Epidemiological investigation and ß-lactam antibiotic resistance of Riemerella anatipestifer isolates with waterfowl origination in Anhui Province, China.

Riemerella anatipestifer (R. anatipestifer) is a highly pathogenic and complex serotypes waterfowl pathogen with inherent resistance to multiple antibiotics. This study was aimed to investigate the antibiotic resistance characteristics and genomic features of R. anatipestifer isolates in Anhui Province, China in 2023. A total of 287 cases were analysed from duck farms and goose farms, and the R. anatipestifer isolates were subjected to drug resistance tests for 30 antimicrobials. Whole genome sequencing (WGS) and bioinformatics analysis were performed on the bacterial genomes, targeting the ß-lactam resistance genes. The results showed that a total of 74 isolates of R. anatipestifer were isolated from 287 cases, with a prevalence of 25.8%. The antimicrobial susceptibility testing (AST) revealed that all the 74 isolates were resistant to multiple drugs, ranging from 13 to 26 kinds of drugs. Notably, these isolates showed significant resistance to aminoglycosides and macrolides, which are also commonly used in clinical practices. Data revealed the presence of several ß-lactamase-related genes among the isolates, including a novel blaRASA-1 variant (16.2%), the class A extended-spectrum ß-lactamase blaRAA-1 (12.2%), and a blaOXA-209 variant (98.6%). Functional analysis of the variants blaRASA-1 and blaOXA-209 showed that the blaRASA-1 variant exhibited activity against various ß-lactam antibiotics while their occurrence in R. anatipestifer were not common. The blaOXA-209 variant, on the other hand, did not perform any ß-lactam antibiotic resistance. Furthermore, we observed that blaRAA-1 could undergo horizontal transmission among different bacteria via the insertion sequence IS982. In conclusion, this study delves into the high prevalence of R. anatipestifer infection in waterfowl in Anhui, China. The isolated strains exhibit severe drug resistance issues, closely associated with the prevalence of antibiotic resistance genes (ARG). Additionally, our research investigates the ß-lactam antibiotic resistance mechanism in R. anatipestifer.

Development and application of a low-priced duplex quantitative PCR assay based on SYBR Green I for the simultaneous detection of porcine deltacoronavirus and porcine sapelovirus.

Porcine deltacoronavirus (PDCoV) and porcine sapelovirus (PSV) are two viruses that can cause diarrhoea in pigs and bring great economic loss to the pig industry. In this research, a duplex real-time quantitative polymerase chain reaction (qPCR) assay based on SYBR Green I was developed to simultaneously detect PDCoV and PSV. No specific melting peaks were found in other porcine diarrhoea-associated viruses, indicating that the method developed in this study had good specificity. The detection limits of PDCoV and PSV were 1.0 × 101 copies µl-1 and 1.0 × 102 copies µl-1, respectively. The duplex real-time qPCR assay tested two hundred and three (203) intestinal and faecal samples collected from diarrhoeal and asymptomatic pigs. The positive rates of PDCoV and PSV were 20.2% and 23.2%, respectively. The co-infection rate of PDCoV and PSV was 13.8%. To evaluate the accuracy of the developed method, conventional PCR and singular TaqMan real-time qPCR assays for PDCoV/PSV were also used to detect the samples. The results showed that the duplex real-time qPCR assay was consistent with the singular assays, but its sensitivity was higher than conventional PCR methods. This duplex real-time qPCR assay provides a rapid, sensitive and reliable method in a clinic to simultaneously detect PDCoV and PSV.

Porcine ß-defensin-2 alleviates aflatoxin B1 induced intestinal mucosal damage via ROS-Erk1/2 signaling pathway.

Aflatoxin B1 (AFB1) is a highly toxic fungal toxin that causes severe damage to animal intestines. Porcine beta-defensin-2 (pBD-2) is a well-studied antimicrobial peptide in pigs that can protect animal intestines and improve productivity. This study aimed to investigate the molecular mechanisms of pBD-2 in alleviating AFB1-induced oxidative stress and intestinal mucosal damage using porcine intestinal epithelial cells (IPEC-J2 cells) and Kunming (KM) mice. The maximum destructive concentration of AFB1 for IPEC-J2 cells and the optimal therapeutic concentration of pBD-2 were determined by CCK-8 and RT-qPCR. We then investigated the oxidative stress and intestinal damage induced by AFB1 and the alleviating effect of pBD-2 by detecting changes of reactive oxygen species (ROS), inflammatory cytokines, tight junction proteins (TJPs) and mucin. Finally, the molecular mechanism of pBD-2 mitigates AFB1-induced oxidative stress and intestinal mucosal damage were explored by adding ROS and Erk1/2 pathway inhibitors to comparative analysis. In vivo, the therapeutic effect of pBD-2 on AFB1-induced intestinal damage was analyzed from aspects such as average daily gain (ADG), pathological damage, inflammation, and mucosal barrier in KM mice. The study found that low doses of pBD-2 promoted cell proliferation and prevented AFB1-induced cell death, and pBD-2 significantly restored the feed conversion rate and ADG of KM mice reduced by long-term exposed AFB1. Increasing the intracellular ROS and the expression and phosphorylation of Erk1/2, AFB1 promoted inflammation by altering inflammatory cytokines TNF-α, IL-1ß, IL-6, and IL-8, and disrupted the mucosal barrier by interfering with Claudin-3, Occludin, and MUC2, while pBD-2 significantly reduced ROS and decreased the expression and phosphorylation of Erk1/2 to restored their expression to alleviate AFB1-induced oxidative stress and intestinal mucosal damage in IPEC-J2 cells and the small intestine of mice.

Expression of codon-optimized PDCoV-RBD protein in baculovirus expression system and immunogenicity evaluation in mice.

Porcine deltacoronavirus (PDCoV) is a global epidemic enteropathogenic coronavirus that mainly infects piglets, and causes huge losses to the pig industry. However, there are still no commercial vaccines available for PDCoV prevention and controlment. Receptor-binding domain (RBD) is located at the S1 subunit of PDCoV and is the major target for developing viral inhibitor and vaccine. In this study, the characteristics of the RBD were analyzed by bioinformatic tools, and codon optimization was performed to efficiently express the PDCoV-RBD protein in the insect baculovirus expression system. The purified PDCoV-RBD protein was obtained and fully emulsified with CPG2395 adjuvant, aqueous adjuvant and Al(OH)3 adjuvant, respectively, to develop vaccines. The humoral and cellular immune responses were assessed on mice. The results showed that both the RBD/CPG2395 and RBD/aqueous adjuvant could induce stronger immune responses in mice than that of RBD/Al(OH)3. In addition, the PDCoV challenge infection was conducted and the RBD/CPG2395 could provide better protection against PDCoV in mice. Our study showed that the RBD protein has good antigenicity and can be used as a protective antigen, which provided a basis for the development of the PDCoV vaccine.

Porcine ß-defensin-2 alleviates AFB1-induced intestinal mucosal injury by inhibiting oxidative stress and apoptosis.

Aflatoxin B1 (AFB1) is the most toxic mycotoxin contaminant, which is widely present in crops and poses a major safety hazard to animal and human health. To alleviate the cytotoxic effects of AFB1 on the intestine, we tested the protective effects of porcine ß-defensin-2 (pBD-2). Results demonstrated that pBD-2 inhibited oxidative stress induced by AFB1 via decreasing the levels of ROS and enhancing the expression of antioxidant factors SOD-2 and NQO-1. In addition, pBD-2 attenuated AFB1-induced intestinal porcine epithelial cell line-J2 (IPEC-J2) injury through blocking mitochondria-mediated apoptosis. In vivo, pBD-2 treatment restored the intestinal mucosal structure and reduced the expression levels of apoptosis factors caspase-3 and Bax/Bcl-2. In conclusion, these results indicated that pBD-2 can alleviate AFB1-induced intestinal mucosal injury by inhibiting oxidative stress and mitochondria-mediated apoptosis. This study provides an effective strategy in developing pBD-2 as green feed additive to prevent AFB1 damage to animals.

Unique Fluorescent Protein-Encoded Microbeads with Supramaximal Encoding Capacity and High Sensitivity for Multiplex Bioassays.

Fluorescence-encoded microbeads (FEBs) have been widely used as a critical component in multiplexed biomolecular assays. Here, we propose a simple, sustainable, low-cost, and safe strategy for preparing FEBs by assembling fluorescent proteins (FPs) onto magnetic microbeads (MBs) via chemical coupling. Combining the type of FP, the concentration of FP, and the size of the magnetic microbeads as encoding elements, an ultralarge encoding capacity with 506 barcodes was obtained. We demonstrate that the FP-based FEBs have good stability during long-term storage and tolerate the use of an organic solution. Multiplex detection of femtomolar ssDNA molecules was achieved via flow cytometry, and the detection procedure is simple and fast because it does not require amplification and washing strategies. The advantages of this advanced method for multiplex detections including high sensitivity, specificity, accuracy, repeatability, rapidity, and cost-effectiveness show a broad application prospect in basic and applied research fields such as disease diagnosis, food safety, environmental protection, proteomics, genomics, and drug screening.

The Coexistence of Bacterial Species Restructures Biofilm Architecture and Increases Tolerance to Antimicrobial Agents.

Chronic infections caused by polymicrobial biofilms are often difficult to treat effectively, partially due to the elevated tolerance of polymicrobial biofilms to antimicrobial treatments. It is known that interspecific interactions influence polymicrobial biofilm formation. However, the underlying role of the coexistence of bacterial species in polymicrobial biofilm formation is not fully understood. Here, we investigated the effect of the coexistence of Enterococcus faecalis, Escherichia coli O157:H7, and Salmonella enteritidis on triple-species biofilm formation. Our results demonstrated that the coexistence of these three species enhanced the biofilm biomass and led to restructuring of the biofilm into a tower-like architecture. Furthermore, the proportions of polysaccharides, proteins, and eDNAs in the extracellular matrix (ECM) composition of the triple-species biofilm were significantly changed compared to those in the E. faecalis mono-species biofilm. Finally, we analyzed the transcriptomic profile of E. faecalis in response to coexistence with E. coli and S. enteritidis in the triple-species biofilm. The results suggested that E. faecalis established dominance and restructured the triple-species biofilm by enhancing nutrient transport and biosynthesis of amino acids, upregulating central carbon metabolism, manipulating the microenvironment through "biological weapons," and activating versatile stress response regulators. Together, the results of this pilot study reveal the nature of E. faecalis-harboring triple-species biofilms with a static biofilm model and provide novel insights for further understanding interspecies interactions and the clinical treatment of polymicrobial biofilms. IMPORTANCE Bacterial biofilms possess distinct community properties that affect various aspects of our daily lives. In particular, biofilms exhibit increased tolerance to chemical disinfectants, antimicrobial agents, and host immune responses. Multispecies biofilms are undoubtedly the dominant form of biofilms in nature. Thus, there is a pressing need for more research directed at delineating the nature of multispecies biofilms and the effects of the properties on the development and survival of the biofilm community. Here, we address the effects of the coexistence of Enterococcus faecalis, Escherichia coli, and Salmonella enteritidis on triple-species biofilm formation with a static model. In combination with transcriptomic analyses, this pilot study explores the potential underlying mechanisms that lead to the dominance of E. faecalis in triple-species biofilms. Our findings provide novel insights into the nature of triple-species biofilms and indicate that the composition of multispecies biofilms should be a key consideration when determining antimicrobial treatments.

Microorganisms@aMIL-125 (Ti): An Amorphous Metal-Organic Framework Induced by Microorganisms and Their Applications.

Amorphous metal-organic framework (aMOF)-based materials have attracted considerable attention as an emerging class of nanomaterials. Herein, novel microorganisms@aMIL-125 (Ti) composites including yeast@aMIL-125 (Ti), PCC 6803@aMIL-125 (Ti), and Escherichia coli@aMIL-125 (Ti) composites were respectively synthesized by self-assembling aMOFs on the microorganisms' surface. The functional groups on the microorganisms' surface induced structural defects and participated in the formation of aMIL-125 (Ti) composites. Finally, the application of microorganisms@aMIL-125 (Ti) composites for the removal of glyphosate from aqueous solution was selected as a model reaction to illustrate their potential for environmental protection. The present method is not only economical but also has other advantages including ease of operation, environmentally friendly assay, and high adsorption. The maximum adsorption capacity of aMIL-125 (Ti) was 1096.25 mg g-1, which was 1.74 times that of crystalline MIL-125 (Ti). Therefore, the microorganisms@aMOFs composites will have broad application prospects in energy storage, drug delivery, catalysis, adsorbing toxic substances, sensing, encapsulating and delivering enzymes, and in other fields.

Aflatoxin B1 disrupts the intestinal barrier integrity by reducing junction protein and promoting apoptosis in pigs and mice.

With the growing diversity and complexity of diet, animals and humans are at risk of exposure to aflatoxin B1 (AFB1), which is a well-known contaminant in the food chain that causes various toxicological effects. The intestine acts as the first barrier against external contaminants, but the effect of AFB1 on intestinal barrier has not been determined. This study aimed to evaluate AFB1 on the intestinal barrier function in vitro and in vivo. In vitro, porcine jejunal epithelial cells (IPEC-J2) were treated with increasing concentrations of AFB1 (10-60 mg/L). In vivo, Kunming (KM) mice were used as controls or gavaged with 1% dimethyl sulfoxide (110 mg/kg b.w.) and AFB1 (0.3 mg/kg b.w.) for 28 days. In IPEC-J2 cells, the cell viability decreased with increasing mycotoxin concentrations, and the viability of IPEC-J2 cells decreased significantly (P < 0.05) when the AFB1 concentrations were greater than 30 mg/L. In addition, quantitative real-time PCR, Western blot analysis, and immunofluorescence results show that AFB1 can downregulate the tight junction proteins and increase the expression levels of Caspase-3 and the ratio of Bax/Bcl-2, suggesting that AFB1 was cytotoxic to IPEC-J2. In vivo, the ratio of villus height to crypt depth, the intestinal wall thickness, the number of intestinal villus per 1000 µm in the jejunum, the expression levels of ZO-1, Claudin-3, Occludin, MUC2, and Caspase-3, and the ratio of Bax/Bcl-2 were significantly affected in mice exposed to AFB1. In vitro and in vivo results showed that the effects of exposure to AFB1 on the intestinal function in the jejunum of KM mice and in the IPEC-J2 was similar, suggesting that AFB1 may adversely affect animal intestine.

Biomimetic Assembly of Spore@ZIF-8 Microspheres for Vaccination.

Vaccines have been one of the most powerful weapons to defend against infectious diseases for a long time now. Subunit vaccines are of increasing importance because of their safety and effectiveness. In this work, a Bacillus amyloliquefaciens spore@zeolitic imidazolate framework-8 (ZIF-8) vaccine platform is constructed. The ovalbumin (OVA) is encapsulated in the ZIF-8 shells as a model antigen to form a spore@OVA@ZIF-8 (SOZ) composite. The assembly of ZIF-8 improves the loading content of OVA on the spores and provides OVA with long-term protection. The SOZ composite enhances the immunization efficacy in multiple ways, such as facilitation of antigen uptake and lysosome escape, stimulation of dendritic cells to mature and secrete cytokines, boosting of antibody production and formation of an antigen depot. This platform shows several advantages including easy preparation, cost-effectiveness, long life, convenience of transportation and storage, and no need for the cold chain. These findings may have promising implications for the rational design of safe and effective spore-based composite vaccine platforms.

Development and immunogenicity evaluation of porcine deltacoronavirus inactivated vaccine with different adjuvants in mice.

Porcine deltacoronavirus (PDCoV) is a novel coronavirus that causes diarrhea in pigs of various ages, especially in suckling piglets, and there are no effective measures to prevent and control PDCoV currently. In this study, two adjuvants Al(OH)3 and ODN2395 working through different mechanisms were used to prepare inactivated PDCoV vaccines, and the immune effects of PDCoV inactivated vaccines were assessed in mice. From the results, we found that both PDCoV/Al(OH)3 vaccine and PDCoV/2395 vaccine could induce IgG and neutralizing antibodies with high levels in mice. At the same time, cytokines of IFN-γ, IL-4 and chemokine ligand of CXCL13 in serum were significantly increased after immunization, and reached the highest levels in PDCoV/2395 vaccine group, which suggested that PDCoV/2395 could promote the production of both Th1 and Th2 polarized cytokines. In addition, histopathological observations showed that vaccination helped mice resist PDCoV infection. These results indicated that both the two inactivated vaccines have good immune effects. Moreover, the PDCoV/2395 vaccine worked better than the PDCoV/Al(OH)3 vaccine for PDCoV/2395 having the good ability to induce both humoral and cellular immunogenicity. The PDCoV/2395 inactivated vaccine developed in this study might be an effective tool for the prevention of PDCoV infection.

Light-Regulated Natural Fluorescence of the PCC 6803@ZIF-8 Composite as an Encoded Microsphere for the Detection of Multiple Biomarkers.

The use of color-encoded microspheres for a bead-based assay has attracted increasing attention for high-throughput multiplexed bioassays. A fluorescent PCC 6803@ZIF-8 composite was prepared as a bead-based assay platform by a self-assembled zeolitic imidazolate framework (ZIF-8) on the surface of inactivated PCC 6803 cells. The composite fluorescence owing to the presence of pigment proteins in PCC 6803 could be gradually bleached with the prolongation of the ultraviolet light irradiation time. The composites with different fluorescence intensities were therefore obtained as encoded microspheres for the multiplexed assay. ZIF-8 provides a stable, rigid shell and a large specific surface area for composites, which prevent the composites from breakage during use and storage, simplify the protein immobilization procedure, reduce non-specific adsorption, and enhance the detection sensitivity. The encoded composites were successfully used to detect multiple DNA insertion sequences of Mycobacterium tuberculosis. The presented strategy offers an innovative color-encoding method for high-throughput multiplexed bioassays without the need of using chemically synthesized fluorescent materials.

E. coli@UiO-67 composites as a recyclable adsorbent for bisphenol A removal.

E. coli@UiO-67 composites were obtained using an effective and simple self-assembly method. The composites showed unique properties as a remarkable and recyclable adsorbent for the efficient removal of bisphenol A (BPA) from water with a high adsorption capacity (402.930 mg g-1). The increase in pore size is a key factor why E. coli@UiO-67 composites maintained high capacity. The reason might be due to that the composites with large pore sizes and defects could effectively improve mass transport and active molecular metal sites. The adsorption of BPA is a chemisorption process due to the Zr-OH groups in UiO-67 exhibit affinity toward BPA molecules, π-π interaction, and electrostatic attraction. The adsorption efficiency remained at 82.5% after 15 cycles without any remarkable changes in the PXRD patterns of E. coli@UiO-67. Moreover, the use of microorganism-loading MOFs could reduce the cost to at least 50% and minimize secondary pollution through nanoscale MOFs usage reduction. The developed composites have advantages, including low-cost, high adsorption capacity, easy to be separated and regenerated from aqueous solution, a large number of cycles, short adsorption equilibrium time, and stability, showing excellent application prospects. The presented strategy would be a potentially promising way to produce novel MOFs-based adsorbents with high-performance to control environmental pollution from wastewater.

Microorganism@UiO-66-NH2 Composites for the Detection of Multiple Colorectal Cancer-Related microRNAs with Flow Cytometry.

High-throughput analyses of multitarget markers can facilitate rapid and accurate clinical diagnosis. Suspension array assays, a flow cytometry-based analysis technology, are among some of the most promising multicomponent analysis methods for clinical diagnostics and research purposes. These assays are appropriate for examining low-volume, complex samples having trace amounts of analytes due to superior elimination of background. Physical shape is an important and promising code system, which uses a set of visually distinct patterns to identify different assay particles. Here, we presented a morphology recognizable suspension arrays based on the microorganisms with different morphologies. In this study, UiO-66-NH2 (UiO stands for University of Oslo) metal-organic frameworks (MOFs), was wrapped on the microorganism surface to form an innovative class of microorganism@UiO-66-NH2 composites for suspension array assays. The use of microorganisms endowed composites barcoding ability with their different morphology and size. Meanwhile, the UiO-66-NH2 provided a stable rigid shell, large specific surface area, and metal(IV) ions with multiple binding sites, which could simplify the protein immobilization procedure and enhance detection sensitivity. With this method, simultaneous detection of three colorectal cancer-related microRNA (miRNA), including miRNA-21, miRNA-17, and miRNA-182, could be easily achieved with femtomolar sensitivity by using a commercial flow cytometer. The synergy between microorganisms and MOFs make the composites a prospective barcoding candidate with excellent characteristics for multicomponent analysis, offering great potential for the development of high throughput and accurate diagnostics.

Rapid Self-Assembly of Au Nanoparticles on Rigid Mesoporous Yeast-Based Microspheres for Sensitive Immunoassay.

A simple, rapid, inexpensive, eco-friendly, and high-throughput biological strategy for the preparation of functional microspheres on a yeast-cell platform was introduced. Microspheres prepared through the treatment of yeast cells with formaldehyde and decoating buffer exhibited excellent characteristics, such as superior mechanical strength, high sulfhydryl group content, and mesoporous structure. Au nanoparticles (NPs) easily and rapidly self-assembled onto the surfaces of the yeast-based microspheres within 5 min to form rigid yeast@Au microspheres with high monodispersity and uniformity. The rapid formation of yeast@Au microspheres mainly involved the enhancement of sulfhydryl groups and mesoporosity. The yeast@Au microspheres were successfully used in a flow cytometry immunoassay to detect Pseudorabies viral infection events. Signal-to-noise ratio was enhanced by approximately 49.4-fold. The presence of Au NPs on the yeast-based microspheres greatly improved sensitivity by decreasing noise through reducing nonspecific adsorption, highly enhancing the fluorescence signal caused by the surface plasmon resonance effect, and increasing the coupling efficiency of the capture protein. The presented method was used to analyze 81 clinical swine serum specimens. The results obtained by this developed method were compared to those of commercial diagnostic kits. The sensitivity, specificity, and efficiency of the developed method were 92.31, 88.24, and 88.89%, respectively. The excellent characteristics of the yeast@Au microspheres illustrate its great potential for high-throughput immunoassay applications in the fields of disease diagnosis, environmental analysis, and food safety.

Intestinal absorption differences of major bioactive compounds of Gegenqinlian Decoction between normal and bacterial diarrheal mini-pigs in vitro and in situ.

Intestinal condition plays an important role in drug absorption and metabolism, thus the effects of varied gastrointestinal diseases such as infectious diarrhea on the intestinal function are crucial for drug absorption. However, due to the lack of suitable models, the differences of absorption and metabolism of drugs between the diarrheal and normal intestines are rarely reported. Thus, in this study, Escherichia coli diarrhea model was induced in mini-pigs and single-pass intestinal perfusion and intestinal mucosal enzyme metabolism experiments were conducted. A simple and rapid ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to determine the concentrations of 9 major components in Gegen Qinlian decoction (GQD). Samples were pretreated by protein precipitation with methanol and naringin and prednisolone were used as internal standards. The validated method demonstrated adequate sensitivity, selectivity, and process efficiency for the bioanalysis of 9 compounds. Results of intestinal perfusion showed that puerarin, daidzein, daidzin and baicalin and berberine were absorbed faster in diarrheal jejunum than in normal intestines (p < 0.05). However, puerarin, daidzin and liquiritin were metabolized more slowly in diarrheal intestine after incubation compared with the normal group (p < 0.05). The concentrations of daidzein in both perfusion and metabolism and wogonin in metabolism were significantly increased (p < 0.05). In conclusion, absorption and metabolism of GQD were significantly different between the diarrheal and normal intestines, which suggest that bacterial diarrheal mini-pigs model can be used in the intestinal absorption study and is worthy to be applied in the other intestinal absorption study of anti- diarrheal drugs.

Soybean Milk Inhibits Absorption and Intestinal Transmembrane Transport of Gegen in Rats.

Puerariae Lobatae Radix, known as Gegen in Chinese, is widely used to treat cardiovascular diseases, diabetes, and many other chronic illnesses. Flavonoids are the main active components in Gegen and are found in high concentrations in soybeans. Few studies, however, have focused on the effects of flavonoid-rich food on the absorption of Gegen. Here, we report an in vivo pharmacokinetic study on rats to explore the effects of soybean milk on the absorption of Gegen and an in vitro Ussing chamber study of puerarin intestinal transmembrane absorption. Area under the plasma concentration-time curve (AUC0-t ) and maximum plasma concentration (Cmax) values of puerarin in rats were significantly decreased after drinking soybean milk, when taking Gegen decoction or a Gegen patent medicine (P < 0.01). In the Ussing chamber experiment, cumulative transmission (Qtn) after 2 h and apparent permeability coefficient (Papp) were lower in the puerarin-daidzin and puerarin-soybean milk solution groups than in the puerarin group. Daidzin in soybean milk inhibited the transmembrane transport of puerarin, resulting in decreased bioavailability of puerarin in Gegen. The results of this study strongly suggest that Gegen should not be taken with flavonoid-rich food, particularly soybean products.

Comparative pharmacokinetics of eight major bioactive components in normal and bacterial diarrhea mini-pigs after oral administration of Gegen Qinlian Decoction.

Healthy animals are most widely used in current pharmacokinetic(PK) studies. However, neglecting the effects of specific diseases on drug absorption results in the PK parameters of those experiments not accurately reflecting in vivo drug concentration changes during treatment. In this study, an E. coli infective diarrheal minipig model was applied to explore the pharmacokinetics of Gegen Qinlian decoction (GQD). A simple and rapid ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to determine the concentrations of the eight GQD components in minipig plasma after intragastric administration of GQD. The PK parameters of the main GQD components in normal and model minipigs after oral administration of GQD were compared. There were statistically significant differences (p<0.05) in the pharmacokinetic parameters of Puerarin, Wogonin and Daidzein involving the AUC0-t, Cmax, MRT(0-t), t1/2z between normal and model minipigs. Results showed that bacterial diarrhea had a great impact on the biological availability of the main ingredients in GQD. More importantly, the results obtained suggest that the bacterial diarrheal minipig model can be successfully applied in PK studies and may be used in other PK studies of drugs targeting intestinal disease.

A novel fluorimetric method for laccase activities measurement using Amplex Red as substrate.

In this paper, a novel fluorescence-based method for laccase assay was presented. The method was based on the transformation of Amplex Red into a highly fluorescent and colored resorufin catalyzed by laccase in the presence of O2. The catalysis and transformation mechanism were investigated in detail. The kinetic parameters of the Amplex Red catalysis by laccase were determined using the Lineweaver-Burk equation. Vmax and Km were estimated to be 15.63µmolmin-1 and 76.88µmolL-1, respectively. Under optimal conditions, a good linear correlation was found between fluorescence intensity and laccase activities within 5.62-702UL-1 (r=0.9992), with a detection limit of 1.76UL-1 (S/N=3). A series of repeatability measurements (351UL-1 laccase) gave reproducible results with a relative standard deviation (RSD) of 1.9% (n=11). The recoveries ranged from 93.7% to 100.0% after standard additions. Common existing species such as Mg2+, Zn2+, Ni2+, Al3+, Co2+, Cd2+, K+, Ca2+, Na+, Fe3+, Li+, Cu2+, Mn2+, Fe2+, l-lysine, glycine, glucose, phenol, humic acid, lignin peroxidase, manganese peroxidase alkaline phosphatase, cellulose, glucose oxidase, urease, catalase, invertase, and horseradish peroxidase did not significantly exhibit interference. The test solution (i.e., Amplex Red stock solution) could stabilize at least three months via storage in dark at 4±0.1°C. These results confirmed that the laccase-Amplex Red system was stable and reproducible with strong anti-interference ability and good selectivity, suggesting that this method can has great potential in practical applications for the assay of laccase activity. The proposed method was further successfully used to detect laccase activities in 38 soil samples. We noticed that the laccase activity significantly correlated with total nitrogen content (r=0.559; p<0.01) of soil, indicating laccase activity assay holds great promise as an index of soil analysis. These findings indicate that this presented method has great perspective in ecological investigation and fundamental research of soil environment.