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Photoacoustic (PA) imaging has proved versatile for many biomedical applications from drug delivery tracking to disease diagnostics and postoperative surveillance. It recently emerged as a tool for accurate and real-time heparin monitoring to avoid bleeding complications associated with anticoagulant therapy. However, molecular-dye-based application is limited by high concentration requirements, photostability, and a strong background hemoglobin signal. We developed polydopamine nanocapsules (PNCs) viasupramolecular templates and loaded them with molecular dyes for enhanced PA-mediated heparin detection. Depending on surface charge, the dye-loaded PNCs undergo disassembly or aggregation upon heparin recognition: both experiments and simulation have revealed that the increased PA signal mainly results from dye-loaded PNC–heparin aggregation. Importantly, Nile blue (NB)-loaded PNCs generated a 10-fold higher PA signal than free NB dye, and such PNC enabled the direct detection of heparin in a clinically relevant therapeutic window (0–4 U/mL) in whole human blood (R2= 0.91). Furthermore, the PA signal of PNC@NB obtained from 17 patients linearly correlated with ACT values (R2= 0.73) and cumulative heparin (R2= 0.83). This PNC-based strategy for functional nanocapsules offers a versatile engineering platform for robust biomedical contrast agents and nanocarriers.

Recent experiments indicate that the C-Jun amino-terminal kinase-interacting protein 1 (JIP1) binds to and activates the c-Jun N-terminal kinase (JNK) protein. JNK is an integral part of cell apoptosis, and misregulation of this process is a causative factor in diseases such as Alzheimer’s disease (AD), obesity, and cancer. It has also been shown that JIP1 may increase the phosphorylation of tau by facilitating the interaction between the tau protein and JNK, which could also be a causative factor in AD. Very little is known about the structure and dynamics of JIP1; however, the amino acid composition of the first 350 residues suggests that it contains an intrinsically disordered region. Molecular dynamics (MD) simulations using AMBER 14 were used to study the structure and dynamics of a functionally active JIP1 10mer fragment to better understand the solution behavior of the fragment. Two microseconds of unbiased MD was performed on the JIP1 10mer fragment in 10 different seeds for a total of 20 μs of simulation time, and from this, seven structurally stable conformations of the 10mer fragment were identified via classical clustering. The 10mer ensemble was also used to build a Markov state model (MSM) that identified four metastable states that encompassed six of the seven conformational families identified by classical dimensional reduction. Based on this MSM, conformational interconversions between the four states occur via two dominant pathways with probability fluxes of 55 and 44% for each individual pathway. Transitions between the initial and final states occur with mean first passage times of 31 (forward) and 16 (reverse) μs.

Shigellais the causative agent of bacillary dysentery and is responsible for an estimated 165 million infections and 600,000 deaths annually. Like many Gram-negative pathogens, Shigellarelies on a type three secretion system (T3SS) to initiate and sustain infection by directly injecting effector proteins into host cells. Protein secretion through the needle-like injectisome and overall Shigellavirulence rely on the T3SS ATPase Spa47, making it a likely means for T3SS regulation and an attractive target for therapeutic small molecule inhibitors. Here, we utilize a recently solved 2.15 Å crystal structure of Spa47 to computationally screen 7.6 million drug-like compounds for candidates which avoid the highly conserved active site by targeting a distal, but critical, interface between adjacent protomers of the Spa47 homohexamer. Ten of the top inhibitor candidates were characterized, identifying novel Spa47 inhibitors that reduce in vitroATPase activity by as much as 87.9 ± 10.5% with IC50’s as low as 25 ± 20 μM and reduce in vivo ShigellaT3SS protein secretion by as much as 94.7 ± 3.0%. Kinetic analyses show that the inhibitors operate through a noncompetitive mechanism that likely supports the inhibitors’ low cytotoxicity, as they avoid off-target ATPases involved in either Shigellaor mammalian cell metabolism. Interestingly, the inhibitors display nearly identical inhibition profiles for Spa47 and the T3SS ATPases EscN from E. coliand FliI from Salmonella.Together, the results of this study provide much-needed insight into T3SS ATPase inhibition mechanisms and a strong platform for developing broadly effective cross-pathogen T3SS ATPase inhibitors.

Optimizing DNA polymerases for a broad range of tasks requires an understanding of the factors influencing polymerase fidelity, but many details of polymerase behavior remain unknown, especially in the presence of mismatched nascent base pairs. Using molecular dynamics, the large fragment of Bacillus stearothermophilusDNA polymerase I is simulated in the presence of all 16 possible standard nucleoside triphosphate-template (dNTP-dN) pairs, including four Watson–Crick pairs and 12 mismatches. The precatalytic steps of nucleotide addition from nucleotide insertion to immediately preceding catalysis are explored using three starting structures representing different stages of nucleotide addition. From these simulations, interactions between dNTPs and the DNA–protein complex formed by the polymerase are elucidated. Patterns of large-scale conformational shifts, classification of nucleotide pairs based on composition, and investigation of the roles of residues interacting with dNTPs are completed on 50+ μs of simulation. The role of molecular dynamics in studies of polymerase behavior is discussed.