How Well Can You Tailor the Charge of Lipid Vesicles?

Knowledge and control of surface charge or potential is important for tailoring colloidal interactions. In this work, we compare widely used zeta potential (ζ) measurements of charged lipid vesicle surface potential to direct measurements using the surface force apparatus (SFA). Our measurements show good agreement between the two techniques. On varying the fraction of anionic lipids dimyristoylphosphatidylserine (DMPS) or dimyristoylphosphatidylglycerol (DMPG) mixed with zwitterionic dimyristoylphosphatidylcholine (DMPC) from 0 to 100 mol % we observed a near-linear increase in membrane surface charge or potential up to 20-30 mol % charged lipids beyond which charge saturation occurred in physiological (high) salt conditions. Similarly, in low salt concentrations, a linear increase in charge/potential was found but only up to ∼5-10 mol % charged lipids beyond which the surface charge or potential leveled off. While a lower degree of ionization is expected due to the lower dielectric constant (ε ∼ 4) of the lipid acyl chain environment, increasing intramembrane electrostatic repulsion between neighboring charged lipid head groups at higher charge loading contributes to charge suppression. Measured potentials in physiological salt solutions were consistent with predictions using the Gouy-Chapman-Stern-Grahame (GCSG) model of the electrical double layer with Langmuir binding of counterions, but in low salt conditions, the model significantly overestimated the surface charge/potential. The much lower ionization in low salt (maximum ∼1-2% of total lipids ionized) instead was consistent with counterion condensation at the bilayer surface which limited the charge that could be obtained. The strong interplay between membrane composition, lipid headgroup ionization, electrolyte concentration, and solution pH complicates exact prediction and tuning of membrane surface charge for applications. However, the theoretical frameworks used here can provide guidelines to understand this interplay and establish a range of achievable potentials for a system and predict the response to triggers like pH and salt concentration changes.

Validation of inflammatory genetic variants associated with long-term cancer related fatigue in a large breast cancer cohort.

BACKGROUND: Studies to date have reported several associations between single nucleotide polymorphisms (SNPs) and cancer related fatigue (CRF), but have been limited by small sample sizes, missing adjustment for relevant covariates or multiple testing, as well as varying CRF definitions, i.e. time and method of assessment. This study aimed to validate previously reported associations using the largest independent breast cancer sample to date and to evaluate further functional cytokine variants in relation to total CRF and all relevant CRF subdomains (physical, cognitive, and affective CRF). METHOD: 45 candidate SNPs in inflammatory pathway genes were selected based on previous reports (16 SNPs) or regulatory function (29 SNPs). Breast cancer patients recruited between 2002 and 2005 provided information on CRF at first follow-up (FU1) (N = 1389) and second follow-up (FU2) (N = 950), a median of 6.2 years and 11.7 years respectively after diagnosis. SNP associations were assessed using linear regression models on CRF scores separately for FU1 and FU2. Additionally, patients with persistent fatigue (fatigued at both time-points) were compared to those never fatigued using logistic regression models (N = 684). All analyses were adjusted for relevant covariates. Secondary analyses were conducted for CRF subdomains. RESULTS: For total CRF none of the previously reported associations were confirmed after correction for multiple testing. The p-value distribution of all SNPs was not different than the one expected by chance. Analyses of CRF subdomains yielded a significant association between TNF-α rs3093662 and persistent physical CRF (Odds Ratio (OR) = 3.23, 95% Confidence Interval (CI) = 1.71-6.10, p = 0.0003). CONCLUSION: We were unable to confirm previously reported findings, suggesting that individual SNPs are unlikely to be of clinical utility. Further investigations in well powered studies are warranted, which consider genetic heterogeneity according to subdomains of CRF.

Membrane texture induced by specific protein binding and receptor clustering: active roles for lipids in cellular function.

Biological membranes are complex, self-organized structures that define boundaries and compartmentalize space in living matter. Composed of a wide variety of lipid and protein molecules, these responsive surfaces mediate transmembrane signaling and material transport within the cell and with its environment. It is well known that lipid membrane properties change as a function of composition and phase state, and that protein-lipid interactions can induce changes in the membrane's properties and biochemical response. Here, molecular level changes in lipid organization induced by multivalent toxin binding were investigated using grazing incidence X-ray diffraction. Structural changes to lipid monolayers at the air-water interface and bilayers at the solid-water interface were studied before and after specific binding of cholera toxin to membrane embedded receptors. At biologically relevant surface pressures, protein binding perturbed lipid packing within monolayers and bilayers resulting in topological defects and the emergence of a new orientationally textured lipid phase. In bilayers this altered lipid order was transmitted from the receptor laden exterior membrane leaflet to the inner leaflet, representing a potential mechanism for lipid mediated outside-in signaling by multivalent protein binding. It is further hypothesized that cell-surface micro-domains exhibiting this type of lipid order may serve as nucleation sites for vesicle formation in clathrin independent endocytosis of cholera toxin.

A fixed-target platform for serial femtosecond crystallography in a hydrated environment.

For serial femtosecond crystallography at X-ray free-electron lasers, which entails collection of single-pulse diffraction patterns from a constantly refreshed supply of microcrystalline sample, delivery of the sample into the X-ray beam path while maintaining low background remains a technical challenge for some experiments, especially where this methodology is applied to relatively low-ordered samples or those difficult to purify and crystallize in large quantities. This work demonstrates a scheme to encapsulate biological samples using polymer thin films and graphene to maintain sample hydration in vacuum conditions. The encapsulated sample is delivered into the X-ray beam on fixed targets for rapid scanning using the Roadrunner fixed-target system towards a long-term goal of low-background measurements on weakly diffracting samples. As a proof of principle, we used microcrystals of the 24 kDa rapid encystment protein (REP24) to provide a benchmark for polymer/graphene sandwich performance. The REP24 microcrystal unit cell obtained from our sandwiched in-vacuum sample was consistent with previously established unit-cell parameters and with those measured by us without encapsulation in humidified helium, indicating that the platform is robust against evaporative losses. While significant scattering from water was observed because of the sample-deposition method, the polymer/graphene sandwich itself was shown to contribute minimally to background scattering.

Crystallization of ApoA1 and ApoE4 nanolipoprotein particles and initial XFEL-based structural studies.

Nanolipoprotein particles (NLPs), also called "nanodiscs", are discoidal particles with a patch of lipid bilayer corralled by apolipoproteins. NLPs have long been of interest due to both their utility as membrane-model systems into which membrane proteins can be inserted and solubilized and their physiological role in lipid and cholesterol transport via HDL and LDL maturation, which are important for human health. Serial femtosecond crystallography (SFX) at X-ray free electron lasers (XFELs) is a powerful approach for structural biology of membrane proteins, which are traditionally difficult to crystallize as large single crystals capable of producing high-quality diffraction suitable for structure determination. To facilitate understanding of the specific role of two apolipoprotein/lipid complexes, ApoA1 and ApoE4, in lipid binding and HDL/LDL particle maturation dynamics and develop new SFX methods involving NLP membrane protein encapsulation, we have prepared and crystallized homogeneous populations of ApoA1 and ApoE4 NLPs. Crystallization of empty NLPs yields semi-ordered objects that appear crystalline and give highly anisotropic and diffuse X-ray diffraction, similar in characteristics to fiber diffraction. Several unit cell parameters were approximately determined for both NLPs from these measurements. Thus, low-background, sample conservative methods of delivery are critical. Here we implemented a fixed target sample delivery scheme utilizing the Roadrunner fast-scanning system and ultra-thin polymer/graphene support films, providing a low-volume, low-background approach to membrane protein SFX. This study represents initial steps in obtaining structural information for ApoA1 and ApoE4 NLPs and developing this system as a supporting scaffold for future structural studies of membrane proteins crystalized in a native lipid environment.

Direct measurement of a tethered ligand-receptor interaction potential.

Many biological recognition interactions involve ligands and receptors that are tethered rather than rigidly bound on a cell surface. A surface forces apparatus was used to directly measure the force-distance interaction between a polymer-tethered ligand and its receptor. At separations near the fully extended tether length, the ligands rapidly lock onto their binding sites, pulling the ligand and receptor together. The measured interaction potential and its dynamics can be modeled with standard theories of polymer and colloidal interactions.

Impact of polymer tether length on multiple ligand-receptor bond formation.

The promoters of cell adhesion are ligands, which are often attached to flexible tethers that bind to surface receptors on adjacent cells. Using a combination of Monte Carlo simulations, diffusion reaction theory, and direct experiments (surface force measurements) of the biotin-streptavidin system, we have quantified polymer chain dynamics and the kinetics and spatial range of tethered ligand-receptor binding. The results show that the efficiency of strong binding does not depend solely on the molecular architecture or binding energy of the receptor-ligand pair, nor on the equilibrium configuration of the polymer tether, but rather on its "rare" extended conformations.

Molecular dynamics simulations of polystyrene brushes in dry conditions and in toluene solution.

The properties of polystyrene brushes in dry conditions and in toluene solution are studied as a function of grafting density using molecular dynamics simulations. Both, individual brushes and double layers of opposing brushes are considered, the structural properties of which were found to be similar. The density profiles show very pronounced density oscillations which extend up to approximately 1.8 nm and fall into two groups of three peaks each. These features are observed regardless of grafting density and solvent conditions. In the absence of solvent, the chains undergo a transition from an oblate to a spherical shape as the grafting density increases. In contrast, in good solvent, the chains remain spherical independent of the grafting density. Solvation also increases the extension of the polystyrene chains roughly by a factor 2.5. Isotropic and two-dimensional radial distribution functions are used to characterize the structure of the polystyrene brushes. Toluene is observed to form up to four layers at the base of the grafted chains irrespective of grafting density.

Part I: an x-ray scattering study of cholera toxin penetration and induced phase transformations in lipid membranes.

Cholera toxin is a highly efficient biotoxin, which is frequently used as a tool to investigate protein-membrane interactions and as a reporter for membrane rafts. Cholera toxin binds selectively to gangliosides with highest affinity to GM(1). However, the mechanism by which cholera toxin crosses the membrane remains unresolved. Using x-ray reflectivity and grazing incidence diffraction, we have been able to monitor the binding and penetration of cholera toxin into a model lipid monolayer containing the receptor GM(1) at the air-water interface. Very high toxin coverage was obtained allowing precise measurements of how toxin binding alters lipid packing. Grazing incidence x-ray diffraction revealed the coexistence of two monolayer phases after toxin binding. The first was identical to the monolayer before toxin binding. In regions where toxin was bound, a second membrane phase exhibited a decrease in order as evidenced by a larger area per molecule and tilt angle with concomitant thinning of the monolayer. These results demonstrate that cholera toxin binding induces the formation of structurally distinct, less ordered domains in gel phases. Furthermore, the largest decrease in lateral order to the monolayer occurred at low pH, supporting a low endosomal pH in the infection pathway. Surprisingly, at pH = 8 toxin penetration by the binding portion of the toxin, the B(5) pentamer, was also observed.

Part II: diffraction from two-dimensional cholera toxin crystals bound to their receptors in a lipid monolayer.

The structure of cholera toxin (CTAB(5)) bound to its putative ganglioside receptor, galactosyl-N-acetylgalactosaminyl (N-acetyl-neuraminyl) galactosylglucosylceramide (GM(1)), in a lipid monolayer at the air-water interface has been studied utilizing grazing incidence x-ray diffraction. Cholera toxin is one of very few proteins to be crystallized in two dimensions and characterized in a fully hydrated state. The observed grazing incidence x-ray diffraction Bragg peaks indicated cholera toxin was ordered in a hexagonal lattice and the order extended 600-800 A. The pentameric binding portion of cholera toxin (CTB(5)) improved in-plane ordering over the full toxin (CTAB(5)) especially at low pH. Disulfide bond reduction (activation of the full toxin) also increased the protein layer ordering. These findings are consistent with A-subunit flexibility and motion, which cause packing inefficiencies and greater disorder of the protein layer. Corroborative out-of-plane diffraction (Bragg rod) analysis indicated that the scattering units in the cholera layer with CTAB(5) shortened after disulfide bond reduction of the A subunit. These studies, together with Part I results, revealed key changes in the structure of the cholera toxin-lipid system under different pH conditions.

Quantitative study of 10 Hz operation of a soft x-ray laser-energy stability and target considerations.

A soft x-ray laser from Ni-like Mo, pumped in grazing incidence (GRIP), is analyzed with regard to high repetition rate operation. Reliable lasing is obtained, but with significant energy fluctuations attributed mainly to beam pointing jitter from the pump laser. Two modes of operation are compared: continuously moving target and stationary target. With a moving target the soft X-ray output is constant on average, whereas the repeated use of the same target position leads to a pulse energy which increases for several tens of shots. This effect might be caused by improved guiding of the pump laser in the formed groove and the removal, through laser ablation, of the oxide layer on the target surface.

Neutron and X-ray scattering studies of cholera toxin interactions with lipid monolayers at the air-liquid interface.

Using neutron/X-ray reflectivity and X-ray grazing incidence diffraction (GID), we have characterized the structure of mixed DPPE:GM(1) lipid monolayers before and during the binding of cholera toxin (CTAB(5)) or its B subunit (CTB(5)). Structural parameters such as the density and thickness of the lipid layer, extension of the GM(1) oligosaccharide headgroup, and orientation and position of the protein upon binding are reported. Both CTAB(5) and CTB(5) were measured to have approximately 50% coverage when bound to the lipid monolayer. X-ray GID experiments show that both the lipid monolayer and the cholera toxin layer are crystalline. The effects of X-ray beam damage have been assessed and the monolayer/toxin structure does not change with time after protein binding has saturated.

X-ray and neutron surface scattering for studying lipid/polymer assemblies at the air-liquid and solid-liquid interfaces.

Simple mono- and bilayers, built of amphiphilic molecules and prepared at air-liquid or solid-liquid interfaces, can be used as models to study such effects as water penetration, hydrocarbon chain packing, and structural changes due to head group modification. In the paper, we will discuss neutron and X-ray reflectometry and grazing incidence X-ray diffraction techniques used to explore structures of such ultra-thin organic films in different environments. We will illustrate the use of these methods to characterize the morphologies of the following systems: (i) polyethylene glycol-modified distearoylphosphatidylethanolamine monolayers at air-liquid and solid-liquid interfaces; and (ii) assemblies of branched polyethyleneimine polymer and dimyristoylphophatidylcholine lipid at solid-liquid interfaces.

Direct visualization of cavitation and damage in ultrathin liquid films.

The Surface Forces Apparatus technique was used to observe, at the nanoscopic level, the rapid growth and disappearance of vapor cavities between two moving solid surfaces while simultaneously monitoring their effects on the deformations and damage produced on the surfaces. Both approach-separation and shear motions were studied. In both geometries, as the relative velocity between the two surfaces increases, they deform 'elastohydrodynamically'; in some regions the surfaces become flattened (corresponding to the build up of positive or compressive pressure in the adjacent liquid) while in other regions the surfaces become pointed (corresponding to negative or tensile pressure). Vapor cavities nucleate at these pointed regions once the relative velocity of the two surfaces exceeds a certain critical value. The sudden nucleation and growth of a cavity in a thin liquid film is seen to be a much more violent event than its subsequent collapse, and surface damage due to cavitation occurs during the inception rather than during the much smoother collapse of cavities. These studies are the first to directly visualize cavity formation and associated damage in ultrathin lubricating films, and provide a fairly detailed picture of both of these mechanisms and the relationship between them. The picture that emerges in complex and different from the currently accepted model, in which damage is caused during the collapse of cavities rather than their growth. The results should have a bearing on all phenomena where cavitation and cavitation damage occur in thin lubricating films.

[Percutaneous tracheostomy with use of a laryngeal mask]. / Alternative Punktionstracheotomie mit Hilfe der Larynxmaske.

The percutaneous tracheostomy is a conventional procedure in patients undergoing long term ventilation on ICU. It both facilitates weaning and reduces the ventilation and tracheal tube associated risks.Usually the tracheostomy is accomplished via the tracheal tube. The alternative implies extubation and reinsertion of a laryngeal mask. This method itself affords a better overview for the bronchoscoping person accompanied by a lower risk for cuff or bronchoscope lesions. An accidental extubation as well as an injuring of the vocal cords (because of the inflated cuff during accidental extubation) appears impossible in this method.This paper gives a general survey on indication, contraindication, advantages and disadvantages of the percutaneous tracheostomy via laryngeal mask. We also described the procedure itself, step by step.

[Extracorporeal life support given to a 16-year-old girl with cystic fibrosis, candida pneumonia and acute respiratory distress syndrome]. / Extrakorporale Membranoxygenierung bei einer jungen Patientin mit ARDS durch eine Pilzpneumonie bei Mukoviszidose.

HISTORY: Small bowel resection had to be performed because of an acute ileus in a 16-year old girl with mucoviscidosis. Severe respiratory insufficiency developed and she was transferred to the intensive care unit. INVESTIGATIONS: The clinical signs of a severe ARDS were demonstrated: Horowitz index < 200, pO (2) 57 mm Hg, FiO (2) 1,0, pCO (2) 82 mm Hg. Candida serology was positive (titer 1 : 5120), and there was a leukocytosis (20 000/µl), hypalbuminemia (14 g/l) and elevation of C-reactive protein (190 mg/l). TREATMENT AND COURSE: Because all non invasive treatment options had failed to improve the patient's condition, an extracorporal membrane oxygenation (ECMO) device was connected. Seven days later, after the pulmonary situation had improved, the device was successfully removed; the patient was discharged in a satisfactory condition after another month. CONCLUSION: ECMO is a another treatment option for serious ARDS in infection-related worsening of pulmonary cystic fibrosis.