Lemon essential oil nanoemulsions: Potential natural inhibitors against Escherichia coli.

Lemon essential oil (LEO) is a common natural antibacterial substance, and encapsulating LEO into nanoemulsions (NEs) can improve their stability and broaden its application. Our study aimed to investigate the bacterial inhibitory effect of LEO-NEs against Escherichia coli (E. coli). Results showed that the minimum inhibitory concentration (MIC) of LEO-NEs was 6.25 mg/mL, and the time-kill curve showed that E. coli were significantly killed by LEO-NEs after 5 h of treatment at 1MIC. Flow-cytometry analysis showed that LEO-NEs adversely affected the cell-membrane depolarisation, cell-membrane integrity, and efflux pump function of E. coli. Confocal laser scanning microscopy demonstrated that 8MIC of LEO-NEs induced changes in the cell-membrane permeability and cell-wall integrity of E. coli. Proteomic results suggested that the mode of action LEO-NEs against E. coli was to enhance bacterial chemotaxis and significantly inhibit ribosomal assembly. They may also affect butyric acid, ascorbic acid and aldehyde metabolism, and sulphur-relay system pathways. In conclusion, LEO-NEs had potential application as a natural antibacterial agent for the control of E. coli in the food industry.

Identification of potential predictive biomarkers and biological pathways and the correction with immune infiltration in the activation of Crohn's disease.

Crohn's disease (CD), a subtype of inflammatory bowel disease (IBD), has increasing prevalence in the world. Due to the lack of cure strategy, most patients with CD develop progressive disease companying with a series of serious complications. Therefore, exploring molecular mechanism differences between active and inactive CD will help in the screening of predict markers and therapeutic targets. In this study, we analyzed differentially expressed genes (DEGs) and molecular pathways through between active and inactive CD patients. In addition, the abundance of 22 immune cell types were assessed by using the CIBERSORT. The hub DEGs were screened out by the CytoHubba in Cytoscape, followed by the least absolute shrinkage and selection operator (LASSO) regression. Finally, the clinical predictive model was constructed by binary logistic regression model. The diagnostic efficacy was tested by receiver operating characteristic (ROC) curve and verified in independent datasets. The results showed that there were 137 DEGs between the active and inactive CD. Most of them were involved in regulating the immunity process. In addition, the decreased abundance of CD8 T cells and the increased abundance of M0, M1 macrophages, and neutrophils were closely related to CD activation. CXCL9, C3AR1, IL1B, and TLR4 were the hub gene and can be applied to the prediction of CD activation. Our results provided important targets for the prediction of CD activation and the selection of therapeutic targets.

Molecular basis of P[II] major human rotavirus VP8* domain recognition of histo-blood group antigens.

Initial cell attachment of rotavirus (RV) to specific cell surface glycan receptors, which is the essential first step in RV infection, is mediated by the VP8* domain of the spike protein VP4. Recently, human histo-blood group antigens (HBGAs) have been identified as receptors or attachment factors for human RV strains. RV strains in the P[4] and P[8] genotypes of the P[II] genogroup share common recognition of the Lewis b (Leb) and H type 1 antigens, however, the molecular basis of receptor recognition by the major human P[8] RVs remains unknown due to lack of experimental structural information. Here, we used nuclear magnetic resonance (NMR) spectroscopy-based titration experiments and NMR-derived high ambiguity driven docking (HADDOCK) methods to elucidate the molecular basis for P[8] VP8* recognition of the Leb (LNDFH I) and type 1 HBGAs. We also used X-ray crystallography to determine the molecular details underlying P[6] recognition of H type 1 HBGAs. Unlike P[6]/P[19] VP8*s that recognize H type 1 HBGAs in a binding surface composed of an α-helix and a ß-sheet, referred as the "ßα binding site", the P[8] and P[4] VP8*s bind Leb HBGAs in a previously undescribed pocket formed by the edges of two ß-sheets, referred to as the "ßß binding site". Importantly, the P[8] and P[4] VP8*s retain binding capability to non-Leb type 1 HBGAs using the ßα binding site. The presence of two distinct binding sites for Leb and non-Leb HBGA glycans in the P[8] and P[4] VP8* domains suggests host-pathogen co-evolution under structural and functional adaptation of RV pathogens to host glycan polymorphisms. Assessment and understanding of the precise impact of this co-evolutionary process in determining RV host ranges and cross-species RV transmission should facilitate improved RV vaccine development and prediction of future RV strain emergence and epidemics.

Linalool Nanoemulsion Preparation, Characterization and Antimicrobial Activity against Aeromonas hydrophila.

Aeromonas hydrophila is one of the most important aquatic pathogens causing huge economic losses to aquaculture. Linalool, a vital ingredient of a variety of essential oils, was proved as a good antimicrobial agent in our previous studies. However, the low solubility and volatility of Linalool obstruct its application in the field of aquatic drugs. Thus, in this study, Linalool nano-emulsion (LN) was prepared to solve these obstructions. We investigated the physicochemical properties, antibacterial activity, and mode of action of LN against A. hydrophila. LN with different medium chain triglycerides (MCT) concentrations were prepared by ultrasonic method. The results showed that the emulsion droplet size of LN was the smallest when MCT was not added to the formulation. Nano-emulsions are usually less than 500 nm in diameter. In our study, LN in this formulation were spherical droplet with a diameter of 126.57 ± 0.85 nm and showed good stability. LN showed strong antibacterial activity, the MIC and MBC values were 0.3125% v/v and 0.625% v/v, respectively. The bacterial population decreased substantially at 1 × MIC of LN. LN exhibited disruptive effect on cell membranes by scanning electron microscope (SEM) and transmission electron microscope (TEM). The present study provided a formulation of Linalool nano-emulsion preparation. Moreover, the good antibacterial activity of LN showed in our study will promote the application of Linalool for the control and prevention of A. hydrophila in aquaculture.

Genome-Wide Identification and Expression Profile Analysis of WRKY Family Genes in the Autopolyploid Saccharum spontaneum.

WRKY is one of the largest transcription factor families in plants and plays important roles in the regulation of developmental and physiological processes. To date, the WRKY gene family has not been identified in Saccharum species because of its complex polyploid genome. In this study, a total of 294 sequences for 154 SsWRKY genes were identified in the polyploid Saccharum spontaneum genome and then named on the basis of their chromosome locations, including 13 (8.4%) genes with four alleles, 29 (18.8%) genes with three alleles and 41 (26.6%) genes with two alleles. Among them, 73.8% and 16.0% of the SsWRKY genes originated from segmental duplications and tandem duplications, respectively. The WRKY members exhibited conserved gene structures and amino acid sequences among the allelic haplotypes, which were accompanied by variations in intron sizes. Phylogenetic and collinearity analyses revealed that 27 SsWRKYs originated after the split of sorghum and Saccharum, resulting in a significantly higher number of WRKYs in sugarcane than in the proximal diploid species sorghum. The analysis of RNA-seq data revealed that SsWRKYs' expression profiles in 46 different samples including different developmental stages revealed distinct temporal and spatial patterns with 52 genes expressed in all tissues, four genes not expressed in any tissues and 21 SsWRKY genes likely to be involved in photosynthesis. The comprehensive analysis of SsWRKYs' expression will provide an important and valuable foundation for further investigation of the regulatory mechanisms of WRKYs in physiological roles in sugarcane S. spontaneum.

Norovirus GII.17 Natural Infections in Rhesus Monkeys, China.

Noroviruses are a leading viral cause of acute gastroenteritis among humans. During the 2014-15 epidemic season, norovirus GII.17 was detected in rhesus monkeys in China. Genetic, structural, and challenge studies revealed virus mutations and verified the infections. Thus, cross-species transmission may occur, and monkeys may be a virus reservoir.

Glycan Specificity of P[19] Rotavirus and Comparison with Those of Related P Genotypes.

The P[19] genotype belongs to the P[II] genogroup of group A rotaviruses (RVs). However, unlike the other P[II] RVs, which mainly infect humans, P[19] RVs commonly infect animals (pigs), making P[19] unique for the study of RV diversity and host ranges. Through in vitro binding assays and saturation transfer difference (STD) nuclear magnetic resonance (NMR), we found that P[19] could bind mucin cores 2, 4, and 6, as well as type 1 histo-blood group antigens (HBGAs). The common sequences of these glycans serve as minimal binding units, while additional residues, such as the A, B, H, and Lewis epitopes of the type 1 HBGAs, can further define the binding outcomes and therefore likely the host ranges for P[19] RVs. This complex binding property of P[19] is shared with the other three P[II] RVs (P[4], P[6], and P[8]) in that all of them recognized the type 1 HBGA precursor, although P[4] and P[8], but not P[6], also bind to mucin cores. Moreover, while essential for P[4] and P[8] binding, the addition of the Lewis epitope blocked P[6] and P[19] binding to type 1 HBGAs. Chemical-shift NMR of P[19] VP8* identified a ligand binding interface that has shifted away from the known RV P-genotype binding sites but is conserved among all P[II] RVs and two P[I] RVs (P[10] and P[12]), suggesting an evolutionary connection among these human and animal RVs. Taken together, these data are important for hypotheses on potential mechanisms for RV diversity, host ranges, and cross-species transmission. IMPORTANCE: In this study, we found that our P[19] strain and other P[II] RVs recognize mucin cores and the type 1 HBGA precursors as the minimal functional units and that additional saccharides adjacent to these units can alter binding outcomes and thereby possibly host ranges. These data may help to explain why some P[II] RVs, such as P[6] and P[19], commonly infect animals but rarely humans, while others, such as the P[4] and P[8] RVs, mainly infect humans and are predominant over other P genotypes. Elucidation of the molecular bases for strain-specific host ranges and cross-species transmission of these human and animal RVs is important to understand RV epidemiology and disease burden, which may impact development of control and prevention strategies against RV gastroenteritis.

Intranasal P particle vaccine provided partial cross-variant protection against human GII.4 norovirus diarrhea in gnotobiotic pigs.

UNLABELLED: Noroviruses (NoVs) are the leading cause of nonbacterial acute gastroenteritis worldwide in people of all ages. The P particle is a novel vaccine candidate derived from the protruding (P) domain of the NoV VP1 capsid protein. This study utilized the neonatal gnotobiotic pig model to evaluate the protective efficacies of primary infection, P particles, and virus-like particles (VLPs) against NoV infection and disease and the T cell responses to these treatments. Pigs either were vaccinated intranasally with GII.4/1997 NoV (VA387)-derived P particles or VLPs or were inoculated orally with a GII.4/2006b NoV variant. At postinoculation day (PID) 28, pigs either were euthanized or were challenged with the GII.4/2006b variant and monitored for diarrhea and virus shedding for 7 days. The T cell responses in intestinal and systemic lymphoid tissues were examined. Primary NoV infection provided 83% homologous protection against diarrhea and 49% homologous protection against virus shedding, while the P particle and VLP vaccines provided cross-variant protection (47% and 60%, respectively) against diarrhea. The protection rates against diarrhea are significantly inversely correlated with T cell expansion in the duodenum and are positively correlated with T cell expansion in the ileum and spleen. The P particle vaccine primed for stronger immune responses than VLPs, including significantly higher numbers of activated CD4+ T cells in all tissues, gamma interferon-producing (IFN-γ+) CD8+ T cells in the duodenum, regulatory T cells (Tregs) in the blood, and transforming growth factor ß (TGF-ß)-producing CD4+ CD25- FoxP3+ Tregs in the spleen postchallenge, indicating that P particles are more immunogenic than VLPs at the same dose. In conclusion, the P particle vaccine is a promising vaccine candidate worthy of further development. IMPORTANCE: The norovirus (NoV) P particle is a vaccine candidate derived from the protruding (P) domain of the NoV VP1 capsid protein. P particles can be easily produced in Escherichia coli at high yields and thus may be more economically viable than the virus-like particle (VLP) vaccine. This study demonstrated, for the first time, the cross-variant protection (46.7%) of the intranasal P particle vaccine against human NoV diarrhea and revealed in detail the intestinal and systemic T cell responses by using the gnotobiotic pig model. The cross-variant protective efficacy of the P particle vaccine was comparable to that of the VLP vaccine in pigs (60%) and to the homologous protective efficacy of the VLP vaccine in humans (47%). NoV is now the leading cause of pediatric dehydrating diarrhea, responsible for approximately 1 million hospital visits for U.S. children and 218,000 deaths in developing countries. The P particle vaccine holds promise for reducing the disease burden and mortality.

Association between smoking, smoking cessation and serum α-klotho levels among American adults: National Health and Nutrition Examination Survey.

α-klotho is an anti-aging protein. The correlation between smoking, smoking cessation and serum α-klotho levels remains controversial. The aim of this study was to investigate the association between smoking, smoking cessation and serum α-klotho levels. This cross-sectional study finally included 4877 participants, aged 40-79 years, who participated in the National Health and Nutrition Examination Survey studies from 2013 to 2016. Of these, 2312 (47.4%) were men and 894 (18.3%) were current smokers, and the mean age of the participants was 57.8±10.7 years. Multivariate linear regression modeling was used to assess the association between smoking, smoking cessation and serum α-klotho levels. After adjustment for multiple confounders, this study observed that smoking was negatively associated with serum α-klotho levels (ß: -58.3; 95% confidence interval CI: -82.0 to -34.6; p<0.001), whereas smoking cessation was positively associated with serum α-klotho levels (ß: 52.3; 95% CI: 24.1 to 80.6; p<0.001). In subgroup and interaction analyses, p-value for the interaction between smoking and race on serum klotho levels was found to be less than 0.001. The correlation between smoking, smoking cessation and serum α-klotho levels remained stable after propensity score matching (ß: -54.1; 95% CI: -81.5 to -26.7; p<0.001, ß: 54.8; 95% CI: 24.2 to 85.4; p<0.001). In a large sample population, the present study found that smoking, smoking cessation and serum α-klotho levels were associated in opposite directions.

Median infectious dose of human norovirus GII.4 in gnotobiotic pigs is decreased by simvastatin treatment and increased by age.

Human noroviruses (NoVs), a major cause of viral gastroenteritis, are difficult to study due to the lack of a cell-culture and a small-animal model. Pigs share with humans the types A and H histo-blood group antigens on the intestinal epithelium and have been suggested as a potential model for studies of NoV pathogenesis, immunity and vaccines. In this study, the effects of age and a cholesterol-lowering drug, simvastatin, on the susceptibility of pigs to NoV infection were evaluated. The median infectious dose (ID50) of a genogroup II, genotype 4 (GII.4) 2006b variant was determined. The ID50 in neonatal (4-5 days of age) pigs was ≤2.74×10(3) viral RNA copies. In older pigs (33-34 days of age), the ID50 was 6.43×10(4) but decreased to <2.74×10(3) in simvastatin-fed older pigs. Evidence of NoV infection was obtained by increased virus load in the intestinal contents, cytopathological changes in the small intestine, including irregular microvilli, necrosis and apoptosis, and detection of viral antigen in the tip of villi in duodenum. This GII.4 variant was isolated in 2008 from a patient from whom a large volume of stool was collected. GII.4 NoVs are continuously subjected to selective pressure by human immunity, and antigenically different GII.4 NoV variants emerge every 1-2 years. The determination of the ID50 of this challenge virus is valuable for evaluation of protection against different GII.4 variants conferred by NoV vaccines in concurrence with other GII.4 variants in the gnotobiotic pig model.

Spike protein VP8* of human rotavirus recognizes histo-blood group antigens in a type-specific manner.

Rotaviruses (RVs), an important cause of severe diarrhea in children, have been found to recognize sialic acid as receptors for host cell attachment. While a few animal RVs (of P[1], P[2], P[3], and P[7]) are sialidase sensitive, human RVs and the majority of animal RVs are sialidase insensitive. In this study, we demonstrated that the surface spike protein VP8* of the major P genotypes of human RVs interacts with the secretor histo-blood group antigens (HBGAs). Strains of the P[4] and P[8] genotypes shared reactivity with the common antigens of Lewis b (Le(b)) and H type 1, while strains of the P[6] genotype bound the H type 1 antigen only. The bindings between recombinant VP8* and human saliva, milk, or synthetic HBGA oligosaccharides were demonstrated, which was confirmed by blockade of the bindings by monoclonal antibodies (MAbs) specific to Le(b) and/or H type 1. In addition, specific binding activities were observed when triple-layered particles of a P[8] (Wa) RV were tested. Our results suggest that the spike protein VP8* of RVs is involved in the recognition of human HBGAs that may function as ligands or receptors for RV attachment to host cells.

Human pulmonary microvascular endothelial cells support productive replication of highly pathogenic avian influenza viruses: possible involvement in the pathogenesis of human H5N1 virus infection.

Highly pathogenic avian influenza (HPAI) H5N1 viruses continue to cause sporadic human infections with a high fatality rate. Respiratory failure due to acute respiratory distress syndrome (ARDS) is a complication among hospitalized patients. Since progressive pulmonary endothelial damage is the hallmark of ARDS, we investigated host responses following HPAI virus infection of human pulmonary microvascular endothelial cells. Evaluation of these cells for the presence of receptors preferred by influenza virus demonstrated that avian-like (α2-3-linked) receptors were more abundant than human-like (α2-6-linked) receptors. To test the permissiveness of pulmonary endothelial cells to virus infection, we compared the replication of selected seasonal, pandemic (2009 H1N1 and 1918), and potentially pandemic (H5N1) influenza virus strains. We observed that these cells support productive replication only of HPAI H5N1 viruses, which preferentially enter through and are released from the apical surface of polarized human endothelial monolayers. Furthermore, A/Thailand/16/2004 and A/Vietnam/1203/2004 (VN/1203) H5N1 viruses, which exhibit heightened virulence in mammalian models, replicated to higher titers than less virulent H5N1 strains. VN/1203 infection caused a significant decrease in endothelial cell proliferation compared to other subtype viruses. VN/1203 virus was also found to be a potent inducer of cytokines and adhesion molecules known to regulate inflammation during acute lung injury. Deletion of the H5 hemagglutinin (HA) multibasic cleavage site did not affect virus infectivity but resulted in decreased virus replication in endothelial cells. Our results highlight remarkable tropism and infectivity of the H5N1 viruses for human pulmonary endothelial cells, resulting in the potent induction of host inflammatory responses.

Predicting susceptibility to norovirus GII.4 by use of a challenge model involving humans.

BACKGROUND: GII.4 is the predominant norovirus genotype worldwide. Challenge models involving humans have shown the association of human histo-blood group antigens (HBGAs) and susceptibility to infection with Norwalk virus (GI.1 norovirus), but the association of HBGAs and infection with other noroviruses is based on results of epidemiological studies. We performed the first GII.4 challenge study involving humans and prospectively evaluated the relationship between HBGAs and norovirus infection and associated illness. METHODS: Forty healthy adults (23 secretors and 17 nonsecretors of HBGAs) were challenged with 5 10(4) reverse-transcription polymerase chain reaction (RT-PCR) units of GII.4 norovirus. Subjects were assessed daily for clinical illness, and stool specimens were evaluated for norovirus by RT-PCR. Infection was defined by detection of norovirus and/or seroconversion to GII.4 antibody. RESULTS: Of the 23 secretors, 16 (70%) were infected with norovirus, 13 (57%) became ill (characterized by vomiting and/or diarrhea), and 12 (52%) developed norovirus-associated illness. In contrast, only 1 nonsecretor (5.9%) became ill, and another nonsecretor shed virus for a single day (P < .001 for each variable, compared with secretors). Infection occurred in secretors regardless of ABO blood group. Illness was mild to moderate in severity and lasted 1-3 days. CONCLUSIONS: Secretor status determined the susceptibility to norovirus GII.4 challenge. This human challenge model should be useful for evaluating norovirus vaccines and antiviral agents. Clinical trials registration. NCT01322503.

Linalool controls the viability of Escherichia coli by regulating the synthesis and modification of lipopolysaccharide, the assembly of ribosome, and the expression of substrate transporting proteins.

Escherichia coli (E. coli) is a Gram-negative bacterium and some pathogenic types may cause serious diseases, foods or food environments were the primary routes for its infection. Citrus aurantium L. var. amara Engl., a variety of sour orange, were used as a kind of non-conventional edible plant in China, but its antimicrobial activity and mechanisms were not well studied. Thus, in this study, EO from the flower of Citrus aurantium L. var. amara Engl. (CAEO) were studied as a kind of natural antimicrobial agent to control E. coli, our results showed that both of CAEO and its main component (linalool) exhibited strong antibacterial efficacy. Further, transcriptomic and proteomic analysis were carried out to explore cell response under linalool treatment and the main results included: (1) The synthesis and modification of lipopolysaccharide (LPS) was significantly influenced. (2) Ribosomal assembly and protein synthesis were significantly inhibited. (3) The expression of proteins related to the uptake of several essential substances was significantly changed. In all, our results would supply a theoretical basis for the proper use of CAEO and linalool as a promising antimicrobial agent to prevent and control E. coli infection in the future.

Norovirus P particle, a novel platform for vaccine development and antibody production.

The norovirus P particle is an octahedral nanoparticle formed by 24 copies of the protrusion (P) domain of the norovirus capsid protein. This P particle is easily produced in Escherichia coli, extremely stable, and highly immunogenic. There are three surface loops per P domain, making a total of 72 loops per particle, and these are potential sites for foreign antigen presentation for immune enhancement. To prove this concept, a small peptide (His tag, 7 amino acids [aa]) and a large antigen (rotavirus VP8, 159 aa) were inserted into one of the loops. Neither insertion affects P particle formation, while both antigens were presented well on the P particle surface. The immune-enhancement effect of the P particle was demonstrated by significantly increased antibody titers induced by the P particle-presented antigens compared to the titers induced by free antigens. In addition, the measured neutralization antibody titers and levels of protection against rotavirus shedding in mice immunized with the VP8 chimeric P particles were significantly higher than those of mice immunized with the free VP8 antigen. Sera from P particle-VP8 chimera-vaccinated animals also blocked norovirus virus-like particle (VLP) binding to the histo-blood group antigen (HBGA) receptors. From these data, the P particle appears to be an excellent vaccine platform for antigen presentation. The readily available three surface loops and the great capacity for foreign antigen insertion make this platform attractive for wide application in vaccine development and antibody production. The P particle-VP8 chimeras may serve as a dual vaccine against both rotavirus and norovirus.

Effect of grape seed extract on human norovirus GII.4 and murine norovirus 1 in viral suspensions, on stainless steel discs, and in lettuce wash water.

The anti-norovirus (anti-NoV) effect of grape seed extract (GSE) was examined by plaque assay for murine norovirus 1 (MNV-1), cell-binding reverse transcription-PCR for human NoV GII.4, and saliva-binding enzyme-linked immunosorbent assay for human NoV GII.4 P particles, with or without the presence of interfering substances (dried milk and lettuce extract). GSE at 0.2 and 2 mg/ml was shown to reduce the infectivity of MNV-1 (>3-log PFU/ml) and the specific binding ability of NoV GII.4 to Caco-2 cells (>1-log genomic copies/ml), as well as of its P particles to salivary human histo-blood group antigen receptors (optical density at 450 nm of >0.8). These effects were decreased as increasing concentrations of dried milk (0.02 and 0.2%) or lettuce extract were added. Under an electron microscope, human NoV GII.4 virus-like particles showed inflation and deformation after treatment with GSE. Under conditions that simulated applications in the food industry, the anti-NoV effect of GSE using MNV-1 as a target organism was shown to be limited in surface disinfection (<1-log PFU/ml, analyzed in accordance with EN 13697:2001). However, a 1.5- to 2-log PFU/ml reduction in MNV-1 infectivity was noted when 2 mg of GSE/ml was used to sanitize water in the washing bath of fresh-cut lettuce, and this occurred regardless of the chemical oxygen demand (0 to 1,500 mg/ml) of the processing water.

Tannic acid inhibited norovirus binding to HBGA receptors, a study of 50 Chinese medicinal herbs.

Noroviruses (NoVs) are the leading cause of viral acute gastroenteritis affecting people of all ages worldwide. The disease is difficult to control due to its widespread nature and lack of an antiviral or vaccine. NoV infection relies on the interaction of the viruses with histo-blood group antigens (HBGAs) as host receptors. Here we investigated inhibition effects of Chinese medicinal herbs against NoVs binding to HBGAs for potential antivirals against NoVs. Blocking assays was performed using the NoV protrusion (P) protein as NoV surrogate and saliva as HBGAs. Among 50 clinically effective Chinese medicinal herbs against gastroenteritis diseases, two herbs were found highly effective. Chinese Gall blocked NoV P dimer binding to type A saliva at IC(50)=5.35 µg/ml and to B saliva at IC(50)=21.7 µg/ml. Similarly, Pomegranate blocked binding of NoV P dimer to type A saliva at IC(50)=15.59 µg/ml and B saliva at IC(50)=66.67 µg/ml. Literature data on preliminary biochemistry analysis showed that tannic acid is a common composition in the extracts of the two herbs, so we speculate that it might be the effective compound and further studies using commercially available, highly purified tannic acid confirmed the tannic acid as a strong inhibitor in the binding of NoV P protein to both A and B saliva (IC(50)≈0.1 µM). In addition, we tested different forms of hydrolysable tannins with different alkyl esters, including gallic acid, ethyl gallate, lauryl gallate, octyl gallate and propyl gallate. However, none of these tannins-derivatives revealed detectable inhibiting activities. Our data suggested that tannic acid is a promising candidate antiviral against NoVs.

Preparation, Characterization, and Antioxidant Activity of Nanoemulsions Incorporating Lemon Essential Oil.

Lemon essential oil (LEO) is a kind of citrus essential oil with antioxidant, anti-inflammatory, and antimicrobial activities, but low water solubility and biological instability hinder its industrial application. In this study, LEO was nanoemulsified to solve these problems. The preparation procedure of lemon essential oil nanoemulsions (LEO-NEs) was optimized, and the physicochemical characterization and antioxidant activities were explored. Single-factor experiments (SFEs) and response surface methodology (RSM) were conducted for the effects on the mean droplet size of LEO-NEs. Five factors of SFE which may influence the droplet size were identified: HLB value, concentration of essential oil, concentration of surfactant, ultrasonic power, and ultrasonic time. On the basis of the SFE, the RSM approach was used to optimize the preparation procedure to obtain LEO-NEs with the smallest droplet size. LEO-NEs exhibited good antioxidant activity when the HLB value was 13, content of surfactant was 0.157 g/mL, ultrasonic time was 23.50 min, and ultrasonic power was 761.65 W. In conclusion, these results can provide a good theoretical basis for the industrial application of lemon essential oil.

Essential Oils From Citrus unshiu Marc. Effectively Kill Aeromonas hydrophila by Destroying Cell Membrane Integrity, Influencing Cell Potential, and Leaking Intracellular Substances.

Aeromonas hydrophila is one of the important pathogenic bacteria in aquaculture causing serious losses every year. Essential oils are usually used as natural antimicrobial agents to reduce or replace the use of antibiotics. The aim of this study was to evaluate the antibacterial activity and explore the mechanisms of essential oil from satsuma mandarin (Citrus unshiu Marc.) (SMEO) against A. hydrophila. The results of the gas chromatography-mass spectrometer demonstrated that SMEO contains 79 chemical components with the highest proportion of limonene (70.22%). SMEO exhibited strong antibacterial activity against A. hydrophila in vitro, the diameter of the inhibition zone was 31.22 ± 0.46 mm, and the MIC and MBC values were all 1% (v/v). Intracellular material release, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and flow cytometry analysis revealed the dynamic antibacterial process of SMEO, the morphological changes of bacterial cells, and the leakage process of intracellular components. These results demonstrated that SMEO disrupted the extracellular membrane permeability. Our study demonstrated that SEMO has the potential to be used to control and prevent A. hydrophila infections in aquaculture.

Genetic and phenotypic characterization of GII-4 noroviruses that circulated during 1987 to 2008.

The predominance and continual emergence of new variants in GII-4 noroviruses (NVs) in recent years have raised questions about the role of host immunity and histo-blood group antigens (HBGAs) in NV evolution. To address these questions, we performed a genetic and phenotypic characterization of GII-4 variants circulating in the past decade (1998 to 2008). Ninety-three GII-4 sequences were analyzed, and of them, 16 strains representing 6 genetic clusters were selected for further characterization. The HBGA binding properties were determined by both saliva- and oligosaccharide-binding assays using P particles as a model of NV capsid. The antigenic properties were also examined by enzyme immunoassay (EIA), Western blot analysis, and receptor blocking assay, using P-particle-specific antibodies from immunized mice and GII-4 virus-infected patients. Our results showed that 15 of the 16 GII-4 viruses bound to saliva of all A, B, and O secretors. Oligosaccharide binding assays yielded largely consistent results, although the binding affinities to some oligosaccharides varied among some strains. The only nonbinder had a mutation in the binding site. While antigenic variations were detected among the 16 strains, significant cross-blocking on the HBGA binding was also noted. Sequence alignment revealed high conservation of HBGA binding interfaces with some variations in adjacent regions. Taken together, our data suggested that the ability of GII-4 to recognize different secretor HBGAs persisted over the past decade, which may explain the predominance of GII-4 over other genotypes. Our data also indicated that both the host immunity and HBGAs play a role in NV evolution. While host immunity may continue driving NV for antigenic change, the functional selection by the HBGAs tends to lock the architecture of the capsid/HBGA interfaces and allows only limited variations outside the HBGA binding sites. A potential outcome of such counterselection between theses two factors in NV evolution is discussed.