ABSTRACT: Influenza Classification from Short Reads with VAPOR Facilitates Robust Mapping Pipelines and Zoonotic Strain Detection for Routine Surveillance Applications
Project description:The lung response to inhalation exposure to oil vapor particles was investigated in a rat model. Adult male Sprague-Dawley rats were exposed by whole-body inhalation to air or an aerosol containing oil vapor particles at concentrations of 300 ppm, 6 hours/day for 1 day (shot-term) or 300 ppm, 6 hours/day, 4 days/week for 4 weeks (long-term). The control and oil vapor exposed rats were euthanized at post-exposure time intervals of 1 and 28 days and lung toxicity determined. Analysis of bronchoalveolar lavage parameters of toxicity such as lactate dehydrogenase activity, oxidant generation, and inflammation did not reveal any significant lung toxicity in the oil vapor exposed rats. Approximately 50 genes each were found significantly differentially expressed in both the short- and long-term exposure groups of the rats at the one-day post-exposure time interval. The data obtained from the present study demonstrated that oil vapor inhalation exposure, under the exposure conditions employed in the present study, did not result in any significant lung toxicity in the rats despite the gene expression changes detected.
Project description:Determining agreement in classification between platforms and procurement methods requires a variety of methods. We have shown that centroid-based algorithms are robust classifiers for breast cancer subtype assignment across platforms (microarray and qRT-PCR data) and procurement conditions (fresh frozen and formalin-fixed, paraffin-embedded tissues). On a gene-by-gene basis, we found that the standard deviation, dynamic range, and concordance correlation coefficient are important parameters to assess individual primer set performance across procurement methods. Our strategy for primer set validation and classification have applications in routine clinical practice for stratifying breast cancers and other tumor types Keywords: reference x sample
Project description:We examined microRNA expression profiles in amygdala (AMY), nucleus accumbens (NAC) and prefrontal cortex (PFC) of male C57BL/6J mice exposed to 4 cycles of chronic intermittent ethanol (CIE) vapor. Animals were sacrificed at 0, 8, and 120 hr following the last ethanol exposure.
Project description:Nicotine and cigarette smoking have been previously shown to inhibit the differentiation potential and miRNA expression of human periodontal ligament derived stem cells (PDLSCs). Electronic cigarette (EC) vapor also contains nicotine, as well as other non-nicotine compounds, and, like cigarette smoking, EC vaping results in similar routes of toxic exposure for PDLSCs. The effect of exposure on PDLSC miRNA expression is unknown. To determine these effects, cultured PDLSCs were exposed to media supplemented with 1 uM nicotine EC vapor extract for 72h, during which media was changed every 24h. To produce vapor extract, an EC filled with 36 mg/ml nicotine, 50%/50% (w/v) PG/VG, non-flavored e-liquid (American E-liquid) was vaped using an automated vaping robot programmed with experienced e-cigarette user patterns (i.e. puff volume: 70 ml, duration: 3s, frequency: 20s) and resulting vapor was extracted via a liquid impinger. 1 uM of pure (-)-nicotine liquid (Sigma Aldrich) was used as a positive control. Total miRNA was collected with the mirVana miRNA isolation kit. miRNA concentration and quality was measured by spectrophotometry and expression was analyzed with Affymetrix GeneChip miRNA 4.0 Arrays. Results were normalized against human-positive and negative chip controls using Robust Multi-Array Average Normalization.
Project description:Novel tobacco vapor product, generating vapor without combusting tobacco leaves, has been developed expecting the number and quantity of chemicals in the vapor of these products to be reduced compared to conventional combustible cigarettes. However, if the lower chemical levels correlate with lower toxicity remained to be clarified. Here we examined the difference of conventional cigarette smoke (CS) and novel tobacco vapor product (NTV) using cultured cancer cell line A549 and normal bronchial epithelium cell line BEAS-2B. 0.5% of 3R4F which is conventional CS markedly decreased cell proliferation of both A549 and BEAS-2B, however Ploom TECH or Ploom TECH+ which are commercially available NTV did not affect cell growth. To clarify the cause of decreased cell proliferation, Tunnel assay was performed and clarified that apoptosis was observed in both A549 and BEAS-2B after 24hours after exposure to 3R4F. To further explore the effect of CS to epigenetics, we performed western blotting Histone H2A phosphorylation which is known to correlate transcriptional regulation. Only 3R4F decreased histone H2A phosphorylation of both A549 and BEAS-2B. Then we examined alterations of gene expression after 3R4F treatment of A549 cell. 339, 107, 103 genes which were upregulated more than 2fold were observed in 3R4F, Ploom TECH or Ploom TECH+ treated A549 cell, respectively. Among 339 genes which was upregulated 3R4F, we focused EGR1, FOS, and FOSB gene since they were upregulated more than100 fold. We confirmed this upregulation using RTqPCR. These data suggest that Cigarette smoke but not novel tobacco vapor product cause epigenetic disruption and cell apoptosis possibly by elevating genes such as EGR1.
Project description: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.
Project description:A 7-month inhalation study in C57BL/6 mice was conducted to evaluate long-term respiratory toxicity of e-vapor aerosols compared to cigarette smoke and to assess the impact of smoking cessation or switching to an e-vapor product after 3 months of exposure to 3R4F cigarette smoke (CS). In this study, we performed a chronic inhalation (4 h/day, 5 d/week, up to 7 months) study in C57BL/6 mice using a commercial (MarkTen®) e-vapor product and a combustible reference cigarette (3R4F) using a Switching and Cessation study design. A commercial e-vapor product (MarkTen® device [version 2.6.8]; “Test Red”) was supplied by Altria Client Services LLC (Richmond, VA, USA). The Test Red formulation was composed of aerosol formers (propylene glycol [PG] and vegetable glycerol [VG]), ~4% nicotine by weight, and flavors (non-menthol). The 3R4F commercial reference cigarettes were purchased from the University of Kentucky (Lexington, KY). HEPA filtered air at the testing facility (Battelle, West Jefferson, OH) was used as Sham Control. General procedures for animal care and housing met the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) recommendations and requirements stated in the “Guide for Care and Use of Laboratory Animals” [National Research Council (NRC)] and approved by the Institutional Animal Care and Use Committee (IACUC). Female C57BL/6 mice were received from Charles River Kingston (Stone Ridge, NY). Test atmosphere was generated from smoking machines and delivered to the mice through a nose-only exposure system. The modified Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) Reference Method 81 regimen (55/30/5: a 55 ± 0.3 mL puff volume, a puff every 30 seconds, a 5-second puff duration) was used to generate e-vapor aerosol for 130 puffs/cartridge. Mainstream smoke from 3R4F cigarette was generated using a modified Health Canada Intense regimen (55/30/2: a 55 ± 0.3 mL puff volume, a puff every 30 seconds, a 2-second puff duration, and a near-square puff profile) for 8 puffs/cigarette. Female C57BL/6 mice (~10 weeks old) were randomly assigned based on body weight to one of five exposure groups: Sham Control, 3R4F CS, Test Red, Switching, and Cessation. Mice were exposed to 3R4F CS (550 µg/L TPM) or e-vapor aerosols (Test Red; 1100 µg/L TPM) via nose-only inhalation up to 4 h/day, 5 d/week for up to 7 months. After the first 3 months of exposure, groups of 3R4F CS mice were subjected to exposures of: (1) Test Red aerosol (“Switching”) or (2) filtered air (“Cessation”), while a group of mice continued to be exposed to 3R4F CS. Here, the protein expression data for lung tissue assessed by iTRAQ®-based quantitative proteomics is reported.
Project description:The current study was performed for analysis of the biological effects of vapor from novel tobacco vapor product in comparison with 3R4F cigarette smoke. This study was performed using a three-dimensional culture system composed of an air-liquid interface culture of primary normal human bronchial epithelial cells (MucilAir). The MucilAir tissues were subjected to 17 days of exposure to the aqueous extract of novel tobacco product vapor or 3R4F cigarette smoke. The number of differentially expressed genes increased in MucilAir tissues exposed to aqueous extract of each test product dependent on exposure duration. The number of differentially expressed genes was lower in the tissues exposed to aqueous extract of novel tobacco product vapor compared to the tissues exposed to aqueous extract of 3R4F cigarette smoke.
Project description:Cigarette smoking causes serious diseases, including lung cancer, heart disease, and emphysema. While cessation remains the most effective approach to minimize smoking-related disease, alternative non-combustible tobacco-derived nicotine containing products may reduce disease risks among those unable or unwilling to quit. E-vapor aerosols typically contain significantly lower levels of smoke-related harmful and potentially harmful constituents; however, health risks of long-term inhalation exposures are unknown. We designed a 7-month inhalation study in C57BL/6 mice to evaluate long-term respiratory toxicity of e-vapor aerosols compared to cigarette smoke and to assess the impact of smoking cessation or switching to an e-vapor product after 3 months of exposure to 3R4F cigarette smoke (CS). There were no significant changes in in-life observations (body weights, clinical signs) in e-vapor groups compared to the Sham Control. The 3R4F CS group showed reduced respiratory function during exposure and had lower body weight and showed transient signs of distress post-exposure. Following 7 months of exposure, e-vapor aerosols resulted in no or minimal increase in pulmonary inflammation, while exposure to 3R4F CS led to impairment of lung function and caused marked lung inflammation and emphysematous changes. Biological changes observed in the Switching group were similar to the Cessation group. 3R4F CS exposure dysregulated lung and nasal tissue transcriptome, while these molecular effects were substantially lower in the e-vapor group. Results from this study demonstrate that in comparison with 3R4F CS, e-vapor aerosols induce substantially lower biological responses including pulmonary inflammation and emphysema, and that complete switching from CS to e-vapor products significantly reduces biological changes associated with cigarette smoke in C57BL/6 mice.
Project description:Zoonotic influenza A viruses of avian origin can cause severe disease in individuals, or even global pandemics, and thus pose a threat to human populations. Waterfowl and shorebirds are believed to be the reservoir for all influenza A viruses, but this has recently been challenged by the identification of novel influenza A viruses in bats. The major bat influenza A virus envelope glycoprotein, haemagglutinin, does not bind the canonical influenza A virus receptor, sialic acid or any other glycan, despite its high sequence and structural homology with conventional haemagglutinins. This functionally uncharacterized plasticity of the bat influenza A virus haemagglutinin means the tropism and zoonotic potential of these viruses has not been fully determined. Here we show, using transcriptomic profiling of susceptible versus non-susceptible cells in combination with genome-wide CRISPR-Cas9 screening, that the major histocompatibility complex class II (MHC-II) human leukocyte antigen DR isotype (HLA-DR) is an essential entry determinant for bat influenza A viruses. Genetic ablation of the HLA-DR α-chain rendered cells resistant to infection by bat influenza A virus, whereas ectopic expression of the HLA-DR complex in non-susceptible cells conferred susceptibility. Expression of MHC-II from different bat species, pigs, mice or chickens also conferred susceptibility to infection. Notably, the infection of mice with bat influenza A virus resulted in robust virus replication in the upper respiratory tract, whereas mice deficient for MHC-II were resistant. Collectively, our data identify MHC-II as a crucial entry mediator for bat influenza A viruses in multiple species, which permits a broad vertebrate tropism.