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Abstract Background Anxiety disorders (ADs) are a complex group of mental disorders and majorly contribute to the global health-related burden. Symptoms and clinical management differ widely depending on the specific diagnosis. There is a need for new, more effective pharmacological treatments for these patients as many patients do not respond to treatment and treatment is not available for several types of AD. The increased interest in the potential effects of cannabidiol (CBD) on symptoms of AD has led to several preclinical and clinical studies that suggest that CBD may be effective in some patients with AD. However, it remains unclear whether and how CBD can be used in the clinical management of ADs due to a lack of sufficiently robust clinical evidence. Comparative evaluation This narrative review provides a critical analysis of the current state of the art for ADs and summarizes six recently completed and 22 currently ongoing clinical trials investigating the effects of CBD on ADs or anxiety. The aim was to examine whether the ongoing trials are likely to provide the necessary solid evidence, or whether new studies with more robust design parameters can help to overcome the prevailing lack of solid clinical data for this CBD indication. Most of the trials reviewed are considered exploratory and do not focus on specific types of clinical anxiety or ADs as the primary condition studied. Participant numbers, CBD dose, treatment duration, and CBD formulation vary widely among the studies, and all but two are single-center studies. Conclusion For an effective clinical management of ADs using CBD, there is a need for sufficiently powered and appropriately designed clinical trials (RCT, multicenter, defined doses and exposure monitoring, robust primary outcomes) investigating the effect of CBD in specific ADs, such as social anxiety disorder and panic disorder, or in post-traumatic stress disorder.

Abstract Cigarette smoking is a major preventable cause of morbidity and mortality. While quitting smoking is the best option, switching from cigarettes to non-combustible alternatives (NCAs) such as e-vapor products is a viable harm reduction approach for smokers who would otherwise continue to smoke. A key challenge for the clinical assessment of NCAs is that self-reported product use can be unreliable, compromising the proper evaluation of their risk reduction potential. In this cross-sectional study of 205 healthy volunteers, we combined comprehensive exposure characterization with in-depth multi-omics profiling to compare effects across four study groups: cigarette smokers (CS), e-vapor users (EV), former smokers (FS), and never smokers (NS). Multi-omics analyses included metabolomics, transcriptomics, DNA methylomics, proteomics, and lipidomics. Comparison of the molecular effects between CS and NS recapitulated several previous observations, such as increased inflammatory markers in CS. Generally, FS and EV demonstrated intermediate molecular effects between the NS and CS groups. Stratification of the FS and EV by combustion exposure markers suggested that this position on the spectrum between CS and NS was partially driven by non-compliance/dual use. Overall, this study highlights the importance of in-depth exposure characterization before biological effect characterization for any NCA assessment study.

Abstract Background The primary components driving the current commercial fascination with cannabis products are phytocannabinoids, a diverse group of over 100 lipophilic secondary metabolites derived from the cannabis plant. Although numerous phytocannabinoids exhibit pharmacological effects, the foremost attention has been directed towards Δ9-tetrahydrocannabinol (THC) and cannabidiol, the two most abundant phytocannabinoids, for their potential human applications. Despite their structural similarity, THC and cannabidiol diverge in terms of their psychotropic effects, with THC inducing notable psychological alterations. There is a clear need for accurate and rapid THC measurement methods that offer dependable, readily accessible, and cost-effective analytical information. This review presents a comprehensive view of the present state of alternative technologies that could potentially facilitate the creation of portable devices suitable for on-site usage or as personal monitors, enabling non-intrusive THC measurements. Method A literature survey from 2017 to 2023 on the development of portable technologies and commercial products to detect THC in biofluids was performed using electronic databases such as PubMed, Scopus, and Google Scholar. A systematic review of available literature was conducted using Preferred Reporting Items for Systematic. Reviews and Meta-analysis (PRISMA) guidelines. Results Eighty-nine studies met the selection criteria. Fifty-seven peer-reviewed studies were related to the detection of THC by conventional separation techniques used in analytical laboratories that are still considered the gold standard. Studies using optical (n = 12) and electrochemical (n = 13) portable sensors and biosensors were also identified as well as commercially available devices (n = 7). Discussion The landscape of THC detection technology is predominantly shaped by immunoassay tests, owing to their established reliability. However, these methods have distinct drawbacks, particularly for quantitative analysis. Electrochemical sensing technology holds great potential to overcome the challenges of quantification and present a multitude of advantages, encompassing the possibility of miniaturization and diverse modifications to amplify sensitivity and selectivity. Nevertheless, these sensors have considerable limitations, including non-specific interactions and the potential interference of compounds and substances existing in biofluids. Conclusion The foremost challenge in THC detection involves creating electrochemical sensors that are both stable and long-lasting while exhibiting exceptional selectivity, minimal non-specific interactions, and decreased susceptibility to matrix interferences. These aspects need to be resolved before these sensors can be successfully introduced to the market.

Abstract The use of cannabidiol (CBD) for therapeutic purposes is receiving considerable attention, with speculation that CBD can be useful in a wide range of conditions. Only one product, a purified form of plant-derived CBD in solution (Epidiolex), is approved for the treatment of seizures in patients with Lennox-Gastaut syndrome, Dravet syndrome, or tuberous sclerosis complex. Appraisal of the therapeutic evidence base for CBD is complicated by the fact that CBD products sometimes have additional phytochemicals (like tetrahydrocannabinol (THC)) present, which can make the identification of the active pharmaceutical ingredient (API) in positive studies difficult. The aim of the present review is to critically review clinical studies using purified CBD products only, in order to establish the upcoming indications for which purified CBD might be beneficial. The areas in which there is the most clinical evidence to support the use of CBD are in the treatment of anxiety (positive data in 7 uncontrolled studies and 17 randomised controlled trials (RCTs)), psychosis and schizophrenia (positive data in 1 uncontrolled study and 8 RCTs), PTSD (positive data in 2 uncontrolled studies and 4 RCTs) and substance abuse (positive data in 2 uncontrolled studies and 3 RCTs). Seven uncontrolled studies support the use of CBD to improve sleep quality, but this has only been verified in one small RCT. Limited evidence supports the use of CBD for the treatment of Parkinson’s (3 positive uncontrolled studies and 2 positive RCTs), autism (3 positive RCTs), smoking cessation (2 positive RCTs), graft-versus-host disease and intestinal permeability (1 positive RCT each). Current RCT evidence does not support the use of purified oral CBD in pain (at least as an acute analgesic) or for the treatment of COVID symptoms, cancer, Huntington’s or type 2 diabetes. In conclusion, published clinical evidence does support the use of purified CBD in multiple indications beyond epilepsy. However, the evidence base is limited by the number of trials only investigating the acute effects of CBD, testing CBD in healthy volunteers, or in very small patient numbers. Large confirmatory phase 3 trials are required in all indications.

Abstract A growing body of evidence links gut microbiota changes with inflammatory bowel disease (IBD), raising the potential benefit of exploiting metagenomics data for non-invasive IBD diagnostics. The sbv IMPROVER metagenomics diagnosis for inflammatory bowel disease challenge investigated computational metagenomics methods for discriminating IBD and nonIBD subjects. Participants in this challenge were given independent training and test metagenomics data from IBD and nonIBD subjects, which could be wither either raw read data (sub-challenge 1, SC1) or processed Taxonomy- and Function-based profiles (sub-challenge 2, SC2). A total of 81 anonymized submissions were received between September 2019 and March 2020. Most participants’ predictions performed better than random predictions in classifying IBD versus nonIBD, Ulcerative Colitis (UC) versus nonIBD, and Crohn’s Disease (CD) versus nonIBD. However, discrimination between UC and CD remains challenging, with the classification quality similar to the set of random predictions. We analyzed the class prediction accuracy, the metagenomics features by the teams, and computational methods used. These results will be openly shared with the scientific community to help advance IBD research and illustrate the application of a range of computational methodologies for effective metagenomic classification.

The resurgence of cannabis (Cannabis sativa L.) has been propelled by changes in the legal framework governing its cultivation and use, increased demand for hemp-derived products, and studies recognizing the industrial and health benefits of hemp. This has led to the creation of novel high-cannabidiol, low-Δ9-tetrahydrocannabinol varieties, enabling hemp crop expansion worldwide. This review elucidates the recent implications for hemp cultivation in Europe, with a focus on the legislative impacts on the cultivation practices, prospective breeding efforts, and dynamic scientific landscape surrounding this crop. We also review the current cultivars’ cannabinoid composition of the European hemp market and its major differences with that of the United States.

Abstract During the coronavirus disease (COVID-19) pandemic, wearing face masks in public spaces became mandatory in most countries. The risk of self-contamination when handling face masks, which was one of the earliest concerns, can be mitigated by adding antiviral coatings to the masks. In the present study, we evaluated the antiviral effectiveness of sodium chloride deposited on a fabric suitable for the manufacturing of reusable cloth masks using techniques adapted to the home environment. We tested eight coating conditions, involving both spraying and dipping methods and three salt dilutions. Influenza A H3N2 virus particles were incubated directly on the salt-coated materials, collected, and added to human 3D airway epithelial cultures. Live virus replication in the epithelia was quantified over time in collected apical washes. Relative to the non-coated material, salt deposits at or above 4.3 mg/cm2 markedly reduced viral replication. However, even for larger quantities of salt, the effectiveness of the coating remained dependent on the crystal size and distribution, which in turn depended on the coating technique. These findings confirm the suitability of salt coating as antiviral protection on cloth masks, but also emphasize that particular attention should be paid to the coating protocol when developing consumer solutions.

IntroductionAlpha-synuclein (α-Syn) aggregation, transmission, and contribution to neurotoxicity represent central mechanisms underlying Parkinson’s disease. The plant alkaloid “nicotine” was reported to attenuate α-Syn aggregation in different models, but its precise mode of action remains unclear.MethodsIn this study, we investigated the effect of 2-week chronic nicotine treatment on α-Syn aggregation, neuroinflammation, neurodegeneration, and motor deficits in D-line α-Syn transgenic mice. We also established a novel humanized neuronal model of α-Syn aggregation and toxicity based on treatment of dopaminergic neurons derived from human induced pluripotent stem cells (iPSC) with α-Syn preformed fibrils (PFF) and applied this model to investigate the effects of nicotine and other compounds and their modes of action.Results and discussionOverall, our results showed that nicotine attenuated α-Syn-provoked neuropathology in both models. Moreover, when investigating the role of nicotinic acetylcholine receptor (nAChR) signaling in nicotine’s neuroprotective effects in iPSC-derived dopaminergic neurons, we observed that while α4-specific antagonists reduced the nicotine-induced calcium response, α4 agonists (e.g., AZD1446 and anatabine) mediated similar neuroprotective responses against α-Syn PFF-provoked neurodegeneration. Our results show that nicotine attenuates α-Syn-provoked neuropathology in vivo and in a humanized neuronal model of synucleinopathy and that activation of α4β2 nicotinic receptors might mediate these neuroprotective effects.

Abstract Background Selection of optimal computational strategies for analyzing metagenomics data is a decisive step in determining the microbial composition of a sample, and this procedure is complex because of the numerous tools currently available. The aim of this research was to summarize the results of crowdsourced sbv IMPROVER Microbiomics Challenge designed to evaluate the performance of off-the-shelf metagenomics software as well as to investigate the robustness of these results by the extended post-challenge analysis. In total 21 off-the-shelf taxonomic metagenome profiling pipelines were benchmarked for their capacity to identify the microbiome composition at various taxon levels across 104 shotgun metagenomics datasets of bacterial genomes (representative of various microbiome samples) from public databases. Performance was determined by comparing predicted taxonomy profiles with the gold standard. Results Most taxonomic profilers performed homogeneously well at the phylum level but generated intermediate and heterogeneous scores at the genus and species levels, respectively. kmer-based pipelines using Kraken with and without Bracken or using CLARK-S performed best overall, but they exhibited lower precision than the two marker-gene-based methods MetaPhlAn and mOTU. Filtering out the 1% least abundance species—which were not reliably predicted—helped increase the performance of most profilers by increasing precision but at the cost of recall. However, the use of adaptive filtering thresholds determined from the sample’s Shannon index increased the performance of most kmer-based profilers while mitigating the tradeoff between precision and recall. Conclusions kmer-based metagenomic pipelines using Kraken/Bracken or CLARK-S performed most robustly across a large variety of microbiome datasets. Removing non-reliably predicted low-abundance species by using diversity-dependent adaptive filtering thresholds further enhanced the performance of these tools. This work demonstrates the applicability of computational pipelines for accurately determining taxonomic profiles in clinical and environmental contexts and exemplifies the power of crowdsourcing for unbiased evaluation.

The poor physicochemical properties of cannabidiol (CBD) hamper its clinical development. The aim of this review was to examine the literature to identify novel oral products and delivery strategies for CBD, while assessing their clinical implications and translatability. Evaluation of the published literature revealed that oral CBD strategies are primarily focused on lipid-based and emulsion solutions or encapsulations, which improve the overall pharmacokinetics (PK) of CBD. Some emulsion formulations demonstrate more rapid systemic delivery. Variability in the PK effects of different oral CBD products is apparent across species. Several novel administration routes exist for CBD delivery that may offer promise for specific indications. For example, intranasal administration and inhalation allow quick delivery of CBD to the plasma and the brain, whereas transdermal and transmucosal administration routes deliver CBD systemically more slowly. There are limited but promising data on novel delivery routes such as intramuscular and subcutaneous. Very limited data show that CBD is generally well distributed across tissues and that some CBD products enable increased delivery of CBD to different brain regions. However, evidence is limited regarding whether changes in CBD PK profiles and tissue distribution equate to superior therapeutic efficacy across indications and whether specific CBD products might be suited to particular indications.

The COVID-19 (Coronavirus disease 2019) global pandemic has upended the normal pace of society at multiple levels—from daily activities in personal and professional lives to the way the sciences operate. Many laboratories have reported shortage in vital supplies, change in standard operating protocols, suspension of operations because of social distancing and stay-at-home guidelines during the pandemic. This global crisis has opened opportunities to leverage internet of things, connectivity, and artificial intelligence (AI) to build a connected laboratory automation platform. However, laboratory operations involve complex, multicomponent systems. It is unrealistic to completely automate the entire diversity of laboratories and processes. Recently, AI technology, particularly, game simulation has made significant strides in modeling and learning complex, multicomponent systems. Here, we present a cloud-based laboratory management and automation platform which combines multilayer information on a simulation-driven inference engine to plan and optimize laboratory operations under various constraints of COVID-19 and risk scenarios. The platform was used to assess the execution of two cell-based assays with distinct parameters in a real-life high-content screening laboratory scenario. The results show that the platform can provide a systematic framework for assessing laboratory operation scenarios under different conditions, quantifying tradeoffs, and determining the performance impact of specific resources or constraints, thereby enabling decision-making in a cost-effective manner. We envisage the laboratory management and automation platform to be further expanded by connecting it with sensors, robotic equipment, and other components of scientific operations to provide an integrated, end-to-end platform for scientific laboratory automation.

Background: Halitosis is the general term used to describe any disagreeable odor in exhaled air, regardless of whether the odorous substances originate from oral or non-oral sources. Previous research has strongly associated tobacco smoking in the development of halitosis, as it increases the synthesis of toxic volatile sulfur compounds in diseased periodontal pockets. In this review, we summarize the etiopathology and epidemiology of halitosis as well as the current evidence on the impact of smoking by means of a meta-analysis. Methods: PubMed and Embase were searched to identify publications that reported halitosis in smokers and nonsmokers. Meta-analyses were performed if a sufficient number (n ≥ 3) of articles were available that evaluated the same outcome. Results: The meta-analyses showed that there was an increased risk of halitosis in current smokers versus nonsmokers (odds ratios). These results were consistent both in fixed and random effects models. Even though the interstudy heterogeneity was high (I2 = 91%), sensitivity analysis by limiting the number of studies yielded similar results, with no-to-moderate heterogeneity (I2 = 0–65%). The analysis comparing ever smokers with never smokers showed no significant difference in the risk of halitosis in ever smokers. The same effect was observed when upon stratifying the analyses on the basis of ascertainment of halitosis (self-reported or measured by a Halimeter). Conclusions: Halitosis is a common condition which can affect the quality of life of those affected. The results from this literature review and meta-analysis show that current smokers are more likely to suffer from halitosis, even if they are less likely to report it.

Multiple sclerosis (MS) is an inflammatory autoimmune disease associated with genetic and environmental factors. Cigarette smoking is harmful to health and may be one of the risk factors for MS. However, there have been no systematic investigations under controlled experimental conditions linking cigarette smoke (CS) and MS. The present study is the first inhalation study to correlate the pre-clinical and pathological manifestations affected by different doses of CS exposure in a mouse experimental autoimmune encephalomyelitis (EAE) model. Female C57BL/6 mice were whole-body exposed to either fresh air (sham) or three concentrations of CS from a reference cigarette (3R4F) for 2 weeks before and 4 weeks after EAE induction. The effects of exposure on body weight, clinical symptoms, spinal cord pathology, and serum biochemicals were then assessed. Exposure to low and medium concentrations of CS exacerbated the severity of symptoms and spinal cord pathology, while the high concentration had no effect relative to sham exposure in mice with EAE. Interestingly, the clinical chemistry parameters for metabolic profile as well as liver and renal function (e.g. triglycerides and creatinine levels, alkaline phosphatase activity) were lower in these mice than in naïve controls. Although the mouse EAE model does not fully recapitulate the pathology or symptoms of MS in humans, these findings largely corroborate previous epidemiological findings that exposure to CS can worsen the symptoms and pathology of MS. Furthermore, the study newly highlights the possible correlation of clinical chemistry findings such as metabolism and liver and renal function between MS patients and EAE mice.

Anatabine, an alkaloid present in plants of the Solanaceae family (including tobacco and eggplant), has been shown to ameliorate chronic inflammatory conditions in mouse models, such as Alzheimer’s disease, Hashimoto’s thyroiditis, multiple sclerosis, and intestinal inflammation. However, the mechanisms of action of anatabine remain unclear. To understand the impact of anatabine on cellular systems and identify the molecular pathways that are perturbed, we designed a study to examine the concentration-dependent effects of anatabine on various cell types by using a systems pharmacology approach. The resulting dataset, consisting of measurements of various omics data types at different time points, was analyzed by using multiple computational techniques. To identify concentration-dependent activated pathways, we performed linear modeling followed by gene set enrichment. To predict the functional partners of anatabine and the involved pathways, we harnessed the LINCS L1000 dataset’s wealth of information and implemented integer linear programming on directed graphs, respectively. Finally, we experimentally verified our key computational predictions. Using an appropriate luciferase reporter cell system, we were able to demonstrate that anatabine treatment results in NRF2 (nuclear factor-erythroid factor 2-related factor 2) translocation, and our systematic phosphoproteomic assays showed that anatabine treatment results in activation of MAPK signaling. While there are certain areas to be explored in deciphering the exact anti-inflammatory mechanisms of action of anatabine and other NRF2 activators, we believe that anatabine constitutes an interesting molecule for its therapeutic potential in NRF2-related diseases.

Abstract The extracellular matrix regulates cell survival, proliferation, and differentiation. In vitro two-dimensional cell experiments are typically performed on a plastic plate or a substrate of a single extracellular matrix constituent such as collagen or calcium phosphate. As these approaches do not include extracellular matrix proteins or growth factors, they fail to mimic a complex cell microenvironment. The cell-derived matrix is an alternative platform for better representing the in vivo microenvironment in vitro. Standard decellularization of a cell-derived matrix is achieved by combining chemical and physical methods. In this study, we compared the decellularization efficacy of several methods: ammonium hydroxide, sodium dodecyl sulfate (SDS), or Triton X-100 with cold or heat treatment on a matrix of Saos-2 cells. We found that the protocols containing SDS were cytotoxic during recellularization. Heat treatment at 47 °C was not cytotoxic, removed cellular constituents, inactivated alkaline phosphatase activity, and maintained the levels of calcium deposition. Subsequently, we investigated the differentiation efficiency of a direct bone coculture system in the established decellularized Saos-2 matrix, an inorganic matrix of calcium phosphate, and a plastic plate as a control. We found that the decellularized Saos-2 cell matrix obtained by heat treatment at 47 °C enhanced osteoclast differentiation and matrix mineralization better than the inorganic matrix and the control. This simple and low-cost method allows us to create a Saos-2 decellularized matrix that can be used as an in vivo-like support for the growth and differentiation of bone cells.

Abstract The molecular mechanisms of IBD have been the subject of intensive exploration. We, therefore, assembled the available information into a suite of causal biological network models, which offer comprehensive visualization of the processes underlying IBD. Scientific text was curated by using Biological Expression Language (BEL) and compiled with OpenBEL 3.0.0. Network properties were analysed by Cytoscape. Network perturbation amplitudes were computed to score the network models with transcriptomic data from public data repositories. The IBD network model suite consists of three independent models that represent signalling pathways that contribute to IBD. In the “intestinal permeability” model, programmed cell death factors were downregulated in CD and upregulated in UC. In the “inflammation” model, PPARG, IL6, and IFN-associated pathways were prominent regulatory factors in both diseases. In the “wound healing” model, factors promoting wound healing were upregulated in CD and downregulated in UC. Scoring of publicly available transcriptomic datasets onto these network models demonstrated that the IBD models capture the perturbation in each dataset accurately. The IBD network model suite can provide better mechanistic insights of the transcriptional changes in IBD and constitutes a valuable tool in personalized medicine to further understand individual drug responses in IBD.

Abstract The ongoing outbreak of coronavirus disease of 2019 (COVID-19), triggered by the rapid community spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has brought forward the need for speedy testing and manufacturing of respiratory face masks for global use. Overwhelmed by the fast-growing demand for disposable face masks, many governments have recommended the use of, and elaborated design criteria for, reusable face masks that are meant to slow down the transmission of SARS-CoV-2 in the general population. Using aerosol particle size spectrometers, we performed particle size distribution measurements and calculated the fractional aerosol particle size-selective filtration efficiencies of over 300 fabrics and fabric-assemblies, including chiffon, cotton, synthetics, and various promising layered combinations. We suggest, and experimentally verify, a simple way for estimating the aerosol filtration efficiency of layered fabrics. The analysis herein investigates the relationship between the breathability and filtration properties of fabrics, assemblies of fabrics, and commercial reusable masks from various regions around the globe. In addition, we demonstrate how a hydrophobic coating can provide a statistically significant increase in the fabrics’ filtration efficiency. The insights of this work are crucial to developing non-woven, high-filtration-performance, reusable face masks that can be worn for extended periods of time.

Abstract The SARS-CoV-2 pandemic has challenged researchers at a global scale. The scientific community’s massive response has resulted in a flood of experiments, analyses, hypotheses, and publications, especially in the field of drug repurposing. However, many of the proposed therapeutic compounds obtained from SARS-CoV-2 specific assays are not in agreement and thus demonstrate the need for a singular source of COVID-19 related information from which a rational selection of drug repurposing candidates can be made. In this paper, we present the COVID-19 PHARMACOME, a comprehensive drug-target-mechanism graph generated from a compilation of 10 separate disease maps and sources of experimental data focused on SARS-CoV-2/COVID-19 pathophysiology. By applying our systematic approach, we were able to predict the synergistic effect of specific drug pairs, such as Remdesivir and Thioguanosine or Nelfinavir and Raloxifene, on SARS-CoV-2 infection. Experimental validation of our results demonstrate that our graph can be used to not only explore the involved mechanistic pathways, but also to identify novel combinations of drug repurposing candidates.

Abstract Nicotinic acetylcholine receptors (nAChRs) play a critical role in the neuropharmacology of learning and memory. As such, naturally occurring alkaloids that regulate nAChR activity have gained interest for understanding and potentially improving memory function. In this study, we tested the acute effects of three known nicotinic alkaloids, nicotine, cotinine, and anatabine, in suppressing scopolamine-induced memory deficit in rodents by using two classic memory paradigms, Y-maze and novel object recognition (NOR) in mice and rats, respectively. We found that all compounds were able to suppress scopolamine-induced spatial memory deficit in the Y-maze spontaneous alternation paradigm. However, only nicotine was able to suppress the short-term object memory deficit in NOR, despite the higher doses of cotinine and anatabine used to account for their potential differences in nAChR activity. These results indicate that cotinine and anatabine can uniquely regulate short-term spatial memory, while nicotine seems to have more robust and general role in memory regulation in rodents. Thus, nAChR-activating alkaloids may possess distinct procognitive properties in rodents, depending on the memory types examined.

Abstract Background Inflammatory bowel disease (IBD) is the collective term for chronic immune-mediated diseases of unknown, multifactorial etiology, arising from the interplay between genetic and environmental factors and including two main disease manifestations: ulcerative colitis (UC) and Crohn’s disease. In the last few decades, naturally occurring alkaloids have gained interest because of their substantial anti-inflammatory effects in several animal models of disease. Studies on mouse models of IBD have demonstrated the anti-inflammatory action of the main tobacco alkaloid, nicotine. In addition, anatabine, a minor tobacco alkaloid also present in peppers, tomato, and eggplant presents anti-inflammatory properties in vivo and in vitro. In this study, we aimed to evaluate the anti-inflammatory properties of nicotine and anatabine in a dextran sulfate sodium (DSS) mouse model of UC. Results Oral administration of anatabine, but not nicotine, reduced the clinical symptoms of DSS-induced colitis. The result of gene expression analysis suggested that anatabine had a restorative effect on global DSS-induced gene expression profiles, while nicotine only had limited effects. Accordingly, MAP findings revealed that anatabine reduced the colonic abundance of DSS-associated cytokines and increased IL-10 abundance. Conclusions Our results support the amelioration of inflammatory effects by anatabine in the DSS mouse model of UC, and suggest that anatabine constitutes a promising therapeutic agent for IBD treatment.

In vitro models of the human lung play an essential role in evaluating the toxicity of inhaled compounds and understanding the development of respiratory diseases. Three-dimensional (3D) organotypic models derived from lung basal epithelial cells and grown at the air–liquid interface resemble human airway epithelium in multiple aspects, including morphology, cell composition, transcriptional profile, and xenobiotic metabolism. Whether the different characteristics of basal cell donors have an impact on model characteristics and responses remains unknown. In addition, studies are often conducted with 3D cultures from one donor, assuming a representative response on the population level. Whether this assumption is correct requires further investigation. In this study, we compared the morphology and functionality of 3D organotypic bronchial and small airway cultures from different donors at different weeks after air-lift to assess the interdonor variability in these parameters. The thickness, cell type composition, and transepithelial electrical resistance varied among the donors and over time after air-lift. Cilia beating frequency increased in response to isoproterenol treatment in both culture types, independent of the donor. The cultures presented low basal cytochrome P450 (CYP) 1A1/1B1 activity, but 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment induced CYP1A1/1B1 activity regardless of the donor. In conclusion, lung epithelial cultures prepared from different donors present diverse morphology but similar functionality and metabolic activity, with certain variability in their response to stimulation.

Abstract Background Lung aging is characterized by a number of structural alterations including fibrosis, chronic inflammation and the alteration of inflammatory cell composition. Chronic exposure to cigarette smoke (CS) is known to induce similar alterations and may contribute to premature lung aging. Additionally, aging and CS exposure are associated with transcriptional alterations in the lung. The current work aims to explore the interaction between age- and CS- associated transcriptomic perturbations and develop a transcriptomic clock able to predict the biological age and the impact of external factors on lung aging. Results Our investigations revealed a substantial overlap between transcriptomic response to CS exposure and age-related transcriptomic alterations in the murine lung. Of particular interest is the strong upregulation of immunoglobulin genes with increased age and in response to CS exposure, indicating an important implication of B-cells in lung inflammation associated with aging and smoking. Furthermore, we used a machine learning approach based on Lasso regression to build a transcriptomic age model that can accurately predict chronological age in untreated mice and the deviations associated with certain exposures. Interestingly, CS-exposed-mice were predicted to be prematurely aged in contrast to mice exposed to fresh air or to heated tobacco products (HTPs). The accelerated aging rate associated with CS was reversed upon smoking cessation or switching to HTPs. Additionally, our model was able to predict premature aging associated with thoracic irradiation from an independent public dataset. Conclusions Aging and CS exposure share common transcriptional alteration patterns in the murine lung. The massive upregulation of B-cell restricted genes during these processes shed light on the contribution of cell composition and particularly immune cells to the measured transcriptomic signal. Through machine learning approach, we show that gene expression changes can be used to accurately monitor the biological age and the modulations associated with certain exposures. Our findings also suggest that the premature lung aging is reversible upon the reduction of harmful exposures.

Many flavor ingredients are often used in potentially reduced-risk tobacco products (such as e-vapor products). Although most are “generally recognized as safe (GRAS)” when used in food, there is limited information available on their long-term health effects when delivered by inhalation. While obtaining route-of-exposure-specific toxicological data on flavor ingredients is critical to product evaluation, the large number of individual flavor ingredients available and their potential combinations render classical toxicological assessment approaches impractical, as they may require years of preclinical investigations and thousands of laboratory animals. Therefore, we propose a pragmatic approach in which flavor ingredients are initially assigned to groups of structurally related compounds (Flavor Groups), from which flavor group representatives (FGR) are then selected and tested individually and as a mixture in vitro and in vivo. The premise is that structurally related compounds would have comparable metabolic and biological activity and that the data generated using FGRs could support the toxicological assessment of other structurally related flavor ingredients of their respective Flavor Groups. This approach is explained in a step-wise manner and exemplified by a case study, along with its strengths, limitations as well as recommendations for further confirmatory testing. Once completed, this FGR approach could significantly reduce the time and resources required for filling the data gap in understanding the health risks of many flavor ingredients while also minimizing the need for laboratory animals.

Iota-carrageenan (IC) nasal spray, a medical device approved for treating respiratory viral infections, has previously been shown to inhibit the ability of a variety of respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), to enter and replicate in the cell by interfering with the virus binding to the cell surface. The aim of this study was to further investigate the efficacy and safety of IC in SARS-CoV-2 infection in advanced in vitro models of the human respiratory epithelium, the primary target and entry port for SARS-CoV-2. We extended the in vitro safety assessment of nebulized IC in a 3-dimensional model of reconstituted human bronchial epithelium, and we demonstrated the efficacy of IC in protecting reconstituted nasal epithelium against viral infection and replication of a patient-derived SARS-CoV-2 strain. The results obtained from these two advanced models of human respiratory tract epithelia confirm previous findings from in vitro SARS-CoV-2 infection assays and demonstrate that topically applied IC can effectively prevent SARS-CoV-2 infection and replication. Moreover, the absence of toxicity and functional and structural impairment of the mucociliary epithelium demonstrates that the nebulized IC is well tolerated.

Alkaloids that target nicotinic acetylcholine receptors (nAChR) are of great interest because of the critical role they play in mood and anxiety. However, understanding of the neuropharmacological effects of nicotinic alkaloids, such as cotinine and anatabine, is very limited. In this study, we investigated the neuropharmacological effects of three naturally occurring alkaloids—nicotine, cotinine, and anatabine—in vitro and in vivo. A single injection of nicotine induced anxiolytic-like behavioral features in mice by using the SmartCube® behavioral profiling system, while cotinine and anatabine had no detectable effect. The results were corroborated by using the zebrafish novel tank test (NTT), which showed a profound anxiolytic-like effect induced by multiple doses of nicotine after a single 20-min treatment. When the regulation of dopamine and norepinephrine release—the neurotransmitter systems relevant for anxiety—were examined in vitro, we found that nicotine stimulated the release of both norepinephrine and dopamine, while cotinine and anatabine mainly stimulated the dopamine release. The molecular targets of nicotine were confirmed to be nAChRs with its most potent activities against α4β2 and α6/3β2β3 subtypes in vitro. Anatabine was a weaker agonist for these receptors than nicotine. Cotinine was the least potent nAChR compound, only being able to activate α4β2 and α6/3β2β3 subtypes at high doses and no detectable activities against α3β4 and α7 subtypes at the concentrations tested. The observed effects were unlikely due to the off-target effect, because these alkaloids did not bind or regulate >160 other molecular targets in vitro. Thus, the present results suggest that natural nicotinic alkaloids can induce an anxiolytic-like behavior in nonclinical animal models, potency of which may depend on the activation of various nAChRs and regulation of various neurotransmitter systems. Further investigations would help understand their effects on humans, because non-clinical studies should not be taken as a direct indication for human behavior and nicotine is not risk free.

Halitosis is a health condition which counts cigarette smoking (CS) among its major risk factors. Cigarette smoke can cause an imbalance in the oral bacterial community, leading to several oral diseases and conditions, including intraoral halitosis. Although the best approach to decrease smoking-related health risks is quitting smoking, this is not feasible for many smokers. Switching to potentially reduced-risk products, like electronic vapor products (EVP) or heated tobacco products (HTP), may help improve the conditions associated with CS. To date, there have been few systematic studies on the effects of CS on halitosis and none have assessed the effects of EVP and HTP use. Self-assessment studies have shown large limitations owing to the lack of reliability in the participants' judgment. This has compelled the scientific community to develop a strategy for meaningful assessment of these new products in comparison with cigarettes. Here, we compiled a review of the existing literature on CS and halitosis and propose a 3-layer approach that combines the use of the most advanced breath analysis techniques and multi-omics analysis to define the interactions between oral bacterial species and their role in halitosis both in vitro and in vivo. Such an approach will allow us to compare the effects of different nicotine-delivery products on oral bacteria and quantify their impact on halitosis. Defining the impact of alternative nicotine-delivery products on intraoral halitosis and its associated bacteria will help the scientific community advance a step further toward understanding the safety of these products and their potentiall risks for consumers.

Cigarette smoking causes major preventable diseases, morbidity, and mortality worldwide. Smoking cessation and prevention of smoking initiation are the preferred means for reducing these risks. Less harmful tobacco products, termed modified-risk tobacco products (MRTP), are being developed as a potential alternative for current adult smokers who would otherwise continue smoking. According to a regulatory framework issued by the US Food and Drug Administration, a manufacturer must provide comprehensive scientific evidence that the product significantly reduces harm and the risk of tobacco-related diseases, in order to obtain marketing authorization for a new MRTP. For new tobacco products similar to an already approved predicate product, the FDA has foreseen a simplified procedure for assessing “substantial equivalence”. In this article, we present a use case that bridges the nonclinical evidence from previous studies demonstrating the relatively reduced harm potential of two heat-not-burn products based on different tobacco heating principles. The nonclinical evidence was collected along a “causal chain of events leading to disease” (CELSD) to systematically follow the consequences of reduced exposure to toxicants (relative to cigarette smoke) through increasing levels of biological complexity up to disease manifestation in animal models of human disease. This approach leverages the principles of systems biology and toxicology as a basis for further extrapolation to human studies. The experimental results demonstrate a similarly reduced impact of both products on apical and molecular endpoints, no novel effects not seen with cigarette smoke exposure, and an effect of switching from cigarettes to either MRTP that is comparable to that of complete smoking cessation. Ideally, a subset of representative assays from the presented sequence along the CELSD could be sufficient for predicting similarity or substantial equivalence in the nonclinical impact of novel products; this would require further validation, for which the present use case could serve as a starting point.

The expression of some microRNAs (miRNA) is modulated in response to cigarette smoke (CS), which is a leading cause of major preventable diseases. However, whether miRNA expression is also modulated by the aerosol/extract from potentially reduced-risk products is not well studied. The present work is a meta-analysis of 12 in vitro studies in human organotypic epithelial cultures of the aerodigestive tract (buccal, gingival, bronchial, nasal, and small airway epithelia). These studies compared the effects of exposure to aerosols from electronic vapor (e-vapor) products and heated tobacco products, and to extracts from Swedish snus products (in the present work, will be referred to as reduced-risk products [RRPs]) on miRNA expression with the effects of exposure to CS or its total particulate matter fraction. This meta-analysis evaluated 12 datasets of a total of 736 detected miRNAs and 2775 exposed culture inserts. The t-distributed stochastic neighbor embedding method was used to find similarities across the diversity of miRNA responses characterized by tissue type, exposure type, and product concentration. The CS-induced changes in miRNA expression in gingival cultures were close to those in buccal cultures; similarly, the alterations in miRNA expression in small airway, bronchial, and nasal tissues resembled each other. A supervised clustering was performed to identify miRNAs exhibiting particular response patterns. The analysis identified a set of miRNAs whose expression was altered in specific tissues upon exposure to CS (e.g., miR-125b-5p, miR-132-3p, miR-99a-5p, and 146a-5p). Finally, we investigated the impact of RRPs on miRNA expression in relation to that of CS by calculating the response ratio r between the RRP- and CS-induced alterations at an individual miRNA level, showing reduced alterations in miRNA expression following RRP exposure relative to CS exposure (94 % relative reduction). No specific miRNA response pattern indicating exposure to aerosols from heated tobacco products and e-vapor products, or extracts from Swedish snus was identifiable.

Cigarette smoke (CS) causes adverse health effects and, for smoker who do not quit, modified risk tobacco products (MRTPs) can be an alternative to reduce the risk of developing smoking-related diseases. Standard toxicological endpoints can lack sensitivity, with systems toxicology approaches yielding broader insights into toxicological mechanisms. In a 6-month systems toxicology study on ApoE−/− mice, we conducted an integrative multi-omics analysis to assess the effects of aerosols from the Carbon Heated Tobacco Product (CHTP) 1.2 and Tobacco Heating System (THS) 2.2—a potential and a candidate MRTP based on the heat-not-burn (HnB) principle—compared with CS at matched nicotine concentrations. Molecular exposure effects in the lungs were measured by mRNA/microRNA transcriptomics, proteomics, metabolomics, and lipidomics. Integrative data analysis included Multi-Omics Factor Analysis and multi-modality functional network interpretation. Across all five data modalities, CS exposure was associated with an increased inflammatory and oxidative stress response, and lipid/surfactant alterations. Upon HnB aerosol exposure these effects were much more limited or absent, with reversal of CS-induced effects upon cessation and switching to CHTP 1.2. Functional network analysis revealed CS-induced complex immunoregulatory interactions across the investigated molecular layers (e.g., itaconate, quinolinate, and miR-146) and highlighted the engagement of the heme–Hmox–bilirubin oxidative stress axis by CS. This work exemplifies how multi-omics approaches can be leveraged within systems toxicology studies and the generated multi-omics data set can facilitate the development of analysis methods and can yield further insights into the effects of toxicological exposures on the lung of mice.

The development of reduced-risk products aims to provide alternatives to cigarettes that present less risk of harm for adult smokers. Responsible use of flavoring substances in these products may fulfill an important role in product acceptance. While most flavoring substances used in such products are also used by the food industry and are considered safe when ingested, their impact when inhaled may require further assessment. To aid in such an assessment, a three-step approach combining real-time cellular analysis, phenotypic high-content screening assays, and gene expression analysis was developed and tested in normal human bronchial epithelial cells with 28 flavoring substances commonly used in e-liquid formulations, dissolved individually or as a mixture in a base solution composed of propylene glycol, vegetable glycerin, and 0.6% nicotine. By employing this approach, we identified individual flavoring substances that potentially contribute greatly to the overall mixture effect (citronellol and alpha-pinene). By assessing modified mixtures, we showed that, although cytotoxic effects were found when assessed individually, alpha-pinene did not contribute to the overall mixture cytotoxicity. Most of the cytotoxic effect appeared to be attributable to citronellol, with the remaining substances contributing due to synergistic effects. We developed and used different scoring methods (Tox-Score, Phenotypic Score, and Biological Impact Factor/Network Perturbation Amplitude), ultimately enabling a ranking based on cytotoxicity, phenotypic outcome, and molecular network perturbations. This case study highlights the benefits of testing both individual flavoring substances and mixtures for e-liquid flavor assessment and emphasized the importance of data sharing for the benefit of consumer safety.

Background: Cigarette smoking induces cytochrome P450 1A2 (CYP1A2) expression and activity, while smoking cessation normalizes the levels of this enzyme. The aim of this publication is to summarize the data on CYP1A2 gene expression and activity in preclinical and clinical studies on the Tobacco Heating System (THS), currently marketed as IQOS® with HEETs®, and to summarize the potential effects on CYP1A2 to be expected upon switching to reduced-risk products (RRPs). Methods: We summarized PMI’s preclinical and clinical data on the effects of switching from cigarette smoking to THS. Results: Data from four preclinical mouse and rat studies showed that, upon either cessation of cigarette smoke exposure or switching to THS exposure, the upregulation of CYP1A2 observed with exposure to cigarette smoke reverted close to fresh-air levels. Data from four clinical studies yielded similar results on CYP1A2 activity within a time frame of five days. Furthermore, the effects of switching to THS were similar to those seen after smoking cessation. Conclusions: Because smoking cessation and switching to either electronic cigarettes or THS seem to have similar effects on CYP1A2 activity, the same measures taken for patients treated with narrow therapeutic index drugs that are metabolized by CYP1A2 and who quit smoking should be recommended for those switching to RRPs.

Abstract Background High-throughput gene expression technologies provide complex datasets reflecting mechanisms perturbed in an experiment, typically in a treatment versus control design. Analysis of these information-rich data can be guided based on a priori knowledge, such as networks of related proteins or genes. Assessing the response of a specific mechanism and investigating its biological basis is extremely important in systems toxicology; as compounds or treatment need to be assessed with respect to a predefined set of key mechanisms that could lead to toxicity. Two-layer networks are suitable for this task, and a robust computational methodology specifically addressing those needs was previously published. The NPA package (https://github.com/philipmorrisintl/NPA) implements the algorithm, and a data package of eight two-layer networks representing key mechanisms, such as xenobiotic metabolism, apoptosis, or epithelial immune innate activation, is provided. Results Gene expression data from an animal study are analyzed using the package and its network models. The functionalities are implemented using R6 classes, making the use of the package seamless and intuitive. The various network responses are analyzed using the leading node analysis, and an overall perturbation, called the Biological Impact Factor, is computed. Conclusions The NPA package implements the published network perturbation amplitude methodology and provides a set of two-layer networks encoded in the Biological Expression Language.

There is an increasingly urgent call to shift industrial processes from fossil fuel feedstock to sustainable bio-based resources. This change becomes of high importance considering new budget requirements for a carbon-neutral economy. Such a transformation can be driven by traditionally used plants that are able to produce large amounts of valuable biologically relevant secondary metabolites. Tobacco plants can play a leading role in providing value-added products in remote areas of the world. In this study, we propose a non-exhaustive list of compounds with potential economic interest that can be sourced from the tobacco plant. In order to optimize extraction methodologies, we first analyzed their physico-chemical properties using rapid solubility tests and high-resolution microfractionation techniques. Next, to identify an optimal extraction for a selected list of compounds, we compared 13 different extraction method–solvent combinations. We proceeded with profiling some of these compounds in a total of six varieties from Nicotiana tabacum and Nicotiana rustica species, identifying the optimal variety for each. The estimated expected yields for each of these compounds demonstrate that tobacco plants can be a superior source of valuable compounds with diverse applications beyond nicotine. Among the most interesting results, we found high variability of anatabine content between species and varieties, ranging from 287 to 1699 µg/g. In addition, we found that CGA (1305 µg/g) and rutin (7910 µg/g) content are orders of magnitude lower in the Burley variety as compared to all others.

Cigarette smoking causes adverse health effects that might occur shortly after smoking initiation and lead to the development of inflammation and cardiorespiratory disease. Emerging studies have demonstrated the role of the intestinal microbiome in disease pathogenesis. The intestinal microbiome is susceptible to the influence of environmental factors such as smoking, and recent studies have indicated microbiome changes in smokers. Candidate modified risk tobacco products (CMRTP) are being developed to provide substitute products to lower smoking-related health risks in smokers who are unable or unwilling to quit. In this study, the ApoE–/– mouse model was used to investigate the impact of cigarette smoke (CS) from the reference cigarette 3R4F and aerosols from two CMRTPs based on the heat-not-burn principle [carbon-heated tobacco product 1.2 (CHTP 1.2) and tobacco heating system 2.2 (THS 2.2)] on the intestinal microbiome over a 6-month period. The effect of cessation or switching to CHTP 1.2 after 3 months of CS exposure was also assessed. Next-generation sequencing was used to evaluate the impact of CMRTP aerosols in comparison to CS on microbiome composition and gene expression in the digestive tract of mice. Our analyses highlighted significant gene dysregulation in response to 3R4F exposure at 4 and 6 months. The findings showed an increase in the abundance of Akkermansiaceae upon CS exposure, which was reversed upon cessation. Cessation resulted in a significant decrease in Akkemansiaceae abundance, whereas switching to CHTP 1.2 resulted in an increase in Lactobacillaceae abundance. These microbial changes could be important for understanding the effect of CS on gut function and its relevance to disease pathogenesis via the microbiome.

Adverse outcomes that result from chemical toxicity are rarely caused by dysregulation of individual proteins; rather, they are often caused by system-level perturbations in networks of molecular events. To fully understand the mechanisms of toxicity, it is necessary to recognize the interactions of molecules, pathways, and biological processes within these networks. The developing brain is a prime example of an extremely complex network, which makes developmental neurotoxicity one of the most challenging areas in toxicology. We have developed a systems toxicology method that uses a computable biological network to represent molecular interactions in the developing brain of zebrafish larvae. The network is curated from scientific literature and describes interactions between biological processes, signaling pathways, and adverse outcomes associated with neurotoxicity. This allows us to identify important signaling hubs, pathway interactions, and emergent adverse outcomes, providing a more complete understanding of neurotoxicity. Here, we describe the construction of a zebrafish developmental neurotoxicity network and its validation by integration with publicly available neurotoxicity-related transcriptomic datasets. Our network analysis identified consistent regulation of tumor suppressors p53 and retinoblastoma 1 (Rb1) as well as the oncogene Krüppel-like factor (Klf8) in response to chemically induced developmental neurotoxicity. The developed network can be used to interpret transcriptomic data in a neurotoxicological context.

Monoamine oxidases (MAO) are a valuable class of mitochondrial enzymes with a critical role in neuromodulation. In this study, we investigated the effect of natural MAO inhibitors on novel environment-induced anxiety by using the zebrafish novel tank test (NTT). Because zebrafish spend more time at the bottom of the tank when they are anxious, anxiolytic compounds increase the time zebrafish spend at the top of the tank and vice versa. Using this paradigm, we found that harmane, norharmane, and 1,2,3,4-tetrahydroisoquinoline (TIQ) induce anxiolytic-like effects in zebrafish, causing them to spend more time at the top of the test tank and less time at the bottom. 2,3,6-trimethyl-1,4-naphtoquinone (TMN) induced an interesting mix of both anxiolytic- and anxiogenic-like effects during the first and second halves of the test, respectively. TIQ was unique in having no observable effect on general movement. Similarly, a reference MAO inhibitor clorgyline—but not pargyline—increased the time spent at the top in a concentration-dependent manner. We also demonstrated that the brain bioavailability of these compounds are high based on the ex vivo bioavailability assay and in silico prediction models, which support the notion that the observed effects on anxiety-like behavior in zebrafish were most likely due to the direct effect of these compounds in the brain. This study is the first investigation to demonstrate the anxiolytic-like effects of MAO inhibitors on novel environment-induced anxiety in zebrafish.

Intestinal inflammation is the collective term for immune system-mediated diseases of unknown, multifactorial etiology, with often complex interactions between genetic and environmental factors. To mechanistically investigate the effect of treatment with compounds possessing immunomodulating properties in the context of intestinal inflammation, we developed an immunocompetent in vitro triculture intestinal model consisting of a differentiated intestinal epithelial layer (Caco-2/HT29-MTX) and immunocompetent cells (differentiated THP-1). The triculture mimicked a healthy intestine with stable barrier integrity. Lipopolysaccharide treatment triggered a controlled and reversible inflammatory state, resulting in significant impairment of barrier integrity and release of pro-inflammatory cytokines and chemokines, which are known hallmarks of intestinal inflammation. Treatment with known anti-inflammatory reference compounds (TPCA-1 and budenoside) prevented the induction of an inflammatory state; the decreasing triculture responses to this treatment measured by cytokine release, transepithelial electric resistance (TEER), and epithelial layer permeability proved the suitability of the intestinal model for anti-inflammatory drug screening. Finally, selected tobacco alkaloids (nicotine and anatabine (R/S and S forms)) were tested in the in vitro triculture for their potential anti-inflammatory properties. Indeed, naturally occurring alkaloids, such as tobacco-derived alkaloids, have shown substantial anti-inflammatory effects in several in vitro and in vivo models of inflammation, gaining increasing interest. Similar to the anti-inflammatory reference compounds, one of the tobacco alkaloids under investigation partially prevented the decrease in the TEER and increase in permeability and reduced the release of pro-inflammatory cytokines and chemokines. Taken together, these data confirm that our in vitro model is suitable for screening potential anti-inflammatory compounds in the context of intestinal inflammation.

There is a lack of physiologically relevant in vitro human kidney models for disease modelling and detecting drug-induced effects given the limited choice of cells and difficulty implementing quasi-physiological culture conditions. We investigated the influence of fluid shear stress on primary human renal proximal tubule epithelial cells (RPTECs) cultured in the micro-physiological Vitrofluid device. This system houses cells seeded on semipermeable membranes and can be connected to a regulable pump that enables controlled, unidirectional flow. After 7 days in culture, RPTECs maintained physiological characteristics such as barrier integrity, protein uptake ability, and expression of specific transporters (e.g., aquaporin-1). Exposure to constant apical side flow did not cause cytotoxicity, cell detachment, or intracellular reactive oxygen species accumulation. However, unidirectional flow profoundly affected cell morphology and led to primary cilia lengthening and alignment in the flow direction. The dynamic conditions also reduced cell proliferation, altered plasma membrane leakiness, increased cytokine secretion, and repressed histone deacetylase 6 and kidney injury molecule 1 expression. Cells under flow also remained susceptible to colistin-induced toxicity. Collectively, the results suggest that dynamic culture conditions in the Vitrofluid system promote a more differentiated phenotype in primary human RPTECs and represent an improved in vitro kidney model.

The adverse effects of cigarette smoking are well documented, and the two main strategies for reducing smoking prevalence are prevention of smoking initiation and promotion of smoking cessation. More recently, a third and complementary avenue, tobacco harm reduction has emerged, which is aimed to reduce the burden of smoking-related diseases. This has been enabled by the development of novel products such as electronic cigarettes (e-cigarettes) and heated tobacco products, designed to deliver nicotine with significantly reduced levels of the toxicants that are emitted by cigarettes. Several potential modified risk tobacco products (pMRTP) have been reported to emit significantly less toxicants than cigarettes and significantly reduce toxicant exposure in smokers who switch completely to such products. These are two prerequisites for pMRTPs to reduce harm and the risk of smoking-related disease. However, concerns remain regarding the addictive nature of these products. Smoking addiction is a complex phenomenon involving multiple pharmacological and non-pharmacological factors. Although the main pharmacological substance associated with smoking addiction is nicotine, accumulating evidence suggests that nicotine mostly acts as a primary reinforcer and that other factors are involved in establishing smoking addiction. Inhibition of monoamine oxidases (MAO)—mammalian flavoenzymes with a central role in neurotransmitter metabolism—has also been suggested to be involved in this process. Therefore, we aimed to comparatively investigate the ability of several types of pMRTPs and cigarette smoke (3R4F) to inhibit MAO activity. The results showed that the heated tobacco product Tobacco Heating System (THS) 2.2 and the MESH 1.1 e-cigarette possessed no MAO inhibitory activity while 3R4F significantly inhibits the levels of MAO activity (3R4F MAO-A and B; > 2 μM nicotine). Snus products have similar inhibition profiles as 3R4F but for larger nicotine concentrations (snus MAO-A; ∼68-fold, snus MAO-B; ∼23-fold higher compared to 3R4F). These observations were confirmed by analytical datasets of potential MAO inhibitors emitted by these products. In conclusion, we have demonstrated that specific pMRTPs, namely THS 2.2 and MESH 1.1, have a significantly lower MAO-inhibitory activity than 3R4F. These findings provide a basis for further investigation of the role of MAO inhibitors in cigarette addiction as well as the implications of the findings for abuse liability of pMRTPs in comparison with cigarettes. Keywords: Monoamine oxidase, Harm reduction, Tobacco heating system, E-cigarettes, Snus

As part of emerging tobacco harm reduction strategies, modified risk tobacco products (MRTP) are being developed to offer alternatives that have the potential to reduce the individual risk and population harm compared with smoking cigarettes for adult smokers who want to continue using tobacco and nicotine products. MRTPs are defined as any tobacco products that are distributed for use to reduce harm or the risk of tobacco-related disease associated with commercially marketed tobacco products. One such candidate MRTP is the Tobacco Heating System (THS) 2.2, which does not burn tobacco but instead heats it, thus producing significantly reduced levels of harmful and potentially harmful constituents compared with cigarettes. The clinical assessment of candidate MRTPs requires the development of exposure-response markers to distinguish current smokers from either nonsmokers or former smokers with high specificity and sensitivity. Toward this end, a whole blood-derived gene signature was previously developed and reported. Four randomized, controlled, open-label, three-arm parallel group reduced exposure clinical studies have been conducted with subjects randomized to three arms: switching from cigarettes to THS 2.2, continuous use of cigarettes, or smoking abstinence. These clinical studies had an investigational period of 5 days in confinement, which was followed by an 85-day ambulatory period in two studies. Here we tested the previously developed blood-derived signature on the samples derived from those clinical studies. We showed that in all four studies, the signature scores were reduced consistently in subjects who either stopped smoking or switched to THS 2.2 compared with subjects who continued smoking cigarettes.

Mucociliary clearance (MCC), considered as a collaboration of mucus secreted from goblet cells, the airway surface liquid layer, and the beating of cilia of ciliated cells, is the airways’ defense system against airborne contaminants. Because the process is well described at the molecular level, we gathered the available information into a suite of comprehensive causal biological network (CBN) models. The suite consists of three independent models that represent (1) cilium assembly, (2) ciliary beating, and (3) goblet cell hyperplasia/metaplasia and that were built in the Biological Expression Language, which is both human-readable and computable. The network analysis of highly connected nodes and pathways demonstrated that the relevant biology was captured in the MCC models. We also show the scoring of transcriptomic data onto these network models and demonstrate that the models capture the perturbation in each dataset accurately. This work is a continuation of our approach to use computational biological network models and mathematical algorithms that allow for the interpretation of high-throughput molecular datasets in the context of known biology. The MCC network model suite can be a valuable tool in personalized medicine to further understand heterogeneity and individual drug responses in complex respiratory diseases.

Chronic obstructive pulmonary disease (COPD) is a common inflammatory airway disease predominantly associated with cigarette smoking, and its incidence is increasing worldwide. According to the Global Initiative for Obstructive Lung Disease (GOLD) guidelines, spirometry is used to diagnose the disease. However, owing to its complexity, spirometry alone may not account for the multitude of COPD phenotypes or the early, asymptomatic lung damage seen in younger smokers. In addition, suitable biomarkers enabling early diagnosis, guiding treatment and estimating prognosis are still scarce, although large scale ‘omics analyses have added to the spectrum of potential biomarkers that could be used for these purposes. The aim of the current study was to comprehensively profile patients with mild-to-moderate COPD and compare the profiles to i) a group of currently smoking asymptomatic subjects, ii) a group of healthy former smokers, and iii) a group of healthy subjects that had never smoked. The assessment was conducted at the molecular level using proteomics, transcriptomics, and lipidomics and complemented by a series of measurements of traditional and emerging indicators of lung health (ClinicalTrials.gov identifier: NCT01780298). In this data note, we provide a comprehensive description of the study population’s physiological characteristics including full lung function, lung appearance on chest computed tomography, impulse oscillometry, and exercise tolerance and quality of life (QoL) measures.

The US FDA defines modified risk tobacco products (MRTPs) as products that aim to reduce harm or the risk of tobacco-related disease associated with commercially marketed tobacco products. Establishing a product’s potential as an MRTP requires scientific substantiation including toxicity studies and measures of disease risk relative to those of cigarette smoking. Best practices encourage verification of the data from such studies through sharing and open standards. Building on the experience gained from the OpenTox project, a proof-of-concept database and website (INTERVALS) has been developed to share results from both in vivo inhalation studies and in vitro studies conducted by Philip Morris International R&D to assess candidate MRTPs. As datasets are often generated by diverse methods and standards, they need to be traceable, curated, and the methods used well described so that knowledge can be gained using data science principles and tools. The data-management framework described here accounts for the latest standards of data sharing and research reproducibility. Curated data and methods descriptions have been prepared in ISA-Tab format and stored in a database accessible via a search portal on the INTERVALS website. The portal allows users to browse the data by study or mechanism (e.g., inflammation, oxidative stress) and obtain information relevant to study design, methods, and the most important results. Given the successful development of the initial infrastructure, the goal is to grow this initiative and establish a public repository for 21st-century preclinical systems toxicology MRTP assessment data and results that supports open data principles.

Chronic obstructive pulmonary disease (COPD) is one of the major causes of chronic morbidity and mortality worldwide. The development of markers of COPD onset is hampered by the lack of accessibility to the primary target tissue, and there is a need to consider other sample sources as surrogates for biomarker research. Airborne toxicants pass through the nasal epithelium before reaching the lower airways, and the similarity with bronchial histology makes it an attractive surrogate for lower airways. In this work, we describe the transcriptomics findings from the nasal epithelia of subjects enrolled in a clinical study focusing on the identification of COPD biomarkers. Transcriptomic data were analyzed using the biological network approach that enabled us to pinpoint the biological processes affected in the upper respiratory tract in response to smoking and mild-to-moderate COPD. Our results indicated that nasal and lower airway immune responses were considerably different in COPD subjects and caution should be exercised when using upper airway samples as a surrogate for the lower airway. Nevertheless, the network approach described here could present a sensitive means of identifying smokers at risk of developing COPD.

Current toxicology studies frequently lack measurements at molecular resolution to enable a more mechanism-based and predictive toxicological assessment. Recently, a systems toxicology assessment framework has been proposed, which combines conventional toxicological assessment strategies with system-wide measurement methods and computational analysis approaches from the field of systems biology. Proteomic measurements are an integral component of this integrative strategy because protein alterations closely mirror biological effects, such as biological stress responses or global tissue alterations. Here, we provide an overview of the technical foundations and highlight select applications of proteomics for systems toxicology studies. With a focus on mass spectrometry-based proteomics, we summarize the experimental methods for quantitative proteomics and describe the computational approaches used to derive biological/mechanistic insights from these datasets. To illustrate how proteomics has been successfully employed to address mechanistic questions in toxicology, we summarized several case studies. Overall, we provide the technical and conceptual foundation for the integration of proteomic measurements in a more comprehensive systems toxicology assessment framework. We conclude that, owing to the critical importance of protein-level measurements and recent technological advances, proteomics will be an integral part of integrative systems toxicology approaches in the future.