Pore Formation Process Research Articles

The Influence of Lipid Tail Composition on Bid-Mediated Bax Pore Formation

Physical membrane properties are known to influence the function of proteins interacting with lipid bilayers. In the particular case of mitochondrial outer membrane permeabilization by the protein Bax, it is thought that modifications of membrane properties due to, for example, cholesterol overload or lipid oxidation, might have a crucial influence on the process in cells. But what are the physical reasons behind this influence? In this paper, we examine the role of lipid tail composition in mediating the Bax pore formation process when activated by cleaved Bid. We use different fluorescence methods and a reconstituted system to assess this role on different steps in the process. In complement, we use x-ray scattering and molecular dynamics simulations to evaluate the change in membrane thickness and order with change in lipid tail composition. We show that there is an ideal membrane thickness for Bax pore formation. We also show that lipid disorder is crucial in order to allow for both Bid and Bax insertion into the membrane and for their activation, and therefore pore formation.

Taking Toll on Membranes: Curious Cases of Bacterial β-Barrel Pore-Forming Toxins.

A wide variety of bacterial pathogens secrete a unique class of proteins that attack target cell membranes and form transmembrane oligomeric pores with distinct β-barrel structural scaffolds. Owing to their specific mode of action and characteristic structural assembly, these proteins are termed as β-barrel pore-forming toxins (β-PFTs). The most obvious consequence of such pore-forming activity of bacterial β-PFTs is the permeabilization of cell membranes, which eventually leads to cell death. Bacterial β-PFTs have been studied extensively for nearly past four decades, and their mechanisms of actions have revealed some of the most enigmatic aspects of the protein structure-function paradigm. In most of the cases, β-PFTs are released by the bacteria as water-soluble monomeric precursors, which upon encountering target cell membranes assemble into membrane-inserted oligomeric pores. Structural descriptions are now documented for the water-soluble precursor forms, as well as for the membrane-anchored oligomeric pores of many β-PFTs. These studies have revealed that β-PFTs undergo a series of well-orchestrated structural rearrangements during membrane pore formation. Nevertheless, mechanisms that trigger and regulate distinct steps of the pore-formation processes still remain obscure. Here, we discuss our current understanding regarding structure-function mechanisms in the β-PFT family, with particular emphasis on some of the unsolved issues associated with the β-barrel pore-formation mechanism.

Micro-Blooming: Hierarchically Porous Nitrogen-Doped Carbon Flowers Derived from Metal-Organic Mesocrystals.

Synthesis of 3D flower-like zinc-nitrilotriacetic acid (ZnNTA) mesocrystals and their conformal transformation to hierarchically porous N-doped carbon superstructures is reported. During the solvothermal reaction, 2D nanosheet primary building blocks undergo oriented attachment and mesoscale assembly forming stacked layers. The secondary nucleation and growth preferentially occurs at the edges and defects of the layers, leading to formation of 3D flower-like mesocrystals comprised of interconnected 2D micropetals. By simply varying the pyrolysis temperature (550-1000 °C) and the removal method of in the situ-generated Zn species, nonporous parent mesocrystals are transformed to hierarchically porous carbon flowers with controllable surface area (970-1605 m2 g-1 ), nitrogen content (3.4-14.1 at%), pore volume (0.95-2.19 cm3 g-1 ), as well as pore diameter and structures. The carbon flowers prepared at 550 °C show high CO2 /N2 selectivity due to the high nitrogen content and the large fraction of (ultra)micropores, which can greatly increase the CO2 affinity. The results show that the physicochemical properties of carbons are highly dependent on the thermal transformation and associated pore formation process, rather than directly inherited from parent precursors. The present strategy demonstrates metal-organic mesocrystals as a facile and versatile means toward 3D hierarchical carbon superstructures that are attractive for a number of potential applications.

PROPERTIES OF POROMASS DEPENDING ON THE TYPE OF BINDER AND THE CHARACTERISTICS OF SAMPLES BASED ON THEM

The processes of pore formation on the basis of gypsum binders and the physicomechanical properties of various samples based on them are considered.

Reconfigurable solid-state electrolytes for high performance flexible supercapacitor

To realize high performance and a flexible supercapacitor, it is necessary to address the fundamental issues including low ionic conductivity of solid electrolytes and high interfacial resistance of electrode/electrolyte pairs. Here we present unique solid-state electrolytes by integrating highly engineered nano-porous polyvinyl alcohol (PVA) with super-flat vertically aligned single-walled carbon nanotubes (VA-SWNTs). Highly engineered PVA nano-porous films are fabricated by a generic freeze-thaw process followed by water-miscible solvent treatment in order to create highly controlled nano/microscale pores inside of PVA. Such highly porous PVA films act as both reconfigurable electrolyte template and separator where H3PO4 aqueous solution or ionic liquids can be selectively inserted for a variety of power requirements in flexible electronic applications. Our developed pore formation process is suitable for directly integrating high performance VA-SWNTs electrode as it allows the effective permeation of the polymer electrolyte into nanoscale inter-tube space enabling the easy access and faster transport of ions for higher power capability. This unique entity of reconfigurable electrolyte and nanostructured electrode demonstrates high power and energy densities and remarkable stability after 10,000 charge/discharge cycles.

Mathematical modelling of pore formation in polymers using supercritical fluid media in the Ornstein-Zernike approximation

The paper studies the process of pore formation using supercritical fluid media. A mathematical model of pore formation in polymers in the process of supercritical carbon dioxide decompression has been developed, taking into account fluctuations in the Ornstein-Zernike approximation based on Patel-Teja cubic equation. Calculations and comparison with experimental data are given on the example of pore formation in polystyrene, which showed satisfactory agreement of the theory with experiment. Parameters of the Lennard-Jones pair interaction potential for carbon dioxide in the carbon dioxide-polystyrene system were obtained. The diffusion coefficient of supercritical carbon dioxide in a polymer takes on a different value than the coefficient of self-diffusion of pure carbon dioxide under the same thermodynamic conditions due to a change in the Lenard-Jones potential profile in a polymeric medium. The obtained values of the parameters of the Lennard-Jones equation, namely, the parameters of the intermolecular interaction potential and the maximum value of the attraction energy, make it possible to adequately estimate the value of the diffusion coefficient under given thermodynamic conditions.

Dynamics of pore formation during laser powder bed fusion additive manufacturing

Laser powder bed fusion additive manufacturing is an emerging 3D printing technique for the fabrication of advanced metal components. Widespread adoption of it and similar additive technologies is hampered by poor understanding of laser-metal interactions under such extreme thermal regimes. Here, we elucidate the mechanism of pore formation and liquid-solid interface dynamics during typical laser powder bed fusion conditions using in situ X-ray imaging and multi-physics simulations. Pores are revealed to form during changes in laser scan velocity due to the rapid formation then collapse of deep keyhole depressions in the surface which traps inert shielding gas in the solidifying metal. We develop a universal mitigation strategy which eliminates this pore formation process and improves the geometric quality of melt tracks. Our results provide insight into the physics of laser-metal interaction and demonstrate the potential for science-based approaches to improve confidence in components produced by laser powder bed fusion.

Insights into gasdermin pore formation from the structure of a pre‐pore

Downstream of inflammasome activation, gasdermin D (GSDMD) mediates IL‐1β secretion and pyroptosis by forming membrane pores. Structural investigation into GSDMD pore formation will provide new mechanistic knowledge about the GSDM family and facilitate the treatment of GSDMD‐associated diseases. We hypothesize a dynamic model where upon caspase cleavage of autoinhibited GSDMD, GSDMD‐NTs dissociate from GSDMD‐CTs for lipid binding and oligomerization into a pre‐pore, followed by β‐barrel insertion to form a membrane pore. In light of our recently resolved structure of the mouse GSDMA3 membrane pore, we aim to determine the structure of a GSDM pre‐pore. We arrest the pore formation process at the pre‐pore stage by generating a GSDMA3 mutant defective in membrane insertion while functional in lipid binding and oligomerization. Cryo‐electron microscopy (Cryo‐EM) analysis reveals a pre‐pore conformation where GSDMA3‐NT subunits oligomerize into a soluble ring without extended transmembrane β strands.Support or Funding InformationThis work was supported by US NIH grant DP1HD087988 (H.W.), R01Al124491 (H.W.), and Charles A. King Trust Postdoctoral Fellowship Program (J.R.). We thank the University of Massachusetts Cryo‐EM Core Facility and NCI National Cryo‐EM Facility for data collection.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Implication of Cholesterol in Regulating the Pore‐formation Mechanism of Vibrio Cholerae Cytolysin, a β‐barrel Pore‐forming toxin

Vibrio cholerae cytolysin (VCC) is a β‐barrel pore‐forming toxin with potent cell‐killing cytolytic/cytotoxic activity. To exert it's cytolytic effect, VCC binds to the target cell membranes and forms transmembrane oligomeric β‐barrel pores. Earlier studies have indicated that cholesterol might play a critical role in regulating the mode of action of VCC. However, exact mechanistic basis of the process still remains unknown. In the present study, we have explored and examined the roles of cholesterol in the different steps of VCC mode of action. Employing the membrane lipid bilayer of lipid vesicles or liposomes containing varying amount of cholesterol, we have shown that cholesterol governs the efficient interaction of the toxin with the membrane lipid bilayer that in turn facilitates subsequent oligomeric pore‐formation process. We have also studied the physiological relevance of membrane cholesterol in the context of the biomembranes of the eukaryotic cells. Our results have shown that the depletion of membrane cholesterol from erythrocytes compromises the pore‐forming activity of VCC. In the biomembrane of erythrocytes, where accessory interactions are available, binding of VCC is not affected to any significant extent. However, upon depletion of membrane cholesterol in the erythrocytes membranes, membrane‐bound fraction of VCC remains trapped in the form of abortive oligomeric assembly, and does not form the functional pores. Our study has also revealed that VCC shows a marked tendency to associate with the cholesterol‐rich membrane micro‐domains on erythrocytes that, in turn, appears to facilitate cholesterol‐dependent pore‐formation mechanism of the toxin. In sum, our results suggest that the presence of cholesterol in the target membranes play critical role for the pore‐formation mechanism of VCC.Support or Funding InformationWe acknowledge the support through funding from the Department of Biotechnology (DBT), Govt. of India [DBT grant no. BT/HRD/NBA/37/01/2014 (x)] and also the support through funding under the Centre of Excellence (COE) in Frontier Areas of Science and Technology (FAST) program of the Ministry of Human Resource Development, Govt. of India, in the area of protein science, design, and engineering (to K.C.). We also thank the Indian Institute of Science Education and Research Mohali for the support.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Free energy analysis of membrane pore formation process in the presence of multiple melittin peptides

Understanding the molecular mechanism underlying pore formation in lipid membranes by antimicrobial peptides is of great importance in biological sciences as well as in drug design applications. Melittin has been widely studied as a pore forming peptide, though the molecular mechanism for pore formation is still illusive. We examined the free energy barrier for the creation of a pore in lipid membranes with and without multiple melittin peptides. It was found that six melittin peptides significantly stabilized a pore, though a small barrier (a few kBT) for the formation still existed. With five melittin peptides or fewer, the pore formation barrier was much higher, though the established pore was in a local energy minimum. Although seven melittins effectively reduced the free energy barrier, a single melittin peptide left the pore after a long time MD simulation probably because of the overcrowded environment around the bilayer pore. Thus, it is highly selective for the number of melittin peptides to stabilize the membrane pore, as was also suggested by the line tension evaluations. The free energy cost required to insert a single melittin into the membrane is too high to explain the one-by-one insertion mechanism for pore formation, which also supports the collective melittin mechanism for pore formation.

Structure Formation in the SiO2–Na2O–H2O System during the Fabrication of Foam Glass Ceramics by Extrusion

This paper presents a method for the fabrication of a foam glass ceramic, an inorganic heat-insulating material, by extruding a mixture of opal–cristobalite rock and a sodium hydroxide solution through calibrated holes. The method ensures excellent mixture homogenization and allows mixing of components and granulation of the mixture to be combined in a single step. As a result of active reactions of amorphous phases in the opal–cristobalite rock with sodium hydroxide in the SiO2–Na2O–H2O system, the average density of the material decreases by a factor of 1.5. This ensures an economic benefit: a proportional reduction in the percentage of sodium hydroxide, an expensive component. Using electron microscopy, we have assessed the effect of temperature on structure formation in the material. The pore formation process begins at 200°C and is accompanied, up to 500°C, by the formation of a loose structure in the form of an inhomogeneous network. In the range 400–500°C, the material undergoes a transition. Above 600°C, a cellular structure begins to form. As a result, the materials synthesized at 850°C have an average density from 0.38 to 0.57 g/cm3 and compressive strength from 3.1 to 5.9 MPa.

Attempts to design porous carbon monoliths using porous concrete as a template - Part II: Some aspects of the pore formation mechanism

Abstract Porous concrete has been proven as a promising template to prepare porous carbon monoliths because it can easily be formed to any geometric shape. To optimize the pore size distribution the template-assisted route can be combined with steps of the classical carbon activation such as the carbonization in the presence of zinc chloride. For the rational designing of such routes, profound knowledge of the pore-formation process is necessary. This is however complicated because the pore formation is the result of a complex interplay of different sub-processes. Here, we present approaches to estimate the contributions of these sub-processes to the overall porosity in a quantitative way. For that, the role of the template, the gasification reactions as well as the activating agent has been elucidated by elaborate analysis of nitrogen adsorption data.

Computer Modeling of the Process of Synthesis of Porous Glass-Composite Materials

The operational properties of thermal insulation materials directly depend on their structure. The processes of pore formation occur inside the material after high-temperature treatment, which makes their studying more expensive and complicated. On the basis of experimental data, a computer simulation program for the process of pore formation has been developed, which makes it possible to simulate the processes of changing the structure of foam glass during heat treatment without repeated laboratory experiments.

FOAMED GYPSUM MATERIALS WITH FLUORIDE ADDITIVE

Purpose: The aim of this work is to investigate compositions and operational procedures for gypsum foamed materials based on acid fluoride and define conditions for the formation of the foamed structure and ways to improve the strength of foamed samples. Materials/Methodology/Approach: A capacity of sulphuric acid to interact with calcium carbonate with release of carbon dioxide is laid for this research. The hallmark of this study is acid fluoride used as a recycled material in which the residual acid adsorbs on anhydrite grains. Also, liquid glass and aluminum oxide nanofibrer are added to the composition. In this research, standard procedures and physicochemical techniques are used for the detection of properties of constructional materials. X-ray diffraction and differential thermal analyses are carried out to study the chemical composition of acid fluoride. The production of heat-insulating material includes preliminary dosage of initial components (acid fluoride, liquid glass, stabilized dispersion of aluminum nanofiber, sodium carbonate and water); blending of these components during 1–2 min followed by the gypsum addition; hardening in chambers for 24 h at 40–60 °C; pouring the ready mixture in divided molds. Standard equipment can be used for this production process. Research findings: The additional use of liquid glass and aluminum oxide nanofiber provides high physical and mechanical properties of samples. The high strength of material is insured by hydration of gypsum and acid fluoride followed by the chemical reaction due to the hardening catalyst. Heat-insulating properties appear due to porosity of product achieved by the release of carbon dioxide and oxygen during the interaction between acid fluoride and sodium carbonate as well as by the dispersion of aluminum oxide nanofiber accompanied by pore formation. Liquid glass and aqueous dispersion of nanofiber are very important for the material composition. The hardness increase occurs due to the formation of calcium silicates of different valency and their hydrates and due to the addition of nanofiber stabilized by sodium hydroxide of aluminum oxide representing a specific proportion of aluminum oxide and AlOOH. As a result of nanoparticle-Ca ion interaction, calcium aluminates and hydrated aluminates form. Calcium silicates and aluminates form the main space frame for the structure of gypsum stone. Additionally, Na 2 SO 4 hardener forms during the interaction of the blend components, i.e. the system is capable to autocatalysis. Also, the appeared non-soluble and slightly soluble products reinforce the stone structure, such that the initial, large gas release which provides low density and pore formation, does not lead to a sharp strength decrease, thereby ensuring the sufficient quality of material. When the oxide film of aluminum fiber is damaged, its violent reaction occurs with aqueous mixture components with hydrogen release. Practical implications: The obtained results can be used in construction of low-rise building and manufacturing partitions for rooms and flats. Originality: Hydration and pore formation processes are described for hardening gypsum materials based on acid fluoride. These processes allow controlling the mixture composition and properties of binding and binding-containing constructional materials. The authors suggest dependencies for control for the composition, structure and properties of composite, heat-insulating materials. The authors show that it is possible to render a targeted effect on the structure formation observed in complex additives which assist in the production of effective walling materials.

Cell Softness Prevents Cytolytic T Cell Killing of Tumor-Repopulating Cells

Biomechanics is a fundamental feature of a cell. The manner by which a mechanical feature could affect immune evasion of tumor cells remains an enigma. Here we show that although cytotoxic T lymphocytes (CTL) can effectively destroy stiff differentiated tumor cells, they fail to kill tumor-repopulating cells (TRC), whose softness effectively impedes the pore formation process through perforin released from CTLs. Mechanistically, perforin interacting with nonmuscle myosin heavy chain 9 transmits forces to fewer F-actins in soft TRCs, thus inducing a weak contractile force which is inadquate for pore formation by perforin. Stiffening soft TRCs confers perforin the drilling ability, leading to CTLs killing the TRCs, thus achieving a better tumor immunotherapeutic outcome. More importantly, overcoming the mechanical softness also enhances the killing of human TRCs by human CTLs. These findings provide mechanics-based immunotherapeutic strategies with potential clinical applications.

Visualization of perforin/gasdermin/complement-formed pores in real cell membranes using atomic force microscopy

Different types of pores ubiquitously form in cell membranes, leading to various types of cell death that profoundly influence the fate of inflammation and the disease status. However, these pores have never truly been visualized to date. Atomic force microscopy (AFM), which is emerging as a powerful tool to analyze the mechanical properties of biomolecules and cells, is actually an excellent imaging platform that allows biological samples to be visualized by probing surface roughness at the level of atomic resolution. Here, membrane pore structures were clearly visualized using AFM. This visualization not only describes the aperture and depth of the pore complexes but also highlights differences among the pores formed by perforin and gasdermins in tumor cell membranes and by complement in immune cell membranes. Additionally, this type of visualization also reveals the dynamic process of pore formation, fusion, and repair.

Elementary processes of antimicrobial peptide PGLa-induced pore formation in lipid bilayers

Antimicrobial peptide PGLa induces the leakage of intracellular content, leading to its bactericidal activity. However, the elementary process of PGLa-induced leakage remains poorly understood. Here, we examined the interaction of PGLa with lipid bilayers using the single giant unilamellar vesicle (GUV) method. We found that PGLa induced membrane permeation of calcein from GUVs comprised of dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol (DOPG) and its rate increased with time to reach a steady value, indicating that PGLa induced pores in the bilayer. The binding of PGLa to the GUV membrane raised its fractional area change, δ. At high PGLa concentrations, the time course of δ showed a two-step increase; δ increased to a value, δ1, which was constant for an extended period before increasing to another constant value, δ2, that persisted until aspiration of the GUV. To reveal the distribution of PGLa, we investigated the interaction of a mixture of PGLa and carboxyfluorescein (CF) -labeled PGLa (CF-PGLa) with single GUVs. The change of the fluorescence intensity of the GUV rim, I, over time showed a two-step increase from a steady value, I1, to another, I2, concomitant with the entering of CF-PGLa into the lumen of the GUV prior to AF647 leakage. The simultaneous measurement of δ and I indicated that their time courses were virtually the same and the ratios (δ2/δ1 and I2/I1) were almost 2. These results indicated that CF-PGLa translocated across the bilayer before membrane permeation. Based on these results, the elementary processes of the PGLa-induced pore formation were discussed.

Growing a gasdermin pore in membranes of pyroptotic cells.

Inflammasome‐activated caspase‐1, caspase‐11, caspase‐4, and caspase‐5 cleave GSDMD to unleash its N‐terminal gasdermin‐N domain (GSDMD N term ) that perforates the plasma membrane to execute pyroptosis and stimulate inflammation. The mechanism underlying GSDMD N term pore formation is unclear. Mulvihill et al use high‐resolution atomic force microscopy (AFM) to analyze the dynamic pore formation process of GSDMD N term . GSDMD N term protomers are inserted into the lipid membrane to assemble arc‐ or slit‐shaped oligomers that can incorporate additional protomers and grow into large and stable ring‐shaped oligomers to form pores.

OXIDATION OF DISPERSE CARBON MATERIALS

It is proposed to consider the process of carbon materials oxidative activation from the positions of topochemical reactions involving chemisorption of the activating agent (oxidizer) on the material surface active centers followed by chemical interaction. Such an approach makes it possible to control the process of creating a carbon material with the desired characteristics of the porous space. It is assumed that the oxidizer chemisorption active centers are amorphous carbon, which is localized on the material crystallites boundaries. The change in the length of these boundaries will lead to a change in the process rate. It is shown that the number of such active centers on the carbon material surface depends on the size of the crystallites. It will have a significant impact not only on the rate of activation, but also on the possibility of the process flow on the surface or with porosity formation. Mathematical models describing the carbon sample specific surface changing in the oxidation process are proposed. They allow quantifying the proportion of carbon that is oxidized on the sample surface or with pores formation, as well as quantifying the number of pores. It is shown that the ratio of pore formation and surface oxidation processes depends on the oxidation temperature, the oxidizer nature and its flow rate. The proportion of porosity formation decreases with the increase in the oxidant flow rate and the increase in temperature. It was experimentally shown that in order to obtain a material with a more developed porous space and a high specific surface it is preferable to use carbon dioxide as an oxidizing agent.

Effects of Peptide Charge, Orientation, and Concentration on Melittin Transmembrane Pores

Melittin is a short cationic peptide that exerts cytolytic effects on bacterial and eukaryotic cells. Experiments suggest that in zwitterionic membranes, melittin forms transmembrane toroidal pores supported by four to eight peptides. A recently constructed melittin variant with a reduced cationic charge, MelP5, is active at 10-fold lower concentrations. In previous work, we performed molecular dynamics simulations on the microsecond timescale to examine the supramolecular pore structure of a melittin tetramer in zwitterionic and partially anionic membranes. We now extend that study to include the effects of peptide charge, initial orientation, and number of monomers on the pore formation and stabilization processes. Our results show that parallel transmembrane orientations of melittin and MelP5 are more consistent with experimental data. Whereas a MelP5 parallel hexamer forms a large stable pore during the 5-μs simulation time, a melittin hexamer and an octamer are not fully stable, with several monomers dissociating during the simulation time. Interaction-energy analysis shows that this difference in behavior between melittin and MelP5 is not due to stronger electrostatic repulsion between neighboring melittin peptides but to peptide-lipid interactions that disfavor the isolated MelP5 transmembrane monomer. The ability of melittin monomers to diffuse freely in the 1,2-dimyristoyl-SN-glycero-3-phosphocholine membrane leads to dynamic pores with varying molecularity.