Novel Acyl Thiourea-Based Hydrophobic Tagging Degraders Exert Potent Anti-Influenza Activity through Two Distinct Endonuclease Polymerase Acidic-Targeted Degradation Pathways.

The spread of the influenza virus has caused devastating pandemics and huge economic losses worldwide. Antiviral drugs with diverse action modes are urgently required to overcome the challenges of viral mutation and drug resistance, and targeted protein degradation strategies constitute excellent candidates for this purpose. Herein, the first degradation of the influenza virus polymerase acidic (PA) protein using small-molecule degraders developed by hydrophobic tagging (HyT) technology to effectively combat the influenza virus was reported. The SAR results revealed that compound 19b with Boc2-(L)-Lys demonstrated excellent inhibitory activity against A/WSN/33/H1N1 (EC50 = 0.015 µM) and amantadine-resistant strain (A/PR/8/H1N1), low cytotoxicity, high selectivity, substantial degradation ability, and good drug-like properties. Mechanistic studies demonstrated that the proteasome system and autophagic lysosome pathway were the potential drivers of these HyT degraders. Thus, this study provides a powerful tool for investigating the targeted degradation of influenza virus proteins and for antiviral drug development.

The bZIP transcription factor MpbZIP9 regulates anthocyanin biosynthesis in Malus 'Pinkspire' fruit.

Malus 'Pinkspire' is regulated by abscisic acid (ABA), which results in a red colour, but the regulatory relationship between ABA and anthocyanin synthesis has not been determined. The key factors affecting the colour change of M. 'Pinkspire' peel were investigated during the periods of significant colour changes during fruit ripening. The results showed that the transcription factor MpbZIP9 associated with ABA was screened by transcriptomic analysis. MpbZIP9 expression was consistent with the trend of structural genes expression for anthocyanin synthesis in the peel during fruit ripening, as well as with changes in the content of ABA, which is a positive regulator. A yeast one-hybrid assay showed that MpbZIP9 can directly bind to the promoter of MpF3'H. Dual luciferase reporter gene assays and GUS staining experiments showed that MpbZIP9 significantly activate MpF3'H expression. In addition, overexpression of the MpbZIP9 significantly enhanced anthocyanin accumulation and the expression of genes involved in anthocyanin synthesis. In contrast, virus-induced silencing of the MpbZIP9 significantly reduced the expression of structural genes involved in anthocyanin synthesis. These results suggest that the MpbZIP9 transcription factor can regulate the synthesis of peel anthocyanin and is a positive regulator that promotes anthocyanin biosynthesis by activating MpF3'H expression.

Rational design and optimization of acylthioureas as novel potent influenza virus non-nucleoside polymerase inhibitors.

Evidence suggests that rapidly evolving virus subvariants risk rendering current vaccines and anti-influenza drugs ineffective. Hence, exploring novel scaffolds or new targets of anti-influenza drugs is of great urgency. Herein, we report the discovery of a series of acylthiourea derivatives produced via a scaffold-hopping strategy as potent antiviral agents against influenza A and B subtypes. The most effective compound 10m displayed subnanomolar activity against H1N1 proliferation (EC50 = 0.8 nM) and exhibited inhibitory activity toward other influenza strains, including influenza B virus and H1N1 variant (H1N1, H274Y). Additionally, druggability evaluation revealed that 10m exhibited favorable pharmacokinetic properties and was metabolically stable in liver microsome preparations from three different species as well as in human plasma. In vitro and in vivo toxicity studies confirmed that 10m demonstrated a high safety profile. Furthermore, 10m exhibited satisfactory antiviral activity in a lethal influenza virus mouse model. Moreover, mechanistic studies indicated that these acylthiourea derivatives inhibited influenza virus proliferation by targeting influenza virus RNA-dependent RNA polymerase. Thus, 10m is a potential lead compound for the further exploration of treatment options for influenza.

Development of Highly Efficient Estrogen Receptor ß-Targeted Near-Infrared Fluorescence Probes Triggered by Endogenous Hydrogen Peroxide for Diagnostic Imaging of Prostate Cancer.

Hydrogen peroxide is one of the most important reactive oxygen species, which plays a vital role in many physiological and pathological processes. A dramatic increase in H2O2 levels is a prominent feature of cancer. Therefore, rapid and sensitive detection of H2O2 in vivo is quite conducive to an early cancer diagnosis. On the other hand, the therapeutic potential of estrogen receptor beta (ERß) has been implicated in many diseases including prostate cancer, and this target has attracted intensive attention recently. In this work, we report the development of the first H2O2-triggered ERß-targeted near-infrared fluorescence (NIR) probe and its application in imaging of prostate cancer both in vitro and in vivo. The probe showed good ERß selective binding affinity, excellent H2O2 responsiveness and near infrared imaging potential. Moreover, in vivo and ex vivo imaging studies indicated that the probe could selectively bind to DU-145 prostate cancer cells and rapidly visualizes H2O2 in DU-145 xenograft tumors. Mechanistic studies such as high-resolution mass spectrometry (HRMS) and density functional theory (DFT) calculations indicated that the borate ester group is vital for the H2O2 response turn-on fluorescence of the probe. Therefore, this probe might be a promising imaging tool for monitoring the H2O2 levels and early diagnosis studies in prostate cancer research.

Development of a sensitive phage-mimotope and horseradish peroxidase based electrochemical immunosensor for detection of O,O-dimethyl organophosphorus pesticides.

In this work, a green, harmless and signal-amplified electrochemical immunosensor based on phage-mimotope M31 (C-P-D-G-N-H-V-P-F-C) and horseradish peroxidase (HRP) was constructed for detecting O,O-dimethyl organophosphorus pesticides (OPs). The glassy carbon electrode (GCE) was modified by nitrogen and boron doped carbon quantum dots and graphene oxide (NBCQDs@GO) which can provide sufficient surface area and enhance the conductivity of the electrode. The O,O-dimethyl OPs class specific antibody mAb3C9 was assembled onto the NBCQDs@GO and the phage-mimotope M31 competitively bound to mAb3C9 with OPs. Furthermore, large amounts of anti-M13 mAb-HRP were introduced to the electrode through thousands of binding sites on the capsid of phage. HRP can catalyze 4-chloro-1-naphthol (4-CN) to produce insoluble precipitates (Benzo-4-chlorhexanedione, 4-CD). Hence, the concentration of OPs can be quantified by measuring impedance signal with electrochemical impedance spectrum (EIS). Under the optimal detection conditions, the 50% inhibitory concentration (IC50) and limits of detection (LODs) values of 9 O,O-dimethyl OPs were in range of 0.989-4.017 ng/mL and 0.003-0.014 ng/mL, respectively. The recovery rates of spiked OPs in cucumber, cabbage and lettuce were 88.20-112.50% with coefficient of variation from 2.97 to 15.64%. Therefore, the immunosensor showed very good sensitivity and demonstrating potential application for the detection of O,O-dimethyl OPs in food samples.

Rapid and sensitive noncompetitive immunoassay for detection of aflatoxin B1 based on anti-immune complex peptide.

Noncompetitive immunoassays for small molecules are generally considered to be more sensitive than competitive ones. In this study, a phage-peptide against immune complex of aflatoxin B1 (AFB1) and nanobody Nb28 was obtained by phage-display technology. The phage-peptide was labeled with peroxidase and used to develop a direct noncompetitive magnetic-chemiluminescent enzyme-linked immunoassay (Nc-MCLEIA) for AFB1. The 50% signal saturation concentration (SC50) and limit of detection (LOD) of Nc-MCLEIA for AFB1 were 0.089 and 0.006 ng/mL, respectively. Compared with competitive immunoassays developed by the Nb28, the sensitivity and efficiency of Nc-MCLEIA were greatly improved. The recoveries of AFB1 from spiked corn, rice, flour, peanut, peanut oil and corn oil samples ranged from 83.8% to 119.2% with coefficient of variable under 8.9%. Furthermore, parallel analysis of natural corn, rice and flour samples by Nc-MCLEIA and HPLC (high performance liquid chromatography) proved that the Nc-MCLEIA was reliable.

Development of a noncompetitive magnetic-phage anti-immunocomplex assay for detecting of organophosphorus pesticides with a thiophosphate group.

Organophosphorus pesticides (OPs) are widely used in agriculture and the monitoring of their residues is very important to protect human health. Immunoassays are important tools for the analysis of small molecules. Generally, noncompetitive mode of immunoassay is considered to be more sensitive than competitive mode. In this study, peptides that can identify immunocomplex of OPs were screened from a phage display library. Subsequently, a second-generation peptide library was constructed and peptides with better performance were isolated. Then, a rapid and sensitive noncompetitive magnetic-phage anti-immunocomplex assay (MPHAIA) for OPs was developed based on the best phage-peptide and single chain antibody immunomagnetic beads. The MPHAIA showed broad specificity for OPs with a thiophosphate group. The half-saturated concentration (SC50) values and limits of detection (LODs) of MPHAIA to 12 OPs were ranged from 15.04 to 105.48 ng/mL and 4.07-14.19 ng/mL, respectively. The accuracy and reliability of MPHAIA were verified by gas chromatography-tandem mass spectrometry (GC-MS/MS) parallel analysis of six kinds of OPs in spiked cucumber samples. The recovery rates were in range of 81.2-116.3% with coefficient of variation from 4.1% to 14.1%, which were consistent with the results of GC-MS/MS.

Discovery of aminothiazole derivatives as novel human enterovirus A71 capsid protein inhibitors.

Enterovirus A71 (EV-A71), one of the major pathogens that causes hand, foot and mouth disease (HFMD), has seriously threatened the health and safety of young children. In this study, aminothiazole derivatives were synthesized and screened against EV-A71 in Rhabdomyosarcoma (RD) cells. The best compound (12s), with a biphenyl group, showed activity against EV-A71 (EC50: 0.27 µM) but also against a series of different human enteroviruses without significant cytotoxicity (CC50 > 56.2 µM). Mechanistic studies including time-of-drug-addition assays, viral entry assays and microscale thermophoresis (MST) experiments, showed that 12s binds to EV-A71 capsid and blocks the binding between the viral protein VP1 and the relevant human scavenger receptor class B member 2 (hSCARB2).

Discovery of oseltamivir-based novel PROTACs as degraders targeting neuraminidase to combat H1N1 influenza virus.

Annual and sporadic influenza outbreaks pose a great threat to human health and the economy worldwide. Moreover, the frequent mutation of influenza viruses caused by antigen drift complicates the application of antiviral therapeutics. As such, there is an urgent need for novel antiviral agents to tackle the problem of insufficient efficacy of licensed drugs. Inspired by the success of the newly emerged PROTACs (PROteolysis TArgeting Chimeras) strategy, we report herein the design and synthesis of novel PROTAC molecules based on an oseltamivir scaffold to combat severe annual influenza outbreaks. Among these, several compounds showed good anti-H1N1 activity and efficient influenza neuraminidase (NA) degradation activity. The best compound, 8e, effectively induced influenza NA degradation in a dose-dependent manner and relied on the ubiquitin-proteasome pathway. Moreover, Compound 8e exhibited potent antiviral activity toward both wild-type H1N1 virus and an oseltamivir-resistant strain (H1N1, H274Y). A molecular docking study demonstrated that Compound 8e had good hydrogen-bonding and hydrophobic interactions with both the active sites of NA and Von Hippel-Lindau (VHL) proteins, which could effectively drive the favorable interaction of these two proteins. Thus, as the first report of a successful anti-influenza PROTAC, this proof of concept will greatly widen the application range of the PROTAC technique to antiviral drug discovery.

Platinum-Copper Bimetallic Colloid Nanoparticle Cluster Nanozymes with Multiple Enzyme-like Activities for Scavenging Reactive Oxygen Species.

Fabrication of high-performance artificial antioxidant enzyme (AAE) systems based on a single nanozyme possessing multi-enzymatic activities is fascinating but challenging. Here, polyvinylpyrrolidone (PVP)-platinum-copper nanoparticle clusters (PVP-PtCuNCs) are prepared by a facile one-pot chemical coreduction method. PVP-PtCuNCs possess efficient superoxide dismutase (SOD)-like, peroxidase (POD)-like, and catalase (CAT)-like activities, and the multi-enzymatic activities depend on the bimetal component and cluster structure. Compared with individual platinum nanoparticle clusters (PVP-PtNCs), PVP-PtCuNCs can effectively eliminate reactive oxygen species (ROS) including superoxide anions, hydrogen peroxide, and hydroxyl radicals. The doping of copper not only reduces the usage of Pt content but also improves the catalytic efficiency and versatility effectively through the synergistic effect of bimetal components and the nanocluster structure. The results not only demonstrate that a single bimetallic nanozyme has the potential as an efficient AAE system in the biomedical application but also demonstrate that traditional concepts of structure-activity relationships can be used to fabricate nanozymes with the desired multi-enzymatic activities.

Arginine-rich peptide/platinum hybrid colloid nanoparticle cluster: A single nanozyme mimicking multi-enzymatic cascade systems in peroxisome.

Recently, nanozymes have attracted sustained attention for facilitating next generation of artificial enzymatic cascade systems (ECSs). However, the fabrication of integrated multi-ECSs based on a single nanozyme remains a great challenge. Here, inspired by the biological function and self-assembling ability of arginine (R), we synthesized arginine-rich peptide-Pt nanoparticle cluster (ARP-PtNC) nanozymes that mimic two typical enzymatic cascade systems of uricase/catalase and superoxide dismutase/catalase in natural peroxisome. ARPs containing at least 10 arginine residues contribute to the cluster formation based on hydrogen bonding and coordination. The well-designed peptide-Pt hybrid nanozyme not only possesses excellent uricase-mimicking activity to degrade uric acid effectively, but also serves as a desired scavenger for reactive oxygen species (ROS) harnessing two efficient enzyme cascade catalysis of uricase/catalase and superoxide dismutase/catalase. The surface microenvironment of the hybrid nanozymes provided by arginine-rich peptides and the cluster structure contribute to the efficient multiply enzyme-like activities. Fascinatingly, the hybrid nanozyme can inhibit the formation of monosodium urate monohydrate effectively based on the architecture of ARP-PtNCs. Thus, ARP-PtNC nanozyme has the potential in gout and hyperuricemia therapy. Rational design of ingenious peptide-metal hybrid nanozyme with unique physicochemical surface properties provides a versatile and designed strategy to fabricate multi-enzymatic cascade systems, which opens new avenues to broaden the application of nanozymes in practice.

Protein-mediated wool-ball-like copper sulfide as a multifunctional nanozyme for dual fluorescence "turn-on" sensors of cysteine and silver ions.

Developing a multifunctional nanozyme based biosensor with a convenient approach and high reliability is of vital interest for multiplex detection. In this study, wool-ball-like copper sulfide (WBLCS) was obtained facilely using an amphiphilic protein. The acidic amino acid residues and the amphiphilic properties of protein molecules play cooperative roles in the fabrication of hierarchical nanostructures. Unlike copper sulfide with irregular morphologies, the single component WBLCS acts as a multifunctional nanozyme possessing both superior cysteine oxidase- and peroxidase-mimicking activity. Fascinatingly, the addition of silver ions can significantly enhance the performance of the cascade system at a very low fluorescence substrate concentration. Based on this, dual fluorescence "turn on" sensors of cysteine and silver ions with extremely high sensitivity and selectivity are developed. This is the first report to explore multiple fluorescence "turn on" sensing systems based on one single nanozyme. Hence, the present finding has significant implication towards the design of superstructured nanozymes combining different multi-functionalities at the nanoscale for sensing multiplex target molecules sensitively and selectively in practice.

Highly sensitive phage-magnetic-chemiluminescent enzyme immunoassay for determination of zearalenone.

Here we demonstrate a novel phage-magnetic-chemiluminescent enzyme immunoassay (P-MCLEIA) for detection of zearalenone (ZEN). The P-MCLEIA was more efficient than conventional ELISA through several improvements. In the P-MCLEIA, magnetic nanoparticles were replaced of microplates as solid phases to reduce the whole incubation time within 40 min. Phage-mimotope was replaced of chemosynthetic antigen to improve the sensitivity of immunoassay. Chemiluminescence substrate was replaced of chromogenic substrate to further improve the sensitivity. The IC50 value of P-MCLEIA was 31.4 pg/mL, which was about 11 times lower than that of phage-magnetic-enzyme linked immunosorbent assay (P-MELISA) and 72 times lower than that of conventional ELISA. The LOD of P-MCLEIA was 4.3 pg/mL. Recovery study of P-MCLEIA was performed by analyzing ZEN levels in spiked corn samples, intra- and inter-assay recoveries were 80.0-119.8% and 82.7-112.7%, respectively. Furthermore, parallel analysis of natural corn samples showed a good correlation between the P-MCLEIA and high performance liquid chromatography.

Protein-mediated sponge-like copper sulfide as an ingenious and efficient peroxidase mimic for colorimetric glucose sensing.

Strenuous efforts have been made to develop nanozymes for achieving the performance of natural enzymes to broaden their application in practice, but the fabrication of high-performance and biocompatible nanozymes via facile and versatile approaches has always been a great challenge. Here, sponge-like casein-CuS hybrid has been facilely synthesized in the presence of amphiphilic protein-casein through a simple one-step approach. Casein-CuS hybrid exhibits substrates-dependent peroxidase-like activity. Casein-CuS hybrid exhibits well peroxidase-like activity with 3,3',5,5'-tetramethylbenzidine (TMB) and 1,2-diaminobenzene (OPD) as substrates, and the affinity of OPD towards the hybrid nanozyme is much higher than that of TMB. More importantly, due to the high affinity of OPD and the well biocompatibility of the hybrid nanozyme, a superior enzyme cascade for glucose based on the well cooperative effect of casein-CuS hybrid and glucose oxidase is developed. The proposed glucose sensor exhibits a wide linear range of 0.083 to 75 µM and a detection limit of 5 nM. This suggests the promising utilization of protein-metal hybrid nanozymes as robust and potent peroxidase mimics in the medical, food and environmental detection fields.

Knockdown of long non-coding RNA PCAT1 in glioma stem cells promotes radiation sensitivity.

This study aimed to investigate the function of glioma stem cells (GSCs) and the role of PCAT1. This study dissociated the differences between GSCs and glioma cells in terms of apoptosis rate and γH2AX positive cells levels after radiation. Microarray was carried out to detect that expressed PCAT1, and it was testified by RT-qPCR. After transfection, GSCs were used to investigate the influence of PCAT1 on radiation sensitivity. Sphere-formation capability was first examined. Cell apoptosis rate after radiation of 0 Gy or 6 Gy was analyzed by flow cytometry, and the level of γH2AX positive cells after 6 Gy radiation were compared. CCK8 assay was used to investigate the cell proliferation and RT-qPCR was used to examine miR-129-5p and HMGB1 expression. GSCs exhibited great capability in sphere formation and lower expression in apoptosis and γH2AX positive cells rates after 6 Gy radiation. PCAT1 had higher expression in GSCs. PCAT1 knockdown restrained the sphere-formation ability, increased the apoptosis rate and DNA damage under the treatment of radiation. Moreover, knockdown of PCAT1 inhibited the cell proliferation. In addition, silencing PCAT1 could increase the expression of miR-129-5p and decrease the expression of HMGB1. PCAT1 was overexpressed in GSCs and played a facilitating role in radiation resistance.

[Study on the relationship between tone burst ABR and CE-Chirp ASSR in infants with profound sensorineural hearing loss].

OBJECTIVE: To analyse the correlation between tone burst auditory brainstem response (tone burst auditory brainstem response, tb-ABR) and CE-Chirp voice evoked auditory steady-state response (auditory steady-state response, CE-Chirp ASSR) in infants with profound sensorineural hearing loss. METHODS: A total of 45 infants with profound sensorineural hearing loss underwent threshold tone burst ABR and CE-Chirp ASSR of the frequency response test, response thresholds were recorded in 0.5, 1.0, 2.0 and 4.0 kHz. Whether there was correlation or not existed between two methods were analyzed, SPSS 11.0 statistics software was used. RESULTS: Tone burst ABR and CE-Chirp ASSR could lead to different degrees of threshold in each frequency. Response e elicited threshold percentage mainly concentrated in the 91-100 dBnHL, correlation coefficient between 500-4 000 Hz response threshold elicited rate were: 0.837, 0.913, 0.909, and 0.919, respectively (P < 0.001). The difference of the frequency response threshold test between CE-Chirp ASSR and tone burst ABR were not significant (P > 0.05, Chi square). CONCLUSIONS: The tone burst ABR and CE-Chirp ASSR each frequency have different levels of residual hearing in infants with profound sensorineural hearing loss diagnosed by Click ABR, good correlation exists between tone burst ABR and CE-Chirp ASSR.

Osteogenesis and ototoxicity of a novel preparation of autogenous bone cement: implications for mastoid obliteration.

OBJECTIVE: To study the osteogenesis and ototoxicity of autogenous bone cement. STUDY DESIGN: A randomized, prospective, controlled animal study. SETTING: University laboratory. SUBJECTS AND METHODS: We created a critical defect on the bulla wall of guinea pigs. Fifty-three guinea pigs were divided into 3 groups. The defects were filled with autogenous bone cement (ABC) or autogenous bone dust (ABD) on the left side while the contralateral sides were kept intact as the blank control (BC) group or a defect was induced without obliteration as the operation control (OC) group (groups 1 and 2, n = 25 each). The animals were sacrificed at 2, 4, 8, and 12 weeks postoperatively. Group 3 (n = 3) was the ototoxicity positive control (OPC) group. The critical defects were evaluated for bone repair by histologic observation and computed tomography (CT), and the ototoxicity of the material was assessed by measuring shifts in the hearing threshold and changes in cochlear morphology. Antibiotic susceptibility testing of the material was also performed. RESULTS: Histologic observation and CT scans revealed complete bone repair in the ABC group at 12 weeks postoperatively, but progress was slower in the ABD group. The hearing threshold and cochlear morphology did not differ significantly between the preoperative and 12-week postoperative measurement. Autogenous bone dust inhibited some of the bacterial species commonly associated with chronic suppurative otitis media. CONCLUSIONS: Animals treated with ABC exhibited osteogenesis, with no ototoxicity and bacteriostasis compared with the use of ABD.

De novo dominant mutation of SOX10 gene in a Chinese family with Waardenburg syndrome type II.

OBJECTIVE: Waardenburg syndrome is a rare genetic disorder, inherited as an autosomal dominant trait. The condition is characterized by sensorineural hearing loss and pigment disturbances of the hair, skin, and iris. The de novo mutation in the SOX10 gene, responsible for Waardenburg syndrome type II, is rarely seen. The present study aimed to identify the genetic causes of Waardenburg syndrome type II in a Chinese family. METHODS: Clinical and molecular evaluations were conducted in a Chinese family with Waardenburg syndrome type II. RESULTS: A novel SOX10 heterozygous c.259-260delCT mutation was identified. Heterozygosity was not observed in the parents and sister of the proband, indicating that the mutation has arisen de novo. The novel frameshift mutation, located in exon 3 of the SOX10 gene, disrupted normal amino acid coding from Leu87, leading to premature termination at nucleotide 396 (TGA). The high mobility group domain of SOX10 was inferred to be partially impaired. CONCLUSION: The novel heterozygous c.259-260delCT mutation in the SOX10 gene was considered to be the cause of Waardenburg syndrome in the proband. The clinical and genetic characterization of this family would help elucidate the genetic heterogeneity of SOX10 in Waardenburg syndrome type II. Moreover, the de novo pattern expanded the mutation data of SOX10.