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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]

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. [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]

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]

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]

Aerosol constituent yields have been reported from a wide range of electronic nicotine delivery systems. No comprehensive study has been published on the aerosol constituents generated from the JUUL system. Targeted analyses of 53 aerosol constituents from the four JUUL products currently on the US market (Virginia Tobacco and Menthol flavored e-liquids in both 5.0% and 3.0% nicotine concentration by weight) was performed using non-intense and intense puffing regimens. All measurements were conducted by an ISO 17025 accredited contract research organization. JUUL product aerosol constituents were compared to published values for the 3R4F research cigarette and IQOS Regular and Menthol heated tobacco products. Across the four JUUL products and two puffing regimes, only 10/53 analytes were quantifiable, including only two carbonyls (known propylene glycol or glycerol degradants). The remaining analytes were primary ingredients, nicotine degradants and water. Average analyte reductions (excluding primary ingredients and water) for all four JUUL system aerosols tested were greater than 98% lower than 3R4F mainstream smoke, and greater than 88% lower than IQOS aerosol. In summary, chemical characterization and evaluation of JUUL product aerosols demonstrates a significant reduction in toxicants when compared to mainstream cigarette smoke from 3R4F reference cigarettes or aerosols from IQOS-heated tobacco products. [ABSTRACT FROM AUTHOR]

Adult hippocampal neurogenesis was originally discovered in rodents. Subsequent studies identified the adult neural stem cells and found important links between adult neurogenesis and plasticity, behavior, and disease. However, whether new neurons are produced in the human dentate gyrus (DG) during healthy aging is still debated. We and others readily observe proliferating neural progenitors in the infant hippocampus near immature cells expressing doublecortin (DCX), but the number of such cells decreases in children and few, if any, are present in adults. Recent investigations using dual antigen retrieval find many cells stained by DCX antibodies in adult human DG. This has been interpreted as evidence for high rates of adult neurogenesis, even at older ages. However, most of these DCX-labeled cells have mature morphology. Furthermore, studies in the adult human DG have not found a germinal region containing dividing progenitor cells. In this Dual Perspectives article, we show that dual antigen retrieval is not required for the detection of DCX in multiple human brain regions of infants or adults. We review prior studies and present new data showing that DCX is not uniquely expressed by newly born neurons: DCX is present in adult amygdala, entorhinal and parahippocampal cortex neurons despite being absent in the neighboring DG. Analysis of available RNA-sequencing datasets supports the view that DG neurogenesis is rare or absent in the adult human brain. To resolve the conflicting interpretations in humans, it is necessary to identify and visualize dividing neuronal precursors or develop new methods to evaluate the age of a neuron at the single-cell level. [ABSTRACT FROM AUTHOR]

To assess potential exposure of non-users to exhaled constituents from pod and cartridge electronic nicotine delivery systems (ENDS) products, an environmental clinical study was conducted with (n = 43) healthy adult smokers. Room air concentrations of 34 selected constituents (nicotine, propylene glycol, glycerin, 15 carbonyls, 12 volatile organic compounds, and 4 trace metals) and particle number concentration (0.3 to 25 µm) were compared from use of two ENDS products and conventional cigarettes using room ventilations representative of a residential, an office or a hospitality setting over a 4-h. exposure period. Products used were JUUL ENDS, Virginia Tobacco flavor (Group I), VUSE Solo, Original flavor (Group II) (5.0 and 4.8% nicotine by weight, respectively) and subjects' own conventional cigarettes (Group III). Cumulative 4-h room air sampling and particle counting were performed during prescribed (Groups I and II) and ad libitum product use (all Groups). Conventional cigarette use resulted in significantly more constituents detected and higher 4-h cumulative constituent concentrations compared to use of the ENDS products tested, except for the predominant ENDS ingredients, propylene glycol and glycerin. Use of conventional cigarettes also resulted in greater total particle number concentration than either prescribed or ad libitum use of either of the ENDS used in this study. [ABSTRACT FROM AUTHOR]

Inula racemosa is reported as new to Canada, with occurrences in Ontario and Québec. It is also reported as new for several states in the United States. It was first reported for North America in 2020, based on a 2017 collection in Vermont. Earlier collections from Québec (2004) and Michigan (2014) were subsequently found, however. Its distinctive appearance led to the identification of additional records on the web page iNaturalist, the postings often initially misidentified as the common I. helenium. Almost two dozen North American occurrences are now known from Illinois, New Hampshire, New York, Ontario (the largest cluster of occurrences), Québec, Washington, and Wisconsin. Several of these populations contain hundreds to thousands of plants. All populations are considered to likely be of recent origin. At least some represent escapes from cultivation for medicinal or ornamental purposes. Inula racemosa may show a preference for calcareous substrate and river shore sites in the wild. It does not appear to have substantial potential to be invasive into native plant communities. [ABSTRACT FROM AUTHOR]

We provide an update to the Ontario distribution of False Hop Sedge (Carex lupuliformis), prompted by the first documented report of the species in the Niagara region, which represents a noteworthy eastern range extension in the province. [ABSTRACT FROM AUTHOR]

A key to understanding biological response due to cell exposure to chemical constituents in aerosols is to accurately be able to determine the delivered dose. Deposition efficiency and uniformity of deposition was measured experimentally in the Vitrocell® 24/48 air–liquid-interface (ALI) in vitro exposure system using monodisperse solid fluorescent particles with mass median aerodynamic diameters (MMAD) of 0.51, 1.1, 2.2 and 3.3 µm. Experimental results were compared with computational fluid dynamics (CFD; using both Lagrangian and Eulerian approaches) predicted deposition efficiency and uniformity for a single row (N = 6) of cell culture inserts in the Vitrocell® 24/48 system. Deposited fluorescent monodisperse particles were quantified using fluorescent microscopy and Image J software. Experiments were conducted using a suspension of two particle MMADs with each experiment being conducted a total of three times on different days. The average experimentally measured deposition efficiency ranged from a low of 0.013% for 0.51 µm MMAD particles to a maximum 0.86% for 3.3 µm MMAD particles. There was good agreement between the average experimentally measured and the CFD predicted particle deposition efficiency (regardless of approach) with agreement being slightly better at the smaller MMADs. Experimentally measured and CFD predicted average uniformity of deposition was >45% of the mean and within 15% of the mean for 0.51 µm and 2.2 MMAD µm particles, respectively. Experimentally measured average uniformity of deposition was between 15 and 45% of the mean while CFD predictions were within 15% of the mean for 1.1 and 3.3 µm MMAD particles. The deposition efficiency and uniformity across the cell culture inserts for solid particles should be considered when designing exposure regimens using the Vitrocell® 24/48 ALI in vitro exposure system. Copyright © 2019 American Association for Aerosol Research [ABSTRACT FROM AUTHOR]

Purpose: It is reported that a single bout of exercise can lower insulin responses 12-24 h post-exercise; however, the insulin responses to alternate or consecutive bouts of exercise is unknown. Thus, the purpose of this study was to examine the effect of exercise pattern on post-exercise insulin and glucose responses following a glucose challenge.Methods: Ten male participants (n = 10, mean ± SD, Age 29.5 ± 7.7 years; BMI 25.7 ± 3.0 kg/m2) completed three exercise trials of walking for 60 min at ~ 70% of VO2max. The trials consisted of: three consecutive exercise days (3CON), three alternate exercise days (3ALT), a single bout of exercise (SB), and a no exercise control (R). Twelve to fourteen hours after the last bout of exercise or R, participants completed a 75 g oral glucose tolerance test (OGTT) and blood was collected at 30 min intervals for the measurement of glucose, insulin, and C-peptide.Result: Calculated incremental area under the curve (iAUC) for glucose and C-peptide was not different between the four trials. Insulin iAUC decreased 34.9% for 3CON compared to R (p < 0.01).Conclusion: Three consecutive days of walking at ~ 70% VO2max improved insulin response following an OGTT compared to no exercise. It is possible, that for healthy males, the effect of a single bout of exercise or exercise bouts separated by more than 24 h may not be enough stimulus to lower insulin responses to a glucose challenge. [ABSTRACT FROM AUTHOR]

Background: Purified cannabidiol is a new antiepileptic drug that has recently been approved for use in patients with Lennox-Gastaut and Dravet syndromes, but most published studies have not extended beyond 12-16 weeks.Objective: The objective of this study was to evaluate the long-term safety, tolerability, and efficacy of cannabidiol in children with epilepsy.Methods: Patients aged 1-17 years with refractory epilepsy were enrolled in an open-label prospective study through individual patient and expanded access programs between April 2013 and December 2014. Seizure types were video-electroencephalogram confirmed prior to enrollment. After a 28-day evaluation period, during which baseline seizure frequency was assessed, cannabidiol was given as add-on therapy at 5 mg/kg/day and titrated weekly by 5-mg/kg increments to a dose of 25 mg/kg/day. Blood tests were performed at baseline, after 1, 2, and 3 months, and every 3 months thereafter. Trough concentrations of concomitant antiepileptic drugs were measured at baseline, after 1, 2, and 3 months of therapy, and as clinically indicated afterwards. Concomitant antiepileptic drugs, ketogenic diet ratio, and vagal nerve stimulator settings remained unchanged during the baseline period and the first 3 months of treatment, unless there was a significant increase in plasma concentrations. Seizure frequency was reported daily in seizure diaries by parents or caregivers. Clinical assessments occurred after 15 days of treatment, at 1 month, at 3 months, and every 3 months thereafter. Diaries of seizure frequency and adverse events were reviewed at each visit. The primary efficacy outcome was a reduction in seizure frequency and responders were defined as those patients achieving a > 50% reduction in motor seizures.Results: Twenty-six children were enrolled. Most had genetic epilepsies with daily or weekly seizures and multiple seizure types. All were refractory to prior antiepileptic drugs (range 4-11, mean 7), and were taking two antiepileptic drugs on average. Duration of therapy ranged from 4 to 53 months (mean 21 months). Adverse events were reported in 21 patients (80.8%), including reduced appetite in ten (38.4%), diarrhea in nine (34.6%), and weight loss in eight (30.7%). Four (15.4%) had changes in antiepileptic drug concentrations and three had elevated aspartate aminotransferase and alanine aminotransferase levels when cannabidiol was administered together with valproate. Serious adverse events, reported in six patients (23.1%), included status epilepticus in three, catatonia in two, and hypoalbuminemia in one. Fifteen patients (57.7%) discontinued cannabidiol for lack of efficacy, one because of status epilepticus, and one for severe weight loss. The retention rate declined rapidly in the first 6 months and more gradually thereafter. At 24 months, the number of patients continuing cannabidiol as adjunctive therapy was nine of the original 26 (34.6%). Of these patients, seven (26.9%) had a sustained > 50% reduction in motor seizures, including three (11.5%) who remain seizure free.Conclusion: Over a 4-year period, cannabidiol was effective in 26.9% of children with otherwise refractory epilepsy. It was well tolerated in about 20% of patients, but 80.8% had adverse events, including 23.1% with serious adverse events. Decreased appetite and diarrhea were frequent along with weight loss that became evident only later in the treatment. [ABSTRACT FROM AUTHOR]

We compared early biological changes in mice after inhalation exposures to cigarette smoke or e-vapor aerosols (MarkTen® cartridge with Carrier, Test-1, or Test-2 formulations; 4% nicotine). Female C57BL/6 mice were exposed to 3R4F cigarette smoke or e-vapor aerosols by nose-only inhalation for up to 4 hours/day, 5 days/week, for 3 weeks. The 3R4F and e-vapor exposures were set to match the target nose port aerosol nicotine concentration (∼41 µg/L). Only the 3R4F group showed postexposure clinical signs such as tremors and lethargy. At necropsy, the 3R4F group had significant increases in lung weight and changes in bronchoalveolar lavage parameters, as well as microscopic findings in the respiratory tract. The e-vapor groups had minimal microscopic changes, including squamous metaplasia in laryngeal epiglottis, and histiocytic infiltrates in the lung (Test-2 group only). The 3R4F group had a higher incidence and severity of microscopic findings compared to any e-vapor group. Transcriptomic analysis also showed that the 3R4F group had the highest number of differentially expressed genes compared to Sham Control. Among e-vapor groups, Test-2 group had more differentially expressed genes but the magnitude of gene expression-based network perturbations in all e-vapor groups was ∼94% less than the 3R4F group. On proteome analysis in the lung, differentially regulated proteins were detected in the 3R4F group only. In conclusion, 3-weeks of 3R4F exposure induced molecular and microscopic changes associated with smoking-related diseases in the respiratory tract, while e-vapor exposures showed substantially reduced biological activities. [ABSTRACT FROM AUTHOR]

Round-fruited St. John's-wort (Hypericum sphaerocarpum), a native North American herbaceous, perennial vascular plant, is reported from four sites in southern Ontario, Canada. All four sites are along abandoned railway lines. Although the rich association of native flora suggests native status at one site, H. sphaerocarpum is believed to be introduced elsewhere in its Canadian range in Ontario. [ABSTRACT FROM AUTHOR]

Summary: Objective: To evaluate the clinical efficacy and safety of quinidine in patients with KCNT1‐related epilepsy of infancy with migrating focal seizures (EIMFS) in the infantile period and to compare with the effect of quinidine on mutant channels in vitro. Methods: We identified 4 patients with EIMFS with onset in the neonatal period, pathogenic variants in the KCNT1 gene, and lack of response to AEDs. Patients were prospectively enrolled, treated with quinidine, and monitored according to a predefined protocol. Electroclinical, neuroimaging, and genetic data were reviewed. Two patients had novel variants in the KCNT1 gene that were modeled in Xenopus oocytes with channel properties characterized using electrophysiology recordings. Results: Three of four patients were treated with quinidine early in their disease course, prior to 6 months of age. No significant side effects were noted with quinidine therapy. In addition, there were no significant changes in electroencephalography (EEG)–confirmed seizure burden during therapy, and patients had near hourly seizures before, during, and after treatment. Two patients had previously reported gain‐of‐function mutations, which demonstrated sensitivity to high levels of quinidine in the oocyte assay. Two patients with novel variants, showed characteristic gain‐of‐function and were thus predicted to be pathogenic. Of interest, these variants were essentially insensitive to high levels of quinidine. Significance: Patients had no reported benefit to quinidine therapy despite age at treatment initiation. Pharmacogenetic results in oocytes were consistent with clinical treatment failure in 2 patients, suggesting that single‐dose pharmacologic assessment may be helpful in predicting which patients are exceedingly unlikely to achieve benefit with quinidine. In the 2 patients who had a lack of therapeutic benefit despite sensitivity to high concentrations of quinidine with in vitro oocyte assay, it is likely that the achievable exposure levels in the brain were too low to cause significant in vivo channel blockade. [ABSTRACT FROM AUTHOR]

New neurons continue to be generated in the subgranular zone of the dentate gyrus of the adult mammalian hippocampus. This process has been linked to learning and memory, stress and exercise, and is thought to be altered in neurological disease. In humans, some studies have suggested that hundreds of new neurons are added to the adult dentate gyrus every day, whereas other studies find many fewer putative new neurons. Despite these discrepancies, it is generally believed that the adult human hippocampus continues to generate new neurons. Here we show that a defined population of progenitor cells does not coalesce in the subgranular zone during human fetal or postnatal development. We also find that the number of proliferating progenitors and young neurons in the dentate gyrus declines sharply during the first year of life and only a few isolated young neurons are observed by 7 and 13 years of age. In adult patients with epilepsy and healthy adults (18-77 years; n = 17 post-mortem samples from controls; n = 12 surgical resection samples from patients with epilepsy), young neurons were not detected in the dentate gyrus. In the monkey (Macaca mulatta) hippocampus, proliferation of neurons in the subgranular zone was found in early postnatal life, but this diminished during juvenile development as neurogenesis decreased. We conclude that recruitment of young neurons to the primate hippocampus decreases rapidly during the first years of life, and that neurogenesis in the dentate gyrus does not continue, or is extremely rare, in adult humans. The early decline in hippocampal neurogenesis raises questions about how the function of the dentate gyrus differs between humans and other species in which adult hippocampal neurogenesis is preserved. [ABSTRACT FROM AUTHOR]

The purpose of this study was to test the hypothesis that high-intensity interval exercise (HIE) significantly increases growth hormone (GH) secretion to a greater extent than moderate-intensity continuous exercise (MOD) in young women. Five young, sedentary women (mean ± SD; age: 22.6 ± 1.3 years; BMI: 27.4 ± 3.1 kg/m²) were tested during the early follicular phase of their menstrual cycle on three occasions. For each visit, participants reported to the laboratory at 1700 h, exercised from 1730-1800 h, and remained in the laboratory until 0700 h the following morning. The exercise component consisted of either 30-min of moderate-intensity continuous cycling at 50% of measured peak power (MOD), four 30-s "all-out" sprints with 4.5 min of active recovery (HIE), or a time-matched sedentary control using a randomized, crossover design. The overnight GH secretory profile of each trial was determined from 10-min sampling of venous blood from 1730-0600 h, using deconvolution analysis. Deconvolution GH parameters were log transformed prior to statistical analyses. Calculated GH AUC (0-120 min) was significantly greater in HIE than CON (P = 0.04), but HIE was not different from MOD. Total GH secretory rate (ng/mL/12.5 h) was significantly greater in the HIE than the CON (P = 0.05), but MOD was not different from CON or HIE. Nocturnal GH secretion (ng/mL/7.5 h) was not different between the three trials. For these women, in this pilot study, a single bout of HIE was sufficient to increase 12.5 h pulsatile GH secretion. It remains to be determined if regular HIE may contribute to increased daily GH secretion. [ABSTRACT FROM AUTHOR]

Our objective was to evaluate the suitability of using a capillary aerosol generator (CAG) instead of using e-cigarette devices in 90-day or longer inhalation studies. Aerosol characteristics for both the CAG (which uses heat to produce a condensation aerosol) and e-cigarette generators have been previously reported, but a side-by-side comparison with the identical formulation has not been reported. Aerosols from both devices were analyzed immediately after generation for chemicals in the formulation (propylene glycol [PG], glycerin, water, and nicotine), selected carbonyls (acetaldehyde, acrolein, and formaldehyde) by ultra-performance liquid chromatography with ultraviolet detection (UPLC-UV), and a chemical fingerprint analysis using gas chromatography-mass spectroscopy (GC-MS). Aerosol capture methods for chemical analysis included Cambridge filter pads or two impingers in series each containing solution to trap and stabilize selected carbonyl compounds. Particle size distribution (cascade impactor) and exposure port uniformity (gravimetric) was measured in four rodent inhalation exposure chambers under inhalation study conditions. The aerosol of both generators contained the same known and unknown chemicals. Similar levels of compounds in the formula except for PG were detected in the aerosol of both generators. CAG produced more consistent particulate aerosol than e-cigarette generator and had lower levels of carbonyls primarily due to lower levels of formaldehyde. Exposure port concentrations were consistent and closer to target values with the CAG compared to the e-cigarette aerosol generator. CAG was easier to operate on a daily basis although more difficult to maintain because it required daily cleaning compared to single-use e-cigarettes. CAG was determined to be suitable for use in 90-day or longer inhalation studies. Copyright © 2016 American Association for Aerosol Research [ABSTRACT FROM PUBLISHER]

Objective To evaluate treatment responses in benign familial neonatal epilepsy ( BFNE). Methods We recruited patients with BFNE through a multicenter international collaboration and reviewed electroclinical and genetic details, and treatment response. All patients were tested at minimum for mutations/deletions in the KCNQ2, KCNQ3, and SCN2A genes. Results Nineteen patients were included in this study. A family history of neonatal seizures was positive in 16 patients, and one additional patient had a family history of infantile seizures. Mutations or deletions of KCNQ2 were found in 14, and of KCNQ3 in 2, of the 19 patients. In all patients, seizures began at 2-5 days of life and occurred multiple times per day. Four patients developed status epilepticus. Seizures were focal, alternating between hemispheres, and characterized by asymmetric tonic posturing associated with apnea and desaturation, followed by unilateral or bilateral asynchronous clonic jerking. Twelve of 19 patients were treated with multiple medications prior to seizure cessation. Seventeen of (88%) 19 patients were seizure-free within hours of receiving oral carbamazepine ( CBZ) or oxcarbazepine ( OXC). Earlier initiation of CBZ was associated with shorter hospitalization (p < 0.01). No side effects of CBZ were reported. All patients had normal development and remain seizure-free at a mean follow-up period of 7.8 years (6 months-16 years). Significance This study provides evidence that CBZ is safe and rapidly effective in neonates with BFNE, even in status epilepticus. We propose that CBZ should be the drug of choice in benign familial neonatal seizures. [ABSTRACT FROM AUTHOR]

Sexual dimorphism in common disease is pervasive, including a dramatic male preponderance in autism spectrum disorders (ASDs). Potential genetic explanations include a liability threshold model requiring increased polymorphism risk in females, sex-limited X-chromosome contribution, gene-environment interaction driven by differences in hormonal milieu, risk influenced by genes sex-differentially expressed in early brain development, or contribution from general mechanisms of sexual dimorphism shared with secondary sex characteristics. Utilizing a large single nucleotide polymorphism (SNP) dataset, we identify distinct sex-specific genome-wide significant loci. We investigate genetic hypotheses and find no evidence for increased genetic risk load in females, but evidence for sex heterogeneity on the X chromosome, and contribution of sex-heterogeneous SNPs for anthropometric traits to ASD risk. Thus, our results support pleiotropy between secondary sex characteristic determination and ASDs, providing a biological basis for sex differences in ASDs and implicating non brain-limited mechanisms. [ABSTRACT FROM AUTHOR]

The Eurasian Square-stalked St. John's-wort (Hypericum tetrapterum Fr.: Hypericaceae) was found growing in an open Eastern White Cedar (Thuja occidentalis L.) swamp in Caledon, Regional Municipality of Peel, Ontario. This is the first record for eastern North America; previous North American occurrences have been on the Pacific coast in Vancouver, British Columbia, Canada (1991), and in Wahkiakum County, Washington State, USA (2003). [ABSTRACT FROM AUTHOR]

While several reports have been published using research methods of estimating exposure risk to e-cigarette vapors in nonusers, only two have directly measured indoor air concentrations from vaping using validated industrial hygiene sampling methodology. Our first study was designed to measure indoor air concentrations of nicotine, menthol, propylene glycol, glycerol, and total particulates during the use of multiple e-cigarettes in a well-characterized room over a period of time. Our second study was a repeat of the first study, and it also evaluated levels of formaldehyde. Measurements were collected using active sampling, near real-time and direct measurement techniques. Air sampling incorporated industrial hygiene sampling methodology using analytical methods established by the National Institute of Occupational Safety and Health and the Occupational Safety and Health Administration. Active samples were collected over a 12-hr period, for 4 days. Background measurements were taken in the same room the day before and the day after vaping. Panelists (n = 185 Study 1; n = 145 Study 2) used menthol and non-menthol MarkTen prototype e-cigarettes. Vaping sessions (six, 1-hr) included 3 prototypes, with total number of puffs ranging from 36–216 per session. Results of the active samples were below the limit of quantitation of the analytical methods. Near real-time data were below the lowest concentration on the established calibration curves. Data from this study indicate that the majority of chemical constituents sampled were below quantifiable levels. Formaldehyde was detected at consistent levels during all sampling periods. These two studies found that indoor vaping of MarkTen prototype e-cigarette does not produce chemical constituents at quantifiable levels or background levels using standard industrial hygiene collection techniques and analytical methods. [ABSTRACT FROM PUBLISHER]

A prototype electronic cigaret device and three formulations were evaluated in a 90-day rat inhalation study followed by a 42-day recovery period. Animals were randomly assigned to groups for exposure to low-, mid- and high-dose levels of aerosols composed of vehicle (glycerin and propylene glycol mixture); vehicle and 2.0% nicotine; or vehicle, 2.0% nicotine and flavor mixture. Daily targeted aerosol total particulate matter (TPM) doses of 3.2, 9.6 and 32.0 mg/kg/day were achieved by exposure to 1 mg/L aerosol for 16, 48 and 160 min, respectively. Pre-study evaluations included indirect ophthalmoscopy, virology and bacteriological screening. Body weights, clinical observations and food consumption were monitored weekly. Plasma nicotine and cotinine and carboxyhemoglobin levels were measured at days 28 and 90. After days 28, 56 and 90, lung function measurements were obtained. Biological endpoints after 90-day exposure and 42-day recovery period included clinical pathology, urinalysis, bronchoalveolar fluid (BALF) analysis, necropsy and histopathology. Treatment-related effects following 90 days of exposure included changes in body weight, food consumption and respiratory rate. Dose-related decreases in thymus and spleen weights, and increased BALF lactate dehydrogenase, total protein, alveolar macrophages, neutrophils and lung weights were observed. Histopathology evaluations revealed sporadic increases in nasal section 1–4 epithelial hyperplasia and vacuolization. Following the recovery period, effects in the nose and BALF were persistent while other effects were resolved. The no observed effect level based upon body weight decreases is considered to be the mid-dose level for each formulation, equivalent to a daily TPM exposure dose of approximately 9.6 mg/kg/day. [ABSTRACT FROM PUBLISHER]

We report two populations of Trifolium resupinatum (Reversed Clover, trefle resupine) from southern Ontario, confirming it as established in Canada. This Eurasian and north African species was reported in the late 1800s in New Brunswick and Quebec, where it apparently did not persist. Its distribution across the United States is sporadic. [ABSTRACT FROM AUTHOR]

Summary The evolutionarily conserved miR-302 family of microRNAs is expressed during early mammalian embryonic development. Here, we report that deletion of miR-302a-d in mice results in a fully penetrant late embryonic lethal phenotype. Knockout embryos have an anterior neural tube closure defect associated with a thickened neuroepithelium. The neuroepithelium shows increased progenitor proliferation, decreased cell death, and precocious neuronal differentiation. mRNA profiling at multiple time points during neurulation uncovers a complex pattern of changing targets over time. Overexpression of one of these targets, Fgf15 , in the neuroepithelium of the chick embryo induces precocious neuronal differentiation. Compound mutants between mir-302 and the related mir-290 locus have a synthetic lethal phenotype prior to neurulation. Our results show that mir-302 helps regulate neurulation by suppressing neural progenitor expansion and precocious differentiation. Furthermore, these results uncover redundant roles for mir-290 and mir-302 early in development. [ABSTRACT FROM AUTHOR]

Tall Annual Willowherb (Epilobium brachycarpum C. Presl) is reported from Dryden, Kenora District, Ontario, Canada, based on a 2012 collection from a disturbed habitat where the species was presumably introduced. This is the first northern Ontario record of E. brachycarpum and the first Ontario record in more than 40 years. Previous Ontario records are discussed and mapped, and evidence for and against native status in the province is presented. Identification characteristics are provided, and distribution and habitat elsewhere in North America are discussed. [ABSTRACT FROM AUTHOR]

Evolutionary expansion of the human neocortex underlies many of our unique mental abilities. This expansion has been attributed to the increased proliferative potential of radial glia (RG; neural stem cells) and their subventricular dispersion from the periventricular niche during neocortical development. Such adaptations may have evolved through gene expression changes in RG. However, whether or how RG gene expression varies between humans and other species is unknown. Here we show that the transcriptional profiles of human and mouse neocortical RG are broadly conserved during neurogenesis, yet diverge for specific signalling pathways. By analysing differential gene co-expression relationships between the species, we demonstrate that the growth factor PDGFD is specifically expressed by RG in human, but not mouse, corticogenesis. We also show that the expression domain of PDGFRβ, the cognate receptor for PDGFD, is evolutionarily divergent, with high expression in the germinal region of dorsal human neocortex but not in the mouse. Pharmacological inhibition of PDGFD-PDGFRβ signalling in slice culture prevents normal cell cycle progression of neocortical RG in human, but not mouse. Conversely, injection of recombinant PDGFD or ectopic expression of constitutively active PDGFRβ in developing mouse neocortex increases the proportion of RG and their subventricular dispersion. These findings highlight the requirement of PDGFD-PDGFRβ signalling for human neocortical development and suggest that local production of growth factors by RG supports the expanded germinal region and progenitor heterogeneity of species with large brains. [ABSTRACT FROM AUTHOR]