Prue Talbot

Previous work demonstrated that ectodermal cells exhibit greater susceptibility to SARS-CoV-2 than endodermal and mesodermal cells, raising concerns about potential vulnerability of the developing nervous system. We hypothesized that neural stem cells (NSCs) derived from human ectoderm are susceptible to SARS-CoV-2. Using pseudotyped viral particles representing both wild-type and Omicron spike variants, we confirmed efficient transduction in NSCs, with Omicron variants preferentially utilizing endocytosis-mediated entry. Inhibition of endocytosis with filipin and OcTMAB significantly reduced viral entry across all spike variants. Low glycosylation levels on NSCs facilitated viral entry, and enzymatic removal of glycosylation increased their susceptibility. Ectodermal infection by SARS-CoV-2 raises serious concern for potential teratogenic effects on the nervous system, possibly causing latent or subclinical anomalies not immediately evident at birth. Therefore, future clinical studies and long-term surveillance of infants and children exposed in utero are necessary to investigate and identify potential neurodevelopmental deficits.

STUDY QUESTION: Does vanillin, at concentrations relevant to maternal exposures during vaping, disrupt gastrulation-related processes in human embryonic stem cells (hESCs) by activating TRPV4 channels? SUMMARY ANSWER: Activation of TRPV4 channels by nanomolar concentrations of vanillin promoted an exit from pluripotency and a shift toward endodermal gene expression, while micromolar concentrations induced developmental toxicity, identifying a concentration-dependent mechanism by which vanillin, a flavoring agent in electronic cigarette (EC) aerosols, may pose risks to embryonic development during pregnancy. WHAT IS KNOWN ALREADY: The use of ECs during pregnancy is increasing, potentially driven by perceptions of reduced harm and the appeal of flavoring agents, such as vanillin. However, vanillin activates transient receptor potential (TRP) channels, raising concerns about its impact on early embryonic development. STUDY DESIGN, SIZE, DURATION: hESCs, which model the epiblast stage of development, were treated in vitro for various times with vanillin, a TRPV4 antagonist, a TRPV4 function-blocking antibody, or vanillin combined with either the antagonist or antibody. All experiments were done three times with different passages of stem cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: After variable periods of exposure, the hESC colonies were evaluated for activation of TRPV4 channels and calcium influx, colony growth, colony detachment, increased cell death, mitochondrial dysfunction, colony morphology, gap formation, downregulation of EpCAM, loss of pluripotency, and initiation of differentiation. MAIN RESULTS AND THE ROLE OF CHANCE: We demonstrate that vanillin, at concentrations relevant to maternal EC exposure, significantly (P < 0.05) disrupted gastrulation-related processes in hESCs. hESCs, which model the epiblast during Weeks 2–3 of human development, exhibited TRPV4 channel expression and showed a significant increase in intracellular calcium upon nanomolar-to-micromolar vanillin exposure, as detected by the Fluo-8 calcium dye. Time-lapse imaging and quantitative analysis indicated that micromolar vanillin impaired colony expansion, induced colony detachment, and increased cell death over 72 h, while concurrently reducing mitochondrial reductase activity in the MTT assay. At nanomolar concentrations, vanillin promoted TRPV4 activation, an exit from pluripotency, downregulation of EpCAM cell adhesion protein, intercellular gap formation, and a shift toward endodermal gene expression. All effects were blocked by a TRPV4 antagonist (HC067047), identifying activation of this channel as a critical mediator of vanillin-induced toxicity. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Our data are derived from in vitro experiments, as direct investigation of early human development is ethically and practically limited. Although our findings do not establish embryo lethality or malformation in vivo, the observed loss of pluripotency and SOX17 upregulation, consistent with endodermal induction, at nanomolar concentrations of vanillin are signs of early cellular perturbations that could compromise human development. WIDER IMPLICATIONS OF THE FINDINGS: Nanomolar vanillin concentrations, which our exposure model estimates reach the embryo during early pregnancy in EC users, were sufficient to activate TRPV4 channels in hESCs and alter germ layer specification. Micromolar concentrations produced cytotoxic effects and increased cell death. These findings indicate that vanillin exposure at levels attainable during EC use disrupts intracellular Ca2+ homeostasis and compromises core processes of early human development. Maternal EC use during pregnancy therefore represents a significant and previously under-appreciated risk to early embryogenesis. Clinicians should counsel patients who are pregnant or planning pregnancy about this potential harm, and regulatory agencies should consider mandatory ingredient disclosure on EC packaging. FUNDING: This research was supported by grant number T32IR4848 from the Tobacco-Related Disease Research Program (TRDRP), by grant number EDUC4-12752 from the California Institute of Regenerative Medicine (CIRM), and by UCR Yvonne Danielson Endowed Graduate and Dissertation Completion Fellowship Awards. The content is solely the responsibility of the authors and does not necessarily represent the official view of the TRDRP, CIRM, or UCR. DISCLOSURES: The authors have no conflicts of interest to declare.

Despite concerns regarding the potential health risks associated with EC use, their design, puff count, and e-liquid formulations have continued to evolve. Aldehydes are a key class of thermal degradation byproducts generated during the vaporization of EC fluids. This study tested the hypothesis that aldehyde concentrations would be elevated in used ECs with high puff numbers. Devices from multiple brands were analyzed to assess the impact of extended use on aldehyde formation. E-fluids were extracted from field-collected and user-donated devices and analyzed using gas chromatography/mass spectrometry to quantify 178 flavor chemicals, two synthetic coolants, nicotine, solvents, and aldehydes (methylglyoxal (MGO), glyoxal (GO), formaldehyde, acetaldehyde, acrolein, glyceraldehyde, dihydroxyacetone, propanal, and 5-hydroxymethylfurfural). Pulmonary cells were exposed to MGO and acetaldehyde in submerged cultures and at the air–liquid interface, and their toxic effects were analyzed in multiple assays. E-fluid consumption averaged 77% in ElfBar, 58% in FLUM, and 42% in “Other Brands”, with chemical profiles varying significantly across brands and collection groups. Total flavor chemical concentrations averaged 34 mg/mL ± 21.6 for ElfBar and 9.7 mg/mL ± 7.9 for FLUM, and these concentrations were significantly reduced in vaped fluids. MGO, GO, and formaldehyde concentrations increased significantly in vaped fluids, while acetaldehyde and acrolein both decreased. Glyceraldehyde and dihydroxyacetone were detected only in vaped fluids and were generally elevated in ElfBar compared to FLUM products. MGO and acetaldehyde significantly disrupted the morphology of BEAS-2B cells and increased superoxide production in a concentration-dependent manner. These findings underscore the potential for increases in fluid aldehyde concentration with high-puff number ECs and raise concerns about the inhalation of cytotoxic byproducts generated during puffing. Regulatory policies should consider the influence of puff count on aldehyde generation.

Introduction: The early stages of human embryonic development are challenging to study in pregnant women. Methods: A “disease-in-a-dish” model was utilized to investigate SARS-CoV-2 infection of human embryonic stem cells and the three germ layers (ectoderm, endoderm, and mesoderm). Results: Ectodermal cells showed significantly higher infection rates compared to the other cell types. This increased susceptibility was attributed to three key factors characteristic of the ectoderm: dual viral entry pathways (membrane fusion and endocytosis), elevated TMPRSS2 activity, and a markedly reduced glycocalyx, which facilitated viral access to host cell receptors. Discussion: Our findings provide strong evidence that cells in early post-implantation human embryos are susceptible to SARS-CoV-2 infection. The high level of infection in the ectodermal cells raises concern for potential teratogenic effects, particularly involving the nervous system. Future clinical studies should investigate neurological outcomes in infants born to mothers infected with SARS-CoV-2 during pregnancy.

Hydroxyacetone was previously detected at high concentrations (up to ∼12 mg/mL) in electronic cigarette (EC) aerosols, including those derived from products associated with adverse health effects. Given the limited understanding of its inhalation toxicology, we investigated hydroxyacetone's impact on human airway epithelial cells. Acute exposures at the air-liquid interface (ALI) using 3D EpiAirway tissues─a surrogate for human tracheobronchial epithelium─were analyzed via proteomics. Differential expression analysis identified numerous affected proteins, with enrichment pointing to alterations in mitochondrial function and actin cytoskeletal disruption as major targets. Ingenuity Pathway Analysis (IPA) highlighted "Mitochondrial Dysfunction" and "NRF2-Mediated Oxidative Stress" among top toxicological categories, and "Nuclear Cytoskeletal Signaling" as a key canonical pathway. To validate and extend these findings, submerged cultures of BEAS-2B cells were exposed to hydroxyacetone (0.01-10 mg/mL) and assessed for mitochondrial activity, oxidative stress, and F-actin integrity. At 1 mg/mL, mitochondrial membrane potential and reactive oxygen species (ROS) increased, with elevated hydrogen peroxide detected in the culture medium. At 10 mg/mL, mitochondrial activity declined significantly, accompanied by cell rounding and apoptotic blebbing within 2 h. F-actin destabilization occurred at 1, 3.33, and 10 mg/mL, with cytoplasmic and perinuclear filaments more affected than cortical actin. Findings from ALI and submerged models were concordant, supporting hydroxyacetone-induced mitochondrial stress, oxidative damage, and cytoskeletal disruption. These results suggest that hydroxyacetone concentrations found in EC aerosols may contribute to respiratory toxicity and warrant further investigation.

Background: Acetaldehyde and methylglyoxal are structurally related aldehydes produced by thermal degradation of the electronic cigarette (EC) solvents, propylene glycol and glycerin. Despite their presence in EC aerosols, the biological effects of these aldehydes when inhaled during vaping are largely unknown.Methods: Three-dimensional (3D) human bronchial epithelial tissues (EpiAirway™) were exposed at the air liquid interface (ALI) to aerosols containing acetaldehyde or methylglyoxal at concentrations relevant to human vaping. PBS-exposed tissues served as controls. Comparative proteomic analyses were performed to assess global alterations in protein expression. Based on proteomics data, concentration-response experiments were conducted using BEAS-2B bronchial epithelial cells to evaluate reactive oxygen species, mitochondrial function, and cytoskeletal integrity.Results: ALI exposure to acetaldehyde or methylglyoxal resulted in 79 and 76 differentially expressed proteins (DEPs), respectively, with 51 overlapping proteins exhibiting similar fold change directionality. Ingenuity Pathway Analysis (IPA) Toxicity Lists identified key affected pathways, including mitochondrial dysfunction, fatty acid metabolism, G2/M DNA damage checkpoint regulation, and mitochondrial biogenesis. Gene Ontology (GO) ontology analysis revealed substantial overlap in affected biological processes and cellular components. Findings were further supported and expanded in BEAS-2B cell concentration-response assays, which confirmed mitochondrial impairment, elevated ROS levels, and disrupted cytoskeletal organization. Notably, TRPM8 inhibition attenuated methylglyoxal-induced mitochondrial dysfunction (MTT assay), while both TRPM8 and TRPA1 inhibition partially rescued actin depolymerization.Conclusion: Brief ALI exposure of EpiAirway™ tissues to vaping-relevant concentrations of acetaldehyde or methylglyoxal altered the bronchial epithelial proteome. Complementary concentration-response experiments with submerged BEAS-2B cells confirmed and extended the proteomics data. While both aldehydes exhibited similar proteomic and functional impacts, methylglyoxal was effective at substantially lower concentrations in all assays with some effects partially mediated via TRPA1 and TRPM8 channels.

Hydroxyacetone has been detected at high concentrations (up to ∼12 mg/mL) in electronic cigarette (EC) aerosols, including those derived from products associated with adverse health effects. Given the limited understanding of its inhalation toxicology, we investigated hydroxyacetone’s impact on human airway epithelial cells. Acute exposures at the air–liquid interface (ALI) using 3D EpiAirway tissues—a surrogate for human tracheobronchial epithelium—were analyzed via proteomics. Differential expression analysis identified numerous affected proteins, with enrichment pointing to alterations in mitochondrial function and actin cytoskeletal disruption as major targets. Ingenuity Pathway Analysis (IPA) highlighted “Mitochondrial Dysfunction” and “NRF2-Mediated Oxidative Stress” among top toxicological categories, and “Nuclear Cytoskeletal Signaling” as a key canonical pathway. To validate and extend these findings, submerged cultures of BEAS-2B cells were exposed to hydroxyacetone (0.01–10 mg/mL) and assessed for mitochondrial activity, oxidative stress, and F-actin integrity. At 1 mg/mL, mitochondrial membrane potential and reactive oxygen species (ROS) increased, with elevated hydrogen peroxide detected in the culture medium. At 10 mg/mL, mitochondrial activity declined significantly, accompanied by cell rounding and apoptotic blebbing within 2 hours. F-actin destabilization occurred at 1, 3.33, and 10 mg/mL, with cytoplasmic and perinuclear filaments more affected than cortical actin. Findings from ALI and submerged models were concordant, supporting hydroxyacetone-induced mitochondrial stress, oxidative damage, and cytoskeletal disruption. These results suggest that hydroxyacetone concentrations found in EC aerosols may contribute to respiratory toxicity and warrant further investigation.

Summary: Early stages in human development are difficult to study in pregnant women. We used a “disease-in-a-dish” model to investigate SARS-CoV-2 infection of human embryonic stem cells and the three germ layers. Ectoderm had a significantly higher infection than the other cell types. This was due to: (1) the use of two entry pathways by the ectoderm (fusion and endocytosis), (2) high levels of TMPRSS2 in the ectoderm, and (3) a much-reduced ectodermal glycocalyx, which facilitated viral attachment to the ACE2 receptor. Our findings provide strong evidence that cells in young postimplantation human embryos are susceptible to SARS-CoV-2 infection, which could be embryo lethal or teratogenic in surviving embryos. The high level of infection in ectoderm is a concern as its derivatives may also be affected by SARS-CoV-2. Future clinical work should investigate the functioning of the nervous system in infants born to mothers who had COVID-19 during pregnancy. Highlights: SARS-CoV-2 pseudoparticles infected human embryonic stem cells, endoderm, mesoderm and ectoderm Ectoderm was significantly more susceptible to infection than the other three cell types Factors accounting for increased susceptibility and tissue tropism were identified SARS-CoV-2 virus can adversely affect early stages of human development

Electronic cigarette (e-cigarette) liquids and aerosols contain metals, which can be detrimental to human health. Recently marketed ultrasonic cigarettes (u-cigarettes) claim to be less harmful than e-cigarettes that use heating coils. We quantified chemical elements/metals in multiple flavors of SURGE u-cigarettes, JUUL e-cigarettes, and "Other Brands" of pod-style e-cigarettes. Elements/metals were identified in atomizers of SURGE using a scanning electron microscope/energy-dispersive X-ray spectrometer. Quantitation of elements/metals in fluids and aerosols from SURGE, JUUL and Other Brands was performed using inductively coupled plasma optical emission spectroscopy. U-cigarettes contained a sonicator, unlike e-cigarettes which had heated coils. Sixteen elements were identified in at least one fluid or aerosol sample. Generally, u-cigarette fluids and aerosols had more elements/metals at higher concentrations than aerosols from 4^th generation e-cigarettes. Element concentrations generally increased in fluids after vaping. All products, including SURGE, had silicon in their fluids and aerosols. Nickel, which was present in low concentrations in all fluids except KWIT Stick (up to 66,050 μg/mL), transferred to the aerosols with low efficiency. SURGE, but not e-cigarettes, also had copper and zinc in their fluids, but little transferred to their aerosols. SURGE fluids and aerosols, unlike e-cigarettes, had relatively high concentrations of arsenic and selenium. Arsenic and selenium, which are on the FDA's Harmful and Potentially Harmful List, likely came from poor quality solvents used to produce the e-liquids in SURGE pods and possibly from the sonicator, which heats during use. SURGE u-cigarettes produce aerosols with metals equivalent to heated coil-style e-cigarettes and had high levels of arsenic and selenium, which are a health concern. Regulations limiting arsenic and selenium in these products are needed, and routine surveillance to identify rogue products, such as Kwit Stick, that have abnormally high levels of nickel or other metals could protect human health. https://doi.org/10.1289/EHP15648.