Bactericidal activity of photocatalytic TiO(2) reaction: toward an understanding of its killing mechanism.

When titanium dioxide (TiO(2)) is irradiated with near-UV light, this semiconductor exhibits strong bactericidal activity. In this paper, we present the first evidence that the lipid peroxidation reaction is the underlying mechanism of death of Escherichia coli K-12 cells that are irradiated in the presence of the TiO(2) photocatalyst. Using production of malondialdehyde (MDA) as an index to assess cell membrane damage by lipid peroxidation, we observed that there was an exponential increase in the production of MDA, whose concentration reached 1.1 to 2.4 nmol. mg (dry weight) of cells(-1) after 30 min of illumination, and that the kinetics of this process paralleled cell death. Under these conditions, concomitant losses of 77 to 93% of the cell respiratory activity were also detected, as measured by both oxygen uptake and reduction of 2,3,5-triphenyltetrazolium chloride from succinate as the electron donor. The occurrence of lipid peroxidation and the simultaneous losses of both membrane-dependent respiratory activity and cell viability depended strictly on the presence of both light and TiO(2). We concluded that TiO(2) photocatalysis promoted peroxidation of the polyunsaturated phospholipid component of the lipid membrane initially and induced major disorder in the E. coli cell membrane. Subsequently, essential functions that rely on intact cell membrane architecture, such as respiratory activity, were lost, and cell death was inevitable.     

Photoinduced bactericidal activity of TiO2 films

A decline in CFU of gram-positive and gram-negative bacteria on the surface of UV illuminated TiO2 films (wavelength of 380 nm) is shown. A 29, 45, and 47% decrease in bacterial viability of Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli, respectively, was seen following a 12-min exposition. It was first discovered that the reuse of TiO2 films to test a bacterial suspension for viability removes UV-induced bactericidal activity. However, annealing of TiO2 at a temperature above 400 degrees C restores the photoinduced bactericidal activity to its initial state.     

Conformational Transition of Giant DNA in a Confined Space Surrounded by a Phospholipid Membrane

It has been established that a long DNA molecule exhibits a large discrete conformational change from a coiled state to a highly folded state in aqueous solution, depending on the presence of various condensing agents such as polyamines. In this study, T4 DNA labeled with fluorescent dyes was encapsulated in a cell-sized microdroplet covered with a phospholipid membrane to investigate the conformational behavior of a DNA molecule in such a confined space. Fluorescence microscopy showed that the presence of Mg²⁺ induced the adsorption of DNA onto the membrane inner-surface of a droplet composed of phosphatidylethanolamine, while no adsorption was observed onto a phosphatidylcholine membrane. Under the presence of spermine (tetravalent amine), DNA had a folded conformation in the bulk solution. However, when these molecules were encapsulated in the microdroplet, DNA adsorbed onto the membrane surface accompanied by unfolding of its structure into an extended coil conformation under high concentrations of Mg²⁺. In addition, DNA molecules trapped in large droplets tended not to be adsorbed on the membrane, i.e., no conformational transition occurred. A thermodynamic analysis suggests that the translational entropy loss of a DNA molecule that is accompanied by adsorption is a key factor in these phenomena under micrometer-scale confinement.     

Early adhesion of human mesenchymal stem cells on TiO(2) surfaces studied by single-cell force spectroscopy measurements

Understanding the interactions involved in the adhesion of living cells on surfaces is essential in the field of tissue engineering and biomaterials. In this study, we investigate the early adhesion of living human mesenchymal stem cells (hMSCs) on flat titanium dioxide (TiO(2) ) and on nanoporous crystallized TiO(2) surfaces with the use of atomic force microscopy-based single-cell force spectroscopy measurements. The choice of the substrate surfaces was motivated by the fact that implants widely used in orthopaedic and dental surgery are made in Ti and its alloys. Nanoporous TiO(2) surfaces were produced by anodization of Ti surfaces. In a typical force spectroscopy experiment, one living hMSC, immobilized onto a fibronectine-functionalized tipless lever is brought in contact with the surface of interest for 30 s before being detached while recording force-distance curves. Adhesion of hMSCs on nanoporous TiO(2) substrates having inner pore diameter of 45 nm was lower by approximately 25% than on TiO(2) flat surfaces. Force-distance curves exhibited also force steps that can be related to the pulling of membrane tethers from the cell membrane. The mean force step was equal to 35 pN for a given speed independently of the substrate surface probed. The number of tethers observed was substrate dependent. Our results suggest that the strength of the initial adhesion between hMSCs and flat or nanoporous TiO(2) surfaces is driven by the adsorption of proteins deposited from serum in the culture media.     

Bactericidal effect of Fe2+, ceruloplasmin, and phosphate.

Fe2+, when combined with ceruloplasmin or phosphate, was bactericidal to Escherichia coli at pH 5.0, and when Fe2+, ceruloplasmin, and phosphate were combined, a bactericidal effect was observed under conditions, i.e., short incubation period, in which Fe2+ plus ceruloplasmin and Fe2+ plus phosphate were ineffective. Bactericidal activity increased with the ceruloplasmin or phosphate concentration to a maximum and then decreased as their concentration was further increased. Fe2+ was oxidized in the presence of ceruloplasmin, phosphate, or, in particular, a combination of the two. A bactericidal effect was observed when there was only a partial loss of Fe2+, with more extensive oxidation resulting in a loss of bactericidal activity. The bactericidal effect of Fe2+ plus ceruloplasmin and/or phosphate was unaffected by catalase or superoxide dismutase and was not associated with iodination. Fe-EDTA was also bactericidal at an Fe2+: EDTA molar ratio of 1:0.5, where Fe2+ was partially oxidized. However, in contrast to Fe2+ plus ceruloplasmin and/or phosphate, bactericidal activity was inhibited by catalase and was associated with iodination. Combinations of Fe2+ and Fe3+ were not bactericidal under the conditions employed. A requirement for Fe2+ plus either a product of Fe2+ oxidation or an iron ceruloplasmin and/or phosphate chelate for bactericidal activity is proposed.     

The leucine rich amelogenin protein (LRAP) adsorbs as monomers or dimers onto surfaces

Amelogenin is believed to be involved in controlling the formation of the highly anisotropic and ordered hydroxyapatite crystallites that form enamel. The adsorption behavior of amelogenin proteins onto substrates is very important because protein–surface interactions are critical to its function. We have previously used LRAP, a splice variant of amelogenin, as a model protein for the full-length amelogenin in solid-state NMR and neutron reflectivity studies at interfaces. In this work, we examined the adsorption behavior of LRAP in greater detail using model self-assembled monolayers containing COOH, CH₃, and NH₂ end groups as substrates. Dynamic light scattering (DLS) experiments indicated that LRAP in phosphate buffered saline and solutions containing low concentrations of calcium and phosphate consisted of aggregates of nanospheres. Null ellipsometry and atomic force microscopy (AFM) were used to study protein adsorption amounts and quaternary structures on the surfaces. Relatively high amounts of adsorption occurred onto the CH₃ and NH₂ surfaces from both buffer solutions. Adsorption was also promoted onto COOH surfaces only when calcium was present in the solutions suggesting an interaction that involves calcium bridging with the negatively charged C-terminus. The ellipsometry and AFM studies revealed that LRAP adsorbed onto the surfaces as small subnanosphere-sized structures such as monomers or dimers. We propose that the monomers/dimers were present in solution even though they were not detected by DLS or that they adsorbed onto the surfaces by disassembling or “shedding” from the nanospheres that are present in solution. This work reveals the importance of small subnanosphere-sized structures of LRAP at interfaces.     

Microplate-based assays for the evaluation of antibacterial effects of photocatalytic coatings

Three microplate-based viability assays for assessing the antibacterial effects of photocatalytic coatings were compared to the conventional colony count method. In the experimental design, cultured Escherichia coli were exposed to photocatalysis on various TiO₂ films in the presence of either UVA or visible light. The photocatalytic effects on the bacterial physiology were determined by real-time measurements of metabolic activity (XTT assay), biomass formation in the liquid medium (growth assay), and by assessing membrane integrity (with propidium iodide and SYTO 9 fluorescent nucleic acid binding dyes—BacLight assay). All three methods proved to be more sensitive and reproducible than colony count for the evaluation of the bactericidal effect of photocatalysis, XTT, and growth assay succeeded in detecting differences in both UVA and visible light-activated photocatalytic coatings. BacLight could efficiently detect the visible light-dependent photocatalytic effect on bacteria and identify membrane damage, but resulted inadequate for evaluating the UVA-dependent antibacterial effects. The described microplate-based evaluation methods proved being more effective and rapid than the colony count assay for assessing the antibacterial effect of various photocatalytic coatings.     

Modulation of intestinal absorption of calcium]

1. Absorption of ingested calcium (2 ml of a 10mM CaCl2 solution + 45Ca) by the adult rat was shown to be facilitated by the simultaneous ingestion of an active carbohydrate, L-arabinose. As the carbohydrate concentration is increased from 10 to 200 mM, the adsorption of calcium is maximized at a level corresponding to about twice the control adsorption level. 2. A similar doubling of calcium adsorption is obtained when a 100 mM concentration of any one of a number of other carbohydrates (gluconic acid, mannose, glucosamine, sorbitol, lactose, raffinose, stachyose) is ingested simultaneously with a 10 mM CaCl2 solution. 3. Conversely, the simultaneous ingestion of increasing doses (10 to 100 mM) of phosphate (NaH2PO4) with a 10 mM CaCl2 solution results in decreased 45Ca absorption and retention by the adult rat. 4. The maximum inhibition of calcium adsorption by phosphate is independent of the concentration of the ingested calcium solution (from 5 to 50 mM CaCl2). 5. The simultaneous ingestion of CaCl2 (10 mM) with lactose and sodium phosphate (50 and 10 mM, respectively) shows that the activating effect of lactose upon 45Ca adsorption may be partly dissimulated by the presence of phosphate. 6. These various observations indicate that, within a large concentration range (2 to 50 mM CaCl2), calcium adsorption appears to be a precisely modulated diffusion process. Calcium absorption varies (between minimum and maximum levels) as a function of the state of saturation by the activators (carbohydrates) and inhibitors (phosphate) of the calcium transport system.     

Photocatalytic inactivation of bacteriophages by TiO2-coated glass plates under low-intensity, long-wavelength UV irradiation

The International Organization for Standardization (ISO) was used to evaluate antibacterial activity by titanium dioxide (TiO(2)) photocatalysis since 2006. We evaluated photocatalytic inactivation of Qβ and T4 bacteriophages induced by low-intensity, long-wavelength ultraviolet A (UVA; 0.1 mW cm(-2) and 0.001 mW cm(-2)) irradiation on a TiO(2)-coated glass plate using the ISO methodology. The results indicated that both bacteriophages were inactivated at 0.001 mW cm(-2) UVA. The intensity of UV light, including long-wavelength light (UVA), is very low in an actual indoor environment. Thus, TiO(2) photocatalysis can be beneficial for inactivating viruses in an indoor environment. Experiments using qPCR and bovine serum albumin degradation assume that viral inactivation is caused by outer viral protein disorder and not by viral RNA reduction by reactive oxygen species produced during TiO(2) photocatalysis. Furthermore, we showed that the ISO methodology for standard testing of antibacterial activity by TiO(2) photocatalysis can be applied to assess antiviral activity.     

Resistance of spheroplasts and whole cells of Pseudomonas aeruginosa to bactericidal activity of various biocides: evidence of the membrane implication

To emphasise the role of outer and inner membranes in the resistance of Pseudomonas aeruginosa to bactericidal activity of various disinfectants, spheroplasts and whole cells were compared. Spheroplasts are more sensitive than whole cells to quaternary ammonium compounds such as didecyl dimethyl ammonium bromide (DDAB) and C16-benzalkonium chloride. The outer membrane acts as a barrier to prevent these disinfectants from entering the cell. It seems to have no influence on activities of smaller molecules such as C12, C14-benzalkonium chlorides and sodium dichloroisocyanurate. For tri-sodium phosphate, the presence of outer membrane emphasized the action of the molecule. Moreover, resistance of DDAB-adapted spheroplasts to bactericidal activity of DDAB is higher than the resistance of non-adapted spheroplasts. This suggests that the inner membrane could also play a role in resistance to DDAB.     

COUNTERACTION OF THE INHIBITING EFFECTS OF VARIOUS SUBSTANCES ON NITELLA

When living cells of Nitella are first exposed to (1) phosphate buffer mixture, or (2) phosphoric acid, or (3) hydrochloric acid, or (4) sodium chloride, or (5) sodium borate, and are then placed in a solution of brilliant cresyl blue made up with a borate buffer mixture at pH 7.85, the rate of penetration of the dye into the vacuole is decreased as compared with the rate in the case of cells transferred directly from tap water to the same dye solution. When cells exposed to any one of these solutions are placed in the dye solution made up with phosphate buffer solution at pH 7.85, the rate of penetration of dye into the vacuole is the same as the rate in the case of cells transferred from the tap water to the same dye solution. It is probable that this removal of the inhibiting effect is due primarily to the presence of certain concentration of sodium and potassium ions in the phosphate buffer solution. If a sufficient concentration of sodium ions is added to the dye made up with a borate buffer mixture the inhibiting effect is removed just as it is in the case of the dye made up with the phosphate buffer mixture. The inhibiting effect of some of these substances is found to be removed by the dye containing a sufficient concentration of bivalent cations, or by washing the cells with salts of bivalent cations. The inhibiting effect and its removal are discussed from a theoretical standpoint.     

Chemotaxis of Spirochaeta aurantia: involvement of membrane potential in chemosensory signal transduction.

The effects of valinomycin and nigericin on sugar chemotaxis in Spirochaeta aurantia were investigated by using a quantitative capillary assay, and the fluorescent cation, 3,3'-dipropyl-2,2'-thiodicarbocyanine iodide was used as a probe to study effects of chemoattractants on membrane potential. Addition of a chemoattractant, D-xylose, to cells in either potassium or sodium phosphate buffer resulted in a transient membrane depolarization. In the presence of valinomycin, the membrane potential of cells in potassium phosphate buffer was reduced, and the transient membrane depolarization that resulted from the addition of D-xylose was eliminated. Although there was no detectable effect of valinomycin on motility, D-xylose taxis of cells in potassium phosphate buffer was completely inhibited by valinomycin. In sodium phosphate buffer, valinomycin had little effect on membrane potential or D-xylose taxis. Nigericin is known to dissipate the transmembrane pH gradient of S. aurantia in potassium phosphate buffer. This compound did not dissipate the membrane potential or the transient membrane depolarization observed upon addition of D-xylose to cells in either potassium or sodium phosphate buffer. Nigericin did not inhibit D-xylose taxis in either potassium or sodium phosphate buffer. This study indicates that the membrane potential but not the transmembrane pH gradient of S. aurantia is somehow involved in chemosensory signal transduction.     

Immobilization of yeast ADH by adsorption onto polyaminomethylstyrene

Summary Yeast alcohol dehydrogenase (EC 1.1.1.1) (Y-ADH) was immobilized by adsorption onto polyaminomethylstyrene (PAMS). The adsorption was rapid and yielded a product, the operational and storage stability of which depended on the initial content of enzyme and to a great extent on the composition of the adsorption buffer. The best results were obtained in the presence of 0.05 M phosphate solution, pH 8.8, containing 2 M sodium chloride. The amount of adsorbed ADH correlates with the position of anions in the Hofmeister series. After 1,000 bed volumes of the standard assay mixture have flowed through the column, there is no decrease in activity. It is possible to prepare adsorbates using crude yeast extracts as enzyme source. The stability of the immobilized enzyme is similar to that obtained with crystalline ADH preparations.     

改性纳米TiO2对蓝藻的生理生态影响

在自然光照条件和日光灯光照条件下,用掺1％Ag纳米TiO2、掺5％Fe2O3纳米TiO2、掺10％ZnO纳米TiO2和纯纳米TiO2对铜绿微囊藻（Synechocystis sp．）PCC6803和集胞藻（Microcystis aeruginosa）进行处理,并对2种藻样的叶绿素含量、类胡萝卜素含量、体外超氧自由基含量及超氧化物歧化酶（superoxidase dismutase,SOD）活性进行测定。结果表明,自然光照条件比日光灯光照条件更利于纳米材料光催化反应的进行;改性后的3种纳米材料对蓝藻的抑制效果要好于纯纳米TiO2,其中效果最好的为掺1％Ag纳米TiO2;2种藻样在适应由光催化剂引发光催化而产生的自由基胁迫时,集胞藻的适应能力比铜绿微囊藻更强。     

Wunen, a Drosophila lipid phosphate phosphatase, is required for septate junction-mediated barrier function

Lipid phosphate phosphatases (LPPs) are integral membrane enzymes that regulate the levels of bioactive lipids such as sphingosine 1-phosphate and lysophosphatidic acid. The Drosophila LPPs Wunen (Wun) and Wunen-2 (Wun2) have a well-established role in regulating the survival and migration of germ cells. We now show that wun has an essential tissue-autonomous role in development of the trachea: the catalytic activity of Wun is required to maintain septate junction (SJ) paracellular barrier function, loss of which causes failure to accumulate crucial luminal components, suggesting a role for phospholipids in SJ function. We find that the integrity of the blood-brain barrier is also lost in wun mutants, indicating that loss of SJ function is not restricted to the tracheal system. Furthermore, by comparing the rescue ability of different LPP homologs we show that wun function in the trachea is distinct from its role in germ cell migration.     

改性纳米TiO2 对蓝藻的生理生态影响

在自然光照条件和日光灯光照条件下, 用掺1%Ag纳米TiO2 、掺5%Fe2O3纳米TiO2、掺10%ZnO纳米TiO2和纯纳米TiO2对铜绿微囊藻(Synechocystis sp.)PCC6803和集胞藻(Microcystis aeruginosa)进行处理, 并对2种藻样的叶绿素含量、类胡萝卜素含量、体外超氧自由基含量及超氧化物歧化酶(superoxidase dismutase, SOD)活性进行测定。结果表明, 自然光照条件比日光灯光照条件更利于纳米材料光催化反应的进行; 改性后的3种纳米材料对蓝藻的抑制效果要好于纯纳米TiO2,其中效果最好的为掺1%Ag纳米TiO2; 2种藻样在适应由光催化剂引发光催化而产生的自由基胁迫时, 集胞藻的适应能力比铜 绿微囊藻更强。     

Mg2+-dependent interaction of DNA with eukaryotic cells

Interaction of DNA with eukaryotic cells under conditions similar to those providing DNA adsorption onto liposomes was studied. It was revealed that mouse fibroblasts (line A9) and myeloma cells bind phage and plasmid DNA in 0.3 M sucrose solution containing Mg2+-ions. Additional pretreatment of the cells by trypsin did not affect DNA adsorption efficiency. The major part of the adsorbed DNA recovered by salt treatment of the cells, but 10-15% of DNA was found to be irreversible. Up to 50% of the irreversibly bound DNA molecules retain their linear size after treatment of cells with DNAse I. Efficiencies of DNA adsorption and irreversibly binding depend on the concentration of Mg2+ in the medium. The process of DNA irreversible binding is not inhibited by drugs affecting cell metabolism. It is assumed that DNA adsorbs onto the phospholipid domains of the cell membrane, and part of the adsorbed DNA is taken up into the interior of the cells.     

Ion beam treatment of titanium surfaces for enhancing deposition of hydroxyapatite from solution

Surface coating with hydroxyapatite (HA) is a common way to improve the osseointegration of orthopaedic and dental titanium (Ti)-based materials. The main problems with current techniques are changes in composition during heating and poor adhesion to the surface. An alternative method is deposition of HA onto an activated surface out of a solution. The present work studies the surface treatment involving ion implantation of Na into Ti to induce a modification in chemistry and morphology, showing sodium titanate (Na(2)TiO(3)) incorporated within the surface layer with concentration, depth distribution, and morphology depending on the parameters of the ion implantation. Such ion-implanted Ti surfaces actively induce heterogeneous precipitation of HA from a simulated body fluid containing physiological concentrations of calcium and phosphate ions. This is compared with the activation by NaOH etching. The growth of bone forming cells on the pure Na implanted surface is oriented without an increased bone formation. Cell growth on the NaOH etched surface is reduced. After deposition of HA on both surfaces cell the growth pattern was improved.     

Tellurite uptake by cells of the facultative phototroph Rhodobacter capsulatus is a Delta pH-dependent process

The uptake by light-grown cells of Rhodobacter capsulatus of the highly toxic metalloid oxyanion tellurite (TeO(3)(2-)) was examined. We show that tellurite is rapidly taken up by illuminated cells in a process which is inhibited by the protonophore carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP) and by the K(+)/H(+) exchanger nigericin. Notably, the light-driven membrane potential (Delta psi) is enhanced by K(2)TeO(3)> or =200 microM. Further, tellurite uptake is largely insensitive to valinomycin, strongly repressed by the sulfhydryl reagent N-ethylethylmaleimide (NEM) and competitively inhibited by phosphate. We conclude that tellurite is transported into cells by a Delta pH-dependent, non-electrogenic process which is likely to involve the phosphate transporter (PiT family).     

Photocatalytic inactivation of influenza virus by titanium dioxide thin film

Titanium dioxide (TiO(2)) under ultraviolet (UV) light produces a strong oxidative effect and may therefore be used as a photocatalytic disinfectant. Although many studies on the photocatalytic inactivation of bacteria have been reported, few studies have addressed virus inactivation. In the present study, we demonstrated the inactivation of influenza virus through TiO(2) photocatalysis using TiO(2) nanoparticles immobilized on a glass plate. The influences of the UV intensity, UV irradiation time and bovine serum albumin (BSA) concentration in the viral suspensions on the inactivation kinetics were investigated. Additionally, we also determined whether the International Organization for Standardization (ISO) methodology for the evaluation of antibacterial activity of TiO(2) photocatalysis could be applied to the evaluation of antiviral activity. The viral titers were dramatically reduced by the photocatalytic reaction. Even with a low intensity of UV-A (0.01 mW cm(-2)), a viral reduction of approximately 4-log(10) was observed within a short irradiation time. The viral inactivation kinetics were associated with the exposure time, the UV intensity and the BSA concentration in virus suspensions. These results show that TiO(2) photocatalysis could be used to inactivate the influenza virus. Furthermore, a minor modification of the ISO test method for anti-bacterial effects of TiO(2) photocatalysis could be useful for the evaluation of antiviral activity.