CRISPR/Cas9 targeted mutations of OsDSG1 gene enhanced salt tolerance in rice.

Salinization is one of the leading causes of arable land shrinkage and rice yield decline, recently. Therefore, developing and utilizing salt-tolerant rice varieties have been seen as a crucial and urgent strategy to reduce the effects of saline intrusion and protect food security worldwide. In the current study, the CRISPR/Cas9 system was utilized to induce targeted mutations in the coding sequence of the OsDSG1, a gene involved in the ubiquitination pathway and the regulation of biochemical reactions in rice. The CRISPR/Cas9-induced mutations of the OsDSG1 were generated in a local rice cultivar and the mutant inheritance was validated at different generations. The OsDSG1 mutant lines showed an enhancement in salt tolerance compared to wild type plants at both germination and seedling stages indicated by increases in plant height, root length, and total fresh weight as well as the total chlorophyll and relative water contents under the salt stress condition. In addition, lower proline and MDA contents were observed in mutant rice as compared to wild type plants in the presence of salt stress. Importantly, no effect on seed germination and plant growth parameters was recorded in the CRISRP/Cas9-induced mutant rice under the normal condition. This study again indicates the involvement of the OsDSG1 gene in the salt resistant mechanism in rice and provides a potential strategy to enhance the tolerance of local rice varieties to the salt stress.

The Germin-like protein OsGER4 is involved in promoting crown root development under exogenous jasmonic acid treatment in rice.

In rice (Oryza sativa L.), crown roots (CRs) have many important roles in processes such as root system expansion, water and mineral uptake, and adaptation to environmental stresses. Phytohormones such as auxin, cytokinin, and ethylene are known to control CR initiation and development in rice. However, the role of jasmonic acid (JA) in CR development remained elusive. Here, we report that JA promotes CR development by regulating OsGER4, a rice Germin-like protein. Root phenotyping analysis revealed that exogenous JA treatment induced an increase in CR number in a concentration-dependent manner. A subsequent genome-wide association study and gene expression analyses pinpointed a strong association between the Germin-like protein OsGER4 and the increase in CR number under exogenous JA treatment. The ProGER4::GUS reporter line showed that OsGER4 is a hormone-responsive gene involved in various stress responses, mainly confined to epidermal and vascular tissues during CR primordia development and to vascular bundles of mature crown and lateral roots. Notable changes in OsGER4 expression patterns caused by the polar auxin transport inhibitor NPA support its connection to auxin signaling. Phenotyping experiments with OsGER4 knockout mutants confirmed that this gene is required for CR development under exogenous JA treatment. Overall, our results provide important insights into JA-mediated regulation of CR development in rice.

Enhancing powdery mildew resistance in soybean by targeted mutation of MLO genes using the CRISPR/Cas9 system.

BACKGROUND: Powdery mildew is a major disease that causes great losses in soybean yield and seed quality. Disease-resistant varieties, which are generated by reducing the impact of susceptibility genes through mutation in host plants, would be an effective approach to protect crops from this disease. The Mildew Locus O (MLO) genes are well-known susceptibility genes for powdery mildew in plant. In this study, we utilized the CRISPR/Cas9 system to induce targeted mutations in the soybean GmMLO genes to improve powdery mildew resistance. RESULTS: A dual-sgRNA CRISPR/Cas9 construct was designed and successfully transferred into the Vietnamese soybean cultivar DT26 through Agrobacterium tumefaciens-mediated transformation. Various mutant forms of the GmMLO genes including biallelic, chimeric and homozygous were found at the T0 generation. The inheritance and segregation of CRISPR/Cas9-induced mutations were confirmed and validated at the T1 and T2 generations. Out of six GmMLO genes in the soybean genome, we obtained the Gmmlo02/Gmmlo19/Gmmlo23 triple and Gmmlo02/Gmmlo19/Gmmlo20/Gmmlo23 quadruple knockout mutants at the T2 generation. When challenged with Erysiphe diffusa, a fungus that causes soybean powdery mildew, all mutant plants showed enhanced resistance to the pathogen, especially the quadruple mutant. The powdery mildew severity in the mutant soybeans was reduced by up to 36.4% compared to wild-type plants. In addition, no pleiotropic effect on soybean growth and development under net-house conditions was observed in the CRISPR/Cas9 mutants. CONCLUSIONS: Our results indicate the involvement of GmMLO02, GmMLO19, GmMLO20 and GmMLO23 genes in powdery mildew susceptibility in soybean. Further research should be conducted to investigate the roles of individual tested genes and the involvement of other GmMLO genes in this disease infection mechanism. Importantly, utilizing the CRISPR/Cas9 system successfully created the Gmmlo transgene-free homozygous mutant lines with enhanced resistance to powdery mildew, which could be potential materials for soybean breeding programs.

Disrupting Sc-uORFs of a transcription factor bZIP1 using CRISPR/Cas9 enhances sugar and amino acid contents in tomato (Solanum lycopersicum).

MAIN CONCLUSION: Induced mutations in the SC-uORF of the tomato transcription factor gene SlbZIP1 by the CRISPR/Cas9 system led to the high accumulation of sugar and amino acid contents in tomato fruits. Tomato (Solanum lycopersicum) is one of the most popular and consumed vegetable crops in the world. Among important traits for tomato improvement such as yield, biotic and abiotic resistances, appearance, post-harvest shelf life and fruit quality, the last one seems to face more challenges because of its genetic and biochemical complexities. In this study, a dual-gRNAs CRISPR/Cas9 system was developed to induce targeted mutations in uORF regions of the SlbZIP1, a gene involved in the sucrose-induced repression of translation (SIRT) mechanism. Different induced mutations in the SlbZIP1-uORF region were identified at the T0 generation, stably transferred to the offspring, and no mutation was found at potential off-target sites. The induced mutations in the SlbZIP1-uORF region affected the transcription of SlbZIP1 and related genes in sugar and amino acid biosynthesis. Fruit component analysis showed significant increases in soluble solid, sugar and total amino acid contents in all SlbZIP1-uORF mutant lines. The accumulation of sour-tasting amino acids, including aspartic and glutamic acids, raised from 77 to 144%, while the accumulation of sweet-tasting amino acids such as alanine, glycine, proline, serine, and threonine increased from 14 to 107% in the mutant plants. Importantly, the potential SlbZIP1-uORF mutant lines with desirable fruit traits and no impaired effect on plant phenotype, growth and development were identified under the growth chamber condition. Our result indicates the potential utility of the CRISPR/Cas9 system for fruit quality improvement in tomato and other important crops.

Performance and neural modeling of a compost-based biofilter treating a gas-phase mixture of benzene and xylene.

Biofilter (BF) has been regarded as a versatile gas treatment technology for removing volatile organic compounds (VOCs) from contaminated gas streams. In order for BF to be utilized in the industrial setting, it is essential to conduct research aimed at removing VOC mixtures under different inlet loading conditions, i.e. as a function of the gas flow rate and inlet VOC concentrations. The main aim of this study was to apply artificial neural networks (ANN) and determine the relationship between flow rate (FR), pressure drop (PD), inlet concentration (C), and removal efficiency (RE) in the BF treating gas-phase benzene and xylene mixtures. The ANN model was trained and tested to assess the removal efficiency of benzene (REB) and xylene (REX) under the influence of different FR, PD and C. The model's performance was assessed using a cross-validation method. The REb varied from 20% to >60%, while the REx varied from 10% to 70% during the different experimental phases of BF operation. The causal index (CI) technique was used to determine the sensitivity of the input parameters on the output variables. The ANN model with a topology of 4-4-2 performed the best in terms of predicting the RE profiles of both the pollutants. Furthermore, the effect was more pronounced for xylene because an increase in the benzene concentration reduced xylene removal (CI = -25.7170) more severely than benzene removal. An increase in the xylene concentration had a marginally positive effect on the benzene removal (CI = +0.1178).

Joint Clustering and Resource Allocation Optimization in Ultra-Dense Networks with Multiple Drones as Small Cells Using Game Theory.

In this study, we consider the combination of clustering and resource allocation based on game theory in ultra-dense networks that consist of multiple macrocells using massive multiple-input multiple-output and a vast number of randomly distributed drones serving as small-cell base stations. In particular, to mitigate the intercell interference, we propose a coalition game for clustering small cells, with the utility function being the ratio of signal to interference. Then, the optimization problem of resource allocation is divided into two subproblems: subchannel allocation and power allocation. We use the Hungarian method, which is efficient for solving binary optimization problems, to assign the subchannels to users in each cluster of small cells. Additionally, a centralized algorithm with low computational complexity and a distributed algorithm based on the Stackelberg game are provided to maximize the network energy efficiency (EE). The numerical results demonstrate that the game-based method outperforms the centralized method in terms of execution time in small cells and is better than traditional clustering in terms of EE.

Antioxidant Activities, Anticancer Activity, Physico-Chemistry Characteristics, and Acute Toxicity of Alginate/Lignin Polymer.

Alginate/lignin is a synthetic polymer rich in biological activity and is of great interest. Alginate is extracted from seaweed and lignin is extracted from corn stalks and leaves. In this paper, antioxidant activities of alginate/lignin were evaluated, such as total antioxidant activity, reducing power activity, DPPH free radical scavenging activity, and α-glucosidase inhibition activity. Anticancer activity was evaluated in three cell lines (Hep G2, MCF-7, and NCI H460) and fibroblast. Physico-chemistry characteristics of alginate/lignin were determined through FTIR, DSC, SEM_EDS, SEM_EDS mapping, XRD, XRF, and 1H-NMR. The acute toxicity of alginate/lignin was studied on Swiss albino mice. The results demonstrated that alginate/lignin possessed antioxidant activity, such as the total antioxidant activity, and reducing power activity, especially the α-glucosidase inhibition activity, and had no free radical scavenging activity. Alginate/lignin was not typical in cancer cell lines. Alginate/lignin existed in a thermally stable and regular spherical shape in the investigated thermal region. Six metals, three non-metals, and nineteen oxides were detected in alginate/lignin. Some specific functional groups of alginate and lignin did not exist in alginate/lignin crystal. Elements, such as C, O, Na, and S were popular in the alginate/lignin structure. LD0 and LD100 of alginate/lignin in mice were 3.91 g/kg and 9.77 g/kg, respectively. Alginate/lignin has potential for applications in pharmaceutical materials, functional foods, and supporting diabetes treatment.

Spatiotemporal Patterns of Anthrax, Vietnam, 1990-2015.

Anthrax is a priority zoonosis for control in Vietnam. The geographic distribution of anthrax remains to be defined, challenging our ability to target areas for control. We analyzed human anthrax cases in Vietnam to obtain anthrax incidence at the national and provincial level. Nationally, the trendline for cases remained at ≈61 cases/year throughout the 26 years of available data, indicating control efforts are not effectively reducing disease burden over time. Most anthrax cases occurred in the Northern Midlands and Mountainous regions, and the provinces of Lai Chau, Dien Bien, Lao Cai, Ha Giang, Cao Bang, and Son La experienced some of the highest incidence rates. Based on spatial Bayes smoothed maps, every region of Vietnam experienced human anthrax cases during the study period. Clarifying the distribution of anthrax in Vietnam will enable us to better identify risk areas for improved surveillance, rapid clinical care, and livestock vaccination campaigns.

Enhancing plant growth and biomass production by overexpression of GA20ox gene under control of a root preferential promoter.

Overexpression of GA20 oxidase gene has been a recent trend for improving plant growth and biomass. Constitutive expression of GA20ox has successfully improved plant growth and biomass in several plant species. However, the constitutive expression of this gene causes side-effects, such as reduced leaf size and stem diameter, etc. To avoid these effects, we identified and employed different tissue-specific promoters for GA20ox overexpression. In this study, we examined the utility of At1g promoter to drive the expression of GUS (ß-glucuronidase) reporter and AtGA20ox genes in tobacco and Melia azedarach. Histochemical GUS assays and quantitative real-time-PCR results in tobacco showed that At1g was a root-preferential promoter whose expression was particularly strong in root tips. The ectopic expression of AtGA20ox gene under the control of At1g promoter showed improved plant growth and biomass of both tobacco and M. azedarach transgenic plants. Stem length as well as stem and root fresh weight increased by up to 1.5-3 folds in transgenic tobacco and 2 folds in transgenic M. azedarach. Both tobacco and M. azedarach transgenic plants showed increases in root xylem width with xylem to phloem ratio over 150-200% as compared to WT plants. Importantly, no significant difference in leaf shape and size was observed between At1g::AtGA20ox transgenic and WT plants. These results demonstrate the great utility of At1g promoter, when driving AtGA20ox gene, for growth and biomass improvements in woody plants and potentially some other plant species.

Fecal indicator bacteria diversity and decay in an estuarine mangrove ecosystem of the Xuan Thuy National Park, Vietnam.

Mangroves are complex and dynamic ecosystems that are highly dependent on diverse microbial activities. In this study, laboratory experiments and field studies for fecal indicator bacteria (FIB) decay rates are carried out for the first time in the Xuan Thuy Mangrove Forest Reserve of Vietnam. Results show that there are significant differences in bacterial diversity in the water of mangrove areas that have been deforested compared to those which have been planted. The highest mean total coliform (TC) and Escherichia coli (EC) values were found in the natural mangroves (3,807±2,922 and 964±1133 CFU 100 ml-1, respectively). The results indicated that the source of contamination and seasonal changes affect the abundance of fecal bacteria. These results were exceeding by far the safety guidelines for individual, non-commercial water supplies in most of the samples. In the planted mangrove sampling sites, the highest mean Fecal streptococci (FS) values of 1,520±1,652 CFU 100 ml-1 were found. Microbial die-off rates were calculated over 5 days, and observed to be systematically higher for TC than for EC.

First report of foodborne botulism due to Clostridium botulinum type A(B) from vegetarian home-canned pate in Hanoi, Vietnam.

Even one case of foodborne botulism constitutes a public health emergency. We report a series of cases with delayed treatment due to delayed diagnosis. Clostridium botulinum type A(B) was isolated from vegetarian home-canned pate, but not from stool samples. These are the first recorded cases of foodborne botulism in Hanoi.

Carrageenan of Red Algae Eucheuma gelatinae: Extraction, Antioxidant Activity, Rheology Characteristics, and Physicochemistry Characterization.

Carrageenan is an anionic sulfated polysaccharide that accounts for a high content of red seaweed Eucheuma gelatinae. This paper focused on the extraction, optimization, and evaluation of antioxidant activity, rheology characteristics, and physic-chemistry characterization of ß-carrageenan from Eucheuma gelatinae. The extraction and the optimization of ß-carrageenan were by the maceration-stirred method and the experimental model of Box-Behken. Antioxidant activity was evaluated to be the total antioxidant activity and reducing power activity. The rheology characteristics of carrageenan were measured to be gel strength and viscosity. Physic-chemistry characterization was determined, including the molecular weight, sugar composition, function groups, and crystal structure, through GCP, GC-FID, FTIR, and XRD. The results showed that carrageenan possessed antioxidant activity, had intrinsic viscosity and gel strength, corresponding to 263.02 cps and 487.5 g/cm2, respectively. Antioxidant carrageenan is composed of rhamnose, mannose, glucose, fucose, and xylose, with two molecular weight fractions of 2.635 × 106 and 2.58 × 106 g/mol, respectively. Antioxidant carrageenan did not exist in the crystal. The optimization condition of antioxidant carrageenan extraction was done at 82.35 °C for 115.35 min with a solvent-to-algae ratio of 36.42 (v/w). At the optimization condition, the extraction efficiency of carrageenan was predicted to be 87.56 ± 5.61 (%), the total antioxidant activity and reducing power activity were predicted to 71.95 ± 5.32 (mg ascorbic acid equivalent/g DW) and 89.84 ± 5.84 (mg FeSO4 equivalent/g DW), respectively. Purity carrageenan content got the highest value at 42.68 ± 2.37 (%, DW). Antioxidant carrageenan from Eucheuma gelatinae is of potential use in food and pharmaceuticals.

Effects of Supplemental Light Spectra on the Composition, Production and Antimicrobial Activity of Ocimum basilicum L. Essential Oil.

This study was performed to investigate the effects of different supplemental light spectra and doses (duration and illuminance) on the essential oil of basil (Ocimum basilicum L.) cultivated in the net-house in Vietnam during four months. Ten samples of basil aerial parts were hydrodistilled to obtain essential oils which had the average yields from 0.88 to 1.30% (v/w, dry). The oils analyzed using GC-FID and GC-MS showed that the main component was methyl chavicol (87.4−90.6%) with the highest values found in the oils of basil under lighting conditions of 6 h/day and 150−200 µmol·m−2·s−1. Additional lighting conditions caused the significant differences (p < 0.001) in basil biomass and oil production with the highest values found in the oils of basil under two conditions of (1) 71% Red: 20% Blue: 9.0% UVA in at 120 µmol·m−2·s−1 in 6 h/day and (2) 43.5% Red: 43.5% Blue: 8.0% Green: 5.0% Far-Red at 100 µmol·m−2·s−1 in 6 h/day. The oils of basil in some formulas showed weak inhibitory effects on only the Bacillus subtilis strain. Different light spectra affect the biomass and essential oil production of basil, as well as the concentrations of the major components in the oil.

Structural and genetic insights into a poly-γ-glutamic acid with in vitro antioxidant activity of Bacillus velezensis VCN56.

Poly-γ­glutamic acid (γ­PGA) produced by Bacillus species is a natural biopolymer, which is widely used in various fields including food, pharmaceuticals, and cosmetics. In this study, the screening of 19 Bacillus isolates derived from traditionally fermented foods revealed that Bacillus velezensis VCN56 was the most potent γ­PGA producer. The maximum concentration of crude γ­PGA was 32.9 ± 1.5 g/L in the PGA-3 medium containing glycerol, citric acid, sodium glutamate, NH4Cl, and starch. The resulting γ-PGA was purified and then characterized by HPLC, FTIR, and 1H-NMR analyses. Molecular weight of purified γ­PGA was estimated to be 98 kDa with a polydisperse index of 2.04. Notably, the pure γ­PGA showed significant in vitro antioxidant scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (72.0 ± 1.5%), hydroxyl (81.0 ± 0.6%), and superoxide (43.9 ± 0.8%) radicals at the concentration of 4 mg/mL. Using whole-genome sequencing, the genetic organization of pgs operon responsible for γ­PGA biosynthesis in B. velezensis VCN56 differs from those in other Bacillus genomes. Further genome analysis revealed metabolic pathways for γ-PGA production and degradation. For the first time, the present study provides a better understanding of γ-PGA with a promising antioxidant activity produced by B. velezensis at the phenotypic, biochemical, and genomic levels, which hold potential applications in the foods, cosmetics, and pharmaceutical industries.

Plans for Nationwide Serosurveillance Network in Vietnam.

In recent years, serosurveillance has gained momentum as a way of determining disease transmission and immunity in populations, particularly with respect to vaccine-preventable diseases. At the end of 2017, the Oxford University Clinical Research Unit and the National Institute of Hygiene and Epidemiology held a meeting in Vietnam with national policy makers, researchers, and international experts to discuss current seroepidemiologic projects in Vietnam and future needs and plans for nationwide serosurveillance. This report summarizes the meeting and the plans that were discussed to set up nationwide serosurveillance in Vietnam.

A novel p.A191D matrilin-3 variant in a Vietnamese family with multiple epiphyseal dysplasia: a case report.

BACKGROUND: Multiple epiphyseal dysplasia (MED) is a common skeletal dysplasia that is characterized by variable degrees of epiphyseal abnormality primarily involving the hip and knee joints. Mutations in a gene encoding matrilin-3 (MATN3) have been reported as disease causing of autosomal dominant MED. The current study identified a novel c.572 C > A variant (p.A191D) in exon 2 of MATN3 in a Vietnamese family with MED. CASE PRESENTATION: A standard clinical tests and radiological examination were performed in an 8-year-old Vietnamese girl patient. The clinical examination showed that patient height was under average, with bent lower limbs, limited mobility and dislocation of the joints at both knees. Radiological documentation revealed abnormal cartilage development at the epiphysis of the femur and patella. The patient has a varus deformity of the lower limbs. The patient was diagnosed with autosomal dominant MED using molecular testing in the order of the coding sequences and flanking sequences of five genes: COMP (exons 8-19), MATN3 (exon 2), COL9A2 (exon 3), COL9A3 (exon 3), COL9A1 (exon 8) by Sanger sequencing. A novel heterozygous missense variant (c.572 C > A, p.A191D) in MATN3 was identified in this family, which were not inherited from parents. The p.A191D was predicted and classified as a pathogenic variant. When the two predicted structures of the wild type and mutant matrilin-3 were compared, the p.A191D substitution caused conformational changes near the substitution site, resulting in deformity of the ß-sheet of the single A domain of matrilin- 3. CONCLUSIONS: This is the first Vietnamese MED family attributed to p.A191D matrilin-3 variant, and our clinical, radiological and molecular data suggest that the novel de novo missense variant in MATN3 contributed to MED.

Coral and Coral-Associated Microorganisms: A Prolific Source of Potential Bioactive Natural Products.

Marine invertebrates and their associated microorganisms are rich sources of bioactive compounds. Among them, coral and its associated microorganisms are promising providers of marine bioactive compounds. The present review provides an overview of bioactive compounds that are produced by corals and coral-associated microorganisms, covering the literature from 2010 to March 2019. Accordingly, 245 natural products that possess a wide range of potent bioactivities, such as anti-inflammatory, cytotoxic, antimicrobial, antivirus, and antifouling activities, among others, are described in this review.

Antimicrobial and Cytotoxic Properties of Bioactive Metabolites Produced by Streptomyces cavourensis YBQ59 Isolated from Cinnamomum cassia Prels in Yen Bai Province of Vietnam.

The endophytic actinomycete strain YBQ59 was isolated from Cinnamomum cassia Prels in Yen Bai province (21°53'14″N; 104°35'9″E) of northern Vietnam. Based on analysis of morphological, physiological characteristics and 16S rRNA gene sequence (GenBank Acc. No. MF950891), the strain YBQ59 possessed high similarity to Streptomyces cavourensis subsp. cavourensis strain NRRL 2740, therefore assigned as S. cavourensis YBQ59. The ethyl acetate extract of the YBQ59 culture broth isolated eight pure secondary metabolites, identified as 1-monolinolein (1), bafilomycin D (2), nonactic acid (3), daidzein (4), 3'-hydroxydaidzein (5), 5,11-epoxy-10-cadinanol (6), prelactone B (7), and daucosterol (8). Compounds 1, 3-8 were reported for the first time from S. cavourensis. Compounds 1-5 exhibited antimicrobial activities against both methicillin-resistant Staphylococcus aureus ATCC 33591 (MRSA) and methicillin-resistant Staphylococcus epidermidis ATCC 35984 (MRSE) among which the compound 1 revealed the strongest effects with minimum inhibitory concentrations of 8.5 and 14.6 µg/mL, respectively. The compound 2 showed high potential effect against MRSA (MIC of 11.1 µg/mL) but less effect against MRSE (MIC of 30.3 µg/mL). The cytotoxicity of the compounds 1-7 was investigated against human lung adenocarcinoma EGFR-TKI-resistant cells, among which compounds 1, 2, and 5 exhibited the strong effect against A549 cells with IC50 values of 3.6, 6.7, and 7.8 µM, respectively. Taken together, the experimental findings in this study suggested that the compounds 1 and 2 could be reproducible metabolites applicable for inhibition of both drug-resistant bacteria and cancer cell lines.

Neutralizing immune responses induced by oligomeric H5N1-hemagglutinins from plants.

Plant-based transient expression is an alternative platform to produce hemagglutinin-based subunit vaccines. This production system provides not only fast and effective response in the context of a pandemic but also enables the supply of big volume vaccines at low cost. Crude plant extracts containing influenza hemagglutinin are considered to use as vaccine sources because of avoidance of related purification steps resulting in low cost production allowing veterinary applications. Highly immunogenic influenza hemagglutinins are urgently required to meet these pre-conditions. Here, we present a new and innovative way to generate functional H5 oligomers from avian flu hemagglutinin in planta by the specific interaction of S·Tag and S·Protein. A S·Tag was fused to H5 trimers and this construct was transiently co-expressed in planta with S·Protein-TPs which was multimerized by disulfide bonds via cysteine residues in tailpiece sequences (TP) of IgM antibody. Multimerized S·Protein-TPs serve as bridges/molecular docks to combine S·Tag-fused hemagglutinin trimers to form very large hemagglutinin H5 oligomers. H5 oligomers in the plant crude extract were highly active in hemagglutination resulting in high titers. Immunization of mice with two doses of plant crude extracts containing H5 oligomers after storage for 1 week at 4 °C caused strong immune responses and induced neutralizing specific humoral immune responses in mice. These results allow for the development of cheap influenza vaccines for veterinary application in future.

Nanodiamond enhances immune responses in mice against recombinant HA/H7N9 protein.

BACKGROUND: The continuing spread of the newly emerged H7N9 virus among poultry in China, as well as the possibility of human-to-human transmission, has attracted numerous efforts to develop an effective vaccine against H7N9. The use of nanoparticles in vaccinology is inspired by the fact that most pathogens have a dimension within the nano-size range and therefore can be processed efficiently by the immune system, which leads to a potent immune response. Herein, we report a facile approach to increase antigen size to achieve not only fast but also effective responses against the recombinant HA/H7N9 protein via a simple conjugation of the protein onto the surface of nanodiamond particles. RESULTS: In this study, trimeric Haemagglutinin (H7) that is transiently expressed in N. benthamiana was purified using affinity chromatography, and its trimeric state was revealed successfully by the cross-linking reaction. The trimeric H7 solution was subsequently mixed with a nanodiamond suspension in different ratios. The successful conjugation of the trimeric H7 onto the surface of nanodiamond particles was demonstrated by the changes in size and Zeta-potential of the particles before and after protein coating, Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and Western-blot analysis. Next, biofunction of the protein-nanodiamond conjugates was screened using a haemagglutination assay. A mixture containing 5 µg of trimeric H7 and 60 µg of nanodiamond corresponds to a ratio of 1:12 (w/w) of agglutinated chicken red blood cells at HA titer of 1024, which is 512-fold higher than the HA titer of free trimeric H7. After the 2nd and 3rd immunization in mice, ELISA and Western blot analyses demonstrated that the physical mixture of trimeric H7 protein and nanodiamond (1:12, w/w) elicited statistically significant stronger H7-specific-IgG response demonstrated by higher amounts of H7N9-specific IgG (over 15.4-fold with P < 0.05 after the second immunization). CONCLUSIONS: These results indicated a potential effect inherent to nanodiamond towards modulating immune systems, which should be further evaluated and broadly applied in nanovaccine development.