Two-photon excited-state dynamics of mEGFP-linker-mScarlet-I crowding biosensor in controlled environments.

Macromolecular crowding affects many cellular processes such as diffusion, biochemical reaction kinetics, protein-protein interactions, and protein folding. Mapping the heterogeneous, dynamic crowding in living cells or tissues requires genetically encoded, site-specific, crowding sensors that are compatible with quantitative, noninvasive fluorescence micro-spectroscopy. Here, we carried out time-resolved 2P-fluorescence measurements of a new mEGFP-linker-mScarlet-I macromolecular crowding construct (GE2.3) to characterize its environmental sensitivity in biomimetic crowded solutions (Ficoll-70, 0-300 g L-1) via Förster resonance energy transfer (FRET) analysis. The 2P-fluorescence lifetime of the donor (mEGFP) was measured under magic-angle polarization, in the presence (intact) and absence (enzymatically cleaved) of the acceptor (mScarlet-I), as a function of the Ficoll-70 concentration. The FRET efficiency was used to quantify the sensitivity of GE2.3 to macromolecular crowding and to determine the environmental dependence of the mEGFP-mScarlet-I distance. We also carried out time-resolved 2P-fluorescence depolarization anisotropy to examine both macromolecular crowding and linker flexibility effects on GE2.3 rotational dynamics within the context of the Stokes-Einstein model as compared with theoretical predictions based on its molecular weight. These time-resolved 2P-fluorescence depolarization measurements and conformational population analyses of GE2.3 were also used to estimate the free energy gain upon the structural collapse in crowded environment. Our results further the development of a rational engineering design for bioenvironmental sensors without the interference of cellular autofluorescence. Additionally, these results in well-defined environments will inform our future in vivo studies of genetically encoded GE2.3 towards the mapping of the crowded intracellular environment under different physiological conditions.

N-mustard analogs of S-adenosyl-L-methionine as biochemical probes of protein arginine methylation.

Nucleosomes, the fundamental building blocks of eukaryotic chromatin, undergo post-synthetic modifications and play a major role in the regulation of transcriptional processes. Combinations of these modifications, including methylation, regulate chromatin structure, determining its different functional states and playing a central role in differentiation. The biological significance of cellular methylation, particularly on chromatin, is widely recognized, yet we know little about the mechanisms that link biological methylation events. To characterize and fully understand protein methylation, we describe here novel N-mustard analogs of S-adenosyl-l-methionine (SAM) as biochemical tools to better understand protein arginine methylation events using protein arginine methyltransferase 1 (PRMT1). Specifically, azide- and alkyne-functionalized N-mustard analogs serve as cofactor mimics of SAM and are enzymatically transferred to a model peptide substrate in a PRMT1-dependent fashion. Once incorporated, the resulting alkynes and azides can be modified through chemoselective ligations, including click chemistry and the Staudinger ligation. These results readily demonstrate the feasibility of utilizing N-mustard analogs as biochemical tools to site-specifically label substrates of PRMT1 and serve as an alternative approach to study protein methylation events.

Overweight and obesity in hemophilia: a systematic review of the literature.

CONTEXT: As life expectancy in individuals with congenital hemophilia approaches that of the general population, we hypothesize that public health risks, including overweight and obesity, also follow a similar trend. EVIDENCE ACQUISITION: A search of the literature included terms relating to overweight, sequelae of being overweight, and hemophilia. Studies were included if they reported the frequency or clinical significance of known complications of overweight and obesity, including musculoskeletal disease, aerobic capacity, cardiovascular disease, diabetes, hyperlipidemia, decreased quality of life, and change in pharmacokinetics of infused clotting factor in hemophilia. Recommendations from medical organizations were searched for preventive and management strategies applicable to this population. EVIDENCE SYNTHESIS: Overweight and obesity are now more prevalent in the hemophilia population than previous generations, with rates similar to and, in certain subsets even higher, than that of the general population. Increased BMI leads to limitations in joint range of motion in the general population and even more so in persons with hemophilia. CONCLUSIONS: Overweight and obesity in hemophilia are an increasing problem. Simple steps can be taken to encourage patients to decrease caloric intake and increase physical activity. Prevention and management of overweight, obesity, and their sequelae must be addressed in clinical practice in order to maximize the overall health of the hemophilia population.