Use of pooled samples to assess human exposure to parabens, benzophenone-3 and triclosan in Queensland, Australia

Parabens, benzophenone-3 and triclosan are common ingredients used as preservatives, ultraviolet radiation filters and antimicrobial agents, respectively. Human exposure occurs through consumption of processed food and use of cosmetics and consumer products. The aim of this study was to provide a preliminary characterisation of exposure to selected personal care product chemicals in the general Australian population. De-identified urine specimens stratified by age and sex were obtained from a community-based pathology laboratory and pooled (n= 24 pools of 100). Concentrations of free and total (sum of free plus conjugated) species of methyl, ethyl, propyl and butyl paraben, benzophenone-3 and triclosan were quantified using isotope dilution tandem mass spectrometry; with geometric means 232, 33.5, 60.6, 4.32, 61.5 and 87.7. ng/mL, respectively. Age was inversely associated with paraben concentration, and females had concentrations approximately two times higher than males. Total paraben and benzophenone-3 concentrations are significantly higher than reported worldwide, and the average triclosan concentration was more than one order of magnitude higher than in many other populations. This study provides the first data on exposure of the general Australian population to a range of common personal care product chemical ingredients, which appears to be prevalent and warrants further investigation.

Monitoring temporal changes in use of two cathinones in a large urban catchment in Queensland, Australia

Wastewater analysis was used to examine prevalence and temporal trends in the use of two cathinones, methylone and mephedrone, in an urban population (>200,000 people) in South East Queensland, Australia. Wastewater samples were collected from the inlet of the sewage treatment plant that serviced the catchment from 2011 to 2013. Liquid chromatography coupled with tandem mass spectrometry was used to measure mephedrone and methylone in wastewater sample using direct injection mode. Mephedrone was not detected in any samples while methylone was detected in 45% of the samples. Daily mass loads of methylone were normalized to the population and used to evaluate methylone use in the catchment. Methylone mass loads peaked in 2012 but there was no clear temporal trend over the monitoring period. The prevalence of methylone use in the catchment was associated with the use of MDMA, the more popular analogue of methylone, as indicated by other complementary sources. Methylone use was stable in the study catchment during the monitoring period whereas mephedrone use has been declining after its peak in 2010. More research is needed on the pharmacokinetics of emerging illicit drugs to improve the applicability of wastewater analysis in monitoring their use in the population.

Grain sorghum proteomics: integrated approach toward characterization of endosperm storage proteins in kafirin allelic variants

Grain protein composition determines quality traits, such as value for food, feedstock, and biomaterials uses. The major storage proteins in sorghum are the prolamins, known as kafirins. Located primarily on the periphery of the protein bodies surrounding starch, cysteine-rich beta- and gamma-kafirins may limit enzymatic access to internally positioned alpha-kafirins and starch. An integrated approach was used to characterize sorghum with allelic variation at the kafirin loci to determine the effects of this genetic diversity on protein expression. Reversed-phase high performance liquid chromatography and lab-on-a-chip analysis showed reductions in alcohol-soluble protein in beta-kafirin null lines. Gel-based separation and liquid chromatography-tandem mass spectrometry identified a range of redox active proteins affecting storage protein biochemistry. Thioredoxin, involved in the processing of proteins at germination, has reported impacts on grain digestibility and was differentially expressed across genotypes. Thus, redox states of endosperm proteins, of which kafirins are a subset, could affect quality traits in addition to the expression of proteins.

Topographical, structural and geophysical characterization of fracture zones: implications for groundwater flow and vulnerability

The main objective of this study is to evaluate selected geophysical, structural and topographic methods on regional, local, and tunnel and borehole scales, as indicators of the properties of fracture zones or fractures relevant to groundwater flow. Such information serves, for example, groundwater exploration and prediction of the risk of groundwater inflow in underground construction. This study aims to address how the features detected by these methods link to groundwater flow in qualitative and semi-quantitative terms and how well the methods reveal properties of fracturing affecting groundwater flow in the studied sites. The investigated areas are: (1) the Päijänne Tunnel for water-conveyance whose study serves as a verification of structures identified on regional and local scales; (2) the Oitti fuel spill site, to telescope across scales and compare geometries of structural assessment; and (3) Leppävirta, where fracturing and hydrogeological environment have been studied on the scale of a drilled well. The methods applied in this study include: the interpretation of lineaments from topographic data and their comparison with aeromagnetic data; the analysis of geological structures mapped in the Päijänne Tunnel; borehole video surveying; groundwater inflow measurements; groundwater level observations; and information on the tunnel s deterioration as demonstrated by block falls. The study combined geological and geotechnical information on relevant factors governing groundwater inflow into a tunnel and indicators of fracturing, as well as environmental datasets as overlays for spatial analysis using GIS. Geophysical borehole logging and fluid logging were used in Leppävirta to compare the responses of different methods to fracturing and other geological features on the scale of a drilled well. Results from some of the geophysical measurements of boreholes were affected by the large diameter (gamma radiation) or uneven surface (caliper) of these structures. However, different anomalies indicating more fractured upper part of the bedrock traversed by well HN4 in Leppävirta suggest that several methods can be used for detecting fracturing. Fracture trends appear to align similarly on different scales in the zone of the Päijänne Tunnel. For example, similarities of patterns were found between the regional magnetic trends, correlating with orientations of topographic lineaments interpreted as expressions of fracture zones. The same structural orientations as those of the larger structures on local or regional scales were observed in the tunnel, even though a match could not be made in every case. The size and orientation of the observation space (patch of terrain at the surface, tunnel section, or borehole), the characterization method, with its typical sensitivity, and the characteristics of the location, influence the identification of the fracture pattern. Through due consideration of the influence of the sampling geometry and by utilizing complementary fracture characterization methods in tandem, some of the complexities of the relationship between fracturing and groundwater flow can be addressed. The flow connections demonstrated by the response of the groundwater level in monitoring wells to pressure decrease in the tunnel and the transport of MTBE through fractures in bedrock in Oitti, highlight the importance of protecting the tunnel water from a risk of contamination. In general, the largest values of drawdown occurred in monitoring wells closest to the tunnel and/or close to the topographically interpreted fracture zones. It seems that, to some degree, the rate of inflow shows a positive correlation with the level of reinforcement, as both are connected with the fracturing in the bedrock. The following geological features increased the vulnerability of tunnel sections to pollution, especially when several factors affected the same locations: (1) fractured bedrock, particularly with associated groundwater inflow; (2) thin or permeable overburden above fractured rock; (3) a hydraulically conductive layer underneath the surface soil; and (4) a relatively thin bedrock roof above the tunnel. The observed anisotropy of the geological media should ideally be taken into account in the assessment of vulnerability of tunnel sections and eventually for directing protective measures.

Algorithms for 13C metabolic flux analysis

The metabolism of an organism consists of a network of biochemical reactions that transform small molecules, or metabolites, into others in order to produce energy and building blocks for essential macromolecules. The goal of metabolic flux analysis is to uncover the rates, or the fluxes, of those biochemical reactions. In a steady state, the sum of the fluxes that produce an internal metabolite is equal to the sum of the fluxes that consume the same molecule. Thus the steady state imposes linear balance constraints to the fluxes. In general, the balance constraints imposed by the steady state are not sufficient to uncover all the fluxes of a metabolic network. The fluxes through cycles and alternative pathways between the same source and target metabolites remain unknown. More information about the fluxes can be obtained from isotopic labelling experiments, where a cell population is fed with labelled nutrients, such as glucose that contains 13C atoms. Labels are then transferred by biochemical reactions to other metabolites. The relative abundances of different labelling patterns in internal metabolites depend on the fluxes of pathways producing them. Thus, the relative abundances of different labelling patterns contain information about the fluxes that cannot be uncovered from the balance constraints derived from the steady state. The field of research that estimates the fluxes utilizing the measured constraints to the relative abundances of different labelling patterns induced by 13C labelled nutrients is called 13C metabolic flux analysis. There exist two approaches of 13C metabolic flux analysis. In the optimization approach, a non-linear optimization task, where candidate fluxes are iteratively generated until they fit to the measured abundances of different labelling patterns, is constructed. In the direct approach, linear balance constraints given by the steady state are augmented with linear constraints derived from the abundances of different labelling patterns of metabolites. Thus, mathematically involved non-linear optimization methods that can get stuck to the local optima can be avoided. On the other hand, the direct approach may require more measurement data than the optimization approach to obtain the same flux information. Furthermore, the optimization framework can easily be applied regardless of the labelling measurement technology and with all network topologies. In this thesis we present a formal computational framework for direct 13C metabolic flux analysis. The aim of our study is to construct as many linear constraints to the fluxes from the 13C labelling measurements using only computational methods that avoid non-linear techniques and are independent from the type of measurement data, the labelling of external nutrients and the topology of the metabolic network. The presented framework is the first representative of the direct approach for 13C metabolic flux analysis that is free from restricting assumptions made about these parameters.In our framework, measurement data is first propagated from the measured metabolites to other metabolites. The propagation is facilitated by the flow analysis of metabolite fragments in the network. Then new linear constraints to the fluxes are derived from the propagated data by applying the techniques of linear algebra.Based on the results of the fragment flow analysis, we also present an experiment planning method that selects sets of metabolites whose relative abundances of different labelling patterns are most useful for 13C metabolic flux analysis. Furthermore, we give computational tools to process raw 13C labelling data produced by tandem mass spectrometry to a form suitable for 13C metabolic flux analysis.

Multistage monostable multivibrator using load-coupled regenerative feedback

A monostable multivibrator configuration using a new technique of regenerative feedback is discussed. This circuit provides an elegant alternative in situations wherein several monostable multivibrators have to be connected in tandem.

Transcriptional Silencing of a tRNA1Gly Copy from within a Multigene Family Is Modulated by Distal cis Elements

Individual copies of tRNA1Gly from within the multigene family in Bombyx mori could be classified based on in vitro transcription in homologous nuclear extracts into three categories of highly, moderately, or weakly transcribed genes. Segregation of the poorly transcribed gene copies 6 and 7, which are clustered in tandem within 425 base pairs, resulted in enhancement of their individual transcription levels, but the linkage itself had little influence on the transcriptional status. For these gene copies, when fused together generating a single coding region, transcription was barely detectable, which suggested the presence of negatively regulating elements located in the far flanking sequences. They exerted the silencing effect on transcription overriding the activity of positive regulatory elements. Systematic analysis of deletion, chimeric, and mutant constructs revealed the presence of a sequence element TATATAA located beyond 800 nucleotides upstream to the coding region acting as negative modulator, which when mutated resulted in high level transcription. Conversely, a TATATAA motif reintroduced at either far upstream or far downstream flanking regions exerted a negative effect on transcription. The location of cis-regulatory sequences at such farther distances from the coding region and the behavior of TATATAA element as negative regulator reported here are novel. These element(s) could play significant roles in activation or silencing of genes from within a multigene family, by recruitment or sequestration of transcription factors.

Formation of linear plasmid multimers promoted by the phage lambda Red-system in lon mutants of Escherichia coli

We report here the formation of plasmid linear multimers promoted by the Red-system of phage lambda using a multicopy plasmid comprised of lambda red alpha and red beta genes, under the control of the lambda cI857 repressor. Our observations have revealed that the multimerization of plasmid DNA is dependent on the red beta and recA genes, suggesting a concerted role for these functions in the formation of plasmid multimers. The formation of multimers occurred in a recBCD+ sbcB+ xthA+ lon genetic background at a higher frequency than in the isogenic lon+ host cells. The multimers comprised tandem repeats of monomer plasmid DNA. Treatment of purified plasmid DNA with exonuclease III revealed the presence of free double-chain ends in the molecules. Determination of the size of multimeric DNA, by pulse field gel electrophoresis, revealed that the bulk of the DNA was in the range 50-240 kb, representing approximately 5-24 unit lengths of monomeric plasmid DNA. We provide a conceptual framework for Red-system-promoted formation and enhanced accumulation of plasmid linear multimers in lon mutants of E. coli.

Quantitation and characterization of glutathionyl haemoglobin as an oxidative stress marker in chronic renal failure by mass spectrometry

Objectives: Glutathionyl haemoglobin (GS-Hb) belonging to the class of glutathionylated proteins has been investigated as a possible marker of oxidative stress in different chronic diseases. The purpose of this study was to examine whether glutathionyl haemoglobin can serve as an oxidative stress marker in non-diabetic chronic renal failure patients on different renal replacement therapies (RRT) through its quantitation, and characterization of the specific binding site of glutathione in haemoglobin molecule by mass spectrometric analysis. Design and methods: The study group consisted of non-diabetic chronic renal failure patients on renal replacement therapy (RRT): hemodialysis (HD), continuous ambulatory peritoneal dialysis (CAPD) and renal allograft transplant (Txp) patients. Haemoglobin samples of these subjects were analyzed by liquid chromatography electrospray ionization mass spectrometry for GS-Hb quantitation. Characterization of GS-Hb was done by tandem mass spectrometry. Levels of erythrocyte glutathione (GSH) and lipid peroxidation (as thiobarbituric acid reacting substances) were measured spectrophotometrically, while glycated baernoglobin (HbA1c) was measured by HPLC. Results: GS-Hb levels were markedly elevated in the dialysis group and marginally in the transplant group as compared to the controls. GS-Hb levels correlated positively with lipid peroxidation and negatively with the erythrocyte glutathione levels in RRT groups indicating enhanced oxidative stress. De novo sequencing of the chymotryptic fragment of GS-Hb established that glutathione is attached to Cys-93 of the beta globin chain. Mass spectrometric quantitation of total glycated haemoglobin showed good agreement with HbA1c estimation by conventional HPLC method. Conclusions: Glutathionyl haemoglobin can serve as a clinical marker of oxidative stress in chronic debilitating therapies like RRT. Mass spectrometry provides a reliable analytical tool for quantitation and residue level characterization of different post-translational modifications of haemoglobin. (c) 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Observation of sea-salt fraction in sub-100 nm diameter particles at Cape Grim

Volatility-hygroscopicity tandem differential mobility analyzer measurements were used to infer the composition of sub-100 nm diameter Southern Ocean marine aerosols at Cape Grim in November and December 2007. This study focuses on a short-lived high sea spray aerosol (SSA) event on 7–8 December with two externally mixed modes in the Hygroscopic Growth Factor (HGF) distributions (90% relative humidity (RH)), one at HGF > 2 and another at HGF~1.5. The particles with HGF > 2 displayed a deliquescent transition at 73–75% RH and were nonvolatile up to 280°C, which identified them as SSA particles with a large inorganic sea-salt fraction. SSA HGFs were 3–13% below those for pure sea-salt particles, indicating an organic volume fraction (OVF) of up to 11–46%. Observed high inorganic fractions in sub-100 nm SSA is contrary to similar, earlier studies. HGFs increased with decreasing particle diameter over the range 16–97 nm, suggesting a decreased OVF, again contrary to earlier studies. SSA comprised up to 69% of the sub-100 nm particle number, corresponding to concentrations of 110–290 cm−3. Air mass back trajectories indicate that SSA particles were produced 1500 km, 20–40 h upwind of Cape Grim. Transmission electron microscopy (TEM) and X-ray spectrometry measurements of sub-100 nm aerosols collected from the same location, and at the same time, displayed a distinct lack of sea salt. Results herein highlight the potential for biases in TEM analysis of the chemical composition of marine aerosols.

Patient exposure monitoring and radiation qualities in two-dimensional digital x-ray imaging

The methods for estimating patient exposure in x-ray imaging are based on the measurement of radiation incident on the patient. In digital imaging, the useful dose range of the detector is large and excessive doses may remain undetected. Therefore, real-time monitoring of radiation exposure is important. According to international recommendations, the measurement uncertainty should be lower than 7% (confidence level 95%). The kerma-area product (KAP) is a measurement quantity used for monitoring patient exposure to radiation. A field KAP meter is typically attached to an x-ray device, and it is important to recognize the effect of this measurement geometry on the response of the meter. In a tandem calibration method, introduced in this study, a field KAP meter is used in its clinical position and calibration is performed with a reference KAP meter. This method provides a practical way to calibrate field KAP meters. However, the reference KAP meters require comprehensive calibration. In the calibration laboratory it is recommended to use standard radiation qualities. These qualities do not entirely correspond to the large range of clinical radiation qualities. In this work, the energy dependence of the response of different KAP meter types was examined. According to our findings, the recommended accuracy in KAP measurements is difficult to achieve with conventional KAP meters because of their strong energy dependence. The energy dependence of the response of a novel large KAP meter was found out to be much lower than with a conventional KAP meter. The accuracy of the tandem method can be improved by using this meter type as a reference meter. A KAP meter cannot be used to determine the radiation exposure of patients in mammography, in which part of the radiation beam is always aimed directly at the detector without attenuation produced by the tissue. This work assessed whether pixel values from this detector area could be used to monitor the radiation beam incident on the patient. The results were congruent with the tube output calculation, which is the method generally used for this purpose. The recommended accuracy can be achieved with the studied method. New optimization of radiation qualities and dose level is needed when other detector types are introduced. In this work, the optimal selections were examined with one direct digital detector type. For this device, the use of radiation qualities with higher energies was recommended and appropriate image quality was achieved by increasing the low dose level of the system.

The GAF Domain of the cGMP-Binding, cGMP-Specific Phosphodiesterase (PDE5) Is a Sensor and a Sink for cGMP

We describe here a novel sensor for cGMP based on the GAF domain of the cGMP-binding, cGMP-specific phosphodiesterase 5 (PDE5) using bioluminescence resonance energy transfer (BRET). The wild type GAFa domain, capable of binding cGMP with high affinity, and a mutant (GAFaF163A) unable to bind cGMP were cloned as fusions between GFP and Rluc for BRET2 assays. BRET2 ratios of the wild type GAFa fusion protein, but not GAFaF163A, increased in the presence of cGMP but not cAMP. Higher basal BRET2 ratios were observed in cells expressing the wild type GAFa domain than in cells expressing GAFaF163A. This was correlated with elevated basal intracellular levels of cGMP, indicating that the GAF domain could act as a sink for cGMP. The tandem GAF domains in full length PDE5 could also sequester cGMP when the catalytic activity of PDE5 was inhibited. Therefore, these results describe a cGMP sensor utilizing BRET2 technology and experimentally demonstrate the reservoir of cGMP that can be present in cells that express cGMP-binding GAF domain-containing proteins. PDE5 is the target for the anti-impotence drug sildenafil citrate; therefore, this GAF-BRET2 sensor could be used for the identification of novel compounds that inhibit cGMP binding to the GAF domain, thereby regulating PDE5 catalytic activity.

Irradiation of silicon particle detectors with MeV-protons

Silicon particle detectors are used in several applications and will clearly require better hardness against particle radiation in the future large scale experiments than can be provided today. To achieve this goal, more irradiation studies with defect generating bombarding particles are needed. Protons can be considered as important bombarding species, although neutrons and electrons are perhaps the most widely used particles in such irradiation studies. Protons provide unique possibilities, as their defect production rates are clearly higher than those of neutrons and electrons, and, their damage creation in silicon is most similar to the that of pions. This thesis explores the development and testing of an irradiation facility that provides the cooling of the detector and on-line electrical characterisation, such as current-voltage (IV) and capacitance-voltage (CV) measurements. This irradiation facility, which employs a 5-MV tandem accelerator, appears to function well, but some disadvantageous limitations are related to MeV-proton irradiation of silicon particle detectors. Typically, detectors are in non-operational mode during irradiation (i.e., without the applied bias voltage). However, in real experiments the detectors are biased; the ionising proton generates electron-hole pairs, and a rise in rate of proton flux may cause the detector to breakdown. This limits the proton flux for the irradiation of biased detectors. In this work, it is shown that, if detectors are irradiated and kept operational, the electric field decreases the introduction rate of negative space-charges and current-related damage. The effects of various particles with different energies are scaled to each others by the non-ionising energy loss (NIEL) hypothesis. The type of defects induced by irradiation depends on the energy used, and this thesis also discusses the minimum proton energy required at which the NIEL-scaling is valid.

Detection of alpha(2u)-globulin and its bound putative pheromones in the preputial gland of the Indian commensal rat (Rattus rattus) using mass spectrometry

The role of pheromones and pheromone-binding proteins in the laboratory rat has been extensively investigated. However, we have previously reported that the preputial gland of the Indian commensal rat produces a variety of pheromonal molecules and preputial glands would seem to be the predominant source for pheromonal communication. The presence of pheromone-binding proteins has not yet been identified in the preputial gland of the Indian commensal rat; therefore, the experiments were designed to unravel the alpha(2u)-globulin (alpha 2u) and its bound volatiles in the commensal rat. Total preputial glandular proteins were first fractionated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently analyzed by mass spectrometry. Further, we purified alpha 2u and screened for the presence of bound pheromonal molecules with the aid of gas chromatography/mass spectrometry (GC/MS). A novel alpha 2u was identified with a high score and this protein has not been previously described as present in the preputial gland of Indian commensal rats.This novel alpha 2u was then characterized by tandem mass spectrometry (MS/MS). Peptides with m/z values of 969, 1192, 1303 and 1876 were further fragmented with the aid of MS/MS and generated de novo sequences which provided additional evidence for the presence of alpha 2u in the preputial gland. Finally, we identified the presence of farnesol 1 and 2 bound to alpha 2u. The present investigation confirms the presence of alpha 2u (18.54 kDa) in the preputial gland of the Indian commensal rat and identifies farnesol 1 and 2 as probably involved in chemo-communication by the Indian commensal rat.Copyright (C) 2010 John Wiley & Sons, Ltd.

Identification and characterization of a library of microheterogeneous cyclohexadepsipeptides from the fungus Isaria

Ten new cyclic hexadepsipeptides, six isariins and four isaridins, from the fungus Isaria have been identified and characterized by high-performance liquid chromatography, coupled to tandem electrospray ionization mass spectrometry (LC-ESIMS/MS). The isariins possess a beta-hydroxy acid residue and five alpha-amino acids, while isaridins contain a beta-amino acid, an alpha-hydroxy acid, and four alpha-amino acids. One- and two-dimensional NMR spectroscopy confirmed the chemical identity of some of the isariin fractions. Mass spectral fragmentation patterns of [M + H](+) ions reveal clear diagnostic fragment ions for the isariins and isaridins. Previously described cyclic depsipeptides, isarfelins from Isaria felina (Guo, Y. X.; Liu, Q. H.; Ng, T. B.; Wang H. X. Peptides 2005, 26, 2384), are now reassigned as members of the isaridin family. Examination of isaridin sequences revealed significant similarities with cyclic hexadepsipeptides such as destruxins and roseotoxins. The structure of an isariin (isariin A) investigated by NMR spectroscopy indicated the presence of a hybrid alpha beta C-11 turn, formed by the beta-hydroxy acid and glycine residues and a (D)Leu-(L)Ala type II' beta-turn. Additionally, the inhibitory effect of isariins and an isaridin on the intra-erythrocytic growth of Plasmodium falciparum is presented.