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Infection by multidrug-resistant avian pathogenic Escherichia coli (APEC) in chickens always leads to the uselessness of antibiotics, highlighting the need for alternative antibacterial agents. Sophora flavescens and Coptis chinensis have been a classical combination used together in Traditional Chinese Medicine (TCM) formulas to treat diseases with similar symptoms to colibacillosis for an extended period, but the effect of their active ingredients' combination on APEC infection remains unstudied. The objective of this study was to explore the synergistic effect of matrine and berberine hydrochloride on colibacillosis caused by an isolated multidrug-resistant APEC. In this study, a highly pathogenic E. coli was isolated from the liver of a diseased chicken in a farm suspected of colibacillosis, and it was resistant to multiple antibiotics. The LD50 of the strain was approximately 3.759×108 CFU/mL. The strain harbored several antibiotic resistance genes and virulence genes. Matrine and berberine hydrochloride have synergistic antibacterial effect against the isolated strain in vitro. The combined use of matrine and berberine hydrochloride exhibited synergistic effects in the treatment of APEC infection by regulating the organ indices, improving the pathological situation, decreasing the bacterial load, and regulating the inflammatory factors to enhance the survival rate of chickens in vivo. These results provided a foundation for revealing the effective effects and possible mechanisms of matrine and berberine hydrochloride as potential antimicrobial agents on diseases caused by multidrug-resistant APEC in chickens.

ABSTRACT: Duck hepatitis A virus 1 (DHAV-1) is the primary cause of duck viral hepatitis, leading to sudden mortality in ducklings and significant economic losses in the duck industry. However, little is known about how DHAV-1 affects duckling liver at the molecular level. We conducted an analysis comparing the expression patterns of mRNAs and miRNAs in DHAV-1-infected duckling livers to understand the underlying mechanisms and dynamic changes. We identified 6,818 differentially expressed mRNAs (DEGs) and 144 differentially expressed microRNAs (DEMs) during DHAV-1 infection. Functional enrichment analysis of DEGs and miRNA target genes using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed their potential involvement in innate antiviral immunity, mitophagy, and pyroptosis. We constructed coexpression networks of mRNA-miRNA interactions and confirmed key DEMs (novel-mir333, novel-mir288, novel-mir197, and novel-mir71) using RT-qPCR. Further investigation demonstrated that DHAV-1 activates the RLRs signaling pathway, disrupts mitophagy, and induces pyroptosis. In conclusion, DHAV-1-induced antiviral immunity is closely linked to mitophagy, suggesting it could be a promising therapeutic target.

BackgroundTraditional Chinese medicine (TCM) is widely used in humans and animals, which is very important for health. TCM affects the body ‘s immunity and changes in intestinal flora. This study was conducted to investigate the effects of dietary Hong-bailanshen (HBLS) supplementation in horses on serum biochemical profile, antioxidant enzymes and gut microbiota.MethodsIn this study, five horses were selected. On day 0, 14, 28, blood samples and feces were collected on days 0, 14, and 28 to analyse gut microbiota, serum biochemical and redox indexes.ResultsThe results showed that the addition of HBLS to horse diets significantly decreased the level of alanine aminotransferase, alkaline phosphatase, creatine kinase and malondialdehyde (p < 0.05, p < 0.01) and significantly increased the activity of total antioxidant capacity, superoxide dismutase and catalase (p < 0.05, p < 0.01). Compared with day 14, the levels of alanine aminotransferase, alkaline phosphatase and creatine kinase were significantly decreased; however, the level of catalase was significantly increased in the horses continuously fed with HBLS for 28 days (p < 0.05, p < 0.01). Alpha diversity analysis was performed that chao1 (p < 0.05), observed_specicies, faith’pd and goods_coverage upregulated in the horses fed HBLS. A total of 24 differential genera were detected adding HBLS to diet increased the abundance of Bacillus, Lactobacillaceae, Leuconostocaceae, Christensenellaceae, Peptostreptococcaceae, Faecalibacterium, Erysipelotrichaceae, Pyramidobacter, Sphaerochaeta, WCHB1-25, Bacteria, Oscillospira, and Acetobacteraceae, while reduced Aerococcus, EtOH8, Syntrophomonas, Caulobacter, Bradyrhizobiaceae, W22, Succinivibrionaceae, and Desulfovibrio (p < 0.05, p < 0.01).ConclusionAdding HBLS to the diet could be a potentially effective strategy to improve horses’ health.

ABSTRACT: The modified rougan decoction (MRGD) compound formula has been proven a certain ability to relieve lipopolysaccharide-enrofloxacin (LPS-ENR)-induced liver oxidant injury in chickens. Recent advances have shown that mitochondrial dysfunction affects the development of many diseases, leading to increased interest in exploring its effects. Using LPS-ENR-injured in vivo and in vitro to further evaluate the effects of MRGD on mitochondrial structure and function, and emphasized further investigation of its molecular mechanism. After LPS-ENR treatment, the levels of inflammation and apoptosis markers were increased, along with higher mitochondrial injury. Results showed that MRGD reduced inflammatory factors expression and inhibited the nuclear translocation of NF-κB P65, reducing the inflammatory response in vivo and in vitro. Additionally, MRGD pretreatment inhibited mitochondrial dysfunction, mitochondrial oxidative stress, and mitochondrial pathway apoptosis by maintaining mitochondrial structure and function. Moreover, treatment with the inhibitor EX527 showed that MRGD promoted mitochondrial biogenesis ability through the SIRT1/PGC-1α pathway and interfered with mitochondrial dynamics, and activate Nrf2. In summary, MRGD played a key role in promoting mitochondrial function and thus alleviating hepatocyte apoptosis in vivo and in vitro at least in part.

Abstract Staphylococcus saprophyticus is frequently involved in various difficult-to-treat infections due to the formation of biofilms. To identify useful antibiofilm strategies, this study explored the efficacy and mechanism of baicalin in enhancing the ability of azithromycin against multidrug-resistant Staphylococcus saprophyticus-Liu-2016-Liyang, China-francolin (MDRSS) biofilms in vitro and in vivo. When azithromycin was used in combination with baicalin, the minimum inhibitory concentration in biofilm (MICB) for azithromycin decreased 4- to 512-fold. Compared with the azithromycin and baicalin groups, the combination of azithromycin and baicalin could not reduce the biofilm biomass, but the dispersion rates of biofilm were decreased and the bactericidal ability was increased. Furthermore, the relative transcript levels of WalK/R system-related genes were upregulated by the addition of baicalin or azithromycin plus baicalin compared with that of the azithromycin and blank control groups. The strong correlation relationship between the WalK/R system and the bactericidal index demonstrated that baicalin enhanced the bactericidal effect of azithromycin on MDRSS biofilms by modulating the WalK/R system. In the mouse cutaneous infection model, the combination of azithromycin and baicalin succeeded in eradicating MDRSS and decreasing pathological injuries. This study indicated that baicalin has the potential to be an adjuvant to enhance the antimicrobial activity of azithromycin against MDRSS in the biofilm form by modulating the WalK/R system.

ABSTRACT: Liver injury plays a heavy burden on the chicken industry. Although modified rougan decoction is a prescription for the treatment of liver disease based on the classical prescription of rougan decoction (containing peony and licorice). However, the effect and mechanism of modified rougan decoction on the liver remain unclear. In this study, the effects of the water extracts (MRGD) and the alcohol precipitates of water extracts (MRGDE) against lipopolysaccharide-enrofloxacin (LPS-ENR)-induced hepatotoxicity were discussed in vivo and in vitro. The isolated hepatocytes and 128 one-day-old Hyline chickens were considered research objects. The indices of liver injury and oxidative stress were evaluated by hematoxylin and eosin (H&E) stained and the assay kits, and the nuclear erythroid 2-related factor 2 (Nrf2)/antioxidant responsive element (ARE) pathway was detected by the RT-PCR, western blot, and immunofluorescence tests. All data were analyzed using the IBM SPSS 20.0 software. In vivo, the structural integrity of the liver was maintained, AST, ALT, and MDA levels were decreased, and antioxidant enzymes were increased, confirming that the oxidative stress was reduced and liver injury was alleviated. Correspondingly, MRGD and MRGDE were observed to improve cell viability and decrease lactate dehydrogenase (LDH) in vitro, and the cell oxidative damage was reduced. In addition, the nuclear translocation of Nrf2 was improved significantly, and the mRNA and protein expression levels of the related genes were upregulated. In conclusion, MRGD and MRGDE can exert a protective effect against LPS-ENR-induced hepatotoxicity by activating the Nrf2/ARE pathway, which might be a potential therapeutic prescription for preventing or treating liver injury. Notably, no significant difference was found between the 2 extracts, suggesting that a depth extraction method did not always improve the efficacy of natural medicine. Our results provided new insights into finding effective hepatoprotective medicine.

ObjectivesThe near-miss of two ships is a special situation that ships will encounter, and it can be modeled in terms of the time-dimension perspective for evaluating potential maritime traffic risks.Methods In this paper, a temporal near-miss model is first established using the Fujii ship domain to detect near-miss situations, and the near-miss duration and average duration are then defined. An algorithm for calculating near-miss duration is proposed on the basis of the time synchronization processing of automatic identification system (AIS) data. Furthermore, the near-miss duration is used to analyze the time-dimension characteristics of maritime traffic risks. The Bohai Sea area is used as the research example to verify the model.ResultsThe correlations between the average near-miss duration and ship type and length are determined. According to ship type analysis, the longest average near-miss duration is 252.63 s for an oil tanker and at least 180 s for a passenger ship. According to ship size analysis, the longest average near-miss duration is 272.65 s for a large ship and at least 180 s for a mid-size ship.ConclusionsThe proposed model can be used to accurately calculate near-miss duration, providing a new approach to studying maritime traffic risks from the time-dimension perspective.

A component of licorice polysaccharide (GPS-1) was extracted from licorice, its primary structure was identified and characterized for the first time, and its immunomodulatory activity was studied. Crude licorice polysaccharide was isolated and purified by DEAE sepharose FF ion-exchange column chromatography and Chromdex 200 PG gel filtration column chromatography to obtain a purified Glycyrrhiza polysaccharide named GPS-1. NMR and methylation analysis revealed that GPS-1 is composed of homogalacturonan (HG)-type pectin with 4)-D-GalpA-(1 as the backbone. This study of GPS-1 also examined its significant role in regulating immune activity in vitro and in vivo. As a result, GPS-1 promoted the secretion of IFN-γ and IL-4 in mice and increased the proportion of CD3+CD4+ and CD3+CD8+ T lymphocytes in their spleens. Dendritic cells (DCs) treated with GPS-1 showed promotion of DC maturation, antigen presentation, and phagocytic capacity. The results suggest that GPS-1 is a potential immunomodulator that stimulates the immune system by regulating multiple signaling pathways. Combined with our characterization of the primary structure of GPS-1, the present investigation provides the basis for future study of the form-function relationship of polysaccharides.

Escherichia coli is one of the most common pathogenic bacteria in diarrheal chickens, leading to serious economic losses in the poultry industry. The limited effect of antibiotics on antibiotic-resistant E. coli makes this bacterium a potential threat to human health. Yujin powder (YJP) has been reported as an agent that releases the symptoms caused by E. coli for a long time. The objective of this study is to investigate the effect of Yujin powder (YJP) and its components, Scutellariae Radix (SR) and Baicalin (Bac), anti-against multi-drug-resistant E. coli in vitro and in vivo. A multi-drug-resistant bacteria was isolated and identified from a clinical diarrheal chick. Then, the anti-bacterial effects of drugs were assessed in vitro and in vivo by analyzing the bacteria loads of organs, the levels of endotoxin, TNF-α, IL-1β, and IL-6 of the serum. Results found that the pathogenic E. coli was resistant to 19 tested antibiotics. YJP, SR, and Bac could directly inhibit the growth of this strain at high concentrations in vitro, and presents obvious anti-bacterial effects by reducing the bacterial loads, the release of endotoxin, and inflammation in vivo, which was much more effective than the resistant antibiotic ciprofloxacin. This study demonstrates that those natural medicines have the potential to be used as novel treatments to treat the disease caused by this isolated MDREC strain.

Duck hepatitis A virus type 1 (DHAV) infection causes duck viral hepatitis and results in enormous loss to poultry farming industry. We reported that phosphorylated Codonopsis pilosula polysaccharide (pCPPS) inhibited DHAV genome replication. Here we further explored its underlying antiviral mechanisms. Autophagosomes formation is essential for the genome replication of picornaviruses. In this study, Western blot, confocal microscopy observation, and ELISA methods were performed to analyze polysaccharides' effects on autophagy by the in vitro and in vivo experiments. Results obtained from in vitro and in vivo experiments showed that Codonopsis pilosula polysaccharide did not play a role in regulating autophagy and had no therapeutic effects on infected ducklings. However, pCPPS treatment downregulated LC3-II expression level activated by DHAV and rapamycin, indicating the inhibition of autophagosomes formation. The interdiction of autophagosomes formation resulted in the inhibition of DHAV genome replication. Further study showed that pCPPS treatment reduced the concentration of phosphatidylinositol-3-phosphate (PI3P), an important component of membrane, in cells and serum, and consequently, autophagosomes formation was downregulated. In vivo experiments also verified the therapeutic effect of pCPPS. Phosphorylated Codonopsis pilosula polysaccharide treatment increased the infected ducklings' survival rate and alleviated hepatic injury. Our studies verified the effects of pCPPS against DHAV infection in duck embryo hepatocytes and ducklings and confirmed that phosphorylated modification enhanced the bioactivities of polysaccharides. The results also stated pCPPS's antiviral mechanisms, provided fundamental basis for the development of new anti-DHAV agents.

It is an effective method to analyze the maritime traffic risk using near miss model, but there are no works from the temporal perspective. The duration of near miss shows the maritime traffic risk. The longer the duration is, the higher the risk is. The influence of the ship maneuvering capability, speed, size, and Convention on the International Regulations for Preventing Collisions at Sea (COLREGS) on the safety of ship navigation are considered in this article, the navigation safety domain is used to detect near misses, and the duration of a single near miss is defined, which is the time from target ship sailing into the navigation safety domain of own ship to leaving, and then an algorithm for calculating duration of near misses is proposed. To measure the similarity of two ships' lengths, the similarity coefficient model of ships' lengths is defined. Based on the k -means algorithm, different parameters k are selected to cluster the duration of near misses. According to the clustering results, the connections between the duration of near misses and the ship type, length, and speed were analyzed to identify a series of time characteristics of near misses, that is high frequencies of short-duration near misses and the strong connections between the long-duration near misses, ship length, and ship speed. The method is first proposed for doing research of near misses in temporal dimension. The findings of this article help researchers and maritime traffic management agencies to better understand the importance of maritime traffic risk from the temporal perspective.

An electronic product like a smartphone might involve hundreds of thousands of patents. The risks caused by infringement make it necessary for enterprises to consider patent licensing when outsourcing production activities in the electronics industry. This paper studies the outsourcing strategy considering patent licensing in an electronic product supply chain composed of a patent-holding firm, an original equipment manufacturer (OEM) and a contract manufacturer (CM). In addition to outsourcing manufacturing to the CM, OEM also considers outsourcing the patent license obtaining as well. We construct two outsourcing modes, called Turnkey and Consignment. No matter under which outsourcing mode, the patent-holding firm will charge unit royalty and a fixed fee paid in a lump sum. The results show that the OEM tends to choose Consignment without considering fixed fees, while the CM prefers Turnkey. When considering fixed fees, the patent-holding firm could align their preferences by coordinating fixed fees borne by OEM and CM. What’s more, considering the environmental pollution caused by the production of electronic products, both strategies may lead to higher social welfare than the other strategy.

In recent years, the efficacy of antibiotics has been threatened by the evolution of bacterial resistance. We previously demonstrated that baicalin (Bac) showed synergies with azithromycin (Azm) against Azm-resistant Staphylococcus saprophyticus (ARSS). The aim of this study was to explore the roles of Bac in reversing the resistance of ARSS to Azm. The ARSS was sequentially passaged for 20 days with the sub-MIC (minimum inhibitory concentration) of Bac. The strain that recovered sensitivity to Azm was named Azm-sensitive S. saprophyticus (ASSS). The sub-MIC of Bac reversed the resistance of ARSS to Azm. The MIC of Azm against the ASSS strain was 0.488 mg/l, and it was stable within 20 passages. The highest rate of resistance reversal reached 3.09% after ARSS was exposed to 31.25 mg/l Bac for 20 days. Furthermore, semiquantitative biofilm and RT-PCR assays reflected that the ability of biofilm formation and the transcript levels of msrA, mphC, and virulence-associated genes in the ASSS strain were significantly lower than those of the ARSS strain (p < 0.05). Simultaneously, Azm delayed the start time of death, alleviated the injury of the kidney, and decreased the bacterial burden in organs and cytokine levels in mice infected with ASSS. In contrast, Azm did not have a good therapeutic effect on mice infected with ARSS. Therefore, Bac has the potential to be an agent that reversed the resistance of ARSS to Azm.

ABSTRACT: Licorice is a medicinal and food plant widely used to treat diseases and produce food additives, because of its unique chemical constituents like polysaccharides, flavones, and saponins. Glycyrrhiza Polysaccharides (GPS-1) are water-soluble neutral polysaccharides extracted from licorice. Currently, GPS-1 is administrated to chickens by gavage every d for 14 d to observe the impact of GPS-1 on the Newcastle disease vaccine. To determine the immunity of these chickens to NDV, blood serum levels of hemagglutinin-inhibition (HI) antibody, and immunoglobulins IgA and IgG were measured. Meanwhile, the expression levels of cytokines IL-2, IL-4, IL-17, and IFN-γ were measured to evaluate the degree of immune booster activity. The chickens’ spleen and peripheral blood lymphocytes displayed a significant increase in the proportion of CD4+ and CD8+ T cells after booster treatments with GPS-1. The results indicated that GPS-1 had a significant, dose-dependent, immune-boosting effect which could enhance NDV vaccine immunity in chickens.

BackgroundGlucose oxidase is widely used as a livestock feed additive owing to its beneficial effects on growth performance and antioxidant activity. However, little is known about the effects of the enzyme on intestinal health.MethodsTo investigate the effects of glucose oxidase supplementation on the growth performance, intestinal function, and microbiota composition of broilers fed moldy corn, newly hatched Arbor Acres broilers were each randomly assigned to one of four groups, which were fed a basal diet (CON), a contaminated diet (10% moldy corn) (MC), a basal diet supplemented with 0.01% glucose oxidase (GOD), or a contaminated diet supplemented with 0.01% glucose oxidase (MCG).ResultsWe found that the average weight gain (ADG) of the MC group was significantly lower than those of the CON and GOD groups, and there were no significant differences in ADG between the MCG group and the CON and GOD groups. Intestinal morphology results revealed irregularly arranged villi and microvilli in the ilea from the MC group, whereas those from the other three groups were aligned regularly. Tight-junction protein analysis showed that both ZO-1 expression and claudin-4 expression in the MC group were significantly lower than those in the other groups. Inflammation cytokines analysis showed lower serum concentration of interleukin-10, as well as its mRNA expression in the ileum of the MC group, when compared with those of the other groups. Additionally, we observed lower glutathione peroxidase and total superoxide dismutase activity and higher malonaldehyde concentration in the MC group than those in the MCG group. The α and β diversity of microbiota profiling indicated that the cecal microbiota in the MC group differed from those in the other three groups.ConclusionThe results indicated that glucose oxidase supplementation was able to prevent the adverse effects from mycotoxin exposure on growth performance, antioxidant activity, inflammatory response, intestinal function, and microbiota composition in broilers. We suggested that glucose oxidase supplementation can be used in broilers to mitigate the adverse effects of moldy feed, and its benefits are due to its effect on intestinal microbiota composition.

Abstract Background Duck viral hepatitis (DVH) is an acute disease of young ducklings with no effective veterinary drugs for treatment. Gynostemma pentaphyllum is a well-known traditional Chinese medicine that plays an important role in the treatment of various diseases. Gypenoside (GP), one of the main ingredients of Gynostemma pentaphyllum, was reported with good hepatoprotective effects. However, its low solubility limits its application in the clinics. To improve its solubility and bioactivity, a phosphorylated derivative of gypenoside (pGP) was prepared by the sodium trimetaphosphate-sodium tripolyphosphate (STMP-STPP) method. An infrared spectroscopy method was applied to analyse the structures of GP and pGP. Then, a methyl thiazolyl tetrazolium (MTT) colorimetric assay was applied to study the hepatocyte protective efficacy of these two drugs against duck hepatitis A virus type 1 (DHAV-1) infection, and qPCR, TUNEL labelling and flow cytometry methods were used to study the relevant hepatocyte protective in vitro. Results The infrared spectroscopy detection results showed that the phosphorylation modification of GP was successful. The MTT colorimetric assay results showed that both GP and pGP possessed good hepatocyte protective efficacy in vitro, and pGP performed better than GP when the drug was added before or after virus inoculation. Furthermore, the qPCR results revealed that both drugs could effectively inhibit the adsorption (when adding GP and pGP pre-virus inoculation), replication and release of DHAV-1, and the viral inhibition rate of pGP was greater than that of GP. The subsequent TUNEL labelling and flow cytometry assays showed that both GP and pGP could significantly inhibit duck embryo hepatocyte apoptosis induced by DHAV-1, and the inhibition effect of pGP was much stronger than that of GP. Conclusions GP exerts good hepatocyte protective efficacy not only by inhibiting the proliferation of DHAV-1 but also by inhibiting duck embryonic hepatocyte apoptosis induced by DHAV-1, and phosphorylation modification significantly improves the antiviral and the anti-apoptotic effects of GP. Therefore, pGP has the potential to be developed into a novel drug against DHAV-1 infection.

Duck hepatitis A virus type 1 (DHAV-1) is the main pathogen of duck viral hepatitis, but the efficacy of the licensed commercial vaccine needs to be further improved. Therapeutic measures of specific drugs for DHAV-1-infected ducklings need to be urgently developed. Baicalin possesses good antiviral effects. This study aims to investigate the mechanism of baicalin in protecting hepatic mitochondrial function from DHAV-1. The ELISA method was used to detect changes of hepatic and mitochondrial catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPX), inducible nitric oxide synthase (iNOS), adenosine triphosphate (ATP), and malondialdehyde (MDA) levels in vivo and vitro. Hematoxylin and eosin sections and transmission electron microscopy were used to observe liver pathological changes and mitochondrial structural changes. The changes in mitochondrial membrane potential were detected by JC-1 staining method. Western blot and quantitative real-time PCR were employed to analyze the gene and protein expressions in the nuclear erythroid 2-related factor 2 (Nrf2)/antioxidant responsive element (ARE) pathway in duck embryonic hepatocytes infected with DHAV-1. Results showed the administration of baicalin increased the survival rate of ducklings, and alleviated hepatic damage caused by DHAV-1 by enhancing the antioxidant enzyme activities of the liver and mitochondria, including SOD, GPX, CAT, and reducing lipid peroxidative damage (MDA content) and iNOS activities. The mitochondrial ultrastructure changed and the significant increase of ATP content showed that baicalin maintained the structural integrity and ameliorated mitochondrial dysfunction after DHAV-1 infection. In vitro, DHAV-1 infection led to loss of mitochondrial membrane potential and lipid peroxidation and decreased antioxidative enzyme activities (SOD, GPX) and mitochondrial respiratory chain complex activities (succinate dehydrogenase, cytochrome c oxidase). Baicalin relieved the above changes caused by DHAV-1 and activated the gene and protein expressions of Nrf2, which activated ARE-dependent genes including heme oxygenase-1 (HO-1), nicotinamide adenine dinucleotide phosphate quinone oxidoreductase 1 (NQO1), SOD-1, and GPX-1. In addition, baicalin increased the protein expressions of antioxidative enzymes (SOD, GPX). Hence, baicalin protects the liver against oxidative stress in hepatic mitochondria caused by DHAV-1 via activating the Nrf2/ARE signaling pathway.

Abstract Background Using knowledge-based interpretation to analyze omics data can not only obtain essential information regarding various biological processes, but also reflect the current physiological status of cells and tissue. The major challenge to analyze gene expression data, with a large number of genes and small samples, is to extract disease-related information from a massive amount of redundant data and noise. Gene selection, eliminating redundant and irrelevant genes, has been a key step to address this problem. Results The modified method was tested on four benchmark datasets with either two-class phenotypes or multiclass phenotypes, outperforming previous methods, with relatively higher accuracy, true positive rate, false positive rate and reduced runtime. Conclusions This paper proposes an effective feature selection method, combining double RBF-kernels with weighted analysis, to extract feature genes from gene expression data, by exploring its nonlinear mapping ability.

Staphylococcus saprophyticus (S. saprophyticus) is one of the main pathogens that cause serious infection due to its acquisition of antibiotic resistance. The efflux pump decreases antibiotic abundance, and biofilm compromises the penetration of antibiotics. It has been reported that baicalin is a potential agent to inhibit efflux pumps, biofilm formation, and quorum-sensing systems. The purpose of this study was to investigate whether baicalin can inhibit S. saprophyticus biofilm formation and the quorum-sensing system by inhibiting the MsrA efflux pump. First, the mechanism of baicalin inhibiting efflux was investigated by the ethidium bromide (EtBr) efflux assay, measurement of ATP content, and pyruvate kinase (PK) activities. These results revealed that baicalin significantly reduced the efflux of EtBr, the ATP content, and the activity of PK. Moreover, its role in biofilm formation and the agr system was studied by crystal violet staining, confocal laser scanning microscopy, scanning electron microscopy, and real-time polymerase chain reaction. These results showed that baicalin decreased biofilm formation, inhibited bacterial aggregation, and downregulated mRNA transcription levels of the quorum-sensing system regulators agrA, agrC, RNAIII, and sarA. Correlation analysis indicated that there was a strong positive correlation between the efflux pump and biofilm formation and the agr system. We demonstrate for the first time that baicalin inhibits biofilm formation and the agr quorum-sensing system by inhibiting the efflux pump in S. saprophyticus. Therefore, baicalin is a potential therapeutic agent for S. saprophyticus biofilm-associated infections.

Development phases are important in maturing immune systems, intestinal functions, and metabolism for the construction, structure, and diversity of microbiome in the intestine during the entire life. Characterizing the gut microbiota colonization and succession based on age-dependent effects might be crucial if a microbiota-based therapeutic or disease prevention strategy is adopted. The purpose of this study was to reveal the dynamic distribution of intestinal bacterial and fungal communities across all development stages in yaks. Dynamic changes (a substantial difference) in the structure and composition ratio of the microbial community were observed in yaks that matched the natural aging process from juvenile to natural aging. This study included a significant shift in the abundance and proportion of bacterial phyla (Planctomycetes, Firmicutes, Bacteroidetes, Spirochaetes, Tenericutes, Proteobacteria, and Cyanobacteria) and fungal phyla (Chytridiomycota, Mortierellomycota, Neocallimastigomycota, Ascomycota, and Basidiomycota) across all development stages in yaks. As yaks grew older, variation reduced, and diversity increased as compared to young yaks. In addition, the intestine was colonized by a succession of microbiomes that coalesced into a more mature adult, including Ruminococcaceae_UCG-005, Romboutsia, Prevotellaceae_UCG-004, Blautia, Clostridium_sensu_stricto_1, Ruminococcus_1, Ruminiclostridium_5, Rikenellaceae_RC9_gut_group, Alloprevotella, Acetitomaculum, Lachnospiraceae_NK3A20_group, Bacteroides, Treponema_2, Olsenella, Escherichia-Shigella, Candidatus_Saccharimonas, and fungal communities Mortierella, Lomentospora, Orpinomyces, and Saccharomyces. In addition, microorganisms that threaten health, such as Escherichia-Shigella, Mortierella, Lomentospora and Hydrogenoanaerobacterium, Corynebacterium_1, Trichosporon, and Coprinellus, were enriched in young and old yaks, respectively, although all yaks were healthy. The significant shifts in microflora composition and structure might reflect adaptation of gut microbiome, which is associated with physicochemical conditions changes and substrate availability in the gut across all development periods of yaks.

Biocompatible and biodegradable poly(lactic-co-glycolic acid) (PLGA) has been approved by the US Food and Drug Administration and has frequently been used to develop potential vaccine delivery systems. The immunoregulation and immunopotentiation of Chinese yam polysaccharide (CYP) have been widely demonstrated. In the current study, cell uptake mechanisms in dendritic cells (DCs) were monitored in vitro using confocal laser scanning microscopy, transmission electron microscopy, and flow cytometry. To study a CYP-PLGA nanoparticle-adjuvanted delivery system, CYP and ovalbumin (OVA) were encapsulated in PLGA nanoparticles (CYPPs) to act as a vaccine, and the formulation was tested in immunized mice. The CYPPs more easily underwent uptake by DCs in vitro, and CYPP/OVA could stimulate more effective antigen-specific immune responses than any of the single-component formulations in vivo. Mice immunized using CYPP/OVA exhibited more secretion of OVA-specific IgG antibodies, better proliferation, and higher cytokine secretion by splenocytes and significant activation of CD3+CD4+ and CD3+CD8+ T cells. Overall, the CYPP/OVA formulation produced a stronger humoral and cellular immune response and a mixed Th1/Th2 immune response with a greater Th1 bias in comparison with the other formulations. In conclusion, the data demonstrate that the CYPP-adjuvanted delivery system has the potential to strengthen immune responses and lay the foundation for novel adjuvant design.

Context: The flavonoid prescription baicalin-linarin-icariin-notoginsenoside R1 (BLIN) has a curative effect on duck virus hepatitis (DVH) caused by duck hepatitis A virus type 1 (DHAV-1). However, the mechanism of this curative effect is not understood. Objective: This study investigates the mechanism of the curative effect of BLIN on DVH caused by DHAV-1. We analyzed the anti-DHAV-1 reproduction mechanism and immuno-regulatory effect of BLIN. Materials and methods: The anti-DHAV-1 reproduction effects of BLIN at 20, 10, 5 and 2.5 μg/mL in vitro, as well as the influence of BLIN at 20 μg/mL on DHAV-1 adsorption, replication and release were tested using the qRT-PCR method. The promotion abilities of BLIN at 20, 10, 5 and 2.5 μg/mL on T- and B-lymphocyte proliferation were investigated by the MTT method. IL-2 and IFN-γ levels and total anti-DHAV-1 antibody secretion after treatment with DHAV-1 for 4, 8 and 54 h were determined by ELISA. Results: BLIN showed a dose-dependent DHAV-1 reproduction inhibitory effect. The inhibitory effect was highest at 20 μg/mL, where DHAV-1 adsorption and release were significantly lower. Meanwhile, BLIN at 5 μg/mL significantly increased T and B lymphocyte proliferation. BLIN stimulated total anti-DHAV-1 antibody secretion in ducklings at the dosage of 4 mg per duckling, but did not stimulate IL-2 and IFN-γ secretion significantly. Conclusions: BLIN inhibits DHAV-1 reproduction by suppressing its adsorption and release. Additionally, BLIN promoted the duckling antiviral response.

Context: Duck virus hepatitis (DVH) caused by duck hepatitis A virus type 1 (DHAV-1) is an acute and lethal disease of young ducklings. However, there is still no effective drug to treat DVH. Objective: This study assessed the curative effect on DVH of a flavonoid prescription baicalin-linarin-icariin-notoginsenoside R1 (BLIN) as well as the hepatoprotective and antioxidative effects of BLIN. Materials and methods: MTT method was used to test the anti-DHAV-1 ability of BLIN in vitro. We then treated ducklings by BLIN (3 mg per duckling, once a day for 5 days) to evaluate the in vivo efficacy. To study the hepatoprotective and antioxidative roles of BLIN in its curative effect on DVH, we investigated the hepatic injury evaluation biomarkers and the oxidative stress evaluation indices of the ducklings. Results: On duck embryonic hepatocytes, DHAV-1 inhibitory rate of BLIN at 20 μg/mL was 69.3%. The survival rate of ducklings treated by BLIN was about 35.5%, which was significantly higher than that of virus control (0.0%). After the treatment of BLIN, both the hepatic injury and the oxidative stress of infected ducklings alleviated. At the same time, a significant positive correlation (p

The duck virus hepatitis (DVH) caused by the duck hepatitis virus A (DHAV) has produced extensive economic losses to the duck industry. The currently licensed commercial vaccine has shown some defects and does not completely prevent the DVH. Accordingly, a new alternative treatment for this disease is urgently needed. Previous studies have shown that icariin (ICA) and its phosphorylated derivative (pICA) possessed good anti-DHAV effects through direct and indirect antiviral pathways, such as antioxidative stress. But the antioxidant activity showed some differences between ICA and pICA. The aim of this study is to prove that ICA and pICA attenuate oxidative stress caused by DHAV in vitro and in vivo, and to investigate their mechanism of action to explain their differences in antioxidant activities. In vivo, the dynamic deaths, oxidative evaluation indexes and hepatic pathological change scores were detected. When was added the hinokitiol which showed the pro-oxidative effect as an intervention method, pICA still possessed more treatment effect than ICA. The strong correlation between mortality and oxidative stress proves that ICA and pICA alleviate oxidative stress caused by DHAV. This was also demonstrated by the addition of hydrogen peroxide (H2O2) as an intervention method in vitro. pICA can be more effective than ICA to improve duck embryonic hepatocytes (DEHs) viability and reduce the virulence of DHAV. The strong correlation between TCID50 and oxidative stress demonstrates that ICA and pICA can achieve anti-DHAV effects by inhibiting oxidative stress. In addition, the superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) of ICA and pICA showed significant difference. pICA could significantly inhibit the phosphorylation of p38, extra cellular signal regulated Kinase (ERK 1/2) and c-Jun N-terminal kinase (JNK), which were related to mitogen-activated protein kinases (MAPKs) signaling pathways. Ultimately, compared to ICA, pICA exhibited more antioxidant activity that could regulate oxidative stress-related indicators, and inhibited the phosphorylation of MAPKs signaling pathway.

The principal target organ of duck hepatitis A virus type 1 (DHAV-1) is duckling liver, which is an energy-intensive organ and plays important roles in body's energy metabolism and conversion. As the "power house" of the hepatocytes, mitochondria provide more than 90% of the energy. However, mitochondria are much vulnerable to the oxidative stress for their rich in polyunsaturated fatty acids. Although previous researches have demonstrated that DHAV-1 could induce the oxidative stress in the serum of the infected ducklings, no related study on the mitochondria during the pathological process of DVH has been reported by far. To address this issue, we examined the HE stained tissue pathological slices, detected the hepatic SOD, CAT and GPX activities and MDA contents and analyzed the ATP content, mitochondrial ultrastructure and the mitochondrial SOD, GPX activities and MDA content in the liver tissues. The results showed that the hepatic redox status was significantly disturbed so that causing the mitochondrial dysfunction, ATP depletion and mitochondrial oxidative stress during the process of the DHAV-1 infection, and a prescription formulated with Hypericum japonicum flavone, Radix Rehmanniae Recens polysaccharide and Salvia plebeia flavone (HRS), which had been demonstrated with good anti-oxidative activity in serum, could effectively alleviate the hepatic injury and the oxidative stress in liver tissue induced by DHAV-1 thus alleviating the mitochondrial injury and oxidative stress. In a word, this research discovers the oxidative stress induced mitochondrial dysfunction and oxidative stress during the DVH pathological process and demonstrates HRS exerts good anti-oxidative activity in liver tissue to protect mitochondria against reactive oxygen species (ROS).