Energy coupling and stoichiometry of Zn2+/H+ antiport by the prokaryotic cation diffusion facilitator YiiP.

YiiP from Shewanella oneidensis is a prokaryotic Zn2+/H+ antiporter that serves as a model for the Cation Diffusion Facilitator (CDF) superfamily, members of which are generally responsible for homeostasis of transition metal ions. Previous studies of YiiP as well as related CDF transporters have established a homodimeric architecture and the presence of three distinct Zn2+ binding sites named A, B, and C. In this study, we use cryo-EM, microscale thermophoresis and molecular dynamics simulations to address the structural and functional roles of individual sites as well as the interplay between Zn2+ binding and protonation. Structural studies indicate that site C in the cytoplasmic domain is primarily responsible for stabilizing the dimer and that site B at the cytoplasmic membrane surface controls the structural transition from an inward facing conformation to an occluded conformation. Binding data show that intramembrane site A, which is directly responsible for transport, has a dramatic pH dependence consistent with coupling to the proton motive force. A comprehensive thermodynamic model encompassing Zn2+ binding and protonation states of individual residues indicates a transport stoichiometry of 1 Zn2+ to 2-3 H+ depending on the external pH. This stoichiometry would be favorable in a physiological context, allowing the cell to use the proton gradient as well as the membrane potential to drive the export of Zn2+.

Energy Coupling and Stoichiometry of Zn2+/H+ Antiport by the Cation Diffusion Facilitator YiiP.

YiiP is a prokaryotic Zn2+/H+ antiporter that serves as a model for the Cation Diffusion Facilitator (CDF) superfamily, members of which are generally responsible for homeostasis of transition metal ions. Previous studies of YiiP as well as related CDF transporters have established a homodimeric architecture and the presence of three distinct Zn2+ binding sites named A, B, and C. In this study, we use cryo-EM, microscale thermophoresis and molecular dynamics simulations to address the structural and functional roles of individual sites as well as the interplay between Zn2+ binding and protonation. Structural studies indicate that site C in the cytoplasmic domain is primarily responsible for stabilizing the dimer and that site B at the cytoplasmic membrane surface controls the structural transition from an inward facing conformation to an occluded conformation. Binding data show that intramembrane site A, which is directly responsible for transport, has a dramatic pH dependence consistent with coupling to the proton motive force. A comprehensive thermodynamic model encompassing Zn2+ binding and protonation states of individual residues indicates a transport stoichiometry of 1 Zn2+ to 2-3 H+ depending on the external pH. This stoichiometry would be favorable in a physiological context, allowing the cell to use the proton gradient as well as the membrane potential to drive the export of Zn2+.

DTX-P7, a peptide-drug conjugate, is highly effective for non-small cell lung cancer.

Despite tremendous success of molecular targeted therapy together with immunotherapy, only a small subset of patients can benefit from them. Chemotherapy remains the mainstay treatment for most of tumors including non-small cell lung cancer (NSCLC); however, non-selective adverse effects on healthy tissues and secondary resistance are the main obstacles. Meanwhile, the quiescent or dormant cancer stem-like cells (CSLCs) are resistant to antimitotic chemoradiotherapy. Complete remission can only be realized when both proliferative cancer cells and quiescent cancer stem cells are targeted. In the present research, we constructed a cooperatively combating conjugate (DTX-P7) composed of docetaxel (DTX) and a heptapeptide (P7), which specifically binds to cell surface Hsp90, and assessed the anti-tumor effects of DTX-P7 on non-small cell lung cancer. DTX-P7 preferentially suppressed tumor growth compared with DTX in vivo with a favorable distribution to tumor tissues and long circulation half-life. Furthermore, we revealed a distinctive mechanism whereby DTX-P7 induced unfolded protein response and eventually promoted apoptosis. More importantly, we found that DTX-P7 promoted cell cycle reentry of slow-proliferating CSLCs and subsequently killed them, exhibiting a "proliferate to kill" pattern. Collecitvely, by force of active targeting delivery of DTX via membrane-bound Hsp90, DTX-P7 induces unfolded protein response and subsequent apoptosis by degrading Hsp90, meanwhile awakens and kills the dormant cancer stem cells. Thus, DTX-P7 deserves further development as a promising anticancer therapeutic for treatment of various membrane-harboring Hsp90 cancer types.

Precise force-field-based calculations of octanol-water partition coefficients for the SAMPL7 molecules.

We predicted water-octanol partition coefficients for the molecules in the SAMPL7 challenge with explicit solvent classical molecular dynamics (MD) simulations. Water hydration free energies and octanol solvation free energies were calculated with a windowed alchemical free energy approach. Three commonly used force fields (AMBER GAFF, CHARMM CGenFF, OPLS-AA) were tested. Special emphasis was placed on converging all simulations, using a criterion developed for the SAMPL6 challenge. In aggregate, over 1000 [Formula: see text]s of simulations were performed, with some free energy windows remaining not fully converged even after 1 [Formula: see text]s of simulation time. Nevertheless, the amount of sampling produced [Formula: see text] estimates with a precision of 0.1 log units or better for converged simulations. Despite being probably as fully sampled as can expected and is feasible, the agreement with experiment remained modest for all force fields, with no force field performing better than 1.6 in root mean squared error. Overall, our results indicate that a large amount of sampling is necessary to produce precise [Formula: see text] predictions for the SAMPL7 compounds and that high precision does not necessarily lead to high accuracy. Thus, fundamental problems remain to be solved for physics-based [Formula: see text] predictions.

Zinc binding alters the conformational dynamics and drives the transport cycle of the cation diffusion facilitator YiiP.

YiiP is a secondary transporter that couples Zn2+ transport to the proton motive force. Structural studies of YiiP from prokaryotes and Znt8 from humans have revealed three different Zn2+ sites and a conserved homodimeric architecture. These structures define the inward-facing and outward-facing states that characterize the archetypal alternating access mechanism of transport. To study the effects of Zn2+ binding on the conformational transition, we use cryo-EM together with molecular dynamics simulation to compare structures of YiiP from Shewanella oneidensis in the presence and absence of Zn2+. To enable single-particle cryo-EM, we used a phage-display library to develop a Fab antibody fragment with high affinity for YiiP, thus producing a YiiP/Fab complex. To perform MD simulations, we developed a nonbonded dummy model for Zn2+ and validated its performance with known Zn2+-binding proteins. Using these tools, we find that, in the presence of Zn2+, YiiP adopts an inward-facing conformation consistent with that previously seen in tubular crystals. After removal of Zn2+ with high-affinity chelators, YiiP exhibits enhanced flexibility and adopts a novel conformation that appears to be intermediate between inward-facing and outward-facing states. This conformation involves closure of a hydrophobic gate that has been postulated to control access to the primary transport site. Comparison of several independent cryo-EM maps suggests that the transition from the inward-facing state is controlled by occupancy of a secondary Zn2+ site at the cytoplasmic membrane interface. This work enhances our understanding of individual Zn2+ binding sites and their role in the conformational dynamics that govern the transport cycle.

Prediction of octanol-water partition coefficients for the SAMPL6-[Formula: see text] molecules using molecular dynamics simulations with OPLS-AA, AMBER and CHARMM force fields.

All-atom molecular dynamics simulations with stratified alchemical free energy calculations were used to predict the octanol-water partition coefficient [Formula: see text] of eleven small molecules as part of the SAMPL6-[Formula: see text] blind prediction challenge using four different force field parametrizations: standard OPLS-AA with transferable charges, OPLS-AA with non-transferable CM1A charges, AMBER/GAFF, and CHARMM/CGenFF. Octanol parameters for OPLS-AA, GAFF and CHARMM were validated by comparing the density as a function of temperature, the chemical potential, and the hydration free energy to experimental values. The partition coefficients were calculated from the solvation free energy for the compounds in water and pure ("dry") octanol or "wet" octanol with 27 mol% water dissolved. Absolute solvation free energies were computed by thermodynamic integration (TI) and the multistate Bennett acceptance ratio with uncorrelated samples from data generated by an established protocol using 5-ns windowed alchemical free energy perturbation (FEP) calculations with the Gromacs molecular dynamics package. Equilibration of sets of FEP simulations was quantified by a new measure of convergence based on the analysis of forward and time-reversed trajectories. The accuracy of the [Formula: see text] predictions was assessed by descriptive statistical measures such as the root mean square error (RMSE) of the data set compared to the experimental values. Discarding the first 1 ns of each 5-ns window as an equilibration phase had a large effect on the GAFF data, where it improved the RMSE by up to 0.8 log units, while the effect for other data sets was smaller or marginally worsened the agreement. Overall, CGenFF gave the best prediction with RMSE 1.2 log units, although for only eight molecules because the current CGenFF workflow for Gromacs does not generate files for certain halogen-containing compounds. Over all eleven compounds, GAFF gave an RMSE of 1.5. The effect of using a mixed water/octanol solvent slightly decreased the accuracy for CGenFF and GAFF and slightly increased it for OPLS-AA. The GAFF and OPLS-AA results displayed a systematic error where molecules were too hydrophobic whereas CGenFF appeared to be more balanced, at least on this small data set.

Clinical relevance of gemstone spectral CT in the diagnosis of carotid atherosclerosis.

[This retracts the article DOI: 10.3892/etm.2017.4342.].

Improvement of mitochondrial function mediated the neuroprotective effect of 5-(4-hydroxy-3-dimethoxybenzylidene)-2-thioxo-4-thiazolidinone in rats with cerebral ischemia-reperfusion injuries.

Deficits in mitochondrial function is a critical inducement in the major pathways that drive neuronal cell death in ischemic process particularly. Drugs target to improve the mitochondrial function may be a feasible therapeutic choice in treatment with ischemic diseases. In the present study, we investigated whether 5-(4-hydroxy-3-dimethoxybenzylidene)-2-thioxo-4-thiazolidinone (RD-1), a compound derived from rhodanine, could protect against ischemic neuronal damage via improving mitochondrial function. We tested the neuroprotective effect of RD-1 both in rats modeled by middle cerebral artery occlusion reperfusion in vivo and in primary cortical neurons subjected to hypoxia/reperfusion injury in vitro. Results showed that treatment with RD-1 for 14 days remarkably reduced infarct size, decreased neurological deficit score and accelerated the recovery of somatosensory function in vivo. Meanwhile, RD-1 also increased the cellular viability after 48 h treatment in vitro. In addition, RD-1 protected the primary cortical neurons against mitochondrial damage as evidenced by stabilizing the mitochondrial membrane potential and reducing the overproduction of reactive oxygen species. Furthermore, hypoxia/reperfusion injury induced damaged mitochondrial axonal transport and consequently neurotransmitter release disorder, which were ameliorated by RD-1 treatment. Besides, RD-1 inhibited the downregulation of proteins related with mitochondrial transport and neurotransmitter release induced by ischemic injury both in vivo and in vitro. The obtained data demonstrated the neuroprotective effect of RD-1 and the involved mechanisms were partially attributed to the improvement in mitochondrial function and the synaptic activity. Our study indicated that RD-1 may be a potential therapeutic drug for the ischemic stroke therapy.

Proteasome Inhibitor YSY01A Abrogates Constitutive STAT3 Signaling via Down-regulation of Gp130 and JAK2 in Human A549 Lung Cancer Cells.

Proteasome inhibition interfering with many cell signaling pathways has been extensively explored as a therapeutic strategy for cancers. Proteasome inhibitor YSY01A is a novel agent that has shown remarkable anti-tumor effects; however, its mechanisms of action are not fully understood. Here we report that YSY01A is capable of suppressing cancer cell survival by induction of apoptosis. Paradoxically, we find that YSY01A abrogates constitutive activation of STAT3 via proteasome-independent degradation of gp130 and JAK2, but not transcriptional regulation, in human A549 non-small cell lung cancer cells. The reduction in gp130 and JAK2 can be restored by co-treatment with 3-methyladenine, an early-stage autophagy lysosome and type I/III PI3K inhibitor. YSY01A also effectively inhibits cancer cell migration and lung xenograft tumor growth with little adverse effect on animals. Thus, our findings suggest that YSY01A represents a promising candidate for further development of novel anticancer therapeutics targeting the proteasome.

Clinical relevance of gemstone spectral CT in the diagnosis of carotid atherosclerosis.

A variety of imaging methods can be used in the diagnosis of atherosclerotic plaques. In the present study, we investigated the morphology and composition of atherosclerotic plaque associated with ischemic cerebral infarction by comparing gemstone spectral computed tomography (GSCT) and traditional multi-slice CT (MSCT). In total, 200 patients were enrolled and divided into the experimental group (n=100), which underwent GSCT, and the control group (n=100), which underwent MSCT. All the cases were followed up to observe disease outcomes in patients with different atherosclerotic plaque types, and adverse events in carotid artery stenosis or cerebral infarction were recorded. Compared with traditional MSCT, sensitivity (93.2%), specificity (84.5%), and accuracy (91.0%) of GSCT were significantly higher. We found a correlation between vulnerable plaque of carotid atherosclerotic plaque and the occurrence of cerebral infarction. These results suggest the advantages of GSCT in analyzing atherosclerotic plaque and predicting the risk of ischemic cerebral infarction.

Compound porcine cerebroside and ganglioside injection attenuates cerebral ischemia-reperfusion injury in rats by targeting multiple cellular processes.

BACKGROUND: Compound porcine cerebroside and ganglioside injection (CPCGI) is a neurotrophic drug used clinically to treat certain functional disorders of brain. Despite its extensive usage throughout China, the exact mechanistic targets of CPCGI are unknown. This study was carried out to investigate the protective effect of CPCGI against ischemic neuronal damage in rats with middle cerebral artery occlusion (MCAO) reperfusion injury and to investigate the neuroprotective mechanisms of CPCGI. MATERIALS AND METHODS: Adult male Sprague-Dawley rats were subjected to MCAO surgery for 2 hours followed by reperfusion. The rats were administered CPCGI once a day for 14 days after reperfusion, and behavioral tests were performed 1, 3, 7, and 14 days post MCAO. Hematoxylin-eosin staining was used to measure infarct volume, and immunohistochemical analysis was performed to determine the number of NeuN-positive neurons in the ischemic cortex penumbra. Finally, the relative expression levels of proteins associated with apoptosis (Bcl-2, Bax, and GADD45α), synaptic function (Synaptophysin, SNAP25, Syntaxin, and Complexin-1/2), and mitochondrial function (KIFC2 and UCP3) were determined by Western blot. RESULTS: CPCGI treatment reduced infarct size, decreased neurological deficit scores, and accelerated the recovery of somatosensory function 14 days after MCAO. In addition, CPCGI reduced the loss of NeuN-positive cells in the ischemic cortex penumbra. In the ischemic cortex, CPCGI treatment decreased GADD45α expression, increased the Bcl-2/Bax ratio, augmented Synaptophysin, SNAP25, and Complexin-1/2 expression, and increased the expression of KIFC2 and UCP3 compared with sham rats 14 days after MCAO reperfusion injury. CONCLUSION: CPCGI displays neuroprotective properties in rats subjected to MCAO injury by inhibiting apoptosis and improving synaptic and mitochondrial function.