Here we present an evaluation of the binding affinity prediction accuracy of the free energy calculation method FEP+ on internal active drug discovery projects and on a large new public benchmark set.<br>

A series of benzothiazolium-based hemicyanines (3a-3f) have been synthesized. Evaluation of their photophysical properties shows that they exhibit improved photophysical characteristics. In comparison with the available commercial MitoTrackers, the new probes revealed an enhanced Stokes shift (Δλ ∼ 80 nm) and minimized aggregation for increased sensitivity. The synthesized probes are found to exhibit excellent selectivity for mitochondrial staining in an oligodendrocyte cell line. Probes show almost no fluorescence in aqueous environments, while the fluorescence is increased by ∼10-fold in organic solvents, making it possible for mitochondrial imaging without the need for post-staining washing. Since the absorption peaks of probes are close to the laser wavelengths of 561 and 640 nm on a commercial confocal microscope, e.g.3a exhibits λabs ∼ 620 nm and λem ∼ 702 nm, they could be useful probes for mitochondrial tracking in live cells.

A few HBT-Cy-containing fluorescent probes have been synthesized for lysosome visualization in live cells.

The value of time-dependent toxicity (TDT) data in predicting mixture toxicity was examined. Single chemical (A and B) and mixture (A + B) toxicity tests using Microtox® were conducted with inhibition of bioluminescence (Vibrio fischeri) being quantified after 15, 30 and 45-min of exposure. Single chemical and mixture tests for 25 sham (A1:A2) and 125 true (A:B) combinations had a minimum of seven duplicated concentrations with a duplicated control treatment for each test. Concentration/response (x/y) data were fitted to sigmoid curves using the five-parameter logistic minus one parameter (5PL-1P) function, from which slope, EC25, EC50, EC75, asymmetry, maximum effect, and r2 values were obtained for each chemical and mixture at each exposure duration. Toxicity data were used to calculate percentage-based TDT values for each individual chemical and mixture of each combination. Predicted TDT values for each mixture were calculated by averaging the TDT values of the individual components and regressed against the observed TDT values obtained in testing, resulting in strong correlations for both sham (r2 = 0.989, n = 25) and true mixtures (r2 = 0.944, n = 125). Additionally, regression analyses confirmed that observed mixture TDT values calculated for the 50% effect level were somewhat better correlated with predicted mixture TDT values than at the 25 and 75% effect levels. Single chemical and mixture TDT values were classified into five levels in order to discern trends. The results suggested that the ability to predict mixture TDT by averaging the TDT of the single agents was modestly reduced when one agent of the combination had a positive TDT value and the other had a minimal or negative TDT value.

Neural stem cells (NSCs) provide a strategy to replace damaged neurons following traumatic central nervous system injuries. A major hurdle to translation of this therapy is that direct application of NSCs to CNS injury does not support sufficient neurogenesis due to lack of proper cues. To provide prolonged spatial cues to NSCs IFN-γ was immobilized to biomimetic hydrogel substrate to supply physical and biochemical signals to instruct the encapsulated NSCs to be neurogenic. However, the immobilization of factors, including IFN-γ, versus soluble delivery of the same factor, has been incompletely characterized especially with respect to activation of signaling and metabolism in cells over longer time points. In this study, protein and metabolite changes in NSCs induced by immobilized versus soluble IFN-γ at 7 days were evaluated. Soluble IFN-γ, refreshed daily over 7 days, elicited stronger responses in NSCs compared to immobilized IFN-γ, indicating that immobilization may not sustain signaling or has altered ligand/receptor interaction and integrity. However, both IFN-γ delivery types supported increased βIII tubulin expression in parallel with canonical and noncanonical receptor-signaling compared to no IFN-γ. Global metabolomics and pathway analysis revealed that soluble and immobilized IFN-γ altered metabolic pathway activities including energy, lipid, and amino acid synthesis, with soluble IFN-γ having the greatest metabolic impact overall. Finally, soluble and immobilized IFN-γ support mitochondrial voltage-dependent anion channel (VDAC) expression that correlates to differentiated NSCs. This work utilizes new methods to evaluate cell responses to protein delivery and provides insight into mode of action that can be harnessed to improve regenerative medicine-based strategies.

Here we present an evaluation of the binding affinity prediction accuracy of the free energy calculation method FEP+ on internal active drug discovery projects and on a large new public benchmark set.<br>

Highly bio-compatible probes (LC50 > 75 μM) with large Stokes shifts (Δλ > 150 nm) for quick staining of lysosomes (≈20 min) in live cells.

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