GBPs are essential for immunity against intracellular pathogens, especially for Toxoplasma gondii control. Here, the molecular interactions of murine GBPs (mGBP1/2/3/5/6), homo- and hetero-multimerization properties of mGBP2 and its function in parasite killing were investigated by mutational, Multiparameter Fluorescence Image Spectroscopy, and live cell microscopy methodologies. Control of T. gondii replication by mGBP2 requires GTP hydrolysis and isoprenylation thus, enabling reversible oligomerization in vesicle-like structures. mGBP2 undergoes structural transitions between monomeric, dimeric and oligomeric states visualized by quantitative FRET analysis. mGBPs reside in at least two discrete subcellular reservoirs and attack the parasitophorous vacuole membrane (PVM) as orchestrated, supramolecular complexes forming large, densely packed multimers comprising up to several thousand monomers. This dramatic mGBP enrichment results in the loss of PVM integrity, followed by a direct assault of mGBP2 upon the plasma membrane of the parasite. These discoveries provide vital dynamic and molecular perceptions into cell-autonomous immunity.DOI: http://dx.doi.org/10.7554/eLife.11479.001

The CLAVATA (CLV) and flagellin (flg) signaling pathways act through peptide ligands and closely related plasma membrane-localized receptor-like kinases (RLKs). The plant peptide CLV3 regulates stem cell homeostasis, whereas the bacterial flg22 peptide elicits defense responses. We applied multiparameter fluorescence imaging spectroscopy (MFIS) to characterize the dynamics of RLK complexes in the presence of ligand in living plant cells expressing receptor proteins fused to fluorescent proteins. We found that the CLV and flg pathways represent two different principles of signal transduction: flg22 first triggered RLK heterodimerization and later assembly into larger complexes through homomerization. In contrast, CLV receptor complexes were preformed, and ligand binding stimulated their clustering. This different behavior likely reflects the nature of these signaling pathways. Pathogen-triggered flg signaling impedes plant growth and development; therefore, receptor complexes are formed only in the presence of ligand. In contrast, CLV3-dependent stem cell homeostasis continuously requires active signaling, and preformation of receptor complexes may facilitate this task.

TGR5 is the first identified bile acid-sensing G-protein coupled receptor, which has emerged as a potential therapeutic target for metabolic disorders. So far, structural and multimerization properties are largely unknown for TGR5. We used a combined strategy applying cellular biology, Multiparameter Image Fluorescence Spectroscopy (MFIS) for quantitative FRET analysis, and integrative modelling to obtain structural information about dimerization and higher-order oligomerization assemblies of TGR5 wildtype (wt) and Y111 variants fused to fluorescent proteins. Residue 111 is located in transmembrane helix 3 within the highly conserved ERY motif. Co-immunoprecipitation and MFIS-FRET measurements with gradually increasing acceptor to donor concentrations showed that TGR5 wt forms higher-order oligomers, a process disrupted in TGR5 Y111A variants. From the concentration dependence of the MFIS-FRET data we conclude that higher-order oligomers – likely with a tetramer organization - are formed from dimers, the smallest unit suggested for TGR5 Y111A variants. Higher-order oligomers likely have a linear arrangement with interaction sites involving transmembrane helix 1 and helix 8 as well as transmembrane helix 5. The latter interaction is suggested to be disrupted by the Y111A mutation. The proposed model of TGR5 oligomer assembly broadens our view of possible oligomer patterns and affinities of class A GPCRs.

Based on multisource data from spiral CT and the intraoral scanner, 3D digital casts of maxillary defects were generated using the registration technique. These casts were consistent with conventional stone casts in terms of accuracy and were suitable for clinical use.

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Recent studies in biometric-based identification tasks have shown that deep learning methods can achieve better performance. These methods generally extract the global features as descriptor to represent the original image. Nonetheless, it does not perform well for biometric identification under fine-grained tasks. The main reason is that the single image descriptor contains insufficient information to represent image. In this paper, we present a dual global descriptor model, which combines multiple global descriptors to exploit multi level image features. Moreover, we utilize a contrastive loss to enlarge the distance between image representations of confusing classes. The proposed framework achieves the top2 on the

When excited at 440nm, the emission spectra of riboflavin exhibited maxima at 515nm (delayed fluorescence) and 620nm (phosphorescence). Deconvolution of the spectra to two individual bands characterized by asymmetric lognormal functions (I(n)=I0*exp{-ln(2)(ln[1þ2b(n-np)/D2]/b)}) facilitated the extraction of parameters and the analysis of their temperature dependence. Time resolved intensity decays were fitted using stretch, multi-exponential and distribution (by MEM) models. Riboflavin exhibited a lifetime of 184ms at 77K in glycerol-water. The lifetime decreased slowly below the solvent mixture's Tg (170K) and abruptly above Tg; e.g., an increase of 20K above Tg reduced the lifetime from 100ms to 10ms. This reduction was associated with molecular motions in the matrix. Molecular mobility was also modulated by changing the composition of the matrix, by using components of different molecular sizes or by adding plasticizers. Riboflavin phosphorescence also exhibited good sensitivity towards molecular mobility changes driven by composition. This suggests its use as a GRAS optical probe for molecular mobility and its potential application to optimize matrix composition in food and pharmaceutical products to enhance stability of micro and bioactive components. Continuous monitoring of riboflavin phosphorescence during heating and cooling cycles revealed differences in the delayed luminescence emission spectra, likely due to a higher rate of irreversible photodegradation of riboflavin at high temperatures. If properly characterized, the thermal dependence of riboflavin photodegradation can potentially be operationalized in sensors for temperature abuse.