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We propose and demonstrate a novel scheme for optical nanoparticle characterization, optofluidic force induction (OF2i), which achieves real-time optical counting with single-particle sensitivity, high throughput, and for particle sizes ranging from tens of nanometers to several $\mu$m. The particles to be analyzed flow through a microfluidic channel alongside a weakly focused laser vortex beam, which accomplishes 2D trapping of the particles in the transverse directions and size-dependent velocity changes due to the optical forces in the longitudinal direction. Upon monitoring the trajectories and velocity changes of each individually tracked particle, we obtain detailed information about the number based particle size distribution. A parameter-free model based on Maxwell's equations and Mie theory is shown to provide very good agreement with the experimental results for standardized particles of spherical shape. Our results prove that OF2i can provide a flexible work bench for numerous pharmaceutical and technological applications, as well as for medical diagnostics., Comment: 17 pages, 7 figures

Purpose Inkjet printing has the potential to enable novel personalized and tailored drug therapies based on liposome and lipid nanoparticles. However, due to the significant shear force exerted on the jetted fluids, its suitability for shear-sensitive materials such as liposomes, has not been verified. We have conducted a proof-of-concept study to examine whether the particle concentration and size distribution of placebo liposomes are affected by common inkjet/dispensing technologies. Methods We have subjected three types of liposome-containing fluids (“inks”) to two different commercial dispensing/jetting technologies, which are relevant to most drug printing approaches. The liposome jetting processes were observed in real-time using strobographic imaging techniques. The phospholipid concentrations and particle size distributions were determined before and after jetting via enzymatic colorimetric and dynamic light scattering methods, respectively. Results Our results have shown that the jetting dynamics of the liposome inks are well predicted by the established inkjet printing regime map based on their physical properties and the jetting conditions. Importantly, although significant shear forces were confirmed during jetting, the liposome concentrations and particle size distributions in the collected samples remain largely unaffected. Conclusion These findings, we believe, provide the essential proof-of-concept to encourage further development in this highly topical research area.

Although extensive research is being done to combat SARS-CoV-2, we are yet far away from a robust conclusion or strategy. With an increased amount of vaccine research, nanotechnology has found its way into vaccine technology. Researchers have explored the use of various nanostructures for delivering the vaccines for enhanced efficacy. Apart from acting as delivery platforms, multiple studies have shown the application of inorganic nanoparticles in suppressing the growth as well as transmission of the virus. The present review gives a detailed description of various inorganic nanomaterials which are being explored for combating SARS-CoV-2 along with their role in suppressing the transmission of the virus either through air or by contact with inanimate surfaces. The review further discusses the use of nanoparticles for development of an antiviral coating that may decrease adhesion of SARS-CoV-2. A separate section has been included describing the role of nanostructures in biosensing and diagnosis of SARS-CoV-2. The role of nanotechnology in providing an alternative therapeutic platform along with the role of radionuclides in SARS-CoV-2 has been described briefly. Based on ongoing research and commercialization of this nanoplatform for a viral disease, the nanomaterials show the potential in therapy, biosensing, and diagnosis of SARS-CoV-2.

Black phosphorus is one of the emerging members of two-dimensional (2D) materials which has recently entered the biomedical field. Its anisotropic properties and infrared bandgap have enabled researchers to discover its applicability in several fields including optoelectronics, 3D printing, bioimaging, and others. Characterization techniques such as Raman spectroscopy have revealed the structural information of Black phosphorus (BP) along with its fundamental properties, such as the behavior of its photons and electrons. The present review provides an overview of synthetic approaches and properties of BP, in addition to a detailed discussion about various types of surface modifications available for overcoming the stability-related drawbacks and for imparting targeting ability to synthesized nanoplatforms. The review further gives an overview of multiple characterization techniques such as spectroscopic, thermal, optical, and electron microscopic techniques for providing an insight into its fundamental properties. These characterization techniques are not only important for the analysis of the synthesized BP but also play a vital role in assessing the doping as well as the structural integrity of BP-based nanocomposites. The potential role of BP and BP-based nanocomposites for biomedical applications specifically, in the fields of drug delivery, 3D printing, and wound dressing, have been discussed in detail to provide an insight into the multifunctional role of BP-based nanoplatforms for the management of various diseases, including cancer therapy. The review further sheds light on the role of BP-based 2D platforms such as BP nanosheets along with BP-based 0D platforms—i.e., BP quantum dots in the field of therapy and bioimaging of cancer using techniques such as photoacoustic imaging and fluorescence imaging. Although the review inculcates the multimodal therapeutic as well as imaging role of BP, there is still research going on in this field which will help in the development of BP-based theranostic platforms not only for cancer therapy, but various other diseases.

Despite the increasing prevalence of obesity and diabetes, there is no efficient treatment to combat these epidemics. The adipose organ is the main site for energy storage and plays a pivotal role in whole body lipid metabolism and energy homeostasis, including remodeling and dysfunction of adipocytes and adipose tissues in obesity and diabetes. Thus, restoring and balancing metabolic functions in the adipose organ is in demand. MiRNAs represent a novel class of drugs and drug targets, as they are heavily involved in the regulation of many cellular and metabolic processes and diseases, likewise in adipocytes. In this review, we summarize key regulatory activities of miRNAs in the adipose organ, discuss various miRNA replacement and inhibition strategies, promising delivery systems for miRNAs and reflect the future of novel miRNA-based therapeutics to target adipose tissues with the ultimate goal to combat metabolic disorders.

Excessive expression of subunit 1 of GIRK1 in ER+ breast tumors is associated with reduced survival times and increased lymph node metastasis in patients. To investigate possible tumor-initiating properties, benign MCF10A and malign MCF7 mammary epithelial cells were engineered to overexpress GIRK1 neoplasia associated vital parameters and resting potentials were measured and compared to controls. The presence of GIRK1 resulted in resting potentials negative to the controls. Upon GIRK1 overexpression, several cellular pathways were regulated towards pro-tumorigenic action as revealed by comparison of transcriptomes of MCF10AGIRK1 with the control (MCF10AeGFP). According to transcriptome analysis, cellular migration was promoted while wound healing and extracellular matrix interactions were impaired. Vital parameters in MCF7 cells were affected akin the benign MCF10A lines, but to a lesser extent. Thus, GIRK1 regulated cellular pathways in mammary epithelial cells are likely to contribute to the development and progression of breast cancer.

Background Endogenously expressed microRNAs (miRNAs) have attracted attention as important regulators in post-transcriptionally controlling gene expression of various physiological processes. As miRNA dysregulation is often associated with various disease patterns, such as obesity, miRNA-27a might therefore be a promising candidate for miRNA mimic replacement therapy by inhibiting adipogenic marker genes. However, application of naked nucleic acids faces some limitations concerning poor enzymatic stability, bio-membrane permeation and cellular uptake. To overcome these obstacles, the development of appropriate drug delivery systems (DDS) for miRNAs is of paramount importance. Methods In this work, a triple combination of atomic force microscopy (AFM), brightfield (BF) and fluorescence microscopy was used to trace the cellular adhesion of N-TER peptide-nucleic acid complexes followed by time-dependent uptake studies using confocal laser scanning microscopy (cLSM). To reveal the biological effect of miRNA-27a on adipocyte development after transfection treatment, Oil-Red-O (ORO)- staining was performed to estimate the degree of in lipid droplets accumulated ORO in mature adipocytes by using light microscopy images as well as absorbance measurements. Results The present findings demonstrated that amphipathic N-TER peptides represent a suitable DDS for miRNAs by promoting non-covalent complexation through electrostatic interactions between both components as well as cellular adhesion of the N-TER peptide - nucleic acid complexes followed by uptake across cell membranes and intracellular release of miRNAs. The anti-adipogenic effect of miRNA-27a in 3T3-L1 cells could be detected in mature adipocytes by reduced lipid droplet formation. Conclusion The present DDS assembled from amphipathic N-TER peptides and miRNAs is capable of inducing the anti-adipogenic effect of miRNA-27a by reducing lipid droplet accumulation in mature adipocytes. With respect to miRNA mimic replacement therapies, this approach might provide new therapeutic strategies to prevent or treat obesity and obesity-related disorders.

The present pandemic of SARS-CoV-2 has been a tough task for the whole world to deal with. With the absence of specific drugs or vaccines against SARS-CoV-2, the situation is very difficult to control. Apart from the absence of specific therapies, the lack of knowledge about potential therapeutic targets and individual perception is adding to the complications. The present review describes the novel SARS-CoV-2 structure, surface proteins, asymptomatic and symptomatic transmission in addition to the genotype and phenotype of SARS-CoV-2 along with genetic strains and similarity between SARS, MERS and SARS-CoV-2. Therapeutic strategies such as inhibition of the endocytic pathway and suppressing RNA polymerase activity by metal ions, which could be quite beneficial for controlling COVID-19, are outlined. The drug repurposing for SARS-CoV-2 is discussed in detail along with therapeutic classes such as antivirals, antibiotics, and amino quinolones and their probable role in suppressing SARS-CoV-2 with reference to case studies. The ongoing clinical trials both with respect to drug repurposing and vaccines are summarized along with a brief description. The recent advancements and future perspective of ongoing research for therapy and detection of SARS-CoV-2 are provided. The review, in brief, summarizes epidemiology, therapy and the current scenario for combating SARS-CoV-2., Graphical abstract Unlabelled Image

The heterodimeric human (h) electron-transferring flavoprotein (ETF) transfers electrons from at least 13 different flavin dehydrogenases to the mitochondrial respiratory chain through a non-covalently bound FAD cofactor. Here, we describe the discovery of an irreversible and pH-dependent oxidation of the 8α-methyl group to 8-formyl-FAD (8f-FAD), which represents a unique chemical modification of a flavin cofactor in the human flavoproteome. Furthermore, a set of hETF variants revealed that several conserved amino acid residues in the FAD-binding pocket of electron-transferring flavoproteins are required for the conversion to the formyl group. Two of the variants generated in our study, namely αR249C and αT266M, cause glutaric aciduria type II, a severe inherited disease. Both of the variants showed impaired formation of 8f-FAD shedding new light on the potential molecular cause of disease development. Interestingly, the conversion of FAD to 8f-FAD yields a very stable flavin semiquinone that exhibited slightly lower rates of electron transfer in an artificial assay system than hETF containing FAD. In contrast, the formation of 8f-FAD enhanced the affinity to human dimethylglycine dehydrogenase 5-fold, indicating that formation of 8f-FAD modulates the interaction of hETF with client enzymes in the mitochondrial matrix. Thus, we hypothesize that the FAD cofactor bound to hETF is subject to oxidation in the alkaline (pH 8) environment of the mitochondrial matrix, which may modulate electron transport between client dehydrogenases and the respiratory chain. This discovery challenges the current concepts of electron transfer processes in mitochondria.

Lipids exhibit an extraordinary polymorphism in self-assembled mesophases, with lamellar phases as biologically most relevant representative. To mimic lipid lamellar phases with amphiphilic designer peptides, seven systematically varied short peptides were engineered. Indeed, four peptide candidates (V4D, V4WD, V4WD2, I4WD2) readily self-assembled into lamellae in aqueous solution: small-angle X-ray scattering patterns (SAXS) revealed ordered lamellar structures with a repeat distance of ~4-5 nm. Transmission electron microscopy (TEM) images confirmed the presence of stacked sheets. Two derivatives (V3D and V4D2) remained as loose aggregates dispersed in solution; one peptide (L4WD2) formed twisted tapes with internal lamellae and an antiparallel β-type monomer alignment. To understand the interaction of peptides with lipids they were mixed with phosphatidylcholines. Low peptide concentrations (1.1 mM) induced the formation of a heterogeneous mixture of vesicular structures: large multilamellar vesicles (d-spacing ~6.3 nm) coexisted with oligo- or unilamellar vesicles (~50 nm in diameter) and bicelle-like structures (~45 nm length, ~18 nm width). High peptide concentrations (11 mM) led to unilamellar vesicles (ULV, diameter ~260-280 nm) with a homogeneous mixing of lipids and peptides. SAXS revealed the temperature-dependent fine structure of these ULVs: at 25 °C the bilayer is in a fully interdigitated state (headgroup-to-headgroup distance dhh ~2.9 nm), whereas at 50 °C this interdigitation opens up (dhh ~3.6 nm). Our results highlight the versatility of self-assembled peptide superstructures: subtle changes in the amino acid composition are key design elements in creating peptide- or lipid-peptide nanostructures with the same richness in morphology as known from the lipid-world.

Objectives The design of nanocarriers for local drug administration to the lining mucosa requires a sound knowledge of how nanoparticles (NPs) interact with saliva. This contact determines whether NPs agglomerate and become immobile due to size- and interaction-filtering effects or adsorb on the cell surface and are internalized by epithelial cells. The aim of this study was to examine the behavior of NPs in saliva considering physicochemical NP properties. Materials and methods The salivary pore–size distribution was determined, and the viscosity of the fluid inside of the pores was studied with optical tweezers. Distinct functionalized NPs (20 and 200 nm) were dispersed in saliva and salivary buffers and characterized, and surface-bound MUC5B and MUC7 were analyzed by 1D electrophoresis and immunoblotting. NP mobility was recorded, and cellular uptake studies were performed with TR146 cells. Results The mode diameter of the salivary mesh pores is 0.7 μm with a peak width of 1.9 μm, and pores are filled with a low-viscosity fluid. The physicochemical properties of the NPs affected the colloidal stability and mobility: compared with non-functionalized particles, which did not agglomerate and showed a cellular uptake rate of 2.8%, functionalized particles were immobilized, which was correlated with agglomeration and increased binding to mucins. Conclusion The present study showed that the salivary microstructure facilitates NP adsorption. However, NP size and surface functionalization determine the colloidal stability and cellular interactions. Clinical relevance The sound knowledge of NP interactions with saliva enables the improvement of current treatment strategies for inflammatory oral diseases. Electronic supplementary material The online version of this article (doi:10.1007/s00784-017-2172-5) contains supplementary material, which is available to authorized users.

Atherosclerosis is a major cardiovascular disease worldwide, that could benefit from innovative nanomedicine imaging tools and treatments. In this perspective, we here studied, by fluorescence imaging in ApoE-/- mice, the biodistribution of non-functionalized and RXP470.1-targeted nanostructured lipid carriers (NLC) loaded with DiD dye. RXP470.1 specifically binds to MMP12, a metalloprotease that is over-expressed by macrophages residing in atherosclerotic plaques. Physico-chemical characterizations showed that RXP-NLC (about 105 RXP470.1 moieties/particle) displayed similar features as non-functionalized NLC in terms of particle diameter (about 60-65 nm), surface charge (about &minus, 5 &mdash, &minus, 10 mV), and colloidal stability. In vitro inhibition assays demonstrated that RXP-NLC conserved a selectivity and affinity profile, which favored MMP-12. In vivo data indicated that NLC and RXP-NLC presented prolonged blood circulation and accumulation in atherosclerotic lesions in a few hours. Twenty-four hours after injection, particle uptake in atherosclerotic plaques of the brachiocephalic artery was similar for both nanoparticles, as assessed by ex vivo imaging. This suggests that the RXP470.1 coating did not significantly induce an active targeting of the nanoparticles within the plaques. Overall, NLCs appeared to be very promising nanovectors to efficiently and specifically deliver imaging agents or drugs in atherosclerotic lesions, opening avenues for new nanomedicine strategies for cardiovascular diseases.

Non-coding RNAs (ncRNAs) like microRNAs (miRNAs) or small interference RNAs (siRNAs) with their power to selectively silence any gene of interest enable the targeting of so far 'undruggable' proteins and diseases. Such RNA molecules have gained much attention from biotech and pharmaceutical companies, which led to the first Food and Drug Administration (FDA) approved ncRNA therapeutic in 2018. However, the main barrier in clinical practice of ncRNAs is the lack of an effective delivery system that can protect the RNA molecules from nuclease degradation, deliver them to specific tissues and cell types, and release them into the cytoplasm of the targeted cells, all without inducing adverse effects. For that reason, drug delivery approaches, formulations, technologies and systems for transporting pharmacological ncRNA compounds to achieve a diagnostic or therapeutic effect in the human body are in demand. Here, we review the development of therapeutic lipid-based nanoparticles for delivery of miRNAs, one class of endogenous ncRNAs with specific regulatory functions. We outline challenges and opportunities for advanced miRNA-based therapies, and discuss the complexity associated with the delivery of functional miRNAs. Novel strategies are addressed how to deal with the most critical points in miRNA delivery, such as toxicity, specific targeting of disease sites, proper cellular uptake and endosomal escape of miRNAs. Current fields of application and various preclinical settings involving miRNA therapeutics are discussed, providing an outlook to future clinical approaches. Following the current trends and technological developments in nanomedicine exciting new delivery systems for ncRNA-based therapeutics can be expected in upcoming years.

International audience; The objective of our work was to investigate the effects of different types of nanoparticles on endothelial (HUVEC) and monocytic cell functions. We prepared and tested 14 different nanosystems comprising liposomes, lipid nanoparticles, polymer, and iron oxide nanoparticles. Some of the tested nanosystems contained targeting, therapeutic, or contrast agent(s). The effect of particles (0-400 mu g/mL) on endothelial-monocytic cell interactions in response to TNF-alpha was investigated using an arterial bifurcation model and dynamic monocyte adhesion assay. Spontaneous HUVEC migration (0-100 mu g/mL nanoparticles) and chemotaxis of monocytic cells towards MCP-1 in presence of particles (0-400 mu g/mL) were determined using a barrier assay and a modified Boyden chamber assay, respectively. Lipid nanoparticles dose-dependently reduced monocytic cell chemotaxis and adhesion to activated HUVECs. Liposomal nanoparticles had little effect on cell migration, but one formulation induced monocytic cell recruitment by HUVECs under non-uniform shear stress by about 50%. Fucoidan-coated polymer nanoparticles (25-50 mu g/mL) inhibited HUVEC migration and monocytic cell chemotaxis, and had a suppressive effect on monocytic cell recruitment under non-uniform shear stress. No significant effects of iron oxide nanoparticles on monocytic cell recruitment were observed except lauric acid and human albumin-coated particles which increased endothelial-monocytic interactions by 60-70%. Some of the iron oxide nanoparticles inhibited HUVEC migration and monocytic cell chemotaxis. These nanoparticle-induced effects are of importance for vascular cell biology and function and must be considered before the potential clinical use of some of the analyzed nanosystems in cardiovascular applications.

Cancer cells are reprogrammed to consume large amounts of glucose to support anabolic biosynthetic pathways. However, blood perfusion and consequently the supply with glucose are frequently inadequate in solid cancers. PEPCK-M (PCK2), the mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK), has been shown by us and others to be functionally expressed and to mediate gluconeogenesis, the reverse pathway of glycolysis, in different cancer cells. Serine and ribose synthesis have been identified as downstream pathways fed by PEPCK in cancer cells. Here, we report that PEPCK-M-dependent glycerol phosphate formation from noncarbohydrate precursors (glyceroneogenesis) occurs in starved lung cancer cells and supports de novo glycerophospholipid synthesis. Using stable isotope-labeled glutamine and lactate, we show that PEPCK-M generates phosphoenolpyruvate and 3-phosphoglycerate, which are at least partially converted to glycerol phosphate and incorporated into glycerophospholipids (GPL) under glucose and serum starvation. This pathway is required to maintain levels of GPL, especially phosphatidylethanolamine (PE), as shown by stable shRNA-mediated silencing of PEPCK-M in H23 lung cancer cells. PEPCK-M shRNA led to reduced colony formation after starvation, and the effect was partially reversed by the addition of dioleyl-PE. Furthermore, PEPCK-M silencing abrogated cancer growth in a lung cancer cell xenograft model. In conclusion, glycerol phosphate formation for de novo GPL synthesis via glyceroneogenesis is a newly characterized anabolic pathway in cancer cells mediated by PEPCK-M under conditions of severe nutrient deprivation.

Biological cells are fascinating systems of inconceivable complexity, which fulfil various functions. Among others, cells are able to execute motions, to produce heat, to breathe, to subsist, to grow wand proliferate and to die. Science aims at deciphering the different functionalities and activities of, and inside, the cells and how their different components participate to them. The methods employed are also versatile, as optical approaches by microscopies, modelling and simulations, spectroscopies, thermodynamic measurements and much more, each procuring some pieces of the puzzle. Although the different investigations are laborious and time consuming, research is the only way to disentangle the world at the microscopic level surrounding us. In the present study, we cite a few examples of studies on whole cells and cell components by different neutron scattering techniques to illustrate the modern possibilities. As neutrons are not charged, they have interactions directly with the atomic nuclei and give access to structural as well as dynamical information through coherent and incoherent neutron scattering. These techniques can be applied to the same samples and under identical experimental conditions so that we can gain knowledge on the correlations between structural and dynamical functions. Here, we present applications of neutron experiments to decipher the behaviour of complex biological samples, which study was not possible by other probes. The first example focuses on the molecular basis of the adaptation of cells living under extreme conditions, such as Archaea from the deep sea hydrothermal vents which experience both high temperature and high pressure stresses. Molecular dynamics seems to play a key role for adaptation as it is increased for the proteome of cells from such environment, in contrast to common expectation. In the second example, we exposed endogenous nanoparticles, low density lipoproteins, to high hydrostatic pressure, to shed light on the flexibility and stability of such particles under extreme conditions. Here we found that the native particle was surprisingly resistant to pressure application, concerning both dynamics and structure, while a modified form thereof was not.

Low-density lipoproteins (LDL) are natural lipid transporter in human plasma whose chemically modified forms contribute to the progression of atherosclerosis and cardiovascular diseases accounting for a vast majority of deaths in westernized civilizations. For the development of new treatment strategies, it is important to have a detailed picture of LDL nanoparticles on a molecular basis. Through the combination of X-ray and neutron small-angle scattering (SAS) techniques with high hydrostatic pressure (HHP) this study describes structural features of normolipidemic, triglyceride-rich and oxidized forms of LDL. Due to the different scattering contrasts for X-rays and neutrons, information on the effects of HHP on the internal structure determined by lipid rearrangements and changes in particle shape becomes accessible. Independent pressure and temperature variations provoke a phase transition in the lipid core domain. With increasing pressure an interrelated anisotropic deformation and flattening of the particle are induced. All LDL nanoparticles maintain their structural integrity even at 3000 bar and show a reversible response toward pressure variations. The present work depicts the complementarity of pressure and temperature as independent thermodynamic parameters and introduces HHP as a tool to study molecular assembling and interaction processes in distinct lipoprotein particles in a nondestructive manner. (Less)

Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released from the outer membrane (OM) of Gram-negative bacteria. They have been proposed to possess several biological roles in pathogenesis and interbacterial interactions. Additionally, OMVs have been suggested as potential vaccine candidates against infections caused by pathogenic bacteria like Haemophilus influenzae, a human pathogen of the respiratory tract. Unfortunately, there is still a lack of fundamental knowledge regarding OMV biogenesis, protein sorting into OMVs, OMV size and quantity, as well as OMV composition in H. influenzae. Thus, this study comprehensively characterized and compared OMVs and OMs derived from heterologous encapsulated as well as nonencapsulated H. influenzae strains. Semiquantitative immunoblot analysis revealed that certain OM proteins are enriched or excluded in OMVs suggesting the presence of regulated protein sorting mechanisms into OMVs as well as interconnected OMV biogenesis mechanisms in H. influenzae. Nanoparticle tracking analysis, transmission electron microscopy, as well as protein and lipooligosaccharide quantifications demonstrated that heterologous H. influenzae strains differ in their OMV size and quantity. Lipidomic analyses identified palmitic acid as the most abundant fatty acid, while phosphatidylethanolamine was found to be the most dominant phospholipid present in OMVs and the OM of all strains tested. Proteomic analysis confirmed that H. influenzae OMVs contain vaccine candidate proteins as well as important virulence factors. These findings contribute to the understanding of OMV biogenesis as well as biological roles of OMVs and, in addition, may be important for the future development of OMV based vaccines against H. influenzae infections.

Lipid composition of human low-density lipoprotein (LDL) and its physicochemical characteristics are relevant for proper functioning of lipid transport in the blood circulation. To explore dynamical and structural features of LDL particles with either a normal or a triglyceride-rich lipid composition we combined coherent and incoherent neutron scattering methods. The investigations were carried out under high hydrostatic pressure (HHP), which is a versatile tool to study the physicochemical behavior of biomolecules in solution at a molecular level. Within both neutron techniques we applied HHP to probe the shape and degree of freedom of the possible motions (within the time windows of 15 and 100 ps) and consequently the flexibility of LDL particles. We found that HHP does not change the types of motion in LDL, but influences the portion of motions participating. Contrary to our assumption that lipoprotein particles, like membranes, are highly sensitive to pressure we determined that LDL copes surprisingly well with high pressure conditions, although the lipid composition, particularly the triglyceride content of the particles, impacts the molecular dynamics and shape arrangement of LDL under pressure.

The aim of the present study was the in vivo evaluation of thiomer-coated liposomes for an oral application of peptides. For this purpose, salmon calcitonin was chosen as a model drug and encapsulated within liposomes. Subsequently, the drug loaded liposomes were coated with either chitosan–thioglycolic acid (CS–TGA) or an S-protected version of the same polymer (CS–TGA–MNA), leading to an increase in the particle size of about 500 nm and an increase in the zeta potential from approximately − 40 mV to a maximum value of about + 44 mV, depending on the polymer. Coated liposomes were demonstrated to effectively penetrate the intestinal mucus layer where they came in close contact with the underlying epithelium. To investigate the permeation enhancing properties of the coated liposomes ex vivo, we monitored the transport of fluoresceinisothiocyanate-labeled salmon calcitonin (FITC-sCT) through rat small intestine. Liposomes coated with CS–TGA–MNA showed the highest effect, leading to a 3.8-fold increase in the uptake of FITC-sCT versus the buffer control. In vivo evaluation of the different formulations was carried out by the oral application of 40 μg of sCT per rat, either encapsulated within uncoated liposomes, CS–TGA-coated liposomes or CS–TGA–MNA-coated liposomes, or given as a solution serving as negative control. The blood calcium level was monitored over a time period of 24 h. The highest reduction in the blood calcium level, to a minimum of 65% of the initial value after 6 h, was achieved for CS–TGA–MNA-coated liposomes. Comparing the areas above curves (AAC) of the blood calcium levels, CS–TGA–MNA-coated liposomes led to an 8.2-fold increase compared to the free sCT solution if applied orally in the same concentration. According to these results, liposomes coated with S-protected thiomers have demonstrated to be highly valuable carriers for enhancing the oral bioavailability of salmon calcitonin., Graphical abstract

Soluble proteins are often highly unstable under mixing conditions that involve dynamic contacting between the main liquid phase and a gas phase. The recombinant human growth hormone (rhGH) was recently shown to undergo aggregation into micrometer-sized solid particles composed of non-native (mis- or unfolded) protein, once its solutions were stirred or shaken to generate a continuously renewed air-water interface. To gain deepened understanding and improved quantification of the air-water interface effect on rhGH stability, we analyzed the protein's aggregation rate (r(agg)) at controlled specific air-water surface areas (a(G/L)) established by stirring or bubble aeration. We show that in spite of comparable time-averaged values for a(G/L) (≈ 100 m(2)/m(3)), aeration gave a 40-fold higher r(agg) than stirring. The enhanced r(agg) under aeration was ascribed to faster macroscopic regeneration of free a(G/L) during aeration as compared to stirring. We also show that r(agg) was independent of the rhGH concentration in the range 0.67 - 6.7 mg/mL, and that it increased linearly dependent on the available a(G/L). The nonionic surfactant Pluronic F-68, added in 1.6-fold molar excess over rhGH present, resulted in complete suppression of r(agg). Foam formation was not a factor influencing r(agg). Using analysis by circular dichroism spectroscopy and small-angle X-ray scattering, we show that in the presence of Pluronic F-68 under both stirring and aeration, the soluble protein retained its original fold, featuring native-like relative composition of secondary structural elements. We further provide evidence that the efficacy of Pluronic F-68 resulted from direct, probably hydrophobic protein-surfactant interactions that prevented rhGH from becoming attached to the air-water interface. Surface-induced aggregation of rhGH is suggested to involve desorption of non-native protein from the air-water interface as the key limiting step. Proteins or protein aggregates released back into the bulk liquid appear to be essentially insoluble.

Objective Obesity-related immune mediated systemic inflammation was associated with the development of the metabolic syndrome by induction of the tryptophan (TRP)–kynurenine (KYN) pathway. The study aimed to assess whether this holds true across the lifespan from juvenility to adulthood. Design and Methods Five hundred twenty-seven participants aged between 10 and 65 years were analyzed. Standard anthropometric measures, carotid ultrasound, and laboratory analysis including interleukin-6, ultra-sensitive C-reactive protein, lipids, glucose metabolism, neopterin, TRP, KYN levels, and the KYN/TRP ratio were performed. Results Overweight/obese (ow/ob) adults had significantly increased KYN serum levels and a significantly increased KYN/TRP ratio. In sharp contrast, ow/ob juvenile males aged ≤18 years showed decreased, females similar KYN and KYN/TRP ratio in comparison to their control counterparts. Also, adult ow/ob subjects with metabolic syndrome showed markedly increased KYN/TRP ratios contrary to decreased KYN/TRP ratios in ow/ob juveniles. Abdominal fat content, characterized by age normalized waist circumference, and not body mass index, had the strongest effect for an increase of the KYN/TRP ratio in adults. Conclusions TRP metabolism and obesity-related immune mediated inflammation differs markedly between juveniles and adults. While childhood obesity seems to be dominated by a Th2-driven activation, an accelerated production of Th1-type cytokines may pave the way for later atherosclerotic endpoints.

Aim: We report the physicochemical analysis of nanosystems intended for cardiovascular applications and their toxicological characterization in static and dynamic cell culture conditions. Methods: Size, polydispersity and ζ-potential were determined in 10 nanoparticle systems including liposomes, lipid nanoparticles, polymeric and iron oxide nanoparticles. Nanoparticle effects on primary human endothelial cell viability were monitored using real-time cell analysis and live-cell microscopy in static conditions, and in a flow model of arterial bifurcations. Results & conclusions: The majority of tested nanosystems were well tolerated by endothelial cells up to the concentration of 100 μg/ml in static, and up to 400 μg/ml in dynamic conditions. Pilot experiments in a pig model showed that intravenous administration of liposomal nanoparticles did not evoke the hypersensitivity reaction. These findings are of importance for future clinical use of nanosystems intended for intravascular applications.

Vasoactive intestinal peptide (VIP) conveys various physiological effects in the digestive tract, nervous and cardiovascular system, airways, reproductive system, endocrine system, and more. A family of specific membrane bound receptors, termed VPAC1, VPAC2, and PAC1, bind VIP and trigger the effects. Many of them are of clinical interest. To date more than two thousand publications suggest the use of VIP in diseases like asthma, erectile dysfunction, blood pressure regulation, inflammation, endocrinology, tumours, etc. Despite this considerable potential, the peptide is not regularly used in clinical settings. A key problem is the short half life of inhaled or systemically administered VIP due to rapid enzymatic degradation. This shortcomings could be overcome with stable derivates or improved pharmacokinetics. A promising strategy is to use biocompatible and degradable depots, to protect the peptide from early degradation and allow for controlled release. This review focuses on aspects of clinical applications of VIP and the idea to use formulations based on biodegradable particles, to constitute a dispersible VIP-depot. Smart particle systems protect the peptide from early degradation, and assist the sustainable cell targeting with VIP for therapeutic or imaging purposes.

Biological membranes can be targets for compounds that either disrupt their barrier function or affect protein function via membrane-mediated processes. Biophysical studies on membrane-mimetic systems composed of membrane lipids have contributed substantially to our knowledge on the pertaining membrane physics and aid the development of membrane-specific drugs. Moreover, lipid membranes and, in particular, liposomes are convenient systems for drug delivery. We review some of our recent work that demonstrates the applicability of X-ray scattering to understanding the molecular mechanisms of drug-membrane interactions. In particular we focus on effects of anesthetics, sphingomyelinase, and antimicrobial peptides. We further discuss X-ray scattering as a quality-control tool for liposomal drug-delivery systems.

The effect of protamine on the solubility and deamidation of human growth hormone (hGH) was investigated. Protamine is an extremely basic peptide. It is isolated from sperm cells of salmon where it can build a complex with DNA due to electrostatic interactions. We hypothesize a similar electrostatic effect between negatively charged hGH and positively charged protamine residues. Arising cationic complexes are stabilized by electrostatic repulsion. This stabilizing complexation allows solubilization of hGH down to a pH of 5.4 (pI 5.3) at concentrations of 3.4 mg/ml. The minimal solubilizing molar ratio between hGH and protamine was found to be at least 1:23 (hGH:protamine) by turbidity and dynamic light scattering (DLS) measurements. Complexation was characterized by small-angle X-ray scattering (SAXS) and isothermal titration calorimetry (ITC). Electrostatic binding of protamine to hGH was also observed by a reversal in surface charge, as shown by zeta potential measurements. The presence of protamine did not alter the conformational structure of hGH which was determined by circular dichroism (CD) spectroscopy. A pH of 5.4 is known to slow deamidation of hGH and consequently retardation of hGH deamidation could be detected after 3 months storing by reversed-phase high-performance liquid chromatography (RP-HPLC).

Daniela Frascione,1 Clemens Diwoky,2 Gunter Almer,1,3 Peter Opriessnig,2 Caroline Vonach,1 Kerstin Gradauer,1 Gerd Leitinger,4 Harald Mangge,3 Rudolf Stollberger,2 Ruth Prassl1,51Institute of Biophysics and Nanosystems Research, Austrian Academy of Sciences, Graz, Austria; 2Institute of Medical Engineering, University of Technology, Graz, Austria; 3Clinical Institute for Medical and Chemical Laboratory Diagnosis (CIMCL), Medical University, Graz, Austria; 4Institute of Cell Biology, Histology and Embryology, Medical University, Graz, Austria; 5Ludwig Boltzmann Institute for Lung Vascular Research, Graz, AustriaBackground: Magnetic liposomes (MLs) are phospholipid vesicles that encapsulate magnetic and/or paramagnetic nanoparticles. They are applied as contrast agents for magnetic resonance imaging (MRI). MLs have an advantage over free magnetic nanocores, in that various functional groups can be attached to the surface of liposomes for ligand-specific targeting. We have synthesized PEG-coated sterically-stabilized magnetic liposomes (sMLs) containing ultrasmall superparamagnetic iron oxides (USPIOs) with the aim of generating stable liposomal carriers equipped with a high payload of USPIOs for enhanced MRI contrast.Methods: Regarding iron oxide nanoparticles, we have applied two different commercially available surface-coated USPIOs; sMLs synthesized and loaded with USPIOs were compared in terms of magnetization and colloidal stability. The average diameter size, morphology, phospholipid membrane fluidity, and the iron content of the sMLs were determined by dynamic light scattering (DLS), transmission electron microscopy (TEM), fluorescence polarization, and absorption spectroscopy, respectively. A colorimetric assay using potassium thiocyanate (KSCN) was performed to evaluate the encapsulation efficiency (EE%) to express the amount of iron enclosed into a liposome. Subsequently, MRI measurements were carried out in vitro in agarose gel phantoms to evaluate the signal enhancement on T1- and T2-weighted sequences of sMLs. To monitor the biodistribution and the clearance of the particles over time in vivo, sMLs were injected in wild type mice.Results: DLS revealed a mean particle diameter of sMLs in the range between 100 and 200 nm, as confirmed by TEM. An effective iron oxide loading was achieved just for one type of USPIO, with an EE% between 74% and 92%, depending on the initial Fe concentration (being higher for lower amounts of Fe). MRI measurements demonstrated the applicability of these nanostructures as MRI probes.Conclusion: Our results show that the development of sMLs is strictly dependent on the physicochemical characteristics of the nanocores. Once established, sMLs can be further modified to enable noninvasive targeted molecular imaging.Keywords: magnetic liposomes, fluorescence polarization, biodistribution, MRI contrast agent&nbsp

Radiolabeled PEGylated liposomal nanoparticles (NPs) open new possibilities for a variety of applications including diagnosis, drug delivery, targeted therapy, and monitoring treatment effects. Here we describe the characterization of liposomal NPs (liposomes and micelles) derivatized with the somatostatin analogue tyrosine-3-octreotide as a proof of concept for tumor targeting. NPs were radiolabeled with indium-111, and targeting properties were evaluated in vitro on rat pancreatic tumor cells (AR42J), demonstrating specific binding and IC50 values in the low nanomolar range. Biodistribution studies were performed in Lewis rats and compared to single-photon emission computed tomography images. Moderate tumor uptake was found in xenografted nude mice (b2.5% ID/g tissue) as compared to control. Micelles and liposomes revealed comparable pharmacokinetics and targeting properties. This study provides insight into tumor-targeting characteristics of peptide-derivatized liposomal NPs and can serve as a basis for further improvement of these constructs. From the Clinical Editor: The authors investigated tumor-targeting characteristics of peptide-derivatized liposomal NPs. Similar radiolabeled PEGylated liposomal NPs open new possibilities for a variety of applications including diagnosis, drug delivery, targeted therapy, and treatment monitoring.

Purpose New evidence suggests that the anesthetic effect of parenteral propofol emulsions varies between commercial preparations. We examined and compared different propofol preparations to determine propofol release and binding capacity with regard to plasma lipoproteins and albumin. Methods We created a novel assay consisting of microtiter plates coated with either low-density lipoprotein (LDL) or albumin to analyze propofol binding kinetics. Using high performance liquid chromatography (HPLC), we measured propofol release from the oily phase and the corresponding amount of propofol bound to the plates in a time-dependent manner and at equilibrium conditions attained after 30 min of incubation at 37 °C. The concentrations of free propofol in the aqueous phase of different propofol preparations – Diprivan, and the generic formulations Propofol “Fresenius” (1% and 2% propofol) and Propofol-Lipuro – were analyzed using ultracentrifugation or dialysis for phase separation. Finally, we investigated the effect of isolated lipoprotein fractions on propofol release. Results Propofol bound to LDL-coated plates with approximately twofold higher affinity than to albumin-coated plates. No significant differences in total propofol release were observed between preparations. Moreover, similar amounts of free propofol were observed in the aqueous phase of all products tested (1% propofol preparations: 18 μg/ml; 2% propofol preparations: 35 μg/ml), except for the medium-chain and long-chain triglyceride (MCT/LCT) preparation studied, in which the concentration of free propofol was lower. Lipoproteins had no effect on propofol release, except for high-density lipoprotein (HDL), which triggered almost 100% release from the oily phase at HDL concentrations above 1000 μg/ml. Conclusions No differences were observed between the binding/release capacity and lipoprotein interactions of any of the propofol preparations tested. We propose that clinical observations of inconsistent propofol activity are related to variations in the lipoprotein profile, enzyme activity or genetic disorders of individual patients, rather than to the propofol preparation itself.

Hierarchical self-assembly is a fundamental principle in nature, which gives rise to astonishing supramolecular architectures that offer an inspiration for the development of innovative materials in nanotechnology. Here we present the unique structure of a cone-shaped amphiphilic designer peptide. When tracking its concentration-dependent morphologies, we observed elongated bilayered single tapes at the beginning of the assembly process, which further developed into novel double-helix-like superstructures at increased concentrations. This architecture is characterized by a tight intertwisting of two individual helices, resulting in a periodic pitch size over their total lengths of several hundred nanometers. Solution X-ray scattering data revealed a marked 2-layered internal organization. All these characteristics remained unaltered for the investigated period of almost three months. In their collective morphology the assemblies are integrated into a network with hydrogel characteristics. Such a peptide based structure holds promise for a building block of next-generation nanostructured biomaterials.

Being intimately involved in cholesterol transport and lipid metabolism human low density lipoprotein (LDL) plays a prominent role in atherogenesis and cardiovascular diseases. The receptor-mediated cellular uptake of LDL is triggered by apolipoprotein B-100 (apoB-100), which represents the single protein moiety of LDL. Due to the size and hydrophobic nature of apoB-100, its structure is not well characterized. Here we present a low resolution structure of solubilized apoB-100. We have used small angle neutron scattering in combination with advanced shape reconstruction algorithms to generate a three-dimensional model of lipid-free apoB-100. Our model clearly reveals that apoB-100 is composed of distinct domains connected by flexible regions. The apoB-100 molecule adopts a curved shape with a central cavity. In comparison to LDL-associated apoB-100, the lipid-free protein is expanded, whereas according to spectroscopic data the secondary structure is widely preserved. Finally, the low resolution model was used as a template to reconstruct a hypothetical domain organization of apoB-100 on LDL, including information derived from a secondary structure prediction.

In this paper, a ternary system of albumin-protamine-oligonucleotide nanoparticles (AlPrO-NP) recently developed by Vogel et al. [V. Vogel, D. Lochmann, J. Weyermann, G. Mayer, C. Tziatzios, J.A. van den Broek, W. Haase, D. Wouters, U.S. Schubert, J. Kreuter, A. Zimmer, D. Schubert, Oligonucleotide-protamine-albumin nanoparticles: preparation, physical properties and intracellular processing, J. Controlled Rel. (in press)] which could serve as a potential drug delivery system for antisense oligonucleotides. Former studies of binary protamine-oligonucleotide nanoparticles showed two main disadvantages: (i) aggregation of the particles within a few minutes in the presence of salt; (ii) low intracellular dissociation between protamine and oligonucleotide, especially phosphorothioates. To overcome these problems, human serum albumin (HSA) as a non-toxic, biodegradable macromolecule was introduced as protective colloid. The assembly process of AlPrO-NP was investigated by small angle X-ray scattering (SAXS), fluorescence correlation spectroscopy (FCS), photon correlation spectroscopy (PCS) measurements and scanning electron microscopy (SEM). 'Initial complexes' of HSA and protamine sulphate with a mean hydrodynamic diameter (dh) of about 10-14 nm were found. After adding oligonucleotides (unmodified, phosphorothioate DNA and small interfering RNA), nanoparticles (NPs) were assembled in water and in isotonic media with a dh in a range of 230-320 nm for most preparations. The chemical composition of the particles was investigated by high performance liquid chromatography and fluorescence spectrometry. The whole amount of oligonucleotides (30 microg) was entrapped into the particles at a 1:2 mass ratio (oligonucleotide/protamine). Approximately 7-10% (w/w) of the HSA was bound to the particles. The surface charge of the particles ranged from about +12 to -60 mV depending on the protamine concentration and the ionic conditions. The size and the molecular weight of the components, initial complexes and two model NP preparations were calculated from FCS data. These data verified the PCS, SEM and SAXS measurements.

Mammalian lipoxygenases form a heterogeneous family of lipid peroxidizing enzymes, which have been implicated in synthesis of inflammatory mediators, in cell development and in the pathogenesis of various diseases (atherosclerosis, osteoporosis) with major health political importance. The crystal structures of two plant lipoxygenase isoforms have been solved and X-ray coordinates for an inhibitor complex of the rabbit 15-lipoxygenase-1 are also accessible. Here, we investigated the solution structure of the ligand-free rabbit 15-lipoxygenase-1 by small angle X-ray scattering. From the scattering profiles we modeled the solution structure of the enzyme using two independent ab initio approaches. Preliminary experiments indicated that at low protein concentrations (1mg/ml) and at 10 degrees C the enzyme is present as hydrated monomer. Superposition of the high resolution crystal structure and our low resolution model of the solution structure revealed two major differences. (i) Although the two models are almost perfectly superimposed in the region of the catalytic domain the solution structure is stretched out in the region of the N-terminal beta-barrel domain and exhibits a bigger molecular volume. (ii) There is a central bending of the enzyme molecule in the solution structure, which does not show up in the crystal structure. Both structural peculiarities may be explained by a high degree of motional freedom of the N-terminal beta-barrel domain in aqueous solutions. This interdomain movement may be of functional importance for regulation of the catalytic activity and membrane binding.

Low-density lipoprotein (LDL) is the principal cholesterol transporter in the human blood circulation. The quasi-spherical LDL particles (∼20 nm) are made up of a complex combination of various lipids and a large single amphipathic protein moiety, named apolipoprotein B-100. LDL has a hydrophobic core and an amphiphilic shell. Each particle has a specific phase transition temperature (Tm) corresponding to the melting of the core lipids from an ordered liquid crystalline to a disordered fluid phase.The structural impact of high hydrostatic pressure (HHP) was studied with different types of LDL (native, oxidized and triglyceride rich) with SANS (PSI, Switzerland) and SAXS (Elettra, Italy). The HHP ranged from 50 to 3000 bar. Temperature points were chosen below, on and above Tm.The pair distance distribution functions p(r) of the SAS curves revealed pressure dependent changes of the particle structure seen in the low q-region (∼0.25 nm−1), also reflected by a decrease of Radii of Gyration (Rg) with increasing pressure.Especially the p(r) functions from the SAXS data do not only show an overall particle change but also a highly pressure sensitive inner organization. A triple-peak feature indicating the lamellar lipid organization below Tm was induced by raising the pressure up to 3000 bar. These pressure sensitive lipid layers were observed by scattering intensity changes in SAXS curves at q=1.7 nm−1.The shape alterations could be evidenced by fitting an ellipsoidal model to the SANS curves resulting in a decrease of the ellipsoidal radii under pressure. A new LDL model considering the cross-sectional electron density profile is developed and applied to the SAS data to get a more detailed insight into pressure dependent behavior.

The potential role of human low density lipoprotein (LDL) particles as delivery system for lipophilic, cytotoxic drugs critically depends on their structural integrity. In the present study, LDL particles were loaded with antineoplastic prodrugs, i.e. monooleoyl (MOT)- and dioleoyl (DOT)- thymidine esters by different techniques. Using the reconstitution method MOT shows the highest incorporation efficiency with over 80% of the initial drug associated with LDL. In contrast, for the more lipophilic DOT the incorporation efficiency for reconstitution, dry film as well as dimethylsulfoxide method was extremely low. Structural changes upon drug loading were monitored by differential scanning calorimetry (DSC) and small angle X-ray scattering (SAXS). The results show that the influence of MOT and DOT is predominantly confined to the surface monolayer of LDL seen as a destabilisation of the protein moiety and a small increase in particle diameter. The core lipid region of the LDL-drug complexes remains essentially unaffected, as verified by undisturbed core lipid arrangement and core lipid melting behaviour.

This special issue of the European Biophysics Journal marks the contributions of Peter Laggner to molecular biophysics and X-ray and neutron scattering techniques on the occasion of his 68th birthday. Actually, Peter Laggner is not a person who likes to dwell in the past, or talk of the good old days. ‘‘Why look back, if one can look forward?’’, is one of his most popular quotes. Well, we hope that he will forgive us on this one occasion. So, let us look back and recall some of the past from a rich life with many things to remember, but, to make it bearable, we will use an element of humor. Born in Piberbach, Upper Austria, Peter went to Graz to study chemistry and physics. During his PhD, he started with his so successful combination of small-angle X-ray scattering (SAXS), which he learned from the Austrian pioneer in SAXS, Otto Krakty, and biophysics, taught to him by Anton Holasek, then head of the Medical Biochemistry Institute, University of Graz. For his thesis on ‘‘Structure of Antigen Antibody Complexes in Solution by SAXS’’ Peter had an urgent need for blood. Therefore, this part of his career truly was bloody. Using his own car he would go to the slaughterhouse to fetch swine blood in buckets and then drive back to his laboratory. Occasionally blood spilled over. Who cares? In the laboratory the tedious job of precipitation and centrifugation awaited him, accompanied by anxious moments when the rotor lifted and the centrifuge started to move around. By dawn his eyes were blood-shot, but the proteins were prepared and ‘‘real science’’ could start. Soon afterwards lipoproteins—high-density (HDL) and low-density lipoproteins (LDL)—caught Peter’s scientific interest. Again, blood had to be isolated, and trouble started from the beginning. This time the blood had to be of human origin. Thus, healthy normolipidemic volunteers were required. By chance, PhD students were ‘‘lucky’’ to act as donors for Peter’s blood pool, but the most fascinating thing was, however, to use one’s own blood, as he always told us. Later, lipoprotein subspecies came in from John Chapman in Paris, making life much easier. However, whenever whole blood was harvested in our laboratory, Peter was among the first to put his name on the list of donors, eager to support our common efforts to solve the structure of LDL. One day, early in the morning, Peter arrived at the laboratory with a bottle of warm blood in his hands and laughed, ‘‘Hey girls, I have been at the doctor and thought I could bring you some fresh blood!’’, remembers Ruth Prassl. Peter’s pioneering work on lipoproteins has significantly shaped our current understanding of lipoprotein structure and dynamics. In addition to his research focus on LDL, Peter devoted a large part of his endeavor towards understanding complex biological membranes. Early on, he considered the membrane as a dynamic entity with spatial and temporal fluctuations in its local composition. So, he initiated structural studies on lipid polymorphism and domain formation, long before the term ‘‘membrane rafts’’ became popular. A particularly important study concerns the demonstration of chain interdigitated bilayers in ether-chain lipid membranes. He also pioneered lipid interactions with membrane-active peptides, showing that melittin from bee venom acts on membrane collective properties at concentrations as low as 1/1,000 (peptide/lipid molar ratio). G. Pabst (&) R. Prassl H. Amenitsch M. Rappolt K. Lohner Institute of Biophysics and Nanosystems Research, 8042 Graz, Austria e-mail: georg.pabst@oeaw.ac.at

beta(2)-Glycoprotein I (beta(2)GPI) is a highly glycosylated phospholipid-binding plasma protein comprised of four complement control protein (CCP) domains and a distinct fifth domain. The structural organisation of human and bovine beta(2)GPI in aqueous solution was studied by small-angle X-ray scattering (SAXS). Low-resolution models that match the SAXS experimental data best were independently constructed by three different ab initio 3D-reconstruction algorithms. Similar elongated S-shaped models with distinct side-arms, which were correlated to the position of the carbohydrate chains, were restored from all three algorithms. Due to an additional glycosylation site located on the CCP2 domain of bovine beta(2)GPI a small change in the characteristic SAXS parameters was observed, which coincided with results obtained from SDS-PAGE. In comparison to the human analogue the corresponding restored low-resolution models displayed a similar S-shape with less bending in the middle part. As the experimental SAXS curves fit poorly to the simulated scattering curves calculated from the crystallographic coordinates of human beta(2)GPI, the crystal structure was modified. First, additional carbohydrate residues missing from the crystal structure were modelled. Second, on the basis of the low-resolution models, the J-shaped crystal structure was rotated between CCP3 and CCP2 assuming the greatest interdomain flexibility between these domains. An S-shaped model with a tilt angle of approximately 60 degrees between CCP3 and CCP2 yielded the best fit to the experimental SAXS data. Since there is evidence that beta(2)GPI can adopt different conformations, which reveal distinct differences in autoantibody recognition, our data clearly point to a reorientation of the flexible domains, which may be an essential feature for binding of autoantibodies.

Lamellar lipid structures are the fundamental structural motifs of biological membranes. To mimic this feature we have engineered short amphiphilic designer peptides with the aim to create peptide-based membrane analogs. As peptide scaffolds we have chosen six derivatives of V4WD2 - a peptide composed of four hydrophobic valine residues, followed by tryptophan and two negatively charged hydrophilic aspartic acid residues. The hydrophile-lipophile ratio, tryptophan content, and level of hydrophobicity have been systematically varied.