Brain Expression Levels of Commonly Measured Blood Biomarkers of Neurological Damage Differ with Respect to Sex, Race, and Age.

It is increasingly evident that blood biomarkers have potential to improve the diagnosis and management of both acute and chronic neurological conditions. The most well-studied candidates, and arguably those with the broadest utility, are proteins that are highly enriched in neural tissues and released into circulation upon cellular damage. It is currently unknown how the brain expression levels of these proteins is influenced by demographic factors such as sex, race, and age. Given that source tissue abundance is likely a key determinant of the levels observed in the blood during neurological pathology, understanding such influences is important in terms of identifying potential clinical scenarios that could produce diagnostic bias. In this study, we leveraged existing mRNA sequencing data originating from 2,642 normal brain specimens harvested from 382 human donors to examine potential demographic variability in the expression levels of genes which code for 28 candidate blood biomarkers of neurological damage. Existing mass spectrometry data originating from 26 additional normal brain specimens harvested from 26 separate human donors was subsequently used to tentatively assess whether observed transcriptional variance was likely to produce corresponding variance in terms of protein abundance. Genes associated with several well-studied or emerging candidate biomarkers including neurofilament light chain (NfL), ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1), neuron-specific enolase (NSE), and synaptosomal-associated protein 25 (SNAP-25) exhibited significant differences in expression with respect to sex, race, and age. In many instances, these differences in brain expression align well with and provide a mechanistic explanation for previously reported differences in blood levels.

Dataset including whole blood gene expression profiles and matched leukocyte counts with utility for benchmarking cellular deconvolution pipelines.

OBJECTIVES: Cellular deconvolution is a valuable computational process that can infer the cellular composition of heterogeneous tissue samples from bulk RNA-sequencing data. Benchmark testing is a crucial step in the development and evaluation of new cellular deconvolution algorithms, and also plays a key role in the process of building and optimizing deconvolution pipelines for specific experimental applications. However, few in vivo benchmarking datasets exist, particularly for whole blood, which is the single most profiled human tissue. Here, we describe a unique dataset containing whole blood gene expression profiles and matched circulating leukocyte counts from a large cohort of human donors with utility for benchmarking cellular deconvolution pipelines. DATA DESCRIPTION: To produce this dataset, venous whole blood was sampled from 138 total donors recruited at an academic medical center. Genome-wide expression profiling was subsequently performed via next-generation RNA sequencing, and white blood cell differentials were collected in parallel using flow cytometry. The resultant final dataset contains donor-level expression data for over 45,000 protein coding and non-protein coding genes, as well as matched neutrophil, lymphocyte, monocyte, and eosinophil counts.

Variability in donor leukocyte counts confound the use of common RNA sequencing data normalization strategies in transcriptomic biomarker studies performed with whole blood.

Gene expression data generated from whole blood via next generation sequencing is frequently used in studies aimed at identifying mRNA-based biomarker panels with utility for diagnosis or monitoring of human disease. These investigations often employ data normalization techniques more typically used for analysis of data originating from solid tissues, which largely operate under the general assumption that specimens have similar transcriptome composition. However, this assumption may be violated when working with data generated from whole blood, which is more cellularly dynamic, leading to potential confounds. In this study, we used next generation sequencing in combination with flow cytometry to assess the influence of donor leukocyte counts on the transcriptional composition of whole blood specimens sampled from a cohort of 138 human subjects, and then subsequently examined the effect of four frequently used data normalization approaches on our ability to detect inter-specimen biological variance, using the flow cytometry data to benchmark each specimens true cellular and molecular identity. Whole blood samples originating from donors with differing leukocyte counts exhibited dramatic differences in both genome-wide distributions of transcript abundance and gene-level expression patterns. Consequently, three of the normalization strategies we tested, including median ratio (MRN), trimmed mean of m-values (TMM), and quantile normalization, noticeably masked the true biological structure of the data and impaired our ability to detect true interspecimen differences in mRNA levels. The only strategy that improved our ability to detect true biological variance was simple scaling of read counts by sequencing depth, which unlike the aforementioned approaches, makes no assumptions regarding transcriptome composition.

Donor white blood cell differential is the single largest determinant of whole blood gene expression patterns.

It has become widely accepted that sample cellular composition is a significant determinant of the gene expression patterns observed in any transcriptomic experiment performed with bulk tissue. Despite this, many investigations currently performed with whole blood do not experimentally account for possible inter-specimen differences in cellularity, and often assume that any observed gene expression differences are a result of true differences in nuclear transcription. In order to determine how confounding of an assumption this may be, in this study, we recruited a large cohort of human donors (n = 138) and used a combination of next generation sequencing and flow cytometry to quantify and compare the underlying contributions of variance in leukocyte counts versus variance in other biological factors to overall variance in whole blood transcript levels. Our results suggest that the combination of donor neutrophil and lymphocyte counts alone are the primary determinants of whole blood transcript levels for up to 75% of the protein-coding genes expressed in peripheral circulation, whereas the other factors such as age, sex, race, ethnicity, and common disease states have comparatively minimal influence. Broadly, this infers that a majority of gene expression differences observed in experiments performed with whole blood are driven by latent differences in leukocyte counts, and that cell count heterogeneity must be accounted for to meaningfully biologically interpret the results.

An assessment of nicotine pharmacokinetics and subjective effects of the pulze heated tobacco system compared with cigarettes.

Nicotine delivery and subjective effects are determinants of the ability of potentially less harmful tobacco products such as heated tobacco products (HTPs) to support adult smokers in switching away from cigarettes, and therefore to support tobacco harm reduction. This open-label, randomised, crossover, clinical study in 24 healthy adult smokers study assessed nicotine pharmacokinetics and subjective effects of the Pulze Heated Tobacco System (HTS; Pulze HTP device and three iD stick variants-Intense American Blend, Regular American Blend and Regular Menthol) compared with subjects' usual brand cigarettes (UBC). Cmax and AUCt were highest for UBC and significantly lower for each Pulze HTS variant. Cmax and AUCt were significantly higher for Intense American Blend compared with Regular American Blend, while AUCt was significantly higher for Intense American Blend compared with Regular Menthol. Median Tmax was lowest (i.e., nicotine delivery was fastest) for subjects' usual brand cigarettes and similar across the iD stick variants, although no between-product differences were statistically significant. All study products reduced urges to smoke; this effect was greatest for cigarettes although this was not statistically significant. Product evaluation scores for each Pulze HTS variant in the domains of 'satisfaction', 'psychological reward' and 'relief' were similar, and lower than those for UBC. These data demonstrate that the Pulze HTS effectively delivers nicotine and generates positive subjective effects, including satisfaction and reduced urge to smoke. This supports the conclusion that the Pulze HTS may be an acceptable alternative to cigarettes for adult smokers while having a lower abuse liability than cigarettes.

Twenty-eight day repeated exposure of human 3D bronchial epithelial model to heated tobacco aerosols indicates decreased toxicological responses compared to cigarette smoke.

Tobacco harm reduction (THR) involves providing adult smokers with potentially reduced harm modes of nicotine delivery as alternatives to smoking combustible cigarettes. Heated tobacco products (HTPs) form a category with THR potential due to their ability to deliver nicotine and flavours through heating, not burning, tobacco. By eliminating burning, heated tobacco does not produce smoke but an aerosol which contains fewer and lower levels of harmful chemicals compared to cigarette smoke. In this study we assessed the in vitro toxicological profiles of two prototype HTPs' aerosols compared to the 1R6F reference cigarette using the 3D human (bronchial) MucilAir™ model. To increase consumer relevance, whole aerosol/smoke exposures were delivered repeatedly across a 28 day period (16, 32, or 48 puffs per exposure). Cytotoxicity (LDH secretion), histology (Alcian Blue/H&E; Muc5AC; FoxJ1 staining), cilia active area and beat frequency and inflammatory marker (IL-6; IL-8; MMP-1; MMP-3; MMP-9; TNFα) levels were assessed. Diluted 1R6F smoke consistently induced greater and earlier effects compared to the prototype HTP aerosols across the endpoints, and in a puff dependent manner. Although some significant changes across the endpoints were induced by exposure to the HTPs, these were substantially less pronounced and less frequently observed, with apparent adaptive responses occurring over the experimental period. Furthermore, these differences between the two product categories were observed at a greater dilution (and generally lower nicotine delivery range) for 1R6F (1R6F smoke diluted 1/14, HTP aerosols diluted 1/2, with air). Overall, the findings demonstrate the THR potential of the prototype HTPs through demonstrated substantial reductions in toxicological outcomes in in vitro 3D human lung models.

Curiosity and intentions to use myblu e-cigarettes and an examination of the 'gateway' theory: Data from cross-sectional nationally representative surveys.

Encouraging adult smokers who are uninterested or unwilling to quit, and would otherwise continue to smoke, to transition to potentially less harmful nicotine products such as electronic nicotine delivery systems (ENDS) may positively impact population health. However, counterbalancing this benefit is the societal concern that ENDS may be used by never smokers and youth and serve as a 'gateway' into cigarette smoking. Data were analysed from two independent surveys of the prevalence and perceptions of myblu ENDS use in the United States. Total sample size was 22,232 young adults and 23,264 adults. Being curious to use myblu was 1.6-2.0 times more likely in young adult current smokers than young adult never smokers. This likelihood was 2.8 times greater for adult current smokers compared with adult never smokers in the perceptions survey, while in the prevalence survey, there was no difference between adult current and never smokers. Intentions to use myblu were significantly greater in young adult current smokers compared with young adult never smokers in both surveys and in adults in the prevalence survey. In all surveys and age cohorts, 124 of 45,496 participants (0.1% of the total survey population) reported first using myblu prior to smoking cigarettes and went on to become established smokers. Curiosity and intentions to use myblu were generally higher in current smokers compared with never smokers. There was minimal evidence to suggest the existence of a 'gateway' effect to established cigarette smoking among never-smoking myblu users.

Multiple endpoint in vitro toxicity assessment of a prototype heated tobacco product indicates substantially reduced effects compared to those of combustible cigarette.

This study aimed to compare the aerosol chemistry and in vitro toxicological profiles of two prototype Heated Tobacco Product (p-HTP) variants to the 1R6F Reference Cigarette. In the neutral red uptake screen the p-HTPs were 37-39-fold less potent than 1R6F, in the micronucleus assay, responses to the p-HTPs were 8-22-fold less, and in the Ames test mutagenicity was weak or removed compared to 1R6F. The cardiovascular scratch wound assay revealed 58-fold greater wound healing impairment following exposure to 1R6F smoke extracts than the p-HTPs. Furthermore, in seven cell stress-related high content screening endpoints (cell count, cytochrome c release, mitochondrial membrane potential, GSH depletion, NFkB translocation, phosphorylation of c-jun and phosphorylation of H2AX), at 4 and 24 h, responses were substantially greater to 1R6F smoke extracts at comparable nicotine levels. The reduced in vitro effects of the p-HTPs were attributed to substantial reductions (90-97%) in selected HPHCs measured compared to in 1R6F smoke. The multiple endpoint in vitro assessment approach provides greater mechanistic insight and the first reported toxicological characterisation of these p-HTPs in the literature. Overall, the findings contribute to the growing weight of evidence that HTPs may offer a reduced harm mode of nicotine delivery to adult smokers.

Examination of the impact of myblu electronic nicotine delivery system e-liquid nicotine strength on self-reported measures of dependence.

BACKGROUND: Greater nicotine delivery is associated with higher nicotine concentrations in electronic nicotine delivery system (ENDS) liquids. However, there is a current debate as to whether this leads to increased dependence and mitigates ENDS public health potential. METHODS: Self-reported dependence among users of myblu ENDS containing different nicotine concentrations was examined with data from a multiwave cross-sectional survey of US young adults and adults. Questions examined responses related to dependence measures and participants' most often used myblu ENDS nicotine concentration (low: 0%, 1% and 1.2%; medium: 2%, 2.4% and 2.5%; or high: 3.6% and 4%). RESULTS: A global general linear model using nicotine concentration, age and days myblu that was used in the past 30 revealed a significant difference in PROMIS scores among nicotine concentration groups (F = 4.07, p = 0.02). However, pairwise comparisons to examine which specific groups differed significantly from others showed no significant differences. Logistic regression demonstrated that strong past 30-day cravings to use myblu among participants using high or medium nicotine concentrations were not significantly different from those using a low concentration (ORs 0.66 [0.42, 1.03], p = 0.07 and 0.95 [0.49, 1.82], p = 0.98, respectively). Time to daily first use for high or medium nicotine concentration users was not significantly different from those using a low concentration (ORs 0.89 [0.70, 1.14], p = 0.35 and 0.84 [0.57, 1.25], p = 0.40, respectively). CONCLUSIONS: Use of myblu ENDS with different nicotine concentrations is not associated with differing levels of dependence. Our findings contradict the notion that high ENDS e-liquid nicotine levels generate increased dependence.

Use of deep artificial neural networks to identify stroke during triage via subtle changes in circulating cell counts.

BACKGROUND: The development of tools that could help emergency department clinicians recognize stroke during triage could reduce treatment delays and improve patient outcomes. Growing evidence suggests that stroke is associated with several changes in circulating cell counts. The aim of this study was to determine whether machine-learning can be used to identify stroke in the emergency department using data available from a routine complete blood count with differential. METHODS: Red blood cell, platelet, neutrophil, lymphocyte, monocyte, eosinophil, and basophil counts were assessed in admission blood samples collected from 160 stroke patients and 116 stroke mimics recruited from three geographically distinct clinical sites, and an ensemble artificial neural network model was developed and tested for its ability to discriminate between groups. RESULTS: Several modest but statistically significant differences were observed in cell counts between stroke patients and stroke mimics. The counts of no single cell population alone were adequate to discriminate between groups with high levels of accuracy; however, combined classification using the neural network model resulted in a dramatic and statistically significant improvement in diagnostic performance according to receiver-operating characteristic analysis. Furthermore, the neural network model displayed superior performance as a triage decision making tool compared to symptom-based tools such as the Cincinnati Prehospital Stroke Scale (CPSS) and the National Institutes of Health Stroke Scale (NIHSS) when assessed using decision curve analysis. CONCLUSIONS: Our results suggest that algorithmic analysis of commonly collected hematology data using machine-learning could potentially be used to help emergency department clinicians make better-informed triage decisions in situations where advanced imaging techniques or neurological expertise are not immediately available, or even to electronically flag patients in which stroke should be considered as a diagnosis as part of an automated stroke alert system.

A randomised, open-label, cross-over clinical study to evaluate the pharmacokinetic, pharmacodynamic and safety and tolerability profiles of tobacco-free oral nicotine pouches relative to cigarettes.

RATIONALE: Tobacco harm reduction (THR) involves encouraging adult smokers who would otherwise continue to smoke to transition to less harmful forms of nicotine delivery. These products must offer adult smokers reduced exposure to chemicals associated with tobacco combustion, satisfactory blood plasma nicotine levels and serve as an acceptable alternative. The most recent THR innovation is tobacco-free oral nicotine pouches. OBJECTIVES: This study aimed to compare pharmacokinetic, pharmacodynamic and safety and tolerability profiles of two nicotine pouch variants (ZoneX #2 (5.8 mg nicotine/pouch); ZoneX #3 (10.1 mg nicotine/pouch)) with cigarette to assess the pouches' THR potential. METHODS: This was a controlled use, randomised, open-label, cross-over clinical study with 24 healthy adult traditional tobacco users. Pharmacokinetic (plasma nicotine levels; up to 8 h post-use), pharmacodynamic (urge to smoke, product liking; up to 4 h post-use) and short-term safety and tolerability profiles were assessed. RESULTS: Distinct nicotine pouch pharmacokinetic profiles indicated nicotine absorption via the oral mucosa. Plasma nicotine levels were lower, and time to peak slower, for the nicotine pouches compared to cigarette (Cmax cigarette: 11.6 ng/ml vs. #2: 5.2 ng/ml, p < 0.0001; #3: 7.9 ng/ml, p < 0.0003) (Tmax cigarette: 8.6 min vs. #2: 26 min; #3: 22 min). All products effectively reduced subjects' urge to smoke and presented favourable product liking scores; nicotine pouches were also well tolerated following short-term use (no serious adverse events). CONCLUSIONS: Overall, the assessed ZoneX nicotine pouches may offer an acceptable alternative for adult smokers to achieve satisfactory levels of nicotine delivery and, based on the pharmacokinetic parameters and under the study conditions, likely have a lower abuse liability and addictive potential for current adult smokers compared to continued cigarette smoking. CLINICAL TRIAL IDENTIFIER: NCT04891406 (clinicaltrials.gov).

Use of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes to Predict the Cardiotoxicity Potential of Next Generation Nicotine Products.

Combustible cigarette smoking is an established risk factor for cardiovascular disease. By contrast, the cardiotoxicity potential of non-combustible next generation nicotine products (NGPs), which includes heated tobacco products (HTPs) and electronic vaping products (EVPs), and how this compares relative to combustible cigarettes is currently an area of scientific exploration. As such, there is a need for a rapid screening assay to assess this endpoint. The Cardio quickPredict is a metabolomics biomarker-based assay that uses human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) to screen for potential structural and functional cardiac toxicants based on the changes of four metabolites, lactic acid, arachidonic acid, thymidine, and 2'-deoxycytidine. The study aims were to investigate the cardiotoxicity potential of NGPs compared to cigarettes, in addition to nicotine. To accomplish this, hiPSC-CM were exposed to smoke or aerosol bubbled PBS samples: reference cigarette (1R6F); three variants of HTP; and three EVP variants. The 1R6F bPBS was the most active, having cardiotoxic potential at 0.3-0.6% bPBS (0.4-0.9 µg/mL nicotine), followed by HTP, which displayed cardiotoxic potential at a 10 times higher concentration, 3.3% bPBS (4.1 µg/mL nicotine). Both 1R6F and HTP bPBS (at 10-fold higher concentration than 1R6F) affected all four predictive metabolites, whereas none of the EVP bPBS samples were active in the assay up to the maximal concentration tested (10% bPBS). Nicotine tested on its own was predicted to have cardiotoxic potential at concentrations greater than 80 µg/mL, which is higher than expected physiological levels associated with combustible cigarette smoking. The application of this rapid screening assay to NGP research and the associated findings adds to the weight-of-evidence indicating that NGPs have a tobacco harm reduction potential when compared to combustible cigarettes. Additionally, this technique was shown to be sensitive and robust for the assessment of different NGPs and may be considered as part of a larger overall scientific framework for NGP assessments.

E-Cigarette Aerosol Deposition and Disposition of [11C]Nicotine Using Positron Emission Tomography: A Comparison of Nicotine Uptake in Lungs and Brain Using Two Different Nicotine Formulations.

Smoking is a cause of serious disease in smokers. Electronic cigarettes, delivering aerosolized nicotine, offer adult smokers a potentially less harmful alternative to combustible cigarettes. This explorative PET/CT study investigated the distribution and deposition of inhaled [11C]nicotine using the mybluTM e-cigarette with two nicotine formulations, freebase and lactate salt. Fifteen healthy adult smokers participated in the two-part study to assess the distribution and accumulation of [11C]nicotine in the respiratory pathways and brain. Time-activity data for the respiratory pathways, lungs, oesophagus and brain were derived. 31-36% of both inhaled tracer formulations accumulated in the lung within 15-35 s. [11C]Nicotinefreebase exhibited higher uptake and deposition in the upper respiratory pathways. For [11C]nicotinelactate, brain deposition peaked at 4-5%, with an earlier peak and a steeper decline. A different kinetic profile was obtained for [11C]nicotinelactate with lower tracer uptake and accumulation in the upper respiratory pathways and an earlier peak and a steeper decline in lung and brain. Using nicotine lactate formulations in e-cigarettes may thus contribute to greater adult smoker acceptance and satisfaction compared to freebase formulations, potentially aiding a transition from combustible cigarettes and an acceleration of tobacco harm reduction initiatives.

Reductions in biomarkers of exposure to selected harmful and potentially harmful constituents following exclusive and partial switching from combustible cigarettes to myblu™ electronic nicotine delivery systems (ENDS).

Electronic nicotine delivery systems (ENDS) offer adult combustible cigarette smokers an alternative, potentially reduced harm, mode of nicotine delivery, attributed to fewer and reduced levels of harmful and potentially harmful constituents (HPHCs) in their aerosols compared to cigarette smoke. These two identical, randomised, open label, two-part studies aimed to compare levels of 15 biomarkers of exposure (BoE) to selected HPHCs associated with tobacco smoking in healthy US adult smoker subjects (n = 72). Following 9 days of exclusive use of a range of allocated myblu™ ENDS variants, subjects' levels of 14 non-nicotine BoE were substantially reduced compared to baseline values (combustible cigarette use), in the range of 46-97%. BoE reductions were sustained in subjects who continued myblu use exclusively (n = 25) for a further 5 days, and returned to near baseline levels in subjects who returned to exclusive combustible cigarette use (n = 21). Dual users (n = 24) demonstrated reductions in BoE to a lesser extent than with exclusive myblu use. Measured nicotine equivalents did not significantly change throughout the study. These data suggest exclusive use of ENDS provides adult smokers seeking an alternative to combustible cigarettes with substantial reductions in HPHC exposures whilst achieving satisfying levels of nicotine delivery. Dual use involving substitution of cigarettes may also provide some of this advantage, but to lesser extent. Overall, the data contribute to the weight of evidence that ENDS are an important tool in tobacco harm reduction for adult smokers unwilling to or uninterested in quitting smoking. Study 1: NCT04430634, study 2: NCT04429932, clinicaltrials.gov (10-06-2020).

Newly-identified blood biomarkers of neurological damage are correlated with infarct volume in patients with acute ischemic stroke.

Our group recently performed a genome-wide informatic analysis that highlighted eight brain-enriched proteins with strong potential to serve as blood biomarkers of neurological injury (GFAP, MBP, ß-synuclein, OPALIN, MT-3, SNAP-25, KIF5A, MOBP), including six that have yet to be widely investigated. In this study, our aim was to determine whether the circulating levels of these proteins could be used to approximate the extent of neural tissue damage in ischemic stroke. To address this aim, blood was collected from 43 ischemic stroke patients immediately upon hospital admission. The serum levels of the eight candidate proteins were measured via ELISA, infarct volume was assessed via manual tracing of neuroradiological images, and correlational analysis was performed to examine potential associative relationships. The serum levels of all eight proteins exhibited positive correlations with infarct volume, however the strongest associations were observed in a subset of four proteins known to originate from neurons specifically (MT-3, SNAP-25, KIF5A, ß-synuclein). Combining the serum levels of these neuron-originating proteins using principal components analysis produced a single composite value that was more strongly correlated with infarct volume than the levels of any single protein considered in isolation (r = 0.48, p < 0.001). Measures of these proteins could potentially be used to provide a minimally invasive approximation of lesion size when advanced imaging techniques are not available, or when imaging results are inconclusive.

Diagnostic accuracy of blood biomarkers for Alzheimer's disease and amnestic mild cognitive impairment: A meta-analysis.

OBJECTIVE: To examine the diagnostic accuracy of blood-based biomarkers for detecting Alzheimer's disease (AD) and amnestic mild cognitive impairment (aMCI). METHODS: Seven electronic databases were comprehensively searched for studies evaluating the diagnostic accuracy of blood-based biomarkers for detecting AD or aMCI up to July 31, 2020. The pooled sensitivity, specificity, and the diagnostic odds ratio (DOR) were calculated using a hierarchical summary receiver operating characteristic model. RESULTS: A total of 17 studies (n = 2,083) were included. In differentiating patients with AD from the controls, the DOR was 32.2 for the plasma Aß42 (sensitivity = 88 %, specificity = 81 %), 29.1 for the plasma Aß oligomer (sensitivity = 80 %, specificity = 88 %), and 52.1 for the plasma tau (sensitivity = 90 %, specificity = 87 %). For differentiating aMCI from the controls, the DOR was 60.4 for the plasma Aß42 (sensitivity = 86 %, specificity = 90 %) and 49.1 for the plasma tau (sensitivity = 79 %, specificity = 94 %). The use of ultra-high sensitive technology explained the heterogeneity in the diagnostic performance of blood-based biomarkers (P = .01). CONCLUSIONS: We suggest that blood-based biomarkers are minimally invasive and cost-effective tools for detecting AD; however, the evidence for detecting aMCI was still limited.

Perception of the relative harm of electronic cigarettes compared to cigarettes amongst US adults from 2013 to 2016: analysis of the Population Assessment of Tobacco and Health (PATH) study data.

BACKGROUND: Electronic cigarettes (e-cigarettes) have been characterised as significantly less harmful than cigarettes by many health agencies and regulators globally. In this study, we examined to what extent perceived relative harms of e-cigarettes compared to cigarettes have changed in the USA. METHODS: We analysed the data from the longitudinal and nationally representative, Population Assessment of Tobacco and Health Study to assess the relative perceived harm of e-cigarettes amongst US adults between 2013 and 2016. RESULTS: The proportion of US adults who correctly perceived e-cigarettes as less harmful than cigarettes decreased each year from 41.1% (CI 40.1-42.1%) in 2013-2014, 31.5% (CI 30.8-32.2%) in 2014-2015 and 25.3% (CI 24.6-26.0%) in 2015-2016. Concurrently, the proportion of US adults who perceived e-cigarettes as equally, or more, harmful than cigarettes increased from 53.7% (CI 52.3-55.1%), 64.9% (CI 63.6-66.2%) to 72.7% (CI 71.5-73.9%) respectively. The proportion of US adults who held negative relative harm perceptions of e-cigarettes increased regardless of current smoking or vaping status by 24.6% and 29.6% respectively within 3 years. In Wave 3, the proportion of current smokers who perceived the relative harm of e-cigarettes as less harmful was lower at 29.3% (CI 28.2-30.4%) compared to current e-cigarette users at 43.5% (CI 40.3-46.7%). Former smokers who used e-cigarettes and believed that they were equally, or more, harmful than cigarettes in 2014-2015 had significantly higher rates of smoking relapse in the following year, 29% and 37% (p < 2.2e-16), respectively, compared to those with positive relative harm perceptions who reported relapse rates of 19%. CONCLUSIONS: In this study, the proportion of US adults who incorrectly perceived e-cigarettes as equal to, or more, harmful than cigarettes increased steadily regardless of smoking or vaping status. Current adult smokers appear to be poorly informed about the relative risks of e-cigarettes yet have potentially the most to gain from transitioning to these products. The findings of this study emphasise the urgent need to accurately communicate the reduced relative risk of e-cigarettes compared to continued cigarette smoking and clearly differentiate absolute and relative harms. Further research is required to elucidate why the relative harm of e-cigarettes is misunderstood and continues to deteriorate.

Large-scale informatic analysis to algorithmically identify blood biomarkers of neurological damage.

The identification of precision blood biomarkers which can accurately indicate damage to brain tissue could yield molecular diagnostics with the potential to improve how we detect and treat neurological pathologies. However, a majority of candidate blood biomarkers for neurological damage that are studied today are proteins which were arbitrarily proposed several decades before the advent of high-throughput omic techniques, and it is unclear whether they represent the best possible targets relative to the remainder of the human proteome. Here, we leveraged mRNA expression data generated from nearly 12,000 human specimens to algorithmically evaluate over 17,000 protein-coding genes in terms of their potential to produce blood biomarkers for neurological damage based on their expression profiles both across the body and within the brain. The circulating levels of proteins associated with the top-ranked genes were then measured in blood sampled from a diverse cohort of patients diagnosed with a variety of acute and chronic neurological disorders, including ischemic stroke, hemorrhagic stroke, traumatic brain injury, Alzheimer's disease, and multiple sclerosis, and evaluated for their diagnostic performance. Our analysis identifies several previously unexplored candidate blood biomarkers of neurological damage with possible clinical utility, many of which whose presence in blood is likely linked to specific cell-level pathologic processes. Furthermore, our findings also suggest that many frequently cited previously proposed blood biomarkers exhibit expression profiles which could limit their diagnostic efficacy.

Bioinformatic analysis of brain-specific miRNAs for identification of candidate traumatic brain injury blood biomarkers.

BACKGROUND: Detection of brain-specific miRNAs in the peripheral blood could serve as a surrogate marker of traumatic brain injury (TBI). Here, we systematically identified brain-enriched miRNAs, and tested their utility as TBI biomarkers in the acute phase of care. METHODS: Publically available microarray data generated from 29 postmortem human tissues were used to rank 1,364 miRNAs in terms of their degree of brain-specific expression. Levels of the top six ranked miRNAs were then prospectively measured in serum samples collected from 10 Patients with TBI at hospital admission, as well as from 10 controls. RESULTS: The top six miRNAs identified in our analysis (miR-124-3p, miR-219a-5p, miR-9-5p, miR-9-3p, miR-137, and miR-128-3p) were enriched 70 to 320-fold in brain relative to other tissues, and exhibited dramatically greater brain specificity compared to several miRNAs previously proposed as biomarkers. Furthermore, their levels were elevated in serum from patients with TBI compared to controls, and could collectively discriminate between groups with 90% sensitivity and 100% specificity. Interestingly, subsequent informatic pathway analysis revealed that their target transcripts were enriched for components of signaling pathways active in peripheral organs involved in common post-TBI complications. CONCLUSIONS: The six candidate miRNAs identified in this preliminary study have promise as blood biomarkers of TBI, and could also be molecular contributors to systemic physiologic changes commonly observed post-injury.

Diagnosis of ischemic stroke using circulating levels of brain-specific proteins measured via high-sensitivity digital ELISA.

Limited lower detection ranges associated with traditional immunoassay techniques have prevented the use of brain-specific proteins as blood biomarkers of stroke in the acute phase of care, as these proteins are often only present in circulation at low concentrations. Digital ELISA is a newly developed technique with allows for quantification of proteins in biofluids with up to 1000 times greater sensitivity than conventional ELISA techniques. The purpose of this study was to determine whether the extended lower limits of detection associated with digital ELISA could enable the use of brain-specific proteins as blood biomarkers of ischemic stroke during triage. Blood was sampled from ischemic stroke patients (n = 14) at emergency department admission, as well as from neurologically normal controls matched in terms of risk factors for cardiovascular disease (n = 33). Plasma levels of two brain-specific axonal proteins, neurofilament light chain (NfL) and tau, were measured via digital ELISA, and receiver-operating characteristic analysis was used to determine their ability to discriminate between groups. Plasma levels of NfL and tau were both significantly elevated in stroke patients versus controls, and could respectively discriminate between groups with 92.9% sensitivity / 84.9% specificity, and 85.7% sensitivity / 54.6% specificity. Furthermore, adjustment of measured NfL and Tau levels according to the lower-limits of detection associated with commercially-available conventional ELISA assays resulted in a dramatic and statistically significant decrease in diagnostic performance. Collectively, our results suggest that the increased analytical sensitivity of digital ELISA could enable the use of brain-specific proteins as blood biomarkers of ischemic stroke during triage.