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Introduction The Population Assessment of Tobacco and Health (PATH) Study is a longitudinal cohort study on tobacco use behavior, attitudes and beliefs, and tobacco-related health outcomes, including biomarkers of tobacco exposure in the U.S. population. In this report we provide a summary of urinary nicotine metabolite measurements among adult users and non-users of tobacco from Wave 1 (2013–2014) of the PATH Study. Methods Total nicotine and its metabolites including cotinine, trans-3′-hydroxycotinine (HCTT), and other minor metabolites were measured in more than 11 500 adult participants by liquid chromatography tandem mass spectrometry methods. Weighted geometric means (GM) and least square means from statistical modeling were calculated for non-users and users of various tobacco products. Results Among daily users, the highest GM concentrations of nicotine, cotinine and HCTT were found in exclusive smokeless tobacco users, and the lowest in exclusive e-cigarette users. Exclusive combustible product users had intermediate concentrations, similar to those found in users of multiple products (polyusers). Concentrations increased with age within the categories of tobacco users, and differences associated with gender, race/ethnicity and educational attainment were also noted among user categories. Recent (past 12 months) former users had GM cotinine concentrations that were more than threefold greater than never users. Conclusions These urinary nicotine metabolite data provide quantification of nicotine exposure representative of the entire US adult population during 2013–2014 and may serve as a reference for similar analyses in future measurements within this study. Implications Nicotine and its metabolites in urine provide perhaps the most fundamental biomarkers of recent nicotine exposure. This report, based on Wave 1 of the Population Assessment of Tobacco and Health (PATH) Study, provides the first nationally representative data describing urinary nicotine biomarker concentrations in both non-users, and users of a variety of tobacco products including combustible, e-cigarette and smokeless products. These data provide a urinary biomarker concentration snapshot in time for the entire US population during 2013–2014, and will provide a basis for comparison with future results from continuing, periodic evaluations in the PATH Study.

Introduction The Population Assessment of Tobacco and Health (PATH) Study is a nationally representative cohort of tobacco product users and nonusers. The study’s main purpose is to obtain longitudinal epidemiologic data on tobacco use and exposure among the US population. Aims and Methods Nicotine biomarkers—cotinine (COT) and trans-3′-hydroxycotinine (HCT)—were measured in blood samples collected from adult daily tobacco users and nonusers during Wave 1 of the PATH Study (2013–2014; n = 5012; one sample per participant). Participants’ tobacco product use and exposure to secondhand smoke were categorized based on questionnaire responses. Nonusers were subdivided into never users and recent former users. Daily tobacco users were classified into seven tobacco product use categories: exclusive users of cigarette, smokeless tobacco, electronic cigarette, cigar, pipe, and hookah, as well as polyusers. We calculated sample-weighted geometric mean (GM) concentrations of cotinine, HCT, and the nicotine metabolite ratio (NMR) and evaluated their associations with tobacco use with adjustment for potential confounders. Results The GMs (95% confidence intervals) of COT and HCT concentrations for daily tobacco users were 196 (184 to 208) and 72.5 (67.8 to 77.4) ng/mL, and for nonusers they were 0.033 (0.028 to 0.037) and 0.021 (0.018 to 0.023) ng/mL. Exclusive smokeless tobacco users had the highest COT concentrations of all user groups examined. The GM NMR in daily users was 0.339 (95% confidence interval: 0.330 to 0.350). Conclusions These nationally representative estimates of serum nicotine biomarkers could be the basis for reference ranges characterizing nicotine exposure for daily tobacco users and nonusers in the US adult population. Implications This report summarizes the serum nicotine biomarker measurements in Wave 1 of the PATH Study. We are reporting the first estimates of HCT in serum for daily tobacco users and nonusers in the noninstitutionalized, civilian US adult population; the first nationally representative serum COT estimates for daily exclusive users of different tobacco products and daily polyusers; and the first nationally representative estimate of the serum NMR in daily tobacco users by age, race/ethnicity, and sex.

Tobacco use is the leading preventable cause of premature disease and death in the United States. Approximately, 34 million U.S. adults currently smoke cigarettes. We developed a method for automated sample preparation and liquid chromatography-tandem mass spectrometry quantitation of 14 tobacco-related analytes: nicotine (NICF), cotinine (COTF), trans-3′-hydroxycotinine (HCTF), menthol glucuronide (MEG), anabasine (ANBF), anatabine (ANTF), isonicoteine (ISNT), myosmine (MYOS), beta-nicotyrine (BNTR), bupropion (BUPR), cytisine (CYTI), varenicline (VARE), arecaidine (ARD), and arecoline (ARL). The method includes automated solid-phase extraction using customized positive-pressure functions. The preparation scheme has the capacity to process a batch of 96 samples within 4 h with greater than 88% recovery for all analytes. The 14 analytes, separated within 4.15 min using reversed-phase liquid chromatography, were determined using a triple-quadrupole mass spectrometer with atmospheric-pressure chemical ionization and multiple reaction monitoring in negative and positive ionization modes. Wide quantitation ranges, within 1.2–72,000 ng/mL, were established especially for COTF, HCTF, MEG, and NICF to quantify the broad range of biomarker concentrations found in the U.S. population. The method accuracy is above 90% while the overall imprecision is below 7%. Finally, we tested urine samples from 90 smokers and observed detection rates of over 98% for six analytes with urinary HCTF and MEG concentrations ranging from 200–14,100 and 60–57,100 ng/mL, respectively. This high throughput analytical process can prepare and analyze a sample in 9 min and along with the 14-compound analyte panel can be useful for tobacco-exposure studies, in smoking-cessation programs, and for detecting changes in exposure related to tobacco products and their use.

Anabasine and anatabine are minor alkaloids in tobacco products and are precursors for tobacco-specific nitrosamines (TSNAs). The levels of these two compounds have been used to differentiate tobacco product sources, monitor compliance with smoking cessation programs, and for biomonitoring in TSNA-related studies. The concentrations of urinary anabasine and anatabine were measured in a representative sample of U.S. adults who smoked cigarettes (N = 770) during the 2013–2014 National Health and Nutrition Examination Survey (NHANES) study cycle, which was the first cycle where urinary anabasine and anatabine data became available. Weighted geometric means (GM) and geometric least squares means (LSM) with 95% confidence intervals were calculated for urinary anabasine and anatabine categorized by tobacco-use status [cigarettes per day (CPD) and smoking frequency] and demographic characteristics. Smoking ≥20 CPD was associated with 3.6× higher anabasine GM and 4.8× higher anatabine GM compared with smoking

Background: Determine the overall, sex-, and racially/ethnically-appropriate population-level cotinine and total nicotine equivalents (TNE-2, the molar sum of the two major nicotine metabolites) cut-points to distinguish tobacco users from nonusers across multiple definitions of use (e.g., exclusive vs. polytobacco, and daily vs. non-daily). Methods: Using Wave 1 (2013–2014) of the U.S. Population Assessment of Tobacco and Health (PATH) Study, we conducted weighted Receiver Operating Characteristic (ROC) analysis to determine the optimal urinary cotinine and TNE-2 cut-points, stratified by sex and race/ethnicity. Results: For past 30-day exclusive cigarette users, the cotinine cut-point that distinguished them from nonusers was 40.5 ng/mL, with considerable variation by sex (male: 22.2 ng/mL; female: 43.1 ng/mL) and between racial/ethnic groups (non-Hispanic other: 5.2 ng/mL; non-Hispanic black: 297.0 ng/mL). A similar, but attenuated, pattern emerged when assessing polytobacco cigarette users (overall cut-point = 39.1 ng/mL, range = 5.5 ng/mL–80.4 ng/mL) and any tobacco users (overall cut-point = 39.1 ng/mL, range = 4.8 ng/mL–40.0 ng/mL). Using TNE-2, which is less impacted by racial differences in nicotine metabolism, produced a comparable pattern of results although reduced the range magnitude. Conclusions: Because of similar frequency of cigarette use among polytobacco users, overall cut-points for exclusive cigarette use were not substantially different from cut-points that included polytobacco cigarette use or any tobacco use. Results revealed important differences in sex and race/ethnicity appropriate cut-points when evaluating tobacco use status and established novel urinary TNE-2 cut-points. Impact: These cut-points may be used for biochemical verification of self-reported tobacco use in epidemiologic studies and clinical trials.

We characterize nicotine exposure in the U.S. population by measuring urinary nicotine and its major (cotinine, trans-3′-hydroxycotinine) and minor (nicotine 1′-oxide, cotinine N-oxide, and 1-(3-pyridyl)-1-butanol-4-carboxylic acid, nornicotine) metabolites in participants from the 2015–2016 National Health and Nutrition Examination Survey. This is one of the first U.S. population-based urinary nicotine biomarker reports using the derived total nicotine equivalents (i.e., TNEs) to characterize exposure. Serum cotinine data is used to stratify tobacco non-users with no detectable serum cotinine (−sCOT), non-users with detectable serum cotinine (+sCOT), and individuals who use tobacco (users). The molar concentration sum of cotinine and trans-3′-hydroxycotinine was calculated to derive the TNE2 for non-users. Additionally, for users, the molar concentration sum of nicotine and TNE2 was calculated to derive the TNE3, and the molar concentration sum of the minor metabolites and TNE3 was calculated to derive the TNE7. Sample-weighted summary statistics are reported. We also generated multiple linear regression models to analyze the association between biomarker concentrations and tobacco use status, after adjusting for select demographic factors. We found TNE7 is positively correlated with TNE3 and TNE2 (r = 0.99 and 0.98, respectively), and TNE3 is positively correlated with TNE2 (r = 0.98). The mean TNE2 concentration was elevated for the +sCOT compared with the −sCOT group (0.0143 [0.0120, 0.0172] µmol/g creatinine and 0.00188 [0.00172, 0.00205] µmol/g creatinine, respectively), and highest among users (33.5 [29.6, 37.9] µmol/g creatinine). Non-daily tobacco use was associated with 50% lower TNE7 concentrations (p < 0.0001) compared with daily use. In this report, we show tobacco use frequency and passive exposure to nicotine are important sources of nicotine exposure. Furthermore, this report provides more information on non-users than a serum biomarker report, which underscores the value of urinary nicotine biomarkers in extending the range of trace-level exposures that can be characterized.

Introduction This study examined the predictive relationships between biomarkers of nicotine exposure and 16-item self-reported level of tobacco dependence (TD) and subsequent tobacco use outcomes. Aims and Methods The Population Assessment of Tobacco and Health (PATH) Study surveyed adult current established tobacco users who provided urine biospecimens at Wave 1 (September 2013–December 2014) and completed the Wave 2 (October 2014–October 2015) interview (n = 6872). Mutually exclusive user groups at Wave 1 included: Cigarette Only, E-cigarette Only, Cigar Only, Hookah Only, Smokeless Tobacco Only, Cigarette Plus E-cigarette, multiple tobacco product users who smoked cigarettes, and multiple tobacco product users who did not smoke cigarettes. Total Nicotine Equivalents (TNE-2) and TD were measured at Wave 1. Approximate one-year outcomes included frequency/quantity used, quitting, and adding/switching to different tobacco products. Results For Cigarette Only smokers and multiple tobacco product users who smoked cigarettes, higher TD and TNE-2 were associated with: a tendency to smoke more, smoking more frequently over time, decreased likelihood of switching away from cigarettes, and decreased probability of quitting after one year. For other product user groups, Wave 1 TD and/or TNE-2 were less consistently related to changes in quantity and frequency of product use, or for adding or switching products, but higher TNE-2 was more consistently predictive of decreased probability of quitting. Conclusions Self-reported TD and nicotine exposure assess common and independent aspects of dependence in relation to tobacco use behaviors for cigarette smokers. For other product user groups, nicotine exposure is a more consistent predictor of quitting than self-reported TD. Implications This study suggests that smoking cigarettes leads to the most coherent pattern of associations consistent with a syndrome of TD. Because cigarettes continue to be prevalent and harmful, efforts to decrease their use may be accelerated via conventional means (eg, smoking cessation interventions and treatments), but also perhaps by decreasing their dependence potential. The implications for noncombustible tobacco products are less clear as the stability of tobacco use patterns that include products such as e-cigarettes continue to evolve. TD, nicotine exposure measures, and consumption could be used in studies that attempt to understand and predict product-specific tobacco use behavioral outcomes.

Urinary cotinine is one of the most commonly measured biomarkers reflecting recent exposure to nicotine. In some cases a simple qualitative dichotomization of smokers and non-smokers is all that is required. NicAlert® test strips have been evaluated for this purpose, but other recently introduced, inexpensive single-line test strips have not. In this study we evaluated two such strips with nominal cutoffs of 200 and 10 ng/mL. A total of 800 urine samples with known cotinine concentrations determined by an LC–MS-MS method were examined, including 400 urine samples ranging from 0.23 to more than 24,000 ng/mL by the 200 ng/mL strip, and 400 samples with concentrations 98%. Our results suggest that these simple and inexpensive lateral flow immunoassay test strips can provide useful qualitative estimates of nicotine exposures for appropriate applications within the inherent limitations of sensitivity and precision of the immunoassay test strip format.

Background: Biomarkers of tobacco exposure have a central role in studies of tobacco use and nicotine intake. The most significant exposure markers are nicotine itself and its metabolites in urine. Therefore, it is important to evaluate the performance of laboratories conducting these biomarker measurements. Methods: This report presents the results from a method performance study involving 11 laboratories from 6 countries that are currently active in this area. Each laboratory assayed blind replicates of seven human urine pools at various concentrations on three separate days. The samples included five pools blended from smoker and nonsmoker urine sources, and two additional blank urine samples fortified with pure nicotine, cotinine, and hydroxycotinine standards. All laboratories used their own methods, and all were based on some form of liquid chromatography/tandem mass spectrometry. Results: Overall, good agreement was found among the laboratories in this study. Intralaboratory precision was good, and in the fortified pools, the mean bias observed was < + 3.5% for nicotine, approximately 1.2% for hydroxycotinine, and less than 1% for cotinine (1 outlier excluded in each case). Both indirect and direct methods for analyzing the glucuronides gave comparable results. Conclusions: This evaluation indicates that the experienced laboratories participating in this study can produce reliable and comparable human urinary nicotine metabolic profiles in samples from people with significant recent exposure to nicotine. Impact: This work supports the reliability and agreement of an international group of established laboratories measuring nicotine and its metabolites in urine in support of nicotine exposure studies. Cancer Epidemiol Biomarkers Prev; 27(9); 1083–90. ©2018 AACR.

In this paper, the clock duty cycle distortion (DCD) jitter will be investigated and the results will be compared for two channel configurations: using a general UDIMM topology and using discrete SDRAM component topology. These channel configurations will be simulated and analyzed for ISI effects, such as channel loss and reflection. The outcome of this investigation will show the primary factors that contribute to on-clock DCD in a most common DDR channel configurations. After analysis and comparison, the simulated differential clock DCD data will be provided while changing channel impedance corners due to the substrate manufacturing tolerance for package and PCB in a reflective discrete DRAM component and lossy UDIMM configurations. Finally, the simulation model-to-hardware correlation will be performed for each configuration. The simulated clock waveforms will be correlated with the measured waveforms for each configuration and the simulated differential DDR clock DCD analysis will be verified with the measured clock DCD jitter amount in actual system configurations.

This paper is focused on high speed transceivers system level PDN modeling and analysis to avoid transmitter phase noise. Transmitter phases noise impacts output jitters and eventually falls into failing category for some high-speed protocols. A system level PDN specification is developed based on a good reference board design and all other customers and development kits are evaluated based on reference design.

Background:Given the rapid increase in the popularity of e-cigarettes and the paucity of associated longitudinal health-related data, the need to assess the potential risks of long-term use is essential.Objective:To compare exposure to nicotine, tobacco-related carcinogens, and toxins among smokers of combustible cigarettes only, former smokers with long-term e-cigarette use only, former smokers with long-term nicotine replacement therapy (NRT) use only, long-term dual users of both combustible cigarettes and e-cigarettes, and long-term users of both combustible cigarettes and NRT.Design:Cross-sectional study.Setting:United Kingdom.Participants:The following 5 groups were purposively recruited: combustible cigarette–only users, former smokers with long-term (≥6 months) e-cigarette–only or NRT-only use, and long-term dual combustible cigarette–e-cigarette or combustible cigarette–NRT users (n = 36 to 37 per group; total n = 181).Measurements:Sociodemographic and smoking characteristics were assessed. Participants provided urine and saliva samples and were analyzed for biomarkers of nicotine, tobacco-specific N-nitrosamines (TSNAs), and volatile organic compounds (VOCs).Results:After confounders were controlled for, no clear between-group differences in salivary or urinary biomarkers of nicotine intake were found. The e-cigarette–only and NRT-only users had significantly lower metabolite levels for TSNAs (including the carcinogenic metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol [NNAL]) and VOCs (including metabolites of the toxins acrolein; acrylamide; acrylonitrile; 1,3-butadiene; and ethylene oxide) than combustible cigarette–only, dual combustible cigarette–e-cigarette, or dual combustible cigarette–NRT users. The e-cigarette–only users had significantly lower NNAL levels than all other groups. Combustible cigarette–only, dual combustible cigarette–NRT, and dual combustible cigarette–e-cigarette users had largely similar levels of TSNA and VOC metabolites.Limitation:Cross-sectional design with self-selected sample.Conclusion:Former smokers with long-term e-cigarette–only or NRT-only use may obtain roughly similar levels of nicotine compared with smokers of combustible cigarettes only, but results varied. Long-term NRT-only and e-cigarette–only use, but not dual use of NRTs or e-cigarettes with combustible cigarettes, is associated with substantially reduced levels of measured carcinogens and toxins relative to smoking only combustible cigarettes.Primary Funding Source:Cancer Research UK

Th17 and TfH cells are thought to promote tissue inflammation and autoantibody production, respectively, in autoimmune diseases including rheumatoid arthritis (RA). TfH cells that co-express Th17 markers (CXCR5+Th17) encompass both of these pathogenic functions, and are increased in some human autoimmune settings including juvenile dermatomyositis. We investigated CXCR5+Th17 cells in RA subjects with stable or active disease and before and after TNF inhibitor therapy. CXCR5+Th17 cell frequency was increased in RA compared to healthy controls, but other helper T cell subsets were not different. CXCR5+Th17 cells correlated with disease activity in subjects with active RA prior to initiation of TNF inhibitor therapy. Baseline CXCR5+Th17 cells also correlated with numbers of swollen joints as late as one year post-therapy. CXCR5+Th17 cell frequencies were unaltered by TNF blockade and in fact remained remarkably stable within individuals. We conclude that CXCR5+Th17 cells are not a direct target of TNF blockade and therefore cannot serve as a biomarker of current disease activity. However, basal CXCR5+Th17 cell frequency may indicate underlying differences in disease phenotype between patients and predict ultimate success of TNF inhibitor therapy.

A microchip has been developed on the basis of immno-precipitation approach for fast and sensitive enrichment of low abundant carbonylated proteins. This microfluidic method could enrich molecular biomarkers, which could be further analyzed in the proteomic study of age-related diseases and therapeutic development. In this study, an immunoaffinity-based PDMS micro-device was designed, fabricated, and chemically modified to specifically trap DNP-labeled PTM proteins of low abundance from a complex protein mixture. Carbonylated protein is selected as a representative PTM protein to illustrate the wide application of this immuno-based microchip for other PTMs which could be readily labeled by different antibody groups. Surface characterization methods such as atomic force microscopy and fluorescence microscopy were used to evaluate the construction of glutaraldehyde- and antibody- terminated PDMS substrates in the device fabrication. Quantitative study was also applied to study the target protein capture and elution efficiency of the device. In a testing mixture consisting of smaller amount of test model-In Vitro oxidized cytochrome c and large blocking protein BSA, a high sensitivity and specificity for only carbonylated protein biomarkers was demonstrated using this on-chip immnuoaffinity based extraction/enrichment. For this highly dense 193-post arrays μ-chip, a low abundance of 159 ng of standard in vitro test model- cytochrome c was enriched at flow speed of 5 μL/min within 110 min. We demonstrated that this nascent micro-immunoprecipitation (μ-IP) method is capable for enrichment of biomarkers in protein post-translation modification related diseases and promise great advance in early disease detection.

Ammonia in mainstream smoke is present in both the particulate and vapor phases. The presence of ammonia in the cigarette filler material and smoke is of significance because of the potential role ammonia could have in raising the “smoke pH.” An increased smoke pH could shift a fraction of total nicotine to free-base nicotine, which is reportedly more rapidly absorbed by the smoker. Methods measuring ammonia in smoke typically employ acid filled impingers to trap the smoke. We developed a fast, reliable method to measure ammonia in mainstream smoke without the use of costly and time consuming impingers to examine differences in ammonia delivery. The method uses both a Cambridge filter pad and a Tedlar bag to capture particulate and vapor phases of the smoke. We quantified ammonia levels in the mainstream smoke of 50 cigarette brands from 5 manufacturers. Ammonia levels ranged from approximately 1μg to 23μg per cigarette for ISO smoking conditions and 38μg to 67μg per cigarette for Canadian intense smoking conditions and statistically significance differences were observed between brands and manufacturers. Our findings suggest that ammonia levels vary by brand and are higher under Canadian intense smoking conditions.

Glutathione plays an essential role in the intracellular antioxidant defense against oxidant radicals, especially the •OH radical. To understand the early and progressive cellular changes in the development of Alzheimer's disease (AD), we investigated reduced glutathione/oxidized glutathione (GSH/GSSG) status in a double mutated AD transgenic mouse model (B6.Cg-Tg), which carries Swedish amyloid-β protein precursor mutation (AβPPswe) and exon 9 deletion of the PSEN1 gene. In this study, we quantified and compared both GSH/GSSG and mixed-disulfide (Pr-SSG) levels in blood samples and three anatomic positions in brain (cerebrum, cerebellum, and hippocampus) at 3 age stages (1, 5, and 11 months) of AD transgenic (Tg)/wild type mice. The present study was designed to characterize and provide insight into the glutathione redox state of both brain tissues and blood samples at different disease stages of this Tg model. The level of Pr-SSG increased in all AD brain tissues and blood compared with controls regardless of age. The GSH/GSSG ratio in AD-Tg brain tissue started at a higher value at 1 month, fell at the transitional period of 5 months, right before the onset of amyloid plaques, followed by an increase in GSSG and associated decrease of GSH/GSSG at 11 months. These results suggest that formation of Pr-SSG may be an early event, preceding amyloid plaque appearance, and the data further implies that tissue thiol redox is tightly regulated. Notably, the high basal levels of mixed-disulfides in hippocampus suggest a potential for increased oxidative damage under oxidizing conditions and increased GSSG in this vulnerable region.

Post-translational modifications (PTM) of proteins play essential roles in cellular physiology and disease. The identification of protein substrates and detection of modification site helps understand PTM-mediated regulation in essential biological pathways and functions in various diseases. However, PTM proteins are typically present only at trace levels, making them difficult to identify in mass spectrometry based proteomics. In this paper, we report a novel and sensitive affinity chromatography on the avidin-functionalized poly(methyl methacrylate) (PMMA) microchip for enrichment of nanogram (ng) amount of PTMs. The chemical modification of poly(methyl methacrylate) (PMMA) surfaces yield avidin-terminated PMMA surfaces after UV radiation and consecutive EDC mediated coupling (amide reaction). This functionalized PMMA micro-device was developed to identify and specifically trap biotinylated PTM proteins of low abundance from complex protein mixture. Here we selected carbonylated protein as a representative PTM to illustrate the wide application of this affinity microchip for any PTMs converted into a tractable tag after derivatization. The surface topography, surface functional group mapping and elemental composition changes after each modification step of the treatment process were systematically measured qualitatively and quantitatively by atomic force microscopy, X-ray photoelectron spectroscopy and fluorescence microscopy. Quantitative study of biotinlated carbonylated protein capture recovery and elution efficiency of the device was also studied. We also envision that this subproteome enrichment micro-device can be assembled with other lab-on-a-chip components for follow-up protein analysis.

A method was developed for the quantitative analysis of 30 drugs of abuse and their metabolites in urine, including opiates, barbiturates, amphetamines, cocaine, cannabinoids, phencyclidine, methadone, and benzodiazepines. This method uses solid-phase extraction (SPE) on an Oasis HLB column followed by liquid chromatography-tandem mass spectrometry. Analytes were quantified by multiple reaction monitoring with the deuterated analogues as internal standards, using an atmospheric pressure ionization-electrospray interface. The method was validated by examining specificity, precision, accuracy, linearity, recovery, reproducibility, and detection limits. The limits of detection ranged from 9 pg/mL to 2.29 ng/mL in urine depending on the analyte. The SPE procedure was automated on a RapidTrace workstation to increase analytical throughput, and the results obtained via automated SPE were compared to those obtained by manual SPE to examine carryover effect, precision, accuracy, recovery, and reproducibility. To evaluate method performance, 108 urine samples were collected anonymously and tested for the presence of these drugs.

A stable, protein C activating membrane-mimetic film was produced on a polyelectrolyte multilayer (PEM) by in-situ photopolymerization of a phospholipid assembly containing thrombomodulin (TM). The monoacrylated phospholipid monomer was initially synthesized and prepared as unilamellar vesicles with varying molar concentrations of TM. Notably, in mixed-lipid systems, K m values for protein C activation increased in direct proportion to the mole fraction of polymerizable lipid, which was likely due to reduced membrane mobility after photopolymerization. Membrane-mimetic films were also constructed on planar substrates with predictable surface concentrations of catalytically active TM. Significantly, at a TM surface concentration of 170 fmol/cm 2 , the rate of protein C activation was comparable to that measured for a variety of confluent endothelial cell monolayers. Serial measurements of contact angles and protein C activation confirmed short-term film stability under a variety of in vitro conditions. Moreover, 1 2 5 I labeling of TM demonstrated little change in TM surface concentration over periods of up to 28 days. Significantly, polymeric lipid membranes functionalized with thrombomodulin efficiently inhibited thrombin generation. We believe that the design of membrane-mimetic films that have the capacity to activate the protein C pathway will provide a useful strategy for generating "actively" antithrombogenic surfaces.

A stabilized, membrane-mimetic film was produced on a polyelectrolyte multiplayer (PEM) by in-situ photopolymerization of an acrylate functonalized phospholipid assembly at a solid-liquid interface. The phospholipid monomer was synthesized, prepared as unilamellar vesicles, and fused onto close-packed octadecyl chains as part of an amphiphilic terpolymer anchored onto the PEM by electrostatic interactions. The lipid film displayed an advancing contact angle of ∼ 60°, elemental composition, as determined by X-ray photoelectron spectroscopy, was in agreement with that anticipated for a lipid membrane. Data obtained from both high-resolution scanning electron microscopy and ellipsometry were consistent with the formation of a supported lipid monolayer. In addition, polarized external reflection infrared spectroscopy revealed significant acyl chain ordering induced on lipid fusion and polymerization. Doping the lipid assembly with a fluorescein terminated polymerizable lipid provided visible confirmation of film formation and its stability under a variety of conditions, including shear rates of 2000 sec - 1 . Transport studies demonstrated that the addition of a lipid film significantly reduced barrier permeability for compounds in excess of 70 kD. The ability to coat microbeads (d ∼ 300 μm) with a robust membrane-mimetic film, while preserving encapsulated cell viability is illustrated, thereby establishing a new strategy for modulating the physiochemical and biological properties of immunoisolation barriers for cell transplantation.

The speed of general purpose input output (GPIO) continues to increase as more consumer applications utilize “smart” devices. The low-voltage differential signaling (LVDS) is often times the highest speed that GPIO interface needs to support in the mixture of different single ended signaling pins. Although LVDS is differential and somewhat immune to direct signal coupling from other signals, it is still subject to coupling through a shared power supply noise. A thorough SSN analysis between single ended to differential signals is presented in this paper. To help designing GPIO systems, we have considered different single ended signaling types such as SSTL, LVTTL, etc.

Most sample preparation methods characteristically involve intensive and repetitive labor, which is inefficient when preparing large numbers of samples from population-scale studies.This study presents a robotic system designed to meet the sampling requirements for large population-scale studies. Using this robotic system, we developed and validated a method to simultaneously measure urinary anatabine, anabasine, nicotine and seven major nicotine metabolites: 4-Hydroxy-4-(3-pyridyl)butanoic acid, cotinine-N-oxide, nicotine-N-oxide, trans-3'-hydroxycotinine, norcotinine, cotinine and nornicotine. We analyzed robotically prepared samples using high-performance liquid chromatography (HPLC) coupled with triple quadrupole mass spectrometry in positive electrospray ionization mode using scheduled multiple reaction monitoring (sMRM) with a total runtime of 8.5 min.The optimized procedure was able to deliver linear analyte responses over a broad range of concentrations. Responses of urine-based calibrators delivered coefficients of determination (R(2)) of0.995. Sample preparation recovery was generally higher than 80%. The robotic system was able to prepare four 96-well plate (384 urine samples) per day, and the overall method afforded an accuracy range of 92-115%, and an imprecision of15.0% on average.The validation results demonstrate that the method is accurate, precise, sensitive, robust, and most significantly labor-saving for sample preparation, making it efficient and practical for routine measurements in large population-scale studies such as the National Health and Nutrition Examination Survey (NHANES) and the Population Assessment of Tobacco and Health (PATH) study.

For single-ended signaling DDR4 channels at 3200Mbps, signal and power integrity issues become increasingly challenging with much smaller voltage and timing windows to balance the budget. As systems increase data rate and IO count, supply noise does not scale accordingly. We present a system level signal and power co-simulation analysis to optimize system performance under stringent timing requirement [1]. Signal integrity of DDR4 interface, such as inter-symbol interference ISI, reflection, and signal cross talk, needs to be minimized in order to meet an ever shrinking timing budget. Also, power delivery network (PDN) design becomes very difficult as a result of smaller die size and multilayer complex package design. SI and PI co-design optimization is driven by both channel performance and overall system cost.

8-Hydroxy-2′-deoxyguanosine (8-OHdG) is one of the major forms of oxidative deoxyribonucleic acid (DNA) damage, and is commonly analyzed as an excellent marker of DNA lesions. The purpose of this study was to develop a sensitive method to accurately and rapidly quantify the 8-OHdG by using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). The method involved the use of specific antibody to detect DNA lesions (8-OHdG) and consecutive fluorescence labeling. Next, the urine sample with 8-OHdG fluorescently labeled along with other constituents was resolved by capillary electrophoretic system and the lesion of interest was detected using fluorescence detector. The limit of detection was 0.18 fmol, which is sufficient sensitivity for detection and quantification of 8-OHdG in untreated urine samples. The relative standard deviation (RSD) was found to be 11.32 % for migration time, and 5.52 % for peak area. To demonstrate the utility of this method, the urinary concentration of 8-OHdG in an Alzheimer’s transgenic mouse model was determined. Collectively, our results indicate that this methodology offers great advantages such as high separation efficiency, good selectivity, low limit of detection (LOD), simplicity and low cost of analysis.

We here report an antibody functionalized microimmunoprecipitation (μIP) method used for enrich lowabundant post-translational modified (PTM) proteins. The device is a fabricated by inert, nontoxic and disposable polydimethylsiloxane (PDMS) using a silane-based chemical modification protocol, which yield an antibody-terminated PDMS surfaces. In this study, the μIP device is specifically designed for purification of carbonylated protein, a representative example here to illustrate the potential applications for any other PTMs, which could be immuno-tagged by specific antibodies. The test model- in vitro oxidized Bovine Serum Albumin (BSA) was first derivitized by dinitrophenylhydrazide (DNPH) and then captured by the anti-DNP immobilized on this μIP device. The surface functional group mapping was systematically analyzed and validated by fluorescence microscopy. Quantitative study of DNP-derivatized carbonylated protein capture recovery and elution efficiency of the device was also studied. We also envision that this proteome enrichment μIP device can be assembled with other lab-on-a-chip components, such as microelectrophoresis or micro-chromatographic devices for follow-up protein analysis. This selective enrichment of modified proteins greatly facilitates the study of low abundant protein biomarkers discovery.

We report a proof of principle study for the use of oxalyldihydrazide as a crosslinker for enrichment of carbonylated proteins within a microfluidic chip. Surface modification steps are characterized and analyzed using analytical techniques. We use in vitro oxidized bovine serum albumin as a model protein for testing the selectivity and specificity of the methodology for carbonylated proteins. We demonstrate high specificity for our target protein, obtaining 100% capture of our target molecule during the first three minutes of continuous injection. After 30 minutes of continuous loading, the device captures half of the targeted molecules. Elution of the proteins is confirmed qualitatively. Under the flow conditions used in this experiment, nearly 300 ng of proteins can be enriched prior to chip saturation. This report suggests the validity of using immobilized oxalyldihydrazide on a microchip as an enrichment methodology for low abundance proteins in a sample.

Protein S-nitrosylation, a reverse post translational modification (PTM) of cysteine, has been considered as biomarkers in many diseases. Microfluidic has been widely used for development of novel tools for separation of protein mixture. In this report, we have demonstrated a twodimensional micro-electrophoresis (2D μ-CE) separation of nitrosylated proteins in human colon epithelial adenocarcinoma cells (HT-29). Sodium dodecyl sulphate micro-capillary gel electrophoresis (SDS μ-CGE) and microemulsion electrokinetic chromatography (MEEKC) were used for the first and second dimension of separation electrophoresis, respectively. The effective separation lengths for both dimensions were 10 mm, and electrokinetic injection and separation were used with field strengths at 400 V/cm. To our knowledge, this is the first report on 2D profiling of nitrosylated proteins in the biological samples.

Microfluidic devices are used in several engineering fields ranging from biomedical to chemical to engineering for applications such as micro reactor, target molecular enriching and cell capturing. With regard to related applications, microfluidic devices offer advantages such as high surface area to volume ratio, increased mass transfer coefficient and portability in addition to their requirement of low analytes. Affinity based microfluidic devices with microscale posts have high compactness and mass transfer coefficient. In order to maximize the benefits offered by employing microfluidic devices, it is important to apply parametric study in the device designing work. This study is aimed at studying the operating and geometric parameters of microfluidic devices with square/rectangular microscale posts. The geometric parameters, such as aspect ratio of the microposts used, could possibly decide the performance of the device. Operating parameters studied are Reynolds number, Peclet number, Damköhler number, and equilibrium reaction constant. These parameters encompass the influence of velocity, diffusivity, density, viscosity, hydraulic diameter, inlet concentration of species and absorption/desorption reaction constants. This work theoretically analyzes the influence of the above mentioned parameters using COMSOL Multiphysics 4.2.a. The governing equations of microfluidic devices, i.e. Navier-Stokes equations and the advection-diffusion equation, subjected to the above mentioned operating parameters, are solved to obtain the velocity profile, pressure drop and concentration profile of the species. The metric used for analyzing the influence of each operating parameter is the capture efficiency, i.e. the ratio of outlet concentration to inlet concentration as well the pressure drop. The results of this study would improve the design of microfluidic devices used for chemical reactions as well as that used for protein enrichment.

S-nitrosylation (also referred to as nitrosation), a reversible post translational modification (PTM) of cysteine, plays an important role in cellular functions and cell signalling pathways. Nitrosylated proteins are considered as biomarkers of aging and Alzheimer's disease (AD). Microfluidics has been widely used for development of novel tools for separation of protein mixtures. Here we demonstrate two-dimensional micro-electrophoresis (2D μ-CE) separations of nitrosylated proteins from the human colon epithelial adenocarcinoma cells (HT-29) and AD transgenic mice brain tissues. Sodium dodecyl sulphate micro-capillary gel electrophoresis (SDS μ-CGE) and microemulsion electrokinetic chromatography (MEEKC) were used for the first and second dimensional separations, respectively. The effective separation lengths for both dimensions were 10 mm, and electrokinetic injection was used with field strength at 200 V cm(-1). After 80 s separation in the first CGE dimension, fractions were successfully transferred to a second MEEKC dimension for a short 10 s separation. We first demonstrate this 2D μ-CE separation by resolving five standard proteins with molecular weight (MW) ranging from 20 to 64 kDa. We also present a high peak capacity 3D landscape image of nitrosylated proteins from HT-29 cells before and following menadione (MQ) treatment to induce oxidative stress. Additionally, to illustrate the potential of the 2D μ-CE separation method for rapid profiling of oxidative stress-induced biomarkers implicated in AD disease, the nitrosylated protein fingerprints from 11-month-old AD transgenic mice brain and their age matched controls were also generated. To our knowledge, this is the first report on 2D profiling of nitrosylated proteins in biological samples on a microchip. The characteristics of this biomarker profiling will potentially serve as the screening for early detection of AD.

To date, prediction of Alzheimer's disease (AD) is mainly based on clinical criteria because no well-established biochemical biomarkers for routine clinical diagnosis of AD currently exist. We developed an approach to aid in the early diagnosis of AD by using principal component analysis (PCA)-based spectral analysis of oxidized protein electrophoretic profiling. We found that the combination of capillary electrophoresis and PCA analysis of S-glutathionylation distribution characterization can be used in the sample classification and molecular weight (Mw) prediction. The comparison of leave-one-out AD versus non-AD gives the sensitivity of 100% and 93.33% in brain tissues and blood samples, respectively, while the specificity of 100% in brain and 90.0% in blood samples. Our findings demonstrate that PCA of S-glutathionylation electrophoretic profiling detects AD pathology features, and that the molecular weight based electrophoretic profiling of blood and brain S-glutathionylated proteins are sensitive to change, even at the early stage of the disease. Our results offer a previously unexplored diagnostic approach by using electrophoretic characteristics of oxidized proteins to serve as a predictor of AD progression and early stage screening.

S-nitrosylated proteins are biomarkers of oxidative damage in aging and Alzheimer's disease (AD). Here, we report a new method for detecting and quantifying nitrosylated proteins by capillary gel electrophoresis with laser induced fluorescence detection (CGE-LIF). Dylight 488 maleimide was used to specifically label thiol group (SH) after switching the S-nitrosothiol (S-NO) to SH in cysteine using the "fluorescence switch" assay. In vitro nitrosylation model-BSA subjected to S-nitrosoglutathione (GSNO) optimized the labeling reactions and characterized the response of the LIF detector. The method proves to be highly sensitive, detecting 1.3 picomolar (pM) concentration of nitrosothiols in nanograms of proteins, which is the lowest limit of detection of nitrosothiols reported to date. We further demonstrated the direct application of this method in monitoring protein nitrosylation damage in MQ mediated human colon adenocarcinoma cells. The nitrosothiol amounts in MQ treated and untreated cells are 14.8±0.2 and 10.4±0.5 pmol/mg of proteins, respectively. We also depicted nitrosylated protein electrophoretic profiles of brain cerebrum of 5-month-old AD transgenic (Tg) mice model. In Tg mice brain, 15.5±0.4 pmol of nitrosothiols/mg of proteins was quantified while wild type contained 11.7±0.3 pmol/mg proteins. The methodology is validated to quantify low levels of S-nitrosylated protein in complex protein mixtures from both physiological and pathological conditions.

S-glutathionylation (Pr–SSG) is a specific post-translational modification of cysteine residues by the addition of glutathione. S-Glutathionylated proteins induced by oxidative or nitrosative stress play an essential role in understanding the pathogenesis of the aging and age-related disorder, such as Alzheimer’s disease (AD). The purpose of this research is to develop a novel and ultrasensitive method to accurately and rapidly quantify the Pr–SSG by using capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF). The derivatization method is based on the specific reduction of protein-bound S-glutathionylation with glutaredoxin (Grx) and labeling with thiol-reactive fluorescent dye (Dylight 488 maleimide). The experiments were performed by coupling the derivatization method with CGE-LIF to study electrophoretic profiling in in vitro oxidative stress model–S-glutathionylated bovine serum albumin (BSA-SSG), oxidant-induced human colon adenocarcinoma (HT-29) cells, brain tissues, and whole blood samples from an AD transgenic (Tg) mouse model. The results showed almost an eightfold increase in S-glutathionyl abundance when subjecting HT-29 cells in an oxidant environment, resulting in Pr–SSG at 232 ± 10.64 (average ±SD; n = 3) nmol/mg. In the AD–Tg mouse model, an initial quantitative measurement demonstrated the extent of protein S-glutathionylation in three brain regions (hippocampus, cerebellum, and cerebrum), ranging from 1 to 10 nmol/mg. Additionally, we described our developed method to potentially serve as a highly desirable diagnostic tool for monitoring S-glutathionylated protein profile in minuscule amount of whole blood. The whole blood samples for S-glutathionyl expression of 5-month-old AD–Tg mice are quantified as 16.3 μmol/L (=7.2 nmol/mg protein). Altogether, this is a fast, easy, and accurate method, reaching the lowest limit of Pr–SSG detection at 1.8 attomole (amol) level, reported to date.

The nature of this project is to equip and support research in nanoengineered systems for biomedical, bioenvironmental, and bioenergy applications. Funds provided by the Department of Energy (DoE) under this Congressional Directive were used to support two ongoing research projects at Louisiana Tech University in biomedical, bioenvironmental, and bioenergy applications. Two major projects (Enzyme Immobilization for Large Scale Reactors to Reduce Cellulosic Ethanol Costs, and Nanocatalysts for Coal and Biomass Conversion to Diesel Fuel) and to fund three to five additional seed projects were funded using the project budget. The project funds also allowed the purchase and repair of sophisticated research equipment that will support continued research in these areas for many years to come. Project funds also supported faculty, graduate students, and undergraduate students, contributing to the development of a technically sophisticated work force in the region and the State. Descriptions of the technical accomplishments for each funded project are provided. Biofuels are an important part of the solution for sustainable transportation fuel and energy production for the future. Unfortunately, the country's appetite for fuel cannot be satisfied with traditional sugar crops such as sugar cane or corn. Emerging technologies are allowing cellulosic biomass (wood, grass, stalks, etc.) tomore » also be converted into ethanol. Cellulosic ethanol does not compete with food production and it has the potential to decrease greenhouse gas (GHG) emissions by 86% versus current fossil fuels (current techniques for corn ethanol only reduce greenhouse gases by 19%). Because of these advantages, the federal government has made cellulosic ethanol a high priority. The Energy Independence and Security Act of 2007 (EISA) requires a minimum production of at least 16 billion gallons of cellulosic ethanol by 2022. Indeed, the Obama administration has signaled an ambitious commitment of achieving 2 billion gallons of cellulosic ethanol by 2013. Louisiana is well positioned to become a national contributor in cellulosic ethanol, with an excellent growing season, a strong pulp/paper industry, and one of the nation's first cellulosic ethanol demonstration plants. Dr. Palmer in Chemical Engineering at Louisiana Tech University is collaborating with Drs. Lvov and Snow in Chemistry and Dr. Hegab in Mechanical Engineering to capitalize on these advantages by applying nanotechnology to improve the cellulosic ethanol processes. In many of these processes, expensive enzymes are used to convert the cellulose to sugars. The nanotechnology processes developed at Louisiana Tech University can immobilize these enzymes and therefore significantly reduce the overall costs of the process. Estimates of savings range from approximately $32 million at each cellulosic ethanol plant, to $7.5 billion total if the 16 billion gallons of cellulosic ethanol is achieved. This process has the advantage of being easy to apply in a large-scale commercial environment and can immobilize a wide variety or mixture of enzymes for production. Two primary objectives with any immobilization technique are to demonstrate reusability and catalytic activity (both reuse of the immobilized enzyme and reuse of the polymer substrate). The scale-up of the layering-by-layering process has been a focus this past year as some interesting challenges in the surface chemistry have become evident. Catalytic activity of cellulase is highly dependent upon how the feed material is pretreated to enhance digestion. Therefore, efforts this year have been performed this year to characterize our process on a few of the more prevalent pretreatment methods.« less

Streak camera is an important diagnostic device in the studies of laser plasma interaction, the detailed structure of photo reaction from material science to biochemistry, and in the measurement of the longitudinal distribution of a beam in accelerators. In this paper, we report on a new method which can potentially improve the temporal resolution of a streak camera down to femtoseconds. This method uses a time-dependent acceleration field to defocus the photo electrons longitudinally. This not only reduces the time dispersion distortion caused by initial energy spread but also mitigates the effects from the space- charge forces of photo electrons. An illustration of the method shows significant improvement of the modulation transfer function (MFT) compared with the conventional design.

The performance of multi-gigahertz interconnect systems is adversely impacted by non-ideal physical effects such as attenuation, crosstalk, impedance mismatches, intersymbol interference, and by parameter variations due to process and environmental changes. Many of these non-ideal physical effects can be mitigated by selecting proper signaling topologies and techniques, by using judicious design rules, and by applying advanced circuitry and signal processing techniques. However, the parameter variations due to process and environmental conditions are more difficult to overcome, and their impact on a system needs to be quantified in order to ensure robust system operation under the worst-case operating conditions. This paper describes the design and characterization methodology used to develop robust multi-gigahertz systems using conventional interconnect and packaging technologies. It focuses on studying the sensitivity of the performance of the system to parameter variations due to process and environmental changes. The simulation and measurement results of high-speed equalized channels are presented. The performance of the equalized channels is also verified by comparing the measured and simulated eye diagrams for various values of equalization coefficients for data rates above 6.4 GHz. Finally, the sensitivity of equalization taps to parameter variations is also studied.

As the clock speed increases and rise time decreases, an accurate I/O driver model is crucial for robust system designs. Due to the runtime limitations, a full-blown driver circuit model is impractical for those designs that require numerous simulation runs. Take the high-speed Rambus memory channel design as an example. Thousands of data read and data write simulations were performed with various patterns, configurations, and comer models. Many complex interconnect models are included in the simulation decks, in the form of frequency-dependent, lossy, and coupled transmission lines [1]. The only nonlinear model that is present is either the memory controller circuitry for data write or RDRAM driver circuitry for data read simulations. This paper describes a reduced, but equivalent, RDRAM driver model that significantly speeds up the simulation time for Rambus memory channel designs. The concept of using a reduced model in place of a full-circuit model is not new [2]. This paper, however, chooses a model that consists of a simple level-1 transistor in parallel with a voltage-controlled current source. A systematic approach is shown that curvefits the I-V curve by a level-1 transistor and matches the load line by a current source. The reduced model thus obtained results in time-domain waveforms that are nearly identical to the full-circuit's. Finally, it is noted that the same procedures can be applied to derive reduced models for many nonlinear circuits.

The wire-bond ball-grid-array (BGA) package is of interest because of its low cost and high pin counts. For better electrical performance, the coupling in the wire-bond regions needs to be contained. This paper shows a double-swizzle design that reduces coupling by ∼40% from a reference single-swizzle design. The smaller coupling was achieved through the proper assignment of ground wires. Test packages with shorted load were built, and the measurements were done by connecting either a vector network analyzer (VNA) or a time-domain reflectometer (TDR) to the balls of the package. The coupled models of bond wires, fanouts, traces, and plating stubs were then extracted from the measured data by a customized extractor. The extracted models gave some insights into the package. The attenuation was larger than expected. Some spikes in the measured S11 plots were attributed to the coupling. A package model without coupling would not be able to capture these spikes. Yet there were other spikes that were unexplained. They were finally tracked down to be caused by the Vdd plane and routing. Connecting decoupling capacitors between Vdd and ground balls moved the resonance spikes to higher frequencies. Shorting the Vdd and ground balls eliminated these mysterious spikes altogether.

This paper shows a generalized optimizer that interacts with an external circuit simulator to extract frequency-dependent lossy coupled transmission line models from TDR or VNA measurements. The optimizer's architectural flow, and such concepts as global and local iterations, weighting function, time-windowing and piecewise-linear source models are mentioned. Several examples, including a complex BGA package model, are used to demonstrate its effectiveness in extracting model parameters and correlating waveforms.

We report a proof of principle study for the use of oxalyldihydrazide as a crosslinker for enrichment of carbonylated proteins within a microfluidic chip. Surface modification steps are characterized and analyzed using analytical techniques. We use oxidized cytochrome c as our model protein and demonstrate the chip's ability to capture carbonylated targets. After 100 min of continuous loading, the chip is capable of capturing 7.5 μg of carbonylated protein. All the proteins captured are eluted out of the chip using the elution protocol. Finally, we demonstrate the chip's specificity for oxidized targets by mixing oxidized cytochrome c and TRITC-BSA, with cytochrome c in low abundance. The results show that the chip is efficient at finding its target when unoxidized proteins are present. This is the first report to suggest the use of immobilized oxalyldihydrazide on a microchip as an enrichment methodology for low abundance proteins in a sample.