Nondimensional frequency scaling of aerodynamically-tensioned membranes

Membrane wings have applications that involve low Reynolds number flyers such as micro air vehicles. The time-averaged and time-dependent deformations of the membrane affect the aerodynamic characteristics of the wing, primarily in the region beyond the maximum aerodynamic efficiency of the wing. This paper investigates an appropriate nondimensional vibration frequency scaling of a spanwise tensioned membrane with free (unattached) leading and trailing edges at low Reynolds numbers relative to nondimensional aeroelastic parameters. Silicone rubber membranes with varying spanwise pre-tension, aerodynamic tension (due to wing angle-of-attack and flow dynamic pressure), modulus of elasticity, span, and thickness are studied. Experimental results are compared to a proposed scaling that simplifies the aerodynamic loading as a uniform pressure distribution acting on the membrane. Data is further compared and discussed relative to previous published results of membrane wings with finite wing spans (three-dimensional flow) and fixed (rigid) leading edges.     

Membranes,peptides, and disease: Unraveling the mechanisms of viral proteins with solid state nuclear magnetic resonance spectroscopy

The interplay between peptides and lipid bilayers drives crucial biological processes. For example, a critical step in the replication cycle of enveloped viruses is the fusion of the viral membrane and host cell endosomal membrane, and these fusion events are controlled by viral fusion peptides. Thus such membrane-interacting peptides are of considerable interest as potential pharmacological targets. Deeper insight is needed into the mechanisms by which fusion peptides and other viral peptides modulate their surrounding membrane environment, and also how the particular membrane environment modulates the structure and activity of these peptides. An important step toward understanding these processes is to characterize the structure of viral peptides in environments that are as biologically relevant as possible. Solid state nuclear magnetic resonance (ssNMR) is uniquely well suited to provide atomic level information on the structure and dynamics of both membrane-associated peptides as well as the lipid bilayer itself; further ssNMR can delineate the contribution of specific membrane components, such as cholesterol, or changing cellular conditions, such as a decrease in pH on membrane-associating peptides. This paper highlights recent advances in the study of three types of membrane associated viral peptides by ssNMR to illustrate the more general power of ssNMR in addressing important biological questions involving membrane proteins.     

Treatment by serum up-conversion nanoparticles in the fluoride matrix changes the mechanism of cell death and the elasticity of the membrane

Nanoparticles are increasingly being used for treatment and diagnostic purposes, but their effects on cells is not fully understood. Here, the interaction of fluorescent up-conversion nanoparticles (UpC-NPs) with neutrophils was investigated by imaging and measurement of membrane-cytosceletal elasticity by atomic force microscopy. It was found that UpC-NPs induce the death of neutrophils mainly by necrosis, and to a smaller extent by a novel process called ‘mummification'. Necrosis occurs by gradual loss of intracellular contents and nuclei, 45–110 min after exposure to UpC-NPs. Mummification is apparent as an increase in the rigidity of the neutrophils' membrane and acquisition of a characteristic bumpy shape with numerous protrusions; this structure does not change during atomic force microscopy scanning. Coating UpC-NPs with protein by incubation with serum leads to (1) formation of nanoparticle aggregates in the nm and μm size range, (2) a reduction in toxicity, (3) reduced mummification of neutrophils, and (4) no significant reduction of the elasticity of the membrane-cytoskeletal complex of neutrophils 30 min after exposure to coated UpC-NPs. The study shows that serum proteins greatly curb the toxicity of nanoparticles and reveals mummification as a novel mechanism of UpC-NP-induced cell death.     

Study and optimization of the ultrasound-enhanced cleaning of an ultrafiltration ceramic membrane through a combined experimental–statistical approach

Membrane fouling is one of the main drawbacks of ultrafiltration technology during the treatment of dye-containing effluents. Therefore, the optimization of the membrane cleaning procedure is essential to improve the overall efficiency. In this work, a study of the factors affecting the ultrasound-assisted cleaning of an ultrafiltration ceramic membrane fouled by dye particles was carried out. The effect of transmembrane pressure (0.5, 1.5, 2.5 bar), cross-flow velocity (1, 2, 3 m s⁻¹), ultrasound power level (40%, 70%, 100%) and ultrasound frequency mode (37, 80 kHz and mixed wave) on the cleaning efficiency was evaluated. The lowest frequency showed better results, although the best cleaning performance was obtained using the mixed wave mode.A Box–Behnken Design was used to find the optimal conditions for the cleaning procedure through a response surface study. The optimal operating conditions leading to the maximum cleaning efficiency predicted (32.19%) were found to be 1.1 bar, 3 m s⁻¹ and 100% of power level.Finally, the optimized response was compared to the efficiency of a chemical cleaning with NaOH solution, with and without the use of ultrasound. By using NaOH, cleaning efficiency nearly triples, and it improves up to 25% by adding ultrasound.     

Synthesis of L-Lysine and L-Glutamic Acid Derivatives with Long Alkyl Chains and Polycondensation of Langmuir-Blodgett Films

Two esters of L-lysine and L-glutamic acid containing long alkyl groups were synthesized and their polycondensation in monolayers and multilayers was investigated. The pressure-area isotherms of the ester of L-lysine depend markedly on the time of residence at the air-water interface. The change of FT-IR spectra of the deposited film, which can be lifted as a Z-type film, indicates that polycondensation can occur in the monolayer at 10°C without any treatment. The spectrum of the film cast from chloroform hardly changed with time. These results lead to the conclusion that a regular arrangement of monomer molecules in the monolayer, where the amino and ester carbonyl groups are concentrated, is more suitable for the polycondensation. The ester of L-glutamic acid can also form stable monolayers which can be easily deposited on a hydrophobic plate as a Y-type film by the Blodgett technique. The polycondensation of multilayers under an atmosphere of triethylamine was investigated by IR spectroscopy. It indicates that the condensation in multilayers proceeds via intermolecular and intramolecular reactions, by which poly(L-glutamate) derivatives and 2-pyrrolidone derivatives are formed, respectively. The condensation in the bulk crystalline powder gives exclusively the 2-pyrrolidone derivative by intramolecular reaction. These results suggest that the monomer molecules in the multilayers are favorably aligned for the intermolecular reaction, in contrast to the situation in the bulk crystalline powder.     

Performance of hydrophobic interaction ligands for human membrane‐bound catechol‐O‐methyltransferase purification

Despite of membrane catechol‐O‐methyltransferase (MBCOMT, EC 2.1.1.6) physiological importance on catecholamines’ O‐methylation, no studies allowed their total isolation. Therefore, for the first time, we compare the performance of three hydrophobic adsorbents (butyl‐, epoxy‐, and octyl‐Sepharose) in purification of recombinant human COMT (hMBCOMT) from crude Brevibacillus choshinensis cell lysates to develop a sustainable chromatographic process. Hydrophobic matrices were evaluated in terms of selectivity and hMBCOMT's binding and elution conditions. Results show that hMBCOMT's adsorption was promoted on octyl and butyl at ≤375 mM NaH₂PO_4, while on epoxy higher concentrations (>850 mM) were required. Additionally, hMBCOMT's elution was promoted on epoxy, butyl, and octyl using respectively 0.1–0.5, 0.25–1, and 1% of Triton X‐100. On butyl media, a stepwise strategy using 375 and 0 mM NaH₂PO₄, followed by three elution steps at 0.25, 0.7 and 1% Triton X‐100, allowed selective hMBCOMT isolation. In conclusion, significant amounts of MBCOMT were purified with high selectivity on a single chromatography procedure, despite its elution occurs on multiple peaks. Although successful applications of hydrophobic interaction chromatography in purification of membrane proteins are uncommon, we proved that traditional hydrophobic matrices can open a promising unexplored field to fulfill specific requirements for kinetic and pharmacological trials.     

Evaluation of the combinative application of SDS and sodium deoxycholate to the LC–MS‐based shotgun analysis of membrane proteomes

SDS and sodium deoxycholate (SDC) as two representative detergents have been widely used in LC–MS/MS‐based shotgun analysis of membrane proteomes. However, some inherent disadvantages limit their applications such as interference with MS analysis or their weak ability to disrupt membranes. To address this, the combinative application of SDS and SDC was developed and evaluated in our study, which comprehensively used the strong ability of SDS to lyse membranes and solubilize hydrophobic membrane proteins, and the high efficiencies of an optimized acetone precipitation method and SDC in sample clean‐up, protein recovery, and redissolution and digestion of precipitated proteins. The comparative study using a rat‐liver‐membrane‐enriched sample showed that, compared with other three commonly used methods including the filter‐aided sample preparation strategy, the combinative method not only increased the identified number of total proteins, membrane proteins, and integral membrane proteins by an average of 19.8, 23.9, and 24.8%, respectively, but also led to the identification of the highest number of matching peptides. All these results demonstrate that the method yielded better recovery and reliability in the identification of the proteins especially highly hydrophobic integral membrane proteins than the other three methods, and thereby has more potential in shotgun membrane proteomics.     

Reproducible method to enrich membrane proteins with high purity and high yield for an LC‐MS/MS approach in quantitative membrane proteomics

The proportionately low abundance of membrane proteins hampers their proteomic analysis, especially for a quantitative LC‐MS/MS approach. To overcome this limitation, a method was developed that consists of one cell disruption step in a hypotonic reagent using liquid nitrogen, one isolation step using a low speed centrifugation, and three wash steps using high speed centrifugation. Pellets contained plasma, nuclear, and mitochondrial membranes, including their integral, peripheral, and anchored membrane proteins. The reproducibility of this method was verified by protein assay of four separate experiments with a CV of 7.7%, and by comparative LC‐MS/MS label‐free quantification of individual proteins between two experiments with 99% of the quantified proteins having a CV ≤30%. Western blot and LC‐MS/MS results of markers for cytoplasm, nucleus, mitochondria, and their membranes indicated that the enriched membrane fraction was highly pure by the absence of, or presence of trace amounts of, nonmembrane marker proteins. The average yield of membrane proteins was 237 μg/10 million HT29‐MTX cells. LC‐MS/MS analysis of the membrane‐enriched sample resulted in the identification of 2597 protein groups. In summary, the developed method is reproducible, produces a highly pure membrane fraction, and generates a high yield of membrane proteins.     

Electric migration of α‐hemolysin in supported n‐bilayers: A model for transmembrane protein microelectrophoresis

Proteome analysis involves separating proteins as a preliminary step toward their characterization. This paper reports on the translational migration of a model transmembrane protein (α‐hemolysin) in supported n‐bilayers (n, the number of bilayers, varies from 1 to around 500 bilayers) when an electric field parallel to the membrane plane is applied. The migration changes in direction as the charge on the protein changes its sign. Its electrophoretic mobility is shown to depend on size and charge. The electrophoretic mobility varies as 1/R², with R the equivalent geometric radius of the embedded part of the protein. Measuring mobilities at differing pH in our system enables us to determine the pI and the charge of the protein. Establishing all these variations points to the feasibility of electrophoretic transport of a charged object in this medium and is a first step toward electrophoretic separation of membrane proteins in n‐bilayer systems.     

基于微流控技术的蛋白质结晶及其筛选方法的研究进展简

微流控技术以其高通量、低消耗和集成化等优点成为蛋白质结晶微型化研究的重要手段. 本文综述了基于微流控技术的蛋白质结晶技术和方法,主要包括微泵微阀、液滴(Droplet)、滑动芯片(SlipChip)以及液滴实验室(DropLab)等技术. 此外,还针对当前膜蛋白在结构生物学研究中的重要地位,综述了应用于膜蛋白结晶的微流控技术的研究进展.