Enhanced synergistic effects from multiple iron oxide nanoparticles encapsulated within nitrogen-doped carbon nanocages for simple and label-free visual detection of blood glucose.

Hollow-structured carbon materials play a crucial role in research of biosensors, energy storage and nanomedicine as a kind of material with advantages like high surface area, tunable pore volume, excellent mechanical properties, and good biocompatibility. Herein, we developed a simple, facile and controllable method for synthesis of Fe3O4 nanoparticles encapsulated in hollow carbon nanocages (FNHCs) with SiO2 nanospheres as a sacrificial template. Owing to the unique structure of multiple Fe3O4 nanoparticles cores integrated with N-doped carbon nanocages, the as-synthesized FNHCs exhibited greatly enhanced peroxidase mimicking activity with extremely high signal-to-noise ratio of ∼91 fold. Also, it was found that the FNHCs possessed a higher peroxidase-like activity than that of other similar-structured Fe3O4 architectures (e.g. Fe3O4@C NPs). The resulting steady-state kinetic curve demonstrated the enzymatic activity of FNHCs with classic Michaelis-Menton kinetics following a ping-pong mechanism. On the basis of the superior enzymatic activity, the FNHCs performed as a high-efficiency peroxidase mimic, realizing facile, label-free, highly sensitive/selective colorimetric detection of H2O2 and glucose. Furthermore, the colorimetric sensor successfully determined glucose in patients' serum samples with high accuracy and precision, suggesting great potential for real applications.

TLR2 Ligand Pam3CSK4 Regulates MMP-2/9 Expression by MAPK/NF-κB Signaling Pathways in Primary Brain Microvascular Endothelial Cells.

Blood-brain barrier (BBB) destruction is associated with a variety of neurological diseases. Brain microvascular endothelial cells (BMECs) are the key constituent of BBB. Both matrix metalloproteinases-2/9 (MMP-2/9) and toll-like receptor-2 (TLR2) are coexpressed in BMECs and have been shown to play important roles in BBB breakdown. It is unknown whether TLR2 can regulate MMP-2/9 in BMECs. In this study, Pam3CSK4 was used to activate TLR2, and the expression of MMP-2/9 and tight junctions (TJs) in BBB was measured by quantitative real-time PCR and western blotting. Phosphoproteins were determined by western blotting. The inhibitors of mitogen-activated protein kinases (MAPKs) and NF-κB were used to identify the signaling pathways by which TLR2 regulates the expression of MMP-2/9 in BMECs. This study showed that Pam3CSK4 upregulated the mRNA and protein expression of MMP-9 and downregulated MMP-2 and TJ expression in BMECs simultaneously. Pam3CSK4 also induced the phosphorylation of MAPKs and NF-κB signaling pathways in BMECs. MMP-9 expression was found to decrease by pretreatment with inhibitors of ERK1/2 and JNK but not p38. However, the mRNA and protein expression of MMP-2 and MMP-9 increased after addition of a NF-κB inhibitor. Our results indicated that Pam3CSK4 was able to upregulate MMP-9 expression through ERK1/2 and JNK signaling pathways, but the NF-κB signaling pathway negatively regulated the effect of TLR2 on MMP-2 and MMP-9 expression in BMECs. The finding provides novel insight into the molecular mechanism of MMP-2/9 expression in BMECs.

Gold@Halloysite nanotubes-chitin composite hydrogel with antibacterial and hemostatic activity for wound healing.

Infection and healing of wounds after injury has always been an unavoidable problem in daily life, so design of a biomaterial with antibacterial and good wound healing properties is highly needed. Herein, a wound healing hydrogel dressing with halloysite clay and chitin as the main components was prepared, which combines the advantages of the biomacromolecule and clay. Halloysite nanotubes (HNTs) are extremely biocompatible clay materials with a hollow tubular structure, and the inner and outer surfaces of HNTs are composed of SiOx and AlOx layers with different charges. Au nanoparticles with diameter in 5-10 nm were filled into the HNTs' lumen to endow photothermal effect of the clay materials. Au@HNTs was then mixed with chitin solution to prepare flexible composite hydrogel by crosslinking by epichlorohydrin. The antibacterial properties, biocompatibility and hemostatic properties of the hydrogel material were investigated by antibacterial experiments, cell experiments, mouse liver and tail hemostatic experiments. After infecting the back wound of mice with Staphylococcus aureus, the hydrogel was applied to the wound to further verify the killing effect on bacteria and wound healing effect of the hydrogel material in vivo. The Au@HNTs-chitin composite hydrogel exhibits high antibacterial and hemostatic activity as well as promoting wound healing function with low cytotoxicity. This study is significant for the development of high-performance wound dressings based on two commonly used biocompatible materials, which shows promising application in wound sterilization and healing.

Sustainable, High-Performance, and Biodegradable Plastics Made from Chitin.

A high-performance biodegradable plastic was made from a chitin KOH/urea solution. The solution was transferred into a hydrogel by cross-linking using epichlorohydrin and ethanol immersion, and a chitin bioplastic was finally prepared by drying in a mold at 40 °C. The solution concentration positively impacts viscosity, crystallinity, and smoothness. A 4% chitin bioplastic exhibits high barrier properties, flame retardancy, high-temperature resistance, mechanical properties (tensile strength up to 107.1 MPa), and soil degradation properties. The chitin bioplastic can be completely degraded by microorganisms in 7 weeks. In addition, biosafety tests suggest that chitin is safe for cells and crops (wheat and mung beans). The chitin bioplastic was further applied to containers, straws, cups, and photoprotection, and it was found that the water resistance and transparency were comparable to those of commercial polypropylene plastics. Due to the excellent performance, safety, and sustainability of the chitin bioplastic, it is expected to become a good substitute for conventional fossil fuel-based plastics.

[In vivo study on the effects of intercellular adhesion operon of Staphylococcus epidermidis on the inflammation associated with bacteria-fungal mixed biofilm].

OBJECTIVE: To study the effect of intercellular adhesion (ica) operon of Staphylococcus epidermidis on the inflammation associated with mixed biofilm of Staphylococcus epidermidis and Candida albicans on endotracheal tube material in rabbits. METHODS: The standard strains of Staphylococcus epidermidis RP62A (ica operon positive, positive group) and ATCC12228 (ica operon negative, negative group) were taken to prepare a bacterial solution with a concentration of 1×10 6 CFU/mL, respectively. Then, the two bacterial solutions were mixed with the standard strain of Candida albicans ATCC10231 of the same concentration to prepare a mixed culture solution at a ratio of 1∶1, respectively. The mixed culture solution was incubated with endotracheal tube material for 24 hours. The formation of mixed biofilm on the surface of the material was observed by scanning electron microscope. Thirty New Zealand rabbits, aged 4-6 months, were divided into two groups ( n=15), and the endotracheal tube materials of the positive group and the negative group that were incubated for 24 hours were implanted beside the trachea. The body mass of rabbits in the two groups was measured before operation and at 1, 3, and 7 days after operation. At 1, 3, and 7 days after operation, the levels of interleukin 1ß (IL-1ß), IL-6, tumor necrosis factor α (TNF-α), and monocytechemotactic protein 1 (MCP-1) were detected by using an ELISA test kit. At 7 days after operation, the formation of mixed biofilm on the surface of the endotracheal tube materials was observed by scanning electron microscope, the inflammation and infiltration of tissues around the materials were observed by HE staining, and the bacterial infections in heart, lung, liver, and kidney were observed by plate colony counting method. RESULTS: Scanning electron microscope observation showed that the mixed biofilm structure was obvious in the positive group after 24 hours in vitro incubation, but no mixed biofilm formation was observed in the negative group. In vivo studies showed that there was no significant difference in body mass between the two groups before operation and at 1, 3, and 7 days after operation ( P>0.05). Compared with the negative group, the levels of MCP-1 and IL-1ß at 1 day, and the levels of IL-1ß, MCP-1, IL-6, and TNF-α at 3 and 7 days in the positive group all increased, with significant differences ( P<0.05). Scanning electron microscope observation showed that a large amount of Staphylococcus epidermis and mixed biofilm structure were observed in the positive group, and a very small amount of bacteria was observed in the negative group with no mixed biofilm structure. HE staining of surrounding tissue showed inflammatory cell infiltration in both groups, and neutrophils and lymphocytes were more in the positive group than in the negative group. There was no significant difference in the number of bacterial infections in heart and liver between the two groups ( P>0.05). The number of bacterial infections in lung and kidney in the positive group was higher than that in negative group ( P<0.05). CONCLUSION: In the mixed infection of Staphylococcus epidermidis and Candida albicans, the ica operon may strengthen the structure of the biofilm and the spread of the biofilm in vivo, leading to increased inflammatory factors, and the bacteria are difficult to remove and persist.

Mechanistic study on the inhibition of Staphylococcus epidermidis biofilm by agrC-specific binding polypeptide.

BACKGROUND: Considering the wide-spread misuse of antibiotics, the development of new antibacterial drugs may effectively prevent the emergence of antibiotic resistance in bacteria. The understanding of the mechanism underlying the Staphylococcus epidermidis agrC-specific binding polypeptide-mediated inhibition of S. epidermidis biofilm formation may supply ideas for the development of new antibacterial drugs. METHODS: S. epidermidis cells were cultured with different concentrations (0, 100, 200, 400, 800, and 1,600 µg/mL) of agrC-specific binding polypeptide (N1) and blank (N0). Crystal violet staining was performed to test the formation of biofilms and to determine the best concentration of agrC-specific binding polypeptides, and the bacterial inhibitory concentration was also determined. At different time points (6, 12, 18, 24, and 30 h), XTT assay was used to measure bacterial viability, and the real-time quantitative polymerase chain reaction was performed to measure the expression of atlE, icaA, fbe, and icaR genes. The sulfuric acid-phenol method was used to determine polysaccharide intercellular adhesin (PIA) levels. RESULTS: The biofilm formation ability of S. epidermidis was the lowest after treatment with 800 µg/mL agrC-specific binding polypeptide. After 6 h of culture, agrC-specific binding polypeptide upregulated the expression of atlE, icaA, fbe, and icaR and increased the bacterial viability. However, the polypeptide downregulated the expression of atlE, icaA, fbe, and icaR and inhibited S. epidermidis growth and PIA formation after 12 h of culture. Although agrC-specific binding polypeptide upregulated the expression of atlE, icaA, fbe, and icaR after 18 h, they inhibited bacterial growth and PIA formation. CONCLUSIONS: Thus, agrC-specific binding polypeptide could downregulate the expression of atlE, icaA, fbe, and icaR and inhibit PIA formation by S. epidermidis after 12 h, demonstrating its transient inhibitory effects on the biofilm formation ability of S. epidermidis. Its effective concentration was 800 µg/mL.

[Establishment of Method for Detecting Red Blood Cell Osmotic Fragility by Flow Cytometry].

OBJECTIVE: To establish a new method for detection of red blood cell osmotic fragility by using flow cytometry. METHODS: The hypotension salt solution of different concentrations (0.70 ml normal saline+0.3 ml deionized water, 0.60 ml normal saline+0.40 ml deionized water and 0.55 ml normal saline+0.45 ml deionized water) were prepared with normal saline and deionized water, in which the red blood cells were suspended, and the residual red blood cells were detected by flow cytometer. RESULTS: There was no significant difference in percentage of residual red blood cells between different time points detected by flow cytometer in 3 different hypotonic salt solutions. The percentage of residual red blood cells in B+C+D+E+F+G detected time region was different among 3 NaCl dilution groups. The percentage of residual red blood cells in normal control was lower than that in hemoglobinopathy group. The percentage of residual red blood cells in hereditary spherocytosis (HS) group was obviously lower than that in hemoglobinopathy and normal control groups. The comparison of 3 different dilution concentrations found that the second concentration (0.60 ml normal saline+0.40 ml deionized water) is more suitable to screen HS by FC500 flow cytometer. CONCLUSION: The detection of red cell osmotic fragility by using flow cytometry is a simple, rapid, objective and economic way that can be an effective screening method for diagnose the HS.

[FUNCTION OF INTERCELLULAR ADHESION A, FIBRINOGEN BINDING PROTEIN, AND ACCUMULATION-ASSOCIATED PROTEIN GENES IN FORMATION OF STAPHYLOCOCCUS EPIDERMIDIS-CANDIDA ALBICANS MIXED SPECIES BIOFILMS].

OBJECTIVE: To explore the function of intercellular adhesion A (icaA), fibrinogen binding protein (fbe), and accumulation-associated protein (aap) genes in formation of Staphylococcus epidermidis-Candida albicans mixed species biofilms. METHODS: The experiment was divided into 3 groups: single culture of Staphylococcus epidermidis ATCC35984 (S. epidermidis group) or Candida albicans ATCC10231 (C. albicans group), and co-culture of two strains (mixed group) to build in vitro biofilm model. Biofilm mass was detected by crystal violet semi-quantitative adherence assay at 2, 4, 6, 8, 12, 24, 48, and 72 hours after incubation. XTT assay was performed to determine the growth kinetics in the same time. Scanning electron microscopy (SEM) was used to observe the ultrastructure of the biofilms after 24 and 72 hours of incubation. The expressions of icaA, fbe, and aap genes were analyzed by real-time fluorescent quantitative PCR. RESULTS: Crystal violet semi-quantitative adherence assay showed that the biofilms thickened at 12 hours in the S. epidermidis and mixed groups; after co-cultured for 72 hours the thickness of biofilm in mixed group was more than that in the S. epidermidis group, and there was significant difference between 2 groups at the other time (P < 0.05) except at 72 hours (P > 0.05). In C. albicans group, the biofilm started to grow at 12 hours of cultivation, but the thickness of the biofilm was significantly lower than that in the mixed group in all the time points (P < 0.05). XTT assay showed that the overall growth speed in the mixed group was greater than that in the C. albicans group, and it was greater than that in the S. epidermidis group at 48 hours; there was no significant difference in the growth speed between the mixed groups and the S. epidermidis group in the other time points (P > 0.05) except at 12 hours (P < 0.05). The absorbance (A) value in the mixed group was lower than that in the S. epidermidis group at 2 and 4 hours, but no significant difference was shown (P > 0.05); the A value of mixed group was significantly higher than that of the C. albicans group after 6 hours (P < 0.05). SEM observation showed that mature biofilms with complex structure formed in all groups. The real-time fluorescent quantitative PCR showed the expressions of fbe, icaA, and aap genes in mixed group increased 1.93, 1.52, and 1.46 times respectively at 72 hours compared with the S. epidermidis group (P < 0.05). CONCLUSION: Mixed species biofilms have more complex structure and are thicker than single species biofilms of Staphylococcus epidermidis or Candida albicans, which is related to increased expressions of the icaA, fbe, and aap genes of Staphylococcus epidermidis.

[Effect of brominated furanones on biofilm formation of Escherichia coli on polyvinyl chloride material].

OBJECTIVE: To study the influence of brominated furanones on the biofilm formation of Escherichia coli on the polyvinyl chloride (PVC) material, and to provide new ideas for the research of surface modification of materials and clinical treatment of biomaterial centered infection. METHODS: Three brominated furanones with representative chemical structure were chosen and coated on the surface modification of PVC materials, respectively [furanone 1: 3, 4-dibromo-5-hydroxy-furanone; furanone 2: 4-bromo-5-(4-methoxyphenyl)-3-(methylamino)-furanone; furanone 3: 3, 4-dibromo-5, 5-bis (4-methylphenyl)-2 (5H)-furanone]. All the modificated PVC materials and Escherichia coli were co-cultivated. The PVC material soaked with 75% ethanol for 5 minutes and Escherichia coli were co-cultivated together as the control group. The thickness of bacterial community and bacterial community quantity in the unit area on PVC materials were measured by confocal laser scanning microscope (CLSM), and the surface structure of biofilm formation was observed by scanning electron microscope (SEM). RESULTS: The CLSM showed that the thickness of bacterial community and the bacterial community quantity in the unit area of PVC materials was significantly less (P < 0.05) in furanone 3 group than in control group, but no significant difference (P > 0.05) was found between furanone 1, furanone 2 groups and control group. SEM showed that the quantity of bacterial community in the unit area of PVC materials surface in furanone 3 group was fewer than that in control group at 6 hours; the biofilm structure on PVC materials surface formed at 18 hours in control group, furanone 1 group, and furanone 2 group, but there was no mature biofilm structure on PVC materials surface in furanone 3 group at 18 hours. CONCLUSION: The impact of different brominated furanones on Escherichia coli biofilm formation on the surface of PVC materials is different, 3, 4-dibromo-5, 5-bis (4-methylphenyl)-2 (5H)-furanone can inhibit Escherichia coli biofilm formation on the surface of PVC material. Polyvinyl chloride