Partitioning recombinant chitinase from Nicotiana benthamiana by an aqueous two-phase system based on polyethylene glycol and phosphate salts.

The objectives of this study were to purify 42 kDa chitinase derived from Trichoderma asperellum SH16 produced in Nicotiana benthamiana by a polyethylene glycol (PEG)/salt aqueous two-phase system (ATPS). The specific activities of the crude chitinase and the partially purified chitinase from N. benthamiana were about 251 unit/mg and 386 unit/mg, respectively. The study found the 300 g/L PEG 6000 + 200 g/L potassium phosphate (PP) and 300 g/L PEG 6000 + 150 g/L sodium phosphate (SP) systems had the highest partitioning efficiency for each salt in primary extraction. However, among the two types of salt, PP displayed higher efficiency than SP, with a partitioning coefficient K of 4.85 vs. 3.89, a volume ratio V of 2.94 vs. 2.68, and a partitioning yield Y of approximately 95 % vs. 83 %. After back extraction, the enzymatic activity of purified chitinase was up to 834 unit/mg (PP) and 492 unit/mg (SP). The purification factors reached 3.32 (PP) and 1.96 (SP), with recovery yields of about 59 % and 61 %, respectively. SDS-PAGE and zymogram analysis showed that the recombinant chitinase was significantly purified by using ATPS. The purified enzyme exhibited high chitinolytic activity, with the hydrolysis zone's diameter being around 2.5 cm-3 cm. It also dramatically reduced the growth of Sclerotium rolfsii; the colony diameter after treatment with 60 unit of enzyme for 104 spores was only about 1 cm, compared to 3.5 cm in the control. The antifungal effect of chitinase suggests that this enzyme has great potential for applications in agricultural production as well as postharvest fruit and vegetable preservation.

Bioproduction of eriodictyol by Escherichia coli engineered co-culture.

Eriodictyol (ED) is a flavonoid in the flavanones subclass. It is abundantly present in a wide range of medicinal plants, citrus fruits, and vegetables. In addition, ED owns numerous importantly medicinal bioactivities such as inhibition of proliferation, metastasis and induction of apoptosis in glioma cells or inhibition of glioblastoma migration, and invasion. This study described the heterologous production of ED by E. coli based co-culture engineering system from the simple carbon substrate D-glucose. Two E. coli strains were engineered and functioned as constitutive components of biological system. Specifically, the first strain (upstream module) contained genes for synthesis of p-coumaric acid (pCA) from D-glucose. And, the second strain (downstream module) consisted of genes for the synthesis of ED from pCA. The highest yield in ED production was achieved 51.5 ± 0.4 mg/L using stepwise optimal culture conditions, while monoculture was achieved 21.3 ± 0.2 mg/L only. In conclusion, co-culture was the most efficient alternative approach for the synthesis of ED and other natural products.

Assessment of a psychiatric intervention at community level for people who inject drugs in a low-middle income country: the DRIVE-Mind cohort study in Hai Phong, Viet Nam.

BACKGROUND: Access to psychiatric care for people who inject drugs (PWID) is limited/absent and stigmatized in most low-middle-income countries (LMICs). Innovative interventions are needed. We aimed to describe and assess the impact of a community-based psychiatric intervention among PWID in Hai Phong, Vietnam. METHODS: In a cohort study with one year psychiatric follow-up, PWID diagnosed with a psychotic disorder, a major depressive episode, or suicide risk, were recruited from the wider Drug-Related Infections in ViEtnam (DRIVE) project in the city of Hai Phong. The community-based psychiatric intervention included specialized follow-up (free consultations with psychiatrists, free medication, referral to mental health department for hospitalization when necessary) and support from community-based organisations (case management, harm reduction, administrative support, linkage to HIV care, methadone maintenance treatment and mental health support). The main outcome was reduction/remission of symptoms. Access to and retention in psychiatric care, quality-of-life and stigmatization were also measured pre and post-intervention. FINDINGS: Among the 1212 participants screened from March to May 2019, 271 met the inclusion criteria, 233 (86.3%) accepted the intervention and 170 completed the follow-up (72.9%). At inclusion, 80.6% were diagnosed with current depression, 44.7% with psychotic disorder and 42.4% with suicide risk. After a one-year follow-up, these proportions dropped to 15.9%, 21.8%, and 22.9% respectively. Quality-of-life and perceived stigma related to mental health were also significantly improved, while drug use decreased only marginally. INTERPRETATION: Community-based psychiatric interventions are both feasible and efficient in the Vietnamese context. Similar interventions should be implemented and evaluated in other, different LMICs. FUNDING: : This work was supported by grants from NIDA (US) (#DA041978) and ANRS (France) (#13353). The funding agencies had no role in designing the research, data analyses, or preparation of the report.

De novo whole-genome assembly and discovery of genes involved in triterpenoid saponin biosynthesis of Vietnamese ginseng (Panax vietnamensis Ha et Grushv.).

Vietnamese ginseng (Panax vietnamensis Ha et Grushv.), also known as Ngoc Linh ginseng, is a high-value herb in Vietnam. Vietnamese ginseng has been proven to be effective in enhancing the immune system, human memory, anti-stress, anti-inflammatory, anti-cancer, and prevent aging. The present study reports the first draft whole-genome of Vietnamese ginseng and the identification of potential genes involved in the triterpenoid metabolic pathway. De novo whole-genome assembly was performed successfully from a data of approximately 139 Gbps of 394,802,120 high quality reads to generate 9815 scaffolds with an N50 value of 572,722 bp from the leaf of Vietnamese ginseng. The assembled genome of Vietnamese ginseng is 3,001,967,204 bp long containing 79,374 gene models. Among them, there are 55,012 genes (69.30%) were annotated by various public molecular biology databases. The potential genes involved in triterpenoid saponin biosynthesis in Vietnamese ginseng and their metabolic pathway were also predicted." Three genes encoding squalene monooxygenase isozymes in Vietnamese ginseng were cloned, sequenced and characterized. Moreover, expression levels of several key genes involved in terpenoid biosynthesis in different parts of Vietnamese ginseng were also analyzed. The SSR markers were detected by various programs from both of assembly full dataset of Vietnamese ginseng genome and predicted genes. The present work provided important data of the draft whole-genome of Vietnamese ginseng for further studies to understand the role of genes involved in ginsenoside biosynthesis and their metabolic pathway at the molecular level of this rare medicinal species. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01076-1.

Expression of 42 kDa chitinase of Trichoderma asperellum (Ta-CHI42) from a synthetic gene in Escherichia coli.

Chitinases are enzymes that catalyze the degradation of chitin, a major component of the cell walls of pathogenic fungi and cuticles of insects, gaining increasing attention for the control of fungal pathogens and insect pests. Production of recombinant chitinase in a suitable host can result in a more pure product with less processing time and a significantly larger yield than that produced by native microorganisms. The present study aimed to express the synthetic chi42 gene (syncodChi42), which was optimized from the chi42 gene of Trichoderma asperellum SH16, in Escherichia coli to produce 42 kDa chitinase (Ta-CHI42); then determined the activity of this enzyme, characterizations and in vitro antifungal activity as well as its immunogenicity in mice. The results showed that Ta-CHI42 was overexpressed in E. coli. Analysis of the colloidal chitin hydrolytic activity of purified Ta-CHI42 on an agar plate revealed that this enzyme was in a highly active form. This is a neutral chitinase with pH stability in a range of 6-8 and has an optimum temperature of 45°C with thermal stability in a range of 25-35°C. The chitinolytic activity of Ta-CHI42 was almost completely abolished by 5 mM Zn2+ or 1% SDS, whereas it remained about haft under the effect of 1 M urea, 1% Triton X-100 or 5 mM Cu2+. Except for ions such as Mn2+ and Ca2+ at 5 mM that have enhanced chitinolytic activity; 5 mM of Na+, Fe2+ or Mg2+ ions or 1 mM EDTA negatively impacted the enzyme. Ta-CHI42 at 60 U/mL concentration strongly inhibited the growth of the pathogenic fungus Aspergillus niger. Analysis of western blot indicated that the polyclonal antibody against Ta-CHI42 was greatly produced in mice. It can be used to analyze the expression of the syncodChi42 gene in transgenic plants, through immunoblotting assays, for resistance to pathogenic fungi.

Systemic and Oral Immunogenicity of Porcine Epidemic Diarrhea Virus Antigen Fused to Poly-Fc of Immunoglobulin G and Expressed in ΔXT/FT Nicotiana benthamiana Plants.

Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family has become increasingly probelmatic in the pig farming industry. Currently, there are no effective, globally applicable vaccines against PEDV. Here, we tested a recombinant PEDV vaccine candidate based on the expression of the core neutralising epitope (COE) of PEDV conjugated to polymeric immunoglobulin G scaffold (PIGS) in glycoengineered Nicotiana be nthamiana plants. The biological activity of COE-PIGS was demonstrated by binding to C1q component of the complement system, as well as the surface of antigen-presenting cells (APCs) in vitro. The recombinant COE-PIGS induced humoral and cellular immune responses specific for PEDV after both systemic and mucosal vaccination. Altogether, the data indicated that PEDV antigen fusion to poly-Fc could be a promising vaccine platform against respiratory PEDV infection.

Effects of various polysaccharides (alginate, carrageenan, gums, chitosan) and their combination with prebiotic saccharides (resistant starch, lactosucrose, lactulose) on the encapsulation of probiotic bacteria Lactobacillus casei 01 strain.

The probiotics are extremely sensitive to various environmental factors, which imposes limitation on their health and functional effectiveness. Thus, development of delivery system for protection of viable cells while passing through different stages of the human digestion system is key factor in application of probiotic products. In our study, the effects of several polysaccharides such as alginate, κ-carrageenan, locust bean gum, gellan gum, xanthan gum and their combination with various prebiotic components (resistant starch, lactulose, lactosucrose) on encapsulation of probiotic Lactobacillus casei 01 strain were studied. Both regular and unregular beads with size distributions from 2 mm up to 5 mm were obtained. The encapsulation efficiencies varied from 64.4% up to 79%. Based on the texture's profiles, the capsules can be grouped into 5 clusters with squared Euclidean distance 3.5. Meanwhile, the starch-alginate and the lactosucrose LS55L - alginate beads were found to be the most stable and to have massive textural properties, whereas the gellan gum - xanthan gum and the chitosan coated alginate beads emerged as the softest. Encapsulation significantly improved the degree of gastric tolerance of probiotic cells even in the presence of pepsin. The INFOGEST in vitro digestion protocol was adapted to investigate the protection effects of different capsules. The highest survival (with loss rate of lower than 1 log CFU/g) was observed in the case of the cells encapsulated in starch-alginate beads. Moreover, the alginate microcapsules combined with lactosucrose LS55L also provided very promising shield for probiotics from the low pH of gastric conditions. Our findings suggest that incorporation of prebiotics into alginate-base encapsulation would be good idea in development of micro delivery systems that helps the survival of probiotics and their delivery to the target sites of action in human body.

An efficient protocol for in vitro regeneration of peanut (Arachis hypogaea L ) cultivar L14

The present work aims to establish an efficient protocol for in vitro regeneration of peanut (Arachis hypogaea) cultivar L14. The study showed that de-embryonated cotyledon was a suitable explant for shoot multiplication on MS medium containing 4 mg/L BAP. The highest number of shoots per explant obtained after 4 weeks of culture was up to 6.8 shoots. Shoots in vitro were able to produce a large number of approximately 11 roots on MS medium supplemented with 0.5 mg/L NAA. These results will be very useful in establishing an in vitro regeneration protocol for peanut cultivar L14 during gene transfer in the next studies to improve their disease resistance.

Daily heroin injection and psychiatric disorders: A cross-sectional survey among People Who Inject Drugs (PWID) in Haiphong, Vietnam.

BACKGROUND: Psychiatric comorbidities are frequent among people who inject drugs, they are associated with a poorer prognosis and need to be addressed. Their interaction with daily heroin injection requires clarification. METHODS: A cross-sectional survey was conducted among PWID recruited in the city of Haiphong, Vietnam, by respondent-driven sampling. The inclusion criteria were age 18 or older and current injection drug use, verified by skin marks and positive urine tests for heroin or methamphetamine. Data on socio-demographic characteristics, drug use, sexual behaviour and access to treatment were collected using face-to-face questionnaires by trained interviewers. PWID were screened by trained psychiatrists for depression, psychotic disorder and suicidality, using the MINI questionnaire. RESULTS: 418 participants were included in the analyses. All were injected heroin users, 21 % were diagnosed with a current major depressive disorder, 15 % with a current psychotic disorder and 12 % presented a suicide risk. In the bivariate analyses, regular meth use, cannabis use and ketamine use were positively associated with presenting at least one psychiatric condition while daily heroin injection and being currently treated with methadone were negatively associated. In the multivariate model, poly-substance use was positively associated with depression (methamphetamine and drinking in addition to heroin) and psychotic disorder (methamphetamine and/or hazardous drinking in addition to heroin) while daily heroin injection and current methadone treatment were negatively and independently associated with depression and psychotic syndrome. CONCLUSIONS: Our survey confirms the burden of methamphetamine use and the protective effect of methadone but also a possible protective effect of daily heroin injection.

Decrease in Choroidal Vascularity Index of Haller's layer in diabetic eyes precedes retinopathy.

INTRODUCTION: The study aimed to evaluate Choroidal Vascularity Index (CVI) of Haller's and Sattler's layers and their relationships with choroidal and retinal thickness, volumes measured on enhanced depth imaging-optical coherence tomography (OCT) scans in the eyes of patients without diabetes, patients with diabetes with no diabetic retinopathy (DR) and patients with diabetes and DR. RESEARCH DESIGN AND METHODS: Retrospective analysis of 165 eyes from 84 Singapore Indian Eye Study-2 study participants (group 1: no diabetes, group 2: diabetes with no DR and group 3: with DR). Groups 1 and 2 were matched by age and gender from group 3. RESULTS: In the eyes of patients with diabetes without DR, the macular CVI of Haller's but not Sattler's layer was significantly reduced compared with eyes of patients without diabetes. Eyes with >5 years of diabetes have significantly decreased CVI of Sattler's layers (mean difference=0.06 ± 0.10, p=0.04) and also decreased subfoveal choroidal volume (mean difference=0.89 ± 0.16 mm3, p=0.02), compared with those with ≤5 years of diabetes. CONCLUSION: Diabetic eyes without DR had significantly lower CVI of macular Haller's layer than those of healthy controls. With a longer duration of diabetes, CVI of subfoveal Sattler's layer and choroidal volume continue to decrease, irrespective of diabetic control, suggesting that early diabetic choroidopathy mainly affects larger choroidal veins initially before medium-sized arterioles. The CVI of macular Haller's layer could potentially be used as a marker on spectral domain OCT imaging in newly diagnosed patients with diabetes for the onset of DR and as a possible prognostication tool in diabetic eyes. Future prospective longitudinal studies in diabetic eyes would be useful in establishing the relationship between CVIs of Haller's and Sattler's layer with visual acuity as a marker of photoreceptor health and visual prognosis.

Improved expression of porcine epidemic diarrhea antigen by fusion with cholera toxin B subunit and chloroplast transformation in Nicotiana tabacum.

The porcine epidemic diarrhea virus (PEDV) belongs to the coronavirus family, which causes acute diarrhea in pigs with higher mortality in piglets less than 2 weeks old. The PEDV is one of the major concerns of the pig industry around the world, including Asian countries and Noth America since first identified in Europe. Currently, there is no PEDV licensed vaccine to effectively prevent this disease. This study was performed for the development of a mucosal PEDV vaccine and B subunit of cholera toxin (CTB) as a carrier was employed to surpass the tolerogenic nature of GALT and induce potent immune responses against the target antigen fused to CTB. An epitope (S1D) alone or conjugated with CTB was constructed into the tobacco chloroplasts expression vector which is controlled under the chloroplast rRNA operon promoter with T7g10 5' UTR and the psbA 3'UTR as a terminator. The homoplastomic lines were obtained by third round screening via organogenesis from the leaf tissues which were verified by PCR with antigen and chloroplast specific primers and then confirmed by Southern blot analysis. While the expression level of the S1D alone as detected by Western blotting was approximately 0.07% of total soluble protein, the CTB-S1D fusion protein was expressed up to 1.4%. The fusion protein showed binding to the intestinal membrane GM1-ganglioside receptor, demonstrating its functionality. The result shows that the highest expression of S1D could be achieved by fusion with a stable CTB protein and chloroplast transformation. Furthermore, the CTB-S1D expressed in chloroplasts of Nicotiana tabacum cv. Maryland could be assembled to pentameric form which increases the possibility to develop a mucosal vaccine against PEDV.

Lactic acid fermentation of apricot juice by mono- and mixed cultures of probiotic Lactobacillus and Bifidobacterium strains.

Apricot is a popular fruit in the world with rich in carbohydrates, vitamins and elements as well as has high antioxidant capacity. In this study, fermentation of this juice by mono- and mixed cultures was investigated. All tested strains exhibited good growth properties on apricot juice without any nutrient supplementation. In monoculture fermentation, 7.2, 7.25, 7.06 and 7.16 log (cfu/mL h) cell yields were observed for Bifidobacterium lactis Bb-12, Bifidobacterium longum Bb-46, Lactobacillus casei 01 and Lactobacillus acidophilus La-5 strains, respectively, and higher cell yields were obtained in the mixed culture fermentation. The antioxidant capacity increased slightly during fermentation. The concentration of acetic acid (27-48 mM) were about doubled in cases of the mixed culture fermentations than of monoculture fermentations (18-30 mM), while the levels of lactic acid were similar (70-90 mM). The relatively high values of these properties offer the potential for development of novel probiotic apricot juice.

Six-Year Incidence and Risk Factors of Age-Related Macular Degeneration in Singaporean Indians: The Singapore Indian Eye Study.

We aimed to determine the 6-year incidence and risk factors of age-related macular degeneration (AMD) in first and second generations of Singaporean Indians. Baseline examination was conducted in 2007-9 and 6-year propsective follow-up examination of this Indian population in 2013-5. All participants underwent interviews with questionnaires and comprehensive medical and eye examinations. Incidence was age-standardized to Singaporean 2010 census. Risk factors associated with AMD incidence were assessed and compared between first and second generations of immigrants. Among 2200 persons who participated in the follow-up examination (75.5% response rate), gradable fundus photographs were available in 2105. The 6-year age-standardized incidences of early and late AMD were 5.26% and 0.51% respectively. Incident early AMD was associated with cardiovascular disease history (HR 1.59, 95% CI 1.04-2.45), underweight body mass index (BMI) (HR 3.12, 95% CI 1.37-7.14) (BMI of <18.5 vs 18.51-25 kg/m2), heavy alcohol drinking (HR 3.14 95% CI 1.25-7.89) and ARMS2 rs3750847 homozygous genetic loci carrier (HR 2.52, 95% CI 1.59-3.99). We found a relatively low incidence of early AMD in this Singaporean Indian population compared to Caucasian populations. Both first and second-generation Indian immigrants have similar incidence and risk factor patterns for early AMD.

Associations between sleep duration, sleep quality and diabetic retinopathy.

BACKGROUND: Abnormal durations of sleep have been associated with risk of diabetes. However, it is not clear if sleep duration is associated with diabetic retinopathy (DR). METHODS: In a cross-sectional study, we included 1,231 (Malay, n = 395; Indian, n = 836) adults (mean age 64.4 ± 9.0 years, 50.4% female) with diabetes from the second visit of two independent population-based cohort studies (2011-15) in Singapore. Self-reported habitual sleep duration was categorized as short (<6 h), normal (6≤ h <8), and long (≥8 h). Questionnaires were administered to detect risk of obstructive sleep apnea (OSA), excessive daytime sleepiness, and insomnia, all of which may indicate poor quality of sleep. The associations between sleep-related characteristics with moderate DR and vision-threatening DR (VTDR) were analysed using logistic regression models adjusted for potential confounders. RESULTS: Prevalence of moderate DR and VTDR in the study population were 10.5% and 6.3% respectively. The mean duration of sleep was 6.4 ± 1.5 h. Compared to normal sleep duration, both short and long sleep durations were associated with moderate DR with multivariable odds ratio (95% confidence interval) of 1.73 (1.03-2.89) and 2.17 (1.28-3.66) respectively. Long sleep duration (2.37 [1.16-4.89]), high risk of OSA (2.24 [1.09-4.75]), and excessive daytime sleepiness (3.27 [1.02-10.30]) were separately associated with VTDR. CONCLUSION: Sleep duration had a U-shaped association with moderate DR; long sleep duration, excessive daytime sleepiness and high risk of OSA were positively associated with VTDR.

Development of the FitSight Fitness Tracker to Increase Time Outdoors to Prevent Myopia.

PURPOSE: To develop a fitness tracker (FitSight) to encourage children to increase time spent outdoors. To evaluate the wear pattern for this tracker and outdoor time pattern by estimating light illumination levels among children. METHODS: The development of the FitSight fitness tracker involved the designing of two components: (1) the smartwatch with custom-made FitSight watch application (app) to log the instant light illuminance levels the wearer is exposed to, and (2) a companion smartphone app that synchronizes the time outdoors recorded by the smartwatch to smartphone via Bluetooth communication. Smartwatch wear patterns and tracker-recorded daily light illuminance levels data were gathered over 7 days from 23 Singapore children (mean ± standard deviation age: 9.2 ± 1.4 years). Feedback about the tracker was obtained from 14 parents using a three-level rating scale: very poor/poor/good. RESULTS: Of the 14 parents, 93% rated the complete "FitSight fitness tracker" as good and 64% rated its wearability as good. While 61% of 23 children wore the watch on all study days (i.e., 0 nonwear days), 26% had 1 nonwear day, and 4.5% children each had 3, 4, and 5 nonwear days, respectively. On average, children spent approximately 1 hour in light levels greater than 1000 lux on weekdays and 1.3 hours on weekends (60 ± 46 vs. 79 ± 53 minutes, P = 0.19). Mean number of outdoor "spurts" (light illuminance levels >1000 lux) per day was 8 ± 3 spurts with spurt duration of 34 ± 32 minutes. CONCLUSION: The FitSight tracker with its novel features may motivate children to increase time outdoors and play an important role in supplementing community outdoor programs to prevent myopia. TRANSLATIONAL RELEVANCE: If the developed noninvasive, wearable, smartwatch-based fitness tracker, FitSight, promotes daytime outdoor activity among children, it will be beneficial in addressing the epidemic of myopia.

Viral hemorrhagic septicemia virus glycoprotein production in tobacco.

Viral hemorrhagic septicemia virus (VHSV) causes mortality in numerous marine and freshwater fish species resulting in heavy losses in fish farming. The glycoprotein gene of VHSV was fused with the cholera toxin B subunit (CTB) and expressed transiently in leaf tissues of Nicotiana benthamiana via the agroinfiltration method. The glycoprotein gene was divided into two parts to improve assembly of CTB fusion proteins (CTB-VHSV99-235 and CTB-VHSV258-417). Production of CTB fusion proteins was confirmed in the agroinfiltrated leaf tissue by western blot analysis. The plant-produced CTB fusion proteins showed biological activity to GM1-ganglioside, a receptor for biologically active CTB, on GM1-ELISA. The expression level of the CTB-VHSV fusion proteins was 0.86% (CTB-VHSV99-235) and 0.93% (CTB-VHSV258-417) of total proteins in agroinfiltrated leaf tissue, as determined by GM1-ELISA. These results suggest that Agrobacterium-mediated transient expression of CTB fusion antigens of VHSV is a rapid and convenient method and demonstrate the feasibility of using agroinfiltrated plant leaf tissues expressing CTB-fusion antigens as a plant-based vaccine to prevent VHSV infection.

Expression and assembly of cholera toxin B subunit and domain III of dengue virus 2 envelope fusion protein in transgenic potatoes.

The rates of mosquito-transmitted dengue virus infection in humans have increased in tropical and sub-tropical areas. Domain III of dengue envelope protein (EDIII) is involved in cellular receptor binding and induces serotype-specific neutralizing antibodies. EDIII fused to the B subunit of Vibrio cholera (CTB-EDIII) was expressed in potatoes to develop a plant-based vaccine against dengue virus type 2. CTB-EDIII fused to an endoplasmic reticulum (ER) retention signal, SEKDEL, was introduced into potatoes by A. tumefaciens-mediated gene transformation. The integration of the CTB-EDIII fusion gene into the nuclear genome of transgenic plants was confirmed by genomic DNA polymerase chain reaction (PCR), and mRNA transcripts of CTB-EDIII were detected. CTB-EDIII fusion protein was expressed in potato tubers and assembled into a pentameric form capable of binding monosialotetrahexosylganglioside (GM1). The level of expression was determined to be ∼0.005% of total soluble protein in potato tubers. These results suggest that dengue virus antigen could be produced in potatoes, raising the possibility that edible plants are employed in mucosal vaccines for protection against dengue infection.

Immunogenicity of an S1D epitope from porcine epidemic diarrhea virus and cholera toxin B subunit fusion protein transiently expressed in infiltrated Nicotiana benthamiana leaves.

Porcine epidemic diarrhea virus (PEDV) belongs to the Coronaviridae family and causes acute enteritis in pigs. A fragment of the large spike glycoprotein, termed the S1D epitope (aa 636-789), alone and fused with cholera toxin B subunit, were independently cloned into plant expression vectors, yielding plasmids pMYV717 and pMYV719, respectively. Plant expression vectors were transformed into Agrobacterium tumefaciens and subsequently infiltrated into Nicotiana benthamiana leaves. The highest expression level of S1D was found at 2 days post infiltration (dpi), reached 0.04 % of total soluble protein, and rapidly decreased thereafter. The expression and assembly of CTB-S1D fusion protein were confirmed by Western blot and GM1-ELISA. The highest expression level of CTB-S1D fusion protein was 0.07 % of TSP at 4 dpi, with a rapid decrease thereafter. In the presence of p19 protein from tomato bushy stunt virus, the S1D and CTB-S1D protein levels peaked at 6 dpi and were fourfold to sevenfold higher than in the absence of p19, respectively. After oral administration of transiently expressed CTB-S1D fusion protein, or with bacterial cholera toxin or rice callus expressing mutant cholera toxin 61F, mice exhibited significantly greater serum IgG and sIgA levels against bacterial CTB and S1D antigen, peaking at week 6. Transiently expressed CTB-S1D fusion protein will be administered orally to pigs to assess the immune response against PEDV.

Dengue virus E glycoprotein production in transgenic rice callus.

Dengue is a disease caused by dengue virus and represents the most important arthropod-borne viral disease in humans. Dengue virus enters host cells via binding of envelope glycoprotein (E) to a receptor. In this study, plant expression vectors containing native and synthetic glycoprotein E genes (sE) modified based on plant-optimized codon usage and fused with an ER retention signal were constructed under control of the rice amylase 3D promoter expression system. Plant expression vectors were introduced into rice callus (Oryza sativa L. cv. Dongin) via particle bombardment-mediated transformation. The integration and expression of target genes were confirmed in the transgenic callus by genomic DNA PCR and Northern blot analyses, respectively. The plant-codon optimized sE gene with an ER retention signal showed high protein production levels based on Western blot analysis of approximately 18.5 ug/g dried calli weight by immunoblot-based densitometric analysis. These results suggest that the plant-codon optimized sE gene with an ER retention signal was highly produced in the transgenic rice callus.