IntroductionExtracellular vesicles (EV) are phospholipid bilayer-enclosed vesicles recognized as new mediators in intercellular communication and potential biomarkers of disease. They are found in many body fluids and mainly studied in fractions isolated from blood plasma in view of their potential in medicine. Due to the limitations of available analytical methods, morphological information on EV in fresh plasma is still rather limited.ObjectivesTo image EV and determine the morphology, structure and size distribution in fresh plasma by cryo-electron microscopy (cryo-EM).MethodsFresh citrate- and ethylenediaminetetraacetic acid (EDTA)-anticoagulated plasma or EV isolated from these plasmas were rapidly cryo-immobilized by vitrification and visualized by cryo-EM.ResultsEV isolated from fresh plasma were highly heterogeneous in morphology and size and mostly contain a discernible lipid bilayer (lipid vesicles). In fresh plasma there were 2 types of particles with a median diameter of 30 nm (25–260 nm). The majority of these particles are electron dense particles which most likely represent lipoproteins. The minority are lipid vesicles, either electron dense or electron lucent, which most likely represent EV. Lipid vesicles were occasionally observed in close proximity of platelets in citrate and EDTA-anticoagulated platelet-rich plasma. Cryo-electron tomography (cryo-ET) was employed to determine the 3D structure of platelet secretory granules.ConclusionsCryo-EM is a powerful technique that enables the characterization of EV in fresh plasma revealing structural details and considerable morphological heterogeneity. Only a small proportion of the submicron structures in fresh plasma are lipid vesicles representing EV.

DNA and synthetic poly(styrenesulfonate) (PSS) solutions without excess simple salt were investigated with small-angle neutron scattering. For both polyelectrolytes, the transition from the rod to the coil regime was covered by an appropriate choice of molecular weights. The polymer, polymer−counterion, and counterion partial structure functions were obtained using contrast variation. For PSS, the single-chain scattering (form function) was observed from samples with zero-average polyion scattering length density contrast. The PSS polymer structure can be described by a locally rodlike configuration, but the projected monomer repeat distance 0.17 nm is smaller than the value expected for a fully stretched (trans) conformation. The PSS persistence length is of order 10 nm and does not agree with any theoretical analysis based on either the bending rigidity of a wormlike chain or modern variational results. The interpolymer structure was derived and compared with results based on the random-phase approximation. Poor agreement was observed, due to the high linear polyion charge density and, hence, strong electrostatic coupling. For highly charged linear polyelectrolytes, it was shown that from the full set of partial structure functions information on the radial counterion profile can be obtained without resorting to a model describing chain correlations. For PSS and DNA, the data agree with the counterion distribution obtained from the classical Poisson−Boltzmann theory and the cylindrical cell model, if the momentum transfer is far greater than the inverse persistence length.

Microparticles, also known as microvesicles, found in blood plasma, urine, and most other body fluids, may serve as valuable biomarkers of diseases such as cardiovascular diseases, systemic inflammatory disease, thrombosis, and cancer. Unfortunately, the detection and quantification of microparticles are hampered by the microscopic size of these particles and their relatively low abundance in blood plasma. The use of a combination of microfluidics and atomic force microscopy to detect microparticles in blood plasma circumvents both problems. In this study, capture of a specific subset of microparticles directly from blood plasma on antibody-coated mica surface is demonstrated. The described method excludes isolation and washing steps to prepare microparticles, improves the detection sensitivity, and yields the size distribution of the captured particles. The majority of the captured particles have a size ranging from 30 to 90 nm, which is in good agreement with prior results obtained with microparticles immediately isolated from fresh plasma. Furthermore, the qualitative shape of the size distribution of microparticles is shown not to be affected by high-speed centrifugation or the use of the microfluidic circuit, demonstrating the relative stable nature of microparticles ex vivo.Electronic supplementary materialThe online version of this article (doi:10.1007/s10544-012-9642-y) contains supplementary material, which is available to authorized users.

Nucleation is the rate-limiting step in protein crystallization. Introducing heterogeneous substrates may in some cases lower the energy barrier for nucleation and thereby facilitate crystal growth. To date, the mechanism of heterogeneous protein nucleation remains poorly understood. In this study, the nucleating properties of fragments of human hair in crystallization experiments have been investigated. The four proteins that were tested, lysozyme, glucose isomerase, a polysaccharide-specific Fab fragment and potato serine protease inhibitor, nucleated preferentially on the hair surface. Macrocrystals and showers of tiny crystals of a few hundred nanometres thickness were obtained also under conditions that did not produce crystals in the absence of the nucleating agent. Cryo-electron diffraction showed that the nanocrystals diffracted to at least 4 A resolution. The mechanism of heterogeneous nucleation was studied using confocal fluorescent microscopy which demonstrated that the protein is concentrated on the nucleating surface. A substantial accumulation of protein was observed on the sharp edges of the hair's cuticles, explaining the strong nucleating activity of the surface.