The study of ever more complex biomolecular assemblies implicated in human health and disease is facilitated by a suite of complementary biophysical methods. Pulse dipolar electron paramagnetic resonance spectroscopy (PDS) is a powerful tool that provides highly precise geometric constraints in frozen solutions; however, the drive toward PDS at physiologically relevant sub-μM concentrations is limited by the currently achievable concentration sensitivity. Recently, PDS using a combination of nitroxide- and CuII-based spin labels allowed measuring a 500 nM concentration of a model protein. Using commercial instrumentation and spin labels, we demonstrate CuII-CuII and nitroxide-nitroxide PDS measurements at protein concentrations below previous examples reaching 500 and 100 nM, respectively. These results demonstrate the general feasibility of sub-μM PDS measurements at short to intermediate distances (∼1.5 to 3.5 nm), and are of particular relevance for applications where the achievable concentration is limiting.Both decabromodiphenyl ether (BDE 209) and decabromodiphenyl ethane (DBDPE) are still produced in large quantities in China, especially in the Shandong Province closed to the Bohai Sea (BS). This study conducted a comprehensive investigation of the distribution and budget of brominated flame retardants (BFRs) in the BS. BDE 209 was the predominant BFR in most of the investigated rivers flowing into the BS, although DBDPE exceeded BDE 209 in certain rivers as a result of the replacement of BDE 209 with DBDPE in North China. The spatial distributions of BFRs in the rivers were controlled by the proximity of the BFR manufacturing base and the extent of urbanization. BFRs' spatial distribution in the BS was influenced by a combination of land-based pollution sources, environmental parameters (e.g., suspended particulate matter, particulate organic carbon, and particulate black carbon), and hydrodynamic conditions. The spatial variation trend of BDE 209/DBDPE ratios in various environmental media provided useful information. Vertically, the BDE 209/DBDPE ratio decreased from the seawater surface layer to the sediment, indicating their differential transport in the BS. A multi-box mass balance model and analysis of BDE 209 showed that degradation was the primary sink of BFRs in seawater (∼68%) and surface sediment (∼72%) in the BS.Electrostatic interactions play crucial roles in protein function. Measuring pKa value perturbations upon complex formation or self-assembly of e.g. amyloid fibrils gives valuable information about the effect of electrostatic interactions in those processes. Site-specific pKa value determination by solution NMR spectroscopy is challenged by the high molecular weight of amyloid fibrils. Here we report a pH increase during fibril formation of α-synuclein, observed using three complementary experimental methods pH electrode measurements in water; colorimetric changes of a fluorescent indicator; and chemical shift changes for histidine residues using solution state NMR spectroscopy. A significant pH increase was detected during fibril formation in water, on average by 0.9 pH units from 5.6 to 6.5, showing that protons are taken up during fibril formation. The pH upshift was used to calculate the average change in the apparent pKaave value of the acidic residues, which was found to increase by at least 1.1 unit due to fibril formation. Metropolis Monte Carlo simulations were performed on a comparable system that also showed a proton uptake due to fibril formation. Fibril formation moreover leads to a significant change in proton binding capacitance. Parallel studies of a mutant with five charge deletions in the C-terminal tail revealed a smaller pH increase due to fibril formation, and a smaller change (0.5 units on average) in the apparent pKaave values of the acidic residues. We conclude that the proton uptake during the fibril formation is connected to the high density of acidic residues in the C-terminal tail of α-synuclein.Extracellular electrical stimulation (ES) can provide electrical potential from outside the cell membrane, but it is often ineffective due to interference from external factors such as culture medium resistance and membrane capacitance. To address this, we developed a vertical nanowire electrode array (VNEA) to directly provide intracellular electrical potential and current to cells through nanoelectrodes. Using this approach, the cell membrane resistivity and capacitance could be excluded, allowing effective ES. Human fetal neural stem cells (hfNSCs) were cultured on the VNEA for intracellular ES. selleck Combining the structural properties of VNEA and VNEA-mediated ES, transient nanoscale perforation of the electrode was induced, promoting cell penetration and delivering current to the cell. Intracellular ES using VNEA improved the neuronal differentiation of hfNSCs more effectively than extracellular ES and facilitated electrophysiological functional maturation of hfNSCs because of the enhanced voltage-dependent ion-channel activity. The results demonstrate that VNEA with advanced nanoelectrodes serves as a highly effective culture and stimulation platform for stem-cell neurogenesis.Targeting the genetic material without destruction is a priority to develop safe anticancer drugs. Histone deacetylase 8 (HDAC8), which is proved to be involved in carcinogenesis, is an enzyme associated with the chromatin for post-translational deacetylation of acetylated lysine. In this study, HDAC8 co-crystallized with the intermediate state tetrapeptide Trapoxin A (TA) inhibitor and the holoenzyme are utilized to find their conformational ensembles. Furthermore, the co-crystallized intermediate gem-diolate TA was used to find optimum interactions with the active site residues by conventional molecular dynamics (MD) simulation and QM/MM umbrella sampling. Finally, the intermediate state of the acetyl-l-lysine substrate was explored by QM/MM steered MD and compared to the binding of the intermediate state of the inhibitor. This research showed that HDAC8 is flexible and exists in conformational ensembles in its holoenzyme state. Binding of the intermediate state TA stabilizes its conformation. The optimum binding to the active site of HDAC8 for structures of gem-diolate TA (intermediate state) and acetyl-l-lysine (intermediate state) was determined according to the corresponding energy profiles.