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A key objective for herbicide research is to develop new compounds with improved bioactivity. Protoporphyrinogen IX oxidase (PPO) is an essential target for herbicide discovery. Here, we report using an in silico structure-guided optimization approach of our previous lead compound 1 and designed and synthesized a new series of compounds 2-6. Systematic bioassays led to the discovery of a highly potent compound 6g, 1-methyl-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-yn-1-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione, which exhibited an excellent and wide spectrum of weed control at the rates of 30-75 g ai/ha by the postemergence application and is relatively safe on maize at 75 g ai/ha. Additionally, the Ki value of 6g to Nicotiana tabacum PPO (NtPPO) was found to be 2.5 nM, showing 3-, 12-, and 18-fold higher potency relative to compound 1 (Ki = 7.4 nM), trifludimoxazin (Ki = 31 nM), and flumioxazin (Ki = 46 nM), respectively. Furthermore, molecular simulations further suggested that the thieno[2,3-d]pyrimidine-2,4-dione moiety of 6g could form a more favorable π-π stacking interaction with the Phe392 of NtPPO than the heterocyclic moiety of compound 1. This study provides an effective strategy to obtain enzyme inhibitors with improved performance through molecular simulation and structure-guided optimization.

Protoporphyrinogen oxidase (PPO, EC 1.3.3.4) is an effective target for green herbicide discovery. In this work, we reported the unexpected discovery of a novel series of N-phenylaminomethylthioacetylpyrimidine-2,4-diones (2-6) as promising PPO inhibitors based on investigating the reaction intermediates of our initially designed N-phenyluracil thiazolidinone (1). An efficient one-pot procedure that gave 41 target compounds in good to high yields was developed. Systematic Nicotiana tabacum PPO (NtPPO) inhibitory and herbicidal activity evaluations led to identifying some compounds with improved NtPPO inhibition potency than saflufenacil and good post-emergence herbicidal activity at 37.5-150 g of ai/ha. Among these analogues, ethyl 2-((((2-chloro-4-fluoro-5-(3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1(2H)-yl)phenyl)amino)methyl)thio)acetate (2c) (Ki = 11 nM), exhibited excellent weed control at 37.5-150 g of ai/ha and was safe for rice at 150 g of ai/ha, indicating that compound 2c has the potential to be developed as a new herbicide for weed management in paddy fields. Additionally, our molecular simulation and metabolism studies showed that the side chains of compound 2c could form a hydrogen-bond-mediated seven-membered ring system; substituting a methyl group at R1 could reinforce the hydrogen bond of the ring system and reduce the metabolic rate of target compounds in planta.

In order to evaluate 7-sulfonamide benzoxadiazole (SBD) derivatives for the development of fluorescent probes, herein we investigated the thiolysis reactivity and selectivity of a series of SBD compounds with different atoms (N/O/S/Se) at the 4-position. Both SBD-amine and SBD-ether are stable toward biothiols in buffer (pH 7.4), while SBD-selenoether can react efficiently with biothiols GSH/Hcy, Cys, and H2S to produce SBD-SG/S-Hcy, SBD-NH-Cys, and SBD-SH, respectively, with three different sets of spectral signals. Therefore, the SBD-selenoether compounds should be useful platforms for the differentiation of these biothiols. Though SBD-alkylthioether shows much lower reactivity than SBD-selenoether, SBD-arylthioether is a tunable motif and structural modifications at the aryl moiety enable the rate of thiol-mediated thiolysis to be modified. To this end, an ER-targeted GSH-selective fluorescent probe 7 was rationally designed via thiolysis of SBD-arylthioether. Compared with control probe SBD-Cl, probe 7 exhibits improved GSH selectivity and better biocompatibility. In total, this study highlights that the modification at the 4-position of SBD is an efficient strategy for the development of new fluorescent probes with tunable reactivity and selectivity.

Specific and expeditious identification and enrichment of target proteins in living cells is often a challenging task. The hexahistidine (6His) tag is frequently used to label artificially engineered proteins produced in prokaryotic or eukaryotic cells. Utilizing the interaction between 6His-tag and nitrilotriacetic acid (NTA) mediated by divalent metal ions (Ni2+, Cu2+, Zn2+ or Co2+), we designed and synthesized a series of Nap-G/Biotin/ANA-FFpYGK-NTA probes that, assisted by alkaline phosphatase (ALP), self-assemble into nanofibers. The probe consists of an NTA group that specifically binds to 6His-tag, an FFpY group that promotes self-assembly facilitated by ALP, and a hydrophobic (Nap-G/ANA/Biotin) capping group for various applications. We demonstrate that the ANA-FFpYGK-NTA(Ni2+) nanofibers are fit for real-time tracking of His-tagged protein in living cells, and the Biotin-FFpYGK-NTA(Ni2+) nanofibers are for isolating His-tagged proteins and other proteins that they interact with.

Protoporphyrinogen oxidase (PPO, EC 1.3.3.4) is an important target for green agrochemical discovery. Herein, a novel N-phenylisoxazoline-thiadiazolo[3,4-a]pyridazine herbicidal active scaffold was designed by the scaffold hybridization strategy. Systematic structural optimization enabled the discovery of a series of derivatives with excellent weed control at 9.375-150 g ai/ha by the post-emergent application. Some derivatives exhibited improved Nicotiana tabacum PPO (NtPPO)-inhibitory activity than fluthiacet-methyl. Of these, 2b, with Ki = 21.8 nM, displayed higher weed control than fluthiacet-methyl at the rate of 12-75 g ai/ha, and selective to maize at 75 g ai/ha. In planta, 2b was converted into a bioactive metabolite 5 (Ki = 4.6 nM), which exhibited 4.6-fold more potency than 2b in inhibiting the activity of NtPPO. Molecular dynamics simulation explained that 5 formed stronger π-π interaction with Phe392 than that of 2b. This work not only provides a promising lead compound for weed control in maize fields but is also helpful to understand the molecular mechanism and basis of the designed hybrids.

H2S is a well-known toxic gas and also a gaseous signaling molecule involved in many biological processes. Advanced chemical tools that can regulate H2S levels in vivo are useful for understanding H2S biology as well as its potential therapeutic effects. To this end, we have developed a series of 7-nitro-1,2,3-benzoxadiazole (NBD) amines as potential H2S scavengers. The kinetic studies of thiolysis reactions revealed that incorporation of positively-charged groups onto the NBD amines greatly increased the rate of the H2S-specific thiolysis reaction. We demonstrate that these reactions proceed effectively, with second order rate constants (k2) of >116 M−1 s−1 at 37 °C for NBD-S8. Additionally, we demonstrate that NBD-S8 can effectively scavenge enzymatically-produced and endogenous H2S in live cells. Furthering the biological significance, we demonstrate NBD-S8 mediates scavenging of H2S in mice., We demonstrate that positively-charged NBD amines can effectively scavenge biological H2S in live cells and in mice.

Potassium supported on commercial activated carbon (K/AC) catalysts were prepared and used for catalytic fast pyrolysis (CFP) of walnut shell to produce phenolic-rich bio-oil. Pyrolysis–gas chromatography/mass spectrometry (Py–GC/MS) and fixed-bed CFP tests were performed to reveal the influences of pyrolytic temperature, potassium loading and catalyst-to-biomass (C-to-B) ratio on the product distribution. The Py–GC/MS results showed that the K5.79/AC catalyst in which the potassium loading was 5.79% exhibited the maximal improvement on the yield of phenolic compounds. Pyrolytic temperature significantly affected the total phenolic yield, which achieved the maximum value at 450 °C whether with or without catalysts. Moreover, the C-to-B ratio also dramatically affected the yield of phenolic compounds. The total phenolic yield raised with increasing C-to-B ratio, whereas the excessive C-to-B ratio (higher than 3:1) would lead to an obvious decrease in total phenolic yield. Overall, the yield of total phenolic compounds achieved the maximum value of 145.65 mg/g (14.57 wt%) at 450 °C under C-to-B ratio of 3:1 in Py–GC/MS runs. Meanwhile, the lab-scale experiments obtained the maximal total phenolic yield of 135.13 mg/g (13.51 wt%) at 450 °C under C-to-B ratio of 1:3.

The development of advanced bioorthogonal reactions for detection and labeling of biomolecules is significant in chemical biology. Recently, researchers have found that multifluorinated aryl azides hold great potential for the development of improved bioorthogonal reactions. The fluorine atom can be a perfect substituent group because of its properties of excellent electronegativity and small steric hindrance. In this Minireview, we discuss recent developments of improved hydrogen sulfide (H2 S) fluorescence probes, fast strain-promoted azide-alkyne cycloaddition (SPAAC) and nonhydrolysis Staudinger reactions based on the use of multifluorinated aryl azides. Additionally, kinetic studies and biological applications of these reactions are also presented.

Cyclic dinucleotides (CDNs) are known to activate stimulator of interferon genes (STING) and induce type I interferon responses, therefor possess great potentials to be of immunotherapeutic value for cancers and infectious diseases. However, the existence of different single nucleotide polymorphism (SNP) of human STING (hSTING) gene poses an obstacle to achieve broad-spectrum activation by CDNs. We reported here the design and synthesis of a total of 36 CDNs, representing all structural variations, that contain four bases (A, G, C, U) and two linkage directions (2′-5′-linked and 3′-5′-linked phosphodiester). Through systematic evaluation of IFN-β induction with a dual-luciferase reporter assay, we discovered that wild type hSTING and two isoforms (HAQ and AQ) showed strong response while hSTING-R232H and R293Q exhibited the relatively weak response to CDNs stimulation. For the first time, we found that the c[G(2′,5′)U(2′,5′)] showed excellent activity against all five hSTING variants even equivalent to the endogenous ligand c[G(2′,5′)A(3′,5′)]. Furthermore, we have also demonstrated that 3′-3′ CDNs with two 3′-5′ phosphodiesters showed higher serum and hydrolase stability than 2′-2′ CDNs with two 2′-5′ phosphodiesters and 2′-3′ CDNs with one 2′-5′ and one 3′-5′ phosphodiester. It is very interesting to note that 2′-2′ CDNs has been found for the first time to show strong activity. These findings will stimulate our exploration for the new functional role of CDNs, and provide guidelines to design CDNs based hSTING targeted drugs.

Strigolactones (SLs) are plant hormones that play various roles in plant physiology, including provoking the germination of parasitic weeds Orobanche and Striga. A family of α/β-hydrolases have been proposed to be the SL receptor proteins. Effective assays for measuring the activity of SL receptors could promote the development of SL-related biology and chemistry. In this study, we developed a new approach called pharmacophore-linked probe virtual screening (PPVS). Its application yielded an effective "off-on" probe named Xilatone Red (XLR). This probe showed a broad spectrum and excellent sensitivity toward SL receptors, including ShD14 (Striga D14), for which the detection limit was determined to be in the micromolar range, outperforming that of the commercial fluorogenic agonist Yoshimulactone Green (YLG). Upon hydrolysis by SL receptors, XLR provided fluorogenic and colorimetric signaling responses. Furthermore, XLR could induce germination of Phelipanche aegyptiaca seeds and prevent Arabidopsis max4-1 branching defects at micromolar concentrations. Our molecular simulations revealed the essential factors in the molecular perception of XLR. We anticipate that this study can prompt the discovery of high-performance SL agonists/antagonists to combat parasitic weeds.

Fluorinated RNA molecules, particularly 2'-F RNA, have found a wide range of applications in RNA therapeutics, RNA aptamers, and ribozymes and as 19F NMR probes for elucidating RNA structure. Owing to the instability of 4'-F ribonucleosides, synthesis of 4'-F-modified RNA has long been a challenge. In this study, we developed a strategy for synthesizing a 4'-F-uridine (4'FU) phosphoramidite, and we used it to prepare 4'-F RNA successfully. In the context of an RNA strand, 4'FU, which existed in a North conformation, was reasonably stable and resembled unmodified uridine well. The 19F NMR signal of 4'FU was sensitive to RNA secondary structure, with a chemical shift dispersion as large as 4 ppm (compared with

Many reagents have been developed for cysteine-specific protein modification. However, few of them allow for multi-functionalization of a single Cys residue and disulfide bridging bioconjugation. Herein, we report 3-bromo-5-methylene pyrrolones (3Br-5MPs) as a simple, robust, and versatile class of reagents for cysteine-specific protein modification. These compounds can be facilely synthesized via a one-pot mild reaction and they show comparable tagging efficiency but higher cysteine specificity than the maleimide counterparts. The addition of cysteine to 3Br-5MPs generates conjugates that are amenable to secondary addition by another thiol or cysteine, making 3Br-5MPs valuable for multi-functionalization of a single cysteine and disulfide bridging bioconjugation. The labeling reaction and subsequent treatments are mild enough to produce stable and active protein conjugates for biological applications., Many reagents have been developed for cysteine-specific protein modification. However, few of them allow for multi-functionalization of a single Cys residue and disulfide bridging bioconjugation. Here the authors report 3-bromo-5-methylene pyrrolones as a simple, robust and versatile class of reagents for cysteine-specific protein modification.

We developed a new strategy to overcome the MDR of etoposide using self-assembling nanofibers. Compared with the original etoposide, the inhibitory activity of Nap-GFFpYK-etoposide1/2 against murine Lewis lung cancer or breast cancer cells was increased 10 times, and 20 times on these cells with artificially overexpressed MDR1. Our method to synthesize and separate etoposide isomers provides a new strategy for the modification of this drug.

Protoporphyrinogen oxidase (PPO, EC 1.3.3.4) is a promising target for herbicide discovery. Search for new compounds with novel chemotypes is a key objective for agrochemists. Here, we describe the...

Acetohydroxyacid synthase (AHAS) exists in plants and many microorganisms (including gut flora) but not in mammals, making it an attractive drug target. Fluorescent-based methods should be practical for high-throughput screening of inhibitors. Herein, we describe the development of the first AHAS fluorogenic assay based on an intramolecular charge transfer (ICT)-based fluorescent probe. The assay is facile, sensitive, and continuous and can be applied toward various AHASs from different species, AHAS mutants, and crude cell lysates. The fluorogenic assay was successfully applied for (1) high-throughput screening of commerical herbicides toward different AHASs for choosing matching herbicides, (2) identification of a Soybean AHAS gene with broad-spectrum herbicide resistance, and (3) identification of selective inhibitors toward intestinal-bacterial AHASs. Among the AHAS inhibitors, an active agent was found for selective inhibition of obesity-associated Ruminococcus torques growth, implying the possibility of AHAS inhibitors for the ultimate goal toward antiobesity therapeutics. The fluorogenic assay opens the door for high-throughput programs in AHAS-related fields, and the design principle might be applied for development of fluorogenic assays of other synthases.

Phenol is an important phenolic compound widely used in chemical industry. Lignin in lignocellulosic biomass materials has been recognized as a renewable source for phenol production due to its aromatic nature. In the present study, phenol was selectively prepared from fast pyrolysis of biomass using a recyclable magnetic solid base catalyst (K3PO4/Fe3O4, MSB) in an atmosphere with low hydrogen content. The study focused on the influence of pyrolysis atmosphere, pyrolysis temperature, catalyst content, and biomass type on the product distribution, as well as the recycle performance of MSB in consecutive pyrolysis experiments. Results suggested that the synergistic effect of MSB catalyst and hydrogen atmosphere significantly improved the formation of phenol. In a hydrogen atmosphere at MSB to biomass ratio of 1, the maximal phenol yield of 4.3 wt% with the selectivity of 17.6% was obtained from poplar wood pyrolysis at 550°C. Pyrolysis experiments with different biomass materials suggested that the phenol formation was positively correlated with biomass lignin content. A three consecutive run experiment indicated that the MSB catalyst had excellent performance on recovery and recycle. In addition, possible synergistic mechanism between MSB and hydrogen atmosphere was proposed.

The fast pyrolysis mechanism of xylose, xylobiose and xylan was investigated with combined fast pyrolysis experiments and quantum chemistry calculations. The results indicate that acyclic d -xylose is an important intermediate for the formation of the major pyrolytic products in xylose pyrolysis, i.e., 1,4-anhydro- d -xylopyranose (ADX), furfural (FF) and hydroxyacetaldehyde (HAA). ADX derives from the successive cyclization of d -xylose which is the only source for ADX formation. 3-Deoxy-xylose is the most important intermediate for FF formation. HAA results from the C C bond scission by retro-aldol and cyclic Grob fragment. In addition, the five-membered intermediate derived from the ring-contraction reaction can transform into FF with high selectivity. The dehydrated xylose is also another source for HAA formation. The fast pyrolysis mechanism of xylobiose is similar to that of xylose, and thus similar pyrolytic product distribution can be obtained from them. However, the pyrolytic products of xylan differ significantly from those of xylose and xylobiose, which is ascribed to its high degree of polymerization and the branches in it.

In continuation of our search for potent protoporphyrinogen IX oxidase (PPO, EC 1.3.3.4) inhibitors, we designed and synthesized a series of novel herbicidal cycloalka[d]quinazoline-2,4-dione-benzoxazinones. The bioassay results of these synthesized compounds indicated that most of the compounds exhibited very strong Nicotiana tabacum PPO (NtPPO) inhibition activity. More than half of the 37 synthesized compounds displayed over 80% control of all three tested broadleaf weeds at 37.5-150 g ai/ha by postemergent application, and a majority of them showed no phytotoxicity toward at least one kind of crop at 150 g ai/ha. Promisingly, 17i (Ki = 6.7 nM) was 6 and 4 times more potent than flumioxazin (Ki = 46 nM) and trifludimoxazin (Ki = 31 nM), respectively. Moreover, 17i displayed excellent, broad-spectrum herbicidal activity, even at levels as low as 37.5 g ai/ha, and it was determined to be safe for wheat at 150 g ai/ha in postemergent application, indicating the great potential for 17i development as a herbicide for weed control in wheat fields.

To selectively produce monocyclic aromatic hydrocarbons (MAHs) with low amount of polycyclic aromatic hydrocarbons (PAHs), modified HZSM-5 catalysts (Mo2N/HZSM-5, Ti(SO4)2/HZSM-5 and Ti(SO4)2-Mo2N/HZSM-5) were prepared and employed for ex-situ catalytic fast pyrolysis (CFP) of pine wood. Both analytical pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) and lab-scale pyrolysis experiments were conducted to investigate the effects of catalyst, pyrolysis temperature and catalyst-to-pine ratio on the distribution of aromatic hydrocarbon products. Py-GC/MS results showed that the maximal MAHs yields from the HZSM-5, Mo2N/HZSM-5, Ti(SO4)2/HZSM-5 and Ti(SO4)2-Mo2N/HZSM-5 catalysts were 7.57 wt%, 8.11 wt%, 8.71 wt% and 12.53 wt% at 650 °C and catalyst-to-pine ratio of 7. Corresponding PAHs yields from the four catalysts were 2.55 wt%, 0.97 wt%, 1.47 wt% and 1.10 wt%, which clearly indicated that the Ti(SO4)2-Mo2N/HZSM-5 significantly increased MAHs formation and decreased PAHs formation as compared with HZSM-5. Furthermore, lab-scale experiments were conducted to quantitatively determine the pyrolytic product distribution. Results showed that Ti(SO4)2-Mo2N/HZSM-5 catalyst increased the yield of liquid and gas products, and decreased solid products as compared with HZSM-5. Under optimal conditions of 650 °C and catalyst-to-pine ratio of 4/3, the actual yields of MAHs and PAHs from Ti(SO4)2-Mo2N/HZSM-5 catalyst were 9.71 wt% and 1.45 wt%, respectively. In addition, stability tests confirmed that the Ti(SO4)2-Mo2N/HZSM-5 catalyst possessed better effects on MAHs production as well as anti-deactivation than the HZSM-5.

Eukaryotic genomic DNA is packed into chromatin, whose fundamental structural unit is the nucleosome. As DNA-histone protein complexes, nucleosomes show different properties toward exogenous and endogenous DNA-damaging agents. This review summarizes nucleosome DNA damage due to different sources, including alkylating agents, radicals, UV radiation and reactive DNA damage intermediates. In most cases, the histone core protects the associated DNA against damage via its structure and/or scavenging of damaging agents. In contrast, histones react with damaged DNA and, in some instances, catalyze DNA damage in the nucleosome. The biological consequence of nucleosome DNA damage and future prospects in this field are briefly discussed.

The delivery of siRNAs to selectively target cells poses a great challenge in RNAi-based cancer therapy. The lack of suitable cell-targeting methods seriously restricts the advance in delivering siRNAs to extrahepatic tissues. Based on prostate-specific membrane antigen (PSMA)-targeting ligands, we have synthesized a series of lysine-urea-glutamate (KUE)-siRNA conjugates and verified their effective cell uptake and gene silencing properties in prostate cancers. The results indicated that the KUE-siRNA conjugates could selectively enter PSMA+ LNCaP cells, eventually down-regulating STAT3 expression. Based on post-synthesis modification and receptor-mediated endocytosis, this strategy of constructing ligand-siRNA conjugates might provide a general method of siRNA delivery for cell-targeted gene silencing.

Compounds with a nitrobenzoxadiazole (NBD) skeleton exhibit prominent useful properties including environmental sensitivity, high reactivity toward amines and biothiols (including H(2)S) accompanied by distinct colorimetric and fluorescent changes, fluorescence-quenching ability, and small size, all of which facilitate biomolecular sensing and self-assembly. Amines are important biological nucleophiles, and the unique activity of NBD ethers with amines has allowed for site-specific protein labelling and for the detection of enzyme activities. Both H(2)S and biothiols are involved in a wide range of physiological processes in mammals, and misregulation of these small molecules is associated with numerous diseases including cancers. In this review, we focus on NBD-based synthetic probes as advanced chemical tools for biomolecular sensing. Specifically, we discuss the sensing mechanisms and selectivity of the probes, the design strategies for multi-reactable multi-quenching probes, and the associated biological applications of these important constructs. We also highlight self-assembled NBD-based probes and outline future directions for NBD-based chemosensors. We hope that this comprehensive review will facilitate the development of future probes for investigating and understanding different biological processes and aid the development of potential theranostic agents.

Understanding the role of H2 S in host defense mechanisms against RNA viruses may provide opportunities for the development of antivirals to combat viral infections. Here, we have developed a green-emitting fluorogenic probe, which exhibits a large fluorescence response at 520 nm (>560-fold) when treated with 100 μM H2 S for 1 h. It is highly selective for H2 S over biothiols (>400-fold F/F0 ) and has a detection limit of 12.9 nM. We demonstrate the application of the probe for endogenous H2 S detection in vivo for the understanding of its roles in antiviral host defense. Such virus-induced H2 S inhibits viral replication by reducing gene expression of RNA-dependent RNA polymerase (RdRp) and coat protein (CP). Additionally, a H2 S donor GYY4137 showed significantly antiviral activity as ribavirin, a broad-spectrum drug against RNA viruses. Furtherly, we propose a possible molecular mechanism for the TMV-induced H2 S biogenesis. This work provides a proof-of-principle in support of further studies identifying endogenous H2 S and its donors as potential antivirals toward RNA viruses.

Protoporphyrinogen IX oxidase (PPO) is the last common enzyme in chlorophyll and heme biosynthesis pathways. In human, point mutations on PPO are responsible for the dominantly inherited disorder disease, Variegate Porphyria (VP). Of the VP-causing mutation site, the Arg59 is by far the most prevalent VP mutation residue identified. Multiple sequences alignment of PPOs shows that the Arg59 of human PPO (hPPO) is not conserved, and experiments have shown that the equivalent residues in PPO from various species are essential for enzymatic activity. In this work, it was proposed that the Arg59 performs its function by forming a hydrogen-bonding (HB) network around it in hPPO, and we investigated the role of the HB network via site-directed mutagenesis, enzymatic kinetics and computational studies. We found the integrity of the HB network around Arg59 is important for enzyme activity. The HB network maintains the substrate binding chamber by holding the side chain of Arg59, while it stabilizes the micro-environment of the isoalloxazine ring of FAD, which is favorable for the substrate-FAD interaction. Our result provides a new insight to understanding the relationship between the structure and function for hPPO that non-conserved residues can form a conserved element to maintain the function of protein.

Nanoparticles, that can be enriched in the tumor microenvironment and deliver the payloads into cancer cells, are desirable carriers for theranostic agents in cancer diagnosis and treatment. However, efficient targeted delivery and enhanced endocytosis for probes and drugs in theranostics are still major challenges. Here, a nanoparticle, which is capable of charge reversal from negative to positive in response to matrix metalloproteinase 9 (MMP9) in tumor microenvironment is reported. This nanoparticle is based on a novel charge reversible amphiphilic molecule consisting of hydrophobic oleic acid, MMP9-cleavable peptide, and glutamate-rich segment (named as OMPE). The OMPE-modified cationic liposome forms an intelligent anionic nanohybrid (O-NP) with enhanced endocytosis through surface charge reversal in response to MMP9 in vitro. Successfully, O-NP nanohybrid performs preferential accumulation and enhances the endocytosis in MMP9-expressing xenografted tumors in mouse models, which improve the sensitivity of diagnosis agents and the antitumor effects of drugs in vivo by overcoming their low solubility and/or nonspecific enrichment. These results indicate that O-NP can be a promising delivery platform for cancer diagnosis and therapy.

The direct and indirect competition time-resolved fluorescence immunoassays (dc-TRFIA, ic-TRFIA) were established by combining the autofluorescence properties of lanthanide europium (Eu) with the monoclonal antibody of oxyfluorfen. The purified Eu antibody was optimized and the conditions such as the working concentration of the Eu antibody, monoclonal antibody, and working buffer were optimized. In the optimal condition, the IC50 of dc-TRFIA was 10.27 ng/mL, the lowest detection limit IC10 was 0.071 ng/mL, the detection range (IC10-IC90) was 0.071–1074.3 ng/mL, and the detection range (IC10-IC90) and IC50 of ic-TRFIA were 0.024–504.6 and 2.76 ng/mL, respectively. The comparison showed that the sensitivity and detection limit of ic-TRFIA were superior to dc-TRFIA. The cross reaction (CR) tests showed that the CR with other oxyfluorfen structure analogs was y = 0.975x – 0.4446, R2 = 0.9901), which proved that the TRFIA method established in this study had high reliability and accuracy and could be used in environment and agricultural products for rapid detection of oxyfluorfen residues.

Caspase-3/8 are key members of the cysteine-aspartyl protease family with pivotal roles in apoptosis. We have designed and synthesized self-assembling probes, Nap-GFFpYDEVD-AFC and Nap-GFFpYIETD-AFC, with fluorescence 'turn-on' properties for real-time monitoring of Caspase-3/8 activity in living cells.

8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) is a biomarker of oxidative DNA damage and can be repaired by hOGG1 and APE1 via the base excision repair (BER) pathway. In this work, we studied coordinated BER of 8-oxodGuo by hOGG1 and APE1 in nucleosome core particles and found that histones transiently formed DNA-protein cross-links (DPCs) with active repair intermediates such as 3′-phospho-α,β-unsaturated aldehyde (PUA) and 5′-deoxyribosephosphate (dRP). The effects of histone participation could be beneficial or deleterious to the BER process, depending on the circumstances. In the absence of APE1, histones enhanced the AP lyase activity of hOGG1 by cross-linking with 3′-PUA. However, the formed histone-PUA DPCs hampered the subsequent repair process. In the presence of APE1, both the AP lyase activity of hOGG1 and the formation of histone-PUA DPCs were suppressed. In this case, histones could catalyse removal of the 5′-dRP by transiently cross-linking with the active intermediate. That is, histones promoted the repair by acting as 5′-dRP lyases. Our findings demonstrate that histones participate in multiple steps of 8-oxodGuo repair in nucleosome core particles, highlighting the diverse roles that histones may play during DNA repair in eukaryotic cells.

Root parasitic weeds such as Striga spp. have caused significant losses in agriculture production worldwide. The seed germination of the weeds depends on strigolactones (SLs) that target a series of HYPOSENSITIVE TO LIGHT/KARRIKIN INSENSITIVE2 in Striga hermonthica (ShHTL) proteins. In the present study, 60 ShHTL7 mutants were constructed, and the equilibrium dissociation constants for GR24 (a synthetic SL analogue, commonly used as a standard in SL germination studies) against these mutants were measured by surface plasmon resonance. Based on these data, the SL binding pocket residues were distinguished. Of them, some specific residues for ShHTL7 were found, such as T142, T190, and M219. A model showing quite well internal and external predictive abilities was established by the mutation-dependent biomacromolecular quantitative structure-activity relationship method. It provided an expanded understanding for GR24 binding to a series of ShHTL receptors and should help design broad-spectrum agrochemicals with cross interactions with several members of SL receptors.

The nucleosome is the basic structural repeating unit of chromatin. DNA damage and cell apoptosis release nucleosomes into the blood circulatory system, and increased levels of circulating nucleosomes have been observed to be related to inflammation and autoimmune diseases. However, how circulating nucleosomes trigger immune responses has not been fully elucidated. cGAS (cGMP-AMP synthase) is a recently discovered pattern recognition receptor that senses cytoplasmic double-stranded DNA (dsDNA). In this study, we employed in vitro reconstituted nucleosomes to examine whether extracellular nucleosomes can gain access to the cytoplasm of mammalian cells to induce immune responses by activating cGAS. We showed that nucleosomes can be taken up by various mammalian cells. Additionally, we found that in vitro reconstituted mononucleosomes and oligonucleosomes can be recognized by cGAS. Compared to dsDNA, nucleosomes exhibit higher binding affinities to cGAS but considerably lower potency in cGAS activation. Incubation of monocytic cells with reconstituted nucleosomes leads to limited production of type I interferons and proinflammatory cytokines via a cGAS-dependent mechanism. This proof-of-concept study reveals the cGAS-dependent immunogenicity of nucleosomes and highlights the potential roles of circulating nucleosomes in autoimmune diseases, inflammation, and antitumour immunity.

Chemical labelling can significantly extend the structures and functions of proteins for advanced applications. Herein, a highly efficient bench-stable reagent diazo-azide was designed and synthesized for the incorporation of tetrafluorinated aromatic azides into proteins via the diazonium coupling. The diazo-azide-labelled proteins could be further functionalized via the nonhydrolysis Staudinger reaction to achieve fluorescence labelling, PEGylation and biotinylation. The whole protein labelling processes were catalysis-free and could be finished within several hours under the mild conditions. To this end, we have prepared thickened viral nanoparticles with controllable diameters.

The combined usage of two bioorthogonal reactions can provide hetero-bifunctional molecules under physiological conditions for various applications. Based on the Nonhydrolysis Staudinger Reaction (NSR), we design and develop a bisazido linker 1 for chemoselective dual-functionalization without the need of protection using catalyst-free and one-pot procedures. The NSR is much faster with tetrafluorinated aromatic azide than that the Staudinger-Bertozzi or SPAAC ligation with alkyl azide, as revealed by HPLC analysis and fluorescence kinetics. Based on the tandem NSR and Staudinger-Bertozzi ligation, we prepare a molecular beacon 7 from 1 in one-pot synthesis with a recovery yield of 32%. When a faster SPAAC ligation is used instead of the Staudinger-Bertozzi ligation, compound 8 is prepared in the tandem NSR and SPAAC reactions with a recovery yield of 59%. As a proof-of-concept study, the tandem NSR and SPAAC ligation is further used to produce a FRET-based dyad in living cells, as revealed by dual-color bioimaging. This work shows that NSR can be combined with other bioorthogonal reactions without the need of protection in one-pot.

The RNA-guided CRISPR/Cas9 system could cleave double-stranded DNA at the on-target sites but also induce off-target mutations in unexpected genomic regions. The base-pairing interaction of sgRNA with off-target DNA was still not well understood and also lacked a direct cell-based assay. Herein we developed a fast target DNA mutagenesis-based fluorescence assay to directly detect the Cas9 activity at different off-target sites in living cells. The results showed that Cas9 nuclease had low tolerance to the nucleotide mismatches in the binding region adjacent to PAM sites, and a tradeoff between Cas9 activity and specificity was also observed compared with the high-fidelity Cas9 variant. The combination of computer-based predictions and this target DNA mutagenesis-based fluorescence assay could further provide accurate off-target prediction guidance to minimize off-target effects to enable safer genome engineering.

Herein, we report the synthesis of 4'-C-trifluoromethyl (4'-CF3) thymidine (T4'-CF3) and its incorporation into oligodeoxynucleotides (ODNs) through solid-supported DNA synthesis. The 4'-CF3 modification leads to a marginal effect on the deoxyribose conformation and a local helical structure perturbation for ODN/RNA duplexes. This type of modification slightly decreases the thermal stability of ODN/RNA duplexes (-1 °C/modification) and leads to improved nuclease resistance. Like the well-known phosphorothioate (PS) modification, heavy 4'-CF3 modifications enable direct cellular uptake of the modified ODNs without any delivery reagents. This work highlights that 4'-CF3 modified ODNs are promising candidates for antisense-based therapeutics, which will, in turn, inspire us to develop more potent modifications for antisense ODNs and siRNAs.

Biothiols are implicated in different physiological and pathological processes, while abnormal levels of biothiols are related to numerous diseases. It is essential to discriminatively detect these molecules and explore their inherent transformation in living biological samples. Herein, dual-reactive and dual-quenching fluorescent probes based on thiolysis of NBD (7-nitro-1,2,3-benzoxadiazole) thioether/ether/amine for separate detection of H2S and Cys/Hcy were rationally designed. Probe 2 was able to sense H2S (λex/em = 405/455 nm) and Cys/Hcy (λex/em = 470/550 nm) in the presence of GSH in aqueous buffer. Moreover, the probe not only could be applied for visualization of exogenous H2S and Cys in living cells but also successfully showcased the potential to verify the biosynthesis of endogenous H2S from Cys.

The acetohydroxyacid synthase (AHAS) holoenzyme catalyzes the first step of branch-chain amino acid biosynthesis and is essential for plants and bacteria. It consists of a regulatory subunit (RSU) and a catalytic subunit (CSU). The allosteric mechanism of the AHAS holoenzyme has remained elusive for decades. Here, we determined the crystal structure of the AHAS holoenzyme, revealing the association between the RSU and CSU in an A2B2 mode. Structural analysis in combination with mutational studies demonstrated that the RSU dimer forms extensive interactions with the CSU dimer, in which a conserved salt bridge between R32 and D120 may act as a trigger to open the activation loop of the CSU, resulting in the activation of the CSU by the RSU. Our study reveals the activation mechanism of the AHAS holoenzyme.

8-Oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodGuo) is a common primary product of cellular oxidative DNA damage. 8-OxodGuo is more readily oxidized than 2’-deoxyguanosine (dG); a two-electron oxidation generates a highly reactive intermediate (OG(ox)), which forms covalent adducts with nucleophiles, including OH(−), free amines, and the side chains of amino acids such as lysine. We determined here that K(3)Fe(CN)(6) oxidation of 8-oxodGuo in nucleosome core particles (NCPs) produces high yields, quantitative (i.e. 100%) in some cases, of DNA-protein cross-links (DPCs). The efficiency of DPC formation was closely related to 8-oxodGuo base pairing and location within the NCP and was only slightly decreased by adding the DNA-protective polyamine spermine to the system. Using NCPs that contained histone mutants, we determined that DPCs result predominantly from OG(ox) trapping by the N-terminal histone amine. The DPCs were stable under physiological conditions and therefore could have important biological consequences. For instance, the essentially quantitative yield of DPCs at some positions within NCPs would reduce the yield of the mutagenic DNA lesions spiroiminodihydantoin and guanidinohydantoin produced from the common intermediate OG(ox), which in turn would affect mutation signatures of oxidative stress in a position dependent manner. In summary, our findings indicate that site-specific incorporation of 8-oxodGuo into NCPs, followed by its oxidation, leads to DPCs with an efficiency depending on 8-oxodGuo location and orientation. Given that 8-oxodGuo formation is widespread in genomic DNA and that DPC formation is highly efficient, DPCs may occur in eukaryotic cells and may affect several important biological processes.

Activated carbon (AC) prepared by physical activation with steam (named as AC-H2O) was employed for catalytic fast pyrolysis of bagasse in a hydrogen atmosphere to selectively produce 4-ethyl phenol (4-EP), a valuable phenolic compound. Lab-scale fast pyrolysis experiments were conducted to quantitatively determine the pyrolytic products and investigate several factors on the 4-EP production, including pyrolysis atmosphere, AC type, catalytic pyrolysis temperature, and AC-to-bagasse ratio. The results indicated that the AC-H2O (activated by H2O) catalyst exhibited much better performance on 4-EP production than other ACs prepared by different activation methods. Compared with the inert atmosphere, the presence of hydrogen atmosphere could significantly promote the 4-EP formation with the yield increase by over 36%. The maximal 4-EP yield of 3.55 wt% with the selectivity of 17.42% was obtained under AC-to-bagasse ratio of 1.5 at 330 °C. In addition, the gas product produced by the pyrolysis process could be recycled to provide a hydrogen atmosphere without an additional hydrogen supply.

RNA cleavage via internal transesterification is a fundamental reaction involved in RNA processing and metabolism, and the regulation thereof. Herein, the influence of ribose conformation on this reaction was investigated with conformationally constrained ribonucleotides. RNA cleavage rates were found to decrease in the order South-constrained ribonucleotide > native ribonucleotide ≫ North-constrained counterpart, indicating that the ribose conformation plays an important role in modulating RNA cleavage via internal transesterification.

Sulfuric acid (H2SO4) is widely used as a strong acid catalyst in biomass pyrolysis process, and exhibits prominent catalytic effects on the pyrolytic reactions and product distribution. In this study, the fundamental catalytic mechanism of H2SO4 on cellulose pyrolysis process was investigated via combined experimental and computational methods. Both thermographic analysis (TGA) and pyrolysis–gas chromatography/mass spectrometry (Py–GC/MS) experiments were performed to reveal the pyrolytic characteristics and product distribution in the H2SO4-catalyzed pyrolysis of cellulose. In addition, quantum chemistry methods were employed to build the reaction models and investigate the major H2SO4-assisted reactions in initial cellulose pyrolysis process, i.e., depolymerization of the cellulose chain, bridged dehydration, ring-opening, ring-contraction and dehydration of the free hydroxyl groups. The results indicate that the H2SO4-assisted depolymerization via C1-O1 bond scission and bridged dehydration reactions take place in a two-step mechanism involving a sulfate ester intermediate. While other reactions occur via a one-step mechanism involving only hydroxyl group or both hydroxyl and sulfonic groups of H2SO4. The activation energies of above reactions are all decreased by adding H2SO4, and thus lowering the degradation temperature of cellulose. Among all initial pyrolytic reactions, dehydration reactions which are inconspicuous in the non-catalytic process, become very important in the catalytic process, because their activation energies are decreased dramatically by adding H2SO4 (from 322.7 to 372.1 to 152.2–237.7 kJ/mol). The promoting effect on the dehydration reactions plays a vital role in the increased char yield and significant change of organic volatile product distribution. Through the promoted dehydration reactions, unsaturated structures of cellulose are formed, which is favorable for the formation of certain dehydrated products at the expense of depolymerized and ring scission products.

A series of Mo2N/HZSM-5 and transition metal modified Mo2N/HZSM-5 catalysts were prepared for the catalytic upgrading of pine wood-derived pyrolytic vapors for the selective production of monocyclic aromatic hydrocarbons (MAHs), while restraining the formation of polycyclic aromatic hydrocarbons (PAHs). Pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) experiments were performed to determine the effects of several factors on selective MAHs production, including Mo2N loading on HZSM-5, transition metal (Fe, Ce, La, Cu, Cr) modification of Mo2N/HZSM-5, pyrolysis temperature, and catalyst-to-biomass ratio. In addition, quantitative experiments were conducted to determine the actual yields of major aromatic hydrocarbons and the source of aromatic hydrocarbons from basic biomass components. Results indicated that among the various catalysts, the Ce-10%Mo2N/HZSM-5 exhibited the best performance on promoting the formation of MAHs and restraining the generation of PAHs. Under the optimal conditions, the actual yields of MAHs and PAHs from Ce-10%Mo2N/HZSM-5 catalytic process were 99.8mg/g and 7.5mg/g, while those from HZSM catalyst were only 77.2mg/g and 23.7mg/g respectively. Furthermore, the possible catalytic mechanism of the Ce-Mo2N/HZSM-5 catalyst was proposed based on the catalyst characterization.

Bright and hydrosoluble fluorescent dyes with large Stokes shifts are widely used as molecular probes and light-emitting markers in chemistry and biology. In this report, one-step synthesis of a bright green-emitting coumarin (Φ = 0.52, λem = 505 nm) was achieved from available reagents under neat conditions. The novel dye 1 showed very large Stokes shift (Δλ = 138 nm) due to strong intramolecular charge transfer (ICT) effect, which was confirmed by density functional theory (DFT) calculations. Dye 1 was hydrosoluble and displayed strong emission at a wide range of pH from 3 to 8. Importantly, the secondary amine in 1 made it extremely convenient for futher bioconjugation. Representative derivatives were showcased for various successful applications of fluorogenic enzyme assay, biolabelling and mitochondrial staining.