Evaluation of the antimicrobial function of Ginkgo biloba exocarp extract against clinical bacteria and its effect on Staphylococcus haemolyticus by disrupting biofilms.

ETHNOPHARMACOLOGICAL RELEVANCE: The fruit of Ginkgo biloba L. (Ginkgo nuts) has been used for a long time as a critical Chinese medicine material to treat cough and asthma, as well as a disinfectant. Similar records were written in the Compendium of Materia Medica (Ben Cao Gang Mu, pinyin in Chinese) and Sheng Nong's herbal classic (Shen Nong Ben Cao Jing, pinyin in Chinese). Recent research has shown that Ginkgo biloba exocarp extract (GBEE) has the functions of unblocking blood vessels and improving brain function, as well as antitumour activity and antibacterial activity. GBEE was shown to inhibit methicillin-resistant Staphylococcus aureus (MRSA) biofilm formation as a traditional Chinese herb in our previous report in this journal. AIM OF THE STUD: yThe antibiotic resistance of clinical bacteria has recently become increasingly serious. Thus, this study aimed to investigate the Ginkgo biloba exocarp extract (GBEE) antibacterial lineage, as well as its effect and mechanism on S. haemolyticus biofilms. This study will provide a new perspective on clinical multidrug resistant (MDR) treatment with ethnopharmacology herbs. METHODS: The microbroth dilution assay was carried out to measure the antibacterial effect of GBEE on 13 types of clinical bacteria. Bacterial growth curves with or without GBEE treatment were drawn at different time points. The potential targets of GBEE against S. haemolyticus were screened by transcriptome sequencing. The effects of GBEE on bacterial biofilm formation and mature biofilm disruption were determined by crystal violet staining and scanning electron microscopy. The metabolic activity of bacteria inside the biofilm was assessed by colony-forming unit (CFU) counting and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2HY-tetrazolium bromide (MTT) assay. Quantitative polymerase chain reaction (qPCR) was used to measure the gene expression profile of GBEE on S. haemolyticus biofilm-related factors. RESULTS: The results showed that GBEE has bacteriostatic effects on 3 g-positive (G+) and 2 g-negative (G-) bacteria among 13 species of clinical bacteria. The antibacterial effect of GBEE supernatant liquid was stronger than the antibacterial effect of GBEE supernviaould-like liquid. GBEE supernatant liquid inhibited the growth of S. epidermidis, S. haemolyticus, and E. faecium at shallow concentrations with minimum inhibitory concentrations (MICs) of 2 µg/ml, 4 µg/ml and 8 µg/ml, respectively. Genes involved in quorum sensing, two-component systems, folate biosynthesis, and ATP-binding cassette (ABC) transporters were differentially expressed in GBEE-treated groups compared with controls. Crystal violet, scanning electron microscopy (SEM) and MTT assays showed that GBEE suppressed S. haemolyticus biofilm formation in a dose-dependent manner. Moreover, GBEE supernatant liquid downregulated cidA, cidB and atl, which are involved in cell lysis and extracellular DNA (eDNA) release, as well as downregulated the cbp, ebp and fbp participation in encoding cell-surface binding proteins. CONCLUSIONS: GBEE has an excellent antibacterial effect on gram-positive bacteria and also inhibits the growth of gram-negative bacteria, such as A. baumannii (carbapenem-resistant Acinetobacter baumannii) CRABA and S. maltophilia. GBEE inhibits the biofilm formation of S. haemolyticus by altering the regulation and biofilm material-related genes, including the release of eDNA and cell-surface binding proteins.

Ginkgo biloba exocarp extracts inhibit S. aureus and MRSA by disrupting biofilms and affecting gene expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginkgo biloba L. fruit, also known as Bai Guo, Ya Jiao Zi (in pinyin Chinese), and ginkgo nut (in English), has been used for many years as an important material in Chinese traditional medicine to treat coughs and asthma and as a disinfectant, as described in the Compendium of Materia Medica (Ben Cao Gang Mu, pinyin in Chinese), an old herbal book. Ginkgo nuts are used to treat phlegm-associated asthma, astringent gasp, frequent urination, gonorrhoea and turgidity; consumed raw to reduce phlegm and treat hangovers; and used as a disinfectant and insecticide. A similar record was also found in Sheng Nong's herbal classic (Shen Nong Ben Cao Jing, pinyin in Chinese). Recent research has shown that Ginkgo biloba L. exocarp extract (GBEE) can unblock blood vessels and improve brain function and exhibits antitumour and antibacterial activities. AIM OF STUDY: To investigate the inhibitory effect of Ginkgo biloba L. exocarp extract (GBEE) on methicillin-resistant S. aureus (MRSA) biofilms and assess its associated molecular mechanism. MATERIALS AND METHODS: The antibacterial effects of GBEE on S. aureus and MRSA were determined using the broth microdilution method. The growth curves of bacteria treated with or without GBEE were generated by measuring the CFU (colony forming unit) of cultures at different time points. The effects of GBEE on bacterial biofilm formation and mature biofilm disruption were determined by crystal violet staining. Quantitative polymerase chain reaction (qPCR) was used to measure the effects of GBEE on the gene expression profiles of MRSA biofilm-related factors at 6, 8, 12, 16 and 24 h. RESULTS: The minimum inhibitory concentration (MIC) of GBEE on S. aureus and MRSA was 4 µg/mL, and the minimum bactericidal concentration (MBC) was 8 µg/ml. Moreover, GBEE (4-12 µg/mL) inhibited S. aureus and MRSA biofilm formation in a dose-dependent manner. Interestingly, GBEE also destroyed mature biofilms of S. aureus and MRSA at 12 µg/ml. The expression of the MRSA biofilm-associated factor icaA and sarA were downregulated after 6 h of treatment with GBEE, while sigB was downregulated after 12 h. MeanwhileMeanwhile, icaR was upregulated at 12 h. In addition, GBEE also downregulated the virulence gene hld and inhibited the synthesis of staphyloxanthin. CONCLUSIONS: GBEE has excellent antibacterial effects against S. aureus and MRSA and inhibits their biofilm-forming ability by altering related gene expression.

Siderophore Production by Rhizosphere Biological Control Bacteria Brevibacillus brevis GZDF3 of Pinellia ternata and Its Antifungal Effects on Candida albicans.

Brevibacillus brevis GZDF3 is a gram-positive, plant growth-promoting rhizosphere bacterium (PGPR) isolated from the rhizosphere soil of Pinellia ternata (an important herb in traditional Chinese medicine). The GZDF3 strain produces certain active compounds, such as siderophores, which are the final metabolite products of non-ribosomal peptide synthetase (NRPS) and independent non-ribosomal peptide synthetase (NIS) activity. With the present study, we attempted to investigate the siderophore production characteristics and conditions of Bacillus sp. GZDF3. The antibacterial activity of the siderophores on pathogenic fungi was also investigated. Optimal conditions for the synthesis of siderophores were determined by single factor method, using sucrose 15 g/l, asparagine 2 g/l, 32°C, and 48 h. The optimized sucrose asparagine medium significantly increased the production of siderophores, from 27.09% to 54.99%. Moreover, the effects of different kinds of metal ions on siderophore production were explored here. We found that Fe3+ and Cu2+ significantly inhibited the synthesis of siderophores. The preliminary separation and purification of siderophores by immobilized-metal affinity chromatography (IMAC) provides strong antibacterial activity against Candida albicans. The synergistic effect of siderophores and amphotericin B was also demonstrated. Our results have shown that the GZDF3 strain could produce a large amount of siderophores with strong antagonistic activity, which is helpful in the development of new biological control agents.

Fumonisin B1 Affects the Biophysical Properties, Migration and Cytoskeletal Structure of Human Umbilical Vein Endothelial Cells.

Fumonisin B1 (FB1) is an important mycotoxin in nature and is a serious threat to human and animal health, but its specific target and molecular mechanism of the toxicity and potential carcinogenicity remain unclear. In this study, we first detected the effects of FB1 on the cell viability, biophysical properties, migration ability, and reactive oxygen species (ROS) of human umbilical vein endothelial cells (HUVECs). Subsequently, changes in the cytoskeletal structure and its binding proteins were analyzed by immunofluorescence and real-time PCR, respectively. The results showed that FB1 could inhibit the viability of HUVECs in a dose-dependent manner. After treatment of HUVECs with FB1, the hypotonic resistance, cell surface charges, cell membrane fluidity, and migration ability were weakened, whereas the ROS levels were significantly increased. Moreover, the cytoskeletal structure of the HUVECs was significantly changed, and the mRNA expression of some important actin-binding proteins was altered. Therefore, this study revealed that FB1 can affect the migration and cytoskeletal structure of HUVECs, which provides a new perspective for further understanding the molecular mechanisms of FB1 toxicity.

Clinical Implications of Tumor-Infiltrating Immune Cells in Breast Cancer.

The immune infiltration of tumors is closely related to clinical outcomes. The composition of tumor-infiltrating immune cells (TIICs) can serve as biomarkers for predicting response to treatment and survival in different patient subgroups in terms of chemotherapy and immunotherapy. This study is focused on investigating the clinical implications of TIICs in breast cancer patients. We performed several in silico analyses of gene expression profiles in 2976 nonmetastatic tumor samples. CIBERSORT was used to estimate the proportion of 22 immune cell types to analyze their correlation with overall survival (OS) and disease-free survival (DFS) in different breast cancer subtypes and stages. Our results showed that a higher fraction of plasma cells in estrogen receptor (ER)-positive breast cancer patients indicated an increase in DFS (hazard ratio [HR]=0.66, 95% confidence interval [CI] 0.54~0.82, p<0.01), while a decreased OS was correlated with a greater number of M0 macrophages (HR=2.02, 95% CI 1.27~3.30, p=0.01) and regulatory T cells (HR=1.90, 95% CI 1.20~3.02, p=0.02). In ER-negative or progesterone receptor (PR)-negative subtypes or in a combined subtype, the increase in activated memory CD4+ T cells was correlated with increased DFS (HR=0.46, 95% CI 0.33~0.63, p<0.01). In all breast cancer patients, a higher proportion of M0 macrophages indicated a decreased DFS (HR=1.67, 95% CI 1.22~2.27, p<0.01), while increased OS was associated with relatively larger fractions of resting memory CD4+ T cells (HR=0.70, 95% CI 0.55~0.90, p=0.02) and γδ T cells (HR=0.66, 95% CI 0.51~0.85, p<0.01). Therefore, this study revealed that the composition of TIICs is different in patients with various subtypes of breast cancer and is directly related to prognosis, suggesting that TIICs are important participants in tumor progression and may, potentially be used for future diagnosis and treatment.

A Mathematical Modelling of Initiation of Dendritic Cells-Induced T Cell Immune Response.

Dendritic cells (DCs) are the most potent specialized antigen-presenting cells as now known, which play a crucial role in initiating and amplifying both the innate and adaptive immune responses. Immunologically, the motilities and T cell activation capabilities of DCs are closely related to the resulting immune responses. However, due to the complexity of the immune system, the dynamic changes in the number of cells during the peripheral tissue (e.g. skin and mucosa) immune response induced by DCs are still poorly understood. Therefore, this study simulated dynamic number changes of DCs and T cells in this process by constructing several ordinary differential equations and setting the initial conditions of the functions and parameters. The results showed that these equations could simulate dynamic numerical changes of DCs and T cells in peripheral tissue and lymph node, which was in accordance with the physiological conditions such as the duration of immune response, the proliferation rates and the motilities of DCs and T cells. This model provided a theoretical reference for studying the immunologic functions of DCs and practical guidance for the clinical DCs-based therapy against immune-related diseases.

Biophysical Properties and Motility of Human Mature Dendritic Cells Deteriorated by Vascular Endothelial Growth Factor through Cytoskeleton Remodeling.

Dendritic cells (DCs), the most potent antigen-presenting cells, play a central role in the initiation, regulation, and maintenance of the immune responses. Vascular endothelial growth factor (VEGF) is one of the important cytokines in the tumor microenvironment (TME) and can inhibit the differentiation and functional maturation of DCs. To elucidate the potential mechanisms of DC dysfunction induced by VEGF, the effects of VEGF on the biophysical characteristics and motility of human mature DCs (mDCs) were investigated. The results showed that VEGF had a negative influence on the biophysical properties, including electrophoretic mobility, osmotic fragility, viscoelasticity, and transmigration. Further cytoskeleton structure analysis by confocal microscope and gene expression profile analyses by gene microarray and real-time PCR indicated that the abnormal remodeling of F-actin cytoskeleton may be the main reason for the deterioration of biophysical properties, motility, and stimulatory capability of VEGF-treated mDCs. This is significant for understanding the biological behavior of DCs and the immune escape mechanism of tumors. Simultaneously, the therapeutic efficacies may be improved by blocking the signaling pathway of VEGF in an appropriate manner before the deployment of DC-based vaccinations against tumors.

An antibody that confers plant disease resistance targets a membrane-bound glyoxal oxidase in Fusarium.

Plant germplasm resources with natural resistance against globally important toxigenic Fusarium are inadequate. CWP2, a Fusarium genus-specific antibody, confers durable resistance to different Fusarium pathogens that infect cereals and other crops, producing mycotoxins. However, the nature of the CWP2 target is not known. Thus, investigation of the gene coding for the CWP2 antibody target will likely provide critical insights into the mechanism underlying the resistance mediated by this disease-resistance antibody. Immunoblots and mass spectrometry analysis of two-dimensional electrophoresis gels containing cell wall proteins from Fusarium graminearum (Fg) revealed that a glyoxal oxidase (GLX) is the CWP2 antigen. Cellular localization studies showed that GLX is localized to the plasma membrane. This GLX efficiently catalyzes hydrogen peroxide production; this enzymatic activity was specifically inhibited by the CWP2 antibody. GLX-deletion strains of Fg, F. verticillioides (Fv) and F. oxysporum had significantly reduced virulence on plants. The GLX-deletion Fg and Fv strains had markedly reduced mycotoxin accumulation, and the expression of key genes in mycotoxin metabolism was downregulated. This study reveals a single gene-encoded and highly conserved cellular surface antigen that is specifically recognized by the disease-resistance antibody CWP2 and regulates both virulence and mycotoxin biosynthesis in Fusarium species.

An affinity improved single-chain antibody from phage display of a library derived from monoclonal antibodies detects fumonisins by immunoassay.

Fumonisin B analogs, particularly FB1, FB2, and FB3, are major mycotoxins found in cereals. Single-chain fragment variable (scFv) antibodies represent a promising alternative immunoassay system. A phage-displayed antibody library derived from four monoclonal antibodies (mAbs) generated against FB1 was used to screen high binding affinity scFv antibodies; the best candidate was designated H2. Surface plasmon resonance measurements confirmed that the H2 scFv displayed a 82-fold higher binding affinity than its parent mAb. Direct competitive enzyme-linked immunosorbent assay demonstrated that the H2 antibody could competitively bind to free FB1, FB2, and FB3, with an IC50 of 0.11, 0.04, and 0.10 µM, respectively; it had no cross-reactivity to deoxynivalenol, nivalenol and aflatoxin. Validation assays with naturally contaminated samples revealed a linear relationship between the H2 antibody-based assay results and chemical analysis results, that could be expressed as y=1.7072x+5.5606 (R(2)=0.8883). Homology modeling of H2 revealed a favorable binding structure highly complementary to the three fumonisins. Molecular docking analyses suggested that the preferential binding of the H2 scFv to FB2 was due to the presence of a hydrogen radical in its R1 position, leading to a proper electrostatic matching and hydrophobic interaction. The H2 scFv antibody can be used for the rapid, accurate, and specific detection of fumonisin contamination in agricultural samples.

Chicken single-chain antibody fused to alkaline phosphatase detects Aspergillus pathogens and their presence in natural samples by direct sandwich enzyme-linked immunosorbent assay.

A sensitive and specific analytical method to detect ubiquitous aflatoxigenic Aspergillus pathogens is essential for monitoring and controlling aflatoxins. Four highly reactive chicken single-chain variable fragments (scFvs) against soluble cell wall proteins (SCWPs) from Aspergillus flavus were isolated by phage display. The scFv antibody AfSA4 displayed the highest activity toward both A. flavus and A. parasiticus and specifically recognized a surface target of their cell walls as revealed by immunofluorescence localization. Molecular modeling revealed a unique compact motif on the antibody surface mainly involving L-CDR2 and H-CDR3. As measured by surface plasmon resonance, AfSA4 fused to alkaline phosphatase had a higher binding capability and 6-fold higher affinity compared with AfSA4 alone. Immunoblot analyses showed that the fusion had good binding capacity to SCWP components from the two fungal species. Direct sandwich enzyme-linked immunosorbent assays with mouse antiaspergillus monoclonal antibody mAb2A8 generated in parallel as a capture antibody revealed that the detection limit of the two fungi was as low as 10(-3) µg/mL, 1000-fold more sensitive than that reported previously (1 µg/mL). The fusion protein was able to detect fungal concentrations below 1 µg/g of maize and peanut grains in both artificially and naturally contaminated samples, with at least 10-fold more sensitivity than that reported (10 µg/g) thus far. Thus, the fusion can be applied in rapid, simple, and specific diagnosis of Aspergillus contamination in field and stored food/feed commodities.

A phage-displayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains.

Fusarium and its poisonous mycotoxins are distributed worldwide and are of particular interest in agriculture and food safety. A simple analytical method to detect pathogens is essential for forecasting diseases and controlling mycotoxins. This article describes a proposed method for convenient and sensitive detection of Fusarium pathogens that uses the fusion of single-chain variable fragment (scFv) and alkaline phosphatase (AP). A highly reactive scFv antibody specific to soluble cell wall-bound proteins (SCWPs) of F. verticillioides was selected from an immunized chicken phagemid library by phage display. The antibody was verified to bind on the surface of ungerminated conidiospores and mycelia of F. verticillioides. The scFv-AP fusion was constructed, and soluble expression in bacteria was confirmed. Both the antibody properties and enzymatic activity were retained, and the antigen-binding capacity of the fusion was enhanced by the addition of a linker. Surface plasmon resonance measurements confirmed that the fusion displayed 4-fold higher affinity compared with the fusion's parental scFv antibody. Immunoblot analyses showed that the fusion had good binding capacity to the components from SCWPs of F. verticillioides, and enzyme-linked immunosorbent assays revealed that the detection limit of the fungus was below 10(-2) µg mL(-1), superior to the scFv antibody. The fusion protein was able to detect fungal concentrations as low as 10(-3) mg g(-1) of maize grains in both naturally and artificially contaminated samples. Thus, the fusion can be applied in rapid and simple diagnosis of Fusarium contamination in field and stored grain or in food.

Generation of a highly reactive chicken-derived single-chain variable fragment against Fusarium verticillioides by phage display.

Fusarium verticillioides is the primary causal agent of Fusarium ear and kernel rot in maize, producing fumonisin mycotoxins that are toxic to humans and domestic animals. Rapid detection and monitoring of fumonisin-producing fungi are pivotally important for the prevention of mycotoxins from entering into food/feed products. Chicken-derived single-chain variable fragments (scFvs) against cell wall-bound proteins from F. verticillioides were isolated from an immunocompetent phage display library. Comparative phage enzyme-linked immunosorbant assays (ELISAs) and sequencing analyses identified four different scFv antibodies with high sensitivity. Soluble antibody ELISAs identified two highly sensitive scFv antibodies, FvCA3 and FvCA4, with the latter being slightly more sensitive. Three-dimensional modeling revealed that the FvCA4 may hold a better overall structure with CDRH3, CDRL1 and CDRL3 centered in the core region of antibody surface compared with that of other scFvs. Immunofluorescence labeling revealed that the binding of FvCA4 antibody was localized to the cell walls of conidiospores and hyphae of F. verticillioides, confirming the specificity of this antibody for a surface target. This scFv antibody was able to detect the fungal mycelium as low as 10(-2) µg/mL and contaminating mycelium at a quantity of 10(-2) mg/g maize. This is the first report that scFv antibodies derived from phage display have a wide application for rapid and accurate detection and monitoring of fumonisin-producing pathogens in agricultural samples.

Attainment of 15-fold higher affinity of a Fusarium-specific single-chain antibody by directed molecular evolution coupled to phage display.

Fusarium head blight (FHB) caused by Fusarium graminearum infection is a devastating disease of wheat, maize, and other cereals. A previously isolated chicken single-chain Fv antibody (scFv), CWP2, that conferred durable resistance in planta was subjected to directed evolution by error-prone PCR and DNA shuffling, generating a mutated library. Panning of the mutated library against cell wall-bound proteins (CWPs) from F. graminearum by phage display enriched phage clones that were used for a further round of DNA shuffling to construct a combinatorial library comprising 3 × 10(6) variants. Screening of this library by phage display for variants reactive against the CWPs led to the identification of a number of clones. Comparative enzyme-linked immunosorbent assay analyses revealed eight clones exhibiting a higher reactivity than the parent, CWP2, and containing four different single-chain antibody sequences. Surface plasmon resonance measurements confirmed that three mutated scFvs, CWPa, CWPb, and CWPd, displayed 15-fold, 11-fold, and 7-fold higher affinities, respectively, compared with CWP2. Three-dimension modeling of CWPa illustrates a conformational change bringing all six complementary domain regions on the antibody surface in one direction. These results provide promising unique resistance molecules for effective control of FHB and its associated mycotoxins in food/feed chains.

Lower hybrid drift instability in a neutral sheet with O+ ions.

The electromagnetic lower-hybrid drift instability (LHDI) in the intermediate-wavelength regime k_(y)sqrt[rho_(i)rho_(e)] approximately 1 , where k_(y) and rho_(e,i) are the wave vector and the electron and ion gyroradii, respectively, in a thin plasma sheet containing electrons and H+ and O+ ions is examined using kinetic theory. It is shown that the growth rate of the LHDI first decreases and then increases with increase in the O+ content and temperature, with a minimum at a moderate level of the latter. The results can be relevant to understanding magnetic reconnection in the presence of LHDI.

Antibody-mediated prevention of Fusarium mycotoxins in the field.

Fusarium mycotoxins directly accumulated in grains during the infection of wheat and other cereal crops by Fusarium head blight (FHB) pathogens are detrimental to humans and domesticated animals. Prevention of the mycotoxins via the development of FHB-resistant varieties has been a challenge due to the scarcity of natural resistance against FHB pathogens. Various antibodies specific to Fusarium fungi and mycotoxins are widely used in immunoassays and antibody-mediated resistance in planta against Fusarium pathogens has been demonstrated. Antibodies fused to antifungal proteins have been shown to confer a very significantly enhanced Fusarium resistance in transgenic plants. Thus, antibody fusions hold great promise as an effective tool for the prevention of mycotoxin contaminations in cereal grains. This review highlights the utilization of protective antibodies derived from phage display to increase endogenous resistance of wheat to FHB pathogens and consequently to reduce mycotoxins in field. The role played by Fusarium-specific antibody in the resistance is also discussed.