Dedifferentiation, transdifferentiation and reprogramming: three routes to regeneration.

The ultimate goal of regenerative medicine is to replace lost or damaged cells. This can potentially be accomplished using the processes of dedifferentiation, transdifferentiation or reprogramming. Recent advances have shown that the addition of a group of genes can not only restore pluripotency in a fully differentiated cell state (reprogramming) but can also induce the cell to proliferate (dedifferentiation) or even switch to another cell type (transdifferentiation). Current research aims to understand how these processes work and to eventually harness them for use in regenerative medicine.

Crowdsourced benchmarking of taxonomic metagenome profilers: lessons learned from the sbv IMPROVER Microbiomics challenge.

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.

Toxicological assessment of Tobacco Heating System 2.2: Findings from an independent peer review.

Offering safer alternatives to cigarettes, such as e-cigarettes and heated tobacco products, to smokers who are not willing to quit could reduce the harm caused by smoking. Extensive and rigorous scientific studies are conducted to assess the relative risk of such potentially modified risk tobacco products compared with that of smoking cigarettes. In addition to the peer review of publications reporting individual studies, we aimed to gauge the plausibility of the evidence to the scientific community and appreciate likely necessary additions prior to regulatory submission. Therefore, we sponsored a two-tier peer review organized by an independent third party who identified, recruited, and managed 7 panels of 5-12 experts whose identity remains unknown to us. The reviewers had access to all publications and raw data from preclinical and clinical studies via a web portal. The reviewers were asked questions regarding study design, methods, quality of data, and interpretation of results to judge the validity of the conclusions regarding the relative effects of the Tobacco Heating System 2.2 compared with cigarettes. Once their conclusions were submitted, the experts had the opportunity to participate in an anonymized online debate with their fellow panel members. We present here the results obtained from this innovative peer review effort which revealed supportive or very supportive of the study methods and results, and support the robustness of the studies and validity of the conclusions.

Embracing Transparency Through Data Sharing.

Low rates of reproducibility and translatability of data from nonclinical research have been reported. Major causes of irreproducibility include oversights in study design, failure to characterize reagents and protocols, a lack of access to detailed methods and data, and an absence of universally accepted and applied standards and guidelines. Specific areas of concern include uncharacterized antibodies and cell lines, the use of inappropriate sampling and testing protocols, a lack of transparency and access to raw data, and deficiencies in the translatability of findings to the clinic from studies using animal models of disease. All stakeholders-academia, industry, funding agencies, regulators, nonprofit entities, and publishers-are encouraged to play active roles in addressing these challenges by formulating and promoting access to best practices and standard operating procedures and validating data collaboratively at each step of the biomedical research life cycle.

Crowd-Sourced Verification of Computational Methods and Data in Systems Toxicology: A Case Study with a Heat-Not-Burn Candidate Modified Risk Tobacco Product.

Systems toxicology intends to quantify the effect of toxic molecules in biological systems and unravel their mechanisms of toxicity. The development of advanced computational methods is required for analyzing and integrating high throughput data generated for this purpose as well as for extrapolating predictive toxicological outcomes and risk estimates. To ensure the performance and reliability of the methods and verify conclusions from systems toxicology data analysis, it is important to conduct unbiased evaluations by independent third parties. As a case study, we report here the results of an independent verification of methods and data in systems toxicology by crowdsourcing. The sbv IMPROVER systems toxicology computational challenge aimed to evaluate computational methods for the development of blood-based gene expression signature classification models with the ability to predict smoking exposure status. Participants created/trained models on blood gene expression data sets including smokers/mice exposed to 3R4F (a reference cigarette) or noncurrent smokers/Sham (mice exposed to air). Participants applied their models on unseen data to predict whether subjects classify closer to smoke-exposed or nonsmoke exposed groups. The data sets also included data from subjects that had been exposed to potential modified risk tobacco products (MRTPs) or that had switched to a MRTP after exposure to conventional cigarette smoke. The scoring of anonymized participants' predictions was done using predefined metrics. The top 3 performers' methods predicted class labels with area under the precision recall scores above 0.9. Furthermore, although various computational approaches were used, the crowd's results confirmed our own data analysis outcomes with regards to the classification of MRTP-related samples. Mice exposed directly to a MRTP were classified closer to the Sham group. After switching to a MRTP, the confidence that subjects belonged to the smoke-exposed group decreased significantly. Smoking exposure gene signatures that contributed to the group separation included a core set of genes highly consistent across teams such as AHRR, LRRN3, SASH1, and P2RY6. In conclusion, crowdsourcing constitutes a pertinent approach, in complement to the classical peer review process, to independently and unbiasedly verify computational methods and data for risk assessment using systems toxicology.

Methods for making induced pluripotent stem cells: reprogramming à la carte.

Pluripotent stem-cell lines can be obtained through the reprogramming of somatic cells from different tissues and species by ectopic expression of defined factors. In theory, these cells--known as induced pluripotent stem cells (iPSCs)--are suitable for various purposes, including disease modelling, autologous cell therapy, drug or toxicity screening and basic research. Recent methodological improvements are increasing the ease and efficiency of reprogramming, and reducing the genomic modifications required to complete the process. However, depending on the downstream applications, certain technologies have advantages over others. Here, we provide a comprehensive overview of the existing reprogramming approaches with the aim of providing readers with a better understanding of the reprogramming process and a basis for selecting the most suitable method for basic or clinical applications.

Recapitulation of premature ageing with iPSCs from Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal human premature ageing disease, characterized by premature arteriosclerosis and degeneration of vascular smooth muscle cells (SMCs). HGPS is caused by a single point mutation in the lamin A (LMNA) gene, resulting in the generation of progerin, a truncated splicing mutant of lamin A. Accumulation of progerin leads to various ageing-associated nuclear defects including disorganization of nuclear lamina and loss of heterochromatin. Here we report the generation of induced pluripotent stem cells (iPSCs) from fibroblasts obtained from patients with HGPS. HGPS-iPSCs show absence of progerin, and more importantly, lack the nuclear envelope and epigenetic alterations normally associated with premature ageing. Upon differentiation of HGPS-iPSCs, progerin and its ageing-associated phenotypic consequences are restored. Specifically, directed differentiation of HGPS-iPSCs to SMCs leads to the appearance of premature senescence phenotypes associated with vascular ageing. Additionally, our studies identify DNA-dependent protein kinase catalytic subunit (DNAPKcs, also known as PRKDC) as a downstream target of progerin. The absence of nuclear DNAPK holoenzyme correlates with premature as well as physiological ageing. Because progerin also accumulates during physiological ageing, our results provide an in vitro iPSC-based model to study the pathogenesis of human premature and physiological vascular ageing.

The Apoe(-/-) mouse model: a suitable model to study cardiovascular and respiratory diseases in the context of cigarette smoke exposure and harm reduction.

Atherosclerosis-prone apolipoprotein E-deficient (Apoe(-/-)) mice display poor lipoprotein clearance with subsequent accumulation of cholesterol ester-enriched particles in the blood, which promote the development of atherosclerotic plaques. Therefore, the Apoe(-/-) mouse model is well established for the study of human atherosclerosis. The systemic proinflammatory status of Apoe(-/-) mice also makes them good candidates for studying chronic obstructive pulmonary disease, characterized by pulmonary inflammation, airway obstruction, and emphysema, and which shares several risk factors with cardiovascular diseases, including smoking. Herein, we review the results from published studies using Apoe(-/-) mice, with a particular focus on work conducted in the context of cigarette smoke inhalation studies. The findings from these studies highlight the suitability of this animal model for researching the effects of cigarette smoking on atherosclerosis and emphysema.

Effects of cigarette smoke, cessation and switching to a candidate modified risk tobacco product on the liver in Apoe -/- mice--a systems toxicology analysis.

The liver is one of the most important organs involved in elimination of xenobiotic and potentially toxic substances. Cigarette smoke (CS) contains more than 7000 chemicals, including those that exert biological effects and cause smoking-related diseases. Though CS is not directly hepatotoxic, a growing body of evidence suggests that it may exacerbate pre-existing chronic liver disease. In this study, we integrated toxicological endpoints with molecular measurements and computational analyses to investigate effects of exposures on the livers of Apoe(-/- )mice. Mice were exposed to 3R4F reference CS, to an aerosol from the Tobacco Heating System (THS) 2.2, a candidate modified risk tobacco product (MRTP) or to filtered air (Sham) for up to 8 months. THS2.2 takes advantage of a "heat-not-burn" technology that, by heating tobacco, avoids pyrogenesis and pyrosynthesis. After CS exposure for 2 months, some groups were either switched to the MRTP or filtered air. While no group showed clear signs of hepatotoxicity, integrative analysis of proteomics and transcriptomics data showed a CS-dependent impairment of specific biological networks. These networks included lipid and xenobiotic metabolism and iron homeostasis that likely contributed synergistically to exacerbating oxidative stress. In contrast, most proteomic and transcriptomic changes were lower in mice exposed to THS2.2 and in the cessation and switching groups compared to the CS group. Our findings elucidate the complex biological responses of the liver to CS exposure. Furthermore, they provide evidence that THS2.2 aerosol has reduced biological effects, as compared with CS, on the livers of Apoe(-/- )mice.

Evaluation of the Tobacco Heating System 2.2. Part 4: 90-day OECD 413 rat inhalation study with systems toxicology endpoints demonstrates reduced exposure effects compared with cigarette smoke.

The objective of the study was to characterize the toxicity from sub-chronic inhalation of test atmospheres from the candidate modified risk tobacco product (MRTP), Tobacco Heating System version 2.2 (THS2.2), and to compare it with that of the 3R4F reference cigarette. A 90-day nose-only inhalation study on Sprague-Dawley rats was performed, combining classical and systems toxicology approaches. Reduction in respiratory minute volume, degree of lung inflammation, and histopathological findings in the respiratory tract organs were significantly less pronounced in THS2.2-exposed groups compared with 3R4F-exposed groups. Transcriptomics data obtained from nasal epithelium and lung parenchyma showed concentration-dependent differential gene expression following 3R4F exposure that was less pronounced in the THS2.2-exposed groups. Molecular network analysis showed that inflammatory processes were the most affected by 3R4F, while the extent of THS2.2 impact was much lower. Most other toxicological endpoints evaluated did not show exposure-related effects. Where findings were observed, the effects were similar in 3R4F- and THS2.2-exposed animals. In summary, toxicological changes observed in the respiratory tract organs of THS2.2 aerosol-exposed rats were much less pronounced than in 3R4F-exposed rats while other toxicological endpoints either showed no exposure-related effects or were comparable to what was observed in the 3R4F-exposed rats.

Alterations in Serum Polyunsaturated Fatty Acids and Eicosanoids in Patients with Mild to Moderate Chronic Obstructive Pulmonary Disease (COPD).

Smoking is a major risk factor for several diseases including chronic obstructive pulmonary disease (COPD). To better understand the systemic effects of cigarette smoke exposure and mild to moderate COPD-and to support future biomarker development-we profiled the serum lipidomes of healthy smokers, smokers with mild to moderate COPD (GOLD stages 1 and 2), former smokers, and never-smokers (n = 40 per group) (ClinicalTrials.gov registration: NCT01780298). Serum lipidome profiling was conducted with untargeted and targeted mass spectrometry-based lipidomics. Guided by weighted lipid co-expression network analysis, we identified three main trends comparing smokers, especially those with COPD, with non-smokers: a general increase in glycero(phospho)lipids, including triglycerols; changes in fatty acid desaturation (decrease in ω-3 polyunsaturated fatty acids, and an increase in monounsaturated fatty acids); and an imbalance in eicosanoids (increase in 11,12- and 14,15-DHETs (dihydroxyeicosatrienoic acids), and a decrease in 9- and 13-HODEs (hydroxyoctadecadienoic acids)). The lipidome profiles supported classification of study subjects as smokers or non-smokers, but were not sufficient to distinguish between smokers with and without COPD. Overall, our study yielded further insights into the complex interplay between smoke exposure, lung disease, and systemic alterations in serum lipid profiles.

Transcriptional profiling and targeted proteomics reveals common molecular changes associated with cigarette smoke-induced lung emphysema development in five susceptible mouse strains.

BACKGROUND: Mouse models are useful for studying cigarette smoke (CS)-induced chronic pulmonary pathologies such as lung emphysema. To enhance translation of large-scale omics data from mechanistic studies into pathophysiological changes, we have developed computational tools based on reverse causal reasoning (RCR). OBJECTIVE: In the present study we applied a systems biology approach leveraging RCR to identify molecular mechanistic explanations of pathophysiological changes associated with CS-induced lung emphysema in susceptible mice. METHODS: The lung transcriptomes of five mouse models (C57BL/6, ApoE (-/-) , A/J, CD1, and Nrf2 (-/-) ) were analyzed following 5-7 months of CS exposure. RESULTS: We predicted 39 molecular changes mostly related to inflammatory processes including known key emphysema drivers such as NF-κB and TLR4 signaling, and increased levels of TNF-α, CSF2, and several interleukins. More importantly, RCR predicted potential molecular mechanisms that are less well-established, including increased transcriptional activity of PU.1, STAT1, C/EBP, FOXM1, YY1, and N-COR, and reduced protein abundance of ITGB6 and CFTR. We corroborated several predictions using targeted proteomic approaches, demonstrating increased abundance of CSF2, C/EBPα, C/EBPß, PU.1, BRCA1, and STAT1. CONCLUSION: These systems biology-derived candidate mechanisms common to susceptible mouse models may enhance understanding of CS-induced molecular processes underlying emphysema development in mice and their relevancy for human chronic obstructive pulmonary disease.

Toxicity of aerosols of nicotine and pyruvic acid (separate and combined) in Sprague-Dawley rats in a 28-day OECD 412 inhalation study and assessment of systems toxicology.

Toxicity of nebulized nicotine (Nic) and nicotine/pyruvic acid mixtures (Nic/Pyr) was characterized in a 28-day Organization for Economic Co-operation and Development 412 inhalation study with additional transcriptomic and lipidomic analyses. Sprague-Dawley rats were nose-only exposed, 6 h/day, 5 days/week to filtered air, saline, nicotine (50 µg/l), sodium pyruvate (NaPyr, 33.9 µg/l) or equimolar Nic/Pyr mixtures (18, 25 and 50 µg nicotine/l). Saline and NaPyr caused no health effects, but rats exposed to nicotine-containing aerosols had decreased body weight gains and concentration-dependent increases in liver weight. Blood neutrophil counts were increased and lymphocyte counts decreased in rats exposed to nicotine; activities of alkaline phosphatase and alanine aminotransferase were increased, and levels of cholesterol and glucose decreased. The only histopathologic finding in non-respiratory tract organs was increased liver vacuolation and glycogen content. Respiratory tract findings upon nicotine exposure (but also some phosphate-buffered saline aerosol effects) were observed only in the larynx and were limited to adaptive changes. Gene expression changes in the lung and liver were very weak. Nic and Nic/Pyr caused few significant changes (including Cyp1a1 gene upregulation). Changes were predominantly related to energy metabolism and fatty acid metabolism but did not indicate an obvious toxicity-related response. Nicotine exposure lowered plasma lipids, including cholesteryl ester (CE) and free cholesterol and, in the liver, phospholipids and sphingolipids. Nic, NaPyr and Nic/Pyr decreased hepatic triacylglycerol and CE. In the lung, Nic and Nic/Pyr increased CE levels. These data suggest that only minor biologic effects related to inhalation of Nic or Nic/Pyr aerosols were observed in this 28-day study.

Waves of early transcriptional activation and pluripotency program initiation during human preimplantation development.

The events regulating human preimplantation development are still largely unknown owing to a scarcity of material, ethical and legal limitations and a lack of reliable techniques to faithfully amplify the transcriptome of a single cell. Nonetheless, human embryology is gathering renewed interest due to its close relationship with both stem cell biology and epigenetic reprogramming to pluripotency and their importance in regenerative medicine. Carefully timed genome-wide transcript analyses of single oocytes and embryos uncovered a series of successive waves of embryonic transcriptional initiation that start as early as the 2-cell stage. In addition, we identified the hierarchical activation of genes involved in the regulation of pluripotency. Finally, we developed HumER, a database of human preimplantation gene expression, to serve the scientific community. Importantly, our work links early transcription in the human embryo with the correct execution of the pluripotency program later in development and paves the way for the identification of factors to improve epigenetic reprogramming.

Strengths and limitations of microarray-based phenotype prediction: lessons learned from the IMPROVER Diagnostic Signature Challenge.

MOTIVATION: After more than a decade since microarrays were used to predict phenotype of biological samples, real-life applications for disease screening and identification of patients who would best benefit from treatment are still emerging. The interest of the scientific community in identifying best approaches to develop such prediction models was reaffirmed in a competition style international collaboration called IMPROVER Diagnostic Signature Challenge whose results we describe herein. RESULTS: Fifty-four teams used public data to develop prediction models in four disease areas including multiple sclerosis, lung cancer, psoriasis and chronic obstructive pulmonary disease, and made predictions on blinded new data that we generated. Teams were scored using three metrics that captured various aspects of the quality of predictions, and best performers were awarded. This article presents the challenge results and introduces to the community the approaches of the best overall three performers, as well as an R package that implements the approach of the best overall team. The analyses of model performance data submitted in the challenge as well as additional simulations that we have performed revealed that (i) the quality of predictions depends more on the disease endpoint than on the particular approaches used in the challenge; (ii) the most important modeling factor (e.g. data preprocessing, feature selection and classifier type) is problem dependent; and (iii) for optimal results datasets and methods have to be carefully matched. Biomedical factors such as the disease severity and confidence in diagnostic were found to be associated with the misclassification rates across the different teams. AVAILABILITY: The lung cancer dataset is available from Gene Expression Omnibus (accession, GSE43580). The maPredictDSC R package implementing the approach of the best overall team is available at www.bioconductor.org or http://bioinformaticsprb.med.wayne.edu/.

A vascular biology network model focused on inflammatory processes to investigate atherogenesis and plaque instability.

BACKGROUND: Numerous inflammation-related pathways have been shown to play important roles in atherogenesis. Rapid and efficient assessment of the relative influence of each of those pathways is a challenge in the era of "omics" data generation. The aim of the present work was to develop a network model of inflammation-related molecular pathways underlying vascular disease to assess the degree of translatability of preclinical molecular data to the human clinical setting. METHODS: We constructed and evaluated the Vascular Inflammatory Processes Network (V-IPN), a model representing a collection of vascular processes modulated by inflammatory stimuli that lead to the development of atherosclerosis. RESULTS: Utilizing the V-IPN as a platform for biological discovery, we have identified key vascular processes and mechanisms captured by gene expression profiling data from four independent datasets from human endothelial cells (ECs) and human and murine intact vessels. Primary ECs in culture from multiple donors revealed a richer mapping of mechanisms identified by the V-IPN compared to an immortalized EC line. Furthermore, an evaluation of gene expression datasets from aortas of old ApoE-/- mice (78 weeks) and human coronary arteries with advanced atherosclerotic lesions identified significant commonalities in the two species, as well as several mechanisms specific to human arteries that are consistent with the development of unstable atherosclerotic plaques. CONCLUSIONS: We have generated a new biological network model of atherogenic processes that demonstrates the power of network analysis to advance integrative, systems biology-based knowledge of cross-species translatability, plaque development and potential mechanisms leading to plaque instability.

Results and lessons learned from the sbv IMPROVER metagenomics diagnostics for inflammatory bowel disease challenge.

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.

Direct membrane protein-DNA interactions required early in nuclear envelope assembly.

Among the earliest events in postmitotic nuclear envelope (NE) assembly are the interactions between chromatin and the membranes that will fuse to form the NE. It has been proposed that interactions between integral NE proteins and chromatin proteins mediate initial membrane recruitment to chromatin. We show that several transmembrane NE proteins bind to DNA directly and that NE membrane proteins as a class are enriched in long, basic domains that potentially bind DNA. Membrane fractions that are essential for NE formation are shown to bind directly to protein-free DNA, and our data suggest that these interactions are critical for early steps in NE assembly.

ASTD: The Alternative Splicing and Transcript Diversity database.

The Alternative Splicing and Transcript Diversity database (ASTD) gives access to a vast collection of alternative transcripts that integrate transcription initiation, polyadenylation and splicing variant data. Alternative transcripts are derived from the mapping of transcribed sequences to the complete human, mouse and rat genomes using an extension of the computational pipeline developed for the ASD (Alternative Splicing Database) and ATD (Alternative Transcript Diversity) databases, which are now superseded by ASTD. For the human genome, ASTD identifies splicing variants, transcription initiation variants and polyadenylation variants in 68%, 68% and 62% of the gene set, respectively, consistent with current estimates for transcription variation. Users can access ASTD through a variety of browsing and query tools, including expression state-based queries for the identification of tissue-specific isoforms. Participating laboratories have experimentally validated a subset of ASTD-predicted alternative splice forms and alternative polyadenylation forms that were not previously reported. The ASTD database can be accessed at http://www.ebi.ac.uk/astd.

Reduced Chronic Toxicity and Carcinogenicity in A/J Mice in Response to Life-Time Exposure to Aerosol From a Heated Tobacco Product Compared With Cigarette Smoke.

We conducted an inhalation study, in accordance with Organisation for Economic Co-operation and Development Test Guideline 453, exposing A/J mice to tobacco heating system (THS) 2.2 aerosol or 3R4F reference cigarette smoke (CS) for up to 18 months to evaluate chronic toxicity and carcinogenicity. All exposed mice showed lower thymus and spleen weight, blood lymphocyte counts, and serum lipid concentrations than sham mice, most likely because of stress and/or nicotine effects. Unlike THS 2.2 aerosol-exposed mice, CS-exposed mice showed increased heart weight, changes in red blood cell profiles and serum liver function parameters. Similarly, increased pulmonary inflammation, altered lung function, and emphysematous changes were observed only in CS-exposed mice. Histopathological changes in other respiratory tract organs were significantly lower in the THS 2.2 aerosol-exposed groups than in the CS-exposed group. Chronic exposure to THS 2.2 aerosol also did not increase the incidence or multiplicity of bronchioloalveolar adenomas or carcinomas relative to sham, whereas CS exposure did. Male THS 2.2 aerosol-exposed mice had a lower survival rate than sham mice, related to an increased incidence of urogenital issues that appears to be related to congenital factors rather than test item exposure. The lower impact of THS 2.2 aerosol exposure on tumor development and chronic toxicity is consistent with the significantly reduced levels of harmful and potentially harmful constituents in THS 2.2 aerosol relative to CS. The totality of the evidence from this study further supports the risk reduction potential of THS 2.2 for lung diseases in comparison with cigarettes.