+]-dependent [K.sup.+] uptake KtrABE system is essential for the adaptation of Synechocystis to salinity stress and high osmolality. While KtrB forms the [K.sup.+]-translocating pore, the role of the subunits KtrA and KtrE for Ktr function remains elusive. Here, we characterized the role of KtrA and KtrE in Ktr-mediated [K.sup.+] uptake and in modulating [Na.sup.+] dependency. Expression of KtrB alone in a [K.sup.+] uptake-deficient Escherichia coli strain conferred low [K.sup.+] uptake activity that was not stimulated by [Na.sup.+]. Coexpression of both KtrA and KtrE with KtrB increased the [K.sup.+] transport activity in a [Na.sup.+]-dependent manner. KtrA and KtrE were found to be localized to the plasma membrane in Synechocystis. Site-directed mutagenesis was used to analyze the role of single charged residues in KtrB for Ktr function. Replacing negatively charged residues facing the extracellular space with residues of the opposite charge increased the apparent K, for [K.sup.+] in all cases. However, none of the mutations eliminated the [Na.sup.+] dependency of Ktr-mediated [K.sup.+] transport. Mutations of residues on the cytoplasmic side had larger effects on [K.sup.+] uptake activity than those of residues on the extracellular side. Further analysis revealed that replacement of R262, which is well conserved among Ktr/Trk/HKT transporters in the third extracellular loop, by Glu abolished transport activity. The atomic-scale homology model indicated that R262 might interact with E247 and D261. Based on these data, interaction of KtrA and KtrE with KtrB increased the [K.sup.+] uptake rate and conferred [Na.sup.+] dependency. doi: 10.1128/JB.00569-10