Formation of an N-formylkynurenine-derived fluorophore and its use for measuring indoleamine 2,3-dioxygenase 1 activity

Indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan-catabolizing enzyme whose expression by a broad range of clinical tumors is associated with immunosuppression and poor patient outcome. Here we describe a new fluorescence assay for measuring IDO1 activity suitable for high-throughput screening of compound libraries for novel IDO1 inhibitors. This assay is easy to perform, requiring the addition of only one reagent prior to readout. In place of measuring kynurenine, it uses the in situ formation of an N-formylkynurenine-derived fluorophore (NFKPIP) measured at an excitation wavelength of 400 nm and an emission wavelength of 500 nm. The fluorescence intensity of the NFKPIP formed is directly related to the amount of enzyme activity, and the signal is stable over 8 h. This assay has a lower limit of detection, equating to 153 nM N-formylkynurenine, which is over 30-fold lower than the limits of detection of existing assays for IDO1 activity. When we compared the performance of the new assay with that of the published colorimetric absorbance assay in screening the National Cancer Institute Diversity Set III of 1,597 compounds for IDO1 inhibitors, we obtained an identical list of the 25 most active compounds in the two assays. Although 93 compounds (aldehydes, ketones, and aromatic amines) in the library interfered with the absorbance readout, only 18 compounds (conjugated systems and fused cycles) interfered with the readout of the new fluorescence assay. IC₅₀ values determined using the new assay for three known IDO1 inhibitors—1,4-naphthoquinone, 4-amino-N-(3-chloro-4-fluorophenyl)-N’-hydroxy-1,2,5-oxadiazole-3-carboximidamide and 4-phenyl-1H-imidazole—were consistent with their literature values, further validating the new assay for measuring IDO1 activity.

Profiling of the secretome of human cancer cells: Preparation of supernatant for proteomic analysis

Secretomic analysis requires removal of serum proteins from cell‐culture media. We evaluate the proteins washed from cells prepared in bovine serum‐supplemented medium. PBS and serum‐free‐medium (SFM) were the washing solutions. A Bradford assay was used for total protein concentration and a 1D gel and LC‐MS/MS, to assign the protein to human or bovine origin. For both wash solutions, all bovine protein had been removed by the third wash, without compromising the number of living cells. Further washes reduced the number of living cells, especially when using PBS. Proteomic analysis of wash supernatant showed that SFM induced greater lysis of dead cells. Three washes were sufficient to minimize the effects on cell viability, while still removing serum proteins. Washing in SFM resulted in contamination of the wash supernatant with lysed dead cell proteins. Washed cells were incubated in SFM and exposed to ionizing radiation. Analysis of the supernatant showed an increase in human cytoplasmic, plasma membrane, and nuclear protein following irradiation. Secreted proteins were also detected, but in smaller quantities. The significance of these findings extend to in vitro studies of bystander phenomena, since the proteins of lysed dead cells may participate in driving bystander responses.     

Novel C‐acylated triazole derivatives of ravuconazole,an azole antifungal agent

Novel C‐acylated triazoles were synthesized from ravuconazole via bicyclic triazole lactone. The synthesis and antifungal activity of these C‐acylated derivatives are described.     

Ambiphilic diphosphine-borane ligands: metal-->borane interactions within isoelectronic complexes of rhodium, platinum and palladium

Coordination of an ambiphilic diphosphine-borane (DPB) ligand to the RhCl(CO) fragment affords two isomeric complexes. According to X-ray diffraction analysis, each complex adopts a square-pyramidal geometry with trans coordination of the two phosphine buttresses and axial RhB contacts, but the two differ in the relative orientations around the rhodium and boron centres. DFT calculations on the actual complexes provide insight into the influence of the pi-accepting CO co-ligand, compared with previously reported complexes [Rh(mu-Cl)(dpb)]2 and [RhCl(dmap)(dpb)]. In addition, comparison of the nu(CO) frequency of [RhCl(CO)(dpb)] with that of the related borane-free complex [RhCl(CO)(iPr2PPh)2] substantiates the significant electron-withdrawing effect that the sigma-accepting borane moiety exerts on the metal. Valence isoelectronic [PtCl2(dpb)] and [PdCl2(dpb)] complexes have also been prepared and characterized spectroscopically and structurally. The pronounced influence of the transition metal on the magnitude of the M-->B interaction is highlighted by geometric considerations and NBO analyses.     

Artificial construction of the layered Ruddlesden-Popper manganite La2Sr2Mn3O10 by reflection high energy electron diffraction monitored pulsed laser deposition

Pulsed laser deposition has been used to artificially construct the n = 3 Ruddlesden-Popper structure La(2)Sr(2)Mn(3)O(10) in epitaxial thin film form by sequentially layering La(1-x)Sr(x)MnO(3) and SrO unit cells aided by in situ reflection high energy electron diffraction monitoring. The interval deposition technique was used to promote two-dimensional SrO growth. X-ray diffraction and cross-sectional transmission electron microscopy indicated that the trilayer structure had been formed. A site ordering was found to differ from that expected thermodynamically, with the smaller Sr(2+) predominantly on the R site due to kinetic trapping of the deposited cation sequence. A dependence of the out-of-plane lattice parameter on growth pressure was interpreted as changing the oxygen content of the films. Magnetic and transport measurements on fully oxygenated films indicated a frustrated magnetic ground state characterized as a spin glass-like magnetic phase with the glass temperature T(g) ≈ 34 K. The magnetic frustration has a clear in-plane (ab) magnetic anisotropy, which is maintained up to temperatures of 150 K. Density functional theory calculations suggest competing antiferromagnetic and ferromagnetic long-range orders, which are proposed as the origin of the low-temperature glassy state.     

Chemical Bonding and Atomic Structure in Y(2) O(3) :ZrO(2) -SrTiO(3) Layered Heterostructures

Repeating boundaries: The buried interfaces in artificial heterostructures produced by sequential deposition of nanosized units are critical to their properties. With density functional theory it was shown that in Y(2) O(3) :ZrO(2) (YSZ) and SrTiO(3) (STO) heterostructures reconstruction of the interfaces between the component units is required to access the most favorable structure.     

Dynamic alignment in driven magnetohydrodynamic turbulence

Motivated by recent analytic predictions, we report numerical evidence showing that in driven incompressible magnetohydrodynamic turbulence the magnetic- and velocity-field fluctuations locally tend to align the directions of their polarizations. This dynamic alignment is stronger at smaller scales with the angular mismatch between the polarizations decreasing with the scale lambda approximately as theta(lambda) is proportional to lambda(1/4). This can naturally lead to a weakening of the nonlinear interactions and provide an explanation for the energy spectrum E(k) is proportional to k(-3/2) that is observed in numerical experiments of strongly magnetized turbulence.