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Electronic Nicotine Delivery Systems (ENDS) contain numerous volatile aerosol constituents (e.g., nicotine, propylene glycol, flavors, etc.). Past work clearly indicates the temporal and chemical dynamics of ENDS aerosol requires consideration of these volatile constituents when measuring the particle size distribution. An MSP-135-8 Mini MOUDI™ and Electrical Low Pressure Impactor (ELPI®+) were used to measure the particle size distribution of two JUUL ENDS products. Volatile chemicals were measured from each cascade impactor's exhaust airflow to assess their effect on collection efficiency. Similar mass median aerodynamic diameters were obtained for both ENDS products by both cascade impactors, however the geometric standard deviation from the ELPI®+ measurements were larger for both products than measurements using the Mini MOUDI™ impactor. Although the measured mass of volatile chemicals was greater in the exhaust from the Mini MOUDI™ impactor, a greater variety of volatile chemicals were found within the exhaust of the ELPI®+. The greater variety of volatile chemicals correlated with more room air sampling by the ELPI®+. The reduced amount of volatile chemicals measured in the exhaust of the ELPI®+ may be due to their collection by the vacuum oil used in the sintered collection plates of the ELPI®+. Accounting for the measured volatile chemicals improved the recovery efficiency of the Mini MOUDI™ impactor by 2.9–7.5% with the average recovery efficiency exceeding 82% for the two JUUL ENDS Products. In comparison, accounting for the measured volatile constituents increased the recovery efficiency of the ELPI®+ impactor by 0.4% or less, which did not narrow the recovery efficiency range, that based upon the estimated dilution, consistently exceeded the measured mass loss from both JUUL ENDS products.

Simple Summary: Tumors contain cancerous cells with different mutations and different types of non-cancerous cells. It is important to understand the molecular properties of these cells in order to develop effective treatments for cancers. We describe a new strategy for studying tumor heterogeneity and apply it to human astrocytomas, a type of brain tumor. By slicing astrocytomas into a large number of serial sections and analyzing patterns of mutation frequencies, we reconstruct the evolutionary history of cancer cell mutations in each tumor. By comparing these patterns to gene activity measured in the same sections, we identify molecular features that optimally distinguish different types of cancerous and non-cancerous cells and validate these with different techniques, including analysis of individual nuclei. By integrating analyses of multiple molecular species at multiple scales, our strategy provides a powerful approach for precisely deconstructing intratumoral heterogeneity. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 3% 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. [ABSTRACT FROM AUTHOR]

A unique adult human mouth/throat physical model has been developed to study e-cigarette aerosol dynamics during inhalation. The 3D printed physical model was created from the CT scan of a 28 yr. old healthy male. Internal walls of the physical model were lined with a thin layer of cotton gauze that can be saturated with water to replicate the high relative humidity conditions in a human Oral/Pharyngeal cavity. Aerosol hygroscopic growth and deposition inside the physical model from a cartomizer style e-cigarette using a prototype e-liquid formulation was determined by measuring cumulative aerosol mass from five puffs (gravimetric) and for individual constituents (propylene glycol, glycerol, and nicotine) from a single puff (GC/MS analysis). Measurements were taken at constant temperature of 37 °C under both wet and dry inner wall conditions for a for a 3-sec. 55 mL puff. The condition of holding aerosol inside the physical model without airflow for a duration of 3-sec. was also included. For the same puffing conditions of 3-sec puff of 55 mL and a 3-sec. puff hold time, dry wall conditions resulted in a mean aerosol mass loss of 20.9 ± 3.8%, while the total aerosol mass was increased by 150.9 ± 19% under wet wall condition when compared to total aerosol mass entering the physical model entrance. The dramatic increase is due to water vapor uptake by the aerosol particles when flowing through the wetted physical model. Aerosol evolution of individual chemical constituent analysis appeared to vary as a function of volatility. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Introduction: For in vivo inhalation studies, aerosol dosimetry links aerosol exposure and deposited dose within the respiratory tract. Aerosol dosimetry programs like the Multiple Path Particle Deposition model utilize respiratory tract geometry, respiratory physiology, and aerosol properties to predict particle deposition within the respiratory tract. A challenge to wider use of these dosimetry programs for in vivo inhalation studies is the lack of species-specific or strain specific respiratory tract anatomy, and in some cases, strain specific respiratory physiology data. The objective of this work was to develop aerosol dosimetry predictions for the in vivo human disease models of chronic obstructive pulmonary disease (COPD; C57BL/6 mice) cardiovascular disease (CVD; ApoE-/- mice), and lung cancer (AJ mice) using the Multiple Path Particle Deposition dosimetry model. Methods: Microcomputed tomography derived tracheobronchial geometry data were combined with available pulmonary geometry data for C57BL/6, ApoE-/-, and AJ mice and incorporated into the Multiple Path Particle Deposition dosimetry model. Mouse strain specific respiratory physiology literature values were used as input into the Multiple Path Particle Deposition dosimetry model. Results: The resulting particle deposition predictions compared well with the very limited strain specific experimental particle deposition data. Since multiple tracheobronchial geometries and thus pulmonary anatomies were used for C57BL/6 and ApoE-/- mice, an estimate of intrastrain variability in predicted particle deposition was obtained. Discussion and Conclusions: The intrastrain variability in predicted particle deposition for C57BL/6 and ApoE-/- mice was always smaller in the tracheobronchial compared to pulmonary airways. The differences in reported respiratory physiology values for C57BL/6 mice resulted in greater intrastrain variability in predicted particle deposition than observed with the different tracheobronchial geometries and pulmonary anatomies. These mouse strain specific aerosol dosimetry predictions can provide insight into the delivered dose to specific respiratory tract regions that can be correlated with in vivo endpoints. [ABSTRACT FROM AUTHOR]

Introduction: WS-5 is a flavor ingredient that provides a cooling sensation similar to that of menthol but without the characteristic menthol flavor. In the available toxicological evaluations of WS-5 there was no data specific to inhalation exposure. Methods: A 90-days nose-only inhalation study was conducted using Sprague Dawley rats exposed to Filtered air, propylene glycol/glycerin (40/60 by weight; vehicle control), or one of three WS-5 concentrations (0.2, 0.4, or 0.8% by weight) to assess the inhalation toxicity of WS-5. The study design was consistent with the Organization for Economic Development and Cooperation test guideline 413 and included a 28-day and 6-week recovery sacrifices, in addition to the 13-week assessment. Results: Food consumption and body weights were unaffected by WS-5 exposure compared to vehicle control or Filtered air. No WS-5 exposure related alterations were observed in serum chemistry, hematology, coagulation, urinalysis or bronchoalveolar lavage fluid cytology and clinical chemistry. Macroscopic examinations and terminal organ weights revealed no observations associated with exposure to WS-5. Discussion: Histopathology findings of alveolar hemorrhage and mixed cell infiltration at the 28-day necropsy were either not present in the WS-5 exposed groups or were present at similar frequencies in the vehicle control and/or Filtered air groups at the 13-weeks necropsy and thus, were not considered to be vehicle control or WS-5 related. Conclusion: Based on the absence of adverse effects, the no-observed-adverse effect concentration was considered to be 2.5 mg/L of aerosolized 0.8 w% WS-5 that achieved a mean measured exposure concentration of approximately 18 μg WS-5/L [ABSTRACT FROM AUTHOR]

Cellular immunity against viral infection and tumour cells depends on antigen presentation by major histocompatibility complex class I (MHC I) molecules. Intracellular antigenic peptides are transported into the endoplasmic reticulum by the transporter associated with antigen processing (TAP) and then loaded onto the nascent MHC I molecules, which are exported to the cell surface and present peptides to the immune system (1). Cytotoxic T lymphocytes recognize non-self peptides and program the infected or malignant cells for apoptosis. Defects in TAP account for immunodeficiency and tumour development. To escape immune surveillance, some viruses have evolved strategies either to downregulate TAP expression or directly inhibit TAP activity. So far, neither the architecture of TAP nor the mechanism of viral inhibition has been elucidated at the structural level. Here we describe the cryo-electron microscopy structure of human TAP in complex with its inhibitor ICP47, a small protein produced by the herpes simplex virus I. Here we show that the 12 transmembrane helices and 2 cytosolic nucleotide-binding domains of the transporter adopt an inward-facing conformation with the two nucleotide-binding domains separated. The viral inhibitor ICP47 forms a long helical hairpin, which plugs the translocation pathway of TAP from the cytoplasmic side. Association of ICP47 precludes substrate binding and prevents nucleotide-binding domain closure necessary for ATP hydrolysis. This work illustrates a striking example of immune evasion by persistent viruses. By blocking viral antigens from entering the endoplasmic reticulum, herpes simplex virus is hidden from cytotoxic T lymphocytes, which may contribute to establishing a lifelong infection in the host., Inside our body, every nucleated cell has surface 'barcodes' that are surveyed by the immune system. These barcodes are peptides derived from intracellular proteins, presented on the surface by MHC [...]

Author(s): Jan H. Lui [1]; Tomasz J. Nowakowski [1]; Alex A. Pollen [1]; Ashkan Javaherian [1]; Arnold R. Kriegstein (corresponding author) [1]; Michael C. Oldham (corresponding author) [1] Evolutionary expansion [...]

Autosomal dominant mutations of the RNA/DNA binding protein FUS are linked to familial amyotrophic lateral sclerosis (FALS); however, it is not clear how FUS mutations cause neurodegeneration. Using transgenic mice expressing a common FALS-associated FUS mutation (FUS-R521C mice), we found that mutant FUS proteins formed a stable complex with WT FUS proteins and interfered with the normal interactions between FUS and histone deacetylase 1 (HDAC1). Consequently, FUS-R521C mice exhibited evidence of DNA damage as well as profound dendritic and synaptic phenotypes in brain and spinal cord. To provide insights into these defects, we screened neural genes for nucleotide oxidation and identified brain-derived neurotrophic factor (Bdnf ) as a target of FUS-R521C--associated DNA damage and RNA splicing defects in mice. Compared with WT FUS, mutant FUS-R521C proteins formed a more stable complex with Bdnf RNA in electrophoretic mobility shift assays. Stabilization of the FUS/Bdnf RNA complex contributed to Bdnf splicing defects and impaired BDNF signaling through receptor TrkB. Exogenous BDNF only partially restored dendrite phenotype in FUS-R521C neurons, suggesting that BDNF-independent mechanisms may contribute to the defects in these neurons. Indeed, RNA-seq analyses of FUS-R521C spinal cords revealed additional transcription and splicing defects in genes that regulate dendritic growth and synaptic functions. Together, our results provide insight into how gain-of-function FUS mutations affect critical neuronal functions., Introduction Autosomal dominant mutations in RNA/DNA binding protein fused in sarcoma/translocated in liposarcoma (FUS/TLS) have been causally linked to familial ALS (FALS). The main pathological features in FALS with FUS [...]

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. [ABSTRACT FROM AUTHOR]

Author(s): Shawn F. Sorrells [1, 2]; Mercedes F. Paredes [1, 3]; Arantxa Cebrian-Silla [4]; Kadellyn Sandoval [1, 3]; Dashi Qi [5]; Kevin W. Kelley [1]; David James [1]; Simone Mayer [...]