Imaging secondary structure of individual amyloid fibrils of a β2-microglobulin fragment using near-field infrared spectroscopy

Amyloid fibril diseases are characterized by the abnormal production of aggregated proteins and are associated with many types of neuro- and physically degenerative diseases. X-ray diffraction techniques, solid-state magic-angle spinning NMR spectroscopy, circular dichroism (CD) spectroscopy, and transmission electron microscopy studies have been utilized to detect and examine the chemical, electronic, material, and structural properties of amyloid fibrils at up to angstrom spatial resolution. However, X-ray diffraction studies require crystals of the fibril to be analyzed, while other techniques can only probe the bulk solution or solid samples. In the work reported here, apertureless near-field scanning infrared microscopy (ANSIM) was used to probe the secondary structure of individual amyloid fibrils made from an in vitro solution. Simultaneous topographic and infrared images of individual amyloid fibrils synthesized from the #21-31 peptide fragment of β(2)-microglobulin were acquired. Using this technique, IR spectra of the amyloid fibrils were obtained with a spatial resolution of less than 30 nm. It is observed that the experimental scattered field spectrum correlates strongly with that calculated using the far-field absorption spectrum. The near-field images of the amyloid fibrils exhibit much lower scattering of the IR radiation at approximately 1630 cm(-1). In addition, the near-field images also indicate that composition and/or structural variations among individual amyloid fibrils were present.     

Activation of α-keto acid-dependent dioxygenases: application of an {FeNO}7/{FeO2}8 methodology for characterizing the initial steps of O2 activation

The α-keto acid-dependent dioxygenases are a major subgroup within the O(2)-activating mononuclear nonheme iron enzymes. For these enzymes, the resting ferrous, the substrate plus cofactor-bound ferrous, and the Fe(IV)═O states of the reaction have been well studied. The initial O(2)-binding and activation steps are experimentally inaccessible and thus are not well understood. In this study, NO is used as an O(2) analogue to probe the effects of α-keto acid binding in 4-hydroxyphenylpyruvate dioxygenase (HPPD). A combination of EPR, UV-vis absorption, magnetic circular dichroism (MCD), and variable-temperature, variable-field (VTVH) MCD spectroscopies in conjunction with computational models is used to explore the HPPD-NO and HPPD-HPP-NO complexes. New spectroscopic features are present in the α-keto acid bound {FeNO}(7) site that reflect the strong donor interaction of the α-keto acid with the Fe. This promotes the transfer of charge from the Fe to NO. The calculations are extended to the O(2) reaction coordinate where the strong donation associated with the bound α-keto acid promotes formation of a new, S = 1 bridged Fe(IV)-peroxy species. These studies provide insight into the effects of a strong donor ligand on O(2) binding and activation by Fe(II) in the α-keto acid-dependent dioxygenases and are likely relevant to other subgroups of the O(2) activating nonheme ferrous enzymes.     

A continuous DC-insulator dielectrophoretic sorter of microparticles

A lab-on-a-chip device is described for continuous sorting of fluorescent polystyrene microparticles utilizing direct current insulating dielectrophoresis (DC-iDEP) at lower voltages than previously reported. Particles were sorted by combining electrokinetics and dielectrophoresis in a 250 μm wide PDMS microchannel containing a rectangular insulating obstacle and four outlet channels. The DC-iDEP particle flow behaviors were investigated with 3.18, 6.20 and 10 μm fluorescent polystyrene particles which experience negative DEP forces depending on particle size, DC electric field magnitude and medium conductivity. Due to negative DEP effects, particles are deflected into different outlet streams as they pass the region of high electric field density around the obstacle. Particles suspended in dextrose added phosphate buffer saline (PBS) at conductivities ranging from 0.50 to 8.50 mS/cm at pH 7.0 were compared at 6.85 and 17.1 V/cm. Simulations of electrokinetic and dielectrophoretic forces were conducted with COMSOL Multiphysics^® to predict particle pathlines. Experimental and simulation results show the effect of medium and voltage operating conditions on particle sorting. Further, smaller particles experience smaller iDEP forces and are more susceptible to competing nonlinear electrostatic effects, whereas larger particles experience greater iDEP forces and prefer channels 1 and 2. This work demonstrates that 6.20 and 10 μm particles can be independently sorted into specific outlet streams by tuning medium conductivity even at low operating voltages. This work is an essential step forward in employing DC-iDEP for multiparticle sorting in a continuous flow, multiple outlet lab-on-a-chip device.     

Identification of Hypoxic Cells Using an Organotellurium Tag Compatible with Mass Cytometry

Mass cytometry (MC) offers unparalleled potential for the development of highly parameterized assays for characterization of single cells within heterogeneous populations. Current reagents compatible with MC analysis employ antibody‐metal‐chelating polymer conjugates to report on the presence of biomarkers. Here, we expand the utility of MC by developing the first activity‐based probe designed specifically for use with the technology. A compact MC‐detectable telluroether is linked to a bioreductively sensitive 2‐nitroimidazole scaffold, thereby generating a probe sensitive to cellular hypoxia. The probe exhibits low toxicity and is able to selectively label O₂‐deprived cells. A proof‐of‐concept experiment employing metal‐bound DNA intercalators demonstrates that a heterogeneous mixture of cells with differential exposure to O₂ can be effectively discriminated by the quantity of tellurium‐labeling. The organotellurium reagents described herein provide a general approach to the development of a large toolkit of MC‐compatible probes for activity‐based profiling of single cells.     

Discontinuous Galerkin finite element discretization of a strongly anisotropic diffusion operator

The discretization of a diffusion equation with a strong anisotropy by a discontinuous Galerkin finite element method is investigated. This diffusion term is implemented in the tracer equation of an ocean model, thanks to a symmetric tensor that is composed of diapycnal and isopycnal diffusions. The strong anisotropy comes from the difference of magnitude order between both diffusions. As the ocean model uses interior penalty terms to ensure numerical stability, a new penalty factor is required in order to correctly deal with the anisotropy of this diffusion. Two penalty factors from the literature are improved and established from the coercivity property. One of them takes into account the diffusion in the direction normal to the interface between the elements. After comparison, the latter is better because the spurious numerical diffusion is weaker than with the penalty factor proposed in the literature. It is computed with a transformed coordinate system in which the diffusivity tensor is diagonal, using its eigenvalue decomposition. Furthermore, this numerical scheme is validated with the method of manufactured solutions. It is finally applied to simulate the evolution of temperature and salinity due to turbulent processes in an idealized Arctic Ocean. Copyright © 2014 John Wiley & Sons, Ltd.