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Cadherins are calcium dependent adhesion proteins that establish and maintain the intercellular mechanical contact by bridging the gap between adjacent cells. Desmoglein-2 (Dsg2) and desmocollin-2 (Dsc2) are tissue specific cadherin isoforms of the cell-cell contact in cardiac desmosomes. Mutations in the DSG2-gene and in the DSC2-gene are related to arrhythmogenic right ventricular cardiomyopathy (ARVC) a rare but severe heart muscle disease. Here, several possible homophilic and heterophilic binding interactions of wild-type Dsg2, wild-type Dsc2, as well as one Dsg2- and two Dsc2-variants, each associated with ARVC, are investigated. Using single molecule force spectroscopy (SMFS) with atomic force microscopy (AFM) and applying Jarzynski's equality the kinetics and thermodynamics of Dsg2/Dsc2 interaction can be determined. The free energy landscape of Dsg2/Dsc2 dimerization exposes a high activation energy barrier, which is in line with the proposed strand-swapping binding motif. Although the binding motif is not affected by any of the mutations, the binding kinetics of the interactions differ significantly from the wild-type. While wild-type cadherins exhibit an average complex lifetime of approx. 0.3 s interactions involving a variant consistently show - lifetimes that are substantially larger. The lifetimes of the wild-type interactions give rise to the picture of a dynamic adhesion interface consisting of continuously dissociating and (re)associating molecular bonds, while the delayed binding kinetics of interactions involving an ARVC-associated variant might be part of the pathogenesis. Our data provide a comprehensive and consistent thermodynamic and kinetic description of cardiac cadherin binding, allowing detailed insight into the molecular mechanisms of cell adhesion.
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The cardiac muscle consists of individual cardiomyocytes that are mechanically linked by desmosomes. Desmosomal adhesion is mediated by densely packed and organized cadherins which, in presence of Ca2+, stretch out their extracellular domains (EC) and dimerize with opposing binding partners by exchanging an N-terminal tryptophan. The strand-swap binding motif of cardiac cadherins like desmocollin 2 (Dsc2) (and desmoglein2 alike) is highly specific but of low affinity with average bond lifetimes in the range of approximately 0.3 s. Notably, despite this comparatively weak interaction, desmosomes mediate a stable, tensile-resistant bond. In addition, force mediated dissociation of strand-swap dimers exhibit a reduced bond lifetime as external forces increase (slip bond). Using atomic force microscopy based single molecule force spectroscopy (AFM-SMFS), we demonstrate that Dsc2 has two further binding modes that, in addition to strand-swap dimers, most likely play a significant role in the integrity of the cardiac muscle. At short interaction times, the Dsc2 monomers associate only loosely, as can be seen from short-lived force-independent bonds. These ideal bonds are a precursor state and probably stabilize the formation of the self-inhibiting strand-swap dimer. The addition of tryptophan in the measurement buffer acts as a competitive inhibitor, preventing the N-terminal strand exchange. Here, Dsc2 dimerizes as X-dimer which clearly shows a tri-phasic slip-catch-slip type of dissociation. Within the force-mediated transition (catch) regime, Dsc2 dimers switch between a rather brittle low force and a strengthened high force adhesion state. As a result, we can assume that desmosomal adhesion is mediated not only by strand-swap dimers (slip) but also by their precursor states (ideal bond) and force-activated X-dimers (catch bond).
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Cadherinas , Triptófano , Unión Proteica , Triptófano/metabolismo , Cadherinas/metabolismo , Dimerización , Fenómenos Físicos , Adhesión Celular/fisiologíaRESUMEN
Nanoporous carbon nanomembranes (CNMs) created by self-assembled monolayers ideally combine a high water flux and precise ion selectivity for molecular separation and water desalination. However, their practical implementation is often challenged by the availability of large epitaxial substrates, limiting the membrane up-scaling. Here, we report a scalable synthesis of CNMs from poly(4-vinylbiphenyl) (PVBP) spin-coated on SiO2/Si wafers. Electron irradiation of the amorphous PVBP molecular layers induces the formation of a continuous membrane with a thickness of 15 nm and a high density of subnanometer pores, providing a water permeance as high as 530 L m-2 h-1 bar-1, while repelling ions and molecules larger than 1 nm in size. A further introduction of a reinforced porous block copolymer layer enables the fabrication of centimeter-scale CNM composites that efficiently separate organic dyes from water. These results suggest a feasible route for large-scale nanomembrane fabrication.
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Background: Arrhythmogenic cardiomyopathy can be caused by genetic variants in desmosomal cadherins. Since cardiac desmosomal cadherins are crucial for cell-cell-adhesion, their correct localization at the plasma membrane is essential. Methods: Nine desmocollin-2 variants at five positions from various public genetic databases (p.D30N, p.V52A/I, p.G77V/D/S, p.V79G, p.I96V/T) and three additional conserved positions (p.C32, p.C57, p.F71) within the prodomain were investigated in vitro using confocal microscopy. Model variants (p.C32A/S, p.V52G/L, p.C57A/S, p.F71Y/A/S, p.V79A/I/L, p.I96l/A) were generated to investigate the impact of specific amino acids. Results: We revealed that all analyzed positions in the prodomain are critical for the intracellular transport. However, the variants p.D30N, p.V52A/I and p.I96V listed in genetic databases do not disturb the intracellular transport revealing that the loss of these canonical sequences may be compensated. Conclusion: As disease-related homozygous truncating desmocollin-2 variants lacking the transmembrane domain are not localized at the plasma membrane, we predict that some of the investigated prodomain variants may be relevant in the context of arrhythmogenic cardiomyopathy due to disturbed intracellular transport.
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Poly(vinyl alcohol) (PVA) has ice binding and ice nucleating properties. Here, we explore the dependence of the molecular size of PVA on its ice nucleation activity. For this purpose, we studied ice nucleation in aqueous solutions of PVA samples with molar masses ranging from 370 to 145 000 g mol-1, with a particular focus on oligomer samples with low molar mass. The experiments employed a novel microfluidic setup that is a follow-up on the previous WeIzmann Supercooled Droplets Observation on a Microarray (WISDOM) design by Reicher et al. The modified setup introduced and characterized here, termed nanoliter Bielefeld Ice Nucleation ARraY (nanoBINARY), uses droplet microfluidics with droplets (96 ± 4) µm in diameter and a fluorinated continuous oil phase and surfactant. A comparison of homogeneous and heterogeneous ice nucleation data obtained with nanoBINARY to those obtained with WISDOM shows very good agreement, underpinning its ability to study low-temperature ice nucleators as well as homogeneous ice nucleation due to the low background of impurities. The experiments on aqueous PVA solutions revealed that the ice nucleation activity of shorter PVA chains strongly decreases with a decrease in molar mass. While the cumulative number of ice nucleating sites per mass nm of polymers with different molar masses is the same, it becomes smaller for oligomers and completely vanishes for dimer and monomer representatives such as 1,3-butanediol, propan-2-ol, and ethanol, most likely because these molecules become too small to effectively stabilize the critical ice embryo. Overall, our results are consistent with PVA polymers and oligomers acting as heterogeneous ice nucleators.
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The performance of plastic components in water-bearing parts of industrial and household appliances, often in the presence of harsh environments and elevated temperatures, critically relies on the mechanical and thermal polymer stability. In this light, the precise knowledge of aging properties of polymers formulated with dedicated antiaging additive packages as well as various fillers is crucial for long-time device warranty. We investigated and analysed the time-dependent, polymer-liquid interface aging of different industrial performance polypropylene samples in aqueous detergent solution at high temperatures (95 °C). Special emphasis was put on the disadvantageous process of consecutive biofilm formation that often follows surface transformation and degradation. Atomic force microscopy, scanning electron microscopy, and infrared spectroscopy were used to monitor and analyse the surface aging process. Additionally, bacterial adhesion and biofilm formation was characterised by colony forming unit assays. One of the key findings is the observation of crystalline, fibre-like growth of ethylene bis stearamide (EBS) on the surface during the aging process. EBS is a widely used process aid and lubricant enabling the proper demoulding of injection moulding plastic parts. The aging-induced surface-covering EBS layers changed the surface morphology and promoted bacterial adhesion as well as biofilm formation of Pseudomonas aeruginosa.
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Dinuclear copper complexes have been designed for molecular recognition in order to selectively bind to two neighboring phosphate moieties in the backbone of double strand DNA. Associated biophysical, biochemical and cytotoxic effects on DNA were investigated in previous works, where atomic force microscopy (AFM) in ambient conditions turned out to be a particular valuable asset, since the complexes influence the macromechanical properties and configurations of the strands. To investigate and scrutinize these effects in more depth from a structural point of view, cutting-edge preparation methods and scanning force microscopy under ultra-high vacuum (UHV) conditions were employed to yield submolecular resolution images. DNA strand mechanics and interactions could be resolved on the single base pair level, including the amplified formation of melting bubbles. Even the interaction of singular complex molecules could be observed. To better assess the results, the appearance of treated DNA is also compared to the behavior of untreated DNA in UHV on different substrates. Finally, we present data from a statistical simulation reasoning about the nanomechanics of strand dissociation. This sort of quantitative experimental insights paralleled by statistical simulations impressively shade light on the rationale for strand dissociations of this novel DNA interaction process, that is an important nanomechanistic key and novel approach for the development of new chemotherapeutic agents.
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ADN , Desnaturalización de Ácido Nucleico , ADN/química , Emparejamiento Base , Microscopía de Fuerza Atómica/métodosRESUMEN
In this work, palladium-loaded smart membranes made by UV cross-linking of thermoresponsive microgels are prepared to obtain a reusable, catalytically active material which can, for example, be implemented in chemical reactors. The membranes are examined with respect to their coverage of a supporting mesh via atomic force microscopy measurements. Force indentation mapping was performed in the dried, collapsed state and in the swollen state in water to determine the Young modulus. Furthermore, we compare the catalytic activity of the membrane with the corresponding suspended colloidal nanoparticle microgel hybrids. For this purpose, the reduction of 4-nitrophenol is an established model reaction to quantify the catalytic activity by UV-vis spectroscopy. The membrane is embedded inside a continuous stirred tank reactor equipped for continuous monitoring of the reaction progress. Although catalysis with membranes shows lower catalytic activity than freely dispersed particles, membranes allow straightforward separation and recycling of the catalyst. The fabricated membranes in this work show no decrease in catalytic activity between several cycles, unlike free particles. The feasible and durable deposition of catalytically active inter-cross-linked microgel particles on commercial nylon meshes as supporting scaffolds, as demonstrated in this work, is promising for up-scaling of continuous industrial processes.
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Xanthan gum is a polysaccharide that is widely used as a thickening agent in numerous food, cosmetic, and technical applications. Therefore, the knowledge of the molecular interplay that builds up and stabilizes water-binding networks is crucial for the optimization of xanthan thickening performance. Using atomic force microscopy, rheometry, and inductively coupled plasma optical emission spectroscopy, we show a clear correlation between xanthan thickening properties and the ability to form characteristic secondary structures as well as the valence and amount of cations coordinated at the polysaccharide side chain. Based on these findings and the Debye-Hückel theory, we derive an ion-interaction model in which divalent cations mediate bridging of adjacent single polymer strands due to chelate-like coordination building stable secondary structures. We furthermore demonstrate in a cation exchange assay that xanthan secondary structures can be modified in a directed and reversible manner, which, in turn, alters its thickening properties.
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Polímeros , Polisacáridos Bacterianos , Viscosidad , Polisacáridos Bacterianos/química , Microscopía de Fuerza Atómica , Polímeros/químicaRESUMEN
In the light of the SARS-CoV-2 pandemic and growing numbers of bacteria with resistance to antibiotics, the development of antimicrobial coatings is rising worldwide. Inorganic coatings are attractive because of low environmental leakage and wear resistance. Examples for coatings are hot metal dipping or physical vapor deposition of nanometer coatings. Here, magnetron sputtering of various transition metals, such as gold, ruthenium and tantalum, was investigated. Metal films were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM) and energy dispersive X-ray spectroscopy (EDX). We investigated the growth of Pseudomonas aeruginosa isolated from household appliances on different sputter-coated metal surfaces. The fine-grained nanometric structure of these metal coatings was between 14 nm (tantalum) and 26 nm (gold) and the roughness was in a range of 164 pm (ruthenium) to 246 pm (gold). Antibacterial efficacy of metal surfaces followed the order: gold > tantalum > ruthenium. Interestingly, gold had the strongest inhibitory effect on bacterial growth, as analyzed by LIVE/DEAD and CFU assay. High-magnification SEM images showed dead bacteria characterized by shrinkage induced by metal coatings. We conclude that sputtering might be a new application for the development of antimicrobial surfaces on household appliances and or surgical instruments.
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Polymer membranes are widely used in separation processes including desalination1, organic solvent nanofiltration2,3 and crude oil fractionation4,5. Nevertheless, direct evidence of subnanometre pores and a feasible method of manipulating their size is still challenging because of the molecular fluctuations of poorly defined voids in polymers6. Macrocycles with intrinsic cavities could potentially tackle this challenge. However, unfunctionalized macrocycles with indistinguishable reactivities tend towards disordered packing in films hundreds of nanometres thick7-9, hindering cavity interconnection and formation of through-pores. Here, we synthesized selectively functionalized macrocycles with differentiated reactivities that preferentially aligned to create well-defined pores across an ultrathin nanofilm. The ordered structure was enhanced by reducing the nanofilm thickness down to several nanometres. This orientated architecture enabled direct visualization of subnanometre macrocycle pores in the nanofilm surfaces, with the size tailored to ångström precision by varying the macrocycle identity. Aligned macrocycle membranes provided twice the methanol permeance and higher selectivity compared to disordered counterparts. Used in high-value separations, exemplified here by enriching cannabidiol oil, they achieved one order of magnitude faster ethanol transport and threefold higher enrichment than commercial state-of-the-art membranes. This approach offers a feasible strategy for creating subnanometre channels in polymer membranes, and demonstrates their potential for accurate molecular separations.
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Ultrathin membranes with subnanometer pores enabling molecular size-selective separation were generated on surfaces via electron-induced cross-linking of self-assembled monolayers (SAMs). The evolution of p-terphenylthiol (TPT) SAMs on Au(111) surfaces into cross-linked monolayers was observed with a scanning tunneling microscope. As the irradiation dose was increased, the cross-linked regions continued to grow and a large number of subnanometer voids appeared. Their equivalent diameter is 0.5 ± 0.2 nm and the areal density is ≈1.7 × 1017 m-2. Supported by classical molecular dynamics simulations, we propose that these voids may correspond to free volumes inside a cross-linked monolayer.
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Xanthomonas campestris strains are used world-wide for the production of the industrially important exopolysaccharide xanthan. The high industrial relevance of xanthan can be explained by its extraordinary qualities as rheological control agent in aqueous systems and by its stabilizing properties in suspensions and emulsions. The phytopathogen Xanthomonas campestris is a motile bacterium with one polar flagellum. The flagellum is a cost intensive structure, in terms of energy and building block consumption. Based on the assumption that inhibition of the flagellar biosynthesis and related proton driven motility might be beneficial for the xanthan production in Xcc, two genes (fliC and fliM) were mutated to inhibit the motility. Both mutants Xcc JBL007 fliC- and Xcc JBL007 fliM- showed an increased xanthan production. Remarkably, the produced xanthan from both mutants showed enhanced rheological properties. While the chemical composition of the produced xanthan of the initial and both mutant strains did not change, notable differences in persistence length could be measured via atomic force microscopy. Results presented in this study demonstrate the possibility to further improve the xanthan production by Xcc through rational strain design.
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Xanthomonas campestris , Microscopía de Fuerza Atómica , Polisacáridos Bacterianos , Viscosidad , Xanthomonas campestris/genéticaRESUMEN
Despite the prospects of intrinsically porous planar nanomaterials in separation applications, their synthesis on a large scale remains challenging. In particular, preparing water-selective carbon nanomembranes (CNMs) from self-assembled monolayers (SAMs) is limited by the cost of epitaxial metal substrates and molecular precursors with specific chemical functionalities. In this work, we present a facile fabrication of CNMs from polycyclic aromatic hydrocarbons (PAHs) that are drop-cast onto arbitrary supports, including foils and metalized films. The electron-induced carbonization is shown to result in continuous membranes of variable thickness, and the material is characterized with a number of spectroscopic and microscopic techniques. Permeation measurements with freestanding membranes reveal a high degree of porosity, but the selectivity is found to strongly depend on the thickness. While the permeance of helium remains almost the same for 6.5 and 3.0 nm thick CNMs, water permeance increases by 2 orders of magnitude. We rationalize the membrane performance with the help of kinetic modeling and vapor adsorption experiments.
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In this study we show a possibility to produce thermoresponsive, free-standing microgel membranes based on N-isopropylacrylamide (NIPAM) and the UV-sensitive comonomer 2-hydroxy-4-(methacryloyloxy)benzophenone (HMABP). To influence the final network structure and functionality of the membranes, we use different cross-linkers in the microgel syntheses and characterize the resulting structural microgel properties and the swelling behavior by means of AFM, FTIR, and PCS measurements. Varying the cross-linker results in significant changes in the structure and swelling behavior of the individual microgels and has an influence on the incorporation of the comonomer, which is essential for subsequent photochemical membrane formation. We investigate the ion transport through the different membranes by temperature-dependent resistance measurements revealing a sharp increase in resistance when the copolymer microgels reach their collapsed state. The resistance of the membranes can be adjusted by different cross-linkers and the associated incorporation of the comonomer. Furthermore, we show that transferring a reversible cross-linker from a cross-linked state to an un-cross-linked state strongly influences the membrane properties and even reverses the switching behavior, while the mechanical stability of the membrane is maintained.
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Microgeles , Geles , Polímeros , TemperaturaRESUMEN
Poly(N-isopropylacrylamide) (pNIPAM) based copolymer microgels were used to create free-standing, transferable, thermoresponsive membranes. The microgels were synthesized by copolymerization of NIPAM with N-benzylhydrylacrylamide (NBHAM). Monolayers of these colloidal gels were subsequently cross-linked using an electron gun leading to the formation of a connected monolayer. Furthermore, the cross-linked microgel layer is detached from the supporting material by dissolving the substrate. These unique systems can be used as transferable, thermoresponsive coatings and as thermoresponsive membranes. As a proof of principle for the use of such membranes we studied the ion transport through them at different temperatures revealing drastic changes when the lower critical solution temperature of the copolymer microgels is reached.
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In this study we use poly(N-isopropylacrylamide) (PNIPAM) based copolymer microgels to create free-standing, transferable, thermoresponsive membranes. The microgels are synthesized by copolymerization of NIPAM with 2-hydroxy-4-(methacryloyloxy)-benzophenone (HMABP) and spin-coated on Si wafers. After subsequent cross-linking by UV-irradiation, the formed layers easily detach from the supporting material. We obtain free standing microgel membranes with lateral extensions of several millimetres and an average layer thickness of a few hundred nanometres. They can be transferred to other substrates. As one example for potential applications we investigate the temperature dependent ion transport through the membranes via resistance measurements revealing a sharp reversible increase in resistance when the lower critical solution temperature of the copolymer microgels is reached. In addition, prior to cross-linking, the microgels can be decorated with silver nanoparticles and cross-linked afterwards. Such free-standing nanoparticle hybrid membranes are then used as catalytic systems for the reduction of 4-nitrophenol, which is monitored by UV/Vis spectroscopy.
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BACKGROUND: Arrhythmogenic cardiomyopathy (AC) is a heritable myocardial disorder and a major cause of sudden cardiac death. It is typically caused by mutations in desmosomal genes. Desmin gene (DES) variants have been previously reported in AC but with insufficient evidence to support their pathogenicity. METHODS: We aimed to assess a large AC patient cohort for DES mutations and describe a unique phenotype associated with a recurring variant in three families. A cohort of 138 probands with a diagnosis of AC and no identifiable desmosomal gene mutations were prospectively screened by whole-exome sequencing. RESULTS: A single DES variant (p.Leu115Ile, c.343C>A) was identified in 3 index patients (2%). We assessed the clinical phenotypes within their families and confirmed cosegregation. One carrier required heart transplantation, 2 died suddenly, and 1 died of noncardiac causes. All cases had right- and left-ventricular (LV) involvement. LV late gadolinium enhancement was present in all, and circumferential subepicardial distribution was confirmed on histology. A significant burden of ventricular arrhythmias was noted. Desmin aggregates were not observed macroscopically, but analysis of the desmin filament formation in transfected cardiomyocytes derived from induced pluripotent stem cells, and SW13 cells revealed cytoplasmic aggregation of mutant desmin. Atomic force microscopy revealed that the mutant form accumulates into short protofilaments and small fibrous aggregates. CONCLUSIONS: DES p.Leu115Ile leads to disruption of the desmin filament network and causes a malignant biventricular form of AC, characterized by LV dysfunction and a circumferential subepicardial distribution of myocardial fibrosis.
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Cardiomiopatías , Desmina/genética , Fibrosis Endomiocárdica , Disfunción Ventricular Izquierda , Disfunción Ventricular Derecha , Fibrilación Ventricular , Cardiomiopatías/complicaciones , Cardiomiopatías/genética , Cardiomiopatías/patología , Cardiomiopatías/fisiopatología , Cardiomiopatías/terapia , Muerte Súbita Cardíaca , Fibrosis Endomiocárdica/diagnóstico , Fibrosis Endomiocárdica/etiología , Femenino , Estado Funcional , Tamización de Portadores Genéticos/métodos , Pruebas de Función Cardíaca/métodos , Humanos , Masculino , Persona de Mediana Edad , Distrofias Musculares/genética , Distrofias Musculares/patología , Mutación Missense , Miocardio/patología , Reino Unido , Disfunción Ventricular Izquierda/diagnóstico , Disfunción Ventricular Izquierda/etiología , Disfunción Ventricular Derecha/diagnóstico , Disfunción Ventricular Derecha/etiología , Fibrilación Ventricular/diagnóstico , Fibrilación Ventricular/etiologíaRESUMEN
Marine ancestors of freshwater sponges had to undergo a series of physiological adaptations to colonize harsh and heterogeneous limnic environments. Besides reduced salinity, river-lake systems also have calcium concentrations far lower than seawater. Cell adhesion in sponges is mediated by calcium-dependent multivalent self-interactions of sulfated polysaccharide components of membrane-bound proteoglycans named aggregation factors. Cells of marine sponges require seawater average calcium concentration (10 mM) to sustain adhesion promoted by aggregation factors. We demonstrate here that the freshwater sponge Spongilla alba can thrive in a calcium-poor aquatic environment and that their cells are able to aggregate and form primmorphs with calcium concentrations 40-fold lower than that required by marine sponges cells. We also find that their gemmules need calcium and other micronutrients to hatch and generate new sponges. The sulfated polysaccharide purified from S. alba has sulfate content and molecular size notably lower than those from marine sponges. Nuclear magnetic resonance analyses indicated that it is composed of a central backbone of non- and 2-sulfated α- and ß-glucose units decorated with branches of α-glucose. Assessments with atomic force microscopy/single-molecule force spectroscopy show that S. alba glucan requires 10-fold less calcium than sulfated polysaccharides from marine sponges to self-interact efficiently. Such an ability to retain multicellular morphology with low environmental calcium must have been a crucial evolutionary step for freshwater sponges to successfully colonize inland waters.
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Calcio/metabolismo , Polisacáridos/metabolismo , Poríferos/metabolismo , Proteoglicanos/metabolismo , Animales , Calcio/química , Adhesión Celular , Agua Dulce , Polisacáridos/química , Poríferos/citología , Proteoglicanos/químicaRESUMEN
The collective "single-file" motion of water molecules through natural and artificial nanoconduits inspires the development of high-performance membranes for water separation. However, a material that contains a large number of pores combining rapid water flow with superior ion rejection is still highly desirable. Here, a 1.2 nm thick carbon nanomembrane (CNM) made from cross-linking of terphenylthiol (TPT) self-assembled monolayers is reported to possess these properties. Utilizing their extremely high pore density of 1 sub-nm channel nm-2 , TPT CNMs let water molecules rapidly pass, while the translocation of ions, including protons, is efficiently hindered. Their membrane resistance reaches ≈104 Ω cm2 in 1 m Cl- solutions, comparable to lipid bilayers of a cell membrane. Consequently, a single CNM channel yields an ≈108 higher resistance than pores in lipid membrane channels and carbon nanotubes. The ultrahigh ionic exclusion by CNMs is likely dominated by a steric hindrance mechanism, coupled with electrostatic repulsion and entrance effects. The operation of TPT CNM membrane composites in forward osmosis is also demonstrated. These observations highlight the potential of utilizing CNMs for water purification and opens up a simple avenue to creating 2D membranes through molecular self-assembly for highly selective and fast separations.