Because of the power density advantages of fluid power systems, many researchers have developed microactuators using homogeneous electrorheological (ER) fluids (ERFs) for applications to various ...micromachines. An ER valve, as a critical component of the ER actuator, can control ERF flow by the apparent viscosity increase resulting from the applied electric field without any mechanical moving parts. Hence, it is adequate for the miniaturization of a fluidic microactuator. However, it is not easy to integrate rigid ER valves into soft microrobots. To overcome these limitations, we developed a novel elastic ER microarm using flexible ER valves (FERVs) in this study. Each microarm consists of an FERV, a movable chamber, and a displacement constraint element, so that it bends with the inner pressure controlled by the FERV. We proposed and developed a micro-electromechanical system fabrication process for the FERV, movable chamber, and displacement constraint element. By utilizing the proposed method, we successfully fabricate a FERV-integrated microarm. The characteristics of the FERV were experimentally clarified. In addition, the bending motion of the FERV-integrated microarm was demonstrated by experiments and verified by finite-element method simulation. This ER microarm was shown to be feasible for an ER microgripper composed of multiple microarms.
The Bcl-2 family of proteins, consisting of anti-apoptotic and pro-apoptotic members, regulates cell death by controlling mitochondrial membrane permeability that is crucial for apoptotic signal ...transduction. We have recently shown that some of these proteins, such as Bcl-xL, Bax, and Bak, directly modulate the mitochondrial voltage-dependent anion channel (VDAC) and thus regulate apoptogenic cytochrome crelease and potential loss. To elucidate the molecular mechanisms of VDAC regulation by Bcl-2 family proteins, an electrophysiological study was carried out. It was found that VDAC and pro-apoptotic Bax created a large pore, with conductance levels 4- and 10-fold greater than those of the VDAC and Bax channels, respectively. Although the VDAC and Bax channels both show ion selectivity and voltage-dependent modulation of their activity, the VDAC-Bax channel had neither of their properties. Anti-apoptotic Bcl-xL and its BH4 oligopeptide completely closed the VDAC, in contrast to the Bax. Cytochromec passed through a single VDAC-Bax channel but not through the VDAC or Bax channel in a planar lipid bilayer. These data provide direct evidence that VDAC forms a novel large pore together with Bax.
Selective protein export from the endoplasmic reticulum is mediated by COPII vesicles. Here, we investigated the dynamics of fluorescently labelled cargo and non‐cargo proteins during COPII vesicle ...formation using single‐molecule microscopy combined with an artificial planar lipid bilayer. Single‐molecule analysis showed that the Sar1p–Sec23/24p‐cargo complex, but not the Sar1p–Sec23/24p complex, undergoes partial dimerization before Sec13/31p recruitment. On addition of a complete COPII mixture, cargo molecules start to assemble into fluorescent spots and clusters followed by vesicle release from the planar membrane. We show that continuous GTPase cycles of Sar1p facilitate cargo concentration into COPII vesicle buds, and at the same time, non‐cargo proteins are excluded from cargo clusters. We propose that the minimal set of COPII components is required not only to concentrate cargo molecules, but also to mediate exclusion of non‐cargo proteins from the COPII vesicles.
We have developed a quantitative immunoassay chip targeting point-of-care testing. To implement a lateral flow immunoassay, a glass fiber sheet was chosen as the material for the microfluidic channel ...in which the negative charge on the fiber surfaces efficiently generates the electroosmotic flow (EOF). The EOF, in turn, allows controllable bound/free separation of antigen/antibody interactions on the chip and enables precise determination of the antigen concentration. In addition, the defined size of the porous matrix was suitable for the filtration of undesired large particles. We confirmed the linear relationship between the concentration of analyte and the resulting fluorescence intensity from the immunoassay of two model analytes, C-reactive protein (CRP) and insulin, demonstrating that analyte concentration was quantitatively determined within the developed chip in 20 min. The limits of detection were 8.5 ng mL(-1) and 17 ng mL(-1) for CRP and insulin, respectively.
Abstract
PTEN, a 3-phosphatase of phosphoinositide, regulates asymmetric PI(3,4,5)P
3
signaling for the anterior-posterior polarization and migration of motile cells. PTEN acts through posterior ...localization on the plasma membrane, but the mechanism for this accumulation is poorly understood. Here we developed an in vitro single-molecule imaging assay with various lipid compositions and use it to demonstrate that the enzymatic product, PI(4,5)P
2
, stabilizes PTEN’s membrane-binding. The dissociation kinetics and lateral mobility of PTEN depended on the PI(4,5)P
2
density on artificial lipid bilayers. The basic residues of PTEN were responsible for electrostatic interactions with anionic PI(4,5)P
2
and thus the PI(4,5)P
2
-dependent stabilization. Single-molecule imaging in living
Dictyostelium
cells revealed that these interactions were indispensable for the stabilization in vivo, which enabled efficient cell migration by accumulating PTEN posteriorly to restrict PI(3,4,5)P
3
distribution to the anterior. These results suggest that PI(4,5)P
2
-mediated positive feedback and PTEN-induced PI(4,5)P
2
clustering may be important for anterior-posterior polarization.
A Cry46Ab toxin derived from
Bacillus thuringiensis
strain TK-E6 shows mosquitocidal activity against
Culex pipiens pallens
Coquillett (Diptera: Culicidae) larvae as well as preferential cytotoxicity ...against human cancer cells. In
B. thuringiensis
cells, Cry46Ab is produced and accumulates as a protein crystal that is processed into the active 29-kDa toxin upon solubilization in the alkaline environment of the insect midgut. The Cry46Ab protoxin is 30 kDa, and is therefore thought to require an accessory protein such as P20 and/or ORF2 for efficient crystal formation. In the present study, the potency of the 4AaCter-tag was investigated for the production of alkali-soluble inclusion bodies of recombinant Cry46Ab in
Escherichia coli
. The 4AaCter-tag is a polypeptide derived from the C-terminal region of the
B. thuringiensis
Cry4Aa toxin and facilitates the formation of alkali-soluble protein inclusion bodies in
E. coli
. Fusion with the 4AaCter-tag enhanced both Cry46Ab production and the formation of Cry46Ab inclusion bodies. In addition, upon optimization of protein expression procedures, the Cry46Ab–4AaCter inclusion bodies showed mosquitocidal activity and stability in aqueous environments comparable to Cry46Ab without the 4AaCter-tag. Our study suggests that use of the 4AaCter-tag is a straightforward approach for preparing formulations of smaller-sized Cry toxins such as Cry46Ab in
E. coli
.
A change of cytosolic pH 7 to 4 opens the bacterial potassium channel KcsA. However, the overall gating mechanism leading to channel opening, especially the contribution of the cytoplasmic domain, ...remains unsolved. Here we report that deletion of the cytoplasmic domain resulted in changes in channel conductance and gating behavior at pH 4 without channel opening at pH 7. To probe for rearrangements in the cytoplasmic domain during channel opening, amino acid residues were substituted with cysteines and labeled with a fluorophore (tetramethylrhodamine maleimide) that exhibits increased fluorescence intensity upon transfer from a hydrophilic to hydrophobic environment. In all cases channel open probability (Po) was ∼1 at pH 4 and ∼0 at pH 7. Major increases in fluorescence intensity were observed for tetramethylrhodamine maleimide-labeled residues in the cytoplasmic domain as pH changed from 7 to 4, which suggests the fluorophores shifted from a hydrophilic to hydrophobic environment. Dipicrylamide, a lipid soluble quencher, reduced the fluorescence intensities of labeled residues in the cytosolic domain at pH 4. These results reveal that a decrease in pH introduces major conformational rearrangements associated with channel opening in the KcsA cytoplasmic domain.
Secondary aortoduodenal fistula (sADF) is a critical late complication of abdominal aortic repair, requiring complete excision of the infected prosthesis. However, this is a highly invasive procedure ...for the elderly. We describe a case of sADF repair in a 76-year-old woman. Through 18F (fluorine-18)-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography mapping, focal high FDG uptake at the sADF site, right medial limb, and ligated left lateral limb of the prosthesis was detected. The duodenal and prosthetic grafts were partially resected. The proximal and distal anastomotic segments, with no FDG uptake, were retained. The abdominal aorta was reconstructed using a bovine pericardium roll and femorofemoral bypass. Thus, FDG positron emission tomography/computed tomography mapping of the infection site could help in such cases.
Single-channel recording using artificial lipid bilayers is along with the patch-clamp technique a very powerful tool to physiologically and pharmacologically study ion channels. It is particularly ...advantageous in studying channels that are technically difficult to access with a patch pipet. However, the fragility of the bilayers and the difficulty to incorporate ion channels into them significantly compromises measurement efficiency. We have developed a novel method for forming artificial lipid bilayers on a hydrogel surface that significantly improves the measurement efficiency. Bilayers formed almost instantly (<1 s) and were able to incorporate various types of ion channel proteins within a short time (<30 s) enabling multichannel measurements. These results indicate that this method can potentially be applied to developing high-throughput screening devices for drug design.
Many proposals have been made regarding the development of biosensors using single-channel recording with an artificial planar bilayer. The fragile nature of bilayer membranes is the major difficulty ...for the application of the artificial bilayer technique to the development of biosensors. We have developed an apparatus that promptly forms artificial bilayers. This technique is more efficient than other techniques for forming artificial bilayers. Bilayer membranes could be formed within 10
s requiring 1
μl of analyte solution to record single-channel currents using our apparatus. A bilayer was formed by pressing the membrane on an agarose layer with hydraulic pressure. With this novel apparatus, we have recorded single-channel currents of various types of channels such as the BK-channel, the nicotinic receptor channel and the ryanodine receptor channel. The properties of the channels determined with this novel technique agreed well with those determined with conventional techniques.
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