Therapeutic Spp1 silencing in TREM2+ cardiac macrophages suppresses atrial fibrillation.

Atrial fibrillation (AFib) and the risk of its lethal complications are propelled by fibrosis, which induces electrical heterogeneity and gives rise to reentry circuits. Atrial TREM2+ macrophages secrete osteopontin (encoded by Spp1), a matricellular signaling protein that engenders fibrosis and AFib. Here we show that silencing Spp1 in TREM2+ cardiac macrophages with an antibody-siRNA conjugate reduces atrial fibrosis and suppresses AFib in mice, thus offering a new immunotherapy for the most common arrhythmia.

A De Novo Strategy To Improve Pharmacokinetics of Proteins from mRNA Therapeutics via Zwitterionic Polypeptide Fusion.

Achieving therapeutic efficacy in protein replacement therapies requires sustaining pharmacokinetic (PK) profiles, while maintaining the bioactivity of circulating proteins. This is often achieved via PEGylation in protein-based therapies, but it remains challenging for proteins produced in vivo in mRNA-based therapies due to the lack of a suitable post-translational modification method. To address this issue, we integrated a genetically encoded zwitterionic polypeptide, EKP, into mRNA constructs to enhance the PK properties of product proteins. Composed of alternating glutamic acid (E), lysine (K), and proline (P), EKP exhibits unique superhydrophilic properties and low immunogenicity. Our results demonstrate that EKP fusion significantly extends the circulation half-life of proteins expressed from mRNA while preserving their bioactivity using human interferon alpha and Neoleukin-2/15 as examples. This EKP fusion technology offers a new approach to overcoming the current limitations in mRNA therapeutics and has the potential to significantly advance the development of mRNA-based protein replacement therapy.

In vitro to in vivo extrapolation from 3D hiPSC-derived cardiac microtissues and physiologically based pharmacokinetic modeling to inform next-generation arrhythmia risk assessment.

Proarrhythmic cardiotoxicity remains a substantial barrier to drug development as well as a major global health challenge. In vitro human pluripotent stem cell-based new approach methodologies have been increasingly proposed and employed as alternatives to existing in vitro and in vivo models that do not accurately recapitulate human cardiac electrophysiology or cardiotoxicity risk. In this study, we expanded the capacity of our previously established 3D human cardiac microtissue model to perform quantitative risk assessment by combining it with a physiologically based pharmacokinetic model, allowing a direct comparison of potentially harmful concentrations predicted in vitro to in vivo therapeutic levels. This approach enabled the measurement of concentration responses and margins of exposure for 2 physiologically relevant metrics of proarrhythmic risk (i.e. action potential duration and triangulation assessed by optical mapping) across concentrations spanning 3 orders of magnitude. The combination of both metrics enabled accurate proarrhythmic risk assessment of 4 compounds with a range of known proarrhythmic risk profiles (i.e. quinidine, cisapride, ranolazine, and verapamil) and demonstrated close agreement with their known clinical effects. Action potential triangulation was found to be a more sensitive metric for predicting proarrhythmic risk associated with the primary mechanism of concern for pharmaceutical-induced fatal ventricular arrhythmias, delayed cardiac repolarization due to inhibition of the rapid delayed rectifier potassium channel, or hERG channel. This study advances human-induced pluripotent stem cell-based 3D cardiac tissue models as new approach methodologies that enable in vitro proarrhythmic risk assessment with high precision of quantitative metrics for understanding clinically relevant cardiotoxicity.

Integrating gene expression and splicing dynamics across dose-response oxidative modulators.

Toxicological risk assessment increasingly utilizes transcriptomics to derive point of departure (POD) and modes of action (MOA) for chemicals. One essential biological process that allows a single gene to generate several different RNA isoforms is called alternative splicing. To comprehensively assess the role of splicing dysregulation in toxicological evaluation and elucidate its potential as a complementary endpoint, we performed RNA-seq on A549 cells treated with five oxidative stress modulators across a wide dose range. Differential gene expression (DGE) showed limited pathway enrichment except at high concentrations. However, alternative splicing analysis revealed variable intron retention events affecting diverse pathways for all chemicals in the absence of significant expression changes. For instance, diazinon elicited negligible gene expression changes but progressive increase in the number of intron retention events, suggesting splicing alterations precede expression responses. Benchmark dose modeling of intron retention data highlighted relevant pathways overlooked by expression analysis. Systematic integration of splicing datasets should be a useful addition to the toxicogenomic toolkit. Combining both modalities paint a more complete picture of transcriptomic dose-responses. Overall, evaluating intron retention dynamics afforded by toxicogenomics may provide biomarkers that can enhance chemical risk assessment and regulatory decision making. This work highlights splicing-aware toxicogenomics as a possible additional tool for examining cellular responses.

A Case of Non-Syndromic Congenital Cataracts Caused by a Novel MAF Variant in the C-Terminal DNA-Binding Domain-Case Report and Literature Review.

The MAF gene encodes a transcription factor in which pathogenic variants have been associated with both isolated and syndromic congenital cataracts. We aim to review the MAF variants in the C-terminal DNA-binding domain associated with non-syndromic congenital cataracts and describe a patient with a novel, disease-causing de novo missense variant. Published reports of C-terminal MAF variants and their associated congenital cataracts and ophthalmic findings were reviewed. The patient we present and his biological parents had genetic testing via a targeted gene panel followed by trio-based whole exome sequencing. A 4-year-old patient with a history of bilateral nuclear and cortical cataracts was found to have a novel, likely pathogenic de novo variant in MAF, NM_005360.5:c.922A>G (p.Lys308Glu). No syndromic findings or anterior segment abnormalities were identified. We report the novel missense variant, c.922A>G (p.Lys308Glu), in the C-terminal DNA-binding domain of MAF classified as likely pathogenic and associated with non-syndromic bilateral congenital cataracts.

Ocular manifestations of CHARGE syndrome in a pediatric cohort with genotype/phenotype analysis.

CHARGE syndrome is a rare multi-system condition associated with CHD7 variants. However, ocular manifestations and particularly ophthalmic genotype-phenotype associations, are not well-studied. This study evaluated ocular manifestations and genotype-phenotype associations in pediatric patients with CHARGE syndrome. A retrospective chart review included pediatric patients under 20 years-old with clinical diagnosis of CHARGE syndrome and documented ophthalmic examination. Demographics, genetic testing, and ocular findings were collected. Comprehensive literature review enhanced the genotype-phenotype analysis. Forty-two patients (20 male) underwent eye examination at an average age of 9.45 ± 6.52 years-old. Thirty-nine (93%) had ophthalmic manifestations in at least one eye. Optic nerve/chorioretinal colobomas were most common (38 patients), followed by microphthalmia (13), cataract (6), and iris colobomas (4). Extraocular findings included strabismus (32 patients), nasolacrimal duct obstructions (11, 5 with punctal agenesis), and cranial nerve VII palsy (10). Genotype-phenotype analyses (27 patients) showed variability in ocular phenotypes without association to location or variant types. Splicing (10 patients) and frameshift (10) variants were most prevalent. Patients with CHARGE syndrome may present with a myriad of ophthalmic manifestations. There is limited data regarding genotype-phenotype correlations and additional studies are needed.

Passivating the Background of Living Microbes with a Zwitterionic Peptide for Therapies.

Living microbial therapies have been proposed as a course of action for a variety of diseases. However, problematic interactions between the host immune system and the microbial organism present significant clinical concerns. Previously, we developed a genetically encoded superhydrophilic zwitterionic peptide, termed EKP, to mimic low-immunogenic zwitterionic materials, which have been used for the chemical modification of biologics such as protein and nucleic acid drugs to increase their in vivo circulation time and reduce their immunogenicity. Herein, we demonstrate the protective effects of the EKP polypeptide genetically cloaking the surface of Saccharomyces cerevisiae as a model microbe in both in vitro and in vivo systems. First, we show that EKP peptide cloaking suppresses the interactions between yeast cells and their specific antibodies, thereby illustrating its cloaking behavior. Then, we examine the in vitro interactions between EKP peptide surface cloaked yeast cells and murine macrophage cells, which exhibit phagocytotic behavior in the presence of foreign microbes. Our results indicate that EKP cloaking suppresses macrophage interactions and thus reduces phagocytosis. Furthermore, EKP cloaked yeast cells demonstrate a prolonged circulation time in mice in vivo.

Extracellular vesicles incorporating retrovirus-like capsids for the enhanced packaging and systemic delivery of mRNA into neurons.

The blood-brain barrier (BBB) restricts the systemic delivery of messenger RNAs (mRNAs) into diseased neurons. Although leucocyte-derived extracellular vesicles (EVs) can cross the BBB at inflammatory sites, it is difficult to efficiently load long mRNAs into the EVs and to enhance their neuronal uptake. Here we show that the packaging of mRNA into leucocyte-derived EVs and the endocytosis of the EVs by neurons can be enhanced by engineering leucocytes to produce EVs that incorporate retrovirus-like mRNA-packaging capsids. We transfected immortalized and primary bone-marrow-derived leucocytes with DNA or RNA encoding the capsid-forming activity-regulated cytoskeleton-associated (Arc) protein as well as capsid-stabilizing Arc 5'-untranslated-region RNA elements. These engineered EVs inherit endothelial adhesion molecules from donor leukocytes, recruit endogenous enveloping proteins to their surface, cross the BBB, and enter the neurons in neuro-inflammatory sites. Produced from self-derived donor leukocytes, the EVs are immunologically inert, and enhanced the neuronal uptake of the packaged mRNA in a mouse model of low-grade chronic neuro-inflammation.

Hidden hydrophobicity impacts polymer immunogenicity.

Antibodies against poly(ethylene glycol) (PEG) have been found to be the culprit of side reactions and efficacy loss of a number of PEGylated drugs. Fundamental mechanisms of PEG immunogenicity and design principles for PEG alternatives still have not been fully explored. By using hydrophobic interaction chromatography (HIC) under varied salt conditions, we reveal the "hidden" hydrophobicity of those polymers which are generally considered as hydrophilic. A correlation between the hidden hydrophobicity of a polymer and its polymer immunogenicity is observed when this polymer is conjugated with an immunogenic protein. Such a correlation of hidden hydrophobicity vs. immunogenicity for a polymer also applies to corresponding polymer-protein conjugates. Atomistic molecular dynamics (MD) simulation results show a similar trend. Based on polyzwitterion modification and with this HIC technique, we are able to produce extremely low-immunogenic protein conjugates as their hydrophilicity is pushed to the limit and their hydrophobicity is eliminated, breaking the current barriers of eliminating anti-drug and anti-polymer antibodies.

Beyond pharmaceuticals: Fit-for-purpose new approach methodologies for environmental cardiotoxicity testing.

Environmental factors play a substantial role in determining cardiovascular health, but data informing the risks presented by environmental toxicants is insufficient. In vitro new approach methodologies (NAMs) offer a promising approach with which to address the limitations of traditional in vivo and in vitro assays for assessing cardiotoxicity. Driven largely by the needs of pharmaceutical toxicity testing, considerable progress in developing NAMs for cardiotoxicity analysis has already been made. As the scientific and regulatory interest in NAMs for environmental chemicals continues to grow, a thorough understanding of the unique features of environmental cardiotoxicants and their associated cardiotoxicities is needed. Here, we review the key characteristics of as well as important regulatory and biological considerations for fit-for-purpose NAMs for environmental cardiotoxicity. By emphasizing the challenges and opportunities presented by NAMs for environmental cardiotoxicity we hope to accelerate their development, acceptance, and application.

Comparison of Rat Hepatocyte 2D-Monocultures and Hepatocytes Non-Parenchymal Cell Co-Cultures for Assessing Chemical Toxicity.

Liver responses are the most common endpoints used as the basis for setting exposure standards. Liver hepatocytes play a vital role in biotransformation of xenobiotics, but non-parenchymal cells (NPCs) in the liver are also involved in certain liver responses. Development of in vitro systems that more faithfully capture liver responses to reduce reliance on animals is a major focus of New Approach Methodology (NAMs). Since rodent regulatory studies are frequently the sole source safety assessment data, mode-of-action data, and used for risk assessments, in vitro rodent models that reflect in vivo responses need to be developed to reduce reliance on animal models. In the work presented in this paper, we developed a 2-D hepatocyte monoculture and 2-D liver cell co-culture system using rat liver cells. These models were assessed for conditions for short-term stability of the cultures and phenotypic and transcriptomic responses of 2 prototypic hepatotoxicants compounds - acetaminophen and phenobarbital. The optimized multi-cellular 2-D culture required use of freshly prepared hepatocytes and NPCs from a single rat, a 3:1 ratio of hepatocytes to NPCs and growth medium using 50% Complete Williams E medium (WEM) and 50% Endothelial Cell Medium (ECM). The transcriptomic responses of the 2 model systems to PB were compared to previous studies from TG-Gates on the gene expression changes in intact rats and the co-culture model responses were more representative of the in vivo responses. Transcriptomic read-outs promise to move beyond conventional phenotypic evaluations with these in vitro NAMs and provide insights about modes of action.

Motif-based zwitterionic peptides impact their structure and immunogenicity.

The linkage of zwitterionic peptides containing alternating glutamic acid (E) and lysine (K) amino acids exhibits protective effects on protein drugs due to their high hydration capacity. Previously, short EK peptides covering the surface of a protein drug showed significant protective effects and low immunogenicity. However, for high-molecular-weight single-chain (HMWSC) zwitterionic peptides, the incorporation of structure-disrupting amino acids such as proline (P), serine (S), and glycine (G) is necessary to improve their protective ability. Herein, we first probe the immunogenicity of eight EK-containing motif-based peptides, six of which incorporate structure-disrupting amino acids P, S, and G, linked to keyhole limpet hemocyanin (KLH). These studies uncover two sequence motifs, EKS and EKG, which show uniquely higher immunogenicity, while the other motifs, especially those containing P, exhibit lower immunogenicity. Additionally, the structure and dynamics of these sequence motifs are computationally modeled by Rosetta protein predictions and molecular dynamics (MD) simulations to predict properties of higher and lower immunogenicity peptides. These simulations revealed peptides with higher immunogenicity, namely EKS and EKG, exhibit regions of charge imbalance. Then, HMWSC zwitterionic sequences were linked to a typical protein drug, interferon-alpha 2a (IFN), which showed consistent immunogenic behaviors. Finally, epitope mapping and alanine scanning experiments using the serum collected from mice injected with HMWSC sequences also implicated a link between charge imbalance and peptide immunogenicity.

Estimating provisional margins of exposure for data-poor chemicals using high-throughput computational methods.

Current computational technologies hold promise for prioritizing the testing of the thousands of chemicals in commerce. Here, a case study is presented demonstrating comparative risk-prioritization approaches based on the ratio of surrogate hazard and exposure data, called margins of exposure (MoEs). Exposures were estimated using a U.S. EPA's ExpoCast predictive model (SEEM3) results and estimates of bioactivity were predicted using: 1) Oral equivalent doses (OEDs) derived from U.S. EPA's ToxCast high-throughput screening program, together with in vitro to in vivo extrapolation and 2) thresholds of toxicological concern (TTCs) determined using a structure-based decision-tree using the Toxtree open source software. To ground-truth these computational approaches, we compared the MoEs based on predicted noncancer TTC and OED values to those derived using the traditional method of deriving points of departure from no-observed adverse effect levels (NOAELs) from in vivo oral exposures in rodents. TTC-based MoEs were lower than NOAEL-based MoEs for 520 out of 522 (99.6%) compounds in this smaller overlapping dataset, but were relatively well correlated with the same (r 2 = 0.59). TTC-based MoEs were also lower than OED-based MoEs for 590 (83.2%) of the 709 evaluated chemicals, indicating that TTCs may serve as a conservative surrogate in the absence of chemical-specific experimental data. The TTC-based MoE prioritization process was then applied to over 45,000 curated environmental chemical structures as a proof-of-concept for high-throughput prioritization using TTC-based MoEs. This study demonstrates the utility of exploiting existing computational methods at the pre-assessment phase of a tiered risk-based approach to quickly, and conservatively, prioritize thousands of untested chemicals for further study.

NAM-based prediction of point-of-contact toxicity in the lung: A case example with 1,3-dichloropropene.

Time, cost, ethical, and regulatory considerations surrounding in vivo testing methods render them insufficient to meet existing and future chemical safety testing demands. There is a need for the development of in vitro and in silico alternatives to replace traditional in vivo methods for inhalation toxicity assessment. Exposures of differentiated airway epithelial cultures to gases or aerosols at the air-liquid interface (ALI) can assess tissue responses and in vitro to in vivo extrapolation can align in vitro exposure levels with in-life exposures expected to give similar tissue exposures. Because the airway epithelium varies along its length, with various regions composed of different cell types, we have introduced a known toxic vapor to five human-derived, differentiated, in vitro airway epithelial cell culture models-MucilAir of nasal, tracheal, or bronchial origin, SmallAir, and EpiAlveolar-representing five regions of the airway epithelium-nasal, tracheal, bronchial, bronchiolar, and alveolar. We have monitored toxicity in these cultures 24 h after acute exposure using an assay for transepithelial conductance (for epithelial barrier integrity) and the lactate dehydrogenase (LDH) release assay (for cytotoxicity). Our vapor of choice in these experiments was 1,3-dichloropropene (1,3-DCP). Finally, we have developed an airway dosimetry model for 1,3-DCP vapor to predict in vivo external exposure scenarios that would produce toxic local tissue concentrations as determined by in vitro experiments. Measured in vitro points of departure (PoDs) for all tested cell culture models were similar. Calculated rat equivalent inhaled concentrations varied by model according to position of the modeled tissue within the airway, with nasal respiratory tissue being the most proximal and most sensitive tissue, and alveolar epithelium being the most distal and least sensitive tissue. These predictions are qualitatively in accordance with empirically determined in vivo PoDs. The predicted PoD concentrations were close to, but slightly higher than, PoDs determined by in vivo subchronic studies.

Impacts of a Zwitterionic Peptide on its Fusion Protein.

Therapeutic proteins frequently need to be modified with high-molecular-weight molecules to improve their pharmacokinetic properties. The genetic linkage of therapeutic proteins to a high-molecular-weight zwitterionic peptide, termed EKP, offers a promising approach. As with any protein modification, EKP could impact the structural behavior and receptor binding properties of the linked therapeutic protein, thereby altering its bioactivity. To evaluate the effects of EKP on therapeutic proteins, we study the receptor binding properties of high-molecular-weight EKP linked to the growth colony-stimulating factor (GCSF) using the genetically based yeast display platform. We find that yeast-displayed EKP-GCSF and GCSF exhibits similar binding to its receptor GCSF-R, suggesting that EKP does not hinder receptor binding. Furthermore, yeast-displayed EKP-GCSF demonstrates protection against thermal denaturation compared to GCSF. Similarly, to study the structural effects of EKP on GCSF, we employ in silico modeling using alphaFold2 in conjunction with molecular dynamics (MD) simulations. Likewise, in silico modeling reveals that EKP does not alter the structural behavior of GCSF. Finally, we demonstrate the functional benefits of EKP, by which the EKP-GCSF fusion protein produced in Escherichia coli exhibits improved pharmacokinetics and prolonged bioactivity in vivo.

Considerations for Improving Metabolism Predictions for In Vitro to In Vivo Extrapolation.

High-throughput (HT) in vitro to in vivo extrapolation (IVIVE) is an integral component in new approach method (NAM)-based risk assessment paradigms, for rapidly translating in vitro toxicity assay results into the context of in vivo exposure. When coupled with rapid exposure predictions, HT-IVIVE supports the use of HT in vitro assays for risk-based chemical prioritization. However, the reliability of prioritization based on HT bioactivity data and HT-IVIVE can be limited as the domain of applicability of current HT-IVIVE is generally restricted to intrinsic clearance measured primarily in pharmaceutical compounds. Further, current approaches only consider parent chemical toxicity. These limitations occur because current state-of-the-art HT prediction tools for clearance and metabolite kinetics do not provide reliable data to support HT-IVIVE. This paper discusses current challenges in implementation of IVIVE for prioritization and risk assessment and recommends a path forward for addressing the most pressing needs and expanding the utility of IVIVE.

Biological system considerations for application of toxicogenomics in next-generation risk assessment and predictive toxicology.

There is increasing interest in using modern 'omics technologies, such as whole transcriptome sequencing, to inform decisions about human health safety and chemical toxicity hazard. High throughput methodologies using in vitro assays offer a path forward in reducing or eliminating animal testing. However, many aspects of these technologies need assessment before they will gain the trust of regulators and the public as viable alternative test methods for human health and safety. We used a high throughput whole transcriptome sequence assay (TempO-Seq) to assess the use of three widely used cancer cell lines (HepG2, MCF7, and Ishikawa cells) as in vitro systems for determination of cellular modes of action for two well studied compounds with canonical liver responses: ketoconazole and phenobarbital. We evaluated transcriptomic data to infer points of departure for use in risk analyses of compounds. Both compounds displayed shortcomings in evidence for canonical liver-related responses in any cell line, despite a strong dose response in all three. This raises questions about the competence of simple, mono-cultured cancer cell lines as appropriate surrogates for some adverse effects or toxic endpoints. Points of departure derived from benchmark doses were highly consistent across all three cell lines however, indicating the use of transcriptomic BMD analyses for such purposes would be a reliable and consistent approach.

Comparative transcriptomics and eQTL mapping of response to Melampsora americana in selected Salix purpurea F2 progeny.

BACKGROUND: Melampsora spp. rusts are the greatest pathogen threat to shrub willow (Salix spp.) bioenergy crops. Genetic resistance is key to limit the effects of these foliar diseases on host response and biomass yield, however, the genetic basis of host resistance has not been characterized. The addition of new genomic resources for Salix provides greater power to investigate the interaction between S. purpurea and M. americana, species commonly found in the Northeast US. Here, we utilize 3' RNA-seq to investigate host-pathogen interactions following controlled inoculations of M. americana on resistant and susceptible F2 S. purpurea genotypes identified in a recent QTL mapping study. Differential gene expression, network analysis, and eQTL mapping were used to contrast the response to inoculation and to identify associated candidate genes. RESULTS: Controlled inoculation in a replicated greenhouse study identified 19 and 105 differentially expressed genes between resistant and susceptible genotypes at 42 and 66 HPI, respectively. Defense response gene networks were activated in both resistant and susceptible genotypes and enriched for many of the same defense response genes, yet the hub genes of these common response modules showed greater mean expression among the resistant plants. Further, eight and six eQTL hotspots were identified at 42 and 66 HPI, respectively. The combined results of three analyses highlight 124 candidate genes in the host for further analysis while analysis of pathogen RNA showed differential expression of 22 genes, two of which are candidate pathogen effectors. CONCLUSIONS: We identified two differentially expressed M. americana transcripts and 124 S. purpurea genes that are good candidates for future studies to confirm their role in conferring resistance.

The Melampsora americana Population on Salix purpurea in the Great Lakes Region Is Highly Diverse with a Contributory Influence of Clonality.

Shrub willows (Salix spp.) are emerging as a viable lignocellulosic, second-generation bioenergy crop with many growth characteristics favorable for marginal lands in New York State and surrounding areas. Willow rust, caused by members of the genus Melampsora, is the most limiting disease of shrub willow in this region and remains extremely understudied. In this study, genetic diversity, genetic structure, and pathogen clonality were examined in Melampsora americana over two growing seasons via genotyping-by-sequencing to identify single-nucleotide polymorphism markers. In conjunction with this project, a reference genome of rust isolate R15-033-03 was generated to aid in variant discovery. Sampling between years allowed regional and site-specific investigation into population dynamics, in the context of both wild and cultivated hosts within high-density plantings. This work revealed that this pathogen is largely panmictic over the sampled areas, with few sites showing moderate genetic differentiation. These data support the hypothesis of sexual recombination between growing seasons because no genotype persisted across the two years of sampling. Additionally, clonality was determined as a driver of pathogen populations within cultivated fields and single shrubs; however, there is also evidence of high genetic diversity of rust isolates in all settings. This work provides a framework for M. americana population structure in the Great Lakes region, providing crucial information that can aid in future resistance breeding efforts.

RNA-sequencing (transcriptomic) data collected in liver and lung of male and female B6C3F1 mice exposed to various dose levels of 4-methylimidazole for 2, 5, or 28 days.

The National Toxicology Program (NTP) reported that chronic exposure to varying dietary concentrations of 4-methylimidazole (4-MeI) increased lung tumors in female and male mice [1]. In this study, mice (male and female B6C3F1 mice) were either administered 4-MeI by oral gavage (0, 50, 100, 200, or 300 mg/kg/day) for 2 days or exposed for 5 and 28 days to 4-MeI in the diet (0, 150, 300, 1250, or 2500 ppm) and whole transcriptome (RNA-Sequencing) data from 4-MeI-exposed B6C3F1 mice to determine whether changes occurred in the target (lung) and nontarget (liver) tissues. This analysis was conducted to provide information with which to evaluate biological processes affected by exposure to 4-MeI, with a focus on identifying key events that could be used to propose a plausible mode of action (MoA) for mouse lung tumors [2].