Thermodynamics of Proton-Coupled Electron Transfer at Tricopper µ-Oxo/Hydroxo/Aqua Complexes.

Multicopper oxidases (MCOs) utilize a tricopper active site to reduce dioxygen to water through 4H+ 4e- proton-coupled electron transfer (PCET). Understanding the thermodynamics of PCET at a tricopper cluster is essential for elucidating how MCOs harness the oxidative power of O2 while mitigating oxidative damage. In this study, we determined the O-H bond dissociation free energies (BDFEs) and pKa values of a series of tricopper hydroxo and tricopper aqua complexes as synthetic models of the tricopper site in MCOs. Tricopper intermediates on the path of alternating electron and proton transfer (ET-PT-ET-PT-ET) have modest BDFE(O-H) values in the range of 53.0-57.1 kcal/mol. In contrast, those not on the path of ET-PT-ET-PT-ET display much higher (78.1 kcal/mol) or lower (44.7 kcal/mol) BDFE(O-H) values. Additionally, the pKa of bridging OH and OH2 motifs increase by 8-16 pKa units per oxidation state. The same oxidation state changes have a lesser impact on the pKa of N-H motif in the secondary coordination sphere, with an increase of ca. 5 pKa units per oxidation state. The steeper pKa increase of the tricopper center promotes proton transfer from the secondary coordination sphere. Overall, our study shed light on the PCET pathway least prone to decomposition, elucidating why tricopper centers are an optimal choice for promoting efficient oxygen reduction reaction.

Advances in Injectable Hydrogels Based on Diverse Gelation Methods for Biomedical Imaging.

The injectable hydrogels can deliver the loads directly to the predetermined sites and form reservoirs to increase the enrichment and retention of the loads in the target areas. The preparation and injection of injectable hydrogels involve the sol-gel transformation of hydrogels, which is affected by factors such as temperature, ions, enzymes, light, mechanics (self-healing property), and pH. However, tracing the injection, degradation, and drug release from hydrogels based on different ways of gelation is a major concern. To solve this problem, contrast agents are introduced into injectable hydrogels, enabling the hydrogels to be imaged under techniques such as fluorescence imaging, photoacoustic imaging, magnetic resonance imaging, and radionuclide imaging. This review details methods for causing the gelation of imageable hydrogels; discusses the application of injectable hydrogels containing contrast agents in various imaging techniques, and finally explores the potential and challenges of imageable hydrogels based on different modes of gelation.

Gaseous Nitrogen Oxides Catholyte for Rechargeable Redox Flow Batteries.

There is a strong interest in finding highly soluble redox compounds to improve the energy density of redox flow batteries (RFBs). However, the performance of electrolytes is often negatively influenced by high solute concentration. Herein, we designed a high-potential (0.5 V vs. Ag/Ag+ ) catholyte for RFBs, where the charged and discharged species are both gaseous nitrogen oxides (NOx ). These species can be liberated from the liquid electrolyte and stored in a separate gas container, allowing scale-up of storage capacity without increasing the concentration and volume of the electrolyte. The oxidation of NO in the presence of NO3 - affords N2 O3 , and the reduction of N2 O3 regenerates NO and NO3 - , together affording the electrochemical reaction: NO3 - +3 NO⇌2 N2 O3 +e- with a low mass/charge ratio of 152 grams per mole of stored electron. A proof-of-concept NOx symmetric H-cell shows 200 stable cycles over 400 hours with >97 % Coulombic efficiency and negligible capacity decay.

Reductive NO Coupling at Dicopper Center via a [Cu2(NO)2]2+ Diamond-Core Intermediate.

Treatment of a dicopper(I,I) complex with excess amounts of NO leads to the formation of a dicopper dinitrosyl [Cu2(NO)2]2+ complex capable of (i) releasing two equivalents of NO reversibly in 90% yield and (ii) reacting with another equivalent of NO to afford N2O and dicopper nitrosyl oxo species [Cu2(NO)(O)]2+. Resonance Raman characterization of the [Cu2(NO)2]2+ complex shows a 15N-sensitive N═O stretch at 1527.6 cm-1 and two Cu-N stretches at 390.6 and 414.1 cm-1, supporting a symmetric diamond-core structure with bis-µ-NO ligands. The conversion of [Cu2(NO)2]2+ to [Cu2(NO)O]2+ occurs via a rate-limiting reaction with NO and bypasses the dicopper oxo intermediate, a mechanism distinct from that of diFe-mediated NO reduction to N2O.

Revealing the Effects of Endoplasmic Reticulum Stress on Ferroptosis by Two-Channel Real-Time Imaging of pH and Viscosity.

Endoplasmic reticulum (ER) is sensitive to changes in the intracellular environment such as pH and viscosity, and slight changes may trigger stress response. Besides, different from apoptosis and necrosis, ferroptosis is the result of lipid peroxidation accumulation. There is evidence that ferroptosis is closely related to endoplasmic reticulum stress (ERS). However, the possible changes in the pH and viscosity of the ER during the ferroptosis process have not yet been studied. Therefore, we used a new type of ER-targeted dual-excitation fluorescent probe (DSPI-3) to investigate the possible changes of pH and viscosity of ER during the ferroptosis. The novel probe DSPI-3 exhibited a highly sensitive and selective response to pH and viscosity. During the bioimaging process, it was found that the ER acidified and viscosity increased during the ferroptosis process induced by erastin, while the cells treated with ferrostatin-1 did not alter significantly. In addition, when dithiothreitol (DTT) and erastin stimulated the cells at the same time, we discovered that ER was acidified considerably at short notice, but the pH was slightly increased in the later stage. Besides, the change of the viscosity enhanced slowly with the passage of time, and there was a noteworthy decline in the later stage, demonstrating that the DTT-induced ERS accelerated the process of ferroptosis. We hope that this unique fluorescent probe can provide an effective method for studying the relationship between ERS and ferroptosis.

Multiple Proton-Coupled Electron Transfers at a Tricopper Cluster: Modeling the Reductive Regeneration Process in Multicopper Oxidases.

Metal clusters in enzymes carry out the life-sustaining reactions by accumulating multiple redox equivalents in a narrow potential range. This redox potential leveling effect commonly observed in Nature has yet to be reproduced with synthetic metal clusters. Herein, we employ a fully encapsulated synthetic tricopper complex to model the three-electron two-proton reductive regeneration of fully reduced trinuclear copper cluster CuICuICuI(µ2-OH2) (FR) from native intermediate CuIICuIICuII(µ3-O) (NI) in multicopper oxidases (MCOs). The tricopper cluster can access four oxidation states (I,I,I to II,II,II) and four protonation states ([Cu3(µ3-O)]LH, [Cu3(µ3-OH)]L, [Cu3(µ3-OH)]LH, and [Cu3(µ3-OH2)]L, where LH denotes the protonated ligand), allowing mechanistic investigation of proton-coupled electron transfer (PCET) relevant to MCOs. Seven tricopper complexes with discrete oxidation and protonation states were characterized with spectroscopy or X-ray single-crystal diffraction. A stepwise electron transfer-proton transfer (ET-PT) mechanism is established for the reduction of CuIICuIICuII(µ3-O)LH to CuIICuIICuI(µ3-OH)L, while a stepwise PT-ET mechanism is determined for the reduction of CuIICuICuI(µ3-OH)LH to CuICuICuI(µ2-OH2)L. The switch-over from ET-PT to PT-ET mechanism showcases that the tricopper complex can adopt different PCET mechanisms to circumvent high-barrier proton transfer steps. Overall, three-electron two-proton reduction occurs within a narrow potential range of 170 mV, exemplifying the redox potential leveling effect of secondary proton relays in delivering multiple redox equivalents at metal clusters.

A novel ER-targeted two-photon fluorescent probe for monitoring abnormal concentrations of HClO in diabetic mice.

Diabetes is closely related to the presence of excess HClO induced by endoplasmic reticulum stress. Thus, a novel two-photon fluorescent probe was designed and synthesized for the detection of HClO in the endoplasmic reticulum. Significantly, it has been verified that high glucose can indeed induce oxidative stress of the endoplasmic reticulum and produce excessive HClO. Moreover, the probe has also been successfully used in tissue imaging of diabetic mice.

Experimental Study on Timber-Lightweight Concrete Composite Beams with Ductile Bolt Connectors.

A timber-lightweight-concrete (TLC) composite beam connected with a ductile connector in which the ductile connector is made of a stainless-steel bolt anchored with nuts at both ends was proposed. The push-out results and bending performance of the TLC composite specimens were investigated by experimental testing. The push-out results of the shear specimens show that shear-slip curves exhibit good ductility and that their failure can be attributed to bolt buckling accompanied by lightweight concrete cracking. Through the bending tests of ten TLC composite beams and two contrast (pure timber) beams, the effects of different bolt diameters on the strengthening effect of the TLC composite beams were studied. The results show that the TLC composite beams and contrast timber beams break on the timber fiber at the lowest edge of the TLC composite beam, and the failure mode is attributed to bending failure, whereas the bolt connectors and lightweight concrete have no obvious breakage; moreover, the ductile bolt connectors show a good connection performance until the TLC composite beams fail. The ultimate bearing capacities of the TLC composite beams increase 2.03-3.5 times compared to those of the contrast beams, while the mid-span maximum deformation decrease nearly doubled.

RNA Sequencing-Associated Study Identifies GmDRR1 as Positively Regulating the Establishment of Symbiosis in Soybean.

In soybean (Glycine max)-rhizobium interactions, the type III secretion system (T3SS) of rhizobium plays a key role in regulating host specificity. However, the lack of information on the role of T3SS in signaling networks limits our understanding of symbiosis. Here, we conducted an RNA sequencing analysis of three soybean chromosome segment substituted lines, one female parent and two derived lines with different chromosome-substituted segments of wild soybean and opposite nodulation patterns. By analyzing chromosome-linked differentially expressed genes in the substituted segments and quantitative trait loci (QTL)-assisted selection in the substituted-segment region, genes that may respond to type III effectors to mediate plant immunity-related signaling were identified. To narrow down the number of candidate genes, QTL assistant was used to identify the candidate region consistent with the substituted segments. Furthermore, one candidate gene, GmDRR1, was identified in the substituted segment. To investigate the role of GmDRR1 in symbiosis establishment, GmDRR1-overexpression and RNA interference soybean lines were constructed. The nodule number increased in the former compared with wild-type soybean. Additionally, the T3SS-regulated effectors appeared to interact with the GmDDR1 signaling pathway. This finding will allow the detection of T3SS-regulated effectors involved in legume-rhizobium interactions.

Encapsulation of tricopper cluster in a synthetic cryptand enables facile redox processes from CuICuICuI to CuIICuIICuII states.

One-pot reaction of tris(2-aminoethyl)amine (TREN), [CuI(MeCN)4]PF6, and paraformaldehyde affords a mixed-valent [TREN4CuIICuICuI(µ3-OH)](PF6)3 complex. The macrocyclic azacryptand TREN4 contains four TREN motifs, three of which provide a bowl-shape binding pocket for the [Cu3(µ3-OH)]3+ core. The fourth TREN caps on top of the tricopper cluster to form a cryptand, imposing conformational constraints and preventing solvent interaction. Contrasting the limited redox capability of synthetic tricopper complexes reported so far, [TREN4CuIICuICuI(µ3-OH)](PF6)3 exhibits several reversible single-electron redox events. The distinct electrochemical behaviors of [TREN4CuIICuICuI(µ3-OH)](PF6)3 and its solvent-exposed analog [TREN3CuIICuIICuII(µ3-O)](PF6)4 suggest that isolation of tricopper core in a cryptand enables facile electron transfer, allowing potential application of synthetic tricopper complexes as redox catalysts. Indeed, the fully reduced [TREN4CuICuICuI(µ3-OH)](PF6)2 can reduce O2 under acidic conditions. The geometric constraints provided by the cryptand are reminiscent of Nature's multicopper oxidases (MCOs). For the first time, a synthetic tricopper cluster was isolated and fully characterized at CuICuICuI (4a), CuIICuICuI (4b), and CuIICuIICuI (4c) states, providing structural and spectroscopic models for many intermediates in MCOs. Fast electron transfer rates (105 to 106 M-1 s-1) were observed for both CuICuICuI/CuIICuICuI and CuIICuICuI/CuIICuIICuI redox couples, approaching the rapid electron transfer rates of copper sites in MCO.

The identification of novel small-molecule inhibitors targeting WDR5-MLL1 interaction through fluorescence polarization based high-throughput screening.

The protein-protein interaction between WDR5 (WD40 repeat protein 5) and MLL1 (mixed-lineage leukemia 1) is important for maintaining optimal H3K4 methyltransferase activity of MLL1. Dysregulation of MLL1 catalytic function is relevant to mixed-lineage leukemia, and targeting WDR5-MLL1 interaction could be a promising therapeutic strategy for leukemia harboring MLL1 fusion proteins. To date, several peptidomimetic and non-peptidomimetic small-molecule inhibitors targeting WDR5-MLL1 interaction have been reported, yet the discovery walk of new drugs inhibiting MLL1 methytransferase activity is still in its infancy. It's urgent to find other small-molecule WDR5-MLL1 inhibitors with novel scaffolds. In this study, through fluorescence polarization (FP)-based high throughput screening, several small-molecule inhibitors with potent inhibitory activities in vitro against WDR5-MLL1 interaction were discovered. Nuclear Magnetic Resonance (NMR) assays were carried out to confirm the direct binding between hit compounds and WDR5. Subsequent similarity-based analog searching of the 4 hits led to several inhibitors with better activity, among them, DC_M5_2 displayed highest inhibitory activity with IC50 values of 9.63 ±â€¯1.46 µM. Furthermore, a molecular docking study was performed and disclosed the binding modes and interaction mechanisms between two most potent inhibitors and WDR5.

Identification of Selective, Cell Active Inhibitors of Protein Arginine Methyltransferase 5 through Structure-Based Virtual Screening and Biological Assays.

Protein arginine methyltransferase 5 (PRMT5), a type II PRMT enzyme, is reported as an important therapeutic target in leukemia and lymphoma. In the present study, based on the combination of virtual screening and biochemical validations, we discovered a series of small-molecule inhibitors targeting PRMT5. Among those, DC_Y134 exhibited the most potent activity with IC50 value of 1.7 µM and displayed good selectivity against other methyltransferases. Further treatment with DC_Y134 inhibited the proliferation of several hematological malignancy cell lines by causing cell cycle arrest and apoptosis. Western blot assays indicated that DC_Y134 reduced the cellular symmetrically dimethylated levels. In addition, we analyzed the binding mode of DC_Y134 through molecular docking, which revealed that DC_Y134 occupies the binding site of substrate arginine and explained the selectivity of this inhibitor. Taken together, compound DC_Y134 could be used to elucidate the biological roles of PRMT5 and serve as a lead compound for treatment of hematologic malignancies.

Microspheres of Carboxymethyl Chitosan, Sodium Alginate, and Collagen as a Hemostatic Agent in Vivo.

In the search for biocompatible composite microspheres to be used as a hemostatic agent, in a previous study, we designed a novel biomaterial, consisting of composite microspheres containing three natural biological ingredients, carboxymethyl chitosan, sodium alginate and collagen (CSCM). Furthermore, the chemical and physical properties, hemostatic ability, biocompatibility and cytotoxicity were investigated in vitro. In this work, the in vivo hemostatic performance, wound healing, hemocompatibility, histocompatibility, and biodegradability were evaluated by a series of experiments. The results showed that CSCM could both stop bleeding and enhance healing efficiency by accelerating the clotting and the wound closure rate, suggesting that CSCM acts as a hemostat, and enhances wound healing. In addition, the CSCM material had negligible intracutaneous stimulation reactions and no obvious hemolytic reactions. More importantly, CSCM can be degraded in vivo without significant impacts on physiology, biochemistry, and organization. Thus, CSCM may be a useful tool to stop bleeding in emergency conditions in both military and civilian settings.

Discovery of alkyl bis(oxy)dibenzimidamide derivatives as novel protein arginine methyltransferase 1 (PRMT1) inhibitors.

Protein arginine methylation, a post-translational modification critical for a variety of biological processes, is catalyzed by protein arginine N-methyltransferases (PRMTs). In particular, PRMT1 is responsible for over 85% of the arginine methylation in mammalian cells. Dysregulation of PRMT1 is involved in diverse pathological diseases including cancers. However, most current PRMT1 inhibitors are lack of specificity, efficacy, and bioavailability. Herein, a series of alkyl bis(oxy)dibenzimidamide derivatives were identified as selective PRMT1 inhibitors. Among them, the most potent compound corresponds to hexamidine (IC50  = 5.9 ± 1.7 µm), which is an antimicrobial agent. The binding between hexamidine and PRMT1 was further validated by thermal shift assays and nuclear magnetic resonance (NMR) experiments. Molecular docking and NMR assays indicated that hexamidine occupied the substrate binding pocket. Furthermore, hexamidine effectively blocked cell proliferation in cancer cell lines related to PRMT1 overexpression. Taken together, this study has provided a druggable scaffold targeting PRMT1 as well as a new way to repurpose old drugs which is a complementary tool for the discovery of new lead compounds.

Identification of Novel Inhibitors against Coactivator Associated Arginine Methyltransferase 1 Based on Virtual Screening and Biological Assays.

Overexpression of coactivator associated arginine methyltransferase 1 (CARM1), a protein arginine N-methyltransferase (PRMT) family enzyme, is associated with various diseases including cancers. Consequently, the development of small-molecule inhibitors targeting PRMTs has significant value for both research and therapeutic purposes. In this study, together with structure-based virtual screening with biochemical assays, two compounds DC_C11 and DC_C66 were identified as novel inhibitors of CARM1. Cellular studies revealed that the two inhibitors are cell membrane permeable and effectively blocked proliferation of cancer cells including HELA, K562, and MCF7. We further predicted the binding mode of these inhibitors through molecular docking analysis, which indicated that the inhibitors competitively occupied the binding site of the substrate and destroyed the protein-protein interactions between CARM1 and its substrates. Overall, this study has shed light on the development of small-molecule CARM1 inhibitors with novel scaffolds.

Self-assembly of photoswitchable diblock copolymers: salt-induced micellization and the influence of UV irradiation.

Salt-induced self-assemblies of poly(ethylene glycol-block-spiropyran methacrylate) (PEG-b-PSPMA) block copolymers were studied in this research. PEG-b-PSPMA block copolymers were dissolved in a 10 : 1 N,N-dimethyl-formamide (DMF)/water mixture. Upon ultraviolet light (UV) irradiation, the pendant spiropyran (SP) groups in the PSPMA blocks were isomerized into open merocyanine (MC) forms and the addition of inorganic salts (CuCl2, FeCl3 and Zn(CH3COO)2) induced micellization of PEG-b-PSPMA block copolymers in the solutions. In a salt-induced micelle, complexes formed by PSPMA and inorganic ions are in the cores and PEG chains are in the coronae. The reverse conversion of the isomers from MC form to SP form in the dark was studied by UV-vis, and the self-assembled aggregates were analyzed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The self-assembly of PEG-b-PSPMA in aqueous medium was also investigated. In aqueous solution, amphiphilic PEG-b-PSPMA self-assembled into micelles with the hydrophobic PSPMA blocks in the cores and the hydrophilic PEG blocks in the coronae. Upon UV irradiation, the hydrophobic SP units in the cores were isomerized into hydrophilic MC forms. The MC isomers have the attractive MC-MC interactions, and the reversion from MC to SP in the dark is difficult. DLS and TEM results both demonstrated that the micelles self-assembled by PEG-b-PSPMA did not disassemble upon UV irradiation, due to the attractive MC-MC interactions in the cores.