Characterization of titanium dioxide nanoparticles modified with polyacrylic acid and H2O2 for use as a novel radiosensitizer

An induction of polyacrylic acid-modified titanium dioxide with hydrogen peroxide nanoparticles (PAA-TiO₂/H₂O₂ NPs) to a tumor exerted a therapeutic enhancement of X-ray irradiation in our previous study. To understand the mechanism of the radiosensitizing effect of PAA-TiO₂/H₂O₂ NPs, analytical observations that included DLS, FE-SEM, FT-IR, XAFS, and Raman spectrometry were performed. In addition, highly reactive oxygen species (hROS) which PAA-TiO₂/H₂O₂ NPs produced with X-ray irradiation were quantified by using a chemiluminescence method and a EPR spin-trapping method. We found that PAA-TiO₂/H₂O₂ NPs have almost the same characteristics as PAA-TiO₂. Surprisingly, there were no significant differences in hROS generation. However, the existence of H₂O₂ was confirmed in PAA-TiO₂/H₂O₂ NPs, because spontaneous hROS production was observed w/o X-ray irradiation. In addition, PAA-TiO₂/H₂O₂ NPs had a curious characteristic whereby they absorbed H₂O₂ molecules and released them gradually into a liquid phase. Based on these results, the H₂O₂ was continuously released from PAA-TiO₂/H₂O₂ NPs, and then released H₂O₂ assumed to be functioned indirectly as a radiosensitizing factor.     

A novel enzyme-immobilization method for a biofuel cell

Biofuel cells utilizing biocatalysts are attractive alternatives to metal catalyst-based cells because of environmentally friendly cells and their renewability and good operations at room temperatures, even though they provide a low level of electrical power. In this study, the effect of a novel enzyme immobilization method on anodic electrical properties was evaluated under ambient conditions for increasing the power of an enzyme-based biofuel cell. The anodic system employed in the cell contained a gold electrode, pyrroloquinoline quinone (PQQ) as the electron transfer mediator, lactate dehydrogenase (LDH), β-nicotinamide adenine dinucleotide (NAD⁺) as the cofactor, and lactate as the substrate. The anodic electrical properties increased as a result of the novel enzyme-immobilization method. Furthermore, lactate, NAD⁺, or CaCl₂, which can all influence enzyme activation, were used to prevent covalent bond formation near the active site of the LDH during enzyme-immobilization. Protection of the active site of the LDH using this novel enzyme-immobilization method increased its stability, which enabled to increase power production (142 μW/cm²) in a basic enzymatic fuel cell (EFC).     

Laser-beam-triggered microcavitation: a novel method for selective cell destruction

We describe a new method of cell destruction that may have potential for use in antitumor therapy. Cells are loaded by phagocytosis with microparticles (<1 microm) and irradiated with short laser pulses. Absorption of laser energy by the microparticles causes localized vaporization of the fluid surrounding the microparticles, leading to the generation of transient vapor bubbles (microcavitation) around the microparticles. Using cultures of bovine aortic endothelial cells, we demonstrate that induction of intralysosomal microcavitation is an efficient, rapid and selective method of cell killing that is dependent on the number of microparticles, the number of laser pulses, and the fluence of the laser pulses. Cell killing by microcavitation is a very selective process that is restricted to cells containing microparticles, leaving other cells unaffected. Intracytoplasmic release of lysosomal hydrolases is, in part, responsible for cell death, because the protease inhibitors E64d and TLCK diminished cell killing. Using the broad-specificity caspase inhibitor Z-VAD-fmk, we determined that lysosomal hydrolases could induce apoptosis in a caspase-independent manner. We also examined the possibility of microcavitation-induced delayed effects in the cells that survived the treatment. Using flow cytometry, we determined that there was no delayed cell death between 1 and 4 days after microcavitation. Moreover, we did not observe changes in the cell cycle, in expression of the proteins BCL2, HSP70 and HSP27, or in PARP degradation. In conclusion, microcavitation induces rapid and specific cells death (limited only to cells containing microparticles), without producing delayed effects among the surviving cells.     

Targeting of ovarian cancer cell through functionalized gold nanoparticles by novel glypican-3- binding peptide as a ultrasound contrast agents

Gold nanoparticles (AuNPs) are widely studied nanomaterials for their potential employment in advanced biomedical applications, such as selective molecular imaging and targeted drug delivery. AuNPs are generally low cost and highly biocompatible, can be easily functionalized with a wide variety of functional ligands, and have been demonstrated to be effective in enhancing ultrasound contrast at clinical diagnostic frequencies. Therefore, AuNPs might be used as contrast agents in echographic imaging. In this work, we have developed a AuNPs -based system for the in vitro molecular imaging of ovarian carcinoma cells that express high levels of glypican-3 protein (GPC-3) on their surface. In this regard, a novel GPC-3 targeting peptide was designed and conjugated to fluorescent AuNPs nanoparticles. The physicochemical properties, acoustic behavior, and biocompatibility profile of the functionalized AuNPs were characterized. Then, the binding and uptake of both naked and functionalized AuNPs were analyzed by laser scanning confocal microscopy in human HeLa cells (ovarian carcinoma) cell line. The results obtained showed that GPC-3-functionalized fluorescent AuNPs significantly enhanced the ultrasound contrast and were effectively bound and taken up by HeLa cells without affecting their viability.     

Molecular docking,a tool to determine interaction of CuO and TiO2 nanoparticles with human serum albumin

BackgroundWe study the human serum albumin (HSA) protein-CuO nanoparticle interaction to identify the specific binding site of protein with CuO nanoparticles by molecular docking and compared it with HSA-TiO₂ nanoparticle interaction.MethodsThe protein structural data that was obtained using Autodock 4.2.ResultsIn case of CuO np-HSA interaction, the distances from the centre of Subdomain IIIA to Arg-472 is 2.113 Å and Lys 475, Glu 492, Ala 490, Cys 487, Ala 490 are the bound neighbouring residues with Lys 475, Glu 492 at aliphatic region. The binding energy generated was ?1.64 kcal mol^?1. However, for TiO₂ nanoparticle, the binding region is surrounded by Arg 257, Ala 258, Ser 287, His 288, Leu 283, Ala 254, Tyr 150 (subdomain II A) as neighbouring residue. Moreover, Glu 285, Lys 286 forms aliphatic grove for TiO₂-HSA, Ser-287 at the centre region form hydrogen bond with nanoparticle and Leu 283, Leu 284 forming hydrophopobic grove for TiO₂ nanoparticle-HSA interaction. The binding energy generated was ?2.47 kcal mol^?1.ConclusionsAnalysis suggests that CuO bind to suldow site II i.e subdomain III A of HSA protein where as TiO₂ nanoparticle bind to suldow site I i.e subdomain IIA of HSA protein.General significanceThe structural information that derives from this study for CuO and TiO₂ nanoparticles may be useful in terms of both high and low-affinity binding sites when designing these nanoparticles based drugs delivery system.     

A novel Vero cell line for use as a mammalian host-vector system in serum-free medium

We have established a novel cell line from a Vero cell derivative that is useful for expression of exogenous genes and protein production. Parental Vero-317 cells can grow in biotin-containing Eagle's MEM without supplements. By transforming this cell line with replication origin-defective SV40 DNA, which contains a temperature-sensitive tsA58 large T antigen gene, we established the Verots S3 cell line that amplified a SV40-origin containing plasmid. The cell line expressed a human growth hormone (hGH) gene insert with higher efficiency than COS-7 cells in 5% serum-containing MEM and could grow and continue hGH expression in protein-free MEM. However, temperature-sensitive shut down of hGH production was observed not immediately but 3 days after the temperature shift from 33°C to 39.5°C.     

A novel saccharification method of starch using microwave irradiation with addition of activated carbon

Activated carbons were investigated for their heat catalytic effects to improve saccharification of starch by autohydrolysis in water under microwave electromagnetic field, and the results were compared with graphite and carbon nanotubes. The activated carbons with low adsorptive capacity of maltose showed high saccharification rate, while those with high adsorptive capacity exhibited low saccharification. In addition, the former activated carbons decreased the saccharification temperature by 10-30 °C. Maltooligosaccharides produced in the presence of the latter activated carbons were recovered by desorption with 50% aqueous ethanol. The results indicated that both adsorptive capacities of maltooligosaccharides and catalytic effects of hot spots arisen from the uneven surface structure of activated carbons might contribute to the improvement in starch saccharification.     

Combination use of ultrasound irradiation and ionic liquid in enzymatic isomerization of glucose to fructose

The synthetic effect of the combination use of ultrasound irradiation (UI) and ionic liquid (IL) on improving enzyme activity was studied when they were employed in isomerization of glucose to fructose by immobilized glucose isomerase (IGI, produced from streptomyces murinus and immobilized on silica). Both of UI and IL [EMIM][Cl], which was screened as the best medium for this reaction, were found to increase the enzyme activity in isomerization reaction. And a further increase of enzyme activity was observed by combination use of UI and IL. A systematic screening and optimization of the reaction parameters in ILs under UI on the IGI activity were performed. Under the optimum reaction conditions, 45.3% yield of fructose was achieved in 10 h under UI in [EMIM][Cl], compared to only 41.5% yield under stirring in [EMIM][Cl], 44.2% under UI without [EMIM][Cl] and 38.9% under stirring without [EMIM][Cl] in 12 h, respectively. High thermal stability and reusability of IGI was also observed under UI in [EMIM][Cl]. These results indicated that the combination use of UI and IL might be a fast and efficient method for enzymatic isomerization of glucose to fructose.     

A novel method for the intracellular delivery of siRNA using microbubble-enhanced focused ultrasound

Short interfering RNA (siRNA) has attracted much attention for clinical use in various diseases. However, its delivery, especially through the cell membrane, continues to present a challenge. Advances in ultrasound- and ultrasound contrast-agent technologies have made it possible to change transiently the permeability of the cell membrane and, using a focused ultrasound transducer, to narrow and focus the ultrasound energy on a small target, thereby avoiding damage to surrounding tissue. In this in vitro study, we demonstrate that it is possible to deliver siRNA intracellularly via microbubble-enhanced focused ultrasound. Although further optimization is necessary, our novel method for siRNA transduction represents a powerful tool for using siRNA in vivo and possibly in the clinical setting.     

Hydrodynamic deposition: a novel method of cell immobilization.

A novel method of cell immobilization is described. The cell support consists of ceramic microspheres of approximately 50-75 microns diameter. The spheres are hollow, having a wall thickness of 10-15 microns and one entrance (ca. 20 microns diameter). The walls are porous with a mean pore size of approximately 90 nm. When a cell suspension (of S. cerevisiae) is passed through a column of such particles, cells are immobilized. Conditions are devised such that the overwhelming majority of cells are held in the central cavity of the support and not between the particles. Provided turbulence is avoided, the distribution of cells along the column length in the steady state is rather homogeneous. The facts that (a) essentially all particles, regardless of orientation, entrap cells, and (b) nonporous particles also entrap cells with high efficiency, indicate that filtration effects are irrelevant and that heretofore unrecognized hydrodynamic forces are alone responsible for the cell immobilization. Cells can be immobilized to high biomass densities, while the hydrodynamic properties of columns containing such immobilized cells are excellent. We describe an on-line electronic method for the real-time measurement of immobilized cellular biomass. Cell growth (so recorded) and metabolism continue to occur in such particles at high rates. Using the glycolytic production of ethanol by S. cerevisiae as a model reaction, volumetric productivities as great as any published are obtained. Thus the "lobster-pot effect" or "hydrodynamic deposition" represents a novel, promising, and generally applicable method of cell immobilization.     

Laser scanning cytometry: a novel method for the detection of platelet--endothelial cell adhesion

BACKGROUND: Adherence of platelets to endothelial cells may be a significant event in the development of vascular thrombosis. Existing models, which examine platelet-endothelial cell interactions, compromise endothelial cell integrity or use radioactivity to identify platelets that adhere to endothelial cells. We report a novel method for in vitro detection of platelet-endothelial cell adhesion that allows endothelial cells to remain as an intact monolayer and for visualization of individual platelets. METHODS: Fluorescently labeled platelets were incubated with a confluent monolayer of endothelial cells. Laser scanning cytometry (LSC) identified platelets bound to endothelial cells based on their fluorescent signals. RESULTS: LSC detection of platelets reliably reproduced well-described findings of thrombin-induced platelet-endothelial cell adhesion. Results demonstrating reduced adhesion with a glycoprotein IIb-IIIa-specific blocking monoclonal antibody confirmed the specificity of the LSC detection of platelet-endothelial cell adhesion. CONCLUSIONS: LSC is a novel method for detecting platelet--endothelial cell adhesion. Its advantages over other methods are: (a) endothelial cells remain undisturbed and adherent throughout; (b) the ability to detect individual bound platelets and subpopulations; (c) the ability to store images and slides and then relocate, revisualize, and reanalyze individual cells or cell populations of interest; and (d) no radioactivity.     

A novel scale-up method for mammalian cell culture in packed-bed bioreactor

A novel method for the scale-up culture of Chinese hamster ovary (CHO) cells in a packed-bed bioreactor is developed wherein microcarriers, attached with CHO cells in a microcarrier culture system, are inoculated directly into the packed-bed bioreactor. Cells continue to grow after inoculation and the maximum cell density reaches about 2×10⁷ cells ml^–1. The method provides a new technique for the scale-up of a packed-bed culture while decreasing the labour cost and ensuring the safety of operation.     

A novel method for measuring dynamic changes in cell volume.

Many cell types regulate their volume in response to extracellular tonicity changes through a complex series of adaptive mechanisms. Several methods that are presently used to measure cell volume changes include Coulter counters, fluorescent techniques, electronic impedance, and video microscopy. Although these methods are widely used and accepted, there are limitations associated with each technique. This paper describes a new method to measure changes in cell volume based on the principle that fluid flow within a rigid system is well determined. For this study, cos-7 cells were plated to line the inner lumen of a glass capillary and stimulated to swell or shrink by altering the osmolarity of the perfusing solution. The cell capillary was connected in series with a blank reference capillary, and differential pressure changes across each tube were monitored. The advantages of this method include 1) ability to continuously monitor changes in volume during rapid solution changes, 2) independence from cell morphology, 3) presence of physiological conditions with cell surface contacts and cell-cell interactions, 4) no phototoxic effects such as those associated with fluorescent methods, and 5) ability to report from large populations of cells. With this method, we could detect the previously demonstrated enhanced volume regulation of cells overexpressing the membrane phosphoprotein phospholemman, which has been implicated in osmolyte transport.     

Modification of nanocrystalline TiO2 coatings with molecularly imprinted TiO2 for uric acid recognition

Combining the surface modification and molecular imprinting technique, a novel piezoelectric sensing platform with excellent molecular recognition capability was established for the detection of uric acid (UA) based on the immobilization of TiO₂ nanoparticles onto quartz crystal microbalance (QCM) electrode and modification of molecularly imprinted TiO₂ (MIT) layer on TiO₂ nanoparticles. The performance of the fabricated biosensor was evaluated, and the results indicated that the biosensor exhibited high sensitivity in UA detection, with a linear range from 0.04 to 45 μM and a limit of detection of 0.01 μM. Moreover, the biosensor presented high selectivity towards UA in comparison with other interferents. The analytical application of the UA biosensor confirmed the feasibility of UA detection in urine sample.     

Multifactor dimensionality reduction-phenomics: a novel method to capture genetic heterogeneity with use of phenotypic variables

Complex human diseases do not have a clear inheritance pattern, and it is expected that risk involves multiple genes with modest effects acting independently or interacting. Major challenges for the identification of genetic effects are genetic heterogeneity and difficulty in analyzing high-order interactions. To address these challenges, we present MDR-Phenomics, a novel approach based on the multifactor dimensionality reduction (MDR) method, to detect genetic effects in pedigree data by integration of phenotypic covariates (PCs) that may reflect genetic heterogeneity. The P value of the test is calculated using a permutation test adjusted for multiple tests. To validate MDR-Phenomics, we compared it with two MDR-based methods: (1) traditional MDR pedigree disequilibrium test (PDT) without consideration of PCs (MDR-PDT) and (2) stratified phenotype (SP) analysis based on PCs, with use of MDR-PDT with a Bonferroni adjustment (SP-MDR). Using computer simulations, we examined the statistical power and type I error of the different approaches under several genetic models and sampling scenarios. We conclude that MDR-Phenomics is more powerful than MDR-PDT and SP-MDR when there is genetic heterogeneity, and the statistical power is affected by sample size and the number of PC levels. We further compared MDR-Phenomics with conditional logistic regression (CLR) for testing interactions across single or multiple loci with consideration of PC. The results show that CLR with PC has only slightly smaller power than does MDR-Phenomics for single-locus analysis but has considerably smaller power for multiple loci. Finally, by applying MDR-Phenomics to autism, a complex disease in which multiple genes are believed to confer risk, we attempted to identify multiple gene effects in two candidate genes of interest—the serotonin transporter gene (SLC6A4) and the integrin beta 3 gene (ITGB3) on chromosome 17. Analyzing four markers in SLC6A4 and four markers in ITGB3 in 117 white family triads with autism and using sex of the proband as a PC, we found significant interaction between two markers—rs1042173 in SLC6A4 and rs3809865 in ITGB3.     

bcl-2, a novel reguator of cell death

The bcl-2 gene product, a 25 kDa membrane protein residing at mitochondrial, microsomal and nuclear membrane sites within many cell types, is a broad and potent inhibitor of cell death by apoptosis. A family of bcl-2-related genes with death-inhibiting or -promoting activities has recently been described, indicating a potentially quite complex cell death regulatory network at the level of gene expression and protein-protein interactions. The function of bcl-2 may be to regulate a final common pathway in apoptosis. Current hypotheses suggest that oxidative stress, specific proteolytic activity or cell cycle control may be common elements in apoptosis through which bcl-2 exerts its survival function. Based on the extent to which elements of apoptotic pathways overlap with non-apoptotic cellular functions, the physiological role of bcl-2 may also extend to other cellular processes such as differentiation and proliferation.