Complexity of fatty acid distribution inside human macrophages on single cell level using Raman micro-spectroscopy

Macrophages are phagocytic cells which are involved in the non-specific immune defense. Lipid uptake and storage behavior of macrophages also play a key role in the development of atherosclerotic lesions within walls of blood vessels. The allocation of exogenous lipids such as fatty acids in the blood stream dictates the accumulation and quantity of lipids within macrophages. In case of an overexposure, macrophages transform into foam cells because of the large amount of lipid droplets in the cytoplasm. Raman micro-spectroscopy is a powerful tool for studying single cells due to the combination of microscopic imaging with spectral information. With a spatial resolution restricted by the diffraction limit, it is possible to visualize lipid droplets within macrophages. With stable isotopic labeling of fatty acids with deuterium, the uptake and storage of exogenously provided fatty acids can be investigated. In this study, we present the results of time-dependent Raman spectroscopic imaging of single THP-1 macrophages incubated with deuterated arachidonic acid. The polyunsaturated fatty acid plays an important role in the cellular signaling pathway as being the precursor of icosanoids. We show that arachidonic acid is stored in lipid droplets but foam cell formation is less pronounced as with other fatty acids. The storage efficiency in lipid droplets is lower than in cells incubated with deuterated palmitic acid. We validate our results with gas chromatography and gain information on the relative content of arachidonic acid and its metabolites in treated macrophages. These analyses also provide evidence that significant amounts of the intracellular arachidonic acid is elongated to adrenic acid but is not metabolized any further. The co-supplementation of deuterated arachidonic acid and deuterated palmitic acid leads to a non-homogenous storage pattern in lipid droplets within single cells. Figure a

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Boron- versus Nitrogen-Centered Nucleophilic Reactivity of (Cyano)hydroboryl Anions: Synthesis of Cyano(hydro)organoboranes and 2-Aza-1,4-diborabutatrienes

Cyclic alkyl(amino)carbene-stabilized (cyano)hydroboryl anions were synthesized by deprotonation of (cyano)dihydroborane precursors. While they display boron-centered nucleophilic reactivity towards organohalides, generating fully unsymmetrically substituted cyano(hydro)organoboranes, they show cyano-nitrogen-centered nucleophilic reactivity towards haloboranes, resulting in the formation of hitherto unknown linear 2-aza-1,4-diborabutatrienes.     

Neutral-pion photoproduction off protons in the energy range 0.3 GeV<E(gamma)<3 GeV

Single pi(0) photoproduction has been studied with the CB-ELSA experiment at Bonn using tagged photon energies between 0.3 and 3.0 GeV. The experimental setup covers a very large solid angle of approximately 98% of 4pi. Differential cross sections dsigma/dOmega have been measured. Complicated structures in the angular distributions indicate a variety of different resonances being produced in the s channel intermediate state gammap-->N(*)(Delta(*))-->ppi(0). A combined analysis including the data presented in this letter along with other data sets reveals contributions from known resonances and evidence for a new resonance N(2070)D15.     

Multiphase Thermohaline Convection in the Earth’s Crust: I. A New Finite Element – Finite Volume Solution Technique Combined With a New Equation of State for NaCl–H2O

We present a new finite element – finite volume (FEFV) method combined with a realistic equation of state for NaCl–H₂O to model fluid convection driven by temperature and salinity gradients. This method can deal with the nonlinear variations in fluid properties, separation of a saline fluid into a high-density, high-salinity brine phase and low-density, low-salinity vapor phase well above the critical point of pure H₂O, and geometrically complex geological structures. Similar to the well-known implicit pressure explicit saturation formulation, this approach decouples the governing equations. We formulate a fluid pressure equation that is solved using an implicit finite element method. We derive the fluid velocities from the updated pressure field and employ them in a higher-order, mass conserving finite volume formulation to solve hyperbolic parts of the conservation laws. The parabolic parts are solved by finite element methods. This FEFV method provides for geometric flexibility and numerical efficiency. The equation of state for NaCl–H₂O is valid from 0 to 750°C, 0 to 4000 bar, and 0–100 wt.% NaCl. This allows the simulation of thermohaline convection in high-temperature and high-pressure environments, such as continental or oceanic hydrothermal systems where phase separation is common.     

Two-dimensional resonance enhanced multiphoton ionization of H(i)Cl; i = 35, 37: state interactions, photofragmentations and energetics of high energy Rydberg states

Mass spectra were recorded for (2 + n) resonance enhanced multiphoton ionization (REMPI) of HCl as a function of resonance excitation energy in the 88865-89285 cm(-1) region to obtain two-dimensional REMPI data. Band spectra due to two-photon resonance transitions to number of Rydberg states (Ω' = 0, 1, and 2) and the ion-pair state V((1)Σ(+)(Ω' = 0)) for H(35)Cl and H(37)Cl were identified, assigned, and analyzed with respect to Rydberg to ion-pair interactions. Perturbations show as line-, hence energy level-, shifts, as well as ion signal intensity variations with rotational quantum numbers, J', which, together, allowed determination of parameters relevant to the nature and strength of the state interactions as well as dissociation and ionization processes. Whereas near-resonance, level-to-level, interactions are found to be dominant in heterogeneous state interactions (ΔΩ ≠ 0) significant off-resonance interactions are observed in homogeneous interactions (ΔΩ = 0). The alterations in Cl(+) and HCl(+) signal intensities prove to be very useful for spectra assignments. Data relevant to excitations to the j(3)Σ(0(+)) Rydberg states and comparison with (3 + n) REMPI spectra allowed reassignment of corresponding spectra peaks. A band previously assigned to an Ω = 0 Rydberg state was reassigned to an Ω = 2 state (ν(0) = 88957.6 cm(-1)).     

Spectral unmixing and clustering algorithms for assessment of single cells by Raman microscopic imaging

A detailed comparison of six multivariate algorithms is presented to analyze and generate Raman microscopic images that consist of a large number of individual spectra. This includes the segmentation algorithms for hierarchical cluster analysis, fuzzy C-means cluster analysis, and k-means cluster analysis and the spectral unmixing techniques for principal component analysis and vertex component analysis (VCA). All algorithms are reviewed and compared. Furthermore, comparisons are made to the new approach N-FINDR. In contrast to the related VCA approach, the used implementation of N-FINDR searches for the original input spectrum from the non-dimension reduced input matrix and sets it as the endmember signature. The algorithms were applied to hyperspectral data from a Raman image of a single cell. This data set was acquired by collecting individual spectra in a raster pattern using a 0.5-??m step size via a commercial Raman microspectrometer. The results were also compared with a fluorescence staining of the cell including its mitochondrial distribution. The ability of each algorithm to extract chemical and spatial information of subcellular components in the cell is discussed together with advantages and disadvantages.     

Investigation of drug-cyclodextrin complexes by a phase-distribution method: some theoretical and practical considerations

The purpose of the study was to evaluate an octanol-water phase distribution method for investigation of drug/cyclodextrin (D/CD) complexes and to compare stability constant values obtained by this method to values obtained by the phase solubility method. A general equation for determination of 1 : 1 D/CD complex stability constant (K1 : 1) from the slope of a phase-distribution diagram (a diagram of the reciprocal of the apparent partition coefficient vs. the total CD concentration) was derived. The equation accounted for the possible inclusion of the organic solvent in the CD cavity and the gradual saturation of the CD binding with increasing concentration of the guest compound. This method was used to determine K1 : 1 for 2-hydroxypropyl-beta-cyclodextrin (HPbetaCD) complexes of hydrocortisone, prednisolone, diazepam, beta-estradiol and diethylstilbestrol. These values were comparable to K1 : 1 values determined by the phase-solubility method. The phase-distribution method could also be applied to determine stability constants for the neutral and ionic forms of the weakly acidic drugs, naproxen and triclosan and the weakly basic drug lidocaine. The phase-distribution method is a very versatile and fast method and has the advantage, compared to the phase-solubility method, that it only requires very small drug samples. Thus, this method would be suitable for screening of new drug candidates.     

Solvent-dependent ultrafast internal conversion dynamics of n'-apo-beta-carotenoic-n'-acids (n = 8, 10, 12)

The ultrafast internal conversion dynamics of 12'-apo-beta-carotenoic-12'-acid (12'CA), 10'-apo-beta-carotenoic-10'-acid (10'CA) and 8'-apo-beta-carotenoic-8'-acid (8'CA) have been investigated by femtosecond pump-probe spectroscopy. The three apocarotenoic acids were excited to the S(2) state with different excess energies. Time constants tau(1) for the IC process S(1)/ICT --> S(0) were measured by probing the dynamics at 390 nm (S(0) --> S(2)), 575 nm (S(1)/ICT --> S(n)), 850, 860 and 890 nm (S(2) --> S(n) and S(1)/ICT --> S(0)). In nonpolar solvents, the observed reduction of the tau(1) values with increasing conjugation length of the acids is consistent with a reduction of the energy gap between the S(1)/ICT and S(0) states. The values are in good agreement with those of the corresponding apocarotenals studied previously in our groups. In polar solvents, a pronounced reduction of tau(1) values was observed for 12'CA, however the behavior was different from that observed for the respective aldehyde 12'-apo-beta-caroten-12'-al studied previously: First, the degree of tau(1) reduction in methanol was milder for 12'CA (218 --> 55 ps) than for 12'-apo-beta-caroten-12'-al (220 --> 8 ps). Secondly, for 12'CA the plateau of solvent independent tau(1) values extended further into the mid-polar range (up to 0.5 on the Deltaf scale) than previously observed for the 12'-aldehyde. For 10'CA the polarity effect on the tau(1) values was weaker ( approximately 71 ps in n-hexane and 34 ps in methanol) and for 8'CA it disappeared completely ( approximately 24 ps averaged over all solvents). The polarity-induced reduction of tau(1) is likely due to the stabilization of an intramolecular charge transfer state in polar solvents. This S(1)/ICT state is also responsible for the stimulated emission in the near IR, which has been observed in this specific class of carotenoids with a terminal carboxyl group for the first time. The occurrence of stimulated emission in the near IR region is also consistent with the steady-state fluorescence spectra which are reported along with the absorption spectra of these species. Possible reasons for the different behavior of the apocarotenoic acids compared to the respective aldehydes are discussed.