Biology subjects

Lucas, J.

Publications and source records attributed to Lucas, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Patient-Centric Development of a Low-Volume, Low-Viscosity, Once Every 4 Weeks (Q4W) Autoinjector Combination Product for Povetacicept

Biologic therapies delivered subcutaneously (SC) are increasingly moving toward at-home self-administration using drug-device combination products. This shift places greater emphasis on the patient experience, and factors such as dose volume, injection time, dosing frequency, viscosity, injection force, and ease of use can affect treatment burden. This article focuses on the impact of balancing high protein concentration with low viscosity during drug product formulation development for an improved patient experience for povetacicept, an investigational, subcutaneously administered dual B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) inhibitor being developed for the treatment of multiple kidney diseases, including IgA nephropathy (IgAN). For povetacicept, formulation and device considerations were integrated throughout development. Studies evaluated protein concentration, dose volume, viscosity, injection force, injection time, and usability, in addition to standard stability, to develop a low-volume Q4W autoinjector. The selected 175 mg/mL formulation delivers an 80 mg dose in 0.46 mL. The viscosity was no more than 10 cP at 15-25{degrees}C and in a 1 mL prefilled syringe, the average glide force was 6 N, and the injection time at a constant force of 20 N was < 2 seconds. The same prefilled syringe was incorporated into an autoinjector with an injection time of less than 2 seconds in air. We conducted a limited assessment of an approved treatment for IgAN (Voyxact) administered via prefilled syringe (PFS) for effects of viscosity, fill volume and syringe configuration under the same experimental conditions. The viscosity at room temperature was 16 cP, with an average glide force of 27N and injection time of 19 seconds for the 2 mL dose. In conclusion, povetacicept was designed to support convenient and user-friendly at-home self-administration through Q4W dosing, a low injection volume, a low viscosity formulation, rapid delivery, and an easy-to-use autoinjector. This integrated formulation and device development approach demonstrates how patient-centric considerations can be incorporated in combination product development. Together, these attributes have the potential to reduce the practical burden associated with repeated, long-term treatment for IgAN patients.

bioengineering↗

bioRxiv2026

SEPALLATA MADS transcription factors act as key regulators in fertilization efficiency, ovule outer integument growth and mucilage secretory cell differentiation in Arabidopsis

In angiosperms, ovule development requires the activity of the C, D and E classes of MADS genes, which encode key transcriptional regulators of reproductive development. The SEPALLATA (SEP) MADS transcription factors (MTFs), which belong to the E class, act as organizing hubs of MADS heterotetrameric complexes and play an essential role in the development of flower organs. However, the role of the SEP genes in ovule and seed development has been difficult to determine due to redundancy in the subclade, the lack of observable phenotypes in single and double sep1 sep2 mutants and the homeotic conversion of the carpel into sepal or leaf in higher order sep mutants. Here, we engineered a version of SEP3 (SEP3{Delta}M) that encodes a protein lacking the DNA-binding MADS-domain but retains the oligomerization domains needed for MADS protein heterotetramerization. In vitro experiments demonstrated the ability of SEP3{Delta}M to interact with the C and D classes of MTF, reducing the capability of such MADS complex to efficiently bind DNA. sep3{Delta}M plants showed a delay in flower opening and organ maturation and a reduced fertility. The ovules exhibited reduced outer integument growth, and the few seeds that developed showed impaired mucilage secretion upon imbibition. RNA-seq analysis of sep3{Delta}M demonstrated misregulation of genes involved in outer integument and seed coat development. Taken together, these data indicate the key role of SEP3-containing MADS complexes in proper ovule outer integument growth and seed coat development.

plant biology↗

bioRxiv2026

ULTRAPETALA1 remodels PRC2 recruitment to nucleosomes

Polycomb Repressive Complex 2 (PRC2) establishes transcriptional repression through trimethylation of histone H3 lysine 27 (H3K27me3), a modification essential for developmental patterning. Here, we describe a novel plant-specific PRC2 variant (PRC2.3) that employs a distinct nucleosome-targeting mechanism mediated by accessory factor ULTRAPETALA1 (ULT1), which promotes H3K27me3 deposition at over 1,300 developmental genes. The cryo-EM structure of the PRC2SWN-ULT1-nucleosome complex reveals that ULT1 antagonizes the canonical PRC2 binding mode and instead bridges PRC2 to the nucleosome using its own interaction surfaces. ULT1 binds consecutive purines in the nucleosomal DNA and the histone H2A/H2B acidic patch, while enabling PRC2 to accommodate H3K36 modifications. We further show that in planta ULT1 enhances H3K27me3 at purine-rich loci and at genes associated with H3K36 marks, and identify the ULT1-H2A/H2B interface as required for reproductive transition and flower organogenesis. Our findings demonstrate that PRC2 can deploy mechanistically distinct recruitment strategies to control key developmental switches.

Molecular Biology↗

bioRxiv2026

KaryoScope: rapid, alignment-free sequence annotation for the pangenome era

The pangenome era is producing long-read sequencing data and complete genome assemblies (1-3) at a pace that current annotation methods cannot match. Existing tools were each built for a single feature class (repeats, centromeric satellites, or genes) and falter precisely where the genome is most variable and harbours clinically important variation: the centromeres, subtelomeres, and acrocentric short arms. Here we present KaryoScope, an alignment-free method to annotate an assembly at base-pair resolution across any desired feature classes in a single pass, completing in minutes on a standard workstation. Applied to the Human Pangenome Reference Consortium Release 2 assemblies (3), KaryoScope identifies the SST1 macrosatellite as the recurrent sequence at Robertsonian translocation fusion points (4, 5), delivers the first pangenome-wide census of D4Z4 macrosatellite structural diversity at the 4q and 10q subtelomeres relevant to facioscapulohumeral muscular dystrophy (6), and reveals previously uncharacterised centromere structural polymorphism, including chromosome-specific satellite loss and megabase-scale rearrangement validated by fluorescence in situ hybridization. A pre-built KaryoScope database for the human genome is distributed alongside the tool, and additional databases can be built for any reference genome or annotation source. Together, these capabilities bring the most variable regions of the genome within reach for comparative, clinical, and pangenome-scale analysis. KaryoScope is available at https://github.com/barthel-lab/KaryoScope.

bioinformatics↗

bioRxiv2026

Evaluating genome assemblies with HMM-Flagger

HMM-Flagger is a reference-free tool for detecting structural errors in haplotype-resolved genome assemblies based upon the coverage of mapped reads. It models read coverage with a hidden Markov model augmented by a Gaussian autoregressive process, which enables classifying coverage anomalies as erroneous blocks, false duplications, or collapsed blocks. Trained and tested on synthetic misassemblies, it detected synthetic errors using Pacific Biosciences HiFi and Oxford Nanopore Technologies R10 data with F1 scores of 78.4% and 60.4% respectively. When applied to six HG002 assemblies it revealed multiple large misassemblies including false duplications and collapse events in human satellites. Applied to assemblies from the Human Pangenome Reference Consortium (HPRC), HMM-Flagger demonstrated substantial improvements from release 1 (0.94% error rate) to release 2 (0.38%), reflecting technological advances. HMM-Flagger also validated NOTCH2NL assemblies in HPRC release 2 and confirmed the correctness of three novel structural configurations.

bioinformatics↗

bioRxiv2026

CASM potentiates STING-driven NFκB signaling in immune cells

Stimulator of Interferon Gene (STING), a key player of antimicrobial immune responses, has emerged as a promising target to mitigate inflammation and cancer. Following STING activation, proinflammatory molecules and type I Interferons (IFN) are released thus favoring the establishment of effective immune responses and adaptive immunity. Autophagy has been proposed to negatively regulate STING signaling. While STING activation drives microtubule-associated proteins 1A/1B light chain 3B (hereafter referred to as LC3) lipidation, the underlying mechanisms and functional consequences remain however incompletely defined. Especially, the consequences of STING-associated Conjugation of autophagy related (ATG) 8 to Single Membranes (CASM) in the control of immune responses remain elusive. Using innate and adaptive cells specifically inactivated for autophagy or CASM, we found that STING agonists primarily trigger CASM over autophagy. While STING-associated autophagy exerts negative feedback on the STING pathway and downstream type I IFN and pro-inflammatory responses, with different underlying molecular mechanisms between immune cells, STING-driven CASM potentiates NF{kappa}B-associated TNF production. These results overall uncover a new function of CASM and underscore the relevance of both CASM and autophagy in shaping STING signaling

cell biology↗

bioRxiv2025

Beyond binding motif: genomic context predicts transcription factor dependent regulation in Arabidopsis

Transcription Factors (TFs) play a crucial role in the spatiotemporal control of gene expression. Despite the presence of many potential TF binding sites (TFBS) across the genome, TFs do not interact with all of them, and, among those, only a subset leads to actual regulation. In this work, we investigate how the genomic context of a putative TFBS, including the occurrence of binding motifs for other TFs, can be used to predict effective transcriptional regulation by a TF of interest. We focus on LEAFY (LFY), a plant-specific TF and master regulator of flower development. Using available transcriptomes and TF-DNA binding experiments, we identify 1164 LFY binding sites associated with a regulatory response in Arabidopsis thaliana. We then apply a machine learning approach based on properties of the surrounding genomic region, to discriminate these regulatory LFY binding sites from non-regulatory ones. Detailed analysis of the models components reveals that, for LFY, the density and quality of binding sites constitute the most important features but were not sufficient on their own to predict regulatory activity. The presence of binding sites for other transcription factors and the overall richness in TFBS were also essential. These results clarify the nature of the regulatory code by which LFY operates and could serve as a basis for the study of the regulatory elements of other transcription factors. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/655699v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1a1a80org.highwire.dtl.DTLVardef@1c1fee5org.highwire.dtl.DTLVardef@1457164org.highwire.dtl.DTLVardef@176c46_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2025

Rsc1 DNA-binding bromodomain drives RSC activity at A-rich promoters

In yeast, the SWI/SNF chromatin remodeler RSC orchestrates nucleosome positioning at promoters, yet the molecular determinants guiding RSC to chromatin remain unclear. Here, we investigate the RSC subunit Rsc1 in Candida albicans and Saccharomyces cerevisiae. We identify a non-canonical role for its second bromodomain, Rsc1-BD2, which lacks a functional acetyllysine-binding pocket. Instead, Rsc1-BD2, together with the adjacent AT-hook and BAH domains, forms a conserved DNA-binding surface that is critical for RSC-dependent nucleosome remodeling and essential for viability in both species. Genome-wide motif analysis reveals that promoter regions are enriched in polyA sequences, whose distribution correlates with nucleosome positioning and gene expression. We show that Rsc1 preferentially binds polyA motifs and that nucleosome positioning at polyA-rich promoters depends on Rsc1 DNA binding module. These findings establish Rsc1 as a sequence-specific DNA-binding factor that mediates RSC stimulation by polyA motifs at yeast promoters. Overall, this study expands our understanding of the functional diversity of bromodomains and provides insights into the mechanisms governing RSC targeting to chromatin.

molecular biology↗

bioRxiv2024

Highly accurate assembly polishing with DeepPolisher

Accurate genome assemblies are essential for biological research, but even the highest quality assemblies retain errors caused by the technologies used to construct them. Base-level errors are typically fixed with an additional polishing step that uses reads aligned to the draft assembly to identify necessary edits. However, current methods struggle to find a balance between over- and under-polishing. Here, we present an encoder-only transformer model for assembly polishing called DeepPolisher, which predicts corrections to the underlying sequence using Pacbio HiFi read alignments to a diploid assembly. Our pipeline introduces a method, PHARAOH (Phasing Reads in Areas Of Homozygosity), which uses ultra-long ONT data to ensure alignments are accurately phased and to correctly introduce heterozygous edits in falsely homozygous regions. We demonstrate that the DeepPolisher pipeline can reduce assembly errors by half, with a greater than 70% reduction in indel errors. We have applied our DeepPolisher-based pipeline to 180 assemblies from the next Human Pangenome Reference Consortium (HPRC) data release, producing an average predicted Quality Value (QV) improvement of 3.4 (54% error reduction) for the majority of the genome.

bioinformatics↗

bioRxiv2024

LC3-associated phagocytosis is impaired in monocyte-derived macrophages from systemic sclerosis patients

Autophagy is a fundamental catabolic process performed by a network of autophagy related (ATG) proteins. Some ATG proteins coordinate parallel roles in so-called "noncanonical" autophagy such as LC3-associated phagocytosis (LAP). Both autophagy and LAP share key functions in immunity and inflammation and have been linked to autoimmune diseases. Systemic sclerosis (SSc) is an autoimmune disease of unknown etiology characterized by excessive fibrosis in skin and multiple internal organs linked with an aberrant immune activation. Several polymorphisms of genes coding for ATG proteins, particularly in ATG5, are more frequent in SSc patients. We hypothesized that autophagy and/or LAP could be dysregulated in immune cells from SSc patients. No defect of canonical autophagy was found in lymphocytes and monocytes isolated from peripheral blood mononuclear cells of SSc patients. We then generated monocyte-derived macrophages and performed phagocytosis assays to assess LAP activity. While M0 macrophage polarization appears similar than in healthy donors, we showed that LAP is downregulated in SSc patients. We now need to understand the molecular mechanisms underlying LAP dysregulations. Future investigations leading to the discovery of LAP modulating drugs could then open new therapeutic options for SSc treatment. Key messagesO_LIPolymorphisms of autophagy-related genes are associated with several autoimmune and autoinflammatory diseases, including SSc and SLE C_LIO_LIWhile autophagy has been shown to be dysregulated in circulating cells from SLE patients, no information is available for SSc C_LIO_LIWe show here that autophagy is comparable between PBMCs from patients and matched controls C_LIO_LIWe find a strong impartment of LAP, another ATG-dependent mechanism, in monocyte-derived macrophages from SSc patients C_LIO_LIAs LAP is involved in efferocytosis and the regulation of inflammation, we propose that restoring LAP activity could be a therapeutic option to limit fibrosis and inflammation C_LI

immunology↗

bioRxiv2024

Gapless assembly of complete human and plant chromosomes using only nanopore sequencing

The combination of ultra-long Oxford Nanopore (ONT) sequencing reads with long, accurate PacBio HiFi reads has enabled the completion of a human genome and spurred similar efforts to complete the genomes of many other species. However, this approach for complete, "telomere-to-telomere" genome assembly relies on multiple sequencing platforms, limiting its accessibility. ONT "Duplex" sequencing reads, where both strands of the DNA are read to improve quality, promise high per-base accuracy. To evaluate this new data type, we generated ONT Duplex data for three widely-studied genomes: human HG002, Solanum lycopersicum Heinz 1706 (tomato), and Zea mays B73 (maize). For the diploid, heterozygous HG002 genome, we also used "Pore-C chromatin contact mapping to completely phase the haplotypes. We found the accuracy of Duplex data to be similar to HiFi sequencing, but with read lengths tens of kilobases longer, and the Pore-C data to be compatible with existing diploid assembly algorithms. This combination of read length and accuracy enables the construction of a high-quality initial assembly, which can then be further resolved using the ultra-long reads, and finally phased into chromosome-scale haplotypes with Pore-C. The resulting assemblies have a base accuracy exceeding 99.999% (Q50) and near-perfect continuity, with most chromosomes assembled as single contigs. We conclude that ONT sequencing is a viable alternative to HiFi sequencing for de novo genome assembly, and has the potential to provide a single-instrument solution for the reconstruction of complete genomes.

bioinformatics↗

bioRxiv2024

A paternal cardiac lesion induces cardiac adaptation in offspring

BackgroundFollowing cardiac injury, whether the heart is permanently damaged or regenerating, distal organs are subjected to changes in physiological function. It remains largely unknown whether a cardiac lesion can affect gametes and transmit heritable changes to subsequent generations. Here, we report the influence of paternal cardiac injury on the following generation. MethodsWe studied the intergenerational influence of neonatal cardiac injury in the mouse, an animal model capable of regenerating the heart after early life injury. Neonatal male mice were subjected to ventricular cryoinjury, crossed at adulthood, and their sires were compared with litters derived from uninjured male mice. We used echocardiography, histology, and single nuclei RNA-sequencing to thoroughly characterize cardiac morphology, composition, function, and response to cardiac insult. ResultsWe show that paternal cardiac injury affects the heart morphology of offspring under physiological conditions. Furthermore, in response to the same injury, the F1 generation derived from injured fathers shows better systemic and cardiac recovery, with non-pathological left ventricular enlargement and improved cardiac function during the regenerative process. This is accompanied by the activation of the immune system healing program at 3 weeks post-injury, together with enhanced transcription of genes associated with physiological hypertrophy. ConclusionsThe memory of a paternal neonatal lesion can be transmitted to offspring and improve their recovery from a cardiac insult.

developmental biology↗

bioRxiv2023

The ALOG domain defines a new family of plant-specific transcription factors acting during Arabidopsis flower development

The ALOGs (Arabidopsis LIGHT-DEPENDENT SHORT HYPOCOTYLS 1 and Oryza G1) are Transcription Factors (TFs) from an evolutionarily conserved plant-specific family shown to play critical roles in meristem identity, inflorescence architecture and organ boundaries in diverse species from mosses to higher flowering plants. However, the DNA binding-specificity and molecular determinants of protein-DNA interactions of this family were uncharacterized. Using in vitro genome-wide studies, we identified the conserved DNA motif bound by ALOG proteins from the liverwort Marchantia polymorpha and the flowering plants Arabidopsis, tomato and rice. In order to determine the amino acids responsible for DNA-binding specificity, we solved the 2.1[A] structure of the ALOG DNA binding domain in complex with its cognate DNA. The ALOG DBD is an all-alpha helical domain with a structural zinc ribbon insertion and an N-terminal disordered NLS. The NLS sequence forms an integral part of the DNA binding domain and contributes to direct base read-out. To define the function of a group of redundant ALOG proteins in the model plant Arabidopsis thaliana, we generated a series of alog mutants and uncovered their participation in a gene regulatory network involving the other floral regulators LEAFY, BLADE-ON-PETIOLE and PUCHI, all active in defining boundary regions between reproductive meristems and repressing bracts development. Taken together, this work provides the biochemical and structural basis for DNA-binding specificity of an evolutionarily conserved TF family and reveals its role as a key player in defining organ boundaries in Arabidopsis. Significance StatementTranscription Factors (TFs) are key proteins that bind specific regions in the genome and regulate the expression of associated genes. Not all organisms possess the same set of TFs and some, like the ALOGs, are specific to the plant kingdom. These TFs have been shown to play important roles from mosses to flowering plants. However, it was not known what DNA motif they recognize and how they bind DNA. Here we identify this motif, we show it is widely conserved in evolution and we understand how this new type of DNA binding domain works at the structural level. In addition, we also show that several ALOG genes from Arabidopsis share a redundant function within the genetic network underlying correct floral meristem development.

plant biology↗

bioRxiv2023

SEPALLATA-driven MADS transcription factor tetramerization is required for inner whorl floral organ development

MADS genes encode transcription factors that act as master regulators of plant reproduction and flower development. The SEPALLATA (SEP) subfamily is required for the development of floral organs and plays roles in inflorescence architecture and development of the floral meristem. The SEPALLTAs act as organizers of MADS complexes, forming both heterodimers and heterotetramers in vitro. To date, the MADS complexes characterized in angiosperm floral organ development contain at least one SEPALLATA protein. Whether DNA-binding by SEPALLATA-containing dimeric MADS complexes are sufficient for launching floral organ identity programs, however, is not clear as only defects in floral meristem determinacy were observed in tetramerization impaired SEPALLATA mutants. Here, we used a combination of genome-wide binding studies, high resolution structural studies of the SEP3/AGAMOUS tetramerization domain, structure-based mutagenesis and complementation experiments in sep1 sep2 sep3 and sep1 sep2 sep3 ag-4 plants transformed with versions of SEP3 encoding tetramerization mutants. We demonstrate that while SEP3 heterodimers are able to bind DNA both in vitro and in vivo and recognize the majority of SEP3 wild type binding sites genome-wide, tetramerization is not only required for floral meristem determinacy, but also absolutely required for floral organ identity in the second, third and fourth whorls.

plant biology↗

bioRxiv2023

Phased nanopore assembly with Shasta and modular graph phasing with GFAse

As a step towards simplifying and reducing the cost of haplotype resolved de novo assembly, we describe new methods for accurately phasing nanopore data with the Shasta genome assembler and a modular tool for extending phasing to the chromosome scale called GFAse. We test using new variants of Oxford Nanopore Technologies (ONT) PromethION sequencing, including those using proximity ligation and show that newer, higher accuracy ONT reads substantially improve assembly quality.

bioinformatics↗

bioRxiv2022

Plant-TFClass: a structural classification for plant transcription factors

Transcription factors (TFs) bind DNA at specific sequences to regulate gene expression. This universal process is achieved thanks to the DNA-binding domain (DBD) present in each TF. DBDs show a vast diversity of protein folds within and across organisms, ranging from simple long basic alpha helices to complex structural combinations of alpha, beta and loop folds. In mammals, the structural conformation of the DBDs and the way it establishes contact with DNA has been used to organize TFs in a hierarchical classification named TFClass. However, such classification is missing from plants that possess many DBD types absent from mammals. Here, we reviewed the numerous TF DBD 3D-structures and models available for plants to organize all plant TFs types following the TFClass hierarchy (Superclass/Class/Family/Subfamily). We classified most of the 55 recognized plant TF types within the existing TFClass framework. This extended classification led us to add six new classes and 34 new families corresponding to TF DBD structures absent in mammals. Plant-TFClass provides a unique resource for TF and TF binding sites comparison across TF families and across organisms.

plant biology↗

bioRxiv2022

Increased mutation rate and interlocus gene conversion within human segmental duplications.

Single-nucleotide variants (SNVs) within segmental duplications (SDs) have not been systematically assessed because of the difficulty in mapping short-read sequence data to virtually identical repetitive sequences. Using 102 phased human haplotypes, we constructed 1:1 unambiguous alignments spanning high-identity SDs and compared the pattern of SNVs between unique and SD regions. We find that human SNVs are elevated 60% in SDs compared to unique regions. We estimate that at least 23% of this increase is due to interlocus gene conversion (IGC) with >7 Mbp of SD sequence converted on average per human haplotype. We develop a genome-wide map of IGC donors and acceptors, including 498 acceptor and 454 donor hotspots affecting the exons of ~800 protein-coding genes. The latter includes 171 genes that have "relocated" on average 1.61 Mbp in a subset of human haplotypes. Using a coalescent framework, we show that SD regions are evolutionarily older when compared to unique sequences with most of this signal originating from putative IGC loci. SNVs within SDs, however, also exhibit a distinct mutational spectrum where there is a 27.1% increase in transversions that convert cytosine to guanine or the reverse across all triplet contexts. In addition, we observe a 7.6% reduction in the frequency of CpG associated mutations when compared to unique DNA. We hypothesize that these distinct mutational properties help to maintain an overall higher GC content of SD DNA when compared to unique DNA, and we show that these GC-favoring mutational events are likely driven by GC-biased conversion between paralogous sequences.

genomics↗

bioRxiv2022

Flavor Classification/Categorization and Differential Toxicity of Oral Nicotine Pouches (ONPs) in Lung Epithelial Cells

The prevalence of flavored tobacco product usage amongst youth in the United States is partly due to the emergence of non-combustible nicotine-containing products (NCNPs), including oral nicotine pouches (ONPs) and smokeless tobacco products. ONPs are available in various different flavors (mint, fruity, tobacco, dessert, citrus, coffee, wintergreen, and berry) and may use either Tobacco-Derived Nicotine (TDN) or Tobacco-Free Nicotine (TFN). Currently, several brands of ONPs are sold in the U.S and comprise a significant portion of NCNP sales in the U.S. There is a growing concern that flavored ONPs may not only induce oral health effects, but may also induce systemic toxic effects due to nicotine and other ONP byproducts being absorbed into systemic circulation through the oral mucosa. These byproducts can act locally on other tissues and may potentially cause redox dysregulation and heightened inflammatory responses systemically in the respiratory, cardiovascular, and/or renal systems. Hence, we determined the effects of flavored ONPs from four of the most widely sold brands in the U.S in inducing toxicological effects on the respiratory epithelium. Prior to analyzing the effects ONPs, we first classified ONPs sold in the US based on their flavor and the flavor category to which they belong to using a wheel diagram. Subsequently, using human bronchial epithelial cells (16-HBE and BEAS-2B) exposed to extracts of flavored ONPs, we assessed the levels of ONP-induced inflammatory cytokine release (IL-6 and IL-8), cellular Reactive Oxygen Species (ROS) production, and cytotoxicity in the airway epithelium. Our data showed that cells exposed to the lowest concentration treatments showed increased cytotoxicity, differential cellular ROS production, and proinflammatory cytokine release. The most striking response was observed among cells treated with the spearmint ONP, whereas ONPs containing original tobacco and fruity flavors showed varied levels of ROS release in 16-HBE cells. Our data suggest that flavored ONPs are unsafe and likely to cause systemic and local toxicological responses during chronic usage. Our study is a part of ongoing efforts to use in vitro, ex-vivo, and in vivo systems to understand how the usage of various flavored ONPs may cause both oral and pulmonary toxicity, and impact human periodontal health.

pharmacology and toxicology↗