Discovery of a Fungal P450 with an Unusual Two-Step Mechanism for Constructing a Bicyclo[3.2.2]nonane Skeleton.

The successful biomimetic or chemoenzymatic synthesis of target natural products (NPs) and their derivatives relies on enzyme discovery. Herein, we discover a fungal P450 BTG5 that can catalyze the formation of a bicyclo[3.2.2]nonane structure through an unusual two-step mechanism of dimerization and cyclization in the biosynthesis of beticolin 1, whose bicyclo[3.2.2]nonane skeleton connects an anthraquinone moiety and a xanthone moiety. Further investigation reveals that BTG5-T318 not only determines the substrate selectivity but also alters the catalytic reactions, which allows the separation of the reaction to two individual steps, thereby understanding its catalytic mechanism. It reveals that the first heterodimerization undergoes the common oxidation process for P450s, while the second uncommon formal redox-neutral cyclization step is proved as a redox-mediated reaction, which has never been reported. Therefore, this work advances our understanding of P450-catalyzed reactions and paves the way for expansion of the diversity of this class of NPs through synthetic biology.

Fluorophore Label-Free Light-up Near Infrared Deoxyribonucleic Acid Nanosensor for Monitoring Extracellular Potassium Levels.

Fluorescent DNA nanosensors have been widely used due to their unique advantages, among which the near-infrared (NIR) imaging mode can provide deeper penetration depth and lower biological background for the nanosensors. However, efficient NIR quenchers require ingenious design, complex synthesis, and modification, which severely limit the development of NIR DNA nanosensors. Label-free strategies based on G-quadruplex (G4) and NIR G4 dyes were first introduced into in situ extracellular imaging, and a novel NIR sensing strategy for the specific detection of extracellular targets is proposed. The strategy avoids complex synthesis and site-specific modification by controlling the change of the NIR signal through the formation of a G4 nanostructure. A light-up NIR DNA nanosensor based on potassium ion (K+)-sensitive G4 chain PS2.M was constructed to verify the strategy. PS2.M forms a stable G4 nanostructure in the presence of K+ and activates the NIR G4 dye CSTS, thus outputting NIR signals. The nanosensor can rapidly respond to K+ with a linear range of 5-50 mM and has good resistance to interference. The nanosensor with cholesterol can provide feedback on the changes in extracellular K+ concentration in many kinds of cells, serving as a potential tool for the study of diseases such as epilepsy and cancer, as well as the development of related drugs. The strategy can be potentially applied to the NIR detection of a variety of extracellular targets with the help of functional DNAs such as aptamer and DNAzyme.

Synthetic Biology-based Construction of Unnatural Perylenequinones with Improved Photodynamic Anticancer Activities.

The construction of structural complexity and diversity of natural products is crucial for drug discovery and development. To overcome high dark toxicity and poor photostability of natural photosensitizer perylenequinones (PQs) for photodynamic therapy, herein, we aim to introduce the structural complexity and diversity to biosynthesize the desired unnatural PQs in fungus Cercospora through synthetic biology-based strategy. Thus, we first elucidate the intricate biosynthetic pathways of class B PQs and reveal how the branching enzymes create their structural complexity and diversity from a common ancestor. This enables the rational reprogramming of cercosporin biosynthetic pathway in Cercospora to generate diverse unnatural PQs without chemical modification. Among them, unnatural cercosporin A displays remarkably low dark toxicity and high photostability with retention of great photodynamic anticancer and antimicrobial activities. Moreover, it is found that, unlike cercosporin, unnatural cercosporin A could be selectively accumulated in cancer cells, providing potential targets for drug development. Therefore, this work provides a comprehensive foundation for preparing unnatural products with customized functions through synthetic biology-based strategies, thus facilitating drug discovery pipelines from nature.

Hairpin Switches-Based Isothermal Transcription Amplification for Simple, Sensitivity Detection of MicroRNA.

The ability to simply, selectively, and sensitively detect low numbers of miRNAs in clinical samples is highly valuable but remains a challenge. In this work, we present a novel miRNA detection system by using the elaborately designed hairpin switch, where the T7 primer, template, target recognize sequence, and light-up RNA aptamer template are edited and embedded in one single-stranded DNA hairpin structure. In the beginning, the hairpin switch maintained the hairpin structure 1, in which the ds promoter of T7 polymerase was disrupted, thus the transcription reaction of T7 polymerase was inhibited. After binding to the target, the hairpin switch 1 was unfolded and turned to the hairpin structure 2. This switch initiates the in vitro T7 transcription reaction, producing plenty of RNA transcripts containing RNA aptamers. Consequently, transcribed tremendous RNA aptamers lighted up the fluorophore for quantitative analysis. Compared with the existing T7 polymerase-based amplification system, this strategy exhibits several advantages, including simplicity, convenience, and high selectivity and sensitivity. The experimental results demonstrated that we could achieve the quantification of miRNA in buffer and complex biological samples.

Photoenzymatic Enantioselective Synthesis of Oxygen-Containing Benzo-Fused Heterocycles.

New-to-nature biocatalysis in organic synthesis has recently emerged as a green and powerful strategy for the preparation of valuable chiral products, among which chiral oxygen-containing benzo-fused heterocycles are important structural motifs in pharmaceutical industry. However, the asymmetric synthesis of these compounds through radical-mediated methods is challenging. Herein, a novel asymmetric radical-mediated photoenzymatic synthesis strategy is developed to realize the efficient enantioselective synthesis of oxygen-containing benzo-fused heterocycles through structure-guided engineering of a flavin-dependent 'ene'-reductase GluER. It shows that variant GluER-W100H could efficiently produce various benzoxepinones, chromanone and indanone with different benzo-fused rings in high yields with great stereoselectivities under visible light. Moreover, these results are well supported by mechanistic experiments, revealing that this photoenzymatic process involves electron donor-acceptor complex formation, single electron transfer and hydrogen atom transfer. Therefore, we provide an alternative green approach for efficient chemoenzymatic synthesis of important chiral skeletons of bioactive pharmaceuticals.

Full-Length Transcriptome Sequencing Combined with RNA-Seq to Analyze Genes Related to Terpenoid Biosynthesis in Cinnamomum burmannii.

Cinnamomum burmannii is a cinnamomum plant rich in natural D-borneol. Natural D-borneol is a bicycle monoterpenoid compound widely used in the food, pharmaceutical, and cosmetic industries. Therefore, analyzing the biosynthesis mechanism of natural D-borneol in C. burmannii at the molecular level is helpful for directional breeding in the future and further development and utilization of C. burmannii and its related gene resources. In our study, 76 genes related to terpene metabolism were analyzed through third-generation sequencing and second-generation sequencing. Of these genes, 57 were associated with the synthesis of the terpenoid skeleton, and 19 belonged to terpenoid synthase, including four monoterpenoid synthases, seven sesquiterpenoid synthases, and eight diterpenoid synthases. Two genes in diterpenoid synthase were differentially expressed in high D-borneol and low D-borneol plants. It was speculated that these two genes might be related to D-borneol synthesis. How these two genes participate in the synthesis of D-borneol needs further study.

Nanoflare Couple: Multiplexed mRNA Imaging and Logic-Controlled Combinational Therapy.

Theranostics, which combines both diagnostic and therapeutic capabilities in one dose, has always been an intractable challenge in personalized cancer treatment. Herein, a versatile nanotheranostic platform "nanoflare couple (NC)" has been developed for in situ multiplex cancer-related mRNA imaging and subsequent logic-controlled aggregation of gold nanoparticles, leading to gene therapy and photothermal therapy upon irradiation with infrared light. As a proof of concept, TK1 and survivin mRNAs that are highly expressed in most tumor tissues are selected as endogenous cancer indicators and therapy triggers to design the NC. Mice bearing breast cancer cells MCF-7 are prepared as a model to test its efficacy. The in vitro and in vivo assays validate that the NC show the capability for multiplexed mRNA imaging and high efficiency for logic-controlled combinational therapy of breast cancer.

Designing a CRISPR/Cas12a- and Au-Nanobeacon-Based Diagnostic Biosensor Enabling Direct, Rapid, and Sensitive miRNA Detection.

Direct, rapid, sensitive, and selective detection of nucleic acids in complex biological fluids is crucial for medical early diagnosis. We herein combine the trans-cleavage ability of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a with Au-nanobeacon to establish a CRISPR-based biosensor, providing rapid miRNA detection with high speed and attomolar sensitivity. In this strategy, we first report that the trans-cleavage activity of CRISPR/cas12a, which was previously reported to be triggered only by target ssDNA or dsDNA, can be activated by the target miRNA directly. Therefore, this method is direct, i.e., does not need the conversion of miRNA into its complementary DNA (cDNA). Meanwhile, as compared to the traditional ssDNA reporters and molecular beacon (MB) reporters, the Au-nanobeacon reporters exhibit improved reaction kinetics and sensitivity. In this assay, the miRNA-21 could be detected with very high sensitivity in only 5 min. Finally, the proposed strategy enables rapid, sensitive, and selective miRNA determination in complex biological samples, providing a potential tool for medical early diagnosis.

Bimetallic modified halloysite particle electrode enhanced electrocatalytic oxidation for the degradation of sulfanilamide.

The treatment of antibiotics wastewater by electrocatalytic oxidation has attracted much attention. In the paper, a novel halloysite bimetallic (HLS-Cu-Mn) particle electrode material was prepared and a bench-scale electrocatalytic reaction tank was designed. A three-dimensional electrocatalytic oxidation reactor composed of HLS-Cu-Mn and a bench-scale electrocatalytic reaction tank was used to degrade Sulfanilamide (SA) wastewater. Characterization of the synthesized material was conducted with Scanning electron microscopy (SEM), X-ray polycrystalline powder diffractometer (XRD), X-ray photoelectron spectroscopy (XPS), and Brunauer-Emmett-Teller (BET). The electron spin resonance spectroscopy test results confirmed that HLS-Cu-Mn produced a large number of •OH. The electrochemical workstation confirmed that HLS-Cu-Mn had strong electrocatalytic activity and repolarization ability. Under the optimum preparation conditions and degradation process parameters, the removal efficiency of SA and TOC was 99.84% and 88.95% respectively. The method also has good degradation efficiency for aniline, phenol, herbicides, antibiotics, and dyeing wastewater. It was found that 4 main intermediates appeared in the degradation process by Ultra-high performance liquid chromatography/triple tandem quadrupole mass spectrometry (LC-MS). In sum, it was believed that this work provides a new vision and idea for water treatment.

Discovery of the Biosynthetic Pathway of Beticolin 1 Reveals a Novel Non-Heme Iron-Dependent Oxygenase for Anthraquinone Ring Cleavage.

This study used light-mediated comparative transcriptomics to identify the biosynthetic gene cluster of beticolin 1 in Cercospora. It contains an anthraquinone moiety and an unusual halogenated xanthone moiety connected by a bicyclo[3.2.2]nonane. During elucidation of the biosynthetic pathway of beticolin 1, a novel non-heme iron oxygenase BTG13 responsible for anthraquinone ring cleavage was discovered. More importantly, the discovery of non-heme iron oxygenase BTG13 is well supported by experimental evidence: (i) crystal structure and the inductively coupled plasma mass spectrometry revealed that its reactive site is built by an atypical iron ion coordination, where the iron ion is uncommonly coordinated by four histidine residues, an unusual carboxylated-lysine (Kcx377) and water; (ii) Kcx377 is mediated by His58 and Thr299 to modulate the catalytic activity of BTG13. Therefore, we believed this study updates our knowledge of metalloenzymes.

Cell-Surface-Anchored DNA Sensors for Simultaneously Monitoring Extracellular Sodium and Potassium Levels.

The K+ and Na+ levels in cells have a synergistic effect on many biological processes (BPs); therefore, the simultaneous detection of them is important. Here, we propose a novel Y-shaped DNA sensor for simultaneous monitoring of Na+ and K+ in extracellular microenvironments. The designed sensor contributed to the selective response to the above two ions. In addition, it performed the imaging of the above two ions on the cell surface in a real-time, on-site manner, which would shed more light on the association of the Na+/K+ content with regulatory BPs. We believe that this new strategy will be a promising tool to investigate the synergy of Na+/K+ in regulating different BPs.

A 1232 bp upstream sequence of glutamine synthetase 1b from Eichhornia crassipes is a root-preferential promoter sequence.

BACKGROUND: Glutamine synthetase (GS) acts as a key enzyme in plant nitrogen (N) metabolism. It is important to understand the regulation of GS expression in plant. Promoters can initiate the transcription of its downstream gene. Eichhornia crassipes is a most prominent aquatic invasive plant, which has negative effects on environment and economic development. It also can be used in the bioremediation of pollutants present in water and the production of feeding and energy fuel. So identification and characterization of GS promoter in E. crassipes can help to elucidate its regulation mechanism of GS expression and further to control its N metabolism. RESULTS: A 1232 bp genomic fragment upstream of EcGS1b sequence from E. crassipes (EcGS1b-P) has been cloned, analyzed and functionally characterized. TSSP-TCM software and PlantCARE analysis showed a TATA-box core element, a CAAT-box, root specific expression element, light regulation elements including chs-CMA1a, Box I, and Sp1 and other cis-acting elements in the sequence. Three 5'-deletion fragments of EcGS1b upstream sequence with 400 bp, 600 bp and 900 bp length and the 1232 bp fragment were used to drive the expression of ß-glucuronidase (GUS) in tobacco. The quantitative test revealed that GUS activity decreased with the decreasing of the promoter length, which indicated that there were no negative regulated elements in the EcGS1-P. The GUS expressions of EcGS1b-P in roots were significantly higher than those in leaves and stems, indicating EcGS1b-P to be a root-preferential promoter. Real-time Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) analysis of EcGS1b gene also showed higher expression in the roots of E.crassipes than in stems and leaves. CONCLUSIONS: EcGS1b-P is a root-preferential promoter sequence. It can specifically drive the transcription of its downstream gene in root. This study will help to elucidate the regulatory mechanisms of EcGS1b tissue-specific expression and further study its other regulatory mechanisms in order to utilize E.crassipes in remediation of eutrophic water and control its overgrowth from the point of nutrient metabolism.

Dually acid- and GSH-triggered bis(ß-cyclodextrin) as drugs delivery nanoplatform for effective anticancer monotherapy.

The intrinsic poor solubility and limited load capacity of ß-cyclodextrins (ß-CDs) results in reduced bioavailability, rendering the material unsuitable in complex biological environments. In this work, a pair of ß-CDs was methylated and covalently linked with acid-sensitive acylhydrazone and GSH-sensitive disulfide bonds to ensure a precise drug release pattern. The hydrophobic anticancer drug doxorubicin (Dox) was encapsulated inside the hydrophobic core of bis(ß-CD) via hydrophobic association with loading capacity of 24% in weight and a hydrodynamic size of about 100 nm. When exposed to acidic and reductive environments, the acylhydrazone and disulfide bonds were found to be cleaved, resulting in Dox release. Using fluorescence imaging and flow cytometry analysis, the designed bis(ß-CD) were determined to activate the drug release behavior by specific intracellular stimuli (pH and GSH). In vivo studies demonstrated specific drug delivery characteristics and controlled drug release behaviors in the tumor sites, giving rise to high antitumor activity and low toxicity. Taken in concert, this dual stimuli-responsive bis(ß-CD) with superior amphiphilicity and biocompatibility features showed great potential for future clinical applications.

Genome-Wide Analysis of RAV Transcription Factors and Functional Characterization of Anthocyanin-Biosynthesis-Related RAV Genes in Pear.

Related to ABSCISIC ACID INSENSITIVE3/VIVIPAROUS1 (ABI3/VP1, RAV), transcription factors (TFs) belonging to the APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) TF family play critical roles in plant growth, development, and responses to abiotic and biotic stress. In this study, 11 novel RAV TFs were identified in pear (Pyrus bretschneideri Rehd). A phylogenetic analysis revealed that the TFs clustered into three groups with 10 conserved motifs, some of which were group- or subgroup-specific, implying that they are important for the functions of the RAVs in these clades. RAVs in Pyrus and Malus were closely related, and the former showed a collinear relationship. Analysis of their expression patterns in different tissues and at various growth stages and their responses to abiotic and biotic stress suggested that PbRAV6 and PbRAV7 play important roles in drought stress and salt stress, respectively. We investigated the function of RAVs in pear peel coloration using two red pear varieties with different color patterns and applying data from transcriptome analyses. We found that PbRAV6 participates in the regulation of pericarp color. These findings provide insight into a new TF family in pear and a basis for further studies on the response to drought stress and fruit coloration in this commercially important crop.

Intelligent Nanoprobe: Acid-Responsive Drug Release and In Situ Evaluation of Its Own Therapeutic Effect.

The design of an intelligent nanoprobe capable of intracellular controlled release of apoptosis inducers and subsequent high-fidelity imaging of the drug-induced apoptosis is highly desirable for precise cancer treatment. Herein, we report an intelligent nanoprobe that combined therapeutic and imaging functions in one agent. Briefly, a gold nanoparticle is designed to be conjugated with acid-responsive DNA duplexes (Dox intercalates in this region) and caspase-3-specific cleavable peptides (labeled with fluorophore). We demonstrated that the nanoprobe could efficiently deliver an anticancer drug (Dox) into cancer cells and achieve acid-responsive drug release. Furthermore, the apoptotic process was in situ-monitored by detection of fluorescence through the cleavage of the peptide linker by caspase-3, which is one of the executioner caspases involved in apoptosis. This newly developed nanoprobe could serve as a theranostic agent for targeted responsive chemotherapy and also provide feedback apoptosis imaging of the self-therapeutic effect.

Preparation and characterization of molecularly imprinted polymers based on ß-cyclodextrin-stabilized Pickering emulsion polymerization for selective recognition of erythromycin from river water and milk.

Molecularly imprinted polymers were prepared via ß-cyclodextrin-stabilized oil-in-water Pickering emulsion polymerization for selective recognition and adsorption of erythromycin. The synthesized molecularly imprinted polymers were spherical in shape, with diameters ranging from 20 to 40 µm. The molecularly imprinted polymers showed high adsorption capacity (87.08 mg/g) and adsorption isotherm data fitted well with Langmuir model. Adsorption kinetics study demonstrated that the molecularly imprinted polymers acted in a fast adsorption kinetic pattern and the adsorption features of molecularly imprinted polymers followed a pseudo-first-order model. Adsorption selectivity analysis revealed that molecularly imprinted polymers had a much better specificity for erythromycin than that for spiramycin or amoxicillin, and the relative selectivity coefficient values on the bases of spiramycin and amoxicillin were 3.97 and 3.86, respectively. The Molecularly imprinted polymers also showed a satisfactory reusability after four times of regeneration. In addition, molecularly imprinted polymers exhibited good adsorption capacities for erythromycin under complicated environment, that is, river water and milk. These results proved that the as-prepared molecularly imprinted polymers is a potent absorbent for selective recognition of erythromycin, and therefore it may be a promising candidate for practical applications, such as wastewater treatment and detection of erythromycin residues in food.

A nanoprobe for ratiometric imaging of glutathione in living cells based on the use of a nanocomposite prepared from dual-emission carbon dots and manganese dioxide nanosheets.

A ratiometric fluorescence assay for glutathione (GSH) was developed. The novel assay is based on a nanoprobe composed of manganese dioxide nanosheets (MnO2 NS) and dual-emission carbon dots (de-CDs) with intrinsic GSH-response property. After construction of the nanoprobe, two emission peaks of de-CDs were suppressed to varying degrees by MnO2 NS. The suppression was relieved and the two emission peaks recovered proportionally when MnO2 NS was decomposed by GSH, thus realizing the ratiometric assay for micromolar GSH. The intrinsic responsiveness of de-CDs to millimolar GSH broadens the analytical range of the nanoprobe. An appropriate precursor, calcon-carboxylic acid, was screened out to synthesize de-CDs via one-step hydrothermal treatment. The de-CD@MnO2 NS nanoprobe can measure GSH concentrations through the fluorescence intensity ratio between 435 and 516 nm excited at 365 nm. The range of response was from 1 µM to 10 mM and the detection limit reached 0.6 µM (3σ criterion). Benefiting from its good biocompatibility, the proposed nanoprobe has excellent applicability for intracellular GSH imaging.Graphical abstract Schematic representation of glutathione (GSH) ratiometric detection. The nanoprobe is prepared from dual-emission carbon dots (de-CDs) and manganese dioxide nanosheets (MnO2 NS). GSH removes quenching effect by decomposing MnO2 NS and induces intrinsic response of de-CDs, which realizes ratiometric detection.

Antifeedant Activities of Lignans from Stem Bark of Zanthoxylum armatum DC. against Tribolium castaneum.

The speciation of a methanolic extract of Zanthoxylum armatum stem bark has enabled the isolation and characterization of 11 known lignans. Among them, five compounds (6, 8-11) are reported in this plant for the first time. All of the chemical structures were elucidated on the basis of NMR spectral analysis. Additionally, their antifeedant activities against Tribolium castaneum were evaluated scientifically. Among them, asarinin (1), with an EC50 of 25.64 ppm, exhibited a much stronger antifeedant activity than the positive control, toosendanin (EC50 = 71.69 ppm). Moreover, fargesin (2), horsfieldin (3), and magnolone (10), with EC50 values of 63.24, 68.39, and 78.37 ppm, showed almost the same antifeedant activity as the positive control. From the perspective of structure-effectiveness relationship, compounds with the chemical group of methylenedioxy exhibited higher antifeedant activities and have potential to be developed into novel antifeedants or potential lead compounds to protect food and crops in storage.

Chemical Constituents of Supercritical Extracts from Alpinia officinarum and the Feeding Deterrent Activity against Tribolium castaneum.

Alpinia officinarum has been confirmed to possess bioactivities against some pests. In this work, a sample was obtained from A. officinarum rhizomes by supercritical fluid CO2 extraction (SFE). According to GC-MS analysis, the main chemical components for SFE-sample included benzylacetone (26.77%), 1,7-diphenyl-5-hydroxy-3-heptanone (17.78%), guaiacylacetone (10.03%) and benzenepropanal (7.42%). The essential oil of A. officinarum rhizomes (LD50 = 20.71 µg/adult) exhibited more contact toxicity than SFE extract (LD50 = 82.72 µg/adult) against Tribolium castaneum. From SFE extracts, one new compound, 1-phenyl-4-(16,17-dimethyl-9,13-octadiene)-5-isopentenyl-7-(4"-methoxyl-3"-hydroxyl-phenyl)-3-heptanone (3), together with five known compounds identified as 5-hydroxy-1,7-diphenyl-3-heptanone (1), 1,7-diphenyl-4-hepten-3-one (2), galangin (4), galangin-3-methyl ether (5) and pinocembrin (6), were isolated and their feeding deterrent activities against T. castaneum adults were assessed. It was found that compounds 1-6 had feeding deterrent activities against T. castaneum with feeding deterrent indices of 18.21%, 18.94%, 19.79%, 26.99%, 20.34%, and 35.81%, respectively, at the concentration of 1500 ppm. Hence, the essential oil and SFE extracts/compounds of A. officinarum rhizomes represent promising alternatives in the control of T. castaneum adults.