Immersion prime and oral boost vaccination with an inactivated Vibrio harveyi vaccine confers a specific immune response and protection in Asian seabass (Lates calcarifer).

Asian seabass (Lates calcarifer) holds significant economic value in fish farming in the Asia-Pacific region. Vibriosis caused by Vibrio harveyi (Vh) is a severe infectious disease affecting intensive farming of this species, for which prevention strategies by vaccination have been developed. This study investigated an alternative approach to injectable vaccination to prevent vibriosis in Asian seabass juveniles. The strategy begins with an immersion prime vaccination with a heat-inactivated Vh vaccine, followed by two oral booster doses administered at 14- and 28-days post-vaccination (dpv). Expression of five immune genes TNFα, IL1ß, CD4, CD8, and IgM in the head kidney and spleen, along with investigation of anti-Vh antibody response (IgM) in both systemic and mucosal systems, was conducted on a weekly basis. The efficacy of the vaccines was assessed by a laboratory challenge test at 43 dpv. The results showed that the immunized fish displayed higher levels of mRNA transcripts of the immune genes after the immersion prime and the first oral booster dose compared to the control group. The expression levels peaked at 14 and 28 dpv and then declined to baseline at 35 and 42 dpv. Serum specific IgM antibodies were detected as early as 7 dpv (the first time point investigated) and exhibited a steady increase, reaching the first peak at 21 dpv, and a second peak at 35 dpv. Although the antibody levels gradually declined over subsequent weeks, they remained significantly higher than the control group throughout the experiment. A similar antibody response pattern was also observed in the mucosal compartment. The laboratory challenge test demonstrated high protection by injection with 1.65 × 104 CFU/fish, with a relative percent of survival (RPS) of 72.22 ± 7.86 %. In conclusion, our findings highlight the potential of an immersion prime-oral booster vaccination strategy as a promising approach for preventing vibriosis in Asian seabass.

Review of current perspectives and future outlook on bacterial disease prevention through vaccination in Asian seabass (Lates calcarifer).

Asian seabass, Lates calcarifer, is an important aquatic species in mariculture. Intensive farming of this species has faced episodes of bacterial diseases, including those due to vibriosis, scale drop, and muscle necrosis disease, big belly disease, photobacteriosis, columnaris, streptococcosis, aeromoniasis, and tenacibaculosis. Vaccination is one of the most efficient, non-antibiotic, and eco-friendly strategies for protecting fish against bacterial diseases, contributing to aquaculture expansion and ensuring food security. As of now, although numerous vaccines have undergone laboratory research, only one commercially available inactivated vaccine, suitable for both immersion and injection administration, is accessible for preventing Streptococcus iniae. Several key challenges in developing vaccines for Asian seabass must be addressed, such as the current limited understanding of immunological responses to vaccines, the costs associated with vaccine production, forms, and routes of vaccine application, and how to increase the adoption of vaccines by farmers. The future of vaccine development for the Asian seabass industry, therefore, is discussed with these key critical issues in mind. The focus is on improving our understanding of Asian seabass immunity, including maternal immunity, immunocompetence, and immune responses post-vaccination, as well as developing tools to assess vaccine effectiveness. The need for an alignment of fish vaccines with state-of-the-art vaccine technologies employed in human and terrestrial animal healthcare is also discussed. This review also discusses the necessity of providing locally-produced autogenous vaccines, especially for immersion and oral vaccines, to benefit small-scale fish farmers, and the potential benefits that might be extended through changes to current husbandry practices such as the vaccination of broodstock and earlier life stages of their off-spring.

A practical approach to nutritional intervention for people with chronic kidney disease in Vietnam.

BACKGROUND AND OBJECTIVES: A comprehensive nutritional management is necessary for favourable outcomes in patients with chronic kidney disease (CKD). We aimed to assess the changes in nutritional status and disease progression with nutritional management where renal replacement therapy (RRT) was not in place. METHODS AND STUDY DESIGN: A quasi-experiment intervention was conducted on 70 CKD patients at stages 3-5 from July to December 2022. Participants were excluded if they underwent RRT, including dialy-sis (hemodialysis or peritoneal dialysis), or kidney transplantation. The nutritional regimen covered nutrition-al counseling, samples of the dietary menu, and supplement products. We evaluated nutritional status using Subjective Global Assessment (SGA) scale and sub-clinical blood test at T0 (hospital admission) and T1 (two weeks after the admission or 24 hours before the discharge). RESULTS: After the intervention, the number of patients classified as malnutrition or at risk of malnourished reduced significantly (65.7% to 54.3% and 25.7% and 5.7%, respectively). The serum concentration of urea, creatinine and parathyroid hormone decreased remarkably, especially in patients receiving nutritional management. In the intervention group, the dietary pattern provided increased intakes of calcium and iron at T1, while phosphorus, sodium and potassium decreased after follow-up. Nausea/vomiting, loss of appetite, tiredness and sleep disorders were improved in the intervention compared to the control group. CONCLUSIONS: Nutritional therapy enhanced the nutritional sta-tus, and quality of dietary and renal function in CKD patients without RRT. Applying nutrition education and treatment at an early stage can slow CKD progression, which should be applicable elsewhere in Vietnam.

Immunological response of 35 and 42 days old Asian seabass (Lates calcarifer, Bloch 1790) fry following immersion immunization with Streptococcus iniae heat-killed vaccine.

Early disease prevention by vaccination requires understanding when fry fish develop specific immunity to a given pathogen. In this research, we explored the immune responses of Asian seabass (Lates calcarifer) at the stages of 35- and 42- days post-hatching (dph) to an immersive heat-killed Streptococcus iniae (Si) vaccine to determine whether fish can produce specific antibodies against the pathogen. The vaccinated fish of each stage (V35 and V42) were immersed with the Si vaccine at 107 CFU/ml for 3 h, whereas the control groups (C35 and C42) were immersed with tryptic soy broth (TSB) in the same manner. Specific antibodies were measured by enzyme-linked immunosorbent assay (ELISA) before and post-immunization (i.e., 0, 7, and 14 days post-immunization, dpi). Expression of innate (TNFα and IL-1ß) and adaptive (MHCI, MHCII, CD4, CD8, IgM-like, IgT-like, and IgD-like) immune-related genes were evaluated at the same time points with the addition of 1 dpi. The results showed that a subset of immunized fish from both V35 and V42 fry could elicit specific antibodies (IgM) against Si at 14 dpi. All tested innate and adaptive immune genes upregulated at 7 dpi among fish in V35 group. Interestingly, 42 dph fish appeared to respond to the Si vaccine faster than that of 35 dph, as a significant increase in transcripts was observed in CD4, IL-1ß, IgM-like, and IgD-like at 1 dpi; and specific antibody titers of some fish, although not all, were higher than a threshold (p = 0.05) since 7 dpi. In conclusion, this study reveals that 35-42 dph Asian seabass fry can elicit specific immunity to Si immersion vaccine, suggesting that early vaccination of 35 dph fry Asian seabass is feasible.

Enhanced podophyllotoxin production of endophyte Fusarium proliferatum TQN5T by host extract and phenylalanine.

Fermentation technology using endophytes is considered a potential alternative approach for producing pharmaceutical compounds like podophyllotoxin (PTOX). In this study, fungus TQN5T (VCCM 44284) was selected from endophytic fungi isolated from Dysosma versipellis in Vietnam for PTOX production through TLC. The presence of PTOX in TQN5T was further confirmed by HPLC. Molecular identification indicated TQN5T as Fusarium proliferatum with 99.43% identity. This result was asserted by morphological characteristics such as white cottony, filamentous colony, layer and branched mycelium, and clear hyphae septa. Cytotoxic assay indicated both biomass extract and culture filtrate of TQN5T presented strong cytotoxicity on LU-1 and HepG2 with IC50 of 0.11, 0.20, 0.041, and 0071, respectively, implying anti-cancer compounds were accumulated in the mycelium and secreted into the medium. Further, the production of PTOX in TQN5T was investigated in the fermentation condition supplemented with 10 µg/ml of host plant extract or phenylalanine as elicitors. The results revealed a significantly higher amount of PTOX in the PDB + PE and PDB + PA at all studied time points in comparison with PDB (control). Especially, after 168 h of culture, PTOX content in the PDB with plant extract reached the peak with 314 µg/g DW which is 10% higher than the best yield of PTOX in previous studies, denoting F. proliferatum TQN5T as a promising PTOX producer. This is the first study on enhancing the PTOX production in endophytic fungi by supplementing phenylalanine-a precursor for PTOX biosynthesis in plants into fermented media, suggesting a common PTOX biosynthetic pathway between host plant and endophytes. KEY POINTS: • Fusarium proliferatum TQN5T was proven for PTOX production. • Both mycelia extract and spent broth extract of Fusarium proliferatum TQN5T presented strong cytotoxicity on cancer cell lines LU-1 and HepG2. • The supplementation of 10 µg/ml host plant extract and phenylalanine into fermentation media of F. proliferatum TQN5T improved the yield of PTOX.

An efficient Agrobacterium-mediated system based on the pyrG auxotrophic marker for recombinant expression in the filamentous fungus Penicillium rubens.

OBJECTIVES: This work aimed to construct a versatile, effective, and food-grade Agrobacterium tumefaciens-mediated transformation (ATMT) system for recombinant expression in the filamentous fungus Penicillium rubens (also known as Pencillium chrysogenum). RESULTS: In this study, the wild-type P. chrysogenum VTCC 31172 strain was re-classified as P. rubens by a multilocus sequencing analysis. Further, the pyrG gene required for uridine/uracil biosynthesis was successfully deleted in the VTCC 31172 strain by homologous recombination to generate a stable uridine/uracil auxotrophic mutant (ΔpyrG). The growth of the P. rubens ΔpyrG strain could be restored by uridine/uracil supplementation, and a new ATMT system based on the uridine/uracil auxotrophic mechanism was established for this strain. The optimal ATMT efficiency could reach 1750 transformants for 106 spores (equivalent to 0.18%). In addition, supplementation of uridine/uracil at the concentrations of 0.005-0.02% during the co-cultivation process significantly promoted transformation efficiency. Especially, we demonstrated that the pyrG marker and the amyB promoter from the koji mold Aspergillus oryzae were fully functional in P. rubens ΔpyrG. Expression of the DsRed reporter gene under the regulation of the A. oryzae amyB promoter lighted up the mycelium of P. rubens with a robust red signal under fluorescence microscopy. Furthermore, genomic integration of multiple copies of the Aspergillus fumigatus phyA gene under the control of the amyB promoter significantly enhanced phytase activity in P. rubens. CONCLUSIONS: The ATMT system developed in our work provides a safe genetic platform for producing recombinant products in P. rubens without using drug resistance markers.

Structural Basis for Broad Substrate Selectivity of Alcohol Dehydrogenase YjgB from Escherichia coli.

In metabolic engineering and synthetic biology fields, there have been efforts to produce variable bioalcohol fuels, such as isobutanol and 2-phenylethanol, in order to meet industrial demands. YjgB is an aldehyde dehydrogenase from Escherichia coli that shows nicotinamide adenine dinucleotide phosphate (NADP)-dependent broad selectivity for aldehyde derivatives with an aromatic ring or small aliphatic chain. This could contribute to the design of industrial synthetic pathways. We determined the crystal structures of YjgB for both its apo-form and NADP-complexed form at resolutions of 1.55 and 2.00 Å, respectively, in order to understand the mechanism of broad substrate selectivity. The hydrophobic pocket of the active site and the nicotinamide ring of NADP(H) are both involved in conferring its broad specificity toward aldehyde substrates. In addition, based on docking-simulation data, we inferred that π-π stacking between substrates and aromatic side chains might play a crucial role in recognizing substrates. Our structural analysis of YjgB might provide insights into establishing frameworks to understand its broad substrate specificity and develop engineered enzymes for industrial biofuel synthesis.

Study of the underlying mechanisms and consequences of pathogenicity differences between two in vitro selected G1-H9N2 clones originating from a single isolate.

The G1-H9N2 avian influenza virus (AIV) has caused significant economic losses in the commercial poultry industry due to reduced egg production and increased mortality. The field observations have shown that H9N2 viruses circulate and naturally mix with other pathogens and these simultaneous infections can exacerbate disease. To avoid an incorrect virus characterization, due to co-infection, isolates were purified by in vitro plaque assays. Two plaque purified G1-H9N2 clones, selected on different cell types, named MDCK-and CEF-clone in regards to the cell culture used, were studied in vivo, revealing two different virulence phenotypes. Subsequently, the underlying mechanisms were studied. Specifically, the phenotypical outcome of SPF bird infection by the two clones resulted in completely different clinical outcomes. These differences in clinical outcome were used to study the factors behind this output in more detail. Further studies demonstrated that the more severe disease outcome associated with the MDCK-clone involves a strong induction of pro-inflammatory cytokines and a lack of type I interferon production, whereas the mild disease outcome associated with the CEF-clone is related to a greater antiviral cytokine response. The immunosuppressive effect of the MDCK-clone on splenocytes was further demonstrated via ChIFN-γ lack production after ex vivo mitogenic stimulation. Genome sequencing of the two clones identified only four amino acid differences including three in the HA sequence (HA-E198A, HA-R234L, HA-E502D-H9 numbering) and one in the NA sequence (NA-V33M). In the present study, valuable insights on the mechanisms responsible for AI pathogenicity and molecular mechanisms of H9N2 infections in chicken were obtained while highlighting the impact of the cells viruses are grown on their virulence.

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.

An Assessment of Physical Activity and Risk Factors in People Living with Dementia: Findings from a Cross-Sectional Study in a Long-Term Care Facility in Vietnam.

BACKGROUND: People living with dementia (PLWD) may experience substantial cognitive decline as the disease progresses, which interferes with their daily activities. This study aimed to assess physical activity (PA) performance and care dependency (CD) and identify factors related to PA among PLWD. METHODS: We conducted a cross-sectional study in 63 PLWD from National Geriatrics Hospital, Vietnam, from 2021 to 2023. We used the Mini Nutritional Assessment (MNA), International Physical Activity Questionnaire (IPAQ), and Care Dependency Scale (CDS) to assess the nutritional status and the levels of PA and CD, respectively. We used the Mann-Whitney test to compare the differences in the PA types and CD levels between dementia levels and a multivariable logistics regression model to analyze factors related to PA. RESULTS: More than half of the subjects had mild dementia. In total, 35% of the PLWD had a low level of PA, and 46.3% were completely independent of care. The mean score in each CDS aspect of the subjects with moderate/severe dementia was statistically significantly lower than that of those with mild dementia (p ≤ 0.05). Lower dependency (OR = 0.9; 95% CI = 0.88-0.99) and malnutrition (OR = 15.4; 95% CI = 1.18-20.21) were associated with insufficient PA in the PLWD. CONCLUSION: Formal caregivers and healthcare workers should encourage PLWD to perform physical activities at any level and personalize the development of tailored and nutritional care strategies for each individual.

Dehydroascorbate induces plant resistance in rice against root-knot nematode Meloidogyne graminicola.

Ascorbic acid (AsA) is an important antioxidant in plants and regulates various physiological processes. In this study, we show that exogenous treatments with the oxidized form of AsA, that is, dehydroascorbate (DHA), activates induced systemic resistance in rice against the root-knot nematode Meloidogyne graminicola, and investigate the molecular and biochemical mechanisms underlying this phenotype. Detailed transcriptome analysis on roots of rice plants showed an early and robust transcriptional response on foliar DHA treatment, with induction of several genes related to plant stress responses, immunity, antioxidant activity, and secondary metabolism already at 1 day after treatment. Quantitative and qualitative evaluation of H2 O2 levels confirmed the appearance of a reactive oxygen species (ROS) burst on DHA treatment, both at the site of treatment and systemically. Experiments using chemical ROS inhibitors or scavengers confirmed that H2 O2 accumulation contributes to DHA-based induced resistance. Furthermore, hormone measurements in DHA-treated plants showed a significant systemic accumulation of the defence hormone salicylic acid (SA). The role of the SA pathway in DHA-based induced resistance was confirmed by nematode infection experiments using an SA-signalling deficient WRKY45-RNAi line and reverse transcription-quantitative PCR on SA marker genes. Our results collectively reveal that DHA activates induced systemic resistance in rice against the root-knot nematode M. graminicola, mediated through the production of ROS and activation of the SA pathway.

Prevalence of metabolic syndrome and its related factors among Vietnamese people: A systematic review and meta-analysis.

BACKGROUND AND AIMS: Metabolic risk factors are the leading causes of mortality and morbidity in recent decades, yet the burden of metabolic syndrome (MetS) has not been carefully assessed in Vietnam. This review thus aims to examine the prevalence of MetS and its related factors in Vietnam. METHODS: A systematic review was conducted using literature retrieved from PubMed/Medline, Web of Science, Embase, Scopus, and Google Scholar up until July 2021. We selected descriptive and analytical studies which reported the prevalence of MetS and related factors among healthy people aged less than 65 years old rather than morbid people in Vietnam. A meta-analysis with a random-effects model was applied to estimate the pooled prevalence from the included studies. RESULTS: Eighteen studies with 35421 participants were included in the final analysis. The pooled MetS prevalence among the adult population in Vietnam was 16.1% (95% Confidence Interval (CI): 14.1%-18.1%). Higher prevalence was seen among females (17.3%, 95% CI: 13.8%-20.8%). Low level of High Density Lipoprotein-Cholesterol (HDL-C) was the most prevalent component (34.1%), followed by high triglycerides (33.3%). Being female, living in urban areas, having obesity, and having a higher body mass index or body fat percentage were associated with an increased likelihood of having MetS. CONCLUSIONS: MetS was common in the Vietnamese population. Low HDL-C should be considered as an early detectable indicator for MetS screening programs at the population level. Appropriate interventions should be conducted for high-risk groups such as females, those living in urban areas, and obesity.

Simultaneously induced mutations in eIF4E genes by CRISPR/Cas9 enhance PVY resistance in tobacco.

Tobacco is an important commercial crop and a rich source of alkaloids for pharmaceutical and agricultural applications. However, its yield can be reduced by up to 70% due to virus infections, especially by a potyvirus Potato virus Y (PVY). The replication of PVY relies on host factors, and eukaryotic translation initiation factor 4Es (eIF4Es) have already been identified as recessive resistance genes against potyviruses in many plant species. To investigate the molecular basis of PVY resistance in the widely cultivated allotetraploid tobacco variety K326, we developed a dual guide RNA CRISPR/Cas9 system for combinatorial gene editing of two clades, eIF4E1 (eIF4E1-S and eIF4E1-T) and eIF4E2 (eIF4E2-S and eIF4E2-T) in the eIF4E gene family comprising six members in tobacco. We screened for CRISPR/Cas9-induced mutations by heteroduplex analysis and Sanger sequencing, and monitored PVYO accumulation in virus challenged regenerated plants by DAS-ELISA both in T0 and T1 generations. We found that all T0 lines carrying targeted mutations in the eIF4E1-S gene displayed enhanced resistance to PVYO confirming previous reports. More importantly, our combinatorial approach revealed that eIF4E1-S is necessary but not sufficient for complete PVY resistance. Only the quadruple mutants harboring loss-of-function mutations in eIF4E1-S, eIF4E1-T, eIF4E2-S and eIF4E2-T showed heritable high-level resistance to PVYO in tobacco. Our work highlights the importance of understanding host factor redundancy in virus replication and provides a roadmap to generate virus resistance by combinatorial CRISPR/Cas9-mediated editing in non-model crop plants with complex genomes.

Plant-Derived Trimeric CO-26K-Equivalent Epitope Induced Neutralizing Antibodies Against Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea virus (PEDV) is a causative agent of a highly infectious disease with a high mortality rate, especially in newborn piglets in Asian countries resulting in serious economic loss. The development of a rapid, safe, effective and cost-efficient vaccine is crucial to protect pigs against PEDV infection. The COE antigen is regarded to be a major target for subunit vaccine development against PEDV infection. The naturally assembled COE protein forms a homotrimeric structure. In the present study, we successfully produced a trimeric COE protein as a native structure by fusion with the C-terminal isoleucine zipper trimerization (GCN4pII) motif in Nicotiana benthamiana, with a high expression level shown via semi-quantified Western blots. Trimeric COE protein was purified via immobilized metal affinity chromatography (IMAC), and its trimeric structure was successfully demonstrated by a cross-linking reaction, and a native PAGE gel. A crude extract containing the COE trimer was used for evaluating immunogenicity in mice. After 1 and 2 booster immunizations, the crude extract containing trimeric COE elicited elevated PEDV-specific humoral responses, as demonstrated by ELISA and Western blot analyses. Notably, a virus-neutralizing antibody assay indicated that the neutralization activities of sera of mice vaccinated with the crude extract containing COE-GCN4pII were similar to those of mice vaccinated with a commercial vaccine. These results suggest that crude extract containing trimeric COE is a promising plant-based subunit vaccine candidate for PEDV prevention.

A newly constructed Agrobacterium-mediated transformation system revealed the influence of nitrogen sources on the function of the LaeA regulator in Penicillium chrysogenum.

Penicillium chrysogenum is not only an industrially important filamentous fungus for penicillin production, but it also represents as a promising cell factory for production of natural products. Development of efficient transformation systems with suitable selection markers is essential for genetic manipulations in P. chrysogenum. In this study, we have constructed a new and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system with two different selection markers conferring the resistance to nourseothricin and phleomycin for P. chrysogenum. Under the optimized conditions for co-cultivation at 22 °C for 60 h with acetosyringone concentration of 200 µM, the transformation efficiency of the ATMT system could reach 5009 ± 96 transformants per 106 spores. The obtained transformants could be exploited as the T-DNA insertion mutants for screening genes involved in morphogenesis and secondary metabolism. Especially, the constructed ATMT system was applied successfully to generate a knockout mutant of the laeA regulatory gene and relevant complementation strains in a wild strain of P. chrysogenum. Our results indicated that the LaeA regulator controls growth, sporulation, osmotic stress response and antibiotic production in P. chrysogenum, but its function is reliant on nitrogen sources. Furthermore, we showed that the laeA orthologous genes from the citrus postharvest pathogen P. digitatum and from the industrial fungus Aspergillus niger could recover the phenotypic defects in the P. chrysogenum laeA deletion mutant. Conclusively, this work provides a new ATMT system, which can be employed for T-DNA insertional mutagenesis, heterologous gene expression or for molecular inspections of potential genes related to secondary metabolism in P. chrysogenum.