Deconstructing Intratumoral Heterogeneity through Multiomic and Multiscale Analysis of Serial Sections.

Tumors may contain billions of cells, including distinct malignant clones and nonmalignant cell types. Clarifying the evolutionary histories, prevalence, and defining molecular features of these cells is essential for improving clinical outcomes, since intratumoral heterogeneity provides fuel for acquired resistance to targeted therapies. Here we present a statistically motivated strategy for deconstructing intratumoral heterogeneity through multiomic and multiscale analysis of serial tumor sections (MOMA). By combining deep sampling of IDH-mutant astrocytomas with integrative analysis of single-nucleotide variants, copy-number variants, and gene expression, we reconstruct and validate the phylogenies, spatial distributions, and transcriptional profiles of distinct malignant clones. By genotyping nuclei analyzed by single-nucleus RNA-seq for truncal mutations, we further show that commonly used algorithms for identifying cancer cells from single-cell transcriptomes may be inaccurate. We also demonstrate that correlating gene expression with tumor purity in bulk samples can reveal optimal markers of malignant cells and use this approach to identify a core set of genes that are consistently expressed by astrocytoma truncal clones, including AKR1C3, whose expression is associated with poor outcomes in several types of cancer. In summary, MOMA provides a robust and flexible strategy for precisely deconstructing intratumoral heterogeneity and clarifying the core molecular properties of distinct cellular populations in solid tumors.

Skeletal muscle fitness and physiology as determinants of firefighter performance and safety: a narrative review.

Firefighters routinely perform tasks that are reliant on their muscular fitness, which includes muscular strength, power and endurance. Separately, firefighters can present with unique skeletal muscle physiology characteristics due to the strenuous nature of this occupation. This review aims to summarise muscular fitness and physiology as determinants of a firefighter's ability to perform occupation-specific tasks, identify the relevance of both muscular fitness and physiology to a firefighter's risk for sustaining a work-related injury, and address the contributions of muscular fitness and physiology on a firefighter's ability to recover from tasks and their readiness for performing subsequent or future tasks. The presented evidence reveals muscular fitness can determine a firefighter's capacity to perform their job effectively, while also influencing risk for occupational injury. Collectively, this review indicates exercise training emphasising improvements in muscular strength, power, and endurance (i.e. resistance training) should be encouraged in this occupation.

This review addressed muscular fitness and physiology in firefighters. Current evidence suggests firefighter task performance and risk for injury is associated with high levels of muscular fitness. Additionally, firefighters undergo unique changes in muscle morphology and physiology that can negatively affect the ability to safely perform occupation-specific tasks repeatedly.

Principles of peptide selection by the transporter associated with antigen processing.

Our ability to fight pathogens relies on major histocompatibility complex class I (MHC-I) molecules presenting diverse antigens on the surface of diseased cells. The transporter associated with antigen processing (TAP) transports nearly the entire repertoire of antigenic peptides into the endoplasmic reticulum for MHC-I loading. How TAP transports peptides specific for MHC-I is unclear. In this study, we used cryo-EM to determine a series of structures of human TAP, both in the absence and presence of peptides with various sequences and lengths. The structures revealed that peptides of eight or nine residues in length bind in a similarly extended conformation, despite having little sequence overlap. We also identified two peptide-anchoring pockets on either side of the transmembrane cavity, each engaging one end of a peptide with primarily main chain atoms. Occupation of both pockets results in a global conformational change in TAP, bringing the two halves of the transporter closer together to prime it for isomerization and ATP hydrolysis. Shorter peptides are able to bind to each pocket separately but are not long enough to bridge the cavity to bind to both simultaneously. Mutations that disrupt hydrogen bonds with the N and C termini of peptides almost abolish MHC-I surface expression. Our findings reveal that TAP functions as a molecular caliper that selects peptides according to length rather than sequence, providing antigen diversity for MHC-I presentation.

Subchronic inhalation of a novel electronic nicotine delivery system formulation and its corresponding base formulation.

BACKGROUND: Fresh Menthol 3% Nicotine (FM3) is a novel JUUL e-liquid formulation. Its potential toxicity and that of the corresponding base formulation relative to a filtered air (FA) control was studied in a subchronic inhalation study conducted in general accordance with OECD 413. METHODS: Aerosols generated with an intense puffing regime were administered to rats in a nose-only fashion at 1400 µg aerosol collected mass/L on a 6 hour/day basis for 90 days with a 42-day recovery. Exposure atmospheres met target criteria. Systemic exposure was confirmed by plasma measurement of nicotine. RESULTS: No test article-related mortality, clinical signs (other than reversible lower body weight gains in males), clinical pathology or gross findings were noted during this study. No microscopic lesions related to base formulation exposure were identified. Minimal microscopic lesions were observed in the FM3 6-hour exposure group. Microscopic lesions observed in the FM3 6-hour exposure group comprised only minimal laryngeal squamous metaplasia in one male and one female animal. No microscopic lesions related to FM3 exposure remained after the recovery period. CONCLUSION: Exposure atmosphere characterization indicated that conditions were achieved to permit thorough assessment of test articles and results indicate a low order of toxicity for the FM3 Electronic nicotine delivery systems (ENDS) formulation and its base formulation.

Glioblastoma evolution and heterogeneity from a 3D whole-tumor perspective.

Treatment failure for the lethal brain tumor glioblastoma (GBM) is attributed to intratumoral heterogeneity and tumor evolution. We utilized 3D neuronavigation during surgical resection to acquire samples representing the whole tumor mapped by 3D spatial coordinates. Integrative tissue and single-cell analysis revealed sources of genomic, epigenomic, and microenvironmental intratumoral heterogeneity and their spatial patterning. By distinguishing tumor-wide molecular features from those with regional specificity, we inferred GBM evolutionary trajectories from neurodevelopmental lineage origins and initiating events such as chromothripsis to emergence of genetic subclones and spatially restricted activation of differential tumor and microenvironmental programs in the core, periphery, and contrast-enhancing regions. Our work depicts GBM evolution and heterogeneity from a 3D whole-tumor perspective, highlights potential therapeutic targets that might circumvent heterogeneity-related failures, and establishes an interactive platform enabling 360° visualization and analysis of 3D spatial patterns for user-selected genes, programs, and other features across whole GBM tumors.

A Practical Framework for Novel Electronic Nicotine Delivery System Evaluation: Chemical and Toxicological Characterization of JUUL2 Aerosol and Comparison with Reference Cigarettes.

Electronic nicotine delivery systems (ENDSs) are designed as a non-combustible alternative to cigarettes, aiming to deliver nicotine without the harmful byproducts of tobacco combustion. As the category evolves and new ENDS products emerge, it is important to continually assess the levels of toxicologically relevant chemicals in the aerosols and characterize any related toxicology. Herein, we present a proposed framework for characterizing novel ENDS products (i.e., devices and formulations) and determining the reduced risk potential utilizing analytical chemistry and in vitro toxicological studies with a qualitative risk assessment. To demonstrate this proposed framework, long-term stability studies (12 months) analyzing relevant toxicant emissions from six formulations of a next-generation product, JUUL2, were conducted and compared to reference combustible cigarette (CC) smoke under both non-intense and intense puffing regimes. In addition, in vitro cytotoxicity, mutagenicity, and genotoxicity assays were conducted on aerosol and smoke condensates. In all samples, relevant toxicants under both non-intense and intense puffing regimes were substantially lower than those observed in reference CC smoke. Furthermore, neither cytotoxicity, mutagenicity, nor genotoxicity was observed in aerosol condensates generated under both intense and non-intense puffing regimes, in contrast to results observed for reference cigarettes. Following the proposed framework, the results demonstrate that the ENDS products studied in this work generate significantly lower levels of toxicants relative to reference cigarettes and were not cytotoxic, mutagenic, or genotoxic under these in vitro assay conditions.

The carboxy terminus causes interfacial assembly of oleate hydratase on a membrane bilayer.

The soluble flavoprotein oleate hydratase (OhyA) hydrates the 9-cis double bond of unsaturated fatty acids. OhyA substrates are embedded in membrane bilayers; OhyA must remove the fatty acid from the bilayer and enclose it in the active site. Here, we show that the positively charged helix-turn-helix motif in the carboxy terminus (CTD) is responsible for interacting with the negatively charged phosphatidylglycerol (PG) bilayer. Super-resolution microscopy of Staphylococcus aureus cells expressing green fluorescent protein fused to OhyA or the CTD sequence shows subcellular localization along the cellular boundary, indicating OhyA is membrane-associated and the CTD sequence is sufficient for membrane recruitment. Using cryo-electron microscopy, we solved the OhyA dimer structure and conducted 3D variability analysis of the reconstructions to assess CTD flexibility. Our surface plasmon resonance experiments corroborated that OhyA binds the PG bilayer with nanomolar affinity and we found the CTD sequence has intrinsic PG binding properties. We determined that the nuclear magnetic resonance structure of a peptide containing the CTD sequence resembles the OhyA crystal structure. We observed intermolecular NOE from PG liposome protons next to the phosphate group to the CTD peptide. The addition of paramagnetic MnCl2 indicated the CTD peptide binds the PG surface but does not insert into the bilayer. Molecular dynamics simulations, supported by site-directed mutagenesis experiments, identify key residues in the helix-turn-helix that drive membrane association. The data show that the OhyA CTD binds the phosphate layer of the PG surface to obtain bilayer-embedded unsaturated fatty acids.

Deconstructing intratumoral heterogeneity through multiomic and multiscale analysis of serial sections.

Tumors may contain billions of cells including distinct malignant clones and nonmalignant cell types. Clarifying the evolutionary histories, prevalence, and defining molecular features of these cells is essential for improving clinical outcomes, since intratumoral heterogeneity provides fuel for acquired resistance to targeted therapies. Here we present a statistically motivated strategy for deconstructing intratumoral heterogeneity through multiomic and multiscale analysis of serial tumor sections (MOMA). By combining deep sampling of IDH-mutant astrocytomas with integrative analysis of single-nucleotide variants, copy-number variants, and gene expression, we reconstruct and validate the phylogenies, spatial distributions, and transcriptional profiles of distinct malignant clones. By genotyping nuclei analyzed by single-nucleus RNA-seq for truncal mutations, we further show that commonly used algorithms for identifying cancer cells from single-cell transcriptomes may be inaccurate. We also demonstrate that correlating gene expression with tumor purity in bulk samples can reveal optimal markers of malignant cells and use this approach to identify a core set of genes that is consistently expressed by astrocytoma truncal clones, including AKR1C3, whose expression is associated with poor outcomes in several types of cancer. In summary, MOMA provides a robust and flexible strategy for precisely deconstructing intratumoral heterogeneity and clarifying the core molecular properties of distinct cellular populations in solid tumors.

Corrigendum: Analytical approaches for the evaluation of data deficient simulated leachable compounds in ENDS products: a case study.

[This corrects the article DOI: 10.3389/fchem.2023.1212744.].

Determination of chemical constituent yields in e-cigarette aerosol using partial and whole pod collections, a comparative analysis.

Literature reports the chemical constituent yields of electronic nicotine delivery systems (ENDS) aerosol collected using a range of aerosol collection strategies. The number of puffs to deplete an ENDS product varies widely, but collections often consist of data from the first 50-100 puffs. However, it is not clear whether these discrete puff blocks are representative of constituent yields over the life of a pod. We aimed to assess the effect of differing aerosol collection strategies on reported yields for select chemical constituents in the aerosol of closed pod-based ENDS products. Constituents analyzed were chosen to reflect important classes of compounds from the Final Premarket Tobacco Product Application Guidance. Yields were normalized to total device mass loss (DML). Collection strategies that consisted of partial pod collection were valid for determining yields of constituents whose DML normalized yields were consistent for the duration of pod life. These included primary aerosol constituents, such as propylene glycol, glycerol, and nicotine, and whole pod yields could be determined from initial puff blocks. However, changes were observed in the yields of some metals, some carbonyl compounds, and glycidol over pod life in a chemical constituent and product dependent manner. These results suggest that collection strategies consisting of initial puff block collections require validation per chemical constituent/product and are not appropriate for chemical constituents with variable yields over pod life. Whole pod collection increased sensitivity and accuracy in determining metal, carbonyl, and glycidol yields compared to puff block-based collection methodologies for all products tested.

Analytical approaches for the evaluation of data deficient simulated leachable compounds in ENDS products: a case study.

Leachable investigations are routinely undertaken across a range of sectors (e.g., pharmaceuticals, medical devices, etc.) to determine whether chemicals from a container closure system transfer into a product under normal conditions of use. For Electronic Nicotine Delivery Systems (ENDS) the container closure system includes all materials in contact with the e-liquid that is aerosolized and subsequently inhaled by the user. Currently, there is no guidance for conducting leachable studies for ENDS products, however, there are relevant guidance documents for orally inhaled drug products that can be applied to an ENDS container closure system. We present a case study of the analytical investigation of two leachable compounds identified in simulated leachable studies using aged JUULpods filled with unflavored e-liquid (PG/VG/nicotine/benzoic acid). Both compounds had limited toxicological information and were considered data deficient. A qualitative analysis of the aerosol collected from aged commercial JUULpods (Virginia Tobacco and Menthol), using a similar analytical method (LC-MS/MS) used in the simulated leachable studies, showed no trace or detectable levels of either leachable compound. Therefore, this qualitative analysis did not provide semi-quantitative values for the data-deficient leachable compounds necessary to support toxicological risk assessment. Further, no commercial authentic standards or reasonable synthetic route were available due to the molecular size and structural complexity of the compounds. Instead, method limits were established using an alternative approach to standard ICH guidelines. The experimentally determined method limit of quantitation, using spiked samples of simulated leachable e-liquid, provided conservative semi-quantitative values for each data deficient leachable compound in the aerosol that enabled a transfer efficiency from e-liquid to aerosol to be estimated. The transfer efficiency of each leachable compound was experimentally determined to be less than 2% based on the limit of quantitation, which then could be used to define a relevant exposure limit for the toxicological risk assessment. This work details a novel analytical approach for determining the transfer efficiency of data deficient leachable compounds from ENDS container closure systems into the ENDS aerosol to support toxicological health risk assessments.

Comparison of the toxicological potential of two JUUL ENDS products to reference cigarette 3R4F and filtered air in a 90-day nose-only inhalation toxicity study.

Electronic nicotine delivery systems (ENDS) are generally recognized as less harmful alternatives for those who would otherwise continue to smoke cigarettes. The potential toxicity of aerosols generated from JUUL Device and Virginia Tobacco (VT3) or Menthol (ME3) JUULpods at 3.0% nicotine concentration was assessed in rats exposed at target aerosol concentrations of 1400 µg/L for up to 6 h/day on a 5 day/week basis for at least 90 days (general accordance with OECD 413). 3R4F reference cigarette smoke (250 µg/L) and Filtered Air were used as comparators. JUUL ENDS product aerosol exposures at >5x the 3R4F cigarette smoke level resulted in greater plasma nicotine and cotinine levels (up to 2x). Notable cigarette smoke related effects included pronounced body weight reductions in male rats, pulmonary inflammation evidenced by elevated lactate dehydrogenase, pro-inflammatory cytokines and neutrophils in bronchoalveolar lavage fluid, increased heart and lung weights, and minimal to marked respiratory tract histopathology. In contrast, ENDS aerosol exposed animals had minimal body weight changes, no measurable inflammatory changes and minimal to mild laryngeal squamous metaplasia. Despite the higher exposure levels, VT3 and ME3 did not result in significant toxicity or appreciable respiratory histopathology relative to 3R4F cigarette smoke following 90 days administration.

Comparison of biomarkers of exposure among US adult smokers, users of electronic nicotine delivery systems, dual users and nonusers, 2018-2019.

The harm caused by cigarette smoking is overwhelmingly due to byproducts of tobacco combustion. Electronic Nicotine Delivery Systems (ENDS) provide nicotine to users without combustion, and may support tobacco harm reduction among cigarette smokers who would not otherwise quit in the near term. Analyses of Wave 5 of the Population Assessment of Tobacco and Health (PATH) Study compared biomarkers of exposure (BOE) levels for nicotine, 3 metals, 2 tobacco-specific nitrosamines and 14 smoking-related volatile organic compounds in 151 exclusive ENDS users, 1341 exclusive cigarette smokers, 115 dual users (cigarettes and ENDS), and 1846 past 30-day nonusers of tobacco, adjusting for demographics. Nicotine exposure in ENDS users and dual users did not significantly differ from smokers. Among ENDS users, 16 of 18 other BOEs were significantly lower than smokers'; 9 BOEs were not significantly different from nonusers. Among dual users smoking < 10 cigarettes/day, 15 of 18 non-nicotine BOEs were significantly lower than smokers', whereas in dual users smoking ≥ 10 cigarettes per day none of the BOEs significantly differed from smokers'. In this representative sample of US adults, exclusive use of ENDS (vs. cigarette smoking) was associated with much lower exposures to many harmful chemicals associated with smoking-related disease. BOE levels in dual users were directly related to their cigarette consumption. These BOE data provide further evidence that ENDS expose users to substantially lower levels of toxicants than combustible cigarettes, confirming their potential for harm reduction.

Bulk and mosaic deletions of Egfr reveal regionally defined gliogenesis in the developing mouse forebrain.

The epidermal growth factor receptor (EGFR) plays a role in cell proliferation and differentiation during healthy development and tumor growth; however, its requirement for brain development remains unclear. Here we used a conditional mouse allele for Egfr to examine its contributions to perinatal forebrain development at the tissue level. Subtractive bulk ventral and dorsal forebrain deletions of Egfr uncovered significant and permanent decreases in oligodendrogenesis and myelination in the cortex and corpus callosum. Additionally, an increase in astrogenesis or reactive astrocytes in effected regions was evident in response to cortical scarring. Sparse deletion using mosaic analysis with double markers (MADM) surprisingly revealed a regional requirement for EGFR in rostrodorsal, but not ventrocaudal glial lineages including both astrocytes and oligodendrocytes. The EGFR-independent ventral glial progenitors may compensate for the missing EGFR-dependent dorsal glia in the bulk Egfr-deleted forebrain, potentially exposing a regenerative population of gliogenic progenitors in the mouse forebrain.

A macrocyclic peptide inhibitor traps MRP1 in a catalytically incompetent conformation.

Adenosine triphosphate-binding cassette (ABC) transporters, such as multidrug resistance protein 1 (MRP1), protect against cellular toxicity by exporting xenobiotic compounds across the plasma membrane. However, constitutive MRP1 function hinders drug delivery across the blood-brain barrier, and MRP1 overexpression in certain cancers leads to acquired multidrug resistance and chemotherapy failure. Small-molecule inhibitors have the potential to block substrate transport, but few show specificity for MRP1. Here we identify a macrocyclic peptide, named CPI1, which inhibits MRP1 with nanomolar potency but shows minimal inhibition of a related multidrug transporter P-glycoprotein. A cryoelectron microscopy (cryo-EM) structure at 3.27 Å resolution shows that CPI1 binds MRP1 at the same location as the physiological substrate leukotriene C4 (LTC4). Residues that interact with both ligands contain large, flexible sidechains that can form a variety of interactions, revealing how MRP1 recognizes multiple structurally unrelated molecules. CPI1 binding prevents the conformational changes necessary for adenosine triphosphate (ATP) hydrolysis and substrate transport, suggesting it may have potential as a therapeutic candidate.

Key challenges for in vitro testing of tobacco products for regulatory applications: Recommendations for dosimetry.

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to develop recommendations for optimal scientific and technical approaches for conducting in vitro assays to assess potential toxicity within and across tobacco and various next-generation products (NGPs) including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDSs). This publication was developed by a working group of the workshop members in conjunction with the sixth workshop in that series entitled "Dosimetry for conducting in vitro evaluations" and focuses on aerosol dosimetry for aerosol exposure to combustible cigarettes, HTP, and ENDS aerosolized tobacco products and summarizes the key challenges as well as documenting areas for future research.

An Approach to Flavor Chemical Thermal Degradation Analysis.

Toxicological evaluations of flavor chemicals for use in inhalation products that utilize heat for aerosol generation are complicated because of the potential effect heat may have on the flavor chemical. The objective was to develop a thermal degradation technique to screen flavor chemicals as part of a toxicological testing program for their potential use in ENDS formulations. Based upon published data for acetaldehyde, acrolein, and glycidol from ENDS products (common thermal degradants of propylene glycol and glycerin), the pyrolizer temperature was adjusted until a similar ratio of acetaldehyde, acrolein, and glycidol was obtained from a 60/40 ratio (v/v) of glycerin/propylene glycol via GC/MS analysis. For each of 90 flavor chemicals, quantitative measurements of acetaldehyde, acrolein, and glycidol, in addition to semiquantitative non-targeted analysis tentatively identifying chemicals from thermal degradation, were obtained. Twenty flavor chemicals transferred at greater than 99% intact, another 26 transferred at greater than 95% intact, and another 15 flavor chemicals transferred at greater than 90% intact. Most flavor chemicals resulted in fewer than 10-12 tentatively identified thermal degradants. The practical approach to the thermal degradation of flavor chemicals provided useful information as part of the toxicological evaluation of flavor chemicals for potential use in ENDS formulations.

Characterization of a rapid condensate collection apparatus for in vitro assays of electronic nicotine delivery systems.

In vitro testing of Electronic Nicotine Delivery System (ENDS) aerosol condensates is important in evaluating their potential toxicity. Collecting sufficient condensate for these tests is a time consuming and costly procedure. The "triple puff (TP)" is a novel system which collects the aerosol from three ENDS devices sequentially into a single filter pad and impinger. The TP substantially reduces condensate collection time relative to the conventional single ENDS, single puff (SP), device system. Both the TP and SP (using two puffing profiles) were used to generate condensates from JUUL ENDS e-liquid Mint 5.0% (nicotine by weight). Aerosols were collected using the filter pad and ethanol-containing impinger method. Condensates produced with the SP and TP were compared for concentrations of primary constituents and carbonyl compounds as well as for their cytotoxicity (OECD 129), mutagenicity (OECD 471) and genotoxicity (OECD 487). Condensates generated with the SP and TP, regardless of puffing regimen, were very similar chemically and equivalent in the biological assays tested (not cytotoxic, mutagenic, or genotoxic). The TP device significantly reduces production time of ENDS condensates relative to the standard SP method and thus may facilitate further research by reducing the time and effort required to collect ENDS condensates.

Generation of functional human oligodendrocytes from dermal fibroblasts by direct lineage conversion.

Oligodendrocytes, the myelinating cells of the central nervous system, possess great potential for disease modeling and cell transplantation-based therapies for leukodystrophies. However, caveats to oligodendrocyte differentiation protocols ( Ehrlich et al., 2017; Wang et al., 2013; Douvaras and Fossati, 2015) from human embryonic stem and induced pluripotent stem cells (iPSCs), which include slow and inefficient differentiation, and tumorigenic potential of contaminating undifferentiated pluripotent cells, are major bottlenecks towards their translational utility. Here, we report the rapid generation of human oligodendrocytes by direct lineage conversion of human dermal fibroblasts (HDFs). We show that the combination of the four transcription factors OLIG2, SOX10, ASCL1 and NKX2.2 is sufficient to convert HDFs to induced oligodendrocyte precursor cells (iOPCs). iOPCs resemble human primary and iPSC-derived OPCs based on morphology and transcriptomic analysis. Importantly, iOPCs can differentiate into mature myelinating oligodendrocytes in vitro and in vivo. Finally, iOPCs derived from patients with Pelizaeus Merzbacher disease, a hypomyelinating leukodystrophy caused by mutations in the proteolipid protein 1 (PLP1) gene, showed increased cell death compared with iOPCs from healthy donors. Thus, human iOPCs generated by direct lineage conversion represent an attractive new source for human cell-based disease models and potentially myelinating cell grafts.

Nests of dividing neuroblasts sustain interneuron production for the developing human brain.

The human cortex contains inhibitory interneurons derived from the medial ganglionic eminence (MGE), a germinal zone in the embryonic ventral forebrain. How this germinal zone generates sufficient interneurons for the human brain remains unclear. We found that the human MGE (hMGE) contains nests of proliferative neuroblasts with ultrastructural and transcriptomic features that distinguish them from other progenitors in the hMGE. When dissociated hMGE cells are transplanted into the neonatal mouse brain, they reform into nests containing proliferating neuroblasts that generate young neurons that migrate extensively into the mouse forebrain and mature into different subtypes of functional interneurons. Together, these results indicate that the nest organization and sustained proliferation of neuroblasts in the hMGE provide a mechanism for the extended production of interneurons for the human forebrain.