The Level of DING Proteins Is Increased in HIV-Infected Patients: In Vitro and In Vivo Studies

DING proteins constitute an interesting family, owing to their intriguing and important activities. However, after a decade of research, little is known about these proteins. In humans, at least five different DING proteins have been identified, which were implicated in important biological processes and diseases, including HIV. Indeed, recent data from different research groups have highlighted the anti-HIV activity of some DING representatives. These proteins share the ability to inhibit the transcriptional step of HIV-1, a key step of the viral cycle that is not yet targeted by the current therapies. Since such proteins have been isolated from humans, we undertook a comprehensive study that focuses on the relationship between these proteins and HIV-infection in an infectious context. Hence, we developed a home-made ELISA for the quantification of the concentration of DING proteins in human serum. Using this method, we were able to determine the concentration of DING proteins in healthy and HIV-infected patients. Interestingly, we observed a significant increase of the concentration of DING proteins in non treated and treated HIV-infected patients compared to controls. In addition, cell cultures infected with HIV also show an increased expression of DING proteins, ruling out the possible role of antiretroviral treatment in the increase of the expression of DING proteins. In conclusion, results from this study show that the organism reacts to HIV-infection by an overexpression of DING proteins

Live-cell fluorescence lifetime multiplexing using synthetic fluorescent probes

Fluorescence lifetime multiplexing requires fluorescent probes with distinct fluorescence lifetimes but similar spectral properties. Even though synthetic probes for many cellular targets are available for multicolor live-cell fluorescence microscopy, few of them have been characterized for their use in fluorescence lifetime multiplexing. Here we demonstrate that from a panel of 18 synthetic probes, eight pairwise combinations are suitable for fluorescence lifetime multiplexing in living mammalian cell lines. Moreover, combining multiple pairs in different spectral channels enables us to image up to six different biological targets, effectively doubling the number of observable targets. The combination of synthetic probes with fluorescence lifetime multiplexing is thus a powerful approach for live-cell imaging

Environmentally sensitive color‐shifting fluorophores for bioimaging

We introduce color‐shifting fluorophores which reversibly switch between a green and red fluorescent form via an intramolecular spirocyclization. The equilibrium of spirocyclization is environmentally sensitive and can be directly measured by determining the ratio of red to green fluorescence, allowing the generation of ratiometric fluorescent probes and biosensors. Specifically, we develop a ratiometric biosensor for imaging calcium ions (Ca 2+ ) in living cells, ratiometric probes for different proteins and a bioassay for the quantification of nicotinamide adenine dinucleotide phosphate

Engineered HaloTag variants for fluorescence lifetime multiplexing

Self-labeling protein tags such as HaloTag are powerful tools that can label fusion proteins with synthetic fluorophores for use in fluorescence microscopy. Here we introduce HaloTag variants with either increased or decreased brightness and fluorescence lifetime compared with HaloTag7 when labeled with rhodamines. Combining these HaloTag variants enabled live-cell fluorescence lifetime multiplexing of three cellular targets in one spectral channel using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. Additionally, the brightest HaloTag variant showed up to 40% higher brightness in live-cell imaging applications

Compact model of short-channel effects for FDSOI devices including the influence of back-bias and fringing fields for Si and III–V technology

International audienceIn this work, a compact model for short-channel effects is proposed for fully depleted silicon-on-insulator (FDSOI) MOSFETs, which takes into account the impact of body-bias and fringing fields, developed to be suitable for both thin and thick buried oxide (BOX) devices. The model is derived using the Voltage-Doping Transform, confirmed with TCAD simulations and experimental data from the literature. The impact of the BOX thickness on the subthreshold swing and the contribution of the leakiest path position to the DIBL are finally discussed

Synergizing exchangeable fluorophore sabels for multitarget STED microscopy

Investigating the interplay of cellular proteins with optical microscopy requires multitarget labeling. Spectral multiplexing using high-affinity or covalent labels is limited in the number of fluorophores that can be discriminated in a single imaging experiment. Advanced microscopy methods such as STED microscopy additionally demand balanced excitation, depletion, and emission wavelengths for all fluorophores, further reducing multiplexing capabilities. Noncovalent, weak-affinity labels bypass this "spectral barrier" through label exchange and sequential imaging of different targets. Here, we combine exchangeable HaloTag ligands, weak-affinity DNA hybridization, and hydrophophic and protein-peptide interactions to increase labeling flexibility and demonstrate six-target STED microscopy in single cells. We further show that exchangeable labels reduce photobleaching as well as facilitate long acquisition times and multicolor live-cell and high-fidelity 3D STED microscopy. The synergy of different types of exchangeable labels increases the multiplexing capabilities in fluorescence microscopy, and by that, the information content of microscopy images

Probing coenzyme A homeostasis with semisynthetic biosensors

Coenzyme A (CoA) is one of the central cofactors of metabolism, yet a method for measuring its concentration in living cells is missing. Here we introduce the first biosensor for measuring CoA levels in different organelles of mammalian cells. The semisynthetic biosensor is generated through the specific labeling of an engineered GFP–HaloTag fusion protein with a fluorescent ligand. Its readout is based on CoA-dependent changes in Förster resonance energy transfer efficiency between GFP and the fluorescent ligand. Using this biosensor, we probe the role of numerous proteins involved in CoA biosynthesis and transport in mammalian cells. On the basis of these studies, we propose a cellular map of CoA biosynthesis that suggests how pools of cytosolic and mitochondrial CoA are maintained