Novel flavonol derivatives containing benzoxazole as potential antiviral agents: design, synthesis, and biological evaluation.

A series of flavonol derivatives containing benzoxazole were designed and synthesized, and the structures of all the target compounds were determined by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). The structure of X2 was further confirmed by single crystal X-ray diffraction analysis. The results of the bioactivity tests showed that some of the target compounds possessed excellent antiviral activity against tobacco mosaic virus (TMV) in vivo. In particular, the median effective concentration (EC50) values for the curative and protective activities of X17 against TMV were 127.6 and 101.2 µg/mL, respectively, which were superior to those of ningnanmycin (320.0 and 234.6 µg/mL). The results of preliminary mechanism study indicated that X17 had a strong binding affinity for TMV coat protein (TMV-CP), which might hinder the self-assembly and replication of TMV particles. In addition, X17 was able to effectively inhibit tobacco leaf membrane lipid peroxidation and facilitate the removal of O2- from the body, thereby improving the disease resistance of tobacco plants. Therefore, the design and synthesis of flavonol derivatives containing benzoxazole provides value for the development of new antiviral drugs.

Design of a delivery vehicle chitosan-based self-assembling: controlled release, high hydrophobicity, and safe treatment of plant fungal diseases.

BACKGROUND: Traditional pesticides are poorly water-soluble and suffer from low bioavailability. N-succinyl chitosan (NSCS) is a water-soluble chitosan derivative, has been recently used to encapsulate hydrophobic drugs to improve their bioavailability. However, it remains challenging to synthesize pesticides of a wide variety of water-soluble drugs and to scale up the production in a continuous manner. RESULTS: A synthetic method for preparing water-soluble nanopesticides with a polymer carrier was applied. The bioactive molecule BTL-11 was loaded into hollow NSCS to promote drug delivery, improve solubility and anti-fungal activity. The synthesized nanopesticides had well controlled sizes of 606 nm and the encapsulation rate was 80%. The release kinetics, drug toxicity and drug activity were further evaluated. The inhibitory activity of nanopesticides against Rhizoctonia solani (R. solani) was tested in vivo and in vitro. In vivo against R. solani trials revealed that BTL-11 has excellent control efficiency for cultivated rice leaf and sheath was 79.6 and 76.5%, respectively. By contrast, for BTL-11@NSCS NPs, the anti-fungal ability was strongly released and afforded significant control efficiencies of 85.9 and 81.1%. Those effects were significantly better than that of the agricultural fungicide azoxystrobin (51.5 and 66.5%). The proposed mechanism was validated by successfully predicting the synthesis outcomes. CONCLUSIONS: This study demonstrates that NSCS is a promising biocompatible carrier, which can enhance the efficacy of pesticides, synergistically improve plant disease resistance, protect crop growth, and can be used for the delivery of more insoluble pesticides.

Antiviral activity evaluation and action mechanism of myricetin derivatives containing thioether quinoline moiety.

A variety of myricetin derivatives containing thioether quinoline moiety were designed and synthesized. Their structures of title compounds were determined by 1H NMR, 13C NMR, 19F NMR, and HRMS. Single-crystal X-ray diffraction experiments were carried out with B4. Antiviral activity indicated that some of the target compounds exhibited remarkable anti-tobacco mosaic virus (TMV) activity. In particular, compound B6 possessed significant activity. The half maximal effective concentration (EC50) value of the curative activity of compound B6 was 169.0 µg/mL, which was superior to the control agent ningnanmycin (227.2 µg/mL). Meanwhile, the EC50 value of the protective activity of compound B6 was 86.5 µg/mL, which was better than ningnanmycin (179.2 µg/mL). Microscale thermophoresis (MST) indicated that compound B6 had a strong binding capability to the tobacco mosaic virus coat protein (TMV-CP) with a dissociation constant (Kd) value of 0.013 µmol/L, which was superior to that of myricitrin (61.447 µmol/L) and ningnanmycin (3.215 µmol/L). And the molecular docking studies were consistent with the experimental results. Therefore, these novel myricetin derivatives containing thioether quinoline moiety could become potential alternative templates for novel antiviral agents.

Treatment of acute acquired concomitant esotropia.

BACKGROUND: The treatment efficacy of botulinum toxin bilateral medial rectus injections for acute acquired concomitant esotropia (AACE) in adult is not clear. We characterize the effects of botulinum toxin injection in the treatment of AACE, especially in patients over 14 years old, and compared it with surgical treatment. METHODS: In this prospective, nonrandomized, controlled clinical study, patients with AACE in our hospital from March 2017 to March 2020 elected to receive bilateral medial rectus injections of botulinum toxin or to undergo extraocular muscle surgery. Ocular position and stereopsis were evaluated before and after treatment. RESULTS: A total of 60 patients were treated: 40 patients in the botulinum toxin group, and 20 patients in the surgery group. The botulinum toxin group included 31 cases ≥ 14 years of age and 9 cases < 14 years of age. After 1-3 botulinum injections, the cumulative initial success rate was 95% (38/40), and the recurrence rate was 22.5% (9/40). Nine children < 14 years of age were treated successfully, without recurrence. In the surgery group, the initial success rate after surgery was 75% (17/20), and the recurrence rate was 20% (4/20). There was no significant difference between groups in the rate of success rate or the rate of recurrence (P > 0.05). CONCLUSION: The injection of botulinum toxin has a good effect on AACE in adults and children. The outcomes achieved with injected botulinum toxin are similar to those achieved with surgery. TRIAL REGISTRATION: ChiCTR, ChiCTR2000032544 . Registered May 2, 2020, Retrospectively registered.

Predicting the Dielectric Properties of Nanocellulose-Modified Presspaper Based on the Multivariate Analysis Method.

Nanocellulose-modified presspaper is a promising solution to achieve cellulose insulation with better performance, reducing the risk of electrical insulation failures of a converter transformer. Predicting the dielectric properties will help to further design and improvement of presspaper. In this paper, a multivariable method was adopted to determine the effect of softwood fiber on the macroscopic performance of presspaper. Based on the parameters selected using the optimum subset method, a multiple linear regression was built to model the relationship between the fiber properties and insulating performance of presspaper. The results show that the fiber width and crystallinity had an obvious influence on the mechanical properties of presspaper, and fiber length, fines, lignin, and nanocellulose had a significant impact on the breakdown properties. The proposed models exhibit a prediction accuracy of higher than 90% when verified with the experimental results. Finally, the effect of nanocellulose on the breakdown strength of presspaper was taken into account and new models were derived.

Carbonic anhydrase II mediates malignant behavior of pulmonary neuroendocrine tumors.

In normal lung, the predominant cytoplasmic carbonic anhydrase (CA) isozyme (CAII) is highly expressed in amine- and peptide-producing pulmonary neuroendocrine cells where its role involves CO2 sensing. Here, we report robust cytoplasmic expression of CAII by immunohistochemistry in the tumor cells of different native neuroendocrine tumor (NET) types, including typical and atypical carcinoids and small-cell lung carcinomas, and in NET and non-NET tumor cell lines. Because, in both pulmonary neuroendocrine cell and related NETs, the hypercapnia-induced secretion of bioactive serotonin (5-hydroxytryptamine) is mediated by CAII, we investigated the role of CAII in the biological behavior of carcinoid cell line H727 and the type II cell-derived A549 using both in vitro clonogenicity and in vivo xenograft model. We show that short hairpin RNA-mediated down-regulation of CAII resulted in significant reduction in clonogenicity of H727 and A549 cells in vitro, and marked suppression of tumor growth in vivo. CAII-short hairpin RNA cell-derived xenografts showed significantly reduced mitosis (phosphohistone H3 marker) and proliferation associated antigen Ki-67 (Ki67 marker), and significantly increased apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Using an apoptosis gene array, we found no association with caspases 3 and 8, but with a novel association of CAII-mediated apoptosis with specific mitochondrial apoptosis-associated proteins. Furthermore, these xenografts showed a significantly reduced vascularization (CD31 marker). Thus, CAII may play a critical role in NET lung tumor growth, angiogenesis, and survival, possibly via 5-hydroxytryptamine, known to drive autocrine tumor growth. As such, CAII is a potential therapeutic target for the difficult-to-treat lung NETs.

Novel Flavonol Derivatives Containing 1,3,4-Thiadiazole as Potential Antifungal Agents: Design, Synthesis, and Biological Evaluation.

In order to discover novel compounds with excellent agricultural activities, novel flavonol derivatives containing 1,3,4-thiadiazole were synthesized and evaluated for their antifungal activities. The bioassay results showed that some of the target compounds had good antifungal activities against Botrytis cinerea, Phomopsis sp. and Sclerotinia sclerotiorum in vitro. It is worth noting that the half-effective concentration (EC50) value of Y18 against B. cinerea was 2.4 µg/mL, which was obviously superior to that of azoxystrobin (21.7 µg/mL). The curative activity of Y18 at 200 µg/mL (79.9%) was better than that of azoxystrobin (59.1%), and its protective activity (90.9%) was better than that of azoxystrobin (83.9%). Morphological studies by using scanning electron microscopy and fluorescence microscopy revealed that Y18 could affect the normal growth of B. cinerea mycelium. In addition, the mechanism of action studies indicated that Y18 could affect the integrity of cell membranes by inducing the production of endogenous reactive oxygen species and the release of the malondialdehyde content, leading to membrane lipid peroxidation and the release of cell contents. The inhibitory activity of flavonol derivatives containing 1,3,4-thiadiazole on plant fungi is notable, offering significant potential for the development of new antifungal agents.

Synthesis, antibacterial and antifungal activity of myricetin derivatives containing piperidine and amide fragments.

BACKGROUND: Continuous use of synthetic bactericides and fungicides is causing pathogens to develop resistance, resulting in increased use of pesticides and affecting food security. The green pesticides derived from natural products could reduce or avoid 'pesticide hazards' caused by synthetic pesticides as a result of their unique mechanism of action. Therefore, it is of great significance to create green pesticides with novel structures. RESULTS: Herein, 30 novel myricetin derivatives containing piperidine and amide fragments were designed and synthesized using active group splicing. Among them, compound Z30 had excellent inhibitory effect against Xanthomonas oryzae pv. Oryzae (Xoo) with the half effective concentration (EC50 ) of 2.7 µg mL-1 . Compound Z26 not only exhibited better antibacterial activity against Xaxonopodis pv. Citri (Xac) with EC50 of 3.9 µg mL-1 , but also displayed higher antifungal activity against Rhizoctonia solani (Rs) with EC50 of 8.3 µg mL-1 . In vivo experiments proved that Z30 against bacterial blight of rice and Z26 against rice blast exhibits significant protective and curative effect. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed that Z26 and Z30 could change the integrity of cell wall and membrane of pathogen Xoo, Xac and Rs, resulting in cytoplasmic leakage and eventually death. Enzymatic assay, molecular docking and molecular dynamics simulations (MDs) indicated that Z26 could be used as a potential succinate dehydrogenase inhibitor (SDHI). CONCLUSION: Z26 and Z30 significantly reduced the pathogenicity of the pathogens, which provided a new idea and direction for the development of green pesticides. © 2023 Society of Chemical Industry.

Design, Synthesis, and Bioactivity of Chalcone Derivatives Containing Indanone.

A series of chalcone derivatives containing indanone were designed and synthesized by aldehyde-ketone condensation and etherification. The activity test demonstrated that the majority of the compounds had good therapeutic and protective activities against tobacco mosaic virus (TMV) at a concentration of 500 µg/mL when being tested. Among them, the target compounds N2 and N7 showed good therapeutic activities against TMV with EC50 values of 70.7 and 89.9 µg/mL, respectively, which were better than that of ningnanmycin (158.3 µg/mL). N2 and N10 showed better protective activities against TMV with EC50 values of 60.8 and 120.3 µg/mL, which were superior to that of ningnanmycin (175.6 µg/mL). A hydrogen bond interaction was observed between N2 and ARG-341 with a bond length of 3.08 Å and a hydrogen bond was observed between ningnanmycin and ASP-66 with a bond length of 3.72 Å. In contrast, the hydrogen bond length of compound N2 was shorter and its binding was closer. Meanwhile, when the heartleaf tobacco was being treated with N2, its increasing rate of malondialdehyde slowed and its content of defense enzymes significantly increased, again reflecting the good activity of N2 against TMV.

Synthesis and biological activities of novel chalcone derivatives containing pyrazole oxime ethers.

A series of novel chalcone derivatives containing pyrazole oxime ethers were designed and synthesized. The structures of all the target compounds were determined by NMR and HRMS. The structure of H5 was further confirmed via single-crystal X-ray diffraction analysis. The results of biological activity test showed that some of the target compounds exhibited significant antiviral and antibacterial activities. The test results of EC50 value against tobacco mosaic virus showed that H9 had the best curative and protective effect, and the EC50 value of curative activity of H9 was 166.9 µg/mL, which was superior to ningnanmycin (NNM) 280.4 µg/mL, the EC50 value of protective activity of H9 was 126.5 µg/mL, which was superior to ningnanmycin 227.7 µg/mL. Microscale thermophoresis (MST) experiments demonstrated that H9 (Kd = 0.0096 ± 0.0045 µmol/L) exhibited a strong binding ability with tobacco mosaic virus capsid protein (TMV-CP), which was far superior to ningnanmycin (Kd = 1.2987 ± 0.4577 µmol/L). In addition, molecular docking results showed that the affinity of H9 to TMV protein was significantly higher than ningnanmycin. The results of against bacterial activity showed that H17 has a good inhibiting effect against Xanthomonas oryzae pv. oryzae (Xoo), the EC50 value of H17 was 33.0 µg/mL, which was superior to the commercial drugs thiodiazole copper (68.1 µg/mL) and bismerthiazol (81.6 µg/mL), and the antibacterial activity of H17 was verified by scanning electron microscopy (SEM).

Design, synthesis and bioactivity of myricetin derivatives for control of fungal disease and tobacco mosaic virus disease.

A series of myricetin derivatives containing isoxazole were designed and synthesized. All the synthesized compounds were characterized by NMR and HRMS. In terms of antifungal activity, Y3 had a good inhibitory effect on Sclerotinia sclerotiorum (Ss), and the median effective concentration (EC50) value was 13.24 µg mL-1, which was better than azoxystrobin (23.04 µg mL-1) and kresoxim-methyl (46.35 µg mL-1). Release of cellular contents and cell membrane permeability experiments further revealed that Y3 causes the destruction of the cell membrane of the hyphae, which in turn plays an inhibitory role. The anti-tobacco mosaic virus (TMV) activity in vivo showed that Y18 had the best curative and protective activities, with EC50 values of 286.6 and 210.1 µg mL-1 respectively, the effect was better than ningnanmycin. Microscale thermophoresis (MST) data showed that Y18 had a strong binding affinity with tobacco mosaic virus coat protein (TMV-CP), with a dissociation constant (K d) value of 0.855 µM, which was better than ningnanmycin (2.244 µM). Further molecular docking revealed that Y18 interacts with multiple key amino acid residues of TMV-CP, which may hinder the self-assembly of TMV particles. Overall, after the introduction of isoxazole on the structure of myricetin, its anti-Ss and anti-TMV activities have been significantly improved, which can be further studied.

Space Charge Behavior of Thermally Aged Polyethylene Insulation of Track Cables.

The interface of multi-layer insulation is a relatively weak point in the cable system during the long-term high-temperature service. Space charge is prone to continuously accumulate in the interface area, leading to the deterioration of electrical properties and even insulation failure in advance. The knowledge about thermal oxidation of polyethylene (PE) materials at the molecular level is still urgent to explore. Herein, single-layer and double-layer PE insulation, representing the typical insulation structure of frequency-shift pulse voltage track cables, were prepared and thermally aged in the oven for up to 360 h. Thermal, mechanical, electrical, and space charge characterizations were systematically carried out. Thermogravimetric analyzer and oxidation induced temperature (OIT) measurements confirmed that LDPE's thermal-oxidative aging temperature range was the lowest among the three PE groups in the O2 atmosphere. After 360 h thermal aging, the tensile property of HDPE material kept relatively stable, while the elongation at break of the other two groups was lower than 50%. Unaged HDPE exhibits apparent charge injection and migration, leading to the severe electric field distortion of 20%. Noticeable charge accumulation can be observed at the unaged double-layer sample interface due to the mismatching of DC conductivity, which play a significant role in the aged double-layer samples. This work utilizes precise thermal analysis to provide new information about the resistance ability of thermal oxidation of LDPE, FPE, and HDPE, and its influence on space charge behaviors, which is helpful for the insulation design and evaluation in cable applications.

Thermal Aging Properties of 500 kV AC and DC XLPE Cable Insulation Materials.

Despite similar material composition and insulation application, the alternating current (AC) cross-linked polyethylene (XLPE) and direct current (DC) XLPE materials cannot replace each other due to different voltage forms. Herein, this work presents a systematical investigation into the effects of thermal aging on the material composition and properties of 500 kV-level commercial AC XLPE and DC XLPE materials. A higher content of antioxidants in the AC XLPE than in the DC XLPE was experimentally demonstrated via thermal analysis technologies, such as oxidation-induced time and oxidation-induced temperature. Retarded thermal oxidation and suppression of space charge effects were observed in thermally aged AC XLPE samples. On the other hand, the carbonyl index of DC XLPE dramatically rose when thermal aging was up to 168 h. The newly generated oxygen-containing groups provided deep trapping sites (~0.95 eV) for space charges and caused severe electric field distortion (120%) under -50 kV/mm at room temperature in the aged DC XLPE samples. For the unaged XLPE materials, the positive space charge packets were attributed to the residue crosslinking byproducts, even after being treated in vacuum at 70 °C for 24 h. Thus, it was reasoned that the DC XLPE material had a lower crosslinking degree to guarantee fewer crosslinking byproducts. This work offers a simple but accurate method for evaluating thermal oxidation resistance and space charge properties crucial for developing high-performance HVDC cable insulation materials.

Design, Synthesis, and Biological Evaluation of Novel 1,4-Pentadien-3-one Derivatives Containing a Sulfonamide Moiety.

Novel 1,4-pentadien-3-one derivatives containing a sulfonamide moiety were synthesized, and their antifungal, antibacterial, and antiviral activities were verified. These compounds exhibited better activity against five bacteria, with EC50 values ranging from 9.6 to 60.1 µg/mL, prominently, which are superior to those of the commercial agent. A great amount of compounds had excellent fungicidal activity in vitro at 100 µg/mL. Strikingly, compound E6 exhibited moderate activity against Phytophthora litchii than azoxystrobin, with the EC50 value of compound E6 (0.5 µg/mL) drawing near azoxystrobin (0.3 µg/mL). Furthermore, compound E17 had a marked impact on in vivo anti-tobacco mosaic virus, according to the data of microscale thermophoresis, with a Kd value of the intermolecular binding force of 0.002 ± 0.001 µM, which was better than the commercial agent of ningnanmycin (Kd = 0.121 ± 0.031 µM). In addition, the results of these studies suggest that the use of active splicing can improve the biological activity of natural compounds and provide further complement to the development of novel pesticides.

Does education finance reduce the inequality of educational results? The mediation effect of shadow education.

Public education finance in China plays an important role in education equality. This study investigated two mediation effects with a generalized structural equation model that comprised the mediation effect of shadow education at the school, family, and individual levels and the moderating role of education finance. There was a strong association among heterogeneity factors, shadow education, and educational results, with shadow education playing a mediating role in math and English courses. Individual heterogeneity differences had a negative impact on equality in educational results through access to additional shadow education opportunities, while heterogeneous factors were mediated through shadow education, causing financial moderation effects, in turn affecting inequality in educational results. Finally, the moderation degree and direction of education finance varied significantly, with a greater moderation effect on household-level factors that lead to unequal educational results. Targeted efforts are required to regulate shadow education, which is key to the development of the education system.

AuNPs and graphdiyne nanocomposite as robust electrocatalyst for methyl parathion detection in real samples.

The present work describes a simple and rapid synthesis method of gold nanoparticles and graphdiyne (AuNPs@GDY) nanocomposites including porous structure. Moreover, the synthesized AuNPs@GDY material was decorated on the glassy carbon electrode (GCE) with a drop coating method to construct a non-enzymatic electrochemical pesticides sensor. The micro-morphology and elemental composition of the materials were characterized by transmission electron microscopy (TEM) and energy dispersive spectroscopy (EDS). The electrocatalysis and conductivity of the material were studied with cyclic voltammetry (CV) and impedance method, respectively. The properties of the sensor were investigated by CV and differential pulse voltammetry (DPV). The results showed that AuNPs@GDY exhibited excellent electrocatalytic ability for methyl parathion in a wide linear range (from 0.25 ng/mL to 24.43 µg/mL) and low limit of detection value (6.2 pg/mL). Furthermore, the DPV method used in this paper was accurate and sensitive, and could be used for routine quality control of methyl parathion in kiwi fruit and tomato samples.

Moisture Absorption Characteristics of Nanoparticle-Doped Silicone Rubber and Its Influence Mechanism on Electrical Properties.

To further explore the long-term stability of nano-dielectrics, experiments were carried out to investigate the moisture absorption characteristics and electrical properties of silicone rubber (SiR) doped with different inorganic nanoparticles. Thermogravimetric analysis (TGA) is utilized to research the moisture absorption characteristics including mass fraction and binding forms. The trap depth and electron orbitals are calculated by density functional theory to explain the influence mechanism of water molecules on SiR. It is found that the addition of nanoparticles will increase the moisture content of SiR. Additionally, the nano-TiO2-doped SiR absorbs more water and binds with water relatively more loosely than nano-Al2O3. There is a degradation of space charge inhibition capability and breakdown strength after moisture absorption, which might be explained by shallow traps brought by water molecules and the narrowed forbidden bandwidth of SiR.

Spatial-temporal characteristics and influencing factors of atmospheric environmental efficiency in China.

This research constructs a super efficiency slack-based measure (SBM) model based on the Malmquist-Luenberger (ML) index to analyze the atmospheric environmental efficiency (AEE) of 30 provinces in China from 2000 to 2016 and explores the spatial and temporal differences of AEE by using the coefficient of variation method. This paper further analyzes the internal influencing factors of AEE via the ML index decomposition approach and establishes a panel data regression model to explore AEE's influencing factors in China. The results show some regional differences of the AEE level in China, with it the best in the eastern region and followed in order by the western and central region, and these differences exhibit an increasing trend year by year. During the study period, the development trend of AEE in China is similar to that in the eastern and western regions, showing a "W" shape, where in the central region it has a "U" pattern. The conclusion is that technical progress (TC) is the dominant factor affecting AEE, technical efficiency (EC) fails to effectively promote AEE improvement, and TC and EC present varying degrees of influence and different directions of action in the regions. The analysis results show that the influence effect of economic development on AEE presents a "U" pattern of first declining and then rising. The degree of China opening up to the outside world and its carbon dioxide emissions intensity have significant negative effects on AEE, whereas the increase of pollution control input effectively improves AEE.

Crystalline Modification and Its Effects on Dielectric Breakdown Strength and Space Charge Behavior in Isotactic Polypropylene.

Adding nucleating agents (NAs) is one of the most efficient ways to obtain improved mechanical, optical, and thermal properties of isotactic polypropylene (iPP). While it is well appreciated that electrical property is critically affected by crystalline modification, the role between them remains unclear. Here, we address this issue by incorporating commercial α-NA and ß-NA into iPP, both of which exhibit strong nucleation ability, e.g., reducing the size of crystalline agglomerates from 45.3 µm (Pure-iPP) to 2.5 µm (α-iPP) and 7.6 µm (ß-iPP), respectively. Mechanical testing results show that while ß-modification decreases the tensile strength a little, it does enhance the elongation at break (200%) and toughness (25.3% higher), relative to its unfilled counterparts. Moreover, a well-dispersed ß-iPP system obtains a comprehensive improvement of electrical properties, including dielectric breakdown strength, space charge suppression, and internal field distortion under a high external field (-100 kV/mm) due to newly-generated deep charge trapping sites. This crystalline modification strategy is attractive for future development of many engineering insulating polymers.

A truncated hepatitis E virus ORF2 protein expressed in tobacco plastids is immunogenic in mice.

AIM: To cost-effectively express the 23-ku pE2, the most promising subunit vaccine encoded by the E2 fragment comprising of the 3'-portion of hepatitis E virus (HEV) open reading frame 2 (ORF2) in plastids of tobacco (Nicotiana tabacum cv. SR1), to investigate the transgene expression and pE2 accumulation in plastids, and to evaluate the antigenic effect of the plastid-derived pE2 in mice. METHODS: Plastid-targeting vector pRB94-E2 containing the E2 fragment driven by rice psbA promoter was constructed. Upon delivery into tobacco plastids, this construct could initiate homologous recombination in psaB-trnfM and trnG-psbC fragments in plastid genome, and result in transgene inserted between the two fragments. The pRB94-E2 was delivered with a biolistic particle bombardment method, and the plastid-transformed plants were obtained following the regeneration of the bombarded leaf tissues on a spectinomycin-supplemented medium. Transplastomic status of the regenerated plants was confirmed by PCR and Southern blot analysis, transgene expression was investigated by Northern blot analysis, and accumulation of pE2 was measured by ELISA. Furthermore, protein extracts were used to immunize mice, and the presence of the pE2-reactive antibodies in serum samples of the immunized mice was studied by ELISA. RESULTS: Transplastomic lines confirmed by PCR and Southern blot analysis could actively transcribe the E2 mRNA. The pE2 polypeptide was accumulated to a level as high as 13.27 microg/g fresh leaves. The pE2 could stimulate the immunized mice to generate pE2-specific antibodies. CONCLUSION: HEV-E2 fragment can be inserted into the plastid genome and the recombinant pE2 antigen derived is antigenic in mice. Hence, plastids may be a novel source for cost-effective production of HEV vaccines.