Mass spectrometry-based thermal shift assay for protein-ligand binding analysis.

Described here is a mass spectrometry-based screening assay for the detection of protein-ligand binding interactions in multicomponent protein mixtures. The assay utilizes an oxidation labeling protocol that involves using hydrogen peroxide to selectively oxidize methionine residues in proteins in order to probe the solvent accessibility of these residues as a function of temperature. The extent to which methionine residues in a protein are oxidized after specified reaction times at a range of temperatures is determined in a MALDI analysis of the intact proteins and/or an LC-MS analysis of tryptic peptide fragments generated after the oxidation reaction is quenched. Ultimately, the mass spectral data is used to construct thermal denaturation curves for the detected proteins. In this proof-of-principle work, the protocol is applied to a four-protein model mixture comprised of ubiquitin, ribonuclease A (RNaseA), cyclophilin A (CypA), and bovine carbonic anhydrase II (BCAII). The new protocol's ability to detect protein-ligand binding interactions by comparing thermal denaturation data obtained in the absence and in the presence of ligand is demonstrated using cyclosporin A (CsA) as a test ligand. The known binding interaction between CsA and CypA was detected using both the MALDI- and LC-MS-based readouts described here.

Neural mechanisms of context effects on face recognition: automatic binding and context shift decrements.

Although people do not normally try to remember associations between faces and physical contexts, these associations are established automatically, as indicated by the difficulty of recognizing familiar faces in different contexts ("butcher-on-the-bus" phenomenon). The present fMRI study investigated the automatic binding of faces and scenes. In the face-face (F-F) condition, faces were presented alone during both encoding and retrieval, whereas in the face/scene-face (FS-F) condition, they were presented overlaid on scenes during encoding but alone during retrieval (context change). Although participants were instructed to focus only on the faces during both encoding and retrieval, recognition performance was worse in the FS-F than in the F-F condition ("context shift decrement" [CSD]), confirming automatic face-scene binding during encoding. This binding was mediated by the hippocampus as indicated by greater subsequent memory effects (remembered > forgotten) in this region for the FS-F than the F-F condition. Scene memory was mediated by right parahippocampal cortex, which was reactivated during successful retrieval when the faces were associated with a scene during encoding (FS-F condition). Analyses using the CSD as a regressor yielded a clear hemispheric asymmetry in medial temporal lobe activity during encoding: Left hippocampal and parahippocampal activity was associated with a smaller CSD, indicating more flexible memory representations immune to context changes, whereas right hippocampal/rhinal activity was associated with a larger CSD, indicating less flexible representations sensitive to context change. Taken together, the results clarify the neural mechanisms of context effects on face recognition.

Refractive changes after descemet stripping endothelial keratoplasty: a simplified mathematical model.

PURPOSE: To develop a mathematical model that can predict refractive changes after Descemet stripping endothelial keratoplasty (DSEK). METHODS: A mathematical formula based on the Gullstrand eye model was generated to estimate the change in refractive power of the eye after DSEK. This model was applied to four DSEK cases retrospectively, to compare measured and predicted refractive changes after DSEK. RESULTS: The refractive change after DSEK is determined by calculating the difference in the power of the eye before and after DSEK surgery. The power of the eye post-DSEK surgery can be calculated with modified Gullstrand eye model equations that incorporate the change in the posterior radius of curvature and change in the distance between the principal planes of the cornea and lens after DSEK. Analysis of this model suggests that the ratio of central to peripheral graft thickness (CP ratio) and central thickness can have significant effect on refractive change where smaller CP ratios and larger graft thicknesses result in larger hyperopic shifts. This model was applied to four patients, and the average predicted hyperopic shift in the overall power of the eye was calculated to be 0.83 D. This change reflected in a mean of 93% (range, 75%-110%) of patients' measured refractive shifts. CONCLUSIONS: This simplified DSEK mathematical model can be used as a first step for estimating the hyperopic shift after DSEK. Further studies are necessary to refine the validity of this model.

Inter-annual variability of precipitation constrains the production response of boreal Pinus sylvestris to nitrogen fertilization

© 2015 Published by Elsevier B.V.Tree growth resources and the efficiency of resource-use for biomass production determine the productivity of forest ecosystems. In nutrient-limited forests, nitrogen (N)-fertilization increases foliage [N], which may increase photosynthetic rates, leaf area index (L), and thus light interception (IC). The product of such changes is a higher gross primary production and higher net primary production (NPP). However, fertilization may also alter carbohydrate partitioning from below- to aboveground, increasing aboveground NPP (ANPP). We analyzed effects of long-term N-fertilization on NPP, and that of long-term carbon storing organs (NPPS) in a Pinus sylvestris forest on sandy soil, a wide-ranging forest type in the boreal region. We based our analyses on a combination of destructive harvesting, consecutive mensuration, and optical measurements of canopy openness. After eight-year fertilization with a total of 70gNm^-2, ANPP was 27±7% higher in the fertilized (F) relative to the reference (R) stand, but although L increased relative to its pre-fertilization values, IC was not greater than in R. On the seventh year after the treatment initiation, the increase of ANPP was matched by the decrease of belowground NPP (78 vs. 92gCm^-2yr^-1; ~17% of NPP) and, given the similarity of IC, suggests that the main effect of N-fertilization was changed carbon partitioning rather than increased canopy photosynthesis. Annual NPPS increased linearly with growing season temperature (T) in both treatments, with an upward shift of 70.2gCm^-2yr^-1 by fertilization, which also caused greater amount of unexplained variation (r²=0.53 in R, 0.21 in F). Residuals of the NPPS-T relationship of F were related to growing season precipitation (P, r²=0.48), indicating that T constrains productivity at this site regardless of fertility, while P is important in determining productivity where N-limitation is alleviated. We estimated that, in a growing season average T (11.5±1.0°C; 33-year-mean), NPPS response to N-fertilization will be nullified with P 31mm less than the mean (325±85mm), and would double with P 109mm greater than the mean. These results suggest that inter-annual variation in climate, particularly in P, may help explaining the reported large variability in growth responses to fertilization of pine stands on sandy soils. Furthermore, forest management of long-rotation systems, such as those of boreal and northern temperate forests, must consider the efficiency of fertilization in terms of wood production in the context of changes in climate predicted for the region.