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.

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.

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.

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.

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.

Characterization of the Spatial and Temporal Dispersion Differences Between Exhaled E-Cigarette Mist and Cigarette Smoke.

INTRODUCTION: There are fundamental differences between electronic cigarettes (e-cigarettes) and conventional cigarette product categories with regards to potential environmental exposures, notably that e-cigarettes do not contain tobacco or generate side-stream emissions. Here we assess the spatial and temporal patterns of exhaled e-cigarette aerosol at a bystander's position, and compare it with conventional cigarette smoke emissions. METHODS: Smokers were asked to use e-cigarettes or smoke conventional cigarettes in a room-simulating chamber. Volunteers used the products at different distances from a heated mannequin, representing a bystander, and under different room ventilation rates. Aerosol particle concentrations and size distributions at the bystander's position were measured. RESULTS: For both product categories, the particle concentrations registered following each puff were in the same order of magnitude. However, for e-cigarettes the particle concentration returned rapidly to background values within seconds; for conventional cigarettes it increased with successive puffs, returning to background levels after 30-45 minutes. Unlike for the e-cigarette devices tested, such temporal variation was dependent on the room ventilation rate. Particle size measurements showed that exhaled e-cigarette particles were smaller than those emitted during smoking conventional cigarettes and evaporated almost immediately after exhalation, thus affecting the removal of particles through evaporation rather than displacement by ventilation. CONCLUSIONS: Significant differences between emissions from the tested e- and conventional cigarettes are reported. Exhaled e-cigarette particles are liquid droplets evaporating rapidly; conventional cigarette smoke particles are far more stable and linger. IMPLICATIONS: • Several factors potentially influencing particle behavior after exhalation of e-cigarette aerosols or emitted during smoking conventional cigarettes were studied.• Differences in particle size between those exhaled following use of e-cigarettes and those emitted during smoking of conventional cigarettes were observed.• E-cigarette particle concentrations decreased rapidly following exhalation due to evaporation.• The removal of particles following smoking conventional cigarettes was much slower and was dependent on the room ventilation rate.

Snus: a compelling harm reduction alternative to cigarettes.

Snus is an oral smokeless tobacco product which is usually placed behind the upper lip, either in a loose form or in portioned sachets, and is primarily used in Sweden and Norway. The purpose of this review is to examine the reported effects of snus use in relation to specified health effects, namely lung cancer, cardiovascular disease, pancreatic cancer, diabetes, oral cancer and non-neoplastic oral disease. The review also examines the harm reduction potential of snus as an alternative to cigarettes by comparing the prevalence of snus use and cigarette smoking, and the reported incidence of tobacco-related diseases across European Union countries. The scientific literature generally indicates that the use of snus is not a significant risk factor for developing lung cancer, cardiovascular disease, pancreatic cancer or oral cancer. Studies investigating snus use and diabetes have reported that high consumption of snus (estimated as being four or more cans per week) may be associated with a higher risk of developing diabetes or components of metabolic syndrome; however, overall results are not conclusive. Snus use is associated with the presence of non-neoplastic oral mucosal lesions which are reported to heal rapidly once use has stopped. The most recent Eurobarometer data from 2017 reported that Sweden had the lowest prevalence of daily cigarette use in the European Union at 5% whilst daily "oral tobacco" use was reported to be 20%. European data published by the World Health Organisation in 2018 indicated that Sweden had the lowest rate of tobacco-related mortality and the lowest incidence of male lung cancer. Overall, prevalence statistics and epidemiological data indicate that the use of snus confers a significant harm reduction benefit which is reflected in the comparatively low levels of tobacco-related disease in Sweden when compared with the rest of Europe. The available scientific data, including long-term population studies conducted by independent bodies, demonstrates that the health risks associated with snus are considerably lower than those associated with cigarette smoking.

High-throughput profiling of the circulating proteome suggests sexually dimorphic corticosteroid signaling following ischemic stroke.

Increasing evidence suggests that there are innate differences between sexes with respect to stroke pathophysiology; however, the molecular mechanisms underlying these differences remain unclear. In this investigation, we employed a shotgun approach to broadly profile sex-associated differences in the plasma proteomes of a small group of male ( n = 6) and female ( n = 4) ischemic stroke patients. Peripheral blood was sampled during the acute phase of care, and liquid chromatography electrospray ionization mass spectrometry was used to quantify plasma proteins. We observed widespread differences in plasma composition, as 77 out of 294 detected proteins were significantly differentially expressed between sexes. Corticosteroid-binding globulin (CBG), a negative acute-phase reactant that inversely regulates levels of bioactive free cortisol, was the most dramatically differentially regulated, exhibiting 16-fold higher abundance in plasma from women relative to men. Furthermore, functional annotation analysis revealed that the remaining differentially expressed proteins were significantly enriched for those involved in response to corticosteroid signaling. Plasma CBG levels were further examined in an additional group of male ( n = 19) and female ( n = 28) ischemic stroke patients, as well as a group of male ( n = 13) and female ( n = 18) neurologically normal controls. CBG levels were significantly reduced in male stroke patients relative to male controls; however, no differences were observed between female stroke patients and female controls, suggesting that women may exhibit an attenuated cortisol response to stroke. Collectively, our findings reinforce the idea that there are sex-associated differences in stroke pathophysiology and suggest that cortisol signaling should be investigated further as a potential molecular mediator.

Evaluation of the safety profile of an electronic vapour product used for two years by smokers in a real-life setting.

The safety profile of Puritane™, a closed system electronic vapour product (EVP), was evaluated when used by smokers of conventional cigarettes (CCs) for 24 months in a real-life setting. The study was a two-centre ambulatory clinical study with 209 healthy volunteers. Outcome measures included adverse events (AEs), vital signs, electrocardiogram, lung function tests, exposure to nicotine and selected smoke constituents, nicotine withdrawal effects and smoking desire. No serious AEs related to EVP use were observed. The most frequently reported AEs were headache, nasopharyngitis, sore throat and cough, reported by 28.7%, 28.7%, 19.6% and 16.7% of subjects, respectively, which dissipated over time. Small decreases in lung function were not considered clinically relevant. No clinically relevant findings were observed in the other safety parameters. From Month 2, nicotine withdrawal symptoms decreased. Smoking desire and CC consumption steadily decreased over time in all subjects. EVP use was associated with reduced exposure to cigarette smoke constituents, whereas urinary nicotine levels remained close to baseline. Body weight did not increase in CC subjects switching to the EVP. In conclusion, the aerosol of the EVP at study was well tolerated and not associated with any clinically relevant health concerns after usage for up to 24 months.

Circulating leucocytes perpetuate stroke-induced aortic dysfunction.

NEW FINDINGS: What is the central question of this study? Does a stroke event influence aortic endothelial function; and what is the role of peripheral circulating leucocytes in stroke on the vascular reactivity of the aorta? What is the main finding and its importance? In vitro co-culture experiments demonstrated that aortic endothelium-dependent relaxation was impaired when rat aortic rings were co-cultured with leucocytes stimulated with serum from stroke patients. Impaired vascular reactivity was not observed in aortic rings without leucocytes stimulated with serum from stroke patients or age-matched control patients with or without leucocytes. These data suggest that leucocyte-dependent altered aortic endothelium-dependent relaxation with stroke and the systemic consequences of stroke on vascular inflammation may occur in the aorta. Post-stroke inflammation has been linked to poor stroke outcomes. The vascular endothelium senses and responds to circulating factors, in particular inflammatory cytokines. Although stroke-associated local cerebrovascular dysfunction is well reported, the effects of a stroke on conduit artery function are not fully understood. We tested the hypothesis that serum from stroke patients triggers leucocyte-dependent aortic endothelial dysfunction that is associated with elevated concentrations of cytokines. Total leucocytes were isolated from healthy individuals, and the cells were incubated in serum from control subjects or stroke patients for 6 h. The quantity of cytokines in media was determined using an immunoassay. Vascular reactivity was determined by the rat aortic rings that were co-cultured with or without leucocytes and stimulated with serum samples from control subjects or stroke patients. Endothelium-dependent dilatation was significantly impaired in aortic rings co-cultured with leucocytes plus serum from stroke patients (50 ± 30 versus 85 ± 13%, P < 0.05) versus serum from control subjects. In contrast, no difference was observed in aortic function stimulated with serum from control subjects or stroke patients without total leucocytes. Likewise, total leucocyte-derived cytokine concentrations were significantly increased in a time-dependent manner on stimulation with serum from stroke patients (P < 0.05). These observations support the concept that the increased response of leucocytes drives the development of stroke-associated vascular endothelial dysfunction. As such, pharmacologically targeting the source of inflammatory cytokines might alleviate stroke-associated peripheral vascular dysfunction.

Measurement of cardiovascular and pulmonary function endpoints and other physiological effects following partial or complete substitution of cigarettes with electronic cigarettes in adult smokers.

Acute changes in select physiological parameters associated with cardiovascular physiology (systolic and diastolic blood pressure (BP) and heart rate (HR)), pulmonary function (FVC, FEV1, and exhaled CO and NO) and adverse events were measured in 105 clinically confined subjects who were randomized into groups that either completely or partially switched from conventional cigarettes to e-cigarettes or completely discontinued using tobacco and nicotine products altogether. Use of the e-cigarettes for five days under the various study conditions did not lead to higher BP or HR values, negative respiratory health outcomes or serious adverse health events. Reductions in BP and HR vital signs were observed in most of the participants that either ceased tobacco and nicotine products use altogether or switched completely to using e-cigarettes. Pulmonary function tests showed small but non-statistically significant improvements in FVC and FEV1 measurements in most use groups. Statistically significant (p < 0.05) benefits associated with smoking reduction were also noted in exhaled CO and NO levels. All study products were well tolerated. The study findings suggest that there are potential cardiovascular and pulmonary function benefits when smokers switch to using e-cigarette products. This further reinforces the potential that e-cigarettes offer smokers seeking an alternative to conventional tobacco products.

Circulating extracellular DNA levels are acutely elevated in ischaemic stroke and associated with innate immune system activation.

OBJECTIVE: The objective of this work was to assess the ability of peripheral blood cell-free DNA (cfDNA) levels to identify ischaemic stroke early in the acute phase of care, as well as to examine the relationship between peripheral blood cfDNA levels and stroke-induced innate immune system activation. METHODS: Upon emergency department admission, peripheral blood samples were obtained from 43 patients experiencing acute ischaemic stroke and 20 patients identified as stroke mimics. Plasma cfDNA levels were measured using quantitative polymerase chain reaction (qPCR), infarct volume and NIH stroke scale (NIHSS) were used to assess injury severity, and peripheral blood neutrophil count was used as a measure of innate immune system status. RESULTS: Peripheral blood cfDNA levels were significantly elevated in patients suffering stroke relative to those diagnosed as stroke mimics, and could differentiate between groups with 86% (95% CI = 72-95%) sensitivity and 75% (95% CI = 51-91%) specificity. Furthermore, cfDNA levels displayed significant positive associations between both infarct volume and peripheral blood neutrophil count within the stroke group. CONCLUSIONS: These findings suggest that assessment of peripheral blood cfDNA levels may be useful for the identification of ischaemic stroke in the acute care setting, and provide associative evidence that cfDNA is a potential activator of the peripheral innate immune system in response to cerebral ischaemia.

An online method for the analysis of volatile organic compounds in electronic cigarette aerosol based on proton transfer reaction mass spectrometry.

RATIONALE: Due to the recent rapid increase in electronic cigarette (e-cigarette) use worldwide, there is a strong scientific but also practical interest in analyzing e-cigarette aerosols. Most studies to date have used standardized but time-consuming offline technologies. Here a proof-of-concept for a fast online quantification setup based on proton transfer reaction mass spectrometry (PTR-MS) is presented. METHODS: The combination of a novel sampling interface with a time-of-flight PTR-MS instrument specially designed for three scenarios is introduced: (i) mainstream aerosol analysis (aerosol that the user inhales prior to exhalation), and analysis of exhaled breath following (ii) mouth-hold (no inhalation) and (iii) inhalation of e-cigarette aerosols. A double-stage dilution setup allows the various concentration ranges in these scenarios to be accessed. RESULTS: First, the instrument is calibrated for the three principal constituents of the e-cigarettes' liquids, namely propylene glycol, vegetable glycerol and nicotine. With the double-stage dilution the instrument's dynamic range was easily adapted to cover the concentration ranges obtained in the three scenarios: 20-1100 ppmv for the mainstream aerosol characterisation; 4-300 ppmv for the mouth-hold; and 2 ppbv to 20 ppmv for the inhalation experiment. CONCLUSIONS: It is demonstrated that the novel setup enables fast, high time resolution e-cigarette studies with online quantification. This enables the analysis and understanding of any puff-by-puff variations in e-cigarette aerosols. Large-scale studies involving a high number of volunteers will benefit from considerably higher sample throughput and shorter data processing times.

Reductions in biomarkers of exposure (BoE) to harmful or potentially harmful constituents (HPHCs) following partial or complete substitution of cigarettes with electronic cigarettes in adult smokers.

Changes in fifteen urine, blood and exhaled breath BoEs of HPHCs representing classes of compounds reported by FDA to be significant contributors to smoking-associated disease risks were measured in 105 clinical-confined subjects following randomization and a five-day forced-switch from usual brand conventional combustible cigarettes to: (i) exclusive commercial e-cigarette use; (ii) dual-use of commercial e-cigarettes and the subject's usual cigarette brand; or (iii) discontinued use of all tobacco or nicotine products. Levels of urinary biomarkers in subjects that completely substituted their usual cigarette with e-cigarettes were significantly lower (29-95%) after 5 days. Percent reductions in eight of nine urinary BoEs were indistinguishable to smokers who had quit smoking, except for nicotine equivalents, which declined by 25-40%. Dual users who halved self-reported daily cigarette consumption with e-cigarettes exhibited reductions (7-38%) in eight of nine urinary biomarkers, but had increase (1-20%) in nicotine equivalents. Reductions were broadly proportional to the reduced numbers of cigarettes smoked. Dual user urinary nicotine equivalents were slightly higher, but not statistically significant. After 5 days, blood nicotine biomarker levels were lower in the cessation (75-96%) and exclusive use groups (11-83%); with dual users experiencing no significant reductions. All subjects experienced significant decreases in exhaled CO. Decreases in the cessation and exclusive groups ranged from 88-89% and 27-32% in dual users. Exhaled NO increased in the cessation and exclusive groups (46-63% respectively), whereas the dual users experienced minimal changes. Overall, smokers who completely or partially substituted conventional cigarettes with e-cigarettes over five days, experienced reductions in HPHCs.

Interleukin-15 directly stimulates pro-oxidative gene expression in skeletal muscle in-vitro via a mechanism that requires interleukin-15 receptor alpha.

Interleukin-15 (IL-15) signaling is heavily regulated by a high specificity IL-15 binding protein known as interleukin-15 receptor alpha (IL-15Rα). In-vivo disruption of IL-15Rα in the constitutive IL-15Rα knock-out (IL-15RαKO) mouse results in a shift towards an oxidative muscle phenotype characterized by dramatic increases in mitochondrial density. The IL-15RαKO mouse displays elevated levels of IL-15 transcript in muscle tissue, along with increased circulating levels of IL-15. As a result, it has been suggested that loss of IL-15Rα from skeletal muscle enhances muscle IL-15 secretion, and that muscle-derived IL-15 acts in an autocrine fashion to elicit pro-oxidative effects. However, this proposed mechanism of IL-15/IL-15Rα action in skeletal muscle is based primarily on in-vivo associative observations, and has yet to be explored in a direct manner. Thus, our purpose was to assess the immediate influence of IL-15Rα on the capacity of skeletal muscle to secrete and respond to IL-15, and also to determine whether IL-15 has the ability to act directly on skeletal muscle to induce pro-oxidative changes. These aims were addressed in-vitro using primary myogenic cultures derived from IL-15RαKO mice and B6129 controls, as well as cultures of the C2C12 immortalized myogenic cell line. Cultures obtained from IL-15RαKO mice displayed a diminished capacity to secrete IL-15 in relation to B6129 controls. Acute treatment of B6129-derived cultures with recombinant IL-15 increased transcriptional expression of the pro-oxidative genes PGC1α and PPARδ. IL-15 treatment failed to elicit a similar response in cultures generated from IL-15RαKO mice. Chronic treatment of C2C12 cultures with IL-15 during myogenic differentiation resulted in mature myocytes with greater mitochondrial density in relation to vehicle treated controls. Collectively, these results provide evidence that IL-15 has the capacity to act directly on skeletal muscle in a pro-oxidative manner, and that disruption of IL-15Rα ablates the ability of skeletal muscle to secrete and respond to IL-15.

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.

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.

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.

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.

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.