Formation Of Large Pores Research Articles

Beyond Amphiphilic Balance: Changing Subunit Stereochemistry Alters the Pore-Forming Activity of Nylon-3 Polymers.

Amphiphilic nylon-3 polymers have been reported to mimic the biological activities of natural antimicrobial peptides, with high potency against bacteria and minimal toxicity toward eukaryotic cells. Amphiphilic balance, determined by the proportions of hydrophilic and lipophilic subunits, is considered one of the most important features for achieving this activity profile for nylon-3 polymers and many other antimicrobial polymers. Insufficient hydrophobicity often correlates with weak activities against bacteria, whereas excessive hydrophobicity correlates with high toxicity toward eukaryotic cells. To ask whether factors beyond amphiphilic balance influence polymer activities, we synthesized and evaluated new nylon-3 polymers with two stereoisomeric subunits, each bearing an ethyl side chain and an aminomethyl side chain. Subunits that differ only in stereochemistry are predicted to contribute equally to amphiphilic balance, but we observed that the stereochemical difference correlates with significant changes in biological activity profile. Antibacterial activities were not strongly affected by subunit stereochemistry, but the ability to disrupt eukaryotic cell membranes varied considerably. Experiments with planar lipid bilayers and synthetic liposomes suggested that eukaryotic membrane disruption results from polymer-mediated formation of large pores. Collectively, our results suggest that factors other than amphiphilic balance influence the membrane activity profile of synthetic polymers. Subunits that differ in stereochemistry are likely to have distinct conformational propensities, which could potentially lead to differences in the average shapes of polymer chains, even when the subunits are heterochiral. These findings highlight a dimension of polymer design that should be considered more broadly in efforts to improve specificity and efficacy of antimicrobial polymers.

Membrane insertion mechanism and molecular assembly of the bacteriophage lysis toxin ΦX174-E.

The bacteriophage ΦX174 causes large pore formation in Escherichiacoli and related bacteria. Lysis is mediated by the small membrane-bound toxin ΦX174-E, which is composed of a transmembrane domain and a soluble domain. The toxin requires activation by the bacterial chaperone SlyD and inhibits the cell wall precursor forming enzyme MraY. Bacterial cell wall biosynthesis is an important target for antibiotics; therefore, knowledge of molecular details in the ΦX174-E lysis pathway could help to identify new mechanisms and sites of action. In this study, cell-free expression and nanoparticle technology were combined to avoid toxic effects upon ΦX174-E synthesis, resulting in the efficient production of a functional full-length toxin and engineered derivatives. Pre-assembled nanodiscs were used to study ΦX174-E function in defined lipid environments and to analyze its membrane insertion mechanisms. The conformation of the soluble domain of ΦX174-E was identified as a central trigger for membrane insertion, as well as for the oligomeric assembly of the toxin. Stable complex formation of the soluble domain with SlyD is essential to keep nascent ΦX174-E in a conformation competent for membrane insertion. Once inserted into the membrane, ΦX174-E assembles into high-order complexes via its transmembrane domain and oligomerization depends on the presence of an essential proline residue at position 21. The data presented here support a model where an initial contact of the nascent ΦX174-E transmembrane domain with the peptidyl-prolyl isomerase domain of SlyD is essential to allow a subsequent stable interaction of SlyD with the ΦX174-E soluble domain for the generation of a membrane insertion competent toxin.

Mechanical characteristics of metal nanoparticle thin film on flexible substrate exposed to saline solution

The robust and reliable mechanical characteristics of metal nanoparticle (NP) thin films on flexible substrates are important because they operate under tensile, bending, and twisting loads. Furthermore, in wearable printed electronics applications, salty solutions such as sweat and seawater can affect the mechanical reliabilities of devices. In this paper, we investigated the effect of sodium chloride (NaCl) solutions on silver (Ag) NP thin films on flexible polymer substrate. After exposure to NaCl solution of Ag NP thin film, we observed the aggregation behavior between Ag NPs and formation of larger pores in the film due to the removal of organic capping layer from the surface of Ag NPs. The average porosity and 5% deviation strains of Ag NP thin films on the polyimide substrate were dramatically increased and decreased from 2.99% to 9.64% and from 3.94% to 0.87%, respectively, after exposure to NaCl solution for 1 h. Also, we verified a drastic deterioration of the surface adhesion of the Ag NP thin film to the substrate by exposure to NaCl solution. We could observe crack propagation and delamination by in-situ scanning electron microscope imaging. In addition, passivation effect by a parylene layer for preventing the permeation of the saline solution was investigated.

Does Cholinergic Stimulation Affect the P2X7 Receptor-Mediated Dye Uptake in Mast Cells and Macrophages?

Background: Extracellular ATP is a powerful trigger of neuroinflammation by activating immune cells via P2X7 receptors. Acetylcholine and nicotinic agonists inhibit ATP-triggered proinflammatory cytokines via the so-called “cholinergic anti-inflammatory pathway” (CAP). However, it remains unclear as to what stage of ATP-induced signaling cholinergic agents provide this anti-inflammatory effect. Using the specific property of P2X7 receptor to open a pathway permeable to large molecules, associated with activation of inflammasome, we studied the action of cholinergic agents on this key event in CAP activation.Methods: Freshly isolated mouse peritoneal mast cells and primary human macrophages were used. To assess P2X7 channel opening, the permeability to the fluorescent dye YO-PRO1 or ethidium bromide (EtBr) was measured by flow cytometry. Expression of nicotinic receptors was probed in macrophages with the fluorescently labeled α-bungarotoxin or with patch-clamp recordings.Results: ATP opened P2X7 ion channels in mast cells and macrophages permeable to YO-PRO1 or EtBr, respectively. This stimulatory effect in mast cells was inhibited by the specific P2X7 antagonist A839977 confirming that YO-PRO1 uptake was mediated via ATP-gated P2X7 ion channels. Cholinergic agents also slightly induced dye uptake to mast cells but not in macrophages, which expressed functional α7 nicotinic receptors. However, both in mast cells and in macrophages, acetylcholine and nicotine failed to inhibit the stimulatory effect of ATP on dye uptake.Conclusion: These data suggest that in immune cells, cholinergic agents do not act on P2X7 receptor-coupled large pore formation but can mediate the anti-inflammatory effect underlying CAP downstream of ATP-driven signaling.

Microstructural insight into permeability and water retention property of compacted binary silty clay

The durability of silty clay embankments is partially controlled by the moisture migration, which depends on soil hydraulic properties. This paper presents an experimental study of hydraulic properties of compacted binary silty clay. Specimens with different mixing ratios and dry densities were prepared. Scanning electron microscopy and mercury intrusion porosimetry were used to characterise the microstructure of silty clay. Thereafter, falling-head permeability tests and water retention tests were conducted to study the permeability and water retention property, respectively. The results demonstrate that clay particles are dispersed and show preferred arrangements after compaction when the clay content is 100%. As the clay content decreases, the arrangement of clay particles is gradually disturbed because of the existence of silt particles, causing the formation of large pores around silt particles. When the dry density increases, the pores around silt particles significantly decrease. Moreover, the permeability of silty clay decreases but the water retention capacity increases with increasing clay content and dry density. This is because the silty clay with larger clay content and dry density has fewer large pores, which greatly restrains the flow of water. Both the permeability and water retention property of silty clay can be predicted from pore size distribution parameters.

Haemophilus influenzae HP1 Bacteriophage Encodes a Lytic Cassette with a Pinholin and a Signal-Arrest-Release Endolysin.

HP1 is a temperate bacteriophage, belonging to the Myoviridae family and infecting Haemophilus influenzae Rd. By in silico analysis and molecular cloning, we characterized lys and hol gene products, present in the previously proposed lytic module of HP1 phage. The amino acid sequence of the lys gene product revealed the presence of signal-arrest-release (SAR) and muraminidase domains, characteristic for some endolysins. HP1 endolysin was able to induce lysis on its own when cloned and expressed in Escherichia coli, but the new phage release from infected H. influenzae cells was suppressed by inhibition of the secretion (sec) pathway. Protein encoded by hol gene is a transmembrane protein, with unusual C-out and N-in topology, when overexpressed/activated. Its overexpression in E. coli did not allow the formation of large pores (lack of leakage of β-galactosidase), but caused cell death (decrease in viable cell count) without lysis (turbidity remained constant). These data suggest that lys gene encodes a SAR-endolysin and that the hol gene product is a pinholin. HP1 SAR-endolysin is responsible for cell lysis and HP1 pinholin seems to regulate the cell lysis and the phage progeny release from H. influenzae cells, as new phage release from the natural host was inhibited by deletion of the hol gene.

Effects of sodium salt additive to produce ultra lightweight aggregates from industrial sludge-marine clay mix: Laboratory trials

The production of lightweight aggregates from waste is regarded as an environmentally benign means of recycling waste into materials for green building construction, supporting the circular economies of modern societies. In this investigation, aggregates are synthesized from industrial sludge-marine clay mixes using thermal sintering at 1000‒1300 °C with or without the addition of two sodium salts. The sludge-clay 50/50 mix that is fired at 1200 °C and the 0/100 to 50/50 mixes that are fired at 1300 °C yield aggregates with a specific gravity of less than unity (lightweight aggregates). The addition of two sodium salts at 1.5% w/w facilitated the formation of lightweight aggregates. This study reports the first time the production of ultra-lightweight aggregates with a specific gravity of less than 0.35 g/cm3 and sufficiently low water adsorption ratios from a 50/50 mixture with added NaCl and from a 20/80 mix with added Na2CO3 for construction material use. The sodium salt is posited to change the melt properties at the firing temperature used, causing the molten layers to retain their integrity when subjected to gas blasting with the formation of large internal pores and sufficient surface sealing.

Geochemistry effects of supercritical CO2 and H2O on the mesopore and macropore structures of high-rank coal from the Qinshui Basin, China

Abstracts The effects of supercritical CO2 (scCO2) on coal pores play a critical role in the geological storage of CO2 and enhanced coalbed methane recovery (CO2-ECBM). To investigate the effects of scCO2 - H2O on the mesopore - macropore structure of high-rank coals, a series of experimental simulations and analyses were performed. Coal samples from the Qinshui Basin, China were exposed to scCO2 and deionized water for 240 h at 45 °C and 10 MPa and at 80 °C and 20 MPa. The influences on the pore structure were analyzed by performing a mercury intrusion capillary pressure (MICP) measurement. Using a field emission scanning electron microscope (FE-SEM) and an advanced mineral identification control system (AMICS), the geochemical effects of scCO2-H2O on the same minerals and the same pore or fracture (nanometer to micrometer scale) before and after the reaction were investigated. Based on the changes of the minerals, pores and fractures, the types, targets, expressions, and scopes of the geochemical effects were explored. The experimental results show that after the injection of scCO2 into the coal samples, the pores and fractures were newly generated or expanded under the interactions of mineral dissolution-precipitation, clay hydration and swelling, coal swelling, and pyrite oxidation with gypsum precipitation. Moreover, the largest improvement of the pore volume was observed in pores larger than 30 μm or smaller than 100 nm, and higher temperatures and pressures were beneficial to the formation of large pores and fractures. The change in the specific surface area (SSA) was mainly caused by the change in pore volume. The corrosion of carbonate minerals showed the most significant contribution to the pore volume of full-scale macropores and mesopores, while most geochemical effects on other minerals had impacts on the mesopores and the macropores smaller than 1 μm. The coal swelling had the most significant effect on the connectivity of the pores in the coal reservoir.

Mechanical properties and water absorption of steam-cured mortar containing phase change composites

Steam curing is a common method used in the production of precast concrete components. However, the formation of microcracks and large pores during steam curing process are very detrimental to the long-term performance of cement-based materials. The use of phase change materials may be a potential method for improving disadvantages caused by steam curing. Phase change composites are prepared by implanting paraffin into diatomite, expanded graphite, and mixture of diatomite and expanded graphite. They are represented by D/P, EG/P and D/EG/P in turn. The thermal properties and static water contact angle of phase change composite (CPCM) are tested. The mechanical and water absorption properties of steam-cured mortars with CPCM are investigated. Results indicate that the latent heat values of EG/P, D/P and D/EG/P are 80%, 30% and 36% of the values for pure paraffin, respectively. The CPCMs exhibit hydrophobic properties due to the roughness of solid surface. The presence of CPCM greatly improve the water resistance of steam-cured mortar without sacrificing mechanical properties, and steam-cured mortar with 1 vol% D/P has the best performance. The possible reason is that D/P reduces the porosity and improves porous microstructure of steam-cured mortar. The cracks and holes in the mortar matrix can be covered and filled with paraffin from D/P by SEM analysis. There is no interfacial gap between D/P and the mortar matrix through SEM analysis, which promotes the formation of a dense and strong mortar matrix. Therefore, the addition of an appropriate CPCM is helpful to improve the durability of the steam-cured cementitious materials.

P2X7 receptor activity landscape in rat and human glioma cell lines.

P2X7 is a commonly expressed purinergic receptor, which functions as a cation-permeable channel in the plasma membrane. In certain circumstances, the receptor may also form a large transmembrane pore what results in cell death. P2X7 receptors control numerous physiological and pathological cellular processes and their overexpression is often associated with cancer progression. As nucleotides are important signaling molecules in the central nervous system, P2X7 plays also an important but ambiguous role in glioma biology with contrary observations originating from different glioma models. Therefore, the aim of our research was to investigate P2X7 receptor expression and functions in three human (U-87 MG, U-138 MG, U-251 MG) and one rat (C6) glioma cell lines. Although the receptor mRNA and protein were present in all the studied cells, we found profound differences in their level. We also encountered a problem with one human cell lines authenticity (U-87 MG) and excluded it from most of the experiments. Interestingly, there was no clear dependency between P2X7 receptor level, calcium signal and pore formation ability in the studied glioma lines. In U-138 human cell line, the receptor seemed to be inactive, while in U-251 human and C6 rat cell line its activation resulted in calcium influx and large pore formation. However, the viability of studied cells upon the administration of specific P2X7 agonist - BzATP - was not affected for U-138 and U-251, whereas for C6 cells a stimulatory effect was observed. Our results stress the variability of P2X7 signaling in glioma models and the need for future research which would take into account the complicated landscape of the receptor signaling in the brain.

Effects of remoulding and wetting-drying-freezing-thawing cycles on the pore structures of Yanji mudstones

The pore structure governs the physical and mechanical behaviours of geomaterials, thereby affecting the stability of the infrastructures built on these materials. This study investigated the effects of remoulding and wetting-drying-freezing-thawing (WDFT) cycles on the pore structures of two Yanji mudstones using scanning electronic microscopy (SEM), mercury intrusion porosimetry (MIP) and nitrogen adsorption (NA) techniques. The results show that the pores of both the yellow-brown and magenta mudstones become rounder and more disordered after remoulding. Remoulding leads to an increase in the inter-aggregate pore volume and a decrease in the intra-aggregate pore volume because of the exposure of intra-aggregate pores after crushing and the compaction of aggregates during the compression process. The W-D-F-T cycles make the pores of the remoulded yellow-brown mudstone more disordered and flatter and induce an increase in the inter-aggregate pore volume due to the formation of cracks and large pores. Meanwhile, the intra-aggregate pore volume decreases owing to the contraction of aggregates during the drying process. According to the increased inter-aggregate pore volume and decreased intra-aggregate pore volume, an increase in the hydraulic conductivity and a decrease in the shear strength can be expected after remoulding and W-D-F-T cycles, which decrease the stability of mudstone slopes.

Performance Decay of Air Electrode Configuration for Rechargeable Zinc-Air Batteries

The construction of the air electrode structure is critical in the rechargeable zinc-air batteries. So far, the majority of previous articles have been reported on the process in boosting electrocatalytic bifunctional oxygen reaction activity. Nevertheless, the reason that caused the deterioration of air electrodes has been rarely investigated, which dramatically influences cell performance. Herein, self-supported MnO2 bifunctional electrocatalysts on nickel foam are integrated with the gas diffusion layer to form air electrodes. The charge voltage of the as-prepared electrode used in rechargeable zinc-air battery is reduced by 1.82% and the discharge voltage is decreased by 5.25% after 1000 cycles at the current density of 20 mA cm−2 under the mode of 5 min charge and 5 min discharge, which demonstrate the feasibility of the novel electrode structure design. Besides, The X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and electrochemical impedance spectroscopy (EIS) were conducted to analyze the change of composition, structure, and performance. The origin of the decrease of the discharge profile was analyzed. Experimental studies revealed that the main cause of voltage loss is mainly attributed to the destroyed gas diffusion structure (at large current densities) and the activation loss of MnO2 caused by the (oxy)hydroxide film (at small current densities). The destroyed gas diffusion structure gives rise to the poor oxygen transfer and further water flooding due to the formation of large pore caused by oxygen evolution in the charging process and increased hydrophilicity by the carbon oxidation. Meanwhile, the film growing overtop of the electrocatalysts would result in mass transfer and activation losses.

Analysis of fluid permeability in tortuous fibrous bed based on pore radius distribution

The effect of fiber tortuosity on fluid permeability in fibrous beds has been investigated. A particle model is employed in which a fiber is replaced by spherical particles and fiber tortuosity is represented by a bending angle. Assuming a low Reynolds number, the fluid permeability is calculated based on the Stokesian dynamics approach. The results show that the permeability of a tortuous fibrous bed is lower than that of a straight fibrous bed. To examine the permeability dependence on the fiber tortuosity in detail, the pore distribution and pore connectivity are evaluated quantitatively by defining the pore radius. It is found that the tortuous fibers prevent the formation of large pores and their connection in the flow direction, thereby decreasing the permeability.

Catalytic upgrading of lignite pyrolysis volatiles over modified HY zeolites

HY zeolite is modified by hydrothermal and HNO3 treatment and used as the catalyst to upgrade Baiyinghua pyrolysis volatiles on a fixed-bed reactor at 600 °C. The results show that hydrothermal and acid treatment obviously affect zeolite structure and upgrading of pyrolysis volatiles. The dealumination of HY increases with hydrothermal temperature, resulting in the decrease of total surface area and acid amount and the formation of larger pores. However, the content and yield of light tar are enhanced over modified HY, and the cracking of volatiles into gas is retarded. Acid treatment of hydrothermal modified HY at 650 °C removes extra-framework Al, improves surface area and the volatiles cracking. But high acid concentration leads to the destruction of zeolite structure, and is unfavorable to the upgrading. Modified HY by treatment at 650 °C and 0.8 mol/L HNO3 shows the best performance in catalytic volatiles upgrading. About 81.5 wt% light tar content and 61.1% light aromatic hydrocarbons content can be obtained. Especially, the yields of naphthalene and methylnaphthalene are increased by 8.9 and 6.8 times. The content of oxygenated PAHs is reduced by 4.3 times. The improved performance is mainly due to high surface area, large pore diameter and suitable acidity.

Impact of the fermentation broth of Ganoderma lucidum on the quality of Chinese steamed bread

The potential of fermentation broth of Ganoderma lucidum (FBG) in improving the quality of Chinese steamed bread (CSB) was firstly evaluated. The sensory quality scores of CSB treated by FBG are significantly higher than that of CSB in the control, and texture profile analysis also indicates the increase of CSB hardness and chewiness caused by FBG. Observation on micro-structure of CSB shows that formation of larger pores and expansion of starch granules are the important reasons for the improvement of CSB specific volume (volS), and granule expansion is due to that gluten network distributed in CSB is destroyed as a result of cross-linkage of flour proteins catalyzed by laccase, which makes starch granules releasing from the network easily contact with steam or other enzymes during the proofing and steaming of dough. Moreover, FBG contains amylases which not only convert amylopectin to amylose, but also degrade starch to glucose, maltose and polysaccharides, correspondingly resulting in changes of amylose/amylopectin (Ae/An) ratio of flour and CSB volS, and the latter is because more CO2 produced by the yeast during CSB making leads to the larger pore area in crumb. Both hardness and chewiness are determined by the comprehensive effect of protein cross-linkage, Ae/An ratio and volS change, and this viewpoint gives a logical explanation for the effects of 0.025–0.10 ml/g of FBG on hardness and chewiness of CSB.

Pore-graded and conductor- and binder-free FeS2films deposited by spray pyrolysis for high-performance lithium-ion batteries

Abstract

Formation Mechanism of Pores Inside Structure Fabricated by Metal-Based Additive Manufacturing

The powder bed fusion (PBF) technique is a metal-based additive manufacturing (AM) method in which metal powder is deposited on a substrate and melted by selective laser-beam irradiation. Given that the process and parameters of metal-based AM are complicated, there are various problems in high-precision fabrication. One of these is that although metal-based AM can be used for fabrication of high-density parts, pores can easily form inside the fabricated structure owing to process instabilities. Pore formation degrades the mechanical strength of the fabricated structure. Therefore, this study investigated the pore formation mechanism inside a structure fabricated by PBF. Pore suppression by controlling the substrate temperature was also evaluated. Small- and large-sized pores with diameters of 10 μm and more than 50 μm, respectively, were found. Furthermore, differences in pore formation in the cross-section of the fabricated structure were observed owing to a variation in the volume-specific energy density and substrate temperature. At a substrate temperature of 25°C, the number of pores decreased more at the upper position than at the lower position owing to repeated melting and solidification under the laser-beam irradiation. At a substrate temperature of 200°C, the number of pores decreased significantly more than at 25°C. Furthermore, as the substrate temperature increased, the wettability of the molten metal improved, resulting in smaller contact angles of the fabricated structure in the single-line track. In PBF, multiple lines are fabricated in each layer. At low substrate temperatures, interstices were formed between the lines owing to the low wettability of the molten metal. These interstices acted as the origins of pores when the next layer was fabricated. Heating the substrate made the surface of the structure smooth owing to the high wettability of the molten metal and a reduction in the number of pores. Therefore, the formation of large pores could be reduced by controlling the substrate temperature.

Tailoring the molecular sieving properties and thermal stability of carbonized membranes containing polyhedral oligomeric silsesquioxane (POSS)-polyimide via the introduction of norbornene

Two types of POSS (polyhedral oligomeric silsesquioxane)-polymers, (POSS-polyimide-phenyl (POSS-PI-Ph) and POSS-polyimide-phenyl-norbornene (POSS-PI-Ph-Norbornene)), were utilized for the fabrication of highly permeable gas-separation membranes. POSS-PI-Ph and POSS-PI-Ph-Norbornene separation layer for selective gas separation were successfully formed on porous intermediate layer. A POSS-derived membrane calcined at 250–350 °C under N2 showed approximately the same levels of selectivity for He/N2 and CO2/N2 as conventional polyimide membranes, which shows there was no formation of large pores that decreases gas selectivity. The introduction of norbornene, which forms a cross-linked structure by thermally inducing cross-linking at 500 °C under N2, improved gas permeance without decreasing the gas-permeance ratio. A POSS-PI-Ph-Norbornene membrane heat treated at 500 °C showed increases in He permeance that ranged from 1.2 × 10−7 to 4.6 × 10−7 mol m−2 s−1 Pa−1 with He/N2, CO2/N2, He/CF4, and He/SF6 permeance ratios of 38; 8; 2700; and 14000, respectively. The relationship between the activation energy of H2 permeation and the He/H2 permeance ratio in POSS-derived, organosilica, and polymer (polyimide, polyamide, polysulfone) membranes suggests that gas permeation properties depend on the concentration of organic groups (C/Si ratio) in networks, because networks become more flexible when carbon concentration increases and dominates the permeation properties, which corresponds to a solution-diffusion mechanism.

Regulation of SNARE‐Dependent Membrane Fusion by Alpha‐Synuclein

Neurotransmitter release is a foundation of all thoughts in central nervous system. Communication between these neurons is mediated by the release and uptake of neurotransmitters through vesicle fusion at the synapses. The SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment Protein Receptor) proteins are believed to mediate neurotransmitter release by promoting fusion of neurotransmitter laden presynaptic vesicle with the presynaptic membrane. Defects in the regulation of neurotransmission are believed to be the primary cause for many neurological diseases. The key to a better understanding of neurological diseases may lie in a more complete understanding of the molecular machine responsible for synaptic vesicle fusion. Alpha-synuclein (αS) has long been a protein of great interest because of its pathological aggregation in neuronal plaque and Lewy bodies which are associated with Parkinson's Disease and Lewy Bodies Dementia, respectively. Despite its medical significance, the normal function of αS has been elusive. αS is a 14 kDa soluble protein, abundantly present (several μM) in the presynapse. Thus, not surprisingly, αS has been found to play roles in various aspects of vesicle recycling. Very interestingly, however, αS has emerged as an important regulator for SNARE-dependent vesicle fusion. We used an in vitro single-vesicle fusion assay which allowed for the precise variation of the αS concentration. This assay provided the opportunity to dissect the impact of αS on individual vesicle fusion steps such as docking and lipid mixing along the membrane fusion pathway. The assay is effective in analyzing single fusion events in a natural, millisecond timescale between vesicle and a supported-bilayer. Using this assay, we have found that αS enhances vesicle docking by a factor of 10 at excessive 20 μM which is as much as 4 times higher than a normal cellular concentration. Detailed analysis reveals that the effect of αS on SNARE-dependent membrane fusion is largely on vesicle docking with no significant changes in lipid mixing observed. It also suggests that αS plays a role in accelerating and stabilizing large fusion pore formation. αS may be pathologically involved in neurological diseases, but it also is a crucial SNARE regulator by enhancing presynaptic vesicle docking and stabilizing the membrane fusion pore. αS may be pathologically involved in neurological diseases, but it also is a crucial SNARE regulator This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The cell resealing technique for manipulating, visualizing, and elucidating molecular functions in living cells

BackgroundCell-based assays are essential for analyzing molecular functions and spatiotemporal information. The cell resealing technique, in which pore-forming toxins are used to permeabilize cell membranes, enables the delivery of various membrane-impermeable molecules inside cells. Scope of reviewWe review the basics of the resealed cell system, including optimized protocols, assessment of cellular damage, and recovery following permeabilization of the membrane. Additionally, we introduce the streptolysin O (SLO)-type and listeriolysin O (LLO)-type resealing techniques. In SLO, the formation of larger pores (~30 nm) enables the passage of a wider range of molecules. Then, we discuss the advantages and applications of the semi-intact cell system, in which ongoing permeabilization is selected to maintain and analyze a specific cellular environment. Major conclusionsAs confirmed by the effective use of quantitative image analysis, the SLO-type resealing system is successful for establishing and phenotyping diabetic model cells by introducing cytosol from diabetic mice. The LLO-type resealing technique enables the delivery of mid-sized molecules with high efficiency and low damage. As each technique has specific advantages, understanding the characteristics of LLO and SLO is necessary for choosing the appropriate technique. General significanceSLO-type resealing is optimal for creating disease model cells and drug screening, especially lifestyle-related diseases. LLO-type resealing is expected to be suitable for screening mid-sized biological drugs. Semi-intact cells can contribute to elucidating various cellular phenomena that have remained intractable due to their complexity.