Identification of extracellular nanoparticle subsets by nuclear magnetic resonance

Exosomes are a subset of secreted lipid envelope-encapsulated extracellular vesicles (EVs) of 50–150 nm diameter that can transfer cargo from donor to acceptor cells. In the current purification protocols of exosomes, many smaller and larger nanoparticles such as lipoproteins, exomers and microvesicles are typically co-isolated as well. Particle size distribution is one important characteristics of EV samples, as it reflects the cellular origin of EVs and the purity of the isolation. However, most of the physicochemical analytical methods today cannot illustrate the smallest exosomes and other small particles like the exomers. Here, we demonstrate that diffusion ordered spectroscopy (DOSY) nuclear magnetic resonance (NMR) method enables the determination of a very broad distribution of extracellular nanoparticles, ranging from 1 to 500 nm. The range covers sizes of all particles included in EV samples after isolation. The method is non-invasive, as it does not require any labelling or other chemical modification. We investigated EVs secreted from milk as well as embryonic kidney and renal carcinoma cells. Western blot analysis and immuno-electron microscopy confirmed expression of exosomal markers such as ALIX, TSG101, CD81, CD9, and CD63 in the EV samples. In addition to the larger particles observed by nanoparticle tracking analysis (NTA) in the range of 70–500 nm, the DOSY distributions include a significant number of smaller particles in the range of 10–70 nm, which are visible also in transmission electron microscopy images but invisible in NTA. Furthermore, we demonstrate that hyperpolarized chemical exchange saturation transfer (Hyper-CEST) with ¹²⁹Xe NMR indicates also the existence of smaller and larger nanoparticles in the EV samples, providing also additional support for DOSY results. The method implies also that the Xe exchange is significantly faster in the EV pool than in the lipoprotein/exomer pool.

Diffusion and xenon NMR based methods to determine a very broad range of sizes and sub-sets of extracellular vesicles.     

In-beam spectroscopy of 253, 254No

In-beam conversion electron spectroscopy experiments have been performed on the transfermium nuclei ^{253, 254}No using the conversion electron spectrometer SACRED in nearly collinear geometry in conjunction with the gas-filled separator RITU at the University of Jyv?skyl?. The experimental setup is discussed and the spectra are compared to Monte Carlo simulations. The implications for the ground-state configuration of ²⁵³No are discussed. Received: 21 March 2002 / Accepted: 16 May 2002 / Published online: 31 October 2002 RID="a" ID="a"e-mail: rdh@ns.ph.liv.ac.uk RID="b" ID="b"Present address: GANIL, F-14021 Caen, France. RID="c" ID="c"Permanent address: IReS Strasbourg, IN2P3-CNRS, F-67037-Strasbourg, France. RID="d" ID="d"Present address: CEA/DIF DCRE/SDE/LDN F-91680 Bruyeres-le-Chatel. RID="e" ID="e"Present address: Daresbury Laboratory, Daresbury WA4 4AD, UK. RID="f" ID="f"Permanent address: IPN Lyon, IN2P3-CNRS, F-69037 Lyon, France.     

Polypropylene/organoclay nanocomposites compatibilized with hydroxyl‐functional polypropylenes

The properties of polypropylene composites can be tailored through the use of nanoclay fillers. The effectiveness of a metallocene‐catalyzed hydroxyl‐functional polypropylene in the compatibilization of polypropylene layered nanosilicate composites was studied, and the results were compared with those for a commercial maleic anhydride functionalized polypropylene. Polypropylene/organoclay nanocomposites were prepared by melt blending, and two polypropylene/compatibilizer/organoclay ratios, 90/5/5 and 70/20/10, were characterized. The organomodification of the clay was carried out with octadecylamine and N‐methylundecenylamine. The structure of the layered silicate was studied by transmission electron microscopy, wide‐angle X‐ray scattering, and small‐angle X‐ray scattering. The fracture surfaces of the composites and thus the efficiency of the compatibilizers to penetrate the galleries of the organoclays were characterized by scanning electron microscopy, and the melt viscosity was studied by stress‐controlled rotational rheometry. The nanostructure was observed with both alkyl amines used for intercalation. The fillers facilitated the processability of all the composites, consisting of equal amounts of compatibilizer and organoclay filler and, in some of the composites, containing twice as much compatibilizer as organoclay filler. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 1892–1903, 2005     

Cortical sensitivity to periodicity of speech sounds

Previous non-invasive brain research has reported auditory cortical sensitivity to periodicity as reflected by larger and more anterior responses to periodic than to aperiodic vowels. The current study investigated whether there is a lower fundamental frequency (F0) limit for this effect. Auditory evoked fields (AEFs) elicited by natural-sounding 400 ms periodic and aperiodic vowel stimuli were measured with magnetoencephalography. Vowel F0 ranged from normal male speech (113 Hz) to exceptionally low values (9 Hz). Both the auditory N1m and sustained fields were larger in amplitude for periodic than for aperiodic vowels. The AEF sources for periodic vowels were also anterior to those for the aperiodic vowels. Importantly, the AEF amplitudes and locations were unaffected by the F0 decrement of the periodic vowels. However, the N1m latency increased monotonically as F0 was decreased down to 19 Hz, below which this trend broke down. Also, a cascade of transient N1m-like responses was observed in the lowest F0 condition. Thus, the auditory system seems capable of extracting the periodicity even from very low F0 vowels. The behavior of the N1m latency and the emergence of a response cascade at very low F0 values may reflect the lower limit of pitch perception.     

Proton double quantum coherence and cross-relaxation in (NH4)2SnBr6

A proton double quantum coherence signal can be observed exclusively from the T species NH4 groups (with the total spin I = 1) in ammonium compounds at low temperatures by the three-pulse sequence 90x degrees - tpr - 90x degrees - tev - 90x degrees - ta, where tpr and ta are of the magnitude of the inverse line width and tev very short. The usefulness of this pulse sequence, preceded by the additional pulse sequence 90x degrees - t1 -90(-x) degrees - t2 for creating an unbalance between the A and T species magnetizations, was demonstrated by applying it to cross-relaxation studies in (NH4)2SnBr6.     

Design and fabrication of integrated solid-phase extraction-zone electrophoresis microchip

Integrated solid-phase extraction-zone electrophoresis (SPE-ZE) device has been designed and fabricated on microchip. The structures were fabricated by using multiple layers of SU-8 polymer with a novel technique that enables easy alignment and high yield of the chips. SU-8 adhesive bonding has two major advantages: it enables bonding of high aspect ratio pillars and it results in fully SU-8 microchannels with uniform electrokinetic flow properties. The SPE-ZE device has a fluidic reservoir with 15:1 high aspect ratio pillars for bead filters that act as a SPE part in the chip structure. The separation unit is a 25 mm long electrophoresis channel starting from the outlet of SPE reservoir. Argon laser-induced fluorescence (LIF) detector was used to monitor simultaneously the SPE reservoir and the detection site at the end of the electrophoresis channel. Flow characteristics and electric field distributions were simulated with Femlab software. Fluorescein was used as the analyte for detecting the operational performance of the chip. Adsorption, bead rinsing, elution and detection were tested to verify functioning of the chip design.     

Spectral spin diffusion and magnetic dipolar energy in the NMR of 13CH3 compounds

Spin diffusion between ¹³CH₃ groups in solids is studied both theoretically and experimentally. It is shown to be dominated by mutual spin flip–flops of protons belonging to neighbouring methyl groups. Also nonmethyl protons may contribute significantly if present in the sample. The spin–rotational ground state of ¹³CH₃ consists of 16 sublevels. When their populations are used to describe spin diffusion, eight population combinations are shown to be important, two of them corresponding to the ¹³C–proton and proton–proton intra-methyl magnetic dipolar energies, D_c and D_p, respectively. Spin-diffusion transitions modulate these combinations so that a further reduction to two sets of four combinations is possible, with no coupling between the sets. Coupled differential equations are derived to describe the time dependence of the combinations in each set. They are solved numerically and compared with experimental results on a single crystal of aspirin with ¹³C-labelled methyl groups at the carbon resonance. The ¹³C NMR induction signal was observed as a function of time after the preparation either at the carbon resonance (a two-pulse sequence) or at the proton resonance (proton saturation). Usually carbon spectra were computed first and then three of the mentioned population combinations were obtained from the individual spectral components. Some results on the time dependence of D_c were also obtained directly from the amplitude of the out-of-phase induction signal. Theoretical predictions are found to describe semiquantitatively the overall time dependence of these three combinations and especially their variation with different initial conditions, which are discussed in detail. Also the partial transfer of the magnetic dipolar energy between D_c and D_p is nicely explained. Reasons for discrepancies are discussed.     

On the design of reactive systems

The notion of joint actions provides a framework in which the granularity of atomic actions can be refined in the design of concurrent systems. An example of a telephone exchange is elaborated to demonstrate the feasibility of this approach for reactive systems and to illustrate transformations that are justifiable in such a process. Particular problems arise when a refinement would allow new interleavings of semantically relevant events. The meaning of a reactive computation is specified in a way that makes this possible.Dedicated to Peter Naur on the occasion of his 60th birthday     

In-beam conversion electron spectroscopy using the SACRED array

Conversion electron studies of medium-heavy to heavy nuclear mass systems are important where the internal conversion process begins to dominate over gamma-ray emission. The use of a segmented detector array sensitive to conversion electrons has been used to study multiple conversion electron cascades from nuclear transitions. The application of the Silicon Array for ConveRsion Electron Detection (SACRED) for in-beam measurements has successfully been implemented.