Mechanisms of the enhanced antibacterial effect of Ag-TiO2 coatings

It has been demonstrated that Ag-TiO₂ nanocomposite coatings with excellent antimicrobial activity and biocompatibility have the potential to reduce infection problems. However, the mechanism of the synergistic effect of Ag-TiO₂ coatings on antibacterial efficiency is still not well understood. In this study, five types of Ag-TiO₂ nanocomposited coatings with different TiO₂ contents were prepared on a titanium substratum. Leaching tests indicated that the incorporation of TiO₂ nanoparticles into an Ag matrix significantly promoted Ag ion release. Surface energy measurements showed that the addition of TiO₂ nanoparticles also significantly increased the electron donor surface energy of the coatings. Bacterial adhesion assays with Escherichia coli and Staphylococcus aureus demonstrated that the number of adhered bacteria decreased with increasing electron donor surface energy. The increased Ag ion release rate and the increased electron donor surface energy contributed to an enhanced antibacterial efficiency of the coatings.     

纳米-TiO2对变异链球菌和白色假丝酵母菌抗菌机制的研究

目的 采用原子力显微镜对应用抗菌剂纳米Ag-Ti02作用后的口腔两种常见致病菌的分子形貌进行观测,为研究其抑菌机制提供有力的直观影像科学依据和可靠、直观的实验方法.方法 选择两种菌种:白色假丝酵母菌、变形链球菌,采用液体稀释法将纳米Ag-TiO2与两种菌相互作用,分别使用光学显微镜、原子力显微镜观察两种菌的细胞微观形态变化.结果 抗菌剂与两种菌作用后,细菌形态均有不同程度的改变,甚至是死亡.结论 原子力显微镜能直观地显示白色假丝酵母菌,变形链球菌的分子结构,通过本实验在研究纳米Ag-TiO2抗菌剂对白色假丝酵母菌,变形链球菌的抑菌机理形态学改变方面做了进一步的完善.     

Scrambled or bisected mouse eggs and the basis of patterning in mammals

Several findings challenge the notion that specification of cell types and embryonic axes in mammals are rooted entirely in the temporal and spatial relations between cleaving blastomeres. They raise the question as to whether, as in most non-mammalian species, these processes depend on information already present in the egg. However, experiments designed to investigate this possibility directly by perturbing the organization of the zygote or, very recently, by deleting one or other of its polar regions [M. Zernicka-Goetz. Fertile offspring derived from mammalian eggs lacking either animal or vegetal poles. Development 1998;125:4803–4808 (Ref. 1)], have been interpreted to mean that such a role for the egg can be discounted. This conclusion seems premature in view of continuing uncertainty regarding the developmental potential of individual blastomeres in mammals. BioEssays 21:271–274, 1999. © 1999 John Wiley & Sons, Inc.     

Two-step cell patterning on planar and complex curved surfaces by precision spraying of polymers

Controlling adhesion of living animal cells plays a key role in biosensor fabrication, drug-testing technologies, basic biological research, and tissue engineering applications. Current techniques for cell patterning have two primary limitations: (1) they require photolithography, and (2) they are limited to patterning of planar surfaces. Here we demonstrate a simple, precision spraying method for both positive and negative patterning of planar and curved surfaces to achieve cell patterns rapidly and reproducibly. In this method, which we call precision spraying (PS), a polymer solution is aerosolized, focused with sheath airflow through an orifice, and deposited on the substrate using a deposition head to create approximately 25 microm sized features. In positive patterning, adhesive molecules, such as laminin or polyethylenimine (PEI) were patterned on polydimethylsiloxane (PDMS) substrates in a single spraying operation. A variety of animal cell types were found to adhere to the adhesive regions, and avoid the non-adhesive (bare PDMS) regions. In negative patterning, hydrophobic materials, such as polytetrafluoroethylene (PTFE) and PDMS, were patterned on glass substrates. Cells then formed patterns on the exposed glass regions and avoided the hydrophobic regions. Cellular patterns were maintained for up to 2 weeks in the presence of serum, which normally fouls non-adhesive regions. Additionally, we found that precision spraying enabled micropatterning of complex-curved surfaces. Our results show that precision spraying followed by cell plating enables rapid and flexible cellular micropatterning in two simple steps.     

Construction of a triglyceride amperometric biosensor based on chitosan-ZnO nanocomposite film

A method is described for construction of a novel amperometric triglyceride (TG) biosensor based on covalent co-immobilization of lipase, glycerol kinase (GK) and glycerol-3-phosphate oxidase (GPO) onto chitosan (CHIT) and zinc oxide nanoparticles (ZnONPs) composite film deposited on the surface of Pt electrode. The enzymes-ZnONPs-CHIT composite was characterized by X-ray diffraction (XRD), scanning electron microscope (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The sensor showed optimum response within 6 s at pH 7.5 and temperature of 35 °C. The sensor measures current due to electrons generated at 0.4 V against Ag/AgCl from H₂O₂, which is produced from triolein by co-immobilized enzymes. A linear relationship was obtained between a wide triolein concentration range (50-650 mg/dl) and current (mA) under optimum conditions. The biosensor showed high sensitivity, low detection limit (20 mg/dl) and good storage stability (half-life of 7 months at 4 °C). The biosensor was unaffected modified by a number of serum substances at their physiological concentrations. The biosensor was evaluated and employed for determination of TG in sera in apparently healthy subjects and persons suffering from hypertriglyceridemia.     

Study of the attachment of Pseudomonas aeruginosa on gold and modified gold surfaces using surface plasmon resonance

This paper describes how the technique of surface plasmon resonance (SPR) can be utilized to follow (in real time) the attachment of Pseudomonas aeruginosa bacteria on bare gold and gold modified with a self-assembled monolayer (SAM) of mercaptounadecanoic acid. We show that SPR is able to discriminate between the adsorption of live versus dead (thermally shocked) bacteria. Moreover, the SPR distinguishes between the adsorption of wild-type versus mutant bacteria (single gene knockouts), the concentration of the bacterial suspension, and between bacteria adsorbing on SAM-modified and bare gold. SPR is able to measure bacterial adsorption within seconds of the bacterial suspension being introduced. Finally, a qualitative correlation between results from SPR with a crystal violet staining assay for different mutant bacteria was observed.     

Signal amplification on planar and gel-type sensor surfaces in surface plasmon resonance-based detection of prostate-specific antigen

This article describes surface plasmon resonance (SPR)-based detection of prostate-specific antigen (PSA), comparing amplification with colloidal gold (10nm diameter) and latex microspheres (120 nm diameter) on planar- and gel-type sensor surfaces. As matrix, 3% BSA in PBS was used. Experimental data were compared with model calculations that predict the SPR signal that results from covering of the different sensor surfaces with each of the particles used. Amplification with latex particles gave a higher signal than did that with colloidal gold. However, the limit of detection (LOD) attained by latex amplification was not as good as that obtained after gold amplification, and this was unexpected. LOD and sensitivity of the amplified PSA assays when performed with the planar-type sensor disc were equally good or better compared with those when performed with the gel-type sensor disc. Indirect evidence indicates a restricted accessibility of the gel layer on the gel-type sensor toward the colloidal gold. Application of colloidal gold led to a sensitivity increase of approximately three orders of magnitude compared with nonamplified detection. The corresponding LOD was approximately 0.15 ng PSA/ml, which is sufficient for measuring enhanced, clinically relevant PSA levels (>4 ng/ml).     

Adsorption of dansylated amino acids on molecularly imprinted surfaces: a surface plasmon resonance study

Surface plasmon resonance spectroscopy (SPR) was used to measure the adsorption kinetics and isotherms of dansylated amino acids onto surface-confined molecularly imprinted polymer films (MIP-Fs) and the corresponding non-imprinted polymer control films (NIP-Fs). The surface-confined polymer films were grafted from flat gold surfaces using atom transfer radical polymerization (ATRP). This approach allowed uniform nanothin films to be grown, thereby ensuring that the amino acids see a uniform surface during adsorption. N,N'-Didansyl-l-cystine (DDC) and didansyl-l-lysine (DDK) were used as the template molecules to form the MIP-Fs. Adsorption kinetics data were analyzed using single- and dual-site Langmuir adsorption models. It was found that, within the experimental measurement range, adsorption isotherm data were well described by any of four isotherm models: Langmuir, dual-site Langmuir, Freundlich, or Langmuir-Freundlich (LF). The relatively high heterogeneity index values regressed using the Freundlich and LF isotherms suggest the formation of fairly homogeneous MIP-Fs; although Scatchard analysis reveals binding site heterogeneity does exist. Selectivity studies showed that the MIP-Fs display cross-reactivity between DDC and DDK; nevertheless, MIP-Fs prepared against one template showed selectivity for that template. Solution pH and polymer layer thickness were studied as independent parameters to determine their impacts on amino acid adsorption, as monitored by SPR.     

Synthesis, patterning and applications of star-shaped poly(ethylene glycol) biofunctionalized surfaces

Poly(ethylene) glycol (PEG) is an excellent material to modify surfaces to resist non-specific protein adsorption. Linear PEG has been extensively studied both theoretically and experimentally and it has been found that resistance of PEG-coated surfaces to protein adsorption depends mainly on the molecular weight of the polymer and the surface grafting density. End-functionalized star-shaped PEGs allow for interpolymer crosslinking to form a dense layer. An excellent example of such a system consists of a 6-arm PEG/PPG (4 : 1) star polymer functionalized with isocyanate using IPDI. The end functionalization may be further biofunctionalized to recognize specific biomolecules such as streptavidin, His-tagged proteins, amino-terminated oligonucleotides and cell receptors. This functionalization may be patterned into specific geometries using stamping techniques or randomly distributed by statistical reaction of the end group with the biofunctional molecule in solution. The surface preparation uses simple spin-, dip- or spray-coating and produces smooth layers with low background fluorescence. These properties, together with the advantageous chemical properties of PEG, render the surfaces ideal for immobilizing proteins on surfaces with detection limits down to the single molecule level. Proteins immobilized on such surfaces are able to maintain their folded, functional form and are able to completely refold if temporarily exposed to denaturing conditions. Immobilized enzyme molecules were able to perform their function with the same activity as the enzyme in solution. Future directions of using surfaces coated with such crosslinked star polymers in highly sensitive and robust biotechnology applications will be discussed.     

Surface modification of silicon and gold-patterned silicon surfaces for improved biocompatibility and cell patterning selectivity

Clean silicon and gold-patterned silicon platforms were modified with methoxy-polyethylene glycol (M-PEG silane) via a self-assembly technique, which significantly improved their plasma protein resistance capability and cell patterning selectivity. Fibrinogen and IgG were used as model plasma proteins to study the efficacy of PEG layers in resisting protein adsorption. Selective cell patterning on the gold regions of a gold-patterned silicon substrate and tissue compatibility were studied with macrophage and fibroblast cells. The research also revealed how the presence of gold electrodes on a silicon substrate would influence the cell patterning selectivity. Our experimental results showed that the PEG-modified silicon surfaces had a high resistivity to protein and cell attachment and that the PEG-modified gold-patterned silicon surfaces nearly completely eliminated the protein adsorption and cell attachment on silicon. This study provides a new approach to developing biocompatible surfaces for silicon-based BioMEMS devices, particularly for biosensors where a metal-insulator format must be enforced.     

Defining the molecular basis of Arf and Hdm2 interactions.

Understanding the interaction of Arf and Hdm2 has recently become a central issue in cancer biology. In response to hyperproliferative signals, p14(Arf) stabilizes p53 by binding to Hdm2 and inhibits the ubiquitination and subsequent proteosome-dependent degradation of p53. The medical importance of the Arf-Hdm2-p53 regulatory system is highlighted by the finding that either p53 or p14(Arf) are lost or modified in virtually all human cancers. Isolated Arf and Hdm2 domains are dynamically disordered in solution, yet they retain the ability to interact in vitro and in cellular assays. Upon binding, domains of both Arf and Hdm2 undergo a dramatic transition from disordered conformations to extended structures comprised of beta-strands. The presence of domains from both proteins are necessary and sufficient for the formation of the highly stable extended beta structures. We have mapped sites within Arf and Hdm2 that interact at a resolution of five amino acid residues using surface plasmon resonance. Surface plasmon resonance and circular dichroism spectropolarimetry confirm the presence of multiple interaction domains within each protein. Both p14(Arf) (human) and p19(Arf) (mouse) interact with Hdm2 through two short motifs present in their N termini. The Arf interacting region of Hdm2 is also composed of two short sequences located in the central acidic domain, between residues 235-264 and 270-289. The binding-induced structural transition is also induced by short peptides, 15 amino acids in length, that contain the binding motifs. Micro-injection and live cell imaging of proteins tagged with fluorescent labels was used to confirm the in vivo function of the interaction domains. Arf and Hdm2 thus appear to interact through a novel mechanism that exerts control over the cell division cycle. The novel molecular mechanism of interaction and the limited size of the protein domains involved provide opportunities for the development of anticancer therapeutics.     

A mechanically strong, flexible and conductive film based on bacterial cellulose/graphene nanocomposite

A highly flexible nanocomposite film of bacterial cellulose (BC) and graphene oxide (GO) with a layered structure was presented using the vacuum-assisted self-assembly technique. Microscopic and X-ray diffraction measurements demonstrated that the GO nanosheets were uniformly dispersed in the BC matrix. The interactions between BC and GO were studied by Fourier transformation infrared spectroscopy. Compared with pristine BC, the integration of 5 wt% GO resulted in 10% and 20% increase in Young's modulus and tensile strength of the composite film. The electrical conductivity of the composite film containing 1 wt% GO after in situ reduction showed a remarkable increase by 6 orders of magnitude compared with the insulated BC.     

Cell autonomous and non-cell autonomous functions of Otx2 in patterning the rostral brain.

Previous studies have shown that the homeobox gene Otx2 is required first in the visceral endoderm for induction of forebrain and midbrain, and subsequently in the neurectoderm for its regional specification. Here, we demonstrate that Otx2 functions both cell autonomously and non-cell autonomously in neurectoderm cells of the forebrain and midbrain to regulate expression of region-specific homeobox and cell adhesion genes. Using chimeras containing both Otx2 mutant and wild-type cells in the brain, we observe a reduction or loss of expression of Rpx/Hesx1, Wnt1, R-cadherin and ephrin-A2 in mutant cells, whereas expression of En2 and Six3 is rescued by surrounding wild-type cells. Forebrain Otx2 mutant cells subsequently undergo apoptosis. Altogether, this study demonstrates that Otx2 is an important regulator of brain patterning and morphogenesis, through its regulation of candidate target genes such as Rpx/Hesx1, Wnt1, R-cadherin and ephrin-A2.     

Proximal to distal cell communication in the Drosophila leg provides a basis for an intercalary mechanism of limb patterning.

Proximodistal patterning in the Drosophila leg is elaborated from the circular arrangement of the proximal domain expressing escargot and homothorax, and the distal domain expressing Distal-less that are allocated during embryogenesis. The distal domain differentiates multiply segmented distal appendages by activating additional genes such as dachshund. Secreted signaling molecules Wingless and Decapentaplegic, expressed along the anterior-posterior compartment boundary, are required for activation of Distal-less and dachshund and repression of homothorax in the distal domain. However, whether Wingless and Decapentaplegic are sufficient for the circular pattern of gene expression is not known. Here we show that a proximal gene escargot and its activator homothorax regulate proximodistal patterning in the distal domain. Clones of cells expressing escargot or homothorax placed in the distal domain induce intercalary expression of dachshund in surrounding cells and reorient planar cell polarity of those cells. Escargot and homothorax-expressing cells also sort out from other cells in the distal domain. We suggest that inductive cell communication between the proximodistal domains, which is maintained in part by a cell-sorting mechanism, is the cellular basis for an intercalary mechanism of the proximodistal axis patterning of the limb.