Proton-Coupled Electron Transfer upon Oxidation of Tyrosine in a De Novo Protein: Analysis of Proton Acceptor Candidates.

Redox-active residues, such as tyrosine and tryptophan, play important roles in a wide range of biological processes. The α3Y de novo protein, which is composed of three α helices and a tyrosine residue Y32, provides a platform for investigating the redox properties of tyrosine in a well-defined protein environment. Herein, the proton-coupled electron transfer (PCET) reaction that occurs upon oxidation of tyrosine in this model protein by a ruthenium photosensitizer is studied by using a vibronically nonadiabatic PCET theory that includes hydrogen tunneling and excited vibronic states. The input quantities to the analytical nonadiabatic rate constant expression, such as the diabatic proton potential energy curves and associated proton vibrational wave functions, reorganization energy, and proton donor-acceptor distribution functions, are obtained from density functional theory calculations on model systems and molecular dynamics simulations of the solvated α3Y protein. Two possible proton acceptors, namely, water or a glutamate residue in the protein scaffold, are explored. The PCET rate constant is greater when glutamate is the proton acceptor, mainly due to the more favorable driving force and shorter equilibrium proton donor-acceptor distance, although contributions from excited vibronic states mitigate these effects. Nevertheless, water could be the dominant proton acceptor if its equilibrium constant associated with hydrogen bond formation is significantly greater than that for glutamate. Although these calculations do not definitively identify the proton acceptor for this PCET reaction, they elucidate the conditions under which each proton acceptor can be favored. These insights have implications for tyrosine-based PCET in a wide variety of biochemical processes.

Determination of Methamphetamine by High-Performance Liquid Chromatography in Odor-Adsorbent Material Used for Training Drug-Detection Animals.

The objective of the present report was to develop and validate a simple, sensitive, and selective analytical method for the determination of methamphetamine in an odor-adsorbent material (gauze) which was used to improve and standardize the training method used for drug-detection animals. High-performance liquid chromatography (HPLC) was performed using a Spherisorb ODS2 C18 column (200 mm × 4.6 mm, 5 µm), with a mobile phase consisting of a 0.25% methanol/triethylamine aqueous solution (V:V = 20:80), the pH of which was adjusted to 3.1 using glacial acetic acid, at a flow rate of 1.0 mL/min. The column temperature was 25 °C, and the detection of the analytes was performed at a wavelength of 260 nm. Methamphetamine showed good linearity (R2 = 0.9999) in the range of 4.2~83.2 mg/mL. The stability of the test material was good over 24 h. The precision of the method was good, with an average spiked recovery of 86.2% and an RSD of 2.9%. The methamphetamine content in the gauze sample was determined to be 7.8 ± 2.2 µg/sample. A high-performance liquid chromatography (HPLC) method was optimized and validated for the determination of methamphetamine in adsorbent materials (gauze). Validation data in terms of specificity, linearity, the limit of detection and the limit of quantification, reproducibility, precision, stability, and recovery indicated that the method is suitable for the routine analysis of methamphetamine in adsorbent materials (gauze) and provided a basis for training drug-detection animals.

Design and synthesis of multivalent drug delivery system with CA IX inhibitors as ligands.

A multivalent ligand delivery system holds tremendous potential in the field of tumor-targeted drug delivery. It addresses the challenges posed by the low affinity between small molecule ligand receptors and the rapid metabolism of small molecule drug conjugates (SMDCs) in vivo. Notably, existing multivalent ligand systems have demonstrated significant anti-tumor activity in various tumor models. In this study, we have developed a novel multivalent ligand delivery system for SN38, utilizing acetazolamide, a carbonic anhydrase IX (CA IX) inhibitor, as the target ligand. Our multivalent ligand delivery systems exhibited superior metabolic stability and enhanced targeting specificity compared to SMDC molecules. Furthermore, they demonstrated improved anti-proliferation activity, addressing the existing challenges associated with the low receptor affinity and rapid metabolism of SMDCs.

Self-Immolated Nanoadjuvant for In Situ Vaccination Immunotherapy of Colorectal Cancer.

Vaccination immunotherapy has revolutionized cancer treatment modalities. Although the immunomodulatory adjuvant generally employs for potentiating vaccine response, systemic administration may drive immune-related side effects, even immune tolerance. Therefore, tunable immunoadjuvants are highly desirable to simultaneously stimulate the immune response and mitigate systemic toxicity. Self-immolated nanoadjuvants are herein reported to potentiate vaccination immunotherapy of cancer. The nanoadjuvants are engineered by co-assembling an intracellular acidity-ionizable polymeric agonist of toll-like receptor 7/8 resiquimod (R848) and polymeric photosensitizer pyropheophorbide a (PPa). The resultant nanoadjuvants specifically accumulate at the tumor site via passive targeting and are dissociated in the acidic endosome versicles to activate PPa via protonation of the polymer backbone. Upon 671 nm laser irradiation, PPa performed photodynamic therapy to induce immunogenic cell death of tumor cells and subsequently releases R848 in a customized manner, which synergistically activates dendritic cells (DCs), promotes antigen cross-presentation, and eventually recruits cytotoxic T lymphocytes for tumor regression. Furthermore, the synergistic in situ vaccination immunotherapy with immune checkpoint blockade induce sustained immunological memory to suppress tumor recurrence in the rechallenged colorectal tumor model.

Sodium propionate improves cognitive and memory function in mouse models of Alzheimer's disease.

This study was designed to explore whether sodium propionate (SP) alleviates cognitive damage in a mouse model of Alzheimer's disease (AD). We evaluated behavioral and biochemical aspects in an animal model of AD made by intracerebroventricular injection of Aß1-42 peptide. Two-month-old ICR mice were treated with SP or normal saline for 21 days (control group, Aß1-42 group, Aß1-42 + SP50 mg/kg group, Aß1-42 + SP100 mg/kg group, and Aß1-42 + SP200 mg/kg group). Behavioral tests showed that SP alleviated cognitive and memory impairments in AD mice. Moreover, SP treatment significantly suppressed the level of inducible nitric oxide synthase (iNOS) in the hippocampus. Concomitantly, the overexpression of interleukin-1α (IL-1α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) in the hippocampus induced by Aß1-42 was significantly reduced following treatment with SP. In addition, SP was able to increase the levels of synaptophysin (SYN) and postsynaptic dense protein 95 (PSD95). Our study shows that SP could significantly improve Aß1-42-induced spatial learning and memory impairment by reducing neuroinflammation via inhibition of proinflammatory cytokines and iNOS activation and restoring synapse plasticity by increasing synaptically associated protein levels, suggesting that SP has a positive effect and potential for AD therapies.

Engineered bioorthogonal POLY-PROTAC nanoparticles for tumour-specific protein degradation and precise cancer therapy.

PROteolysis TArgeting Chimeras (PROTACs) has been exploited to degrade putative protein targets. However, the antitumor performance of PROTACs is impaired by their insufficient tumour distribution. Herein, we present de novo designed polymeric PROTAC (POLY-PROTAC) nanotherapeutics for tumour-specific protein degradation. The POLY-PROTACs are engineered by covalently grafting small molecular PROTACs onto the backbone of an amphiphilic diblock copolymer via the disulfide bonds. The POLY-PROTACs self-assemble into micellar nanoparticles and sequentially respond to extracellular matrix metalloproteinase-2, intracellular acidic and reductive tumour microenvironment. The POLY-PROTAC NPs are further functionalized with azide groups for bioorthogonal click reaction-amplified PROTAC delivery to the tumour tissue. For proof-of-concept, we demonstrate that tumour-specific BRD4 degradation with the bioorthogonal POLY-PROTAC nanoplatform combine with photodynamic therapy efficiently regress tumour xenografts in a mouse model of MDA-MB-231 breast cancer. This study suggests the potential of the POLY-PROTACs for precise protein degradation and PROTAC-based cancer therapy.

Copackaging photosensitizer and PD-L1 siRNA in a nucleic acid nanogel for synergistic cancer photoimmunotherapy.

Packaging multiple drugs into a nanocarrier with rational design to achieve synergistic cancer therapy remains a challenge due to the intrinsically varied pharmacodynamics of therapeutic agents. Especially difficult is combining small-molecule drugs and macromolecular biologics. Here, we successfully graft pheophorbide A (PPA) photosensitizers on DNA backbone at predesigned phosphorothioate modification sites. The synthesized four PPA-grafted DNAs are assembled into a tetrahedron framework, which further associates with a programmed death ligand-1 (PD-L1) small interfering RNA (siRNA) linker through supramolecular self-assembly to form an siRNA and PPA copackaged nanogel. With dual therapeutic agents inside, the nanogel can photodynamically kill tumor cells and induce remarkable immunogenic cell death. Also, it simultaneously silences the PD-L1 expression of the tumor cells, which substantially promotes the antitumor immune response and leads to an enhanced antitumor efficacy in a synergistic fashion.

Efficacy and Safety of Immune Checkpoint Blockades in the Treatment of Ocular Melanoma: A Systematic Review and Meta-Analysis.

AIM: This meta-analysis aimed to compare the efficacy and safety of immune checkpoint blockade for the treatment of ocular melanoma. METHODS: We searched the PubMed, EMBASE, and Cochrane Library databases up to July 2021. Effect sizes (ESs) and corresponding 95% confidence intervals (CIs) were used to compare the outcomes. Efficacy outcomes included complete response (CR), partial response (PR), stable disease (SD), progressive disease (PD), objective response rate (ORR), overall survival (OS), progression free survival (PFS). Safety outcomes included adverse events (AEs) and serious adverse event (SAEs). RESULTS: A total of 16 eligible articles with 848 ocular melanoma patients were included. ICB treatment significantly improved CR (ES=0.02, 95%CI: 0.00-0.03, P=0.023), PR (ES=0.07, 95%CI: 0.05-0.09, P=0.000), SD (ES=0.31, 95%CI: 0.17-0.46, P=0.000), PD (ES=0.69, 95%CI: 0.61-0.77, P=0.000), ORR (ES=0.10, 95%CI: 0.04-0.15, P=0.000), OS (ES=9.68, 95%CI: 7.28-12.07, P=0.000) and PFS (ES=2.88, 95%CI: 2.69-3.07, P=0.000) in patients with ocular melanoma. Moreover, ICB therapies were associated with reduced AEs (ES=0.48, 95%CI: 0.30-0.67, P=0.000) and SAEs (ES=0.31, 95%CI: 0.18-0.45, P=0.000). CONCLUSIONS: ICB therapy showed good efficacy and safety in treating patients with ocular melanoma.

Frontal-posterior functional imbalance and aberrant function developmental patterns in schizophrenia.

Schizophrenia (SZ) is a neurodevelopmental disorder. There remain significant gaps in understanding the neural trajectory across development in SZ. A major research focus is to clarify the developmental functional changes of SZ and to identify the specific timing, the specific brain regions, and the underlying mechanisms of brain alterations during SZ development. Regional homogeneity (ReHo) characterizing brain function was collected and analyzed on humans with SZ (hSZ) and healthy controls (HC) cross-sectionally, and methylazoxymethanol acetate (MAM) rats, a neurodevelopmental model of SZ, and vehicle rats longitudinally from adolescence to adulthood. Metabolomic and proteomic profiling in adult MAM rats and vehicle rats was examined and bioanalyzed. Compared to HC or adult vehicle rats, similar ReHo alterations were observed in hSZ and adult MAM rats, characterized by increased frontal (medial prefrontal and orbitofrontal cortices) and decreased posterior (visual and associated cortices) ReHo. Longitudinal analysis of MAM rats showed aberrant ReHo patterns as decreased posterior ReHo in adolescence and increased frontal and decreased posterior ReHo in adulthood. Accordingly, it was suggested that the visual cortex was a critical locus and adolescence was a sensitive window in SZ development. In addition, metabolic and proteomic alterations in adult MAM rats suggested that central carbon metabolism disturbance and mitochondrial dysfunction were the potential mechanisms underlying the ReHo alterations. This study proposed frontal-posterior functional imbalance and aberrant function developmental patterns in SZ, suggesting that the adolescent visual cortex was a critical locus and a sensitive window in SZ development. These findings from linking data between hSZ and MAM rats may have a significant translational contribution to the development of effective therapies in SZ.

Verification of a multi-function closed maze for the detection of affective disorder and spatial cognitive impairment in post-weaning socially isolated rats.

OBJECTIVE: To verify a behavioral device for the detection of learning, memory, and affective disorders in post-weaning socially isolated rats. METHODS: We tested the behavioral changes in post-weaning socially isolated rats using a multi-function closed maze, a self-developed behavioral device, against the classical mood disorder detection method, the IntelliCage system and Morris water maze. RESULTS: In the multifunctional closed maze experiment, the spatial learning and memory ability of post-weaning socially isolated rats decreased, which was consistent with the results of the water maze and IntelliCage system. Furthermore, the behavioral changes in the post-weaning socially isolated rats in the multi-function closed maze test were the same as those of the forced swimming and open field tests, indicating that the rats had depression- and anxiety-like behaviors. CONCLUSION: A multi-function closed maze can detect emotional changes, spatial learning ability, and memory ability.

Acidity-Activatable Dynamic Nanoparticles Boosting Ferroptotic Cell Death for Immunotherapy of Cancer.

Immunotherapy shows promising therapeutic potential for long-term tumor regression. However, current cancer immunotherapy displays a low response rate due to insufficient immunogenicity of the tumor cells. To address these challenges, herein, intracellular-acidity-activatable dynamic nanoparticles for eliciting immunogenicity by inducing ferroptosis of the tumor cells are engineered. The nanoparticles are engineered by integrating an ionizable block copolymer and acid-liable phenylboronate ester (PBE) dynamic covalent bonds for tumor-specific delivery of the ferroptosis inducer, a glutathione peroxidase 4 inhibitor RSL-3. The nanoparticles can stably encapsulate RSL-3 inside the hydrophobic core via π-π stacking interaction with the PBE groups at neutral pH (pH = 7.4), while releasing the payload in the endocytic vesicles (pH = 5.8-6.2) by acidity-triggered cleavage of the PBE dynamic covalent bonds. Furthermore, the nanoparticles can perform acid-activatable photodynamic therapy by protonation of the ionizable core, and significantly recruit tumor-infiltrating T lymphocytes for interferon gamma secretion, and thus sensitize the tumor cells to RSL-3-inducible ferroptosis. The combination of nanoparticle-induced ferroptosis and blockade of programmed death ligand 1 efficiently inhibits growth of B16-F10 melanoma tumor and lung metastasis of 4T1 breast tumors, suggesting the promising potential of ferroptosis induction for promoting cancer immunotherapy.

Formation of an unusual glutamine tautomer in a blue light using flavin photocycle characterizes the light-adapted state.

Blue light using flavin (BLUF) photoreceptor proteins are critical for many light-activated biological processes and are promising candidates for optogenetics because of their modular nature and long-range signaling capabilities. Although the photocycle of the Slr1694 BLUF domain has been characterized experimentally, the identity of the light-adapted state following photoexcitation of the bound flavin remains elusive. Herein hybrid quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations of this photocycle provide a nonequilibrium dynamical picture of a possible mechanism for the formation of the light-adapted state. Photoexcitation of the flavin induces a forward proton-coupled electron transfer (PCET) process that leads to the formation of an imidic acid tautomer of Gln50. The calculations herein show that the subsequent rotation of Gln50 allows a reverse PCET process that retains this tautomeric form. In the resulting purported light-adapted state, the glutamine tautomer forms a hydrogen bond with the flavin carbonyl group. Additional ensemble-averaged QM/MM calculations of the dark-adapted and purported light-adapted states demonstrate that the light-adapted state with the imidic acid glutamine tautomer reproduces the experimentally observed spectroscopic signatures. Specifically, the calculations reproduce the red shifts in the flavin electronic absorption and carbonyl stretch infrared spectra in the light-adapted state. Further hydrogen-bonding analyses suggest the formation of hydrogen-bonding interactions between the flavin and Arg65 in the light-adapted state, providing a plausible explanation for the experimental observation of faster photoinduced PCET in this state. These characteristics of the light-adapted state may also be essential for the long-range signaling capabilities of this photoreceptor protein.

Co-Prodrugs of 7-Ethyl-10-hydroxycamptothecin and Vorinostat with in Vitro Hydrolysis and Anticancer Effects.

7-Ethyl-10-hydroxycamptothecin (SN38) and vorinostat (SAHA) are quite promising combination therapy agents applied to the clinical treatment of cancer. In this study, we designed and synthesized a series of novel SN38-SAHA co-prodrugs, which were conjugated by four different amino acids including glycine, alanine, aminobutyric acid, and 6-aminocaproic acid. The hydrolytic reconversion rate to SN38 and SAHA critically depended on the carbon chain length, which were evaluated in PBS (pH 6.0/7.4) and plasma (human/mouse). With decreasing amino acid chain length, the hydrolytic reconversion rate increased gradually. The in vitro cytotoxicity test was evaluated by the sulforhodamine B (SRB) assay on the human lung adenocarcinoma cell line A549 and human colorectal cancer cell line HCT116. With the evaluation of stability and in vitro cytotoxicity, an appropriate linker was found, and the active drug can be released efficiently from compound 3a, which exhibited strong antiproliferative activity in A549 and HCT-116 cell lines correspondingly. These results indicated that the well-designed co-prodrug 3a and this kind of strategy can be a promising approach for anticancer therapy.

Natural silibinin modulates amyloid precursor protein processing and amyloid-ß protein clearance in APP/PS1 mice.

Silibinin has been shown to attenuate cognitive dysfunction and inhibit amyloid-beta (Aß) aggregation in Alzheimer's disease (AD) models. However, the underlying mechanism by which silibinin improves cognition remains poorly understood. In this study, we investigated the effect of silibinin on ß-secretase levels, Aß enzymatic degradation, and oxidative stress in the brains of APP/PS1 mice with cognitive impairments. Oral administration of silibinin for 2 months significantly attenuated the cognitive deficits of APP/PS1 mice in the Y-maze test, novel object recognition test, and Morris water maze test. Biochemical analyses revealed that silibinin decreased Aß deposition and the levels of soluble Aß1-40/1-42 in the hippocampus by downregulating APP and BACE1 and upregulating NEP in APP/PS1 mice. In addition, silibinin decreased the MDA content and increased the activities of the antioxidant enzymes CAT, SOD, and NO. Based on our findings, silibinin is a potentially promising agent for preventing AD-associated Aß pathology.

Synthesis and biological evaluation of paclitaxel and vorinostat co-prodrugs for overcoming drug resistance in cancer therapy in vitro.

Paclitaxel (PTX) is the first-line treatment drug for breast cancer. However, drug resistance after a course of treatment and low selectivity restricted its clinical utility sometimes. In this study, we successfully bound PTX and vorinostat (SAHA) to form co-prodrugs based on the synergistic anticancer effects. The PTX-SAHA co-prodrugs were conjugated by glycine (1a) and succinic acid (1b) respectively and the former has shown better activity in cytotoxicity, cell cycle arrest and western-blot experiments. Therefore, 1a was further prepared to nanomicelles with mPEG2000-PLA1750 as the carrier by using thin film method. PTX-SAHA co-prodrug nanomicelles were spherical with a particle size of 20-100 nm. In vitro drug release test showed 1a nanomicelles had sustained release effect, which could reduce the resistance of PTX. In vitro cytotoxicity was evaluated by SRB assay in HCT-116 cells, MCF-7 cells and drug-resistant MCF-7/ADR cells. The results showed 1a nanomicelles had comparable or even better cytotoxicity than PTX especially in the MCF-7/ADR cells. All the results suggested that PTX-SAHA co-prodrug nanomicelles were promising treatment for PTX resistance cancer.

A series of camptothecin prodrugs exhibit HDAC inhibition activity.

Camptothecin plays an important role in clinical cancer treatment, and its derivatives are a favorite of pharmaceutical chemists. Herein, we have designed a series of camptothecin prodrugs that exhibit histone deacetylase (HDAC) inhibition activity based on the synergy effect between HDAC inhibitors and camptothecin derivatives. With the evaluation of stability in buffers or plasma from human or mouse model, an appropriate linker was found, so the active drug can be released efficiently and compound 21a exhibited strong antiproliferative activity in A549 and HCT-116 cell lines. These results indicated that the well-designed prodrug can be promising in cancer treatment.

The impact of ALDH2 activation by Alda-1 on the expression of VEGF in the hippocampus of a rat model of post-MI depression.

Post-MI depression is a critical clinical problem, the comorbidity of which complicates depression treatment and worsens cardiovascular outcomes. However, which antidepressant is the best to lower the risk of cardiovascular events in persons with depression was still unknown. Recently, it has been proposed that the activation of ALDH2 by Alda-1 can effectively reduce depressive-like behaviors and improve the prognosis of coronary heart disease. In the present study, we investigated the effect of Alda-1 on the expression of VEGF in the hippocampus of a rat model with post-MI depression, as well as the potential treatment mechanism. Alda-1 administration significantly decreased the immobility time and increased the swimming time of the post-MI depression rats in the forced swim test. Moreover, treatment of post-MI depression rats with Alda-1 significantly increased the sucrose preference ratio, as assessed by a sucrose preference test. These behaviors were associated with an increase 5-HT and DA neurotransmitter content, as well as an increase of VEGF levels in the hippocampus of the post-MI depression rats. These results suggest that Alda-1 improves depressive-like behavior in rats after MI by increasing VEGF expression in the hippocampus of rats.

Maleimidation of dextran and the application in designing a dextran-camptothecin conjugate.

Camptothecin analogs, as commonly used chemotherapy drugs, usually have poor water solubility which has limited their use in the clinic. In order to improve the water-solubility of camptothecin, a new dextran derivative Dex-Mal was synthesized and used in designing a dextran-camptothecin conjugate which contained a CTB-sensitive linker. This conjugate could efficiently release the therapeutic drug SN-38 in the presence of cathepsin B and the antiproliferative activity of the conjugate was similar to the approved drug Irinotecan hydrochloride. Furthermore, in the presence of dextran, the conjugate could self-assemble into nanoparticles in water, which could improve the targeting ability through the EPR effect. This provides a potential way to formulate a drug delivery system for camptothecin analogs or other drugs which have poor water solubility.

Neuron-derived CCL2 contributes to microglia activation and neurological decline in hepatic encephalopathy.

BACKGROUND: CCL2 was up-regulated in neurons and involved in microglia activation and neurological decline in mice suffering from hepatic encephalopathy (HE). However, no data exist concerning the effect of neuron-derived CCL2 on microglia activation in vitro. METHODS: The rats were pretreated with CCL2 receptor inhibitors (INCB or C021, 1 mg/kg/day i.p.) for 3 days prior to thioacetamide (TAA) administration (300 mg/kg/day i.p.) for inducing HE model. At 8 h following the last injection (and every 4 h after), the grade of encephalopathy was assessed. Blood and whole brains were collected at coma for measuring CCL2 and Iba1 expression. In vitro, primary neurons were stimulated with TNF-α, and then the medium were collected for addition to microglia cultures with or without INCB or C021 pretreatment. The effect of the medium on microglia proliferation and activation was evaluated after 24 h. RESULTS: CCL2 expression and microglia activation were elevated in the cerebral cortex of rats received TAA alone. CCL2 receptors inhibition improved neurological score and reduced cortical microglia activation. In vitro, TNF-α treatment induced CCL2 release by neurons. Medium from TNF-α stimulated neurons caused microglia proliferation and M1 markers expression, including iNOS, COX2, IL-6 and IL-1ß, which could be suppressed by INCB or C021 pretreatment. The medium could also facilitate p65 nuclear translocation and IκBα phosphorylation, and NF-κB inhibition reduced the increased IL-6 and IL-1ß expression induced by the medium. CONCLUSION: Neuron-derived CCL2 contributed to microglia activation and neurological decline in HE. Blocking CCL2 or inhibiting microglia excessive activation may be potential strategies for HE.