First Report of Neopestalotiopsis clavispora Causing Leaf Spot Disease on Banana (Musa acuminata) in China
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Banana (Musa acuminate L.) is an important tropical fruit in China. In October 2020, a new leaf spot disease was observed on banana plants at an orchard of Zhenkang county (23°45'23.46″ N, 98°48'46.52″ E), Lincang city, Yunnan province, China. The disease incidence was about 1%. The leaf spots occurred sporadically and the percentage of the leaf area covered by lesions was less than 5%. Symptoms on the leaves were initially small, irregular, reddish-brown spots that gradually expanded to fusiform-shaped lesions with a yellow halo and eventually become necrotic, dry, and cracked. To isolate the pathogen, thirty symptomatic leaves (15 mm2) from five plants were surface disinfected in 70% ethanol (10 s) and 0.8% NaClO (2 min), rinsed in sterile water three times, and transferred to potato dextrose agar (PDA) at 28°C for 5 days. Twenty-five colonies formed on the PDA plates were white with cottony aerial mycelium, round with a light orange underside. Abundant black globular acervuli semi-immersed on PDA were observed after a week. Conidia were straight or slightly curved, clavate to spindle, five cells, four septa with dimensions of 17.49 to 34.51 µm × 4.24 to 7.28 µm (avg. 23.83 × 5.62 µm; n=50). The apical and basal cells were hyaline, whereas the three median cells were dark brown. Conidia had a single basal appendage with lengths of 2.95 to 17.7 µm (avg. 7.18 µm; n=50) and two to three apical appendages with lengths of 10.7 to 53.84 µm (avg. 17.36 µm; n=50). These morphological characteristics are consistent with those of Neopestalotiopsis spp. (Maharachchikumbura et al. 2014). To confirm species, single-spore cultures of two representative isolates CATAS-102001 and CATAS-102002 were selected for further identification. The internal transcribed spacer (ITS) region, translation elongation factor 1-α (TEF1-α) and β-tubulin (TUB2) genes of the two isolates were amplified with primers ITS1/ITS4 (White et al. 1990), EF1-728/EF2 (O'Donnell et al. 1998; Carbone and Kohn, 1999) and T1/Bt2b (Glass and Donaldson, 1995; O'Donnell and Cigelnik, 1997), respectively, and sequenced. The sequences were deposited in GenBank (ITS: OM281005 and OM281006; TEF1-α: OM328820 and OM328821; TUB2: OM328818 and OM328819). A maximum likelihood phylogenetic tree was constructed using the MEGA 7.0 (Kumar et al. 2016) based on the concatenated sequences ITS region, EF1-α and TUB2 gene, and the cluster analysis placed the representative isolates CATAS-102001 and CATAS-102002 within a clade comprising Neopestalotiopsis clavispora. The pathogenicity of two isolates was conducted on six 7-leaf-old banana seedlings. Two leaves from each potted plants were stab inoculated by puncturing into 1-mm using a sterilized needle, and stabbing three points at both sides of leaf midrib, and then placing 10 μl conidial suspension (1×106 conidia/ml) on one side of wounded points and the other side of wounded points were inoculated with sterile water as control. Inoculated plants were kept inside a plastic bag for 72 h and maintained in the greenhouse (12 h/12 h light/dark, 28°C, 90% relative humidity). The experiments were repeated twice. Irregular necrotic lesions on inoculated leaves appeared 7 days after inoculation, whereas controls were asymptomatic. The fungus was recovered from inoculated leaves, and its taxonomy was confirmed morphologically and molecularly, fulfilling Koch's postulates. Neopestalotiopsis clavispora has been reported to cause leaf spot on Mangifera indica (Shu et al. 2020), Macadamia integrifolia (Santos et al. 2019) and Ligustrum lucidum (Chen et al. 2020). To our knowledge, this is the first report of N. clavispora on banana in China. The identification of N. clavispora as the causal agent of the observed leaf spot disease on banana is critical to the prevention and control of this disease in the future.Keywords:
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Journal Article Oscillatory Behavior of NiAu Nanocatalyst in Wet Gas Environment Get access Xiaoben Zhang, Xiaoben Zhang Dalian Institute of Chemical Physics (DICP), Chinese Academy of Sciences, Dalian, ChinaUniversity of Chinese Academy of Sciences, Beijing, China Search for other works by this author on: Oxford Academic Google Scholar Fan Zhang, Fan Zhang Dalian Institute of Chemical Physics (DICP), Chinese Academy of Sciences, Dalian, ChinaUniversity of Chinese Academy of Sciences, Beijing, China Search for other works by this author on: Oxford Academic Google Scholar Shaobo Han, Shaobo Han Dalian Institute of Chemical Physics (DICP), Chinese Academy of Sciences, Dalian, China Search for other works by this author on: Oxford Academic Google Scholar Dan Zhou, Dan Zhou DENSsolutions B.V., Delft, The Netherlands Corresponding author: dan.zhou@denssolutions.com; weiliu@dicp.ac.cn Search for other works by this author on: Oxford Academic Google Scholar Wei Liu, Wei Liu Dalian Institute of Chemical Physics (DICP), Chinese Academy of Sciences, Dalian, China Corresponding author: dan.zhou@denssolutions.com; weiliu@dicp.ac.cn Search for other works by this author on: Oxford Academic Google Scholar Wenjie Shen Wenjie Shen Dalian Institute of Chemical Physics (DICP), Chinese Academy of Sciences, Dalian, China Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 28, Issue S1, 1 August 2022, Pages 162–163, https://doi.org/10.1017/S1431927622001544 Published: 01 August 2022
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Open AccessCCS ChemistryRESEARCH ARTICLE1 Feb 2021In Vivo Activation of Pro-Protein Therapeutics via Chemically Engineered Enzyme Cascade Reaction Xiaoti Yang, Jin Chang, Ying Jiang, Qiaobing Xu, Ming Wang and Lanqun Mao Xiaoti Yang Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences (ICCAS), Beijing 100190 University of Chinese Academy of Sciences, Beijing 100049 , Jin Chang Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences (ICCAS), Beijing 100190 University of Chinese Academy of Sciences, Beijing 100049 , Ying Jiang College of Chemistry, Beijing Normal University, Beijing 100875 , Qiaobing Xu Department of Biomedical Engineering, Tufts University, 4 Colby Street, Medford, MA 02155. , Ming Wang *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences (ICCAS), Beijing 100190 University of Chinese Academy of Sciences, Beijing 100049 and Lanqun Mao *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences (ICCAS), Beijing 100190 University of Chinese Academy of Sciences, Beijing 100049 https://doi.org/10.31635/ccschem.020.202000224 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Selective and temporal control over protein activity is of great importance for the advancement of the protein of interest into precise molecular medicine. Simply installing synthetic ligands to proteins for activity regulation, however, is often obscured by either nonspecificity or insufficient efficiency. This study reports a chemical approach in which enzymatic cascade reactions were designed for selective activation of pro-protein both in vitro and in vivo. Specifically, the system consisted of aromatic boronic-acid-modified nanoparticles, reactive oxygen species (ROS)-responsive pro-protein (RNase A-NBC), a small molecule drug, β-Lapachone (β-Lap), and strategically screened synthetic lipids, required for the assembly of the nanocomplexes. Once target-delivered into tumor cells, the reduction of β-Lap produces massive H2O2 in response to NAD(P)H quinone oxidoreductase 1 (NQO1), a tumor-specific enzyme, which triggers further induction by selective chemical modification of ROS-responsive cytosolic protein ribonuclease A (RNase A)-NBC, thus, switching from "inert" pro-protein to active therapeutics, that ultimately prohibit tumor cell growth. Moreover, the designed enzymatic cascade activation of the pro-protein was effective in vivo, displaying superior therapeutic efficacy to either the pro-protein alone or the β-Lap via tumor-targeted delivery and the consequent suppression of the tumor growth. As both RNase A and β-Lap have been evaluated clinically as antitumor therapeutics, our chemical multi-step cascade methodology is, therefore, a promising strategy for selective modulation of pro-protein chemistry in the living system for fundamental investigations, favorable toward potential anticancer applications. Download figure Download PowerPoint Introduction Proteins are essential biomacromolecules that execute diverse functionalities for the regulation of cell fate and life cycle.1 The abnormal expression or dysfunction of intracellular proteins could result in complicated biological consequences and cause various diseases, including cancers.2 Therefore, supplementing disease cells with functional proteins to compensate those that are dysregulated is the most straightforward strategy for cell manipulation and disease treatment.3 The past decade has witnessed great success in developing protein-based biotherapeutics for diverse clinical purposes, including the immune checkpoint inhibitors, PD-1, and PD-L1 antibodies, for cancer immunotherapy.4,5 However, one challenge associated with protein therapy is the lack of control of the protein activity in a selective manner for targeted disease treatment.6–8 In particular, superior selective control of protein activity that stays "inert" until "activation" by exogenous stimuli or endogenous biomarkers of malignant cells is highly desired, but this technique remains extremely limited.9,10 In this regard, pro-proteins that could switch between the "inert" and "active" states under the influence of the endogenous species of disease cells (e.g., tumor metabolites, intracellular enzymes), particularly, those that would show specific response in the intracellular malignant cells microenvironment, are imperative for the advancement of targeted protein therapy.11–14 In the past four years, we and several others have demonstrated highly-efficient designed chemical approaches to control protein conjugation and activity to enable manipulation of cellular function for therapeutic applications.9,15–21 We have reported previously that the conjugation of stimulus-responsive chemical moieties to proteins could deactivate these proteins temporarily and make them (pro-proteins) stay "inert."10,11,22,23 Subsequently, in the presence of either exogenous stimuli or endogenous chemical triggers, the pro-proteins switched to "active" state by removal of the chemical tags. A wide variety of chemical techniques have been used to control protein activity following this strategy; however, their potential for developing pro-protein therapeutics in response to the intracellular microenvironment of the malignant cells is lacking, mostly due to the low effectiveness and alterations in selectivity during proteins switch from "inert" state to "active" state inside the malignant cells, both in vitro and in vivo.12 Natural systems have evolved multiple enzyme-initiated cascade reactions, for instance, post-translational modifications (PTMs) to switch protein activity selectively and the transduction of upstream cell signals into cell phenotypic changes.24 Therefore, we envisioned that regulating the chemical modification of proteins via integration of intracellular environmental factors and enzymatic cascade reactions would be of great appeal for the spatiotemporal control of protein activity in living cells and the development of novel pro-protein therapeutics.25 In this study, we report the first example of controlling the chemical conjugation and activity of pro-protein using tumor-cell-selective enzymatic cascade reactions and the subsequent potential of this cascade system in the development of targeted protein therapy. We show the design of the encapsulated nanoprodrug targeted delivery into the tumor cells and the subsequent cascade of enzymatic reactions that led to the pro-protein activation, as displayed in the illustration of Figure 1. We employed the NAD(P)H quinone oxidoreductase 1 (NQO1), an enzyme that is overexpressed in tumor cells, which catalyzes the futile reduction of β-Lapachone (β-Lap) to generate massive reactive oxygen species (ROS).26,27 We were able to utilize this NQO1 reaction to control the chemical modification of boronic-acid-conjugated ROS-responsive pro-protein, RNase A-NBC, in a cascade manner, to switch its activity from the "inert" to the "active" state (Figure 1). Moreover, to enable an in vivo pro-protein activation within the tumor cells, we designed the cascade system to feature RNase A-NBC, β-Lap, and strategically screened synthetic lipids that could assemble nanoparticles to aid the codelivery of the pro-protein and β-Lap simultaneously into the targeted tumor cells. Once delivered, the reduction of β-Lap could amplify intracellular H2O2 level in response to the cellular specific NQO1, which further switches the "inert" RNase A-NBC to active therapeutics, thereby, bringing about the selective prohibition of the tumor cell growth. Furthermore, the engineered enzymatic cascade reaction displayed superior therapeutic efficacy than either the pro-protein alone or the β-Lap alone delivered in vivo to suppress the tumor growth. As both RNase A and β-Lap have been validated clinically as antitumor therapeutics, our chemical methodology is, therefore, a promising strategy for selective modulation of protein chemistry in the living system for fundamental research purposes and the achievement of potential anticancer applications. Figure 1 | (a) NQO1-catalyzed futile redox cycle of β-Lapachone (β-Lap) to generate massive ROS that activates the pro-protein, RNase A-NBC, in living cells. (b) Nanoparticle formulation facilitates simultaneous delivery of RNase A-NBC and β-Lap into NQO1-overexpressed tumor cells for pro-protein activation in vivo. Schematic illustration of pro-protein activation in vitro and in vivo. Download figure Download PowerPoint Experimental Method Materials and Methods All chemicals used for the organic syntheses, including dicoumarol and bovine pancreatic ribonuclease A (RNAse A), were purchased from Sigma-Aldrich (St. Louis, MO, USA). NAD(P)H quinone oxidoreductase 1 (NQO1), β-Lapachone (β-Lap), and 2,3-dimethoxy 1,4-naphthoquinone (DMNQ) were obtained from Abcam (Boston, USA). 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-Tetrazolium (WST-1) was purchased from BioVision (Milpitas, USA). Nicotinamide adenine dinucleotide (NADH) was purchased from Calbiochem (La Jolla, CA). Chemically-modified RNase A-NBC was prepared by reacting RNase A with an excess amount of 4-nitrophenyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl carbonate.10 The siRNA targeting human NQO1 (CCGUACACAGAUACCUUGA) was synthesized by BIOSYNTHCH (Suzhou, China). All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committees (IUCAC), National Center for Nanoscience and Technology of China (NCNTC). RNase A-NBC activation driven by NQO1-initiated catalytic reduction of β-Lap To verify that the NQO1-catalyzed redox cycle of β-Lap could generate H2O2 to activate RNase A-NBC in situ, RNase A-NBC (13.3 μg/mL) was incubated with β-Lap (0.33 μM) and NQO1 (0.32 ng/mL) in the presence of NADH (1.33 mM) in Dulbecco's phosphate-buffered saline (DPBS) at 37 °C for 2 h. The enzyme activity of the reaction mixture was assayed using a commercial RNase A assay kit and the RNaseAlert Nuclease Detection System (Integrated DNA Technologies, IA, USA). The RNase A detection was monitored using plate reader (BioTek, synergy H1M, USA) at 520 nm emission wavelength, and the results were compared with that of RNase A-NBC reaction without the addition of the β-Lap reducer NQO1, or both NQO1 and the H2O2 inducer β-Lap). Meanwhile, to further confirm the specificity of NQO1-catalyzed β-Lap reduction in restoring RNase A-NBC activity, an inhibitor of NQO1, dicoumarol (250 μM), was mixed with RNase A-NBC, β-Lap, and NQO1 under the same reaction conditions for the RNase A activity assay. Lipid/RNase A-NBC/β-lap nanoparticle preparation To prepare lipid nanoparticle encapsulating RNase A-NBC and β-Lap, lipid nanoparticles, EC16-80, and the polystyrene polymer, 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), were mixed at a weight ratio of 4∶1 in chloroform, to which 1 × 10−7 mol of β-Lap was added. After evaporating the organic solvent under vacuum, the resulted thin layer film was hydrated using DPBS with the aid of bath sonication. Subsequently, 100 μg RNase A-NBC was added into the solution mentioned above along with 200 μg of the polyethylene glycol conjugated phospholipid stabilizer, DSPE-mPEG2000. The final concentration of the lipid in the nanoparticle formulation was 1 mg/mL. The size and morphology of the final micellar EC16-80/RNase A-NBC/β-Lap nanoparticles were characterized using dynamic light scattering (DLS) measurement, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Cell culture All cells used in this study were purchased from National Infrastructure of Cell Line Resource (Beijing, China) and maintained in high-glucose Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C in the presence of 5% CO2. For the intracellular delivery, the cells were subcultured and seeded in 96-well or 48-well plates for 24 h prior to the experiment. DMEM, FBS, and penicillin/streptomycin were purchased from Gibco (NY, USA). Intracellular ROS assay of MCF-7 cells treated with EC16-80/RNase A-NBC/β-Lap nanoparticle The intracellular ROS level of breast cancer MCF-7 cells with and without the formulated nanoparticle (EC16-80/RNase A-NBC/β-Lap) treatment was measured using 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) staining, followed by flow cytometry analysis. MCF-7 cells were seeded in a 24-well plate at a density of 5×104/well and treated with EC16-80/RNase A-NBC/β-Lap nanoparticle (Group 1) or without treatment (Group 2) for 12 h. At the end of the incubation, the cells were harvested, washed by DPBS, stained with DCFDA (1 μM), and immediately subjected to flow cytometry analysis on a Beckman Coulter CytoFLEX (Beckman Coulter, USA). Cell uptake study of EC16-80/RNase A-NBC/β-Lap nanoparticles For the cellular uptake study of EC16-80/RNase A-NBC/β-Lap nanoparticles, the RNase A-NBC was labeled with fluorescein isothiocyanate (FITC) before use. Briefly, 2 mg RNase A-NBC dissolved in 750 μL of sodium bicarbonate solution (0.1 M, pH = 9.5) was mixed with 250 μL of freshly prepared FITC solution (4 mg/mL in DMSO). Then the reaction mixture was stirred at room temperature for 2 h with protection from light. The resulted FITC-labeled RNase A-NBC was purified by size exclusion chromatography (SEC) on a PD-10 desalting column (GE Healthcare, MA, USA). To study the cellular uptake of EC16-80/RNase A-NBC/β-Lap nanoparticles, MCF-7 cells were seeded at a density of 1.5 × 105 cells/well in glass-bottom cell culture dishes (NEST Biotechnology, Wuxi, China) or 2.5 × 104 cells/well in 48-well plates for 24 h before the experiment and then treated with EC16-80/RNase A-NBC/β-Lap nanoparticles (at varying protein concentration, as indicated) for 8 h at 37 °C. At the end of incubation, the cells were washed twice thoroughly with 0.1% heparin solution, followed by confocal laser scanning microscopy (CLSM) imaging on OLYMPUS FV1000-IX81 or by flow cytometry analysis. Intracellular delivery of EC16-80/RNase A-NBC/β-Lap nanoparticles for targeted cancer therapy To verify the delivery of EC16-80/RNase A-NBC/β-Lap nanoparticles that can activate RNase A-NBC in live cells to prohibit tumor cell proliferation, a variety of cancerous cells, including MCF-7 (breast cancer), A549 (human alveolar basal epithelial adenocarcinoma), HeLa (human cervical cancer), and SiHa (human cervical cancer) cell lines, were seeded in 96-well plates at a density of 1 × 104 cells per well 24 h before the prodrug delivery experiment. On the day of the experiment, EC16-80/RNase A-NBC/β-Lap nanoparticles were added directly to the cell cultures, with the final RNase A-NBC concentration increasing from 0.17 to 1.0 μg/mL. After 8 h incubation, the mixtures were replaced with fresh cell culture medium, followed by another 3 days of cell culture. Subsequently, the cell viability was measured by an MTT assay. We excluded the potential of any cytotoxic effects of the empty EC16-80, EC16-80/β-Lap, or EC16-80/RNase A-NBC nanoparticles by adding these individual nanoparticle formulations as controls to each plated cell culture group under the same experimental conditions alongside that of EC16-80/RNase A-NBC/β-Lap nanoparticle (test) system. Then we investigated whether an NQO1-catalyzed cascade reaction could selectively activate RNase A-NBC in cancer cells by adding three noncancerous cell lines, including HEK-293 T (human embryonic kidney SV40 mutant large T antigen), HK-2 (human immortalized proximal tubule epithelial), and NIH-3T3 (murine fibroblast) cells to our four cancer cell lines in the assay. We seeded each of the seven cell types in a 48-well plate at a density of 2.5 × 104 cells/well. The plated cells were treated with the different nanoparticle formulations under the same conditions, and the cell viabilities were measured in each group using an alamarBlue assay. Further, we confirmed the effect of intracellular NQO1 on RNase A-NBC activation in live cells by treating SiHa cells with small interfering (si)NQO1 to study its impact on the knockdown of endogenous NQO1 expression. Briefly, SiHa cells were seeded in 48-well plates at a density of 1.8 × 104 cells/well for 24 h before the delivery experiment. On the day of the experiment, a mixture of siNQO1 and Lipofectamine 2000 (Life Technology, USA) was added directly to SiHa cells at concentrations of 80 nM for siNQO1 and 2.7 μg/mL for Lipofectamine 2000. After 10 h incubation, the mixtures were replaced with fresh cell culture medium for another 6 h. EC16-80/RNase A-NBC/β-Lap nanoparticles were added to the siNQO1 pretreated SiHa cells at final concentrations of 0.4 μg/mL RNase A-NBC and 1.6 μg/mL of EC16-80. After 6 h of further incubation, the mixtures were replaced with fresh cell culture medium, and an alamarBlue assay was performed to measure cell viability. Endogenous NQO1 activity assay We evaluated the endogenous NQO1 expression level in cancerous and noncancerous cells by measuring the activity of NQO1 in the lysates of the seven cell lines under study using a reported method.28 Briefly, each sample of cell lysate containing 25 ng/mL proteins was added to a mixed solution of 10 μM WST-1 tetrazolium salt and 400 μM NADH. Subsequently, 10 μM DMNQ (a redox-cycling reagent) was added to initiate the assay reaction. The absorption of the reaction mixture at 450 nm was monitored for 1 h. In vivo delivery of EC16-80/RNase A-NBC/β-Lap nanoparticles First, we developed MCF-7 tumor-bearing xenograft using 8 × 106 MCF-7 cells suspended in 200 μL DPBS and injecting subcutaneously in the left axilla region of 4-week-old female NuNu nude mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd). Then we investigated an in vivo distribution of EC16-80/FITC-RNase A-NBC/β-Lap nanoparticles by injection of 200 μL of the prodrug into the MCF-7 tumor-bearing mice (n = 3) when the tumor size reached ∼ 500 mm3. The experimental setup with appropriate controls was as follows–Group 1: EC16-80/FITC-RNase A-NBC/β-Lap, Group 2: EC16-80/FITC-RNase A-NBC nanoparticle, and Group 3: free FITC-RNase A-NBC. At 4 h posttreatment, the mice were sacrificed, and the major organs of mice in the different groups were collected for fluorescence imaging on an IVIS small animal imaging system (PerkinElmer, USA). Second, following the in vivo prodrug delivery, tumor growth suppression study was performed when the mice tumor size reached ∼ 100 mm3 in volume. The mice were divided into 4 groups and the prodrug or the controls were injected intravenously, as follows–Group 1: DPBS alone, Group 2: EC16-80/β-Lap nanoparticles, Group 3: EC16-80/RNase A-NBC nanoparticles, and Group 4: EC16-80/β-Lap/RNase A-NBC nanoparticles. The mice in each group received an injection every 2 days until a total of five doses. The composition of each item within the injected nanoparticles was 9 mg/kg EC16-80, 1.1 mg/kg RNase A–NBC, or 0.27 mg/kg β-Lap. Each tumor size and mouse weight was measured in each group every 2 days, and the tumor volume was calculated by the formula 1/2 × lengh (mm) × width2 (mm). At the end of the experiment, blood samples were collected from each mice group, and serum from each group was prepared for hepatocellar function biomarker analysis, including the determination of aspartate transaminase (AST), alanine transaminase (ALT), and total bilirubin using the respective commercial assay kit (Nanjing Jiancheng Bioengineering institute, Nanjing, China). Results and Discussion NQO1-initiated cascade activation of ROS-responsive RNase A-NBC In this study, we selected Ribonuclease A (RNase A) as a model protein because its role in cancer therapy has been evaluated previously in clinical trials and shown to act via the cleavage of intracellular RNA after cellular entry and the subsequent induction of cytotoxic effects.29 Enzymatic activation of RNase A could, therefore, be of great appeal and potential for developing pro-proteins for targeted cancer therapy. The chemically modified "inert" pro-protein, RNase A-NBC (Figure 1a), was prepared by conjugating a ROS-responsive moiety, aromatic boronic acid, to RNase A according to our previous report.10 The chemical conjugation of aromatic boronic acid is a practical approach to develop pro-proteins19 and prodrugs,30 making use of ROS-triggered biorthogonal boronic acid oxidation and cleavage.31 The effectiveness of using endogenous ROS to regulate pro-proteins in vivo, however, might not be sufficiently significant due to the heterogeneity of tumors, which, in turn, display various ROS levels, leading to variations in pro-protein activation. Thus, developing new approaches to activate ROS-responsive pro-proteins in vivo and to advance further its therapeutic potential is highly desirable.8 NQO1 could catalyze a futile redox cycle of β-Lap to generate massive ROS in short periods, for example, > 120 moles of H2O2 per mole of β-Lap in 2 minutes.32 Therefore, we hypothesized that in situ generated H2O2 by NQO1-catalyzed futile reduction of β-Lap could result in a switch of RNase A-NBC from an "inert" state to an "active" state by removing the chemical tag conjugated to the RNase A. We verified this hypothesis by incubating RNase A-NBC (13.3 μg/mL) with NQO1 (0.32 ng/mL) and β-Lap (0.33 μM) for 2 h. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis and RNase A activity assay were performed subsequently to confirm the demodification and the full recovery of the enzyme protein activity. As shown in Supporting Information Figure S1, the molecular weight of RNase A-NBC was decreased from 14,971 to 13,855 Da after a reaction involving NQO1 and β-Lap cotreatment, indicating the efficient and readily removal of NBC groups from RNase A-NBC. Meanwhile, the RNase A activity assay showed extremely weak ribonuclease activity of RNase A-NBC as a result of the chemical modification of an essential lysine residue of RNase A (Figure 2).10 The treatment of RNase A-NBC with NQO1 and β-Lap, however, enhanced the released RNase activity up to 10-fold due to the generation of the active RNase, compared with the nontreated NBC-bound enzyme. This activity enhancement was NQO1 concentration dependence, which was revealed when varying concentrations of NQO1 were incubated with a fixed amount of RNase A-NBC under the same experimental conditions. Moreover, when dicoumarol, a competitive inhibitor of NQO1,32 was added to the reaction mixture composed of RNase A-NBC, NQO1, and β-Lap, no noticeable recovery of RNase activity was noticed (Figure 2), as NQO1 was no longer available to convert the NBC bound enzyme to the active form. This observation confirmed the effectiveness and high selectivity of NQO1-catalyzed β-Lap reduction for the recovery of RNase A activity via the removal of NBC from RNase A-NBC conjugate. Figure 2 | Enzyme activity assay of RNase A-NBC measured on a fluorometer at an emission wavelength of 520 nm over a 1 h time course with and without the treatment of NQO1 or both NQO1 and β-Lap. 13.3 μg/mL RNase A-NBC was incubated with 0.33 μM β-Lap and different concentrations of NQO1 in the presence of NADH. To study the inhibition effect of dicoumarol on NQO1-initiated RNase A-NBC activation, 250 μM dicoumarol was added to the reaction mixture of 13.3 μg/mL RNase A-NBC, 0.33 μM β-Lap, and 0.21 ng/mL NQO1 before incubation. Download figure Download PowerPoint Enzymatic activation of RNase A-NBC in living cells Next, we studied whether the NQO1-catalyzed β-Lap reduction and RNase A-NBC activation were effective in living cells and the potential of active RNase A-NBC to prohibit tumor cell growth. To this end, RNase A-NBC and β-Lap had to be delivered into cells simultaneously to initiate the enzymatic cascade reaction. Since the therapeutic and biomedical applications of proteins are limited mainly by their poor cellular permeability to regulate cell signaling, efficient delivery of proteins is of high demand for the advancement of protein chemistry in order to achieve effective protein therapy.33–37 Herein, we used a cationic lipid, EC16-80, which had recently been developed for effective protein delivery. EC16-80 was screened from a combinatorial library of lipid nanoparticles,23,38,39 for simultaneous delivery of RNase A-NBC and β-Lap into cells (Figure 1b). In the lipid nanoparticle formulation, RNase A-NBC was encapsulated mainly via electrostatic interaction with EC16-80, whereas β-Lap was entrapped in the hydrophobic layer of the lipid nanoparticles (Figure 1b). DLS analysis ( Supporting Information Figure S2) indicated that the EC16-80/RNase A-NBC/β-Lap nanoparticles had a size of ∼ 150 nm in diameter, which was very close to that of the empty EC16-80 nanoparticles, or EC16-80 nanoparticles encapsulated with the RNase A-NBC only or the β-Lap only, suggesting a minimal effect of the coencapsulation of RNase A-NBC and β-Lap on the integrity of the nanoparticle. The zeta-potential of EC16-80/RNase A-NBC/β-Lap nanoparticles decreased from 14 ± 1.7 to 9.8 ± 0.8 mV, compared with that of the empty EC16-80 nanoparticles, indicating that the electrostatic encapsulation of the RNase A-NBC partially neutralized the positive charges of the lipid nanoparticles. Meanwhile, all four types of EC16-80 and RNase A-NBC complexes showed spherical nanoparticle structures, as revealed by SEM (Figure 3) and TEM imaging ( Supporting Information Figure S3) analyses. Figure 3 | SEM images of different lipid/protein nanoparticles: (a) EC16-80 alone, (b) EC16-80/β-Lap nanoparticles, (c) EC16-80/RNase A-NBC nanoparticles, and (d) EC16-80/RNase A-NBC/β-Lap nanoparticles. Scale bar, 1 μm. Download figure Download PowerPoint The intracellular delivery of EC16-80/RNase A-NBC/β-Lap nanoparticles was monitored by transfecting NQO1-overexpressing MCF-7 cells with FITC-labeled RNase A-NBC and characterized further with confocal laser scan imaging (CLSM), as well as flow cytometry. As shown in Figure 4a, the treatment of MCF-7 cells with EC16-80/FITC-RNase A-NBC/β-Lap nanoparticles resulted in a substantial accumulation of green fluorescence in the cytosol. Flow cytometry analysis ( Supporting Information Figure S4) indicated that more than 75% of the MCF-7 cells were transfected with the EC16-80/FITC-RNase A-NBC/β-Lap nanoparticles at a protein concentration of 1.17 μg/mL in a comparable efficiency to that of EC16-80/FITC-RNase A-NBC nanoparticles, indicating a negligible effect of the delivery efficiency of the β-Lap encapsulation on RNase A-NBC. Moreover, we noticed that the intracellular ROS level of MCF-7 cells treated with EC16-80/RNase A-NBC/β-Lap nanoparticles was elevated substantially, compared with the untreated cells or the EC-16-80 alone treated cells or the EC16-80/RNase A-NBC nanoparticles without β-Lap treated cells ( Supporting Information Figure S5). This result was indicative of an intracellularly delivered β-Lap by the EC16-80/RNase A-NBC/β-Lap nanoparticles system, which had undergone NQO1-catalyzed reduction to generate massive ROS that triggered further the oxidative demodification and subsequent activation of RNase A-NBC in the live cells, as discussed further below. Figure 4 | (a) CLSM images of MCF-7 cells treated with EC16-80/FITC-RNase A-NBC/β-Lap nanoparticles (16.7 μg/mL lipid nanoparticle). Scale bar, 10 μm; (b) Viability of MCF-7 cells treated with different lipid nanoparticle formulatio
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Vibrio alginolyticus
Portunus trituberculatus
Marine Biology
Fisheries science
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