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The nicotine metabolite cotinine is widely used to assess the extent of tobacco use in smokers, and secondhand smoke exposure in non-smokers. The ratio of another nicotine metabolite, trans-3′-hydroxycotinine, to cotinine in biofluids is highly correlated with the rate of nicotine metabolism, which is catalyzed mainly by Cytochrome P450 2A6 (CYP2A6). Consequently, this nicotine metabolite ratio is being used to phenotype individuals for CYP2A6 activity and to individualize pharmacotherapies for tobacco addiction. In this paper we describe a highly sensitive liquid chromatography – tandem mass spectrometry method for determination of the nicotine metabolites cotinine and trans-3′-hydroxycotinine in human plasma, urine, and saliva. Lower limits of quantitation range from 0.02 to 0.1 ng/ mL. The extraction procedure is straightforward and suitable for large-scale studies. The method has been applied to several thousand biofluid samples for pharmacogenetic studies and for studies of exposure to low levels of secondhand smoke. Concentrations of both metabolites in urine of non-smokers with different levels of secondhand smoke exposure are presented.

Background: We sought to determine the optimal plasma and urine nicotine metabolites, alone or in combination, to estimate the systemic dose of nicotine after low-level exposure. Methods: We dosed 36 nonsmokers with 100, 200, or 400 μg p.o. of deuterium-labeled nicotine (doses similar to exposure to secondhand smoke) daily for 5 days and then measured plasma and urine nicotine metabolites at various intervals over 24 hours. Results: The strongest correlations with nicotine dose were seen for the sum of four (cotinine + cotinine-glucuronide + trans-3′-hydroxycotinine + 3HC-glucuronide) or six (including also nicotine + nicotine-glucuronide) of the major nicotine metabolites in 24-hour urine collection (r = 0.96), with lesser correlations for these metabolites using spot urines corrected for creatinine at various times of day (r = 0.72-0.80). The sum of plasma cotinine + trans-3′-hydroxycotine was more highly correlated with nicotine dose than plasma cotinine alone (r = 0.82 versus 0.75). Conclusions: Our results provide guidance for the selection of biomarkers to estimate the dose of nicotine taken in low-level (secondhand smoke) tobacco exposure. Impact: This is probably relevant to active smoking as well. Cancer Epidemiol Biomarkers Prev; 19(5); 1160–6. ©2010 AACR.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is tobacco specific and has a longer half-life than other tobacco biomarkers studied thus far. An accurate measurement of the NNAL half-life is important for optimal use to assess exposure to tobacco smoke. We determined the half-life of NNAL in urine in eight daily smokers on a clinical research ward and in five occasional smokers in a real-life environment. Total NNAL in urine was monitored for 14 days in daily smokers after stopping smoking and for up to 60 days in occasional smokers. The average half-life for the terminal phase in the daily smoker group using a two-compartmental body model was 10.3 days (beta phase), and using a noncompartmental model, it was 9.1 days. In the occasional group, these values were 17.6 and 16.0 days, respectively. The alpha-phase half-lives were 14.3 and 27.8 hours for the two groups, respectively. The inter-subject coefficient of variation of the NNAL terminal half-life ranged from 14% to 30%, and the intra-subject coefficient of variation ranged from 3% to 18%. There was very good agreement between the plasma and urinary half-lives in two subjects with plasma analyses: 7.4 versus 7.9 days and 9.2 versus 10.7 days. Mean renal clearance of NNAL was 13 ± 2.3 mL/min. The terminal half-life of NNAL of 10 to 18 days indicates that this biomarker can be used to detect tobacco smoke exposure for 6 to 12 weeks after cessation of exposure and requires a similar time to assess the steady levels of NNAL after switching from one tobacco product to another. (Cancer Epidemiol Biomarkers Prev 2009;18(12):3421–5)

Plasma or saliva cotinine concentrations are used widely as biomarkers of secondhand smoke (SHS) exposure and have been associated with the risk of SHS-related disease. Concentrations of cotinine and other nicotine metabolites are considerably higher in urine than in plasma or saliva, making chemical analysis easier. In addition, urine is often more convenient to collect in some SHS exposure studies. The optimal use of nicotine metabolites in urine, singly or in combination, with or without correction for urine creatinine concentration, to estimate plasma cotinine concentration with low-level nicotine exposure has not been determined.We dosed 36 nonsmokers with 100, 200, or 400 microg deuterium-labeled nicotine (simulating exposure to SHS) by mouth daily for 5 days and then measured plasma and urine cotinine and metabolites at various intervals over 24 hr.A plasma cotinine concentration of 1 ng/ml corresponds on average to a daily intake of 100 microg nicotine. Cotinine concentrations in urine averaged four to five times those in plasma. Correction of urine cotinine for creatinine concentration improved the correlation between urine and plasma cotinine. Measuring multiple cotinine metabolites in urine did not improve the correlation with plasma cotinine, compared with the use of urine cotinine alone.Measurement of urine cotinine corrected for creatinine concentration appears to be the best predictor of plasma cotinine.

Over-allocation of irrigation water has led to widespread environmental degradation in the Murray-Darling Basin (MDB) in Australia, prompting discussions of the water efficiency performance of irrigated industries. There is increasing pressure for irrigators to adopt water efficient practices in line with ecologically sustainable principles, especially with current drought conditions. However, there is great uncertainty surrounding the available practices to improve irrigation efficiency from both ecological and economic standpoints. This paper examines the economic and environmental impact of several possible water efficient practices and government policies, using an interdisciplinary approach combining biophysical modelling and economic modelling. This is done for the case-study of the Mooki catchment in the MDB, located in northern New South Wales, Australia.

The shipping bag used in semiconductor industry is very important as a protection guard of products. It provides good protection for finished products after shipping and the handling by the final users. Among various shipping bags, Moisture Barrier Bag (MBB) is the most widely used type in the semiconductor industry because of its high reliability. However, with the technology advancement, the packing of a large size of IC wafers is inevitable, which brings a new challenge to its resistance to a greater load of shipping and dropping. To reduce the failure rate of MBB leakage during shipment, we evaluate the physical and tensile strength of the shipping bag composed of different films. We significantly improved MBB by optimizing its raw material, which is the first time such approach with plausible results is reported.

Gi-GPCRs, G protein-coupled receptors that signal via Gα proteins of the i/o class (Gαi/o), acutely regulate cellular behaviors widely in mammalian tissues, but their impact on the development and growth of these tissues is less clear. For example, Gi-GPCRs acutely regulate insulin release from pancreatic β cells, and variants in genes encoding several Gi-GPCRs--including the α-2a adrenergic receptor, ADRA2A--increase the risk of type 2 diabetes mellitus. However, type 2 diabetes also is associated with reduced total β-cell mass, and the role of Gi-GPCRs in establishing β-cell mass is unknown. Therefore, we asked whether Gi-GPCR signaling regulates β-cell mass. Here we show that Gi-GPCRs limit the proliferation of the insulin-producing pancreatic β cells and especially their expansion during the critical perinatal period. Increased Gi-GPCR activity in perinatal β cells decreased β-cell proliferation, reduced adult β-cell mass, and impaired glucose homeostasis. In contrast, Gi-GPCR inhibition enhanced perinatal β-cell proliferation, increased adult β-cell mass, and improved glucose homeostasis. Transcriptome analysis detected the expression of multiple Gi-GPCRs in developing and adult β cells, and gene-deletion experiments identified ADRA2A as a key Gi-GPCR regulator of β-cell replication. These studies link Gi-GPCR signaling to β-cell mass and diabetes risk and identify it as a potential target for therapies to protect and increase β-cell mass in patients with diabetes.

The nature of synthetic genetic interactions involving essential genes (those required for viability) has not been previously examined in a broad and unbiased manner. We crossed yeast strains carrying promoter-replacement alleles for more than half of all essential yeast genes1 to a panel of 30 different mutants with defects in diverse cellular processes. The resulting genetic network is biased toward interactions between functionally related genes, enabling identification of a previously uncharacterized essential gene (PGA1) required for specific functions of the endoplasmic reticulum. But there are also many interactions between genes with dissimilar functions, suggesting that individual essential genes are required for buffering many cellular processes. The most notable feature of the essential synthetic genetic network is that it has an interaction density five times that of nonessential synthetic genetic networks2,3, indicating that most yeast genetic interactions involve at least one essential gene.

Mutations in genes encoding the origin recognition complex (ORC) of Saccharomyces cerevisiae affect initiation of DNA replication and transcriptional repression at the silent mating-type loci. To explore the function of ORC in more detail, a screen for genetic interactions was undertaken using large-scale synthetic lethal analysis. Combination of orc2-1 and orc5-1 alleles with the complete set of haploid deletion mutants revealed synthetic lethal/sick phenotypes with genes involved in DNA replication, chromatin structure, checkpoints, DNA repair and recombination, and other genes that were unexpected on the basis of previous studies of ORC. Many of these genetic interactions are shared with other genes that are involved in initiation of DNA replication. Strong synthetic interactions were demonstrated with null mutations in genes that contribute to sister chromatid cohesion. A genetic interaction between orc5-1 and the cohesin mutant scc1-73 suggested that ORC function contributes to sister chromatid cohesion. Thus, comprehensive screening for genetic interactions with a replication gene revealed a connection between initiation of DNA replication and sister chromatid cohesion. Further experiments linked sister chromatid cohesion genes to silencing at mating-type loci and telomeres.

A major metabolite of norepinephrine (NE) in brain is 4-hydroxy-3-methoxyphenylethylene glycol (MHPG). In many species, a large fraction of MHPG formed in brain is converted to the sulfate conjugate. Consequently, MHPG sulfate has been proposed as a biomarker for NE metabolism in the central nervous system. As part of the clinical trials of the monoamine oxidase inhibitor selegiline for treating cocaine addiction, we required a method for measuring urine concentrations of MHPG sulfate. Using a deuterium-labeled analogue as an internal standard, we developed a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/ MS) method for determination of MHPG sulfate in human urine. Sample preparation involves simply diluting 50 microL of urine with 1 mL of ammonium formate buffer and adding the internal standard. The sample is centrifuged, the supernate is transferred to an autosampler vial, and 10 microL is injected into the LC-MS/MS system. Standard curves from 50 to 10,000 ng/mL are generated. Only one sample of 277 clinical samples analyzed had a concentration outside of this range. Precision (coefficient of variation) ranged from 1.9 to 9.7%, and accuracy ranged from 97 to 103% of expected values for controls prepared by spiking sulfatase-treated urine with MHPG sulfate.

To identify the relative importance of peritoneal fibrosis and angiogenesis in peritoneal membrane dysfunction, adenoviral mediated gene transfer of angiostatin, a recognized angiogenesis inhibitor, and decorin, a transforming growth factor-beta-inhibiting proteoglycan, were used in a daily infusion model of peritoneal dialysis. A peritoneal catheter and subcutaneous port were inserted in rats. Five and fourteen d after insertion, adenovirus-expressing angiostatin, decorin, or AdDL70, a null control virus, were administered. Daily infusion of 4.25% Baxter Dianeal was initiated 7 d after catheter insertion and continued until day 35. Three initial doses of lipopolysaccharide were administered on days 8, 10, and 12 to promote an inflammatory response. Net ultrafiltration was used as a measure of membrane function, and peritoneum-associated vasculature and mesenteric collagen content was quantified. Ultrafiltration dysfunction, angiogenesis, and fibrosis were observed in daily infusion control animals. Animals treated with AdAngiostatin demonstrated an improvement in net ultrafiltration (-3.1 versus -7.8 ml for control animals; P = 0.0004) with a significant reduction in vessel density. AdDecorin-treated animals showed a reduction in mesenteric collagen content (1.8 versus 2.9 microg/mg; P = 0.04); however, AdDecorin treatment had no effect on net ultrafiltration. In a rodent model of peritoneal membrane failure, net ultrafiltration was significantly improved and peritoneal-associated blood vessels were significantly reduced by using adenovirus-mediated gene transfer of angiostatin. Decorin, a transforming growth factor-beta-inhibiting proteoglycan, reduced collagen content but did not affect net ultrafiltration. Improvement in the function of the peritoneum as a dialysis membrane after treatment with angiostatin has implications for treatment of peritoneal membrane dysfunction seen in patients on long-term dialysis.

In this paper, an effective baking methodology to remove thin film stability monitor wafer surface contamination is reported. We consider baking temperature, baking time, and time interval between baking and measurement as three key factors for methodology optimization study. A long-time and durative experiment is designed for the obtaining of a suitable and feasible baking condition, in which design of experiment (DOE) is applied for the data analysis and the study of baking effect on wafer thickness change. From DOE analysis, we Anally take the baking condition of 140/ 60/210 as the optimal pre-treatment condition to monitor the stability of thickness metrology tools.

Limitation of cultured rat hepatoma cells for an essential amino acid results in a specific decrease in expression of several genes that are preferentially expressed in the liver, including the serum albumin and transthyretin genes. In the work presented here, we examined whether the coordinate repression of these genes is caused by decreased activity of one or more of the liver-enriched transcription factors, hepatocyte nuclear factor-1 (HNF-1), HNF-3, HNF-4 or C/EBP. To address this question, HepG2 human hepatoma cells were transiently transfected with luciferase reporter constructs containing multiple copies of individual transcription factor binding sites. Limitation for an essential amino acid resulted in specific repression of a construct in which luciferase expression was directed by HNF-1. A single HNF-1 binding site located adjacent to the TATA box plays a major role in transcription directed by the serum albumin promoter in transient transfection assays. Amino acid limitation of cells transfected with an albumin promoter/luciferase reporter construct resulted in specific repression of promoter activity. In addition, bacterial methylation or site-directed mutagenesis of the HNF-1 binding site in the albumin proximal promoter region eliminated the regulation of an albumin promoter-luciferase reporter construct under conditions of amino acid limitation. These results demonstrated that the HNF-1 binding site played a major role in regulation of the albumin promoter by amino acid availability. Deletion analysis of the albumin promoter confirmed regulation through the HNF-1 binding site and also identified a second amino acid regulatory element in the upstream region of the albumin promoter, which has been shown previously to contain a functional binding site for HNF-3. The repression of albumin promoter and HNF-1 reporter constructs in amino acid-limited cells occurred without a change in the DNA binding activity of HNF-1. Moreover, HNF-3 DNA binding activity was also not decreased in amino acid-limited cells. These results suggest that the regulation of transcription by amino acids occurs at the level of transcriptional activation by HNF-1 and HNF-3, rather than by alteration of the DNA binding activity of either factor.

The precise quantitation of smoking during pregnancy is difficult in retrospective studies. Routinely collected blood specimens from newborns, stored as dried blood spots, may provide a low-cost method to objectively measure maternal smoking close to the time of delivery. This article compares cotinine levels in dried blood spots to those in umbilical cord blood to assess cotinine in dried blood spots as a biomarker of maternal smoking close to the time of delivery. The California Genetic Disease Screening Program provided dried blood spots from 428 newborns delivered in 2001-2003 with known umbilical cord blood cotinine levels. Cotinine in dried blood spots was measured in 6.35-mm punches by using liquid chromatography-tandem mass spectrometry (quantitation limit, 3.1 ng/mL). Repeated measures of cotinine in dried blood spots were highly correlated (R2= 0.99, P < 0.001) among 100 dried blood spots with cotinine quantitated in 2 separate punches. Linear regression revealed that cotinine levels in dried blood spots were slightly lower than those in umbilical cord blood and predicted umbilical cord blood cotinine levels well (β = 0.95, R2= 0.80, and P < 0.001 for both cotinine levels in log10 scale). When defining active smoking as a cotinine level of 10 ng/mL or more and using umbilical cord blood cotinine as the criterion standard, we found that measurements of cotinine in dried blood spots had high sensitivity (92.3%) and specificity (99.7%) in the prediction of maternal active smoking. Cotinine levels in dried blood spots are an accurate biomarker of maternal smoking close to the time of delivery. © 2013 The Author. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health. All rights reserved.

Cigarette smoking remains a significant health threat for smokers and nonsmokers alike. Secondhand smoke (SHS) is intrinsically more toxic than directly inhaled smoke. Recently, a new threat has been discovered – Thirdhand smoke (THS) – the accumulation of SHS on surfaces that ages with time, becoming progressively more toxic. THS is a potential health threat to children, spouses of smokers and workers in environments where smoking is or has been allowed. The goal of this study is to investigate the effects of THS on liver, lung, skin healing, and behavior, using an animal model exposed to THS under conditions that mimic exposure of humans. THS-exposed mice show alterations in multiple organ systems and excrete levels of NNAL (a tobacco-specific carcinogen biomarker) similar to those found in children exposed to SHS (and consequently to THS). In liver, THS leads to increased lipid levels and non-alcoholic fatty liver disease, a precursor to cirrhosis and cancer and a potential contributor to cardiovascular disease. In lung, THS stimulates excess collagen production and high levels of inflammatory cytokines, suggesting propensity for fibrosis with implications for inflammation-induced diseases such as chronic obstructive pulmonary disease and asthma. In wounded skin, healing in THS-exposed mice has many characteristics of the poor healing of surgical incisions observed in human smokers. Lastly, behavioral tests show that THS-exposed mice become hyperactive. The latter data, combined with emerging associated behavioral problems in children exposed to SHS/THS, suggest that, with prolonged exposure, they may be at significant risk for developing more severe neurological disorders. These results provide a basis for studies on the toxic effects of THS in humans and inform potential regulatory policies to prevent involuntary exposure to THS.

This paper reports the research of applying the worst stress condition of thick gate oxide LDMOSFET hot carrier reliability. Based on electrical characteristic and hot carrier degradation investigation, the worst stress condition selection and failure mechanism are discussed and then the reasonable stress condition is proposed in this paper.

Although the serotonin (5-hydroxytryptamine, 5-HT) neurotransmitter system has been implicated in modulating executive control processes such as attention, response inhibition, and behavioral flexibility, the contributions of particular serotonin receptors remain unclear. Here, using operant-based behavioral paradigms, we demonstrate that mice with genetically ablated 5-HT2C receptors (2CKO mice) display deficits in executive functions. 2CKO mice were impaired in the acquisition of a visuospatial attention task as assessed in the 5-choice serial reaction time task (5-CSRTT). In this task, 2CKO mice exhibited marked impairment of attentional processes, with normal response inhibition. We assessed dynamic changes in neurotransmitter levels within the nucleus accumbens (NAc) by in vivo microdialysis in task-performing animals. Extracellular dopamine concentrations were elevated in the NAc of 2CKO mice during task performance, indicating that 5-HT2C receptors impact dopamine homeostasis during a visuospatial attention task. These findings raise the possibility that disinhibition of mesolimbic dopamine pathways contributes to impaired attention and perturbed task performance in 2CKO mice. Additionally, in a spatial reversal learning task, 2CKO mice failed to improve their performance over a series of reversals, indicating that intact 5-HT2C receptor signaling is required to accurately respond to repeated changes in reward contingencies. In contrast to the 2CKO phenotype in the 5-CSRTT, wild-type mice treated with the 5-HT2C receptor antagonist SB242084 exhibited diminished response inhibition, suggesting differing effects of acute pharmacological blockade and constitutive loss of 5-HT2C receptor activity. Altogether, these findings provide insights into the serotonergic regulation of executive control processes and suggest that impaired 5-HT2C receptor signaling during development may predispose to executive function disorders.

Background: Smoking tobacco preparations in a water pipe (hookah) is widespread in many places of the world and is perceived by many as relatively safe. We investigated biomarkers of toxicant exposure with water pipe compared with cigarette smoking. Methods: We conducted a crossover study to assess daily nicotine and carcinogen exposure with water pipe and cigarette smoking in 13 people who were experienced in using both products. Results: When smoking an average of 3 water pipe sessions compared with smoking 11 cigarettes per day (cpd), water pipe use was associated with a significantly lower intake of nicotine, greater exposure to carbon monoxide (CO), and a different pattern of carcinogen exposure compared with cigarette smoking, with greater exposure to benzene, and high molecular weight polycyclic aromatic hydrocarbon (PAH), but less exposure to tobacco-specific nitrosamines, 1,3-butadiene, acrolein, acrylonitrile, propylene oxide, ethylene oxide, and low molecular weight PAHs. Conclusions: A different pattern of carcinogen exposure might result in a different cancer risk profile between cigarette and water pipe smoking. Of particular concern is the risk of leukemia related to high levels of benzene exposure with water pipe use. Impact: Smoking tobacco in water pipes has gained popularity in the United States and around the world. Many believe that water pipe smoking is not addictive and less harmful than cigarette smoking. We provide data on toxicant exposure that will help guide regulation and public education regarding water pipe health risk. Cancer Epidemiol Biomarkers Prev; 22(5); 765–72. ©2013 AACR.

Double minutes (DMs) are hallmarks of gene amplification. However, their molecular structure and the mechanisms of formation are largely unknown. To elucidate the structure and underlying molecular mechanism of DMs, we obtained and cloned DMs using microdissection; and degenerated oligonucleotide primed polymerase chain reaction (DOP-PCR) from the ovarian cancer cell line UACC-1598. Two large amplicons, the 284 kb AmpMYCN, originating from locus 2p24.3 and the 391 kb AmpEIF5A2, from locus 3q26.2, were found co-amplified on the same DMs. The two amplicons are joined through a complex 7 kb junction DNA sequence. Analysis of the junction has revealed three de novo created small palindromes surrounding the six breakpoints. Consistent with these observations, we further found that 70% of the 57 reported DM junction sequences have de novo creation of small palindromic sequences surrounding the breakpoints. Together, our findings indicate that de novo-generated small palindromic sequences are characteristic of amplicon boundary junctions on DMs. It is possible that the de novo-generated small palindromic sequences, which may be generated through non-homologous end joining in concert with a novel DNA repair machinery, play a common role in amplicon rejoining and gene amplification.

Background: Smoking tobacco preparations in a water pipe (hookah) is widespread in many places of the world, including the United States, where it is especially popular among young people. Many perceive water pipe smoking to be less hazardous than cigarette smoking. We studied systemic absorption of nicotine, carbon monoxide, and carcinogens from one water pipe smoking session. Methods: Sixteen subjects smoked a water pipe on a clinical research ward. Expired carbon monoxide and carboxyhemoglobin were measured, plasma samples were analyzed for nicotine concentrations, and urine samples were analyzed for the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanol (NNAL) and polycyclic aromatic hydrocarbon (PAH) metabolite biomarker concentrations. Results: We found substantial increases in plasma nicotine concentrations, comparable to cigarette smoking, and increases in carbon monoxide levels that are much higher than those typically observed from cigarette smoking, as previously published. Urinary excretion of NNAL and PAH biomarkers increased significantly following water pipe smoking. Conclusions: Absorption of nicotine in amounts comparable to cigarette smoking indicates a potential for addiction, and absorption of significant amounts of carcinogens raise concerns of cancer risk in people who smoke tobacco products in water pipes. Impact: Our data contribute to an understanding of the health impact of water pipe use. Cancer Epidemiol Biomarkers Prev; 20(11); 2345–53. ©2011 AACR.

using a sensitive plate based ELISA system (MSD). Results: Unlike gamma secretase inhibitors, which demonstrate a “potency shift" when the concentration of substrate is very high as in systems overexpressing APP (Burton et al 2008), the potency of GSMs does not demonstrate a systematic shift in overexpressing systems. The pattern of modulation of As peptides was consistent across model systems and specific to each structurally distinct GSM, with some demonstrating a decrease in As42 and an increase in As38 and others demonstrating a decrease in both As42 and 38. Conclusions: The physiological significance of these alterations in cleavage site is not well understood, but appears to be driven by unique interactions of GSMs with gamma secretase.

The synthesis and extensive biological study of two new tridentates ligands based on pyrazole and triazole are described. The antifungal activity against the budding yeast cells of the newly synthesized compounds was determined. These compounds were toxic to yeast cells. Cell cycle analysis suggested that treatment with these compounds impairs cell division in G1 of the cell cycle. Using yeast-based functional genomics technologies, we found that these compounds tolerance requires DNA repair pathway and SKI complex function. We have also found that the PKC1 heterozygous deletion strain was the most sensitive to these compounds using HaploInsufficiency Profiling, suggesting that the Pkc1 protein may be the target for these compounds. These results strongly suggest that these compounds induce DNA damage and thus exert a different mechanism of action compared to other azole derivatives. These two compounds might therefore represent promising lead compounds for further development of antifungal drugs for human therapy.

A selected ion monitoring method for determination of the tobacco alkaloids anabasine, anatabine, nornicotine, metanicotine, dihydrometanicotine, and 2,3'-bipyridyl in urine of smokers and smokeless tobacco users is described. The method involves conversion of the secondary amine alkaloids to tertiary amine derivatives by reductive alkylation using an aldehyde and sodium borohydride, and chromatography on a 5% phenylmethylsilicone capillary column. These derivatives have good chromatographic properties, allowing determination of concentrations as low as 1 ng/ml. The alkaloid 2,3'-bipyridyl is unaffected by the derivatization procedure and may be determined simultaneously with the other alkaloids. The structural analogues 2-(3-pyridyl)hexahydroazepine, 5-methyldihydrometanicotine, and 6-methyl-2,3'-bipyridyl were synthesized for use as internal standards. Using the method, concentrations and 24 h excretion of anabasine, anatabine, and nornicotine in urine of twenty-two smokers, eight chewing tobacco users, and six oral snuff users were determined and compared with concentrations and excretion of nicotine and its metabolite cotinine. Excretion of nicotine and cotinine was similar in all tobacco users, but excretion of anabasine, anatabine and nornicotine was substantially greater in urine of smokeless tobacco users, presumably due to absence of pyrolysis of these alkaloids in smokeless tobacco products.

Active smoking and secondhand smoke (SHS) exposure can cause or worsen many acute and chronic cardiovascular and pulmonary diseases (1, 2). SHS is a combination of mainstream smoke (exhaled by smokers) and sidestream smoke given off by the burning end of the tobacco product. Even brief exposure to SHS has significant immediate effects on endothelial cell function, inflammation, and lung function (3). Despite public health efforts to discourage smoking, U.S. smoking prevalence has recently plateaued at 20%, and the number of smokers internationally is increasing (4, 5). In addition, even though smoking has been increasingly restricted in public places and workplaces, an estimated 40% of U.S. nonsmokers have biological evidence of significant SHS exposure (6). Thus, cigarette smoke exposure remains an important cause of preventable chronic and acute disease in both smokers and those exposed to SHS. The association between recent tobacco smoke exposure and critical illness has been poorly studied. The lack of accurate methods to quantify exposure to tobacco smoke, and SHS in particular, in the critically ill has been a major hindrance. Historically, studies have commonly used questionnaires to measure exposure to tobacco smoke. This method is particularly difficult to use in the intensive care unit, because most critically ill patients have altered levels of consciousness or are endotracheally intubated or both. When patients are unable to provide a history, surrogate and chart reporting are often substituted; however, these sources can be subject to social desirability bias and are frequently inaccurate, out of date, or missing (7–9). Thus, to study the effects of cigarette smoke exposure on the incidence or outcomes of critical illness, a more quantitative approach to measuring cigarette smoke exposure in critically ill patients is needed. Biomarkers of cigarette smoke exposure quantify the biologically active dose of nicotine and toxins to which patients have been exposed, overcoming the subjectivity and inaccuracy of self-reporting or surrogate reporting. Such markers have been used to distinguish active and passive smokers from nonsmokers in epidemiologic studies and to establish causal relationships between both active smoking and SHS exposure and cardiovascular and lung disease (1, 10, 11). Nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (a potent lung carcinogen), and their metabolites are markers specific for tobacco exposure in smokers, smokeless tobacco users, and nonsmokers exposed to SHS. Determining the optimal biomarker or combination of biomarkers for measuring cigarette smoke exposure in critically ill patients will be key for future studies of this population. The primary objective of our study was to compare cigarette smoke exposure, as measured by biomarkers, to exposure by self-report in a cohort of critically ill patients. A secondary objective was to determine how well biomarkers of exposure taken from different biological specimens (serum, urine, hair, and nails) correlate with each other in this population.

The ability to entrain circadian rhythms to food availability is important for survival. Food-entrained circadian rhythms are characterized by increased locomotor activity in anticipation of food availability (food anticipatory activity). However, the molecular components and neural circuitry underlying the regulation of food anticipatory activity remain unclear. Here we show that serotonin(2C) receptor (5-HT2CR) null mutant mice subjected to a daytime restricted feeding schedule exhibit enhanced food anticipatory activity compared to wild-type littermates, without phenotypic differences in the impact of restricted feeding on food consumption, body weight loss, or blood glucose levels. Moreover, we show that the enhanced food anticipatory activity in 5-HT2CR null mutant mice develops independent of external light cues and persists during two days of total food deprivation, indicating that food anticipatory activity in 5-HT2CR null mutant mice reflects the locomotor output of a food-entrainable oscillator. Whereas restricted feeding induces c-fos expression to a similar extent in hypothalamic nuclei of wild-type and null mutant animals, it produces enhanced expression in the nucleus accumbens and other extrahypothalamic regions of null mutant mice relative to wild-type subjects. These data suggest that 5-HT2CRs gate food anticipatory activity through mechanisms involving extrahypothalamic neural pathways.

The objective of this study was to analyse environmental tobacco smoke (ETS) and PAH metabolites in urine samples of non-occupationally exposed non-smoker adult subjects and to establish relationships between airborne exposures and urinary concentrations in order to (a) assess the suitability of the studied metabolites as biomarkers of PAH and ETS, (b) study the use of 3-ethenypyridine as ETS tracer and (c) link ETS scenarios with exposures to carcinogenic PAH and VOC. Urine samples from 100 subjects were collected and concentrations of monophenolic metabolites of naphthalene, fluorene, phenanthrene, and pyrene and the nicotine metabolites cotinine and trans-3′-hydroxycotinine were measured using liquid chromatography–tandem mass spectrometry (LC-MS/MS) to assess PAH and ETS exposures. Airborne exposures were measured using personal exposure samplers and analysed using GC–MS. These included 1,3-butadiene (BUT), 3-ethenylpyridine (3-EP) (a tobacco-specific tracer derived from nicotine pyrolysis) and PAHs. ETS was reported by the subjects in 30-min time–activity questionnaires and specific comments were collected in an ETS questionnaire each time ETS exposure occurred. The values of 3-EP (N0.25 μg/m3 for ETS) were used to confirm the ETS exposure status of the subject. Concentrations as geometric mean, GM, and standard deviation (GSD) of personal exposures were 0.16 (5.50) μg/m3 for 3-EP, 0.22 (4.28) μg/m3 for BUT and 0.09 (3.03)ng/m3 for benzo(a)pyrene. Concentrations of urinary metabolites were 0.44 (1.70)ng/mL for 1-hydroxypyrene and 0.88 (5.28)ng/mL for cotinine. Concentrations of urinary metabolites of nicotine were lower than in most previous studies, suggesting very low exposures in the ETS-exposed group. Nonetheless,concentrations were higher in the ETS population for cotinine, trans-3′hydroxycotinine, 3-EP, BUT and most high molecular weight PAH, whilst 2-hydroxyphenanthrene, 3+4-hydroxyphenanthrene and 1-hydroxyphenanthrene were only higher in the high-ETS subpopulation. There were not many significant correlations between either personal exposures to PAH and their urinary metabolites, or of the latter with ETS markers. However, it was found that the urinary log cotinine concentration showed significant correlation with log concentrations of 3-EP (R=0.75), BUT (R=0.47), and high molecular weight PAHs (MWN200), especially chrysene (R=0.55) at the p=0.01 level. On the other hand, low correlation was observed between the PAH metabolite 2-naphthol and the parent PAH, gas-phase naphthalene. These results suggest that (1) ETS is a significant source of inhalation exposure to the carcinogen 1,3-butadiene and high molecular weight PAHs, many of which are carcinogenic, and (2) that for lower molecular weight PAHs such as naphthalene, exposure by routes other than inhalation predominate, since metabolite levels correlated poorly with personal exposure air sampling., peer-reviewed

A gas chromatographic method for the determination of the nicotine metabolite trans-3′-hydroxycotinine is described. The method involves conversion of the metabolite to the tert.-butyldimethylsilyl derivative, chromatography on a fused-silica capillary column, and determination using nitrogen—phosphorus detection or electron ionization mass spectrometry with selected ion monitoring. A structural analogue, trans-3-hydroxy-1-methyl-5-(2-pyridyl)pyrrolidin-2-one (trans-3′-hydroxy-ortho-cotinine), was used as an internal standard. Using selected ion monitoring, good precision and accuracy were obtained for determination of trans-3′-hydroxycotinine in urine over the concentration range 10–10 000 ng/ml. There was a good correlation between concentrations determined by selected ion monitoring and by nitrogen—phosphorus detection in urine of smokers, although low concentrations determined using nitrogen—phosphorus detection tended to be somewhat higher, suggesting some interference from urinary constituents. Concentrations and 24-h excretion of trans-3′-hydroxycotinine in the urine of 22 cigarette smokers are reported and compared to concentrations and excretion of nicotine, cotinine, nicotine 1′-N-oxide, nornicotine, and cotinine N-oxide.

The paper appraises the myriad of Murray-Darling Basin-related policies since the early 1990s. It contends that significant environmental improvements could have been achieved at substantially lower cost had decisive action been taken earlier. If the total expenditures in the last two decades had been put solely towards water entitlement buy-backs, an amount of water several times that necessary to significantly improve the health of the Basin would have been acquired.

A method for simultaneous determination of nicotine, its metabolite cotinine, and the stable isotope-labeled analogs nicotine-3',3'-d2 and cotinine-4',4'-d2 in human plasma has been developed. The method utilizes capillary column gas chromatography with detection by electron impact mass spectrometry and selected ion monitoring. Sensitivity is adequate for determination of nicotine and nicotine-d2 at concentrations as low as 1 ng ml-1, and cotinine and cotinine-d2 at concentrations as low as 10 ng ml-1 with good precision and accuracy. The method has been used to compare the elimination kinetics of (S)-nicotine-3',3'-d2 with natural nicotine in human subjects. Total clearance of nicotine-3',3'-d2 was virtually identical to the total clearance of natural nicotine, which validates the use of the deuterium-labeled analog in quantitative studies of nicotine metabolic disposition.

There has now been almost two decades of natural resource management by signatory states under the Murray-Darling Basin Agreement Despite significant public expense, the success of initiatives to improve the Basin’s environmental remains ambiguous. This confusion is partly due to poorly distinguished investment outcomes, a blurring of the transparency of public spending and a lack of accountability of decision makers. The aim of this study is to demonstrate that significant environmental improvements could have been achieved at a much lower cost if decisive action been taken early. The research report outlines the myriad of Murray-Darling Basin related policies and its funding. It also notes the achievements and impediments to program success.

Exposure to secondhand tobacco smoke (SHS) has been linked to increased risk for a number of diseases, including lung cancer. The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is of particular interest due to its potency and its specificity in producing lung tumors in animals. The NNK metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in urine is frequently used as a biomarker for exposure. Due to its long half-life (40-45 days), NNAL may provide a long-term, time-averaged measure of exposure. We developed a highly sensitive liquid chromatography – tandem mass spectrometry (LC-MS/MS) method for determination of NNAL in human urine. The method involves liquid-liquid extraction followed by conversion to the hexanoate ester derivative. This derivative facilitates separation from interfering urinary constituents by extraction and chromatography, and enhances detection with electrospray ionization mass spectrometry. The lower limit of quantitation is 0.25 pg/mL for 5 mL urine specimens. Applications to studies of people with a range of different SHS exposure levels is described.

The radio-contrast agent Angiofil has recently been developed to be predominantely applied in forensic medicine. Angiofil is a liquid radio-contrast agent based on iodine. Its viscosity is easy to adjust by the choice and the concentration of the solvent. Therefore, it is well suited for penetrating vessels of different diameters. The liquid Angiofil avoids the sedimentation of suspensions containing radio-opaque materials such as barium sulfate. The injection of Angiofil into the vascular system of mice post-mortem results in remarkable data showing the vascular trees of tissues and entire organs. Penetration into the surrounding tissue was not observed. Consequently, Angiofil has the potential to reach the performance of the established casting agent Microfil.

Irrigation enterprises can be evaluated from a perspective of technical and environmental efficiency. This study determines the technical and environmental efficiency in irrigated cotton enterprises. This is achieved by investigating efficiency at cotton enterprise in the case study area of the Mooki Catchment, located in northern New South Wales. Deep drainage loss which contributes to salinity is considered as an environmental detrimental output. Using four different specifications of Data Envelopment Analysis relative efficiency rankings are determined for each agricultural area in the catchment. This result is then compared to biophysical characteristics from previous studies to help identify the particular features of an area which may influence outcomes that are both environmentally sound and economically efficient. With the identification of the most and least efficient cotton irrigating areas in the region, policymakers can construct a relative ranking system to best determine policy directions in order to achieve economic and environmental objectives.

The problem of water scarcity has become one of the most controversial topics in Australia over the past decades, with particular focus being the ‘sustainable’ allocation of water between extractive and environmental purposes. Geographical factors are defining the extreme variability in climate and water supply in Australia and, in the past, this was used as a rationale for the construction of large irrigation projects to deliver water to rural, urban, and industrial users. During this ‘expansionary’ phase of Australia’s water use sector, the cost of augmenting supply was relatively low and environmental considerations were secondary to the development imperative. As a result, water resources became over-allocated for extractive uses spurred on by consistent underpricing of water, which indicated a failure to reflect the true cost of water supply. As Australia’s water economy entered a ‘mature’ phase, it was no longer possible to increase supply cheaply as the most easily accessible water resources had already been captured. This was followed by widespread environmental degradation manifested in the Murray- Darling Basin, the nation’s largest river basin which hosts much of Australia’s agricultural production. Consequently, the focus shifted towards demand management, leading to a myriad of regulation aimed at increasing the allocative efficiency of scarce water resources. Towards this end, substantial government funding was injected into the various initiatives throughout the water reform process. Despite the on-going government activities in the area of water reform, the understanding of the actual economic impact and environmental outcomes of various water policies in practice remains limited. In the absence of such understanding, the effectiveness of various government water initiatives is ambiguous and inevitably compromised. The present study addresses this knowledge gap by establishing a method for evaluating the economic and environmental outcomes of environmentally-oriented polices that affect irrigated industries in a catchment. The method is based on an integrated biophysical and economic modelling approach, which enables spatial relationships to be captured accurately allowing a more realistic analysis. Information generated from a computer based biophysical simulation model form the basis of an economic optimisation model with constraints pertaining to environmental targets and water supply limits. The economic model consists of a linear programming and dynamic programming component, and involves the optimisation of resource use from a catchment manager’s perspective, seeking to achieve efficient resource use but at the same time conform to given environmental objectives. This embedded linear and dynamic programming approach was required to determine the optimal intra-seasonal and inter-seasonal water allocation, given various catchment environmental targets. The interdisciplinary approach enables the economic and ecological outcomes of the catchment management policies to be simulated and assessed at a spatially explicit scale, due to the link to Geographical Information Systems (GIS) in the biophysical model. The overall objective was to create a decision-making framework that could be used to determine the least-cost means of meeting environmental targets and resource constraints. The solutions to the analysis are directly applicable to the case study, the Mooki catchment in northern New South Wales (NSW), but with an adaptable framework that can be applied to other catchments. Specific objectives include an evaluation of the possibility of using alternative irrigation systems, as well as an evaluation of the benefits that can be realised by establishing water market, in the light of environmentally-oriented catchment policies for the case study. The economic cost of achieving environmental targets pertaining to environmental flow requirements and salinity reduction, in the form of end-of-valley salinity targets, was explicitly calculated through the economic model. While salinity targets have been set for NSW catchments, the practicality of such targets is in question, given the substantial reductions in water allocation to irrigation activities, which is one of the key contributors to deep-drainage. An additional objective in this study was therefore to investigate the value of having deep drainage targets. A further consideration is the effect of “external agents” in the form of government plans to buyback entitlements from irrigation districts, or the possibility of significant water rights purchases from mining industries. The implications of external water market entrants on the regional agricultural industry were examined.

Background: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life threatening disease often leading to severe bleeding diatheses, and/or miscarriage; although the incidence of miscarriage has not been adequately studied. FNAIT is caused by a maternal immune response against fetal platelet antigens, in which >80% reported cases have antibodies against β3 integrin. Maternal antibodies and Natural Killer (NK) cells may target antigen positive trophoblast cells and cause miscarriage, but this hypothesis has never been explored.In this study, we investigated whether platelet antibodies and NK cells impaired trophoblast invasion, and placental angiogenesis and function. Methods: β3 integrin deficient female mice were immunized with wild-type (WT) platelets and bred with WT males. Placental vascularisation and function were investigated by echography and micro-computered tomography (CT). Angiogenic and inflammatory cytokines, placenta growth factor (PlGF) and fms-like tyrosine receptor levels (Flt-1) were detected by ELISA. Placental function such as nutrient transport was detected by maternal intravenous administration of biotin and its perfusion to the fetus. NK cell phenotype was assessed by FACS. Placenta pathology was investigated by hematoxylin & eosin staining and immuno-histochemistry for cytokeratin-7, NK cell perforin and smooth muscle actin. Apoptosis and decidual remodeling were investigated by TUNEL assays. In vitro, NK cells were co-cultured with trophoblast cell lines and cytotoxicity was detected by flow cytometry. Results and discussion: Growth restriction and fetal loss/miscarriage only occurred in immunized mothers around embryo day E14.5. Placenta of affected fetuses had significantly reduced vascularization and materno-placental perfusion as demonstrated by ultrasound. CT scan also confirmed shallow development of placental sponge capillaries. These observations are validated by significantly reduced biotin perfusion to the fetuses which had a high hypoxia level. Immunized mice exhibited enlarged spleens as well as an increased Th1 and Th17 immune responses. These pro-inflammatory responses may contribute to trophoblast Flt-1 over-expression and a decreased plasma PlGF/sFlt-1 ratio. E.14.5, the end of organogenesis, is concomitant with trophoblast invasion into spiral arteries. The remodeling of spiral arteries lowers maternal vascular resistance and increases utero-placental blood flow which is critical for healthy pregnancy. Expression of trophoblast marker, Cytokeratin 7, was decreased in the placentas of immunized mice, suggesting scanty invasion invasion. Histology of affected placenta confirmed ischemic tissues, as revealed by significantly reduced numbers of maternal red blood cells in the labyrinth. Unexpectedly, decidual NK cell number remained elevated by day E14.5 in the placenta of immunized mice. Importantly activated NK cells released significant amount of perforin in the placenta, which may destroy target cells expressing β3 integrin. These observations support an abnormal remodeling process mediated by maternal antibodies and NK cells, since increased apoptosis was found in the decidua of immunized pregnant mice compared to naive control. Interestingly, maternal intravenous immunoglobulin therapy ameliorated survival of FNAIT fetuses.. To investigate the molecular and cellular mechanism involved, human choriocarcinoma cells (BeWo) were incubated with both murine anti-β3 antibodies and human HPA-1a IgG. Our data demonstrated, for the first time, that anti-platelet antibodies induced over-expression of the anti-angiogenic molecule, Flt-1 by BeWo cells. Conclusion: Our data suggest that maternal β3 integrin antibodies and NK cells impaired placental pro-angiogenic signalling and function. Reduced trophoblast invasion may cause poor materno-placental perfusion and fetus loss/miscarriage in FNAIT. Disclosures No relevant conflicts of interest to declare.

Mutations impacting specific stages of cell growth and division have provided a foundation for dissecting mechanisms that underlie cell cycle progression. We have undertaken an objective examination of the yeast cell cycle through flow cytometric analysis of DNA content in TetO7promoter mutant strains representing 75% of all essential yeast genes. More than 65% of the strains displayed specific alterations in DNA content, suggesting that reduced function of an essential gene in most cases impairs progression through a specific stage of the cell cycle. Because of the large number of essential genes required for protein biosynthesis, G1 accumulation was the most common phenotype observed in our analysis. In contrast, relatively few mutants displayed S-phase delay, and most of these were defective in genes required for DNA replication or nucleotide metabolism. G2 accumulation appeared to arise from a variety of defects. In addition to providing a global view of the diversity of essential cellular processes that influence cell cycle progression, these data also provided predictions regarding the functions of individual genes: we identified four new genes involved in protein trafficking (NUS1, PHS1, PGA2, PGA3), and we found that CSE1 and SMC4 are important for DNA replication.

Over-allocation of irrigation water has led to widespread environmental degradation in the Murray-Darling Basin, prompting discussions of the water efficiency performance of irrigated industries, particularly cotton. There is increasing pressure for irrigators to adopt water efficient practices in line with ecologically sustainable principles, especially with current drought conditions. However, there is great uncertainty surrounding the available measures to improve irrigation efficiency from both ecological and economic standpoints. An integrated biophysical and economic modelling approach is used to determine the optimal allocation of water, irrigation system, source of water, and crop pattern, subject to various environmental and resource targets. Spatially referenced data are used to provide realistic results that are directly applicable to the case study basin in northern New South Wales (NSW). The results can assist in policy design and its implementation to achieve environmental objectives at least cost.

Dietary supplements containing botanical forms of caffeine and ephedra alkaloids have been widely promoted and used in the U.S. for weight loss and athletic enhancement despite a lack of adequate research on the pharmacology of these botanical stimulants. In order to analyze dietary supplements and perform human pharmacokinetic studies, an analytical approach with good precision and accuracy was needed with sufficient sensitivity to detect very low levels of ephedra alkaloids. A liquid chromatography-atmospheric pressure chemical ionization (APCI) tandem mass spectrometry (LC-MS-MS) method was developed for quantitating the various ephedrine-group alkaloids found in dietary supplements that contain Ephedra species, and in plasma and urine of persons consuming these supplements. Using this method, low nanogram-per-milliliter concentrations of ephedrine, pseudoephedrine, norephedrine, norpseudoephedrine, methylephedrine, methylpseudoephedrine, and caffeine can be quantitated in a 12-min LC-MS-MS run.

Objectives Peritoneal membrane changes are related to daily exposure to non physiologic dialysate and recurrent acute inflammation. We modified a daily infusion and inflammation model and evaluated it for fibrotic and angiogenic features. The feasibility of adenovirus-mediated gene transfer in the model was also assessed. Methods Peritoneal catheters were implanted in rats. Over a period of 4 weeks, the animals received a daily infusion of Dianeal 4.25% (Baxter Healthcare Corporation, Deerfield, IL, U.S.A.) with an initial three doses of lipopolysaccharide (LPS) or physiologic saline. Peritoneal fluid was assayed for transforming growth factor beta (TGFβ) and vascular endothelial growth factor (VEGF). Animals were humanely killed at week 5. Net ultrafiltration was then measured, and tissue samples were immunostained for factor VIII. Mesenteric tissue was assayed for hydroxyproline content. Adenovirus-mediated gene transfer of β-galactosidase was assayed by intraperitoneal administration of the virus, 4 days before the end of the experiment. Results Animals treated with either Dianeal or physiologic saline showed peritoneal membrane thickening and increased vascularity. Fibrosis was demonstrated by increased hydroxyproline concentration. Ultrafiltration was impaired. We found increased concentrations of VEGF and TGFβ in the peritoneal fluid of animals treated with LPS and daily infusion. Adenovirus-mediated gene transfer to the peritoneal membrane was demonstrated in the model. Conclusions Exposure to LPS and daily Dianeal or physiologic saline leads to peritoneal fibrosis and neoangiogenesis. Vascularization and glucose transport correlate with ultrafiltration failure. The present animal model mimics changes seen in humans on peritoneal dialysis and may be valuable for evaluating short-term interventions to prevent membrane damage.

The nicotine receptor antagonist mecamylamine has been shown to increase the efficacy of transdermal nicotine as a pharmacotherapy for tobacco addiction. A product for simultaneous transdermal administration of nicotine and mecamylamine is undergoing clinical trials. In order to carry out pharmacokinetic studies, quantitation of low nanogram per milliliter levels of mecamylamine and nicotine was required. This paper describes a method for simultaneous determination of mecamylamine, nicotine, and the nicotine metabolite, cotinine, in human plasma using gas chromatography-mass spectrometry (GC-MS). Limits of quantitation for mecamylamine, nicotine and cotinine are 2, 1 and 2 ng/ml, respectively.