An investigation of the genotoxic potential of a well-characterized yerba mate extract.

Yerba Mate (Ilex paraguariensis) is historically used as a beverage and its extracts are considered traditional medicine in South America. Extract use has been expanding to North American and European markets and the currently available genetic toxicology literature indicate discrepancies in genotoxicity findings for yerba mate. As botanical extract use expands, assumption in safety should be made with caution assuring a good understanding of the test material characterization. Authoritative agencies suggest a two-step paradigm to investigate genotoxicity, and this was implemented to evaluate the safety of yerba mate hydroxycinnamic acid extract. Four OECD compliant assays were employed: bacterial reverse mutation, in vitro micronucleus and a parallel in vivo micronucleus, and comet assay. No evidence of mutagenicity was observed in the in vitro Ames assay, but the results of an in vitro micronucleus study were inconclusive. However, oral gavage treatment of rats for the in vivo micronucleus and comet assays demonstrated negative findings. The results from this battery of tests, supports that this yerba mate hydroxycinnamic acid extract is not anticipated to pose genotoxicity concerns. A high-level comparison of results to other available genotoxicity literature on yerba mate is presented with emphasis on the importance of identity when drawing conclusions on botanicals.

A comparison of two high spatial resolution imaging techniques for determining carbide precipitate type and size in ferritic 9Cr-1Mo steel.

Two high spatial resolution imaging techniques, focused gallium ion beam imaging in conjunction with XeF2 gas (FIB/XeF2) and high-speed atomic force microscopy (HS-AFM), were used to analyse 9Cr-1Mo ferritic steel samples, which had been exposed for extended periods to hot CO2 gas containing traces of CO, H2, H2O and CH4. The carbide precipitates embedded in the metal matrix were observed and their morphology, size and spatial distribution were quantified using these two techniques. The lower resolution of the FIB/XeF2 imaging technique suggested that small carbide precipitates (<50 nm) may be missed, while the existence of a limited flow layer introduced by sample preparation may influence the HS-AFM results. The gallium ion beam was used to remove a thin oxide layer of approximately 50 nm from sample surfaces prior to FIB/XeF2 imaging, avoiding the influence of surface contamination. HS-AFM provided higher resolution (∼5 nm) than FIB/XeF2 imaging. A quantitative comparison of the experimental data confirmed the value of both FIB/XeF2 and HS-AFM for imaging carbide precipitates, while clarifying their strengths and limitations.

The Impact of Alkaliphilic Biofilm Formation on the Release and Retention of Carbon Isotopes from Nuclear Reactor Graphite.

14C is an important consideration within safety assessments for proposed geological disposal facilities for radioactive wastes, since it is capable of re-entering the biosphere through the generation of 14C bearing gases. The irradiation of graphite moderators in the UK gas-cooled nuclear power stations has led to the generation of a significant volume of 14C-containing intermediate level wastes. Some of this 14C is present as a carbonaceous deposit on channel wall surfaces. Within this study, the potential of biofilm growth upon irradiated and 13C doped graphite at alkaline pH was investigated. Complex biofilms were established on both active and simulant samples. High throughput sequencing showed the biofilms to be dominated by Alcaligenes sp at pH 9.5 and Dietzia sp at pH 11.0. Surface characterisation revealed that the biofilms were limited to growth upon the graphite surface with no penetration of the deeper porosity. Biofilm formation resulted in the generation of a low porosity surface layer without the removal or modification of the surface deposits or the release of the associated 14C/13C. Our results indicated that biofilm formation upon irradiated graphite is likely to occur at the pH values studied, without any additional release of the associated 14C.

Potential impurities in drug substances: Compound-specific toxicology limits for 20 synthetic reagents and by-products, and a class-specific toxicology limit for alkyl bromides.

This paper provides compound-specific toxicology limits for 20 widely used synthetic reagents and common by-products that are potential impurities in drug substances. In addition, a 15 µg/day class-specific limit was developed for monofunctional alkyl bromides, aligning this with the class-specific limit previously defined for monofunctional alkyl chlorides. Both the compound- and class-specific toxicology limits assume a lifetime chronic exposure for the general population (including sensitive subpopulations) by all routes of exposure for pharmaceuticals. Inhalation-specific toxicology limits were also derived for acrolein, formaldehyde, and methyl bromide because of their localized toxicity via that route. Mode of action was an important consideration for a compound-specific toxicology limit. Acceptable intake (AI) calculations for certain mutagenic carcinogens assumed a linear dose-response for tumor induction, and permissible daily exposure (PDE) determination assumed a non-linear dose-response. Several compounds evaluated have been previously incorrectly assumed to be mutagenic, or to be mutagenic carcinogens, but the evidence reported here for such compounds indicates a lack of mutagenicity, and a non-mutagenic mode of action for tumor induction. For non-mutagens with insufficient data to develop a toxicology limit, the ICH Q3A qualification thresholds are recommended. The compound- and class-specific toxicology limits described here may be adjusted for an individual drug substance based on treatment duration, dosing schedule, severity of the disease and therapeutic indication.

Top-down design of magnonic crystals from bottom-up magnetic nanoparticles through protein arrays.

We show that chemical fixation enables top-down micro-machining of large periodic 3D arrays of protein-encapsulated magnetic nanoparticles (NPs) without loss of order. We machined 3D micro-cubes containing a superlattice of NPs by means of focused ion beam etching, integrated an individual micro-cube to a thin-film coplanar waveguide and measured the resonant microwave response. Our work represents a major step towards well-defined magnonic metamaterials created from the self-assembly of magnetic nanoparticles.

The oxidative corrosion of carbide inclusions at the surface of uranium metal during exposure to water vapour.

The reaction between uranium and water vapour has been well investigated, however discrepancies exist between the described kinetic laws, pressure dependence of the reaction rate constant and activation energies. Here this problem is looked at by examining the influence of impurities in the form of carbide inclusions on the reaction. Samples of uranium containing 600 ppm carbon were analysed during and after exposure to water vapour at 19 mbar pressure, in an environmental scanning electron microscope (ESEM) system. After water exposure, samples were analysed using secondary ion mass spectrometry (SIMS), focused ion beam (FIB) imaging and sectioning and transmission electron microscopy (TEM) with X-ray diffraction (micro-XRD). The results of the current study indicate that carbide particles on the surface of uranium readily react with water vapour to form voluminous UO(3) · xH(2)O growths at rates significantly faster than that of the metal. The observation may also have implications for previous experimental studies of uranium-water interactions, where the presence of differing levels of undetected carbide may partly account for the discrepancies observed between datasets.

Preparation of location-specific thin foils from Fe-3% Si bi- and tri-crystals for examination in a FEG-STEM.

Bi-crystals and tri-crystals of a nominal Fe-3% Si (wt%) of well-defined orientations have been grown using a floating-zone technique with optical heating. The manufacture of these unique crystals and the preparation technique involved in harvesting thin foils from specific locations for transmission electron microscopy are described in detail. In particular, the grain boundary triple junction has been extracted from the tri-crystal and examined in high-resolution aberration-corrected FEG-STEM instruments. To achieve the necessary resolution, the foils have to be uniformly thin, in the range 50-100 nm over large areas of the specimen. For ferromagnetic materials, there are further challenges arising from the magnetic field interaction, with the electron beam placing significant demands on the aberration correction system. One way to minimise this interaction is to reduce the total mass of magnetic material. To achieve this, an in situ focused ion beam lift-out technique has been combined with an additional precision ion-polishing stage to reproducibly provide thin-foil specimens suitable for high-resolution EELS and EDX analysis. Examination of the foils reveals that the final precision ion-polishing stage removes residual damage arising from the use of focused ion beam milling procedures.

Small Fermi surface pockets in underdoped high temperature superconductors: observation of Shubnikov-de Haas oscillations in YBa2Cu4O8.

We report the observation of Shubnikov-de Haas oscillations in the underdoped cuprate superconductor YBa2Cu4O8 (Y124). For fields aligned along the c axis, the frequency of the oscillations is 660+/-30 T, which corresponds to approximately 2.4% of the total area of the first Brillouin zone. The effective mass of the quasiparticles on this orbit is measured to be 2.7+/-0.3 times the free electron mass. Both the frequency and mass are comparable to those recently observed for ortho-II YBa2Cu3O6.5 (Y123-II). We show that although small Fermi surface pockets may be expected from band-structure calculations in Y123-II, no such pockets are predicted for Y124. Our results therefore imply that these small pockets are a generic feature of the copper oxide plane in underdoped cuprates.

A new detection system for extremely small vertically mounted cantilevers.

Detection techniques currently used in scanning force microscopy impose limitations on the geometrical dimensions of the probes and, as a consequence, on their force sensitivity and temporal response. A new technique, based on scattered evanescent electromagnetic waves (SEW), is presented here that can detect the displacement of the extreme end of a vertically mounted cantilever. The resolution of this method is tested using different cantilever sizes and a theoretical model is developed to maximize the detection sensitivity. The applications presented here clearly show that the SEW detection system enables the use of force sensors with sub-micron size, opening new possibilities in the investigation of biomolecular systems and high speed imaging. Two types of cantilevers were successfully tested: a high force sensitivity lever with a spring constant of 0.17 pN nm(-1) and a resonant frequency of 32 kHz; and a high speed lever with a spring constant of 50 pN nm(-1) and a resonant frequency of 1.8 MHz. Both these force sensors were fabricated by modifying commercial microcantilevers in a focused ion beam system. It is important to emphasize that these modified cantilevers could not be detected by the conventional optical detection system used in commercial atomic force microscopes.

Anomalous scaling for thick electrodeposited films.

Anomalous surface roughness scaling, where both the local and the large-scale roughness show a power-law dependence on the film thickness, has been widely observed. Here we show that the value of the local roughness exponent in the early stages of Cu electrodeposition depends on the deposition potential. However, initial anomalous scaling can lead to two qualitatively different types of behavior for large film thickness (t>/ or =4 microm). For Cu films electrodeposited with forced convection at high potential and current density, the anomalous scaling is transient: the local roughness saturates for the thickest films studied. When Cu films are electrodeposited at similar potential and current density but with reduced convection, no saturation of the local roughness is observed. Instead the film forms overhangs such that the surface height becomes a multivalued function of the lateral position.

The role of the surface chemistry of CoCr alloy particles in the phagocytosis and DNA damage of fibroblast cells.

Surface chemistry of CoCr particles is demonstrated to be fundamental to the process of phagocytosis by fibroblast cells in vitro. Particles preincubated in serum for 5 days and washed in water before addition to cell cultures were phagocytosed less readily than were particles preincubated in minimal essential medium (MEM) for 1 h and washed in water. This was explained by the coating of calcium phosphate and protein on the serum-immersed particles investigated by time-of-flight secondary ion mass spectroscopy. The cells incubated with the serum-immersed particles had a reduced mitotic index when compared with the MEM-immersed particles, indicating that the phagocytosed particles were causing cell cycle arrest. The release of soluble ions measured by electrothermal atomic absorption spectroscopy within the first hour of particle immersion in MEM was identified as the most likely cause for the DNA damage measured by single cell gel electrophoresis ("Comet" assay). Cryofocused ion beam SEM with a spatial resolution of 8 nm was used to cross section cells, to investigate the location of the phagocytosed particles, some of which were found within the nuclear membrane. This paper demonstrated that consideration of the surface chemistry is essential to understand the processes of the effects of orthopedic wear debris.

Angular dependence of domain wall resistivity in artificial magnetic domain structures.

We exploit the ability to precisely control the magnetic domain structure of perpendicularly magnetized Pt/Co/Pt trilayers to fabricate artificial domain wall arrays and study their transport properties. The scaling behavior of this model system confirms the intrinsic domain wall origin of the magnetoresistance, and systematic studies using domains patterned at various angles to the current flow are excellently described by an angular-dependent resistivity tensor containing perpendicular and parallel domain wall resistivities. We find that the latter are fully consistent with Levy-Zhang theory, which allows us to estimate the ratio of minority to majority spin carrier resistivities, rho downward arrow/rho upward arrow approximately 5.5, in good agreement with thin film band structure calculations.

The entrapment of corrosion products from CoCr implant alloys in the deposits of calcium phosphate: a comparison of serum, synovial fluid, albumin, EDTA, and water.

Physical wear of orthopedic implants is inevitable. CoCr alloy samples, typically used in joint reconstruction, corrode rapidly after removal of the protective oxide layer. The behavior of CoCr pellets immersed in human serum, foetal bovine serum (FBS), synovial fluid, albumin in phosphate-buffered saline (PBS), EDTA in PBS, and water were studied using X-ray Photoelectron Spectroscopy (XPS) and Time-of-Flight Secondary Ion Mass Spectroscopy (ToF-SIMS). The difference in the corrosive nature of human serum, water, albumin in PBS and synovial fluid after 5 days of immersion was highlighted by the oxide layer, which was respectively 15, 3.5, 1.5, and 1.5 nm thick. The thickness of an additional calcium phosphate deposit from human serum and synovial fluid was 40 and 2 nm, respectively. Co and Cr ions migrated from the bulk metal surface and were trapped in this deposit by the phosphate anion. This may account for the composition of wear debris from CoCr orthopedic implants, which is known to consist predominantly of hydroxy-phosphate compounds. Known components of synovial fluid including proteoglycans, pyrophosphates, phospholipids, lubricin, and superficial zone protein (SZP), have been identified as possible causes for the lack of significant calcium phosphate deposition in this environment. Circulation of these compounds around the whole implant may inhibit calcium phosphate deposition.

Attitudes to fluorosis and dental caries by a response latency method.

BACKGROUND: Understanding socially relevant attitudes to fluorosis and dental caries is important. Previous studies have concentrated mainly on aesthetic implications. AIMS: To investigate social judgements beyond the aesthetic, made when viewing digitally manipulated extraoral images of dental fluorosis of varying degrees of severity and images of dental caries. METHODOLOGY: Using a response latency technique, which allowed both the direction and strength of attitudes to be measured, 40 volunteers made judgements on 144 image/characteristic combinations. RESULTS: Participants made social judgements which extended beyond the aesthetic to factors such as sociability, reliability and cleanliness. Judgements on mild fluorosis were not markedly different from those made about the same individual with normal enamel, but severe fluorosis had a significant negative impact on social judgements. Untreated dental caries was judged less favourably than normal enamel and mild fluorosis. CONCLUSIONS: Attribution of characteristics that go beyond the aesthetic are significantly influenced by altered tooth appearance.

The effects of calcium phosphate deposition upon corrosion of CoCr alloys and the potential for implant failure.

Physical wear of orthopedic implants is inevitable. CoCr metal samples, typically used in joint reconstruction, corrode rapidly after removal of the protective oxide layer. The behavior of CoCr pellets immersed in human serum, fetal bovine serum (FBS), synovial fluid, and water were studied using time-of-flight secondary ion mass spectroscopy (ToF-SIMS). The differences in the corrosive nature of human serum, FBS, synovial fluid, and water after 5 days immersion were highlighted by the oxide/hydroxide layer, which was, respectively, 25, 10, 1.5, and 3-3.5 nm thick. The thickness of calcium phosphate deposit from human serum, FBS, and synovial fluid was, respectively, 30, 20, and 2 nm. Co and Cr ions migrated from the bulk metal surface and were trapped in the serum deposits, where chromium existed as oxides, hydroxides, and phosphates, whereas the cobalt chemistry was dominated only by phosphates and hydroxides. This may account for the composition of wear debris from CoCr orthopedic implants, which are predominantly hydroxyphosphate compounds. From the literature, proteoglycans, pyrophosphates, phospholipids, lubricin, and superficial zone protein (SZP) have been identified as possible causes for the insignificant deposit of calcium phosphate from synovial fluid. Circulation of these compounds around the whole implant may inhibit calcium phosphate deposition and therefore contribute to osteolysis.

Analysis of boron-10 in soft tissue by dynamic secondary ion mass spectrometry.

We report here a preliminary study in which dynamic secondary ion mass spectrometry (SIMS) has provided images of boron-10 (10B) in biological tissue as used in research into boron neutron capture therapy. Cultured tumour cells incubated in media containing known concentrations of a 10B-containing compound, p-boronophenylalanine (BPA), and intracranial tumour tissue from animals previously injected with BPA were analysed by an in-house constructed SIMS. Investigations were conducted in positive secondary ion detection mode using a 25-keV, 5-nA gallium primary ion source. For calibration purposes, tissue standards were also analysed and their boron-to-carbon signal ratios correlated to bulk boron concentrations measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES). Ion maps of 10B, 12C, 23Na and 39K showing gross tissue and cell features were acquired. SIMS and ICP-AES standard measurements were in good agreement. Tissue regions with high or low 10B concentrations were identified along with 10B hotspots in normal brain areas. Cultured cells revealed the intracellular localization of 10B. SIMS is capable of producing images showing the distribution of 10B at p.p.m. levels in cells and in normal and tumour-bearing brain tissue.

Effects of smokeless tobacco and tumor promoters on cell population growth and apoptosis of B lymphocytes infected with epstein-barr virus types 1 and 2.

The effects of smokeless tobacco purified products 4-(N-methyl-N-nitrosamine)-1-3-pyridinyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN), smokeless tobacco extracts (dry snuff, moist snuff, and loose leaf), and the tumor promoters 12-O-tetradecanoyl phorbol-13-acetate (TPA) and n-butyrate on cell population growth, cell death, and apoptosis were studied in B lymphocyte cell lines harboring Epstein-Barr virus (EBV) type 1 (Raji and X50-7) or type 2 (HR-1K and AG876) and in an EBV-uninfected control lymphocyte cell line (Ramos). Spontaneous apoptosis was present in all EBV-infected cell lines, but at varying levels. Spontaneous and induced apoptosis were generally greater by Student-Newman-Keuls tests in cells harboring EBV type 2 compared to EBV type 1. The greatest effects on cell population growth, cell death, and apoptosis on cells harboring lytic EBV type 1 (X50-7) was with each of the three smokeless tobacco extracts. The greatest effects on cells harboring EBV type 2 was with TPA and n-butyrate. There were no effects of smokeless tobacco extracts on the Raji cell line that harbors EBV type 1 incapable of lytic replication. Smokeless tobacco purified products, NNN and NNK, had no discernible effects. At the concentrations used in these experiments, there appears to be an EBV type-specific response to chemical induction, with greater susceptibility of lytic EBV type 1 to smokeless tobacco extracts and lytic EBV type 2 to TPA and n-butyrate. This EBV type-specific susceptibility to the effects of smokeless tobacco extracts is another phenotypic difference between EBV types. The use of smokeless tobacco products may affect B lymphocytes infected with replication-capable EBV in the oropharynx. The absence of significant effects with NNK and NNN suggests that these properties reside with other compounds present in tobacco extracts.

Evaluation of the effect of smokeless tobacco purified products and extracts on latent Epstein-Barr virus.

Numerous chemical tumor promoters induce latent Epstein Barr virus (EBV) to active replication. The effect of smokeless tobacco purified products N-nitrosonornicotine (NNN), 4-(N-methyl-N-nitrosamine)-1-3-pyridinyl)-1-butanone (NNK), benzo(a)pyrene (BaP), and smokeless tobacco extracts (dry snuff, moist snuff, and loose leaf tobacco) was tested for induction of latent EBV in Raji cells using fluorescence-activated cell sorter flow cytometry detection of the restricted component of EBV early antigen (EA-R). Concentrations of smokeless tobacco purified products or preparations were used that have carcinogenic effects in animal cell lines. There was no discernible effect for the 6-7-day duration of treatment on viability of Raji cells, or on induction of latent EBV in Raji cells. Results were comparable using paraformaldehyde- or acetone-fixed cells. There does not appear to be an in vitro effect of smokeless tobacco constituents on EBV-infected lymphocytes that may contribute to development of oral cancers.

Organ donor index: a benchmark for comparing hospital organ donor rates.

OBJECTIVE: To develop organ donor indices to assess donor rates of individual hospitals. DESIGN: Data from hospital databases were retrospectively reviewed for patient separation ICD-9-CM codes (i.e., diagnostic codes from the International classification of diseases, 9th revision, clinical modification) to identify and categories actual and potential organ donors. Organ donor indices for groups of codes and for individual hospitals were determined by dividing the number of actual donors by the total number of patients who died with the same separation ICD-9-CM codes. SETTING: The three South Australian adult tertiary hospitals in 1988-1995. PATIENTS: The 154 actual organ donors, and all patients aged less than 71 years who died with the same groups of ICD-9-CM codes as the organ donors. RESULTS: Organ donors could be classified by three groups of ICD-9-CM codes specifying diseases or pathological processes that could result in brain death. These groups were head injury (44.2% of donors), cerebrovascular accident (CVA) (42.2%), and eight "other" codes (13.6%). Differences between the head injury donor indices for the three hospitals were not significant (Hospital A, 19.1%; Hospital B, 24%; Hospital C, 21%), but there were significant interhospital differences in donor indices for the CVA group (A, 11.2%; B, 5.7%; C, 5.1%; P < 0.05) and the "other" group (A, 3.6%; B, 0.7%; C, 0.3%; P < 0.001). CONCLUSIONS: ICD-9-CM codes can be used to describe organ donors and hospital populations from which potential organ donors may be found. The casemix-controlled organ donor indices can be used to compare the organ donor rates of individual hospitals and to examine reasons for low rates (other than purely casemix variation).

Characterization of natural Epstein-Barr virus infection and replication in smooth muscle cells from a leiomyosarcoma.

Cells from a leiomyosarcoma tumor (LMS-1) from a patient with the acquired immunodeficiency syndrome (AIDS) were explanted, cultured in vitro, and studied by phase-contrast microscopy for morphologic and growth characteristics, immunostaining for cell markers, EBER in situ hybridization and polymerase chain reaction for detection of Epstein-Barr virus (EBV), and immunostaining for expression of EBV antigens. The cells exhibited very slow growth in vitro, with unusual elliptical and spindle-shaped morphology and fragmentation of the cytoplasm into long, tapering, cytoplasmic processes. Greater than 90% of cells expressed diffuse distribution of the smooth muscle isoform of actin by immunoperoxidase staining. Approximately 25% of cells expressed very bright fluorescence by immunostaining of the smooth muscle isoforms of calponin and actin. The majority of cells demonstrated a weak signal for CD21; approximately 5-10% of cells showed a strong signal that was confined to cell surfaces. The cultured cells harbored EBV, and infectious EBV continued to be detected by polymerase chain reaction and virus culture through several passages in vitro. Several EBV antigens were expressed, including latent antigen EBNA-1, immediate-early antigen BZLF1, early antigen EA-D, and late antigens, including viral capsid antigen p160, gp125, and membrane antigen gp350. Human umbilical cord lymphocytes that were transformed with virus isolated from cultured cells yielded immortalized cell lines that expressed EBV antigens similar to other EBV-transformed lymphocyte cell lines. These results confirm that EBV is capable of lytic infection of smooth muscle cells with expression of a repertoire of latent and replicative viral products and production of infectious virus. EBV infection of smooth muscle cells may contribute to the oncogenesis of leiomyosarcomas.