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The dataset contains leaf venation architecture and functional traits for a phylogenetically diverse set of 122 plant species (including ferns, basal angiosperms, monocots, basal eudicots, asterids, and rosids) collected from the living collections of the University of California Botanical Garden at Berkeley (37.87° N, 122.23° W; CA, USA) from February to September 2021. The sampled species originated from all continents, except Antarctica, and are distributed in different growth forms (aquatic, herb, climbing, tree, shrub). The functional dataset comprises 31 traits (mechanical, hydraulic, anatomical, physiological, economical, and chemical) and describes six main leaf functional axes (hydraulic conductance, resistance and resilience to damages caused by drought and herbivory, mechanical support, and construction cost). It also describes how architecture features vary across venation networks. Our trait dataset is suitable for (1) functional and architectural characterization of plant species; (2) identification of venation architecture-function trade-offs; (3) investigation of evolutionary trends in leaf venation networks; and (4) mechanistic modeling of leaf function. Data are made available under the Open Data Commons Attribution License., (© 2024 The Ecological Society of America.)

Variation in leaf venation network architecture may reflect trade-offs among multiple functions including efficiency, resilience, support, cost, and resistance to drought and herbivory. However, our knowledge about architecture-function trade-offs is mostly based on studies examining a small number of functional axes, so we still lack a more integrative picture of multidimensional trade-offs. Here, we measured architecture and functional traits on 122 ferns and angiosperms species to describe how trade-offs vary across phylogenetic groups and vein spatial scales (small, medium, and large vein width) and determine whether architecture traits at each scale have independent or integrated effects on each function. We found that generalized architecture-function trade-offs are weak. Architecture strongly predicts leaf support and damage resistance axes but weakly predicts efficiency and resilience axes. Architecture traits at different spatial scales contribute to different functional axes, allowing plants to independently modulate different functions by varying network properties at each scale. This independence of vein architecture traits within and across spatial scales may enable evolution of multiple alternative leaf network designs with similar functioning., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Understanding the diversity of gastrointestinal (GI) immune cells, especially in the muscularis propria, is crucial for understanding their role in the maintenance of enteric neurons and smooth muscle and their contribution to GI motility. Here, we present a detailed protocol for isolating single immune cells from the human gastric muscularis propria. We describe steps for tissue preservation, dissection, and dissociation of the muscularis propria. We then detail procedures for magnetic sorting of CD45 + cells and single-cell RNA sequencing (scRNA-seq) analysis. For complete details on the use and execution of this protocol, please refer to Chikkamenahalli et al. 1 ., Competing Interests: Declaration of interests M.G. has received investigator-initiated grant funding from Takeda Pharmaceuticals and clinical trial funding from Alexza and Donga Pharmaceuticals. He serves as a consultant for Alfasigma, Ardelyx, and Evoke Pharmaceuticals., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Astrocytic receptors influence cognitive function and can promote behavioral deficits in disease. These effects may vary based on variables such as biological sex, but it is not known if the effects of astrocytic receptors are dependent on sex. We leveraged in vivo gene editing and chemogenetics to examine the roles of astrocytic receptors in spatial memory and other processes. We show that reductions in metabotropic glutamate receptor 3 (mGluR3), the main astrocytic glutamate receptor in adults, impair memory in females but enhance memory in males. Similarly, increases in astrocytic mGluR3 levels have sex-dependent effects and enhance memory in females. mGluR3 manipulations also alter spatial search strategies during recall in a sex-specific manner. In addition, acute chemogenetic stimulation of G i/o -coupled or G s -coupled receptors in hippocampal astrocytes induces bidirectional and sex-dimorphic effects on memory. Thus, astrocytes are sex-dependent modulators of cognitive function and may promote sex differences in aging and disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Xylem conduits have lignified walls to resist crushing pressures. The thicker the double-wall (T) relative to its diameter (D), the greater the implosion safety. Having safer conduits may incur higher costs and reduced flow, while having less resistant xylem may lead to catastrophic collapse under drought. Although recent studies have shown that conduit implosion commonly occurs in leaves, little is known about how leaf xylem scales T vs D to trade off safety, flow efficiency, mechanical support, and cost. We measured T and D in > 7000 conduits of 122 species to investigate how T vs D scaling varies across clades, habitats, growth forms, leaf, and vein sizes. As conduits become wider, their double-cell walls become proportionally thinner, resulting in a negative allometry between T and D. That is, narrower conduits, which are usually subjected to more negative pressures, are proportionally safer than wider ones. Higher implosion safety (i.e. higher T/D ratios) was found in asterids, arid habitats, shrubs, small leaves, and minor veins. Despite the strong allometry, implosion safety does not clearly trade off with other measured leaf functions, suggesting that implosion safety at whole-leaf level cannot be easily predicted solely by individual conduits' anatomy., (© 2024 The Authors New Phytologist © 2024 New Phytologist Foundation.)

Importance: We analyzed over 22,000 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes of patient samples tested at Mayo Clinic Laboratories during a 2-year period in the COVID-19 pandemic, which included Alpha, Delta, and Omicron variants of concern to examine the roles and relationships of Minnesota virus transmission. We found that Hennepin County, the most populous county, drove the transmission of SARS-CoV-2 viruses in the state after including the formation of earlier clades including 20A, 20C, and 20G, as well as variants of concern Alpha and Delta. We also found that Hennepin County was the source for most of the county-to-county introductions after an initial predicted introduction with the virus in early 2020 from an international source, while other counties acted as transmission "sinks." In addition, major policies, such as the end of the lockdown period in 2020 or the end of all restrictions in 2021, did not appear to have an impact on virus diversity across individual counties., Competing Interests: The authors declare no conflict of interest.

How to identify follicular lymphoma (FL) patients with low disease burden but high risk for early progression is unclear. Building on a prior study demonstrating the early transformation of FLs with high variant allele frequency (VAF) BCL2 mutations at activation-induced cytidine deaminase (AICDA) sites, we examined 11 AICDA mutational targets, including BCL2, BCL6, PAX5, PIM1, RHOH, SOCS, and MYC, in 199 newly diagnosed grade 1 and 2 FLs. BCL2 mutations with VAF ≥20% occurred in 52% of cases. Among 97 FL patients who did not initially receive rituximab-containing therapy, nonsynonymous BCL2 mutations at VAF ≥20% were associated with increased transformation risk (HR 3.01, 95% CI 1.04-8.78, p = 0.043) and a trend toward shorter event-free survival (EFS, median 20 months with mutations versus 54 months without, p = 0.052). Other sequenced genes were less frequently mutated and did not increase the prognostic value of the panel. Across the entire population, nonsynonymous BCL2 mutations at VAF ≥20% were associated with decreased EFS (HR 1.55, 95% CI 1.02-2.35, p = 0.043 after correction for FLIPI and treatment) and decreased overall survival after median 14-year follow-up (HR 1.82, 95% CI 1.05-3.17, p = 0.034). Thus, high VAF nonsynonymous BCL2 mutations remain prognostic even in the chemoimmunotherapy era., (© 2023. The Author(s).)

SARS-CoV-2 has had an unprecedented impact on human health and highlights the need for genomic epidemiology studies to increase our understanding of virus evolution and spread, and to inform policy decisions. We sequenced viral genomes from over 22,000 patient samples tested at Mayo Clinic Laboratories between 2020-2022 and use Bayesian phylodynamics to describe county and regional spread in Minnesota. The earliest introduction into Minnesota was to Hennepin County from a domestic source around January 22, 2020; six weeks before the first confirmed case in the state. This led to the virus spreading to Northern Minnesota, and eventually, the rest of the state. International introductions were most abundant in Hennepin (home to the Minneapolis/St. Paul International (MSP) airport) totaling 45 (out of 107) over the two-year period. Southern Minnesota counties were most common for domestic introductions with 19 (out of 64), potentially driven by bordering states such as Iowa and Wisconsin as well as Illinois which is nearby. Hennepin also was, by far, the most dominant source of in-state transmissions to other Minnesota locations (n=772) over the two-year period. We also analyzed the diversity of the location source of SARS-CoV-2 viruses in each county and noted the timing of state-wide policies as well as trends in clinical cases. Neither the number of clinical cases or major policy decisions, such as the end of the lockdown period in 2020 or the end of all restrictions in 2021, appeared to have impact on virus diversity across each individual county., Competing Interests: Competing interests The authors declare no competing interests.

Aim: Whole-genome methylation sequencing carries both DNA methylation and structural variant information (single nucleotide variant [SNV]; copy number variant [CNV]); however, limited data is available on the reliability of obtaining this information simultaneously from low-input DNA using various library preparation and sequencing protocols. Methods: A HapMap NA12878 sample was sequenced with three protocols (EM-sequencing, QIA-sequencing and Swift-sequencing) and their performance was compared on CpG methylation measurement and SNV and CNV detection. Results: At low DNA input (10-25 ng), EM-sequencing was superior in almost all metrics except CNV detection where all protocols were similar. EM-sequencing captured the highest number of CpGs and true SNVs. Conclusion: EM-sequencing is suitable to detect methylation, SNVs and CNVs from single sequencing with low-input DNA.

Background: Formalin-fixed, paraffin-embedded (FFPE) tissues have many advantages for identification of risk biomarkers, including wide availability and potential for extended follow-up endpoints. However, RNA derived from archival FFPE samples has limited quality. Here we identified parameters that determine which FFPE samples have the potential for successful RNA extraction, library preparation, and generation of usable RNAseq data., Methods: We optimized library preparation protocols designed for use with FFPE samples using seven FFPE and Fresh Frozen replicate pairs, and tested optimized protocols using a study set of 130 FFPE biopsies from women with benign breast disease. Metrics from RNA extraction and preparation procedures were collected and compared with bioinformatics sequencing summary statistics. Finally, a decision tree model was built to learn the relationship between pre-sequencing lab metrics and qc pass/fail status as determined by bioinformatics metrics., Results: Samples that failed bioinformatics qc tended to have low median sample-wise correlation within the cohort (Spearman correlation < 0.75), low number of reads mapped to gene regions (< 25 million), or low number of detectable genes (11,400 # of detected genes with TPM > 4). The median RNA concentration and pre-capture library Qubit values for qc failed samples were 18.9 ng/ul and 2.08 ng/ul respectively, which were significantly lower than those of qc pass samples (40.8 ng/ul and 5.82 ng/ul). We built a decision tree model based on input RNA concentration, input library qubit values, and achieved an F score of 0.848 in predicting QC status (pass/fail) of FFPE samples., Conclusions: We provide a bioinformatics quality control recommendation for FFPE samples from breast tissue by evaluating bioinformatic and sample metrics. Our results suggest a minimum concentration of 25 ng/ul FFPE-extracted RNA for library preparation and 1.7 ng/ul pre-capture library output to achieve adequate RNA-seq data for downstream bioinformatics analysis., (© 2022. The Author(s).)

Understanding the impact of long-term exposure of microorganisms to space is critical in understanding how these exposures impact the evolution and adaptation of microbial life under space conditions. In this work we subjected Nostoc sp. CCCryo 231-06, a cyanobacterium capable of living under many different ecological conditions, and also surviving in extreme ones, to a 23-month stay at the International Space Station (the Biology and Mars Experiment, BIOMEX, on the EXPOSE-R2 platform) and returned it to Earth for single-cell genome analysis. We used microfluidic technology and single cell sequencing to identify the changes that occurred in the whole genome of single Nostoc cells. The variant profile showed that biofilm and photosystem associated loci were the most altered, with an increased variant rate of synonymous base pair substitutions. The cause(s) of these non-random alterations and their implications to the evolutionary potential of single bacterial cells under long-term cosmic exposure warrants further investigation., (© 2022. The Author(s).)

The Nostoc sp. strain CCCryo 231-06 is a cyanobacterial strain capable of surviving under extreme conditions and thus is of great interest for the astrobiology community. The knowledge of its complete genome sequence would serve as a guide for further studies. However, a major concern has been placed on the effects of contamination on the quality of sequencing data without a reference genome. Here, we report the use of microfluidic technology combined with single cell sequencing and de novo assembly to minimize the contamination and recover the complete genome of the Nostoc strain CCCryo 231-06 with high quality. 100% of the whole genome was recovered with all contaminants removed and a strongly supported phylogenetic tree. The data reported can be useful for comparative genomics for phylogenetic and taxonomic studies. The method used in this work can be applied to studies that require high-quality assemblies of genomes of unknown microorganisms., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Background: Archival formalin-fixed, paraffin-embedded (FFPE) tissue samples with clinical and histological data are a singularly valuable resource for developing new molecular biomarkers. However, transcriptome analysis remains challenging with standard mRNA-seq methods as FFPE derived-RNA samples are often highly modified and fragmented. The recently developed 3' mRNA-seq method sequences the 3' region of mRNA using unique molecular identifiers (UMI), thus generating gene expression data with minimal PCR bias. In this study, we evaluated the performance of 3' mRNA-Seq using Lexogen QuantSeq 3' mRNA-Seq Library Prep Kit FWD with UMI, comparing with TruSeq Stranded mRNA-Seq and RNA Exome Capture kit. The fresh-frozen (FF) and FFPE tissues yielded nucleotide sizes range from 13 to > 70% of DV200 values; input amounts ranged from 1 ng to 100 ng for validation., Results: The total mapped reads of QuantSeq 3' mRNA-Seq to the reference genome ranged from 99 to 74% across all samples. After PCR bias correction, 3 to 56% of total sequenced reads were retained. QuantSeq 3' mRNA-Seq data showed highly reproducible data across replicates in Universal Human Reference RNA (UHR, R > 0.94) at input amounts from 1 ng to 100 ng, and FF and FFPE paired samples (R = 0.92) at 10 ng. Severely degraded FFPE RNA with ≤30% of DV200 value showed good concordance (R > 0.87) with 100 ng input. A moderate correlation was observed when directly comparing QuantSeq 3' mRNA-Seq data with TruSeq Stranded mRNA-Seq (R = 0.78) and RNA Exome Capture data (R > 0.67)., Conclusion: In this study, QuantSeq 3' mRNA-Seq with PCR bias correction using UMI is shown to be a suitable method for gene quantification in both FF and FFPE RNAs. 3' mRNA-Seq with UMI may be applied to severely degraded RNA from FFPE tissues generating high-quality sequencing data., (© 2021. The Author(s).)

Background: Benign breast disease (BBD) is a risk factor for breast cancer (BC); however, little is known about the genetic alterations present at the time of BBD diagnosis and how these relate to risk of incident BC., Methods: A subset of a long-term BBD cohort was selected to examine DNA variation across three BBD groups (42 future estrogen receptor-positive (ER+) BC, 36 future estrogen receptor-negative (ER-) BC, and 42 controls cancer-free for at least 16 years post-BBD). DNA extracted from archival formalin fixed, paraffin-embedded (FFPE) tissue blocks was analyzed for presence of DNA alterations using a targeted panel of 93 BC-associated genes. To address artifacts frequently observed in FFPE tissues (e.g., C>T changes), we applied three filtering strategies based on alternative allele frequencies and nucleotide substitution context. Gene-level associations were performed using two types of burden tests and adjusted for clinical and technical covariates., Results: After filtering, the variant frequency of SNPs in our sample was highly consistent with population allele frequencies reported in 1 KG/ExAC (0.986, p < 1e-16). The top ten genes found to be nominally associated with later cancer status by four of 12 association methods(p < 0.05) were MED12, MSH2, BRIP1, PMS1, GATA3, MUC16, FAM175A, EXT2, MLH1 and TGFB1, although these were not statistically significant in permutation testing. However, all 10 gene-level associations had OR < 1 with lower mutation burden in controls compared to cases, which was marginally statistically significant in permutation testing (p = 0.04). Comparing between the three case groups, BBD ER+ cases were closer to controls in mutation profile, while BBD ER- cases were distinct. Notably, the variant burden was significantly higher in controls than in either ER+ or ER- cases. CD45 expression was associated with mutational burden (p < 0.001)., Conclusions: Somatic mutations were more frequent in benign breast tissue from women who did not develop cancer, opening questions of clonal diversity or immune-mediated restraint on future cancer development. CD45 expression was positively associated with mutational burden, most strongly in controls. Further studies in both normal and premalignant tissues are needed to better understand the role of somatic gene mutations and their contribution to future cancer development., (© 2021. The Author(s).)

Electronic nicotine delivery systems (ENDS), or e-cigarettes, are emerging tobacco products that produce aerosols by heating e-liquids, which most often consist of propylene glycol and vegetable glycerin along with various flavoring compounds, bypassing the combustion that occurs in the use of traditional tobacco cigarettes. These products have seen a drastic increase in popularity in recent years both as smoking cessation devices as well as among younger generations, due in large part to the widespread perception among consumers that e-cigs are significantly less harmful to health than traditional tobacco cigarettes. Due to the novelty of ENDS as well as their rapidly increasing use, research into biomarkers of e-cig exposure and toxicity have lagged behind their popularity, leaving important questions about their potential toxicity unanswered. Research into potential biomarkers of acute and chronic e-cig use, and e-cigarette- or vaping-associated lung injury is necessary for informing both clinical and regulatory decision-making. We aim to provide an updated review of recent research into potential circulating, genomic, transcriptomic, and epigenetic biomarkers of exposure to and toxicity of e-cigs. We additionally highlight research areas that warrant additional study to gain a better understanding of health risks associated with ENDS use, as well as to provide validation of existing data and methods for measuring and analyzing e-cig-associated biomarkers in human and animal biofluids, tissues, and cells. This review also highlights ongoing efforts within the WNY Center for Research on Flavored Tobacco for research into novel biomarkers in extracellular vesicles that may be associated with short- and long-term ENDS use.

Electronic cigarette (e-cig) vaping is increasing rapidly in the United States, as e-cigs are considered less harmful than combustible cigarettes. However, limited research has been conducted to understand the possible mechanisms that mediate toxicity and pulmonary health effects of e-cigs. We hypothesized that sub-chronic e-cig exposure induces inflammatory response and dysregulated repair/extracellular matrix (ECM) remodeling, which occur through the α7 nicotinic acetylcholine receptor (nAChRα7). Adult wild-type (WT), nAChRα7 knockout (KO), and lung epithelial cell-specific KO (nAChRα7 CreCC10) mice were exposed to e-cig aerosol containing propylene glycol (PG) with or without nicotine. Bronchoalveolar lavage fluids (BALF) and lung tissues were collected to determine e-cig induced inflammatory response and ECM remodeling, respectively. Sub-chronic e-cig exposure with nicotine increased inflammatory cellular influx of macrophages and T-lymphocytes including increased pro-inflammatory cytokines in BALF and increased SARS-Cov-2 Covid-19 ACE2 receptor, whereas nAChRα7 KO mice show reduced inflammatory responses associated with decreased ACE2 receptor. Interestingly, matrix metalloproteinases (MMPs), such as MMP2, MMP8 and MMP9, were altered both at the protein and mRNA transcript levels in female and male KO mice, but WT mice exposed to PG alone showed a sex-dependent phenotype. Moreover, MMP12 was increased significantly in male mice exposed to PG with or without nicotine in a nAChRα7-dependent manner. Additionally, sub-chronic e-cig exposure with or without nicotine altered the abundance of ECM proteins, such as collagen and fibronectin, significantly in a sex-dependent manner, but without the direct role of nAChRα7 gene. Overall, sub-chronic e-cig exposure with or without nicotine affected lung inflammation and repair responses/ECM remodeling, which were mediated by nAChRα7 in a sex-dependent manner.

Electronic cigarette (e-cig) vaping is increasing rapidly in the United States, as e-cigs are considered less harmful than combustible cigarettes. However, limited research has been conducted to understand the possible mechanism that mediate, toxicity and pulmonary health effects of e-cigs. We hypothesized that sub-chronic e-cig exposure induces inflammatory response and dysregulated repair/extracellular matrix (ECM) remodeling, which occur through the α7 nicotinic acetylcholine receptor (nAChR α7). Adult wild-type (WT), nAChRα7 knockout (KO), and lung epithelial cell-specific KO (nAChRα7 CreCC10) mice were exposed to e-cig aerosol containing propylene glycol (PG) with or without nicotine. Bronchoalveolar lavage fluids (BALF) and lungs tissues were collected to determine e-cig induced inflammatory response and ECM remodeling, respectively. Sub-chronic e-cig exposure with nicotine increased the inflammatory cellular influx of macrophages and T-lymphocytes including increased pro-inflammatory cytokines in BALF and increased ACE2 Covid-19 receptor, whereas nAChR α7 KO mice show reduced inflammatory responses associated with decreased ACE2 receptor. Interestingly, matrix metalloproteinases (MMPs), such as MMP2, MMP8, and MMP9 were altered both at the protein and mRNA transcript levels in female and male, but WT mice exposed to PG alone showed a sex-dependent phenotype. Moreover, MMP12 was increased significantly in male mice exposed to PG with or without nicotine in a nAChR α7-dependent manner. Additionally, sub-chronic e-cig exposure with or without nicotine altered the abundance of ECM proteins, such as collagen and fibronectin significantly in a sex-dependent manner, but without the direct role of nAChR α7 gene. Overall, sub-chronic e-cig exposure with or without nicotine affected lung inflammation and repair responses/ECM remodeling, which were mediated by nAChR α7 in a sex-dependent manner., Competing Interests: Competing interests The authors have declared that no competing interest.

Background: Waterpipe tobacco (WPT) smoking is associated with deleterious effects on cardio-pulmonary systems which may have adverse repercussions in pathophysiology and progression of chronic lung and cardiovascular diseases. We compared the biomarkers of systemic inflammation, lipid mediators, injury/repair and oxidative stress between groups of non-smokers (NS), exclusive WPT smokers (WPS), exclusive cigarette smokers (CS) and dual WPS and CS (DS)., Methods: Two cohorts were recruited. Cohort I consisted of WPS (n=12), CS (n=26), DS (n=10) and NS (n=25). Cohort II consisted of WPS (n=33) and NS (n=24). Plasma and urine samples were collected and analysed for various systemic biomarkers., Results: Compared with NS, plasma levels of inflammatory mediators (interleukin (IL)-6, IL-8, IL1β and tumor necrosis factor-α) were significantly higher in WPS and CS, and were further augmented in DS. Endothelial biomarkers (intracellular adhesion molecule-1, prostaglandin E-2 and metalloproteinase-9) were significantly higher in CS. Most notably, pro-resolving lipid mediator (resolvin E1) and biomarkers of immunity, tissue injury, and repair were significantly lower in WPS and CS. Urinary levels of 8-isoprostane were significantly higher in all smoking groups in cohort I, while 8-isoprostane, myeloperoxidase, receptor for advanced glycation end products (RAGE), En-RAGE and matrix metalloproteinase-9 were significantly higher in all smoking groups in cohort II., Conclusions: Biomarkers of inflammation, oxidative stress, immunity, tissue injury and repair were elevated in WPS and CS groups. Furthermore, concurrent use of WPT and cigarettes is more harmful than cigarette or WPT smoking alone. These data may help inform the public and policy-makers about the dangers of WPT smoking and dual use of tobacco products., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Electronic cigarettes (e-cigs) were introduced as electronic nicotine delivery systems, and have become very popular in the USA and globally. There is a paucity of data on systemic injury biomarkers of vaping in e-cig users that can be used as a noninvasive assessment of vaping-associated lung injuries. We hypothesised that characterisation of systemic biomarkers of inflammation, anti-inflammatory, oxidative stress, vascular and lipid mediators, growth factors, and extracellular matrix breakdown may provide information regarding the toxicity of vaping in e-cig users., Methods: We collected various biological fluids, i.e. plasma, urine, saliva and exhaled breath condensate (EBC), measured pulmonary function and vaping characteristics, and assessed various biomarkers in e-cig users and nonusers., Results: The plasma samples of e-cig users showed a significant increase in biomarkers of inflammation (interleukin (IL)-1β, IL-6, IL-8, IL-13, interferon (IFN)-γ, matrix metalloproteinase-9, intercellular cell adhesion molecule-1) and extracellular matrix breakdown (desmosine), and decreased pro-resolving lipid mediators (resolvin D 1 and resolvin D 2 ). There was a significant increase in growth factor (endothelial growth factor, vascular endothelial growth factor, β-nerve growth factor, platelet-derived growth factor-AA, stem cell factor, hepatocyte growth factor and placental growth factor) levels in plasma of e-cig users versus nonusers. E-cig users showed a significant increase in levels of inflammatory biomarker IFN-γ, oxidative stress biomarker 8-isoprostane and oxidative DNA damage biomarker 8-oxo-dG in urine samples, and of inflammatory biomarker IL-1β in saliva samples. EBC showed a slight increase in levels of triglycerides and 8-isoprostane in e-cig users compared with normal nonusers., Conclusion: E-cig users have increased levels of biomarkers of inflammation and oxidative stress, reduced pro-resolving anti-inflammatory mediators, and endothelial dysfunction, which may act as risk factors for increasing susceptibility to systemic diseases. The identified noninvasive biomarkers can be used for determining e-cig vaping-associated lung injuries, and for regulatory and diagnostic aspects of vaping in humans., Competing Interests: Conflict of interest: K.P. Singh has nothing to disclose. Conflict of interest: G. Lawyer has nothing to disclose. Conflict of interest: T. Muthumalage has nothing to disclose. Conflict of interest: K.P. Maremanda has nothing to disclose. Conflict of interest: N.A. Khan has nothing to disclose. Conflict of interest: S.R. McDonough has nothing to disclose. Conflict of interest: D. Ye has nothing to disclose. Conflict of interest: S. McIntosh has nothing to disclose. Conflict of interest: I. Rahman has nothing to disclose., (Copyright ©ERS 2019.)

Nicotine inhalation via electronic cigarettes (e-cigs) is an emerging concern. However, little is known about the acute toxicity in the lungs following inhalation of nicotine-containing e-cig aerosols. We hypothesized that acute exposure to aerosolized nicotine causes lung toxicity by eliciting inflammatory and dysregulated repair responses. Adult C57BL/6J mice were exposed 2 h daily for 3 days to e-cig aerosols containing propylene glycol (PG) with or without nicotine. Acute exposure to nicotine-containing e-cig aerosols induced inflammatory cell influx (neutrophils and CD8a + T-lymphocytes), and release of pro-inflammatory cytokines in bronchoalveolar lavage fluid in a sex-dependent manner. Inhalation of e-cig aerosol containing PG alone significantly augmented the lung levels of various homeostasis/repair mediators (PPARγ, ADRP, ACTA2, CTNNB1, LEF1, β-catenin, E-cadherin, and MMP2) in a sex-dependent manner when compared to air controls. These findings were accompanied by an increase in protein abundance and altered gene expression of lipogenic markers (PPARγ, ADRP) and myogenic markers (fibronectin, α-smooth muscle actin and β-catenin), suggesting a dysregulated repair response in mouse lungs. Furthermore, exposure to nicotine containing e-cig aerosols or PG alone differentially affected the release of pro-inflammatory cytokines in healthy and COPD human 3D EpiAirway tissues. Overall, acute exposure to nicotine containing e-cig aerosols was sufficient to elicit a pro-inflammatory response and altered mRNA and protein levels of myogenic, lipogenic, and extracellular matrix markers in mouse lung in a sex-dependent manner. Thus, acute exposure to inhaled nicotine via e-cig leads to dysregulated repair and inflammatory responses, which may lead to airway remodeling in the lungs., Competing Interests: Conflicting financial interests: The authors have declared that no conflict of interest exists.

Background: Archived formalin fixed paraffin embedded (FFPE) samples are valuable clinical resources to examine clinically relevant morphology features and also to study genetic changes. However, DNA quality and quantity of FFPE samples are often sub-optimal, and resulting NGS-based genetics variant detections are prone to false positives. Evaluations of wet-lab and bioinformatics approaches are needed to optimize variant detection from FFPE samples., Results: As a pilot study, we designed within-subject triplicate samples of DNA derived from paired FFPE and fresh frozen breast tissues to highlight FFPE-specific artifacts. For FFPE samples, we tested two FFPE DNA extraction methods to determine impact of wet-lab procedures on variant calling: QIAGEN QIAamp DNA Mini Kit ("QA"), and QIAGEN GeneRead DNA FFPE Kit ("QGR"). We also used negative-control (NA12891) and positive control samples (Horizon Discovery Reference Standard FFPE). All DNA sample libraries were prepared for NGS according to the QIAseq Human Breast Cancer Targeted DNA Panel protocol and sequenced on the HiSeq 4000. Variant calling and filtering were performed using QIAGEN Gene Globe Data Portal. Detailed variant concordance comparisons and mutational signature analysis were performed to investigate effects of FFPE samples compared to paired fresh frozen samples, along with different DNA extraction methods. In this study, we found that five times or more variants were called with FFPE samples, compared to their paired fresh-frozen tissue samples even after applying molecular barcoding error-correction and default bioinformatics filtering recommended by the vendor. We also found that QGR as an optimized FFPE-DNA extraction approach leads to much fewer discordant variants between paired fresh frozen and FFPE samples. Approximately 92% of the uniquely called FFPE variants were of low allelic frequency range (< 5%), and collectively shared a "C > T|G > A" mutational signature known to be representative of FFPE artifacts resulting from cytosine deamination. Based on control samples and FFPE-frozen replicates, we derived an effective filtering strategy with associated empirical false-discovery estimates., Conclusions: Through this study, we demonstrated feasibility of calling and filtering genetic variants from FFPE tissue samples using a combined strategy with molecular barcodes, optimized DNA extraction, and bioinformatics methods incorporating genomics context such as mutational signature and variant allelic frequency.

Archival tissues represent a rich resource for clinical genomic studies, particularly when coupled with comprehensive medical records. Use of these in next generation sequencing (NGS) is a priority. Nine formalin-fixed paraffin-embedded (FFPE) DNA extraction methods were evaluated using twelve FFPE samples of varying tissue types. Quality assessment included total yield, percent dsDNA, fragment analysis and multiplex PCR. After assessment, three tissue types from four FFPE DNA methods were selected for NGS downstream evaluation, targeted and whole exome sequencing. In addition, two low input library protocols were evaluated for WES. Analysis revealed average coverage across the target regions for WES was ~20-30X for all four FFPE DNA extraction methods. For the targeted panels, the highest molecular tag coverage was obtained with the Kingfisher FFPE extraction method. The genotype concordance was 99% for the commonly called variant positions between all four extraction methods with the targeted PCR NGS panel and 96% with WES. Assessing quality of extracted DNA aids in selecting the optimal NGS approach, and the choice of both DNA extraction and library preparation approaches can impact the performance of archival tissue in NGS., Competing Interests: The authors have declared that no competing interests exist.

Prostate cancer (PCa) is the second most commonly diagnosed and sixth leading cause of cancer death in American men and one for which no curative therapy exists after metastasis. To meet this need for novel therapies, our laboratory has previously generated conditionally replicating adenovirus (CRAd) vectors expressing the sodium iodide symporter (hNIS). This virus transduced PCa cells and induced functional NIS expression, allowing for noninvasive tumor imaging and combination therapy with radioiodide, referred to as radiovirotherapy. We have now generated two new modified vectors to further improve efficacy. Ad5/3PB-ADP-hNIS and Ad5/3PB-hNIS include a hybrid Ad5/3 fiber knob to improve transduction efficiency, and express NIS from the endogenous major late promoter to restrict NIS expression to target cells. Additionally, Ad5/3PB-ADP-hNIS includes the adenovirus death protein (ADP), which hastens the release of viral particles after assembly. These two vectors specifically induce radioisotope uptake, cytopathic effect, and viral replication in androgen receptor-expressing PCa cell lines with Ad5/3PB-ADP-hNIS showing earlier (131)I uptake and cytolysis at low multiplicity of infection. SPECT-CT imaging of xenograft tumors infected with Ad5/3PB-hNIS showed steady uptake, whereas infection with Ad5/3PB-ADP-hNIS led to increasing uptake, indicating viral spread. Radiovirotherapy of xenograft LNCaP tumors with Ad5/3PB-ADP-hNIS showed the most significant survival extension versus control tumors (p=0.001), but the benefit of radiovirotherapy was not statistically significant compared with virotherapy alone in this model. These results show the potential of Ad5/3PB-ADP-hNIS as a vector for treatment of prostate cancer.

Background: Cholesterol homeostasis is maintained through finely tuned mechanisms regulating intestinal absorption, hepatic biosynthesis and secretion as well as plasma clearance. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted enzyme of the serine protease family that reduces cellular uptake of plasma low-density lipoprotein (LDL) cholesterol by promoting LDL receptor (LDL-R) degradation. Species-specific positive selection has been noted in the LDLR promoter, leading to differential expression of LDLR among primates. Whether PCSK9 experienced significant selective pressure to maintain a functional relationship with its target protein, LDL-R, is unknown., Methodology/principal Findings: We compiled the sequences of the coding regions of PCSK9 from 14 primate species in the clade of Hominoids, Old World monkeys and New World monkeys. To detect selective pressure at the protein level, the ratios of nonsynonymous/synonymous substitution rate (d(N)/d(S)) under different evolutionary models were calculated across the phylogeny of PCSK9. Maximum likelihood analyses of d(N)/d(S) ratios for the aligned coding region sequences among 14 primate species indicated that PCSK9 was subject to a strong functional constraint (i.e., purifying selection). However, positive selection was noted in the functional carboxyl-terminal (C-terminal) domain in many branches across the phylogeny, especially in the lineage leading to the orangutan. Furthermore, at least five positively selected amino acids were detected in this lineage using the branch-site model A. In a sliding-window analysis, several d(N)/d(S) peaks in the C-terminal domain in both the human and the orangutan branches were noted., Conclusions: These results suggest that among primates, differential selective pressure has shaped evolutionary patterns in the functional domains of PCSK9, an important regulator of cholesterol homeostasis.