Nebulisation of receptor-targeted nanocomplexes for gene delivery to the airway epithelium.

PubMed

Manunta, Maria D I; McAnulty, Robin J; Tagalakis, Aristides D; Bottoms, Stephen E; Campbell, Frederick; Hailes, Helen C; Tabor, Alethea B; Laurent, Geoffrey J; O'Callaghan, Christopher; Hart, Stephen L

2011-01-01

Gene therapy mediated by synthetic vectors may provide opportunities for new treatments for cystic fibrosis (CF) via aerosolisation. Vectors for CF must transfect the airway epithelium efficiently and not cause inflammation so they are suitable for repeated dosing. The inhaled aerosol should be deposited in the airways since the cystic fibrosis transmembrane conductance regulator gene (CFTR) is expressed predominantly in the epithelium of the submucosal glands and in the surface airway epithelium. The aim of this project was to develop an optimised aerosol delivery approach applicable to treatment of CF lung disease by gene therapy. The vector suspension investigated in this study comprises receptor-targeting peptides, cationic liposomes and plasmid DNA that self-assemble by electrostatic interactions to form a receptor-targeted nanocomplex (RTN) of approximately 150 nm with a cationic surface charge of +50 mV. The aerodynamic properties of aerosolised nanocomplexes produced with three different nebulisers were compared by determining aerosol deposition in the different stages of a Next Generation Pharmaceutical Impactor (NGI). We also investigated the yield of intact plasmid DNA by agarose gel electrophoresis and densitometry, and transfection efficacies in vitro and in vivo. RTNs nebulised with the AeroEclipse II BAN were the most effective, compared to other nebulisers tested, for gene delivery both in vitro and in vivo. The biophysical properties of the nanocomplexes were unchanged after nebulisation while the deposition of RTNs suggested a range of aerosol aerodynamic sizes between 5.5 µm-1.4 µm cut off (NGI stages 3-6) compatible with deposition in the central and lower airways. RTNs showed their ability at delivering genes via nebulisation, thus suggesting their potential applications for therapeutic interventions of cystic fibrosis and other respiratory disorders.

Transduction of ferret airway epithelia using a pre-treatment and lentiviral gene vector.

PubMed

Cmielewski, Patricia; Farrow, Nigel; Donnelley, Martin; McIntyre, Chantelle; Penny-Dimri, Jahan; Kuchel, Tim; Parsons, David

2014-11-21

The safety and efficiency of gene therapies for cystic fibrosis (CF) need to be assessed in pre-clinical models. Using the normal ferret, this study sought to determine whether ferret airway epithelia could be transduced with a lysophosphatidylcholine (LPC) pre-treatment followed by a VSV-G pseudotyped HIV-1 based lentiviral (LV) vector, in preparation for future studies in CF ferrets. Six normal ferrets (7 -8 weeks old) were treated with a 150 μL LPC pre-treatment, followed one hour later by a 500 μL LV vector dose containing the LacZ transgene. LacZ gene expression in the conducting airways and lung was assessed by X-gal staining after 7 days. The presence of transduction in the lung, as well as off-target transduction in the liver, spleen and gonads, were assessed by qPCR. The levels of LV vector p24 protein bio-distribution in blood sera were assessed by ELISA at 0, 1, 3, 5 and 7 days. The dosing protocol was well tolerated. LacZ gene expression was observed en face in the trachea of all animals. Histology showed that ciliated and basal cells were transduced in the trachea, with rare LacZ transduced single cells noted in lung. p24 levels was not detectable in the sera of 5 of the 6 animals. The LacZ gene was not detected in the lung tissue and no off-target transduction was detected by qPCR. This study shows that ferret airway epithelia are transducible using our unique two-step pre-treatment and LV vector dosing protocol. We have identified a number of unusual anatomical factors that are likely to influence the level of transduction that can be achieved in ferret airways. The ability to transduce ferret airway epithelium is a promising step towards therapeutic LV-CFTR testing in a CF ferret model.

The Human Airway Epithelial Basal Cell Transcriptome

PubMed Central

Wang, Rui; Zwick, Rachel K.; Ferris, Barbara; Witover, Bradley; Salit, Jacqueline; Crystal, Ronald G.

2011-01-01

Background The human airway epithelium consists of 4 major cell types: ciliated, secretory, columnar and basal cells. During natural turnover and in response to injury, the airway basal cells function as stem/progenitor cells for the other airway cell types. The objective of this study is to better understand human airway epithelial basal cell biology by defining the gene expression signature of this cell population. Methodology/Principal Findings Bronchial brushing was used to obtain airway epithelium from healthy nonsmokers. Microarrays were used to assess the transcriptome of basal cells purified from the airway epithelium in comparison to the transcriptome of the differentiated airway epithelium. This analysis identified the “human airway basal cell signature” as 1,161 unique genes with >5-fold higher expression level in basal cells compared to differentiated epithelium. The basal cell signature was suppressed when the basal cells differentiated into a ciliated airway epithelium in vitro. The basal cell signature displayed overlap with genes expressed in basal-like cells from other human tissues and with that of murine airway basal cells. Consistent with self-modulation as well as signaling to other airway cell types, the human airway basal cell signature was characterized by genes encoding extracellular matrix components, growth factors and growth factor receptors, including genes related to the EGF and VEGF pathways. Interestingly, while the basal cell signature overlaps that of basal-like cells of other organs, the human airway basal cell signature has features not previously associated with this cell type, including a unique pattern of genes encoding extracellular matrix components, G protein-coupled receptors, neuroactive ligands and receptors, and ion channels. Conclusion/Significance The human airway epithelial basal cell signature identified in the present study provides novel insights into the molecular phenotype and biology of the stem

Gene transfer and gene mapping in mammalian cells in culture.

PubMed

Shows, T B; Sakaguchi, A Y

1980-01-01

The ability to transfer mammalian genes parasexually has opened new possibilities for gene mapping and fine structure mapping and offers great potential for contributing to several aspects of mammalian biology, including gene expression and genetic engineering. The DNA transferred has ranged from whole genomes to single genes and smaller segments of DNA. The transfer of whole genomes by cell fusion forms cell hybrids, which has promoted the extensive mapping of human and mouse genes. Transfer, by cell fusion, of rearranged chromosomes has contributed significantly to determining close linkage and the assignment of genes to specific chromosomal regions. Transfer of single chromosomes has been achieved utilizing microcells fused to recipient cells. Metaphase chromosomes have been isolated and used to transfer single-to-multigenic DNA segments. DNA-mediated gene transfer, simulating bacterial transformation, has achieved transfer of single-copy genes. By utilizing DNA cleaved with restriction endonucleases, gene transfer is being empolyed as a bioassay for the purification of genes. Gene mapping and the fate of transferred genes can be examined now at the molecular level using sequence-specific probles. Recently, single genes have been cloned into eucaryotic and procaryotic vectors for transfer into mammalian cells. Moreover, recombinant libraries in which entire mammalian genomes are represented collectively are a rich new source of transferable genes. Methodology for transferring mammalian genetic information and applications for mapping mammalian genes is presented and prospects for the future discussed.

Adenovirus-mediated HIF-1α gene transfer promotes repair of mouse airway allograft microvasculature and attenuates chronic rejection

PubMed Central

Jiang, Xinguo; Khan, Mohammad A.; Tian, Wen; Beilke, Joshua; Natarajan, Ramesh; Kosek, Jon; Yoder, Mervin C.; Semenza, Gregg L.; Nicolls, Mark R.

2011-01-01

Chronic rejection, manifested as small airway fibrosis (obliterative bronchiolitis [OB]), is the main obstacle to long-term survival in lung transplantation. Recent studies demonstrate that the airways involved in a lung transplant are relatively hypoxic at baseline and that OB pathogenesis may be linked to ischemia induced by a transient loss of airway microvasculature. Here, we show that HIF-1α mediates airway microvascular repair in a model of orthotopic tracheal transplantation. Grafts with a conditional knockout of Hif1a demonstrated diminished recruitment of recipient-derived Tie2+ angiogenic cells to the allograft, impaired repair of damaged microvasculature, accelerated loss of microvascular perfusion, and hastened denudation of epithelial cells. In contrast, graft HIF-1α overexpression induced via an adenoviral vector prolonged airway microvascular perfusion, preserved epithelial integrity, extended the time window for the graft to be rescued from chronic rejection, and attenuated airway fibrotic remodeling. HIF-1α overexpression induced the expression of proangiogenic factors such as Sdf1, Plgf, and Vegf, and promoted the recruitment of vasoreparative Tie2+ cells. This study demonstrates that a therapy that enhances vascular integrity during acute rejection may promote graft health and prevent chronic rejection. PMID:21606594

Regulation of xanthine dehydrogensase gene expression and uric acid production in human airway epithelial cells

PubMed Central

Huff, Ryan D.; Hsu, Alan C-Y.; Nichol, Kristy S.; Jones, Bernadette; Knight, Darryl A.; Wark, Peter A. B.; Hansbro, Philip M.

2017-01-01

Introduction The airway epithelium is a physical and immunological barrier that protects the pulmonary system from inhaled environmental insults. Uric acid has been detected in the respiratory tract and can function as an antioxidant or damage associated molecular pattern. We have demonstrated that human airway epithelial cells are a source of uric acid. Our hypothesis is that uric acid production by airway epithelial cells is induced by environmental stimuli associated with chronic respiratory diseases. We therefore examined how airway epithelial cells regulate uric acid production. Materials and methods Allergen and cigarette smoke mouse models were performed using house dust mite (HDM) and cigarette smoke exposure, respectively, with outcome measurements of lung uric acid levels. Primary human airway epithelial cells isolated from clinically diagnosed patients with asthma and chronic obstructive pulmonary disease (COPD) were grown in submerged cultures and compared to age-matched healthy controls for uric acid release. HBEC-6KT cells, a human airway epithelial cell line, were grown under submerged monolayer conditions for mechanistic and gene expression studies. Results HDM, but not cigarette smoke exposure, stimulated uric acid production in vivo and in vitro. Primary human airway epithelial cells from asthma, but not COPD patients, displayed elevated levels of extracellular uric acid in culture. In HBEC-6KT, production of uric acid was sensitive to the xanthine dehydrogenase (XDH) inhibitor, allopurinol, and the ATP Binding Cassette C4 (ABCC4) inhibitor, MK-571. Lastly, the pro-inflammatory cytokine combination of TNF-α and IFN-γ elevated extracellular uric acid levels and XDH gene expression in HBEC-6KT cells. Conclusions Our results suggest that the active production of uric acid from human airway epithelial cells may be intrinsically altered in asthma and be further induced by pro-inflammatory cytokines. PMID:28863172

Azithromycin ameliorates airway remodeling via inhibiting airway epithelium apoptosis.

PubMed

Liu, Yuanqi; Pu, Yue; Li, Diandian; Zhou, Liming; Wan, Lihong

2017-02-01

Azithromycin can benefit treating allergic airway inflammation and remodeling. In the present study, we hypothesized that azithromycin alleviated airway epithelium injury through inhibiting airway epithelium apoptosis via down regulation of caspase-3 and Bax/Bcl2 ratio in vivo and in vitro. Ovalbumin induced rat asthma model and TGF-β1-induced BEAS-2B cell apoptosis model were established, respectively. In vivo experiments, airway epithelium was stained with hematoxylin and eosin (HE) and periodic acid-Schiff (PAS) to histologically evaluate the airway inflammation and remodeling. Airway epithelium apoptotic index (AI) was further analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), while expression of apoptosis related gene (Bax, Bcl2, Caspase-3) in lungs were measured by qRT-PCR and western blotting, respectively. In vitro experiments, apoptosis were evaluated by Flow cytometry (FCM) and TUNEL. Above apoptosis related gene were also measured by qRT-PCR and western blotting. Compared with the OVA group, azithromycin significantly reduced the inflammation score, peribronchial smooth muscle layer thickness, epithelial thickening and goblet cell metaplasia (P<0.05), and effectively suppressed AI of airway epithelium (P<0.05). Moreover, the increasing mRNA and protein expressions of Caspase-3 and Bax/Bcl-2 ratio in lung tissue were all significantly decreased in azithromycin-treated rats (P<0.05). In vitro, azithromycin significantly suppressed TGF-β1-induced BEAS-2B cells apoptosis (P<0.05) and reversed TGF-β1 elevated Caspase-3 mRNA level and Bax/Bcl-2 ratio (P<0.05). Azithromycin is an attractive treatment option for reducing airway epithelial cell apoptosis by improving the imbalance of Bax/Bcl-2 ratio and inhibiting Caspase-3 level in airway epithelium. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of genes differentially regulated by vitamin D deficiency that alter lung pathophysiology and inflammation in allergic airways disease.

PubMed

Foong, Rachel E; Bosco, Anthony; Troy, Niamh M; Gorman, Shelley; Hart, Prue H; Kicic, Anthony; Zosky, Graeme R

2016-09-01

Vitamin D deficiency is associated with asthma risk. Vitamin D deficiency may enhance the inflammatory response, and we have previously shown that airway remodeling and airway hyperresponsiveness is increased in vitamin D-deficient mice. In this study, we hypothesize that vitamin D deficiency would exacerbate house dust mite (HDM)-induced inflammation and alterations in lung structure and function. A BALB/c mouse model of vitamin D deficiency was established by dietary manipulation. Responsiveness to methacholine, airway smooth muscle (ASM) mass, mucus cell metaplasia, lung and airway inflammation, and cytokines in bronchoalveolar lavage (BAL) fluid were assessed. Gene expression patterns in mouse lung samples were profiled by RNA-Seq. HDM exposure increased inflammation and inflammatory cytokines in BAL, baseline airway resistance, tissue elastance, and ASM mass. Vitamin D deficiency enhanced the HDM-induced influx of lymphocytes into BAL, ameliorated the HDM-induced increase in ASM mass, and protected against the HDM-induced increase in baseline airway resistance. RNA-Seq identified nine genes that were differentially regulated by vitamin D deficiency in the lungs of HDM-treated mice. Immunohistochemical staining confirmed that protein expression of midline 1 (MID1) and adrenomedullin was differentially regulated such that they promoted inflammation, while hypoxia-inducible lipid droplet-associated, which is associated with ASM remodeling, was downregulated. Protein expression studies in human bronchial epithelial cells also showed that addition of vitamin D decreased MID1 expression. Differential regulation of these genes by vitamin D deficiency could determine lung inflammation and pathophysiology and suggest that the effect of vitamin D deficiency on HDM-induced allergic airways disease is complex. Copyright © 2016 the American Physiological Society.

Bronchial airway gene expression signatures in mouse lung squamous cell carcinoma and their modulation by cancer chemopreventive agents

PubMed Central

Szabo, Eva; Miller, Mark Steven; Lubet, Ronald A.; You, Ming; Wang, Yian

2017-01-01

Due to exposure to environmental toxicants, a “field cancerization” effect occurs in the lung resulting in the development of a field of initiated but morphologically normal appearing cells in the damaged epithelium of bronchial airways with dysregulated gene expression patterns. Using a mouse model of lung squamous cell carcinoma (SCC), we performed transcriptome sequencing (RNA-Seq) to profile bronchial airway gene expression and found activation of the PI3K and Myc signaling networks in cytologically normal bronchial airway epithelial cells of mice with preneopastic lung SCC lesions, which was reversed by treatment with the PI3K Inhibitor XL-147 and pioglitazone, respectively. Activated MYC signaling was also present in premalignant and tumor tissues from human lung SCC patients. In addition, we identified a key microRNA, mmu-miR-449c-5p, whose suppression significantly up-regulated Myc expression in the normal bronchial airway epithelial cells of mice with early stage SCC lesions. We developed a novel bronchial genomic classifier in mice and validated it in humans. In the classifier, Ppbp (pro-platelet basic protein) was overexpressed 115 fold in the bronchial airways of mice with preneoplastic lung SCC lesions. This is the first report that demonstrates Ppbp as a novel biomarker in the bronchial airway for lung cancer diagnosis. PMID:27935865

Advances in Cell and Gene-based Therapies for Cystic Fibrosis Lung Disease

PubMed Central

Oakland, Mayumi; Sinn, Patrick L; McCray Jr, Paul B

2012-01-01

Cystic fibrosis (CF) is a disease characterized by airway infection, inflammation, remodeling, and obstruction that gradually destroy the lungs. Direct delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to airway epithelia may offer advantages, as the tissue is accessible for topical delivery of vectors. Yet, physical and host immune barriers in the lung present challenges for successful gene transfer to the respiratory tract. Advances in gene transfer approaches, tissue engineering, and novel animal models are generating excitement within the CF research field. This review discusses current challenges and advancements in viral and nonviral vectors, cell-based therapies, and CF animal models. PMID:22371844

Nitric oxide enhances Th9 cell differentiation and airway inflammation

PubMed Central

Niedbala, Wanda; Besnard, Anne-Gaelle; Nascimento, Daniele Carvalho; Donate, Paula Barbim; Sonego, Fabiane; Yip, Edwin; Guabiraba, Rodrigo; Chang, Hyun-Dong; Fukada, Sandra Y.; Salmond, Robert J.; Schmitt, Edgar; Bopp, Tobias; Ryffel, Bernhard; Liew, Foo Y.

2014-01-01

Th9 cells protect hosts against helminthic infection but also mediate allergic disease. Here we show that nitric oxide (NO) promotes Th9 cell polarization of murine and human CD4+ T cells. NO de-represses the tumor suppressor gene p53 via nitrosylation of Mdm2. NO also increases p53-mediated IL-2 production, STAT5 phosphorylation and IRF4 expression, all essential for Th9 polarization. NO also increases the expression of TGFβR and IL-4R, pivotal to Th9 polarization. OVA-sensitized mice treated with an NO donor developed more severe airway inflammation. Transferred Th9 cells induced airway inflammation, which was exacerbated by NO and blocked by anti-IL-9 antibody. Nos2−/− mice had less Th9 cells and developed attenuated eosinophilia during OVA-induced airway inflammation compared to wild-type mice. Our data demonstrate that NO is an important endogenous inducer of Th9 cells and provide a hitherto unrecognized mechanism for NO-mediated airway inflammation via the expansion of Th9 cells. PMID:25099390

Nitric oxide enhances Th9 cell differentiation and airway inflammation.

PubMed

Niedbala, Wanda; Besnard, Anne-Gaelle; Nascimento, Daniele Carvalho; Donate, Paula Barbim; Sonego, Fabiane; Yip, Edwin; Guabiraba, Rodrigo; Chang, Hyun-Dong; Fukada, Sandra Y; Salmond, Robert J; Schmitt, Edgar; Bopp, Tobias; Ryffel, Bernhard; Liew, Foo Y

2014-08-07

Th9 cells protect hosts against helminthic infection but also mediate allergic disease. Here we show that nitric oxide (NO) promotes Th9 cell polarization of murine and human CD4(+) T cells. NO de-represses the tumour suppressor gene p53 via nitrosylation of Mdm2. NO also increases p53-mediated IL-2 production, STAT5 phosphorylation and IRF4 expression, all essential for Th9 polarization. NO also increases the expression of TGFβR and IL-4R, pivotal to Th9 polarization. OVA-sensitized mice treated with an NO donor developed more severe airway inflammation. Transferred Th9 cells induced airway inflammation, which was exacerbated by NO and blocked by anti-IL-9 antibody. Nos2(-/-) mice had less Th9 cells and developed attenuated eosinophilia during OVA-induced airway inflammation compared with wild-type mice. Our data demonstrate that NO is an important endogenous inducer of Th9 cells and provide a hitherto unrecognized mechanism for NO-mediated airway inflammation via the expansion of Th9 cells.

Lateral Gene Transfer from the Dead

PubMed Central

Szöllősi, Gergely J.; Tannier, Eric; Lartillot, Nicolas; Daubin, Vincent

2013-01-01

In phylogenetic studies, the evolution of molecular sequences is assumed to have taken place along the phylogeny traced by the ancestors of extant species. In the presence of lateral gene transfer, however, this may not be the case, because the species lineage from which a gene was transferred may have gone extinct or not have been sampled. Because it is not feasible to specify or reconstruct the complete phylogeny of all species, we must describe the evolution of genes outside the represented phylogeny by modeling the speciation dynamics that gave rise to the complete phylogeny. We demonstrate that if the number of sampled species is small compared with the total number of existing species, the overwhelming majority of gene transfers involve speciation to and evolution along extinct or unsampled lineages. We show that the evolution of genes along extinct or unsampled lineages can to good approximation be treated as those of independently evolving lineages described by a few global parameters. Using this result, we derive an algorithm to calculate the probability of a gene tree and recover the maximum-likelihood reconciliation given the phylogeny of the sampled species. Examining 473 near-universal gene families from 36 cyanobacteria, we find that nearly a third of transfer events (28%) appear to have topological signatures of evolution along extinct species, but only approximately 6% of transfers trace their ancestry to before the common ancestor of the sampled cyanobacteria. [Gene tree reconciliation; lateral gene transfer; macroevolution; phylogeny.] PMID:23355531

Occurrence and expression of gene transfer agent genes in marine bacterioplankton.

PubMed

Biers, Erin J; Wang, Kui; Pennington, Catherine; Belas, Robert; Chen, Feng; Moran, Mary Ann

2008-05-01

Genes with homology to the transduction-like gene transfer agent (GTA) were observed in genome sequences of three cultured members of the marine Roseobacter clade. A broader search for homologs for this host-controlled virus-like gene transfer system identified likely GTA systems in cultured Alphaproteobacteria, and particularly in marine bacterioplankton representatives. Expression of GTA genes and extracellular release of GTA particles ( approximately 50 to 70 nm) was demonstrated experimentally for the Roseobacter clade member Silicibacter pomeroyi DSS-3, and intraspecific gene transfer was documented. GTA homologs are surprisingly infrequent in marine metagenomic sequence data, however, and the role of this lateral gene transfer mechanism in ocean bacterioplankton communities remains unclear.

Ethics of Cancer Gene Transfer Clinical Research.

PubMed

Kimmelman, Jonathan

2015-01-01

Translation of cancer gene transfer confronts many familiar-and some distinctive-ethical challenges. In what follows, I survey three major ethical dimensions of cancer gene transfer development. Subheading 1 centers on the ethics of planning, designing, and reporting animal studies. Subheading 2 describes basic elements of human subjects protection as pertaining to cancer gene transfer. In Subheading 3, I describe how cancer gene transfer researchers have obligations to downstream consumers of the evidence they produce.

Occurrence and Expression of Gene Transfer Agent Genes in Marine Bacterioplankton▿

PubMed Central

Biers, Erin J.; Wang, Kui; Pennington, Catherine; Belas, Robert; Chen, Feng; Moran, Mary Ann

2008-01-01

Genes with homology to the transduction-like gene transfer agent (GTA) were observed in genome sequences of three cultured members of the marine Roseobacter clade. A broader search for homologs for this host-controlled virus-like gene transfer system identified likely GTA systems in cultured Alphaproteobacteria, and particularly in marine bacterioplankton representatives. Expression of GTA genes and extracellular release of GTA particles (∼50 to 70 nm) was demonstrated experimentally for the Roseobacter clade member Silicibacter pomeroyi DSS-3, and intraspecific gene transfer was documented. GTA homologs are surprisingly infrequent in marine metagenomic sequence data, however, and the role of this lateral gene transfer mechanism in ocean bacterioplankton communities remains unclear. PMID:18359833

Bioaerosols from a food waste composting plant affect human airway epithelial cell remodeling genes.

PubMed

Chang, Min-Wei; Lee, Chung-Ru; Hung, Hsueh-Fen; Teng, Kuo-Sheng; Huang, Hsin; Chuang, Chun-Yu

2013-12-24

The composting procedure in food waste plants generates airborne bioaerosols that have the potential to damage human airway epithelial cells. Persistent inflammation and repair responses induce airway remodeling and damage to the respiratory system. This study elucidated the expression changes of airway remodeling genes in human lung mucoepidermoid NCI-H292 cells exposed to bioaerosols from a composting plant. Different types of microorganisms were detectable in the composting plant, using the agar culture method. Real-time polymerase chain reaction was used to quantify the level of Aspergillus fumigatus and the profile of remodeling genes. The real-time PCR results indicated that the amount of A. fumigatus in the composting hall was less than 10(2) conidia. The endotoxins in the field bioaerosols were determined using a limulus amebocyte lysate test. The endotoxin levels depended on the type of particulate matter (PM), with coarse particles (2.5-10 μm) having higher endotoxin levels than did fine particles (0.5-2.5 μm). After exposure to the conditioned medium of field bioaerosol samples, NCI-H292 cells showed increased pro-inflammatory interleukin (IL)-6 release and activated epidermal growth factor receptor (EGFR), transforming growth factor (TGF)-β1 and cyclin-dependent kinase inhibitor 1 (p21 WAF1/CIP1) gene expression, but not of matrix metallopeptidase (MMP)-9. Airborne endotoxin levels were higher inside the composting hall than they were in other areas, and they were associated with PM. This suggested that airborne bioaerosols in the composting plant contained endotoxins and microorganisms besides A. fumigatus that cause the inflammatory cytokine secretion and augment the expression of remodeling genes in NCI-H292 cells. It is thus necessary to monitor potentially hazardous materials from bioaerosols in food composting plants, which could affect the health of workers.

Bioaerosols from a Food Waste Composting Plant Affect Human Airway Epithelial Cell Remodeling Genes

PubMed Central

Chang, Ming-Wei; Lee, Chung-Ru; Hung, Hsueh-Fen; Teng, Kuo-Sheng; Huang, Hsin; Chuang, Chun-Yu

2013-01-01

The composting procedure in food waste plants generates airborne bioaerosols that have the potential to damage human airway epithelial cells. Persistent inflammation and repair responses induce airway remodeling and damage to the respiratory system. This study elucidated the expression changes of airway remodeling genes in human lung mucoepidermoid NCI-H292 cells exposed to bioaerosols from a composting plant. Different types of microorganisms were detectable in the composting plant, using the agar culture method. Real-time polymerase chain reaction was used to quantify the level of Aspergillus fumigatus and the profile of remodeling genes. The real-time PCR results indicated that the amount of A. fumigatus in the composting hall was less than 102 conidia. The endotoxins in the field bioaerosols were determined using a limulus amebocyte lysate test. The endotoxin levels depended on the type of particulate matter (PM), with coarse particles (2.5–10 μm) having higher endotoxin levels than did fine particles (0.5–2.5 μm). After exposure to the conditioned medium of field bioaerosol samples, NCI-H292 cells showed increased pro-inflammatory interleukin (IL)-6 release and activated epidermal growth factor receptor (EGFR), transforming growth factor (TGF)-β1 and cyclin-dependent kinase inhibitor 1 (p21WAF1/CIP1) gene expression, but not of matrix metallopeptidase (MMP)-9. Airborne endotoxin levels were higher inside the composting hall than they were in other areas, and they were associated with PM. This suggested that airborne bioaerosols in the composting plant contained endotoxins and microorganisms besides A. fumigatus that cause the inflammatory cytokine secretion and augment the expression of remodeling genes in NCI-H292 cells. It is thus necessary to monitor potentially hazardous materials from bioaerosols in food composting plants, which could affect the health of workers. PMID:24368426

Inhibition by TNF-alpha and IL-4 of cationic lipid mediated gene transfer in cystic fibrosis tracheal gland cells.

PubMed

Bastonero, Sonia; Gargouri, Myriem; Ortiou, Sandrine; Guéant, Jean-Louis; Merten, Marc D

2005-11-01

In vivo, tracheal gland serous cells highly express the cystic fibrosis transmembrane conductance regulator (cftr) gene. This gene is mutated in the lethal monogenic disease cystic fibrosis (CF). Clinical trials in which the human CFTR cDNA was delivered to the respiratory epithelia of CF patients have resulted in weak and transient gene expression. As CF is characterized by mucus inspissation, airway infection, and severe inflammation, we tested the hypothesis that inflammation and especially two cytokines involved in the Th1/Th2 inflammatory response, interleukin 4 (IL-4) and TNFalpha, could inhibit gene transfer efficiency using a model of human CF tracheal gland cells (CF-KM4) and Lipofectamine reagent as a transfection reagent. The specific secretory defects of CF-KM4 cells were corrected by Lipofectamine-mediated human CFTR gene transfer. However, this was altered when cells were pre-treated with IL-4 and TNFalpha. Inhibition of luciferase reporter gene expression by IL-4 and TNFalpha pre-treated CF-KM4 cells was measured by activity and real-time RT-PCR. Both cytokines induced similar and synergistic inhibition of transgene expression and activity. This cytokine-mediated inhibition could be prevented by anti-inflammatory agents such as glucocorticoids but not by non-steroidal (NSAI) agents. This data suggests that an inflammatory context generated by IL-4 and TNFalpha can inhibit human CFTR gene transfer in CF tracheal gland cells and that glucocorticoids may have a protecting action. Copyright (c) 2005 John Wiley & Sons, Ltd.

A Different Microbiome Gene Repertoire in the Airways of Cystic Fibrosis Patients with Severe Lung Disease

PubMed Central

Bacci, Giovanni; Fiscarelli, Ersilia; Taccetti, Giovanni; Dolce, Daniela; Paganin, Patrizia; Morelli, Patrizia; Tuccio, Vanessa; De Alessandri, Alessandra; Lucidi, Vincenzina

2017-01-01

In recent years, next-generation sequencing (NGS) was employed to decipher the structure and composition of the microbiota of the airways in cystic fibrosis (CF) patients. However, little is still known about the overall gene functions harbored by the resident microbial populations and which specific genes are associated with various stages of CF lung disease. In the present study, we aimed to identify the microbial gene repertoire of CF microbiota in twelve patients with severe and normal/mild lung disease by performing sputum shotgun metagenome sequencing. The abundance of metabolic pathways encoded by microbes inhabiting CF airways was reconstructed from the metagenome. We identified a set of metabolic pathways differently distributed in patients with different pulmonary function; namely, pathways related to bacterial chemotaxis and flagellar assembly, as well as genes encoding efflux-mediated antibiotic resistance mechanisms and virulence-related genes. The results indicated that the microbiome of CF patients with low pulmonary function is enriched in virulence-related genes and in genes encoding efflux-mediated antibiotic resistance mechanisms. Overall, the microbiome of severely affected adults with CF seems to encode different mechanisms for the facilitation of microbial colonization and persistence in the lung, consistent with the characteristics of multidrug-resistant microbial communities that are commonly observed in patients with severe lung disease. PMID:28758937

Activation of calcitonin gene-related peptide receptor during ozone inhalation contributes to airway epithelial injury and repair.

PubMed

Oslund, Karen L; Hyde, Dallas M; Putney, Leialoha F; Alfaro, Mario F; Walby, William F; Tyler, Nancy K; Schelegle, Edward S

2009-10-01

The authors investigated the importance of the neuropeptide, calcitonin gene-related peptide (CGRP), in epithelial injury, repair, and neutrophil emigration after ozone exposure. Wistar rats were administered either a CGRP-receptor antagonist (CGRP(8-37)) or saline and exposed to 8 hours of 1-ppm ozone or filtered air with an 8-hour postexposure period. Immediately after exposure, ethidium homodimer was instilled into lungs as a marker of necrotic airway epithelial cells. After fixation, airway dissected lung lobes were stained for 5'-bromo-2'-deoxyuridine, a marker of epithelial proliferation. Positive epithelial cells were quantified in specific airway generations. Rats treated with CGRP(8-37) had significantly reduced epithelial injury in terminal bronchioles and reduced epithelial proliferation in proximal airways and terminal bronchioles. Bronchoalveolar lavage and sections of terminal bronchioles showed no significant difference in the number of neutrophils emigrating into airways in CGRP(8-37)-treated rats. The airway epithelial cell line, HBE-1, showed no difference in the number of oxidant stress positive cells during exposure to hydrogen peroxide and a range of CGRP(8-37) doses, demonstrating no antioxidant effect of CGRP(8-37). We conclude that activation of CGRP receptors during ozone inhalation contributes to airway epithelial injury and subsequent epithelial proliferation, a critical component of repair, but does not influence neutrophil emigration into airways.

Steroid Treatment Reduces Allergic Airway Inflammation and Does Not Alter the Increased Numbers of Dendritic Cells and Calcitonin Gene-Related Peptide-Expressing Neurons in Airway Sensory Ganglia.

PubMed

Le, Duc Dung; Funck, Ulrike; Wronski, Sabine; Heck, Sebastian; Tschernig, Thomas; Bischoff, Markus; Sester, Martina; Herr, Christian; Bals, Robert; Welte, Tobias; Braun, Armin; Dinh, Quoc Thai

2016-01-01

Our previous data demonstrated that allergic airway inflammation induces migration of dendritic cells (DC) into airway sensory jugular and nodose ganglia (jugular-nodose ganglion complex; JNC). Here we investigated the effects of steroid treatment regarding the expression and migration of DC and calcitonin gene-related peptide (CGRP)-immunoreactive neurons of vagal sensory ganglia during allergic airway inflammation. A house dust mite (HDM) model for allergic airway inflammation was used. The mice received 0.3 mg fluticasone propionate per kilogram of body weight in the last 9 days. JNC slices were analyzed on MHC II, the neuronal marker PGP9.5, and the neuropeptide CGRP. Allergic airway inflammation increased the numbers of DC and CGRP-expressing neurons in the JNC significantly in comparison to the controls (DC/neurons: HDM 44.58 ± 1.6% vs. saline 33.29 ± 1.6%, p < 0.05; CGRP-positive neurons/total neurons: HDM 30.65 ± 1.9% vs. saline 19.49 ± 2.3%, p < 0.05). Steroid treatment did not have any effect on the numbers of DC and CGRP-expressing neurons in the JNC compared to HDM-treated mice. The present findings indicate an important role of DC and CGRP-containing neurons in the pathogenesis of allergic airway inflammation. However, steroid treatment did not have an effect on the population of DC and neurons displaying CGRP in the JNC, whereas steroid treatment was found to suppress allergic airway inflammation. © 2015 S. Karger AG, Basel.

Reduced toll-like receptor 4 and substance P gene expression is associated with airway bacterial colonization in children.

PubMed

Grissell, Terry V; Chang, Anne B; Gibson, Peter G

2007-04-01

Neuro-immune interactions are increasingly relevant to human health and disease. The neuropeptide Substance P also has antibacterial activity and bears similarities to the innate immune antibacterial defensins. This suggests possible co-regulation of neuropeptide and innate immune mediators. In this study, non-bronchoscopic bronchoalveolar lavage (BAL) was performed on 69 children. BAL was examined for cellular profile, microbiology (bacteria, virus) and gene expression for TLRs 2, 3, 4; chemokine receptors (CCR3, CCR5, CXCR1); neurotrophins and neurokinin genes (TAC1, TAC3, CGRP, NGF). In children with bacterial colonization (n=10) there was an airway inflammatory response with increased BAL neutrophils, IL-8 protein, and CXCR1 expression. Substance P (TAC1) and TLR4 RNA expression were reduced in children with bacterial colonization. TLR3 mRNA was increased in 7.2% (n=5) children with rhinovirus, and there was a non-significant trend to increased TLR2. There is evidence for co-regulation of neurokinin (TAC1) and TLR4 gene expression in airway cells from children with airway bacterial colonization and their reduced expression may be associated with an impaired bacterial clearance. (c) 2007 Wiley-Liss, Inc.

Interdisciplinary development and implementation of communication checklist for postoperative management of pediatric airway patients.

PubMed

Kim, Sang W; Maturo, Stephen; Dwyer, Danielle; Monash, Bradley; Yager, Phoebe H; Zanger, Kerstin; Hartnick, Christopher J

2012-01-01

The authors describe their multidisciplinary experience in applying the Institute of Health Improvement methodology to develop a protocol and checklist to reduce communication error during transfer of care for postoperative pediatric surgical airway patients. Preliminary outcome data following implementation of the protocol and checklist are also presented. Prospective study from July 1, 2009, to February 1, 2011. Tertiary care center. Subjects. One hundred twenty-six pediatric airway patients who required coordinated care between Massachusetts Eye and Ear Infirmary and Massachusetts General Hospital. Two sentinel events involving airway emergencies demonstrated a critical need for a standardized, comprehensive instrument that would ensure safe transfer of care. After development and implementation of the protocol and checklist, an initial pilot period on the first set of 9 pediatric airway patients was reassessed. Subsequent prospective 11-month follow-up data of 93 pediatric airway patients were collected and analyzed. A multidisciplinary pediatric team developed and implemented a formalized, postoperative checklist and transfer protocol. After implementation of the checklist and transfer protocol, prospective analysis showed no adverse events from miscommunication during transfer of care over the subsequent 11-month period involving 93 pediatric airway patients. There has been very little written in the quality and safety patient literature about coordinating effective transfer of care between the pediatric surgical and medical subspecialty realms. After design and implementation of a simple, electronically based transfer-of-care checklist and protocol, the number of postsurgical pediatric airway information transfer and communication errors decreased significantly.

Widespread of horizontal gene transfer in the human genome.

PubMed

Huang, Wenze; Tsai, Lillian; Li, Yulong; Hua, Nan; Sun, Chen; Wei, Chaochun

2017-04-04

A fundamental concept in biology is that heritable material is passed from parents to offspring, a process called vertical gene transfer. An alternative mechanism of gene acquisition is through horizontal gene transfer (HGT), which involves movement of genetic materials between different species. Horizontal gene transfer has been found prevalent in prokaryotes but very rare in eukaryote. In this paper, we investigate horizontal gene transfer in the human genome. From the pair-wise alignments between human genome and 53 vertebrate genomes, 1,467 human genome regions (2.6 M bases) from all chromosomes were found to be more conserved with non-mammals than with most mammals. These human genome regions involve 642 known genes, which are enriched with ion binding. Compared to known horizontal gene transfer regions in the human genome, there were few overlapping regions, which indicated horizontal gene transfer is more common than we expected in the human genome. Horizontal gene transfer impacts hundreds of human genes and this study provided insight into potential mechanisms of HGT in the human genome.

Finding genes discriminating smokers from non-smokers by applying a growing self-organizing clustering method to large airway epithelium cell microarray data.

PubMed

Shahdoust, Maryam; Hajizadeh, Ebrahim; Mozdarani, Hossein; Chehrei, Ali

2013-01-01

Cigarette smoking is the major risk factor for development of lung cancer. Identification of effects of tobacco on airway gene expression may provide insight into the causes. This research aimed to compare gene expression of large airway epithelium cells in normal smokers (n=13) and non-smokers (n=9) in order to find genes which discriminate the two groups and assess cigarette smoking effects on large airway epithelium cells. Genes discriminating smokers from non-smokers were identified by applying a neural network clustering method, growing self-organizing maps (GSOM), to microarray data according to class discrimination scores. An index was computed based on differentiation between each mean of gene expression in the two groups. This clustering approach provided the possibility of comparing thousands of genes simultaneously. The applied approach compared the mean of 7,129 genes in smokers and non-smokers simultaneously and classified the genes of large airway epithelium cells which had differently expressed in smokers comparing with non-smokers. Seven genes were identified which had the highest different expression in smokers compared with the non-smokers group: NQO1, H19, ALDH3A1, AKR1C1, ABHD2, GPX2 and ADH7. Most (NQO1, ALDH3A1, AKR1C1, H19 and GPX2) are known to be clinically notable in lung cancer studies. Furthermore, statistical discriminate analysis showed that these genes could classify samples in smokers and non-smokers correctly with 100% accuracy. With the performed GSOM map, other nodes with high average discriminate scores included genes with alterations strongly related to the lung cancer such as AKR1C3, CYP1B1, UCHL1 and AKR1B10. This clustering by comparing expression of thousands of genes at the same time revealed alteration in normal smokers. Most of the identified genes were strongly relevant to lung cancer in the existing literature. The genes may be utilized to identify smokers with increased risk for lung cancer. A large sample study is now

Induction of tachykinin gene and peptide expression in guinea pig nodose primary afferent neurons by allergic airway inflammation.

PubMed Central

Fischer, A; McGregor, G P; Saria, A; Philippin, B; Kummer, W

1996-01-01

Substance P (SP), neurokinin A (NKA), and calcitonin gene-related peptide (CGRP) have potent proinflammatory effects in the airways. They are released from sensory nerve endings originating in jugular and dorsal root ganglia. However, the major sensory supply to the airways originates from the nodose ganglion. In this study, we evaluated changes in neuropeptide biosynthesis in the sensory airway innervation of ovalbumin-sensitized and -challenged guinea pigs at the mRNA and peptide level. In the airways, a three- to fourfold increase of SP, NKA, and CGRP, was seen 24 h following allergen challenge. Whereas no evidence of local tachykinin biosynthesis was found 12 h after challenge, increased levels of preprotachykinin (PPT)-A mRNA (encoding SP and NKA) were found in nodose ganglia. Quantitative in situ hybridization indicated that this increase could be accounted for by de novo induction of PPT-A mRNA in nodose ganglion neurons. Quantitative immunohistochemistry showed that 24 h after challenge, the number of tachykinin-immunoreactive nodose ganglion neurons had increased by 25%. Their projection to the airways was shown. Changes in other sensory ganglia innervating the airways were not evident. These findings suggest that an induction of sensory neuropeptides in nodose ganglion neurons is crucially involved in the increase of airway hyperreactivity in the late response to allergen challenge. PMID:8941645

IL-17A Mediates a Selective Gene Expression Profile in Asthmatic Human Airway Smooth Muscle Cells

PubMed Central

Dragon, Stéphane; Hirst, Stuart J.; Lee, Tak H.

2014-01-01

Airway smooth muscle (ASM) cells are thought to contribute to the pathogenesis of allergic asthma by orchestrating and perpetuating airway inflammation and remodeling responses. In this study, we evaluated the IL-17RA signal transduction and gene expression profile in ASM cells from subjects with mild asthma and healthy individuals. Human primary ASM cells were treated with IL-17A and probed by the Affymetrix GeneChip array, and gene targets were validated by real-time quantitative RT-PCR. Genomic analysis underlined the proinflammatory nature of IL-17A, as multiple NF-κB regulatory factors and chemokines were induced in ASM cells. Transcriptional regulators consisting of primary response genes were overrepresented and displayed dynamic expression profiles. IL-17A poorly enhanced IL-1β or IL-22 gene responses in ASM cells from both subjects with mild asthma and healthy donors. Interestingly, protein modifications to the NF-κB regulatory network were not observed after IL-17A stimulation, although oscillations in IκBε expression were detected. ASM cells from subjects with mild asthma up-regulated more genes with greater overall variability in response to IL-17A than from healthy donors. Finally, in response to IL-17A, ASM cells displayed rapid activation of the extracellular signal–regulated kinase/ribosomal S6 kinase signaling pathway and increased nuclear levels of phosphorylated extracellular signal–regulated kinase. Taken together, our results suggest that IL-17A mediated modest gene expression response, which, in cooperation with the NF-κB signaling network, may regulate the gene expression profile in ASM cells. PMID:24393021

Recent events dominate interdomain lateral gene transfers between prokaryotes and eukaryotes and, with the exception of endosymbiotic gene transfers, few ancient transfer events persist

PubMed Central

Katz, Laura A.

2015-01-01

While there is compelling evidence for the impact of endosymbiotic gene transfer (EGT; transfer from either mitochondrion or chloroplast to the nucleus) on genome evolution in eukaryotes, the role of interdomain transfer from bacteria and/or archaea (i.e. prokaryotes) is less clear. Lateral gene transfers (LGTs) have been argued to be potential sources of phylogenetic information, particularly for reconstructing deep nodes that are difficult to recover with traditional phylogenetic methods. We sought to identify interdomain LGTs by using a phylogenomic pipeline that generated 13 465 single gene trees and included up to 487 eukaryotes, 303 bacteria and 118 archaea. Our goals include searching for LGTs that unite major eukaryotic clades, and describing the relative contributions of LGT and EGT across the eukaryotic tree of life. Given the difficulties in interpreting single gene trees that aim to capture the approximately 1.8 billion years of eukaryotic evolution, we focus on presence–absence data to identify interdomain transfer events. Specifically, we identify 1138 genes found only in prokaryotes and representatives of three or fewer major clades of eukaryotes (e.g. Amoebozoa, Archaeplastida, Excavata, Opisthokonta, SAR and orphan lineages). The majority of these genes have phylogenetic patterns that are consistent with recent interdomain LGTs and, with the notable exception of EGTs involving photosynthetic eukaryotes, we detect few ancient interdomain LGTs. These analyses suggest that LGTs have probably occurred throughout the history of eukaryotes, but that ancient events are not maintained unless they are associated with endosymbiotic gene transfer among photosynthetic lineages. PMID:26323756

Distinct microRNA Expression in Human Airway Cells of Asthmatic Donors Identifies a Novel Asthma-associated Gene

EPA Science Inventory

Airway inflammation is the hallmark of asthma and suggests a dysregulation of homeostatic mechanisms. MicroRNAs (miRNAs) are key regulators of gene expression, necessary for the proper function of cellular processes. Here, we tested the hypothesis that differences between healthy...

Evolutionary genomics: transdomain gene transfers.

PubMed

Bordenstein, Seth R

2007-11-06

Biologists have until now conceded that bacterial gene transfer to multicellular animals is relatively uncommon in Nature. A new study showing promiscuous insertions of bacterial endosymbiont genes into invertebrate genomes ushers in a shift in this paradigm.

Identification of horizontally transferred genes in the genus Colletotrichum reveals a steady tempo of bacterial to fungal gene transfer.

PubMed

Jaramillo, Vinicio D Armijos; Sukno, Serenella A; Thon, Michael R

2015-01-02

Horizontal gene transfer (HGT) is the stable transmission of genetic material between organisms by means other than vertical inheritance. HGT has an important role in the evolution of prokaryotes but is relatively rare in eukaryotes. HGT has been shown to contribute to virulence in eukaryotic pathogens. We studied the importance of HGT in plant pathogenic fungi by identifying horizontally transferred genes in the genomes of three members of the genus Colletotrichum. We identified eleven HGT events from bacteria into members of the genus Colletotrichum or their ancestors. The HGT events include genes involved in amino acid, lipid and sugar metabolism as well as lytic enzymes. Additionally, the putative minimal dates of transference were calculated using a time calibrated phylogenetic tree. This analysis reveals a constant flux of genes from bacteria to fungi throughout the evolution of subphylum Pezizomycotina. Genes that are typically transferred by HGT are those that are constantly subject to gene duplication and gene loss. The functions of some of these genes suggest roles in niche adaptation and virulence. We found no evidence of a burst of HGT events coinciding with major geological events. In contrast, HGT appears to be a constant, albeit rare phenomenon in the Pezizomycotina, occurring at a steady rate during their evolution.

[Analysis of horizontal transfer gene of Bombyx mori NPV].

PubMed

Duan, Hai-Rong; Qiu, De-Bin; Gong, Cheng-Liang; Huang, Mo-Li

2011-06-01

For research on genetic characters and evolutionary origin of the genome of baculoviruses, a comprehensive homology search and phylogenetic analysis of the complete genomes of Bombyx mori NPV and Bombyx mori were used. Three horizontally transferred genes (inhibitor of apoptosis, chitinase, and UDP-glucosyltransferase) were identified, and there was evidence that all of these genes were derived from the insect host. The results of analysis showed lots of differences between the features of horizontal transferred genes and the ones of whole genomic genes, such as nucleotide composition, codon usagebias and selection pressure. These results reconfirmed that the horizontally transferred genes are exogenous. The analysis of gene function suggested that horizontally transferred genes acquired from an ancestral host insect can increase the efficiency of baculoviruses transmission.

The chromosomal organization of horizontal gene transfer in bacteria.

PubMed

Oliveira, Pedro H; Touchon, Marie; Cury, Jean; Rocha, Eduardo P C

2017-10-10

Bacterial adaptation is accelerated by the acquisition of novel traits through horizontal gene transfer, but the integration of these genes affects genome organization. We found that transferred genes are concentrated in only ~1% of the chromosomal regions (hotspots) in 80 bacterial species. This concentration increases with genome size and with the rate of transfer. Hotspots diversify by rapid gene turnover; their chromosomal distribution depends on local contexts (neighboring core genes), and content in mobile genetic elements. Hotspots concentrate most changes in gene repertoires, reduce the trade-off between genome diversification and organization, and should be treasure troves of strain-specific adaptive genes. Most mobile genetic elements and antibiotic resistance genes are in hotspots, but many hotspots lack recognizable mobile genetic elements and exhibit frequent homologous recombination at flanking core genes. Overrepresentation of hotspots with fewer mobile genetic elements in naturally transformable bacteria suggests that homologous recombination and horizontal gene transfer are tightly linked in genome evolution.Horizontal gene transfer (HGT) is an important mechanism for genome evolution and adaptation in bacteria. Here, Oliveira and colleagues find HGT hotspots comprising  ~ 1% of the chromosomal regions in 80 bacterial species.

Influence of prehospital airway management on neurological outcome in patients transferred to a heart attack centre following out-of-hospital cardiac arrest.

PubMed

Edwards, Timothy; Williams, Julia; Cottee, Michaela

2018-05-11

To describe the association between prehospital airway management and neurological outcomes in patients transferred by the ambulance service directly to a heart attack centre (HAC) post-return of spontaneous circulation (ROSC). A retrospective observational cohort study in which ambulance records were reviewed to determine prehospital airway management strategy and collect physiological and demographic data. HAC notes were obtained to determine in-hospital management and quantify neurological outcome via the cerebral performance category (CPC) scale. Statistical analyses were performed via χ 2 -test, Mann-Whitney U-test, odds ratios and binomial logistic regression. Two hundred and twenty patients were included between August 2013 and August 2014, with complete outcome data obtained for 209. Median age of patients with complete outcome data was 67 years and 71.3% were male (n = 149). Airway management was provided using a supraglottic airway (SGA) in 72.7% of cases (n = 152) with the remainder undergoing endotracheal intubation (ETI). There was no significant difference in the proportion of patients who had a good neurological outcome (CPC 1 and 2) at discharge between the SGA and ETI groups (P = 0.29). Binomial logistic regression incorporating factors known to influence outcome demonstrated no significant difference in neurological outcomes between the SGA and ETI groups (adjusted OR 0.73, 95% CI 0.34-1.56). In this observational study, there was no significant difference in the proportion of good neurological outcomes in patients managed with SGA versus ETI during cardiac arrest and in the post-ROSC transfer phase. Further research is required to provide more definitive evidence in relation to the optimal airway management strategy in out-of-hospital cardiac arrest. © 2018 Australasian College for Emergency Medicine and Australasian Society for Emergency Medicine.

CRISPR-Cas9-mediated gene knockout in primary human airway epithelial cells reveals a proinflammatory role for MUC18.

PubMed

Chu, H W; Rios, C; Huang, C; Wesolowska-Andersen, A; Burchard, E G; O'Connor, B P; Fingerlin, T E; Nichols, D; Reynolds, S D; Seibold, M A

2015-10-01

Targeted knockout of genes in primary human cells using CRISPR-Cas9-mediated genome-editing represents a powerful approach to study gene function and to discern molecular mechanisms underlying complex human diseases. We used lentiviral delivery of CRISPR-Cas9 machinery and conditional reprogramming culture methods to knockout the MUC18 gene in human primary nasal airway epithelial cells (AECs). Massively parallel sequencing technology was used to confirm that the genome of essentially all cells in the edited AEC populations contained coding region insertions and deletions (indels). Correspondingly, we found mRNA expression of MUC18 was greatly reduced and protein expression was absent. Characterization of MUC18 knockout cell populations stimulated with TLR2, 3 and 4 agonists revealed that IL-8 (a proinflammatory chemokine) responses of AECs were greatly reduced in the absence of functional MUC18 protein. Our results show the feasibility of CRISPR-Cas9-mediated gene knockouts in AEC culture (both submerged and polarized), and suggest a proinflammatory role for MUC18 in airway epithelial response to bacterial and viral stimuli.

Cigarette smoke differentially affects IL-13-induced gene expression in human airway epithelial cells.

PubMed

Mertens, Tinne C J; van der Does, Anne M; Kistemaker, Loes E; Ninaber, Dennis K; Taube, Christian; Hiemstra, Pieter S

2017-07-01

Allergic airways inflammation in asthma is characterized by an airway epithelial gene signature composed of POSTN , CLCA1 , and SERPINB2 This Th2 gene signature is proposed as a tool to classify patients with asthma into Th2-high and Th2-low phenotypes. However, many asthmatics smoke and the effects of cigarette smoke exposure on the epithelial Th2 gene signature are largely unknown. Therefore, we investigated the combined effect of IL-13 and whole cigarette smoke (CS) on the Th2 gene signature and the mucin-related genes MUC5AC and SPDEF in air-liquid interface differentiated human bronchial (ALI-PBEC) and tracheal epithelial cells (ALI-PTEC). Cultures were exposed to IL-13 for 14 days followed by 5 days of IL-13 with CS exposure. Alternatively, cultures were exposed once daily to CS for 14 days, followed by 5 days CS with IL-13. POSTN , SERPINB2 , and CLCA1 expression were measured 24 h after the last exposure to CS and IL-13. In both models POSTN , SERPINB2 , and CLCA1 expression were increased by IL-13. CS markedly affected the IL-13-induced Th2 gene signature as indicated by a reduced POSTN , CLCA1 , and MUC5AC expression in both models. In contrast, IL-13-induced SERPINB2 expression remained unaffected by CS, whereas SPDEF expression was additively increased. Importantly, cessation of CS exposure failed to restore IL-13-induced POSTN and CLCA1 expression. We show for the first time that CS differentially affects the IL-13-induced gene signature for Th2-high asthma. These findings provide novel insights into the interaction between Th2 inflammation and cigarette smoke that is important for asthma pathogenesis and biomarker-guided therapy in asthma. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Horizontal gene transfer between bacteria.

PubMed

Heuer, Holger; Smalla, Kornelia

2007-01-01

Horizontal gene transfer (HGT) refers to the acquisition of foreign genes by organisms. The occurrence of HGT among bacteria in the environment is assumed to have implications in the risk assessment of genetically modified bacteria which are released into the environment. First, introduced genetic sequences from a genetically modified bacterium could be transferred to indigenous micro-organisms and alter their genome and subsequently their ecological niche. Second, the genetically modified bacterium released into the environment might capture mobile genetic elements (MGE) from indigenous micro-organisms which could extend its ecological potential. Thus, for a risk assessment it is important to understand the extent of HGT and genome plasticity of bacteria in the environment. This review summarizes the present state of knowledge on HGT between bacteria as a crucial mechanism contributing to bacterial adaptability and diversity. In view of the use of GM crops and microbes in agricultural settings, in this mini-review we focus particularly on the presence and role of MGE in soil and plant-associated bacteria and the factors affecting gene transfer.

Viral Vectors for in Vivo Gene Transfer

NASA Astrophysics Data System (ADS)

Thévenot, E.; Dufour, N.; Déglon, N.

The transfer of DNA into the nucleus of a eukaryotic cell (gene transfer) is a central theme of modern biology. The transfer is said to be somatic when it refers to non-germline organs of a developed individual, and germline when it concerns gametes or the fertilised egg of an animal, with the aim of transmitting the relevant genetic modification to its descendents [1]. The efficient introduction of genetic material into a somatic or germline cell and the control of its expression over time have led to major advances in understanding how genes work in vivo, i.e., in living organisms (functional genomics), but also to the development of innovative therapeutic methods (gene therapy). The efficiency of gene transfer is conditioned by the vehicle used, called the vector. Desirable features for a vector are as follows: Easy to produce high titer stocks of the vector in a reproducible way. Absence of toxicity related to transduction (transfer of genetic material into the target cell, and its expression there) and no immune reaction of the organism against the vector and/or therapeutic protein. Stability in the expression of the relevant gene over time, and the possibility of regulation, e.g., to control expression of the therapeutic protein on the physiological level, or to end expression at the end of treatment. Transduction of quiescent cells should be as efficient as transduction of dividing cells. Vectors currently used fall into two categories: non-viral and viral vectors. In non-viral vectors, the DNA is complexed with polymers, lipids, or cationic detergents (described in Chap. 3). These vectors have a low risk of toxicity and immune reaction. However, they are less efficient in vivo than viral vectors when it comes to the number of cells transduced and long-term transgene expression. (Naked DNA transfer or electroporation is rather inefficient in the organism. This type of gene transfer will not be discussed here, and the interested reader is referred to the

PCR-based detection of gene transfer vectors: application to gene doping surveillance.

PubMed

Perez, Irene C; Le Guiner, Caroline; Ni, Weiyi; Lyles, Jennifer; Moullier, Philippe; Snyder, Richard O

2013-12-01

Athletes who illicitly use drugs to enhance their athletic performance are at risk of being banned from sports competitions. Consequently, some athletes may seek new doping methods that they expect to be capable of circumventing detection. With advances in gene transfer vector design and therapeutic gene transfer, and demonstrations of safety and therapeutic benefit in humans, there is an increased probability of the pursuit of gene doping by athletes. In anticipation of the potential for gene doping, assays have been established to directly detect complementary DNA of genes that are top candidates for use in doping, as well as vector control elements. The development of molecular assays that are capable of exposing gene doping in sports can serve as a deterrent and may also identify athletes who have illicitly used gene transfer for performance enhancement. PCR-based methods to detect foreign DNA with high reliability, sensitivity, and specificity include TaqMan real-time PCR, nested PCR, and internal threshold control PCR.

Gene transfers can date the tree of life.

PubMed

Davín, Adrián A; Tannier, Eric; Williams, Tom A; Boussau, Bastien; Daubin, Vincent; Szöllősi, Gergely J

2018-05-01

Biodiversity has always been predominantly microbial, and the scarcity of fossils from bacteria, archaea and microbial eukaryotes has prevented a comprehensive dating of the tree of life. Here, we show that patterns of lateral gene transfer deduced from an analysis of modern genomes encode a novel and abundant source of information about the temporal coexistence of lineages throughout the history of life. We use state-of-the-art species tree-aware phylogenetic methods to reconstruct the history of thousands of gene families and demonstrate that dates implied by gene transfers are consistent with estimates from relaxed molecular clocks in Bacteria, Archaea and Eukarya. We present the order of speciations according to lateral gene transfer data calibrated to geological time for three datasets comprising 40 genomes for Cyanobacteria, 60 genomes for Archaea and 60 genomes for Fungi. An inspection of discrepancies between transfers and clocks and a comparison with mammalian fossils show that gene transfer in microbes is potentially as informative for dating the tree of life as the geological record in macroorganisms.

Horizontal Gene Transfer and the History of Life

PubMed Central

Daubin, Vincent; Szöllősi, Gergely J.

2016-01-01

Microbes acquire DNA from a variety of sources. The last decades, which have seen the development of genome sequencing, have revealed that horizontal gene transfer has been a major evolutionary force that has constantly reshaped genomes throughout evolution. However, because the history of life must ultimately be deduced from gene phylogenies, the lack of methods to account for horizontal gene transfer has thrown into confusion the very concept of the tree of life. As a result, many questions remain open, but emerging methodological developments promise to use information conveyed by horizontal gene transfer that remains unexploited today. PMID:26801681

Airway responsiveness to mannitol in asthma is associated with chymase-positive mast cells and eosinophilic airway inflammation.

PubMed

Sverrild, A; Bergqvist, A; Baines, K J; Porsbjerg, C; Andersson, C K; Thomsen, S F; Hoffmann, H J; Gibson, P; Erjefält, J S; Backer, V

2016-02-01

Airway hyperresponsiveness (AHR) to inhaled mannitol is associated with indirect markers of mast cell activation and eosinophilic airway inflammation. It is unknown how AHR to mannitol relates to mast cell phenotype, mast cell function and measures of eosinophilic inflammation in airway tissue. We compared the number and phenotype of mast cells, mRNA expression of mast cell-associated genes and number of eosinophils in airway tissue of subjects with asthma and healthy controls in relation to AHR to mannitol. Airway hyperresponsiveness to inhaled mannitol was measured in 23 non-smoking, corticosteroid-free asthmatic individuals and 10 healthy controls. Mast cells and eosinophils were identified in mucosal biopsies from all participants. Mast cells were divided into phenotypes based on the presence of chymase. mRNA expression of mast cell-associated genes was measured by real-time PCR. The proportion of submucosal MCTC was higher in asthmatic individuals with AHR to mannitol compared with asthmatic individuals without AHR (median: 40.3% vs. 18.7%, P = 0.03). Increased submucosal MCTC numbers were associated with increased levels of mRNA for thymic stromal lymphopoietin (TSLP) and CPA3 in asthmatics. Reactivity to mannitol correlated significantly with eosinophils in submucosa (r(s): 0.56, P = 0.01). Airway hyperresponsiveness to inhaled mannitol is associated with an altered submucosal mast cell profile in asthmatic individuals. This mast cell profile is associated with increased levels of TSLP and CPA3. The degree of AHR to mannitol is correlated with the degree of eosinophilic inflammation in the airway submucosa. © 2015 John Wiley & Sons Ltd.

Pathogenicity Island-Directed Transfer of Unlinked Chromosomal Virulence Genes

PubMed Central

Chen, John; Ram, Geeta; Penadés, José R.; Brown, Stuart; Novick, Richard P.

2014-01-01

Summary In recent decades, the notorious pathogen Staphylococcus aureus has become progressively more contagious, more virulent and more resistant to antibiotics. This implies a rather dynamic evolutionary capability, representing a remarkable level of genomic plasticity, most probably maintained by horizontal gene transfer. Here we report that the staphylococcal pathogenicity islands have a dual role in gene transfer: they not only mediate their own transfer, but they can independently direct the transfer of unlinked chromosomal segments containing virulence genes. While transfer of the island itself requires specific helper phages, transfer of unlinked chromosomal segments does not, so that potentially any pac-type phage will serve. These results reveal that SaPIs can increase the horizontal exchange of accessory genes associated with disease, and may shape pathogen genomes beyond the confines of their attachment sites. PMID:25498143

Methods for Gene Transfer to the Central Nervous System

PubMed Central

Kantor, Boris; Bailey, Rachel M.; Wimberly, Keon; Kalburgi, Sahana N.; Gray, Steven J.

2015-01-01

Gene transfer is an increasingly utilized approach for research and clinical applications involving the central nervous system (CNS). Vectors for gene transfer can be as simple as an unmodified plasmid, but more commonly involve complex modifications to viruses to make them suitable gene delivery vehicles. This chapter will explain how tools for CNS gene transfer have been derived from naturally occurring viruses. The current capabilities of plasmid, retroviral, adeno-associated virus, adenovirus, and herpes simplex virus vectors for CNS gene delivery will be described. These include both focal and global CNS gene transfer strategies, with short- or long-term gene expression. As is described in this chapter, an important aspect of any vector is the cis-acting regulatory elements incorporated into the vector genome that control when, where, and how the transgene is expressed. PMID:25311922

Problems associated with gene transfer and opportunities for microgravity environments

NASA Astrophysics Data System (ADS)

Tennessen, Daniel J.

1997-01-01

The method of crop improvement by gene transfer is becoming increasingly routine with transgenic foods and ornamental crops now being marketed to consumers. However, biological processes of plants, and the physical barriers of current protocols continue to limit the application of gene transfer in many commercial crops. The goal of this paper is to outline the current limitations of gene transfer and to hypothesize possible opportunities for use of microgravity to overcome such limitations. The limitations detailed in this paper include host-range specificity of Agrobacterium mediated transformation, probability of gene insertion, position effects of the inserted genes, gene copy number, stability of foreign gene expression in host plants, and regeneration of recalcitrant plant species. Microgravity offers an opportunity for gene transfer where cell growth kinetics, DNA synthesis, and genetic recombination rates can be altered. Such biological conditions may enhance the ability for recombination of reporter genes and other genes of interest to agriculture. Proposed studies would be useful for understanding instability of foreign gene expression and may lead to stable transformed plants. Other aspects of gene transfer in microgravity are discussed.

A receptor-targeted nanocomplex vector system optimized for respiratory gene transfer.

PubMed

Tagalakis, Aristides D; McAnulty, Robin J; Devaney, James; Bottoms, Stephen E; Wong, John B; Elbs, Martin; Writer, Michele J; Hailes, Helen C; Tabor, Alethea B; O'Callaghan, Christopher; Jaffe, Adam; Hart, Stephen L

2008-05-01

Synthetic vectors for cystic fibrosis (CF) gene therapy are required that efficiently and safely transfect airway epithelial cells, rather than alveolar epithelial cells or macrophages, and that are nonimmunogenic, thus allowing for repeated delivery. We have compared several vector systems against these criteria including GL67, polyethylenimine (PEI) 22 and 25 kd and two new, synthetic vector formulations, comprising a cationic, receptor-targeting peptide K(16)GACSERSMNFCG (E), and the cationic liposomes (L) DHDTMA/DOPE or DOSEP3/DOPE. The lipid and peptide formulations self assemble into receptor-targeted nanocomplexes (RTNs) LED-1 and LED-2, respectively, on mixing with plasmid (D). LED-1 transfected airway epithelium efficiently, while LED-2 and GL67 preferentially transfected alveolar cells. PEI transfected airway epithelial cells with high efficiency, but was more toxic to the mice than the other formulations. On repeat dosing, LED-1 was equally as effective as the single dose, while GL67 was 30% less effective and PEI 22 kd displayed a 90% reduction of efficiency on repeated delivery. LED-1 thus was the only formulation that fulfilled the criteria for a CF gene therapy vector while GL67 and LED-2 may be appropriate for other respiratory diseases. Opportunities for PEI depend on a solution to its toxicity problems. LED-1 formulations were stable to nebulization, the most appropriate delivery method for CF.

A Numerical Study of Heat and Water Vapor Transfer in MDCT-Based Human Airway Models

PubMed Central

Wu, Dan; Tawhai, Merryn H.; Hoffman, Eric A.; Lin, Ching-Long

2014-01-01

A three-dimensional (3D) thermo-fluid model is developed to study regional distributions of temperature and water vapor in three multi-detector row computed-tomography (MDCT)-basedhuman airwayswith minute ventilations of 6, 15 and 30 L/min. A one-dimensional (1D) model is also solved to provide necessary initial and boundary conditionsforthe 3D model. Both 3D and 1D predicted temperature distributions agree well with available in vivo measurement data. On inspiration, the 3D cold high-speed air stream is split at the bifurcation to form secondary flows, with its cold regions biased toward the inner wall. The cold air flowing along the wall is warmed up more rapidly than the air in the lumen center. The repeated splitting pattern of air streams caused by bifurcations acts as an effective mechanism for rapid heat and mass transfer in 3D. This provides a key difference from the 1D model, where heating relies largely on diffusion in the radial direction, thus significantly affecting gradient-dependent variables, such as energy flux and water loss rate. We then propose the correlations for respective heat and mass transfer in the airways of up to 6 generations: Nu=3.504(ReDaDt)0.277, R = 0.841 and Sh=3.652(ReDaDt)0.268, R = 0.825, where Nu is the Nusselt number, Sh is the Sherwood number, Re is the branch Reynolds number, Da is the airway equivalent diameter, and Dt is the tracheal equivalentdiameter. PMID:25081386

Gene expression changes in human small airway epithelial cells exposed to Delta9-tetrahydrocannabinol.

PubMed

Sarafian, Theodore; Habib, Nancy; Mao, Jenny T; Tsu, I-Hsien; Yamamoto, Mitsuko L; Hsu, Erin; Tashkin, Donald P; Roth, Michael D

2005-08-14

Marijuana smoking is associated with inflammation, cellular atypia, and molecular dysregulation of the tracheobronchial epithelium. While marijuana smoke shares many components in common with tobacco, it also contains a high concentration of Delta9-tetrahydrocannabinol (THC). The potential contribution of THC to airway injury was assessed by exposing primary cultures of human small airway epithelial (SAE) cells to THC (0.1-10.0 microg/ml) for either 1 day or 7 days. THC induced a time- and concentration-dependent decrease in cell viability, ATP level, and mitochondrial membrane potential. Using a targeted gene expression array, we observed acute changes (24 h) in the expression of mRNA for caspase-8, catalase, Bax, early growth response-1, cytochrome P4501A1 (CYP1A1), metallothionein 1A, PLAB, and heat shock factor 1 (HSF1). After 7 days of exposure, decrease in expression of mRNA for heat shock proteins (HSPs) and the pro-apoptotic protein Bax was observed, while expression of GADD45A, IL-1A, CYP1A1, and PTGS-2 increased significantly. These findings suggest a contribution of THC to DNA damage, inflammation, and alterations in apoptosis. Treatment with selected prototypical toxicants, 2,3,7,8-tetrachlorodibenznzo-p-dioxin (TCDD) and carbonyl cyanide-p-(trifluoramethoxy)-phenyl hydrazone (FCCP), produced partially overlapping gene expression profiles suggesting some similarity in mechanism of action with THC. THC, delivered as a component of marijuana smoke, may induce a profile of gene expression that contributes to the pulmonary pathology associated with marijuana use.

Gene transfer to promote cardiac regeneration.

PubMed

Collesi, Chiara; Giacca, Mauro

2016-12-01

There is an impelling need to develop new therapeutic strategies for patients with myocardial infarction and heart failure. Leading from the large quantity of new information gathered over the last few years on the mechanisms controlling cardiomyocyte proliferation during embryonic and fetal life, it is now possible to devise innovative therapies based on cardiac gene transfer. Different protein-coding genes controlling cell cycle progression or cardiomyocyte specification and differentiation, along with microRNA mimics and inhibitors regulating pre-natal and early post-natal cell proliferation, are amenable to transformation in potential therapeutics for cardiac regeneration. These gene therapy approaches are conceptually revolutionary, since they are aimed at stimulating the intrinsic potential of differentiated cardiac cells to proliferate, rather than relying on the implantation of exogenously expanded cells to achieve tissue regeneration. For efficient and prolonged cardiac gene transfer, vectors based on the Adeno-Associated Virus stand as safe, efficient and reliable tools for cardiac gene therapy applications.

Horizontal gene transfer of microbial cellulases into nematode genomes is associated with functional assimilation and gene turnover

PubMed Central

2011-01-01

Background Natural acquisition of novel genes from other organisms by horizontal or lateral gene transfer is well established for microorganisms. There is now growing evidence that horizontal gene transfer also plays important roles in the evolution of eukaryotes. Genome-sequencing and EST projects of plant and animal associated nematodes such as Brugia, Meloidogyne, Bursaphelenchus and Pristionchus indicate horizontal gene transfer as a key adaptation towards parasitism and pathogenicity. However, little is known about the functional activity and evolutionary longevity of genes acquired by horizontal gene transfer and the mechanisms favoring such processes. Results We examine the transfer of cellulase genes to the free-living and beetle-associated nematode Pristionchus pacificus, for which detailed phylogenetic knowledge is available, to address predictions by evolutionary theory for successful gene transfer. We used transcriptomics in seven Pristionchus species and three other related diplogastrid nematodes with a well-defined phylogenetic framework to study the evolution of ancestral cellulase genes acquired by horizontal gene transfer. We performed intra-species, inter-species and inter-genic analysis by comparing the transcriptomes of these ten species and tested for cellulase activity in each species. Species with cellulase genes in their transcriptome always exhibited cellulase activity indicating functional integration into the host's genome and biology. The phylogenetic profile of cellulase genes was congruent with the species phylogeny demonstrating gene longevity. Cellulase genes show notable turnover with elevated birth and death rates. Comparison by sequencing of three selected cellulase genes in 24 natural isolates of Pristionchus pacificus suggests these high evolutionary dynamics to be associated with copy number variations and positive selection. Conclusion We could demonstrate functional integration of acquired cellulase genes into the nematode

Horizontal gene transfer of microbial cellulases into nematode genomes is associated with functional assimilation and gene turnover.

PubMed

Mayer, Werner E; Schuster, Lisa N; Bartelmes, Gabi; Dieterich, Christoph; Sommer, Ralf J

2011-01-13

Natural acquisition of novel genes from other organisms by horizontal or lateral gene transfer is well established for microorganisms. There is now growing evidence that horizontal gene transfer also plays important roles in the evolution of eukaryotes. Genome-sequencing and EST projects of plant and animal associated nematodes such as Brugia, Meloidogyne, Bursaphelenchus and Pristionchus indicate horizontal gene transfer as a key adaptation towards parasitism and pathogenicity. However, little is known about the functional activity and evolutionary longevity of genes acquired by horizontal gene transfer and the mechanisms favoring such processes. We examine the transfer of cellulase genes to the free-living and beetle-associated nematode Pristionchus pacificus, for which detailed phylogenetic knowledge is available, to address predictions by evolutionary theory for successful gene transfer. We used transcriptomics in seven Pristionchus species and three other related diplogastrid nematodes with a well-defined phylogenetic framework to study the evolution of ancestral cellulase genes acquired by horizontal gene transfer. We performed intra-species, inter-species and inter-genic analysis by comparing the transcriptomes of these ten species and tested for cellulase activity in each species. Species with cellulase genes in their transcriptome always exhibited cellulase activity indicating functional integration into the host's genome and biology. The phylogenetic profile of cellulase genes was congruent with the species phylogeny demonstrating gene longevity. Cellulase genes show notable turnover with elevated birth and death rates. Comparison by sequencing of three selected cellulase genes in 24 natural isolates of Pristionchus pacificus suggests these high evolutionary dynamics to be associated with copy number variations and positive selection. We could demonstrate functional integration of acquired cellulase genes into the nematode's biology as predicted by theory

A recently transferred cluster of bacterial genes in Trichomonas vaginalis - lateral gene transfer and the fate of acquired genes

PubMed Central

2014-01-01

Background Lateral Gene Transfer (LGT) has recently gained recognition as an important contributor to some eukaryote proteomes, but the mechanisms of acquisition and fixation in eukaryotic genomes are still uncertain. A previously defined norm for LGTs in microbial eukaryotes states that the majority are genes involved in metabolism, the LGTs are typically localized one by one, surrounded by vertically inherited genes on the chromosome, and phylogenetics shows that a broad collection of bacterial lineages have contributed to the transferome. Results A unique 34 kbp long fragment with 27 clustered genes (TvLF) of prokaryote origin was identified in the sequenced genome of the protozoan parasite Trichomonas vaginalis. Using a PCR based approach we confirmed the presence of the orthologous fragment in four additional T. vaginalis strains. Detailed sequence analyses unambiguously suggest that TvLF is the result of one single, recent LGT event. The proposed donor is a close relative to the firmicute bacterium Peptoniphilus harei. High nucleotide sequence similarity between T. vaginalis strains, as well as to P. harei, and the absence of homologs in other Trichomonas species, suggests that the transfer event took place after the radiation of the genus Trichomonas. Some genes have undergone pseudogenization and degradation, indicating that they may not be retained in the future. Functional annotations reveal that genes involved in informational processes are particularly prone to degradation. Conclusions We conclude that, although the majority of eukaryote LGTs are single gene occurrences, they may be acquired in clusters of several genes that are subsequently cleansed of evolutionarily less advantageous genes. PMID:24898731

The ethics of human gene transfer.

PubMed

Kimmelman, Jonathan

2008-03-01

Almost 20 years since the first gene-transfer trial was carried out in humans, the field has made significant advances towards clinical application. Nevertheless, it continues to face numerous unresolved ethical challenges--among them are the question of when to initiate human testing, the acceptability of germline modification and whether the technique should be applied to the enhancement of traits. Although such issues have precedents in other medical contexts, they take on a different character in gene transfer, in part because of the scientific uncertainty and the social context of innovation.

Multi-stage surgery for airway patency after metallic stent removal in benign laryngotracheal airway disease in two adolescents.

PubMed

Coordes, Annekatrin; Todt, Ingo; Ernst, Arne; Seidl, Rainer O

2013-05-01

Laryngotracheal stents may damage the highly complex laryngeal structures, impair voice and swallowing functions and cause tissue ingrowths, thereby necessitating airway patency interventions. In benign airway disease, the number of adolescents with laryngotracheal stents is therefore limited. We present two cases of laryngeal metallic stent placement following benign airway disease. Two adolescents presented with severe dyspnea and self-expandable metallic stent placement after benign laryngotracheal stenoses. Granulation tissue ingrowths required additional surgical interventions every 6-8 weeks to recanalize the stent lumen. We performed multi-stage surgery including removal of the embedded stent, segmental resection of the stenotic area, end-to-end-anastomosis and laryngotracheal reconstruction respectively, to achieve patent airway without tracheal cannulation. Montgomery T-tubes were temporarily inserted to bridge the complex reconstructions. In both adolescents, we achieved successful removal of the embedded stent and patent airway. Bilateral vocal fold paralysis required additional surgery to improve the final airway patency and vocal rehabilitation. Stent removal, segmental resection and laryngotracheal reconstruction provide the achievement of patent airway and allow decannulation. Temporary Montgomery T-tubes bridge complex laryngotracheal reconstructions. In benign laryngeal airway disease, stent placement should be avoided, especially in adolescents. Transfer to a specialist center should be considered prior to metallic stent implantation. In general, self-expanding tracheobronchial stents can be placed in selected patients where surgical interventions are limited. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

The oral administration of bacterial extracts prevents asthma via the recruitment of regulatory T cells to the airways.

PubMed

Navarro, S; Cossalter, G; Chiavaroli, C; Kanda, A; Fleury, S; Lazzari, A; Cazareth, J; Sparwasser, T; Dombrowicz, D; Glaichenhaus, N; Julia, V

2011-01-01

The prevalence of asthma has steadily increased during the last decade, probably as the result of changes in the environment, including reduced microbial exposure during infancy. Accordingly, experimental studies have shown that deliberate infections with live pathogens prevent the development of allergic airway diseases in mice. Bacterial extracts are currently used in children suffering from repeated upper respiratory tract infections. In the present study, we have investigated whether bacterial extracts, commercially available as Broncho-Vaxom (BV), could prevent allergic airway disease in mice. Oral treatment with BV suppressed airway inflammation through interleukin-10 (IL-10)-dependent and MyD88 (myeloid differentiation primary response gene (88))-dependent mechanisms and induced the conversion of FoxP3 (forkhead box P3)(-) T cells into FoxP3(+) regulatory T cells. Furthermore, CD4(+) T cells purified from the trachea of BV-treated mice conferred protection against airway inflammation when adoptively transferred into sensitized mice. Therefore, treatment with BV could possibly be a safe and efficient strategy to prevent the development of allergic diseases in children.

Mutated-leptin gene transfer induces increases in body weight by electroporation and hydrodynamics-based gene delivery in mice.

PubMed

Xiang, Lan; Murai, Atsushi; Muramatsu, Tatsuo

2005-12-01

To investigate whether in vivo gene transfer causes leptin-antagonistic effects on food intake, animal body weight and fat tissue weight, the R128Q mutated-leptin gene, an R to Q substitution at position 128 of mouse leptin, was transferred into mouse liver and leg muscle by electroporation and hydrodynamics-based gene delivery. Mutated-leptin gene transfer by electroporation caused significant increases in body weight at 5 days and after (5.4% increase relative to control; p<0.05). Hydrodynamics-based gene delivery of the mutated-leptin gene also caused an increase in body weight (3.0% increase relative to control; p<0.05). Mutated-leptin gene transfer by electroporation significantly increased the tissue weight of epididymal white fat and neuropeptide Y mRNA expression in the hypothalamus compared with those of the control group 3 weeks after gene transfer (p<0.05). These results suggest that mutated-leptin gene transfer successfully produced leptin-antagonistic effects by modulating the central regulator of energy homeostasis. Also, the extent of leptin-antagonistic effects by electroporation was much higher than hydrodynamics-based gene delivery, with at least single gene transfer.

Gene Transfers Between Distantly Related Organisms

NASA Technical Reports Server (NTRS)

Doolittle, Russell F.

2003-01-01

With the completion of numerous microbial genome sequences, reports of individual gene transfers between distantly related prokaryotes have become commonplace. On the other hand, transfers between prokaryotes and eukaryotes still excite the imagination. Many of these claims may be premature, but some are certainly valid. In this chapter, the kinds of supporting data needed to propose transfers between distantly related organisms and cite some interesting examples are considered.

[Gene transfer agent--a novel and widespread occurrence mechanism of gene exchange in ocean-a review].

PubMed

Cai, Haiyuan

2012-01-01

Gene Transfer Agent (GTA) particles are released by bacteria and resemble small, tailed bacteriophages. GTA particles contain small, random pieces of host DNA rather than GTA structural genes or a phage genome. Gene transfer mediated by GTA is efficient and species specific based on knowledge of currently best studied GTAs produced by 4 anaerobes. Genome sequencing projects have revealed a remarkable distribution of GTA gene clusters in the genomes of marine bacterioplankton, implying GTA may be an important mechanism for horizontal gene transfer in ocean. On basis of characterization of the 4 best studied GTAs, this review described GTAs released by numerically dominant marine bacteria, discussed their properties that were important for horizontal gene transfer in ocean, and gave future perspectives to advance GTA research.

Upper airway gene expression in smokers: the mouth as a "window to the soul" of lung carcinogenesis?

PubMed

Spira, Avrum

2010-03-01

This perspective on Boyle et al. (beginning on page 266 in this issue of the journal) explores transcriptomic profiling of upper airway epithelium as a biomarker of host response to tobacco smoke exposure. Boyle et al. have shown a striking relationship between smoking-related gene expression changes in the mouth and bronchus. This relationship suggests that buccal gene expression may serve as a relatively noninvasive surrogate marker of the physiologic response of the lung to tobacco smoke that could be used in large-scale screening and chemoprevention studies for lung cancer.

Horizontal gene transfer in the acquisition of novel traits by metazoans

PubMed Central

Boto, Luis

2014-01-01

Horizontal gene transfer is accepted as an important evolutionary force modulating the evolution of prokaryote genomes. However, it is thought that horizontal gene transfer plays only a minor role in metazoan evolution. In this paper, I critically review the rising evidence on horizontally transferred genes and on the acquisition of novel traits in metazoans. In particular, I discuss suspected examples in sponges, cnidarians, rotifers, nematodes, molluscs and arthropods which suggest that horizontal gene transfer in metazoans is not simply a curiosity. In addition, I stress the scarcity of studies in vertebrates and other animal groups and the importance of forthcoming studies to understand the importance and extent of horizontal gene transfer in animals. PMID:24403327

The genes of all seven CYP3A isoenzymes identified in the equine genome are expressed in the airways of horses.

PubMed

Tydén, E; Löfgren, M; Hakhverdyan, M; Tjälve, H; Larsson, P

2013-08-01

In the present study, we examined the gene expression of cytochrome P450 3A (CYP3A) isoenzymes in the tracheal and bronchial mucosa and in the lung of equines using TaqMan probes. The results show that all seven CYP3A isoforms identified in the equine genome, that is, CYP3A89, CYP3A93, CYP3A94, CYP3A95, CYP3A96, CYP3A97 and CYP3A129, are expressed in the airways of the investigated horses. Though in previous studies, CYP3A129 was found to be absent in equine intestinal mucosa and liver, this CYP3A isoform is expressed in the airways of horses. The gene expression of the CYP3A isoenzymes varied considerably between the individual horses studied. However, in most of the horses CYP3A89, CYP3A93, CYP3A96, CYP3A97 and CYP3A129 were expressed to a high extent, while CYP3A94 and CYP3A95 were expressed to a low extent in the different parts of the airways. The CYP3A isoenzymes present in the airways may play a role in the metabolic degradation of inhaled xenobiotics. In some instances, the metabolism may, however, result in bioactivation of the xenobiotics and subsequent tissue injury. © 2012 John Wiley & Sons Ltd.

Sphingosine-1-phosphate induces pro-remodelling response in airway smooth muscle cells

PubMed Central

Fuerst, E; Foster, H R; Ward, J P T; Corrigan, C J; Cousins, D J; Woszczek, G

2014-01-01

Background Increased proliferation of airway smooth muscle (ASM) cells leading to hyperplasia and increased ASM mass is one of the most characteristic features of airway remodelling in asthma. A bioactive lipid, sphingosine-1-phosphate (S1P), has been suggested to affect airway remodelling by stimulation of human ASM cell proliferation. Objective To investigate the effect of S1P on signalling and regulation of gene expression in ASM cells from healthy and asthmatic individuals. Methods Airway smooth muscle cells grown from bronchial biopsies of healthy and asthmatic individuals were exposed to S1P. Gene expression was analysed using microarray, real-time PCR and Western blotting. Receptor signalling and function were determined by mRNA knockdown and intracellular calcium mobilization experiments. Results S1P potently regulated the expression of more than 80 genes in human ASM cells, including several genes known to be involved in the regulation of cell proliferation and airway remodelling (HBEGF, TGFB3, TXNIP, PLAUR, SERPINE1, RGS4). S1P acting through S1P2 and S1P3 receptors activated intracellular calcium mobilization and extracellular signal-regulated and Rho-associated kinases to regulate gene expression. S1P-induced responses were not inhibited by corticosteroids and did not differ significantly between ASM cells from healthy and asthmatic individuals. Conclusion S1P induces a steroid-resistant, pro-remodelling pathway in ASM cells. Targeting S1P or its receptors could be a novel treatment strategy for inhibiting airway remodelling in asthma. PMID:25041788

Cystic fibrosis gene therapy: a mutation-independent treatment.

PubMed

Griesenbach, Uta; Davies, Jane C; Alton, Eric

2016-11-01

Since cloning of the disease-causing gene 27 years ago, the development of cystic fibrosis (CF) gene therapy has been pursued. Here, we will summarize key findings with a particular focus on recent developments. Almost 3 decades of research have highlighted the complexity of lung gene transfer and have generated a body of data that has recently led to the completion of a large phase IIB study. This trial has, for the first time, shown that nonviral gene transfer can, albeit modestly, stabilize lung function in CF and provides the impetus for further development of more potent gene transfer agents. Lentiviral vectors, specifically pseudotyped to enable entry into airway epithelial cells have most recently been developed. Persistent expression after a single dose and the ability to be administered repeatedly suggest that these viral vectors hold promise for the treatment of CF; a first-in-man clinical trial will shortly be initiated. Although the development of CF gene therapy has been slower than initially anticipated, recent progress has been encouraging and has renewed the interest of academics and industry to pursue lung gene therapy.

Abiotic gene transfer: rare or rampant?

PubMed Central

Kotnik, Tadej; Weaver, James C.

2016-01-01

Phylogenetic studies reveal that horizontal gene transfer (HGT) plays a prominent role in evolution and genetic variability of life. Five biotic mechanisms of HGT among prokaryotic organisms have been extensively characterized: conjugation, competence, transduction, gene-transfer-agent (GTA) particles, and transitory fusion with recombination, but it is not known whether they can account for all natural HGT. It is even less clear how HGT could have occurred before any of these mechanisms had developed. Here, we consider contemporary conditions and experiments on microorganisms to estimate possible roles of abiotic HGT – currently and throughout evolution. Candidate mechanisms include freeze-and-thaw, microbeads-agitation, and electroporation-based transformation, and we posit that these laboratory techniques have analogues in nature acting as mechanisms of abiotic HGT: freeze-and-thaw cycles in polar waters, sand-agitation at foreshores and riverbeds, and lightning-triggered electroporation in near-surface aqueous habitats. We derive conservative order-of-magnitude estimates for rates of microorganisms subjected to freeze-and-thaw cycles, sand-agitation, and lightning-triggered electroporation, at 1024, 1019, and 1017 per year, respectively. Considering the yield of viable transformants, which is by far the highest in electroporation, we argue this may still favor lightning-triggered transformation over the other two mechanisms. Electroporation-based gene transfer also appears to be the most general of these abiotic candidates, and perhaps even of all known HGT mechanisms. Future studies should provide improved estimates of gene transfer rates and cell viability, currently and in the past, but to assess the importance of abiotic HGT in nature, will likely require substantial progress – also in knowledge of biotic HGT. PMID:27067073

Applying horizontal gene transfer phenomena to enhance non-viral gene therapy

PubMed Central

Elmer, Jacob J.; Christensen, Matthew D.; Rege, Kaushal

2014-01-01

Horizontal gene transfer (HGT) is widespread amongst prokaryotes, but eukaryotes tend to be far less promiscuous with their genetic information. However, several examples of HGT from pathogens into eukaryotic cells have been discovered and mimicked to improve non-viral gene delivery techniques. For example, several viral proteins and DNA sequences have been used to significantly increase cytoplasmic and nuclear gene delivery. Plant genetic engineering is routinely performed with the pathogenic bacterium Agrobacterium tumefaciens and similar pathogens (e.g. Bartonella henselae) may also be able to transform human cells. Intracellular parasites like Trypanosoma cruzi may also provide new insights into overcoming cellular barriers to gene delivery. Finally, intercellular nucleic acid transfer between host cells will also be briefly discussed. This article will review the unique characteristics of several different viruses and microbes and discuss how their traits have been successfully applied to improve non-viral gene delivery techniques. Consequently, pathogenic traits that originally caused diseases may eventually be used to treat many genetic diseases. PMID:23994344

Detecting Horizontal Gene Transfer between Closely Related Taxa

PubMed Central

Adato, Orit; Ninyo, Noga; Gophna, Uri; Snir, Sagi

2015-01-01

Horizontal gene transfer (HGT), the transfer of genetic material between organisms, is crucial for genetic innovation and the evolution of genome architecture. Existing HGT detection algorithms rely on a strong phylogenetic signal distinguishing the transferred sequence from ancestral (vertically derived) genes in its recipient genome. Detecting HGT between closely related species or strains is challenging, as the phylogenetic signal is usually weak and the nucleotide composition is normally nearly identical. Nevertheless, there is a great importance in detecting HGT between congeneric species or strains, especially in clinical microbiology, where understanding the emergence of new virulent and drug-resistant strains is crucial, and often time-sensitive. We developed a novel, self-contained technique named Near HGT, based on the synteny index, to measure the divergence of a gene from its native genomic environment and used it to identify candidate HGT events between closely related strains. The method confirms candidate transferred genes based on the constant relative mutability (CRM). Using CRM, the algorithm assigns a confidence score based on “unusual” sequence divergence. A gene exhibiting exceptional deviations according to both synteny and mutability criteria, is considered a validated HGT product. We first employed the technique to a set of three E. coli strains and detected several highly probable horizontally acquired genes. We then compared the method to existing HGT detection tools using a larger strain data set. When combined with additional approaches our new algorithm provides richer picture and brings us closer to the goal of detecting all newly acquired genes in a particular strain. PMID:26439115

Postnatal airway growth in cystic fibrosis piglets.

PubMed

Adam, Ryan J; Abou Alaiwa, Mahmoud H; Bouzek, Drake C; Cook, Daniel P; Gansemer, Nicholas D; Taft, Peter J; Powers, Linda S; Stroik, Mallory R; Hoegger, Mark J; McMenimen, James D; Hoffman, Eric A; Zabner, Joseph; Welsh, Michael J; Meyerholz, David K; Stoltz, David A

2017-09-01

Mutations in the gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) anion channel cause CF. The leading cause of death in the CF population is lung disease. Increasing evidence suggests that in utero airway development is CFTR-dependent and that developmental abnormalities may contribute to CF lung disease. However, relatively little is known about postnatal CF airway growth, largely because such studies are limited in humans. Therefore, we examined airway growth and lung volume in a porcine model of CF. We hypothesized that CF pigs would have abnormal postnatal airway growth. To test this hypothesis, we performed CT-based airway and lung volume measurements in 3-wk-old non-CF and CF pigs. We found that 3-wk-old CF pigs had tracheas of reduced caliber and irregular shape. Their bronchial lumens were reduced in size proximally but not distally, were irregularly shaped, and had reduced distensibility. Our data suggest that lack of CFTR results in aberrant postnatal airway growth and development, which could contribute to CF lung disease pathogenesis. NEW & NOTEWORTHY This CT scan-based study of airway morphometry in the cystic fibrosis (CF) postnatal period is unique, as analogous studies in humans are greatly limited for ethical and technical reasons. Findings such as reduced airway lumen area and irregular caliber suggest that airway growth and development are CF transmembrane conductance regulator-dependent and that airway growth defects may contribute to CF lung disease pathogenesis. Copyright © 2017 the American Physiological Society.

Interferon response factor 3 is essential for house dust mite-induced airway allergy.

PubMed

Marichal, Thomas; Bedoret, Denis; Mesnil, Claire; Pichavant, Muriel; Goriely, Stanislas; Trottein, François; Cataldo, Didier; Goldman, Michel; Lekeux, Pierre; Bureau, Fabrice; Desmet, Christophe J

2010-10-01

Pattern-recognition receptors (PRRs) are critically involved in the pathophysiology of airway allergy, yet most of the signaling pathways downstream of PRRs implicated in allergic airway sensitization remain unknown. We sought to study the effects of genetic depletion of interferon response factor (IRF) 3 and IRF7, important transcription factors downstream of various PRRs, in a murine model of house dust mite (HDM)-induced allergic asthma. We compared HDM-induced allergic immune responses in IRF3-deficient (IRF3(-/-)), IRF7(-/-), and wild-type mice. Parameters of airway allergy caused by HDM exposure were strongly attenuated in IRF3(-/-), but not IRF7(-/-), mice compared with those in wild-type mice. Indeed, in HDM-exposed IRF3(-/-) mice HDM-specific T(H)2 cell responses did not develop. This correlated with impaired maturation and migration of IRF3(-/-) lung dendritic cells (DCs) on HDM treatment. Furthermore, adoptive transfer of HDM-loaded DCs indicated that IRF3(-/-) DCs had an intrinsic defect rendering them unable to migrate and to prime HDM-specific T(H)2 responses. Intriguingly, we also show that DC function and allergic airway sensitization in response to HDM were independent of signaling by type I interferons, the main target genes of IRF3. Through its role in DC function, IRF3, mainly known as a central activator of antiviral immunity, is essential for the development of T(H)2-type responses to airway allergens. Copyright © 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Efficient delivery of RNA interference oligonucleotides to polarized airway epithelia in vitro

PubMed Central

Ramachandran, Shyam; Krishnamurthy, Sateesh; Jacobi, Ashley M.; Wohlford-Lenane, Christine; Behlke, Mark A.; Davidson, Beverly L.

2013-01-01

Polarized and pseudostratified primary airway epithelia present barriers that significantly reduce their transfection efficiency and the efficacy of RNA interference oligonucleotides. This creates an impediment in studies of the airway epithelium, diminishing the utility of loss-of-function as a research tool. Here we outline methods to introduce RNAi oligonucleotides into primary human and porcine airway epithelia grown at an air-liquid interface and difficult-to-transfect transformed epithelial cell lines grown on plastic. At the time of plating, we reverse transfect small-interfering RNA (siRNA), Dicer-substrate siRNA, or microRNA oligonucleotides into cells by use of lipid or peptide transfection reagents. Using this approach we achieve significant knockdown in vitro of hypoxanthine-guanine phosphoribosyltransferase, IL-8, and CFTR expression at the mRNA and protein levels in 1–3 days. We also attain significant reduction of secreted IL-8 in polarized primary pig airway epithelia 3 days posttransfection and inhibition of CFTR-mediated Cl− conductance in polarized air-liquid interface cultures of human airway epithelia 2 wk posttransfection. These results highlight an efficient means to deliver RNA interference reagents to airway epithelial cells and achieve significant knockdown of target gene expression and function. The ability to reliably conduct loss-of-function assays in polarized primary airway epithelia offers benefits to research in studies of epithelial cell homeostasis, candidate gene function, gene-based therapeutics, microRNA biology, and targeting the replication of respiratory viruses. PMID:23624792

Replacing and Additive Horizontal Gene Transfer in Streptococcus

PubMed Central

Choi, Sang Chul; Rasmussen, Matthew D.; Hubisz, Melissa J.; Gronau, Ilan; Stanhope, Michael J.; Siepel, Adam

2012-01-01

The prominent role of Horizontal Gene Transfer (HGT) in the evolution of bacteria is now well documented, but few studies have differentiated between evolutionary events that predominantly cause genes in one lineage to be replaced by homologs from another lineage (“replacing HGT”) and events that result in the addition of substantial new genomic material (“additive HGT”). Here in, we make use of the distinct phylogenetic signatures of replacing and additive HGTs in a genome-wide study of the important human pathogen Streptococcus pyogenes (SPY) and its close relatives S. dysgalactiae subspecies equisimilis (SDE) and S. dysgalactiae subspecies dysgalactiae (SDD). Using recently developed statistical models and computational methods, we find evidence for abundant gene flow of both kinds within each of the SPY and SDE clades and of reduced levels of exchange between SPY and SDD. In addition, our analysis strongly supports a pronounced asymmetry in SPY–SDE gene flow, favoring the SPY-to-SDE direction. This finding is of particular interest in light of the recent increase in virulence of pathogenic SDE. We find much stronger evidence for SPY–SDE gene flow among replacing than among additive transfers, suggesting a primary influence from homologous recombination between co-occurring SPY and SDE cells in human hosts. Putative virulence genes are correlated with transfer events, but this correlation is found to be driven by additive, not replacing, HGTs. The genes affected by additive HGTs are enriched for functions having to do with transposition, recombination, and DNA integration, consistent with previous findings, whereas replacing HGTs seen to influence a more diverse set of genes. Additive transfers are also found to be associated with evidence of positive selection. These findings shed new light on the manner in which HGT has shaped pathogenic bacterial genomes. PMID:22617954

Airway management after maxillectomy with free flap reconstruction.

PubMed

Brickman, Daniel S; Reh, Douglas D; Schneider, Daniel S; Bush, Ben; Rosenthal, Eben L; Wax, Mark K

2013-08-01

Maxillectomy defects require complex 3-dimensional reconstructions often best suited to microvascular free tissue transfer. Postoperative airway management during this procedure has little discussion in the literature and is often dictated by surgical dogma. The purpose of this article was to review our experience in order to evaluate the effect of airway management on perioperative outcomes in patients undergoing maxillectomy with free flap reconstruction. A retrospective chart review was performed on patients receiving maxillectomy with microvascular reconstruction at 2 institutions between 1999 and 2011. Patient's airways were managed with or without elective tracheotomy at the surgical team's discretion and different perioperative outcomes were measured. The primary outcome was incidence of airway complication including pneumonia and need for further airway intervention. Secondary outcome was measured as factors leading to perioperative performance of the tracheotomy. Seventy-nine of 143 patients received elective tracheotomy perioperatively. The incidence of airway complication was equivalent between groups (10.1% vs 9.4%; p = .89). Patients with cardiopulmonary comorbidities were more likely to receive perioperative tracheotomy (74.1% vs 50.9%; p = .03) without a difference in airway complications. Other patient cofactors did not have an impact on perioperative tracheotomy or airway complication rate. Elective tracheotomy may safely be avoided in a subset of patients undergoing maxillectomy with microvascular reconstruction. Elective tracheotomy should be considered in patients with cardiopulmonary risk factors. Copyright © 2012 Wiley Periodicals, Inc.

Gene transfer to the cerebellum.

PubMed

Louboutin, Jean-Pierre; Reyes, Beverly A S; Van Bockstaele, Elisabeth J; Strayer, David S

2010-12-01

There are several diseases for which gene transfer therapy to the cerebellum might be practicable. In these studies, we used recombinant Tag-deleted SV40-derived vectors (rSV40s) to study gene delivery targeting the cerebellum. These vectors transduce neurons and microglia very effectively in vitro and in vivo, and so we tested them to evaluate gene transfer to the cerebellum in vivo. Using a rSV40 vector carrying human immunodeficiency virus (HIV)-Nef with a C-terminal FLAG epitope, we characterized the distribution, duration, and cell types transduced. Rats received test and control vectors by stereotaxic injection into the cerebellum. Transgene expression was assessed 1, 2, and 4 weeks later by immunostaining of serial brain sections. FLAG epitope-expressing cells were seen, at all times after vector administration, principally detected in the Purkinje cells of the cerebellum, identified as immunopositive for calbindin. Occasional microglial cells were tranduced; transgene expression was not detected in astrocytes or oligodendrocytes. No inflammatory or other reaction was detected at any time. Thus, SV40-derived vectors can deliver effective, safe, and durable transgene expression to the cerebellum.

Horizontal functional gene transfer from bacteria to fishes.

PubMed

Sun, Bao-Fa; Li, Tong; Xiao, Jin-Hua; Jia, Ling-Yi; Liu, Li; Zhang, Peng; Murphy, Robert W; He, Shun-Min; Huang, Da-Wei

2015-12-22

Invertebrates can acquire functional genes via horizontal gene transfer (HGT) from bacteria but fishes are not known to do so. We provide the first reliable evidence of one HGT event from marine bacteria to fishes. The HGT appears to have occurred after emergence of the teleosts. The transferred gene is expressed and regulated developmentally. Its successful integration and expression may change the genetic and metabolic repertoire of fishes. In addition, this gene contains conserved domains and similar tertiary structures in fishes and their putative donor bacteria. Thus, it may function similarly in both groups. Evolutionary analyses indicate that it evolved under purifying selection, further indicating its conserved function. We document the first likely case of HGT of functional gene from prokaryote to fishes. This discovery certifies that HGT can influence vertebrate evolution.

Panspermia and horizontal gene transfer

NASA Astrophysics Data System (ADS)

Klyce, Brig

2009-08-01

Evidence that extremophiles are hardy and ubiquitous is helping to make panspermia a respectable theory. But even if life on Earth originally came from space, biologists assume that the subsequent evolution of life is still governed by the darwinian paradigm. In this review we show how panspermia could amend darwinism and point to a cosmic source for, not only extremophiles but, all of life. This version of panspermia can be called "strong panspermia." To support this theory we will discuss recent evidence pertaining to horizontal gene transfer, viruses, genes apparently older than the Earthly evolution of the features they encode, and primate-specific genes without identifiable precursors.

Allergen-specific Th1 cells fail to counterbalance Th2 cell-induced airway hyperreactivity but cause severe airway inflammation.

PubMed

Hansen, G; Berry, G; DeKruyff, R H; Umetsu, D T

1999-01-01

Allergic asthma, which is present in as many as 10% of individuals in industrialized nations, is characterized by chronic airway inflammation and hyperreactivity induced by allergen-specific Th2 cells secreting interleukin-4 (IL-4) and IL-5. Because Th1 cells antagonize Th2 cell functions, it has been proposed that immune deviation toward Th1 can protect against asthma and allergies. Using an adoptive transfer system, we assessed the roles of Th1, Th2, and Th0 cells in a mouse model of asthma and examined the capacity of Th1 cells to counterbalance the proasthmatic effects of Th2 cells. Th1, Th2, and Th0 lines were generated from ovalbumin (OVA)-specific T-cell receptor (TCR) transgenic mice and transferred into lymphocyte-deficient, OVA-treated severe combined immunodeficiency (SCID) mice. OVA-specific Th2 and Th0 cells induced significant airway hyperreactivity and inflammation. Surprisingly, Th1 cells did not attenuate Th2 cell-induced airway hyperreactivity and inflammation in either SCID mice or in OVA-immunized immunocompetent BALB/c mice, but rather caused severe airway inflammation. These results indicate that antigen-specific Th1 cells may not protect or prevent Th2-mediated allergic disease, but rather may cause acute lung pathology. These findings have significant implications with regard to current therapeutic goals in asthma and allergy and suggest that conversion of Th2-dominated allergic inflammatory responses into Th1-dominated responses may lead to further problems.

Optimizing cardiovascular gene therapy: increased vascular gene transfer with modified adenoviral vectors.

PubMed

Kibbe, M R; Murdock, A; Wickham, T; Lizonova, A; Kovesdi, I; Nie, S; Shears, L; Billiar, T R; Tzeng, E

2000-02-01

Adenovirus is widely used as a vector for gene transfer to the vasculature. However, the efficiency of these vectors can be limited by ineffective viral-target cell interactions. Viral attachment, which largely determines adenoviral tropism, is mediated through binding of the adenoviral fiber coat protein to the Coxsackievirus and adenovirus receptor, while internalization follows binding of the adenoviral RGD motif to alpha(v)-integrin receptors. Modifications of the fiber coat protein sequence have been successful for targeting the adenovirus to more prevalent receptors in the vasculature, including heparan sulfate-containing receptors and alpha(v)-integrin receptors. Modified adenoviral vectors targeted to receptors more prevalent in the vasculature result in an increased transfer efficiency of the virus in vitro and in vivo even in the presence of clinically relevant doses of heparin. We tested 2 modified E1- and E3-deleted Ad5 type adenoviral vectors containing the beta-galactosidase gene. AdZ.F(pK7) contains multiple positively charged lysines in the fiber coat protein that target the adenovirus to heparan sulfate receptors, while AdZ.F(RGD) contains an RGD integrin-binding sequence in the fiber coat protein that allows binding to alpha(v)-integrin receptors. The gene transfer efficiency of these modified viruses was compared in rat aortic smooth muscle cells in vitro and in an in vivo porcine model of balloon-induced arterial injury. Because of the use of heparin during most vascular surgical procedures and the concern that heparin might interfere with the binding of AdZ.F(pK7) to heparan sulfate receptors, the effect of heparin on the in vitro and in vivo transfer efficiency of these 2 modified adenoviruses was evaluated. In vitro infection of rat aortic smooth muscle cells with AdZ.F(pK7) and AdZ.F(RGD) resulted in significantly higher levels of beta-galactosidase expression compared with the unmodified adenovirus (mean +/- SEM, 1766.3 +/- 89.1 and 44

Ultrasound enhances retrovirus-mediated gene transfer.

PubMed

Naka, Toshio; Sakoda, Tsuyoshi; Doi, Takashi; Tsujino, Takeshi; Masuyama, Tohru; Kawashima, Seinosuke; Iwasaki, Tadaaki; Ohyanagi, Mitsumasa

2007-01-01

Viral vector systems are efficient for transfection of foreign genes into many tissues. Especially, retrovirus based vectors integrate the transgene into the genome of the target cells, which can sustain long term expression. However, it has been demonstrated that the transduction efficiency using retrovirus is relatively lower than those of other viruses. Ultrasound was recently reported to increase gene expression using plasmid DNA, with or without, a delivery vehicle. However, there are no reports, which show an ultrasound effect to retrovirus-mediated gene transfer efficiency. Retrovirus-mediated gene transfer systems were used for transfection of 293T cells, bovine aortic endothelial cells (BAECs), rat aortic smooth muscle cells (RASMCs), and rat skeletal muscle myoblasts (L6 cells) with beta-galactosidase (beta-Gal) genes. Transduction efficiency and cell viability assay were performed on 293T cells that were exposed to varying durations (5 to 30 seconds) and power levels (1.0 watts/cm(2) to 4.0 watts/cm(2)) of ultrasound after being transduced by a retrovirus. Effects of ultrasound to the retrovirus itself was evaluated by transduction efficiency of 293T cells. After exposure to varying power levels of ultrasound to a retrovirus for 5 seconds, 293T cells were transduced by a retrovirus, and transduction efficiency was evaluated. Below 1.0 watts/cm(2) and 5 seconds exposure, ultrasound showed increased transduction efficiency and no cytotoxicity to 293T cells transduced by a retrovirus. Also, ultrasound showed no toxicity to the virus itself at the same condition. Exposure of 5 seconds at the power of 1.0 watts/cm(2) of an ultrasound resulted in significant increases in retrovirus-mediated gene expression in all four cell types tested in this experiment. Transduction efficiencies by ultrasound were enhanced 6.6-fold, 4.8-fold, 2.3-fold, and 3.2-fold in 293T cells, BAECs, RASMCs, and L6 cells, respectively. Furthermore, beta-Gal activities were also increased

Horizontal gene transfer in silkworm, Bombyx mori.

PubMed

Zhu, Bo; Lou, Miao-Miao; Xie, Guan-Lin; Zhang, Guo-Qing; Zhou, Xue-Ping; Li, Bin; Jin, Gu-Lei

2011-05-19

The domesticated silkworm, Bombyx mori, is the model insect for the order Lepidoptera, has economically important values, and has gained some representative behavioral characteristics compared to its wild ancestor. The genome of B. mori has been fully sequenced while function analysis of BmChi-h and BmSuc1 genes revealed that horizontal gene transfer (HGT) maybe bestow a clear selective advantage to B. mori. However, the role of HGT in the evolutionary history of B. mori is largely unexplored. In this study, we compare the whole genome of B. mori with those of 382 prokaryotic and eukaryotic species to investigate the potential HGTs. Ten candidate HGT events were defined in B. mori by comprehensive sequence analysis using Maximum Likelihood and Bayesian method combining with EST checking. Phylogenetic analysis of the candidate HGT genes suggested that one HGT was plant-to- B. mori transfer while nine were bacteria-to- B. mori transfer. Furthermore, functional analysis based on expression, coexpression and related literature searching revealed that several HGT candidate genes have added important characters, such as resistance to pathogen, to B. mori. Results from this study clearly demonstrated that HGTs play an important role in the evolution of B. mori although the number of HGT events in B. mori is in general smaller than those of microbes and other insects. In particular, interdomain HGTs in B. mori may give rise to functional, persistent, and possibly evolutionarily significant new genes.

Horizontal gene transfer in silkworm, Bombyx mori

PubMed Central

2011-01-01

Background The domesticated silkworm, Bombyx mori, is the model insect for the order Lepidoptera, has economically important values, and has gained some representative behavioral characteristics compared to its wild ancestor. The genome of B. mori has been fully sequenced while function analysis of BmChi-h and BmSuc1 genes revealed that horizontal gene transfer (HGT) maybe bestow a clear selective advantage to B. mori. However, the role of HGT in the evolutionary history of B. mori is largely unexplored. In this study, we compare the whole genome of B. mori with those of 382 prokaryotic and eukaryotic species to investigate the potential HGTs. Results Ten candidate HGT events were defined in B. mori by comprehensive sequence analysis using Maximum Likelihood and Bayesian method combining with EST checking. Phylogenetic analysis of the candidate HGT genes suggested that one HGT was plant-to- B. mori transfer while nine were bacteria-to- B. mori transfer. Furthermore, functional analysis based on expression, coexpression and related literature searching revealed that several HGT candidate genes have added important characters, such as resistance to pathogen, to B. mori. Conclusions Results from this study clearly demonstrated that HGTs play an important role in the evolution of B. mori although the number of HGT events in B. mori is in general smaller than those of microbes and other insects. In particular, interdomain HGTs in B. mori may give rise to functional, persistent, and possibly evolutionarily significant new genes. PMID:21595916

Twenty Years of European Union Support to Gene Therapy and Gene Transfer.

PubMed

Gancberg, David

2017-11-01

For 20 years and throughout its research programmes, the European Union has supported the entire innovation chain for gene transfer and gene therapy. The fruits of this investment are ripening as gene therapy products are reaching the European market and as clinical trials are demonstrating the safety of this approach to treat previously untreatable diseases.

Bacteriophage-associated gene transfer in pneumococcus: transduction or pseudotransduction?

PubMed Central

Porter, R D; Shoemaker, N B; Rampe, G; Guild, W R

1979-01-01

Lysates of pneumococcal phage PG24 transferred genes from one host to another in a process with many of the properties of generalized transduction, in that the host genes were packaged in DNase-resistant particles that closely resembled infectious phage in physical properties, adsorbed to the recipient cells like phage, and were inhibited by antisera to the phage and by trypsin. However, phage processes did not complete the transfer of host DNA as they did phage DNA. Instead, gene transfer required development of competence and entry of the host DNA by the endonuclease-dependent pathway used for transforming and transfecting DNA. This process often occurred on the assay plate hours after adsorption of the particles to the cells, and the transfer was DNase sensitive if challenged at this time. Phenotypic expression was therefore also delayed. The product of entry was like that in transformation, a single strand of DNA that integrates by formation of a hex-sensitive donor-recipient heteroduplex. Whether this gene transfer process is unique to this system or is only the first one described is not clear. The term "pseudotransduction" may be useful in calling attention to its unexpected features. The DNA of PG24 phage has anomalous physical properties reflecting unusual bases. Images PMID:33154

Horizontal gene transfer in an acid mine drainage microbial community.

PubMed

Guo, Jiangtao; Wang, Qi; Wang, Xiaoqi; Wang, Fumeng; Yao, Jinxian; Zhu, Huaiqiu

2015-07-04

Horizontal gene transfer (HGT) has been widely identified in complete prokaryotic genomes. However, the roles of HGT among members of a microbial community and in evolution remain largely unknown. With the emergence of metagenomics, it is nontrivial to investigate such horizontal flow of genetic materials among members in a microbial community from the natural environment. Because of the lack of suitable methods for metagenomics gene transfer detection, microorganisms from a low-complexity community acid mine drainage (AMD) with near-complete genomes were used to detect possible gene transfer events and suggest the biological significance. Using the annotation of coding regions by the current tools, a phylogenetic approach, and an approximately unbiased test, we found that HGTs in AMD organisms are not rare, and we predicted 119 putative transferred genes. Among them, 14 HGT events were determined to be transfer events among the AMD members. Further analysis of the 14 transferred genes revealed that the HGT events affected the functional evolution of archaea or bacteria in AMD, and it probably shaped the community structure, such as the dominance of G-plasma in archaea in AMD through HGT. Our study provides a novel insight into HGT events among microorganisms in natural communities. The interconnectedness between HGT and community evolution is essential to understand microbial community formation and development.

Horizontal gene transfer is a significant driver of gene innovation in dinoflagellates.

PubMed

Wisecaver, Jennifer H; Brosnahan, Michael L; Hackett, Jeremiah D

2013-01-01

The dinoflagellates are an evolutionarily and ecologically important group of microbial eukaryotes. Previous work suggests that horizontal gene transfer (HGT) is an important source of gene innovation in these organisms. However, dinoflagellate genomes are notoriously large and complex, making genomic investigation of this phenomenon impractical with currently available sequencing technology. Fortunately, de novo transcriptome sequencing and assembly provides an alternative approach for investigating HGT. We sequenced the transcriptome of the dinoflagellate Alexandrium tamarense Group IV to investigate how HGT has contributed to gene innovation in this group. Our comprehensive A. tamarense Group IV gene set was compared with those of 16 other eukaryotic genomes. Ancestral gene content reconstruction of ortholog groups shows that A. tamarense Group IV has the largest number of gene families gained (314-1,563 depending on inference method) relative to all other organisms in the analysis (0-782). Phylogenomic analysis indicates that genes horizontally acquired from bacteria are a significant proportion of this gene influx, as are genes transferred from other eukaryotes either through HGT or endosymbiosis. The dinoflagellates also display curious cases of gene loss associated with mitochondrial metabolism including the entire Complex I of oxidative phosphorylation. Some of these missing genes have been functionally replaced by bacterial and eukaryotic xenologs. The transcriptome of A. tamarense Group IV lends strong support to a growing body of evidence that dinoflagellate genomes are extraordinarily impacted by HGT.

Evaluation of biolistic gene transfer methods in vivo using non-invasive bioluminescent imaging techniques.

PubMed

Xia, Jixiang; Martinez, Angela; Daniell, Henry; Ebert, Steven N

2011-06-02

Gene therapy continues to hold great potential for treating many different types of disease and dysfunction. Safe and efficient techniques for gene transfer and expression in vivo are needed to enable gene therapeutic strategies to be effective in patients. Currently, the most commonly used methods employ replication-defective viral vectors for gene transfer, while physical gene transfer methods such as biolistic-mediated ("gene-gun") delivery to target tissues have not been as extensively explored. In the present study, we evaluated the efficacy of biolistic gene transfer techniques in vivo using non-invasive bioluminescent imaging (BLI) methods. Plasmid DNA carrying the firefly luciferase (LUC) reporter gene under the control of the human Cytomegalovirus (CMV) promoter/enhancer was transfected into mouse skin and liver using biolistic methods. The plasmids were coupled to gold microspheres (1 μm diameter) using different DNA Loading Ratios (DLRs), and "shot" into target tissues using a helium-driven gene gun. The optimal DLR was found to be in the range of 4-10. Bioluminescence was measured using an In Vivo Imaging System (IVIS-50) at various time-points following transfer. Biolistic gene transfer to mouse skin produced peak reporter gene expression one day after transfer. Expression remained detectable through four days, but declined to undetectable levels by six days following gene transfer. Maximum depth of tissue penetration following biolistic transfer to abdominal skin was 200-300 μm. Similarly, biolistic gene transfer to mouse liver in vivo also produced peak early expression followed by a decline over time. In contrast to skin, however, liver expression of the reporter gene was relatively stable 4-8 days post-biolistic gene transfer, and remained detectable for nearly two weeks. The use of bioluminescence imaging techniques enabled efficient evaluation of reporter gene expression in vivo. Our results demonstrate that different tissues show different

Orexin (hypocretin) gene transfer diminishes narcoleptic sleep behavior in mice

PubMed Central

Liu, Meng; Thankachan, Stephen; Kaur, Satvinder; Begum, Suraiya; Blanco-Centurion, Carlos; Sakurai, Takeshi; Yanagisawa, Masashi; Neve, Rachael; Shiromani, Priyattam J.

2008-01-01

Gene transfer has proven to be an effective neurobiological tool in a number of neurodegenerative diseases, but it is not known if it can correct a sleep disorder. Narcolepsy is a neurodegenerative sleep disorder linked to the loss of neurons containing the neuropeptide orexin, also known as hypocretin. Here, a replication-defective herpes simplex virus-1 amplicon-based vector was constructed to transfer the gene for mouse prepro-orexin into mice with a genetic deletion of the orexin gene. After in vitro tests confirmed successful gene transfer into cells, the gene vector was delivered to the lateral hypothalamus of orexin knockout (KO) mice where the orexin peptide was robustly expressed in the somata and processes of numerous neurons, and the peptide product was detected in the cerebrospinal fluid. During the 4-day life-span of the vector the incidence of cataplexy declined by 60%, and the levels of rapid eye movement sleep during the second half of the night were similar to levels in wild-type mice, indicating that narcoleptic sleep–wake behavior in orexin KO mice can be improved by targeted gene transfer. PMID:18973565

Horizontally transferred genes in the genome of Pacific white shrimp, Litopenaeus vannamei

PubMed Central

2013-01-01

Background In recent years, as the development of next-generation sequencing technology, a growing number of genes have been reported as being horizontally transferred from prokaryotes to eukaryotes, most of them involving arthropods. As a member of the phylum Arthropoda, the Pacific white shrimp Litopenaeus vannamei has to adapt to the complex water environments with various symbiotic or parasitic microorganisms, which provide a platform for horizontal gene transfer (HGT). Results In this study, we analyzed the genome-wide HGT events in L. vannamei. Through homology search and phylogenetic analysis, followed by experimental PCR confirmation, 14 genes with HGT event were identified: 12 of them were transferred from bacteria and two from fungi. Structure analysis of these genes showed that the introns of the two fungi-originated genes were substituted by shrimp DNA fragment, two genes transferred from bacteria had shrimp specific introns inserted in them. Furthermore, around other three bacteria-originated genes, there were three large DNA segments inserted into the shrimp genome. One segment was a transposon that fully transferred, and the other two segments contained only coding regions of bacteria. Functional prediction of these 14 genes showed that 6 of them might be related to energy metabolism, and 4 others related to defense of the organism. Conclusions HGT events from bacteria or fungi were happened in the genome of L. vannamei, and these horizontally transferred genes can be transcribed in shrimp. This is the first time to report the existence of horizontally transferred genes in shrimp. Importantly, most of these genes are exposed to a negative selection pressure and appeared to be functional. PMID:23914989

Enhanced gene delivery to the lung using biodegradable polyunsaturated cationic phosphatidylcholine-detergent conjugates.

PubMed

Pierrat, Philippe; Kereselidze, Dimitri; Lux, Marie; Lebeau, Luc; Pons, Françoise

2016-09-10

Lung diseases are among the more representative causes of mortality and morbidity worldwide and gene therapy is considered as a promising therapeutic approach for their treatment. However the design of efficient nucleic acid carriers for airway administration still is a challenge and there is a pressing need for new developments in this field. Herein, new synthetic DNA carriers based on the conjugation of a phospholipid and C12E4, a nonionic detergent, are developed. DNA complexes with phosphatidylcholine-detergent conjugates are administered in mouse airways, and transgene expression and inflammatory activity as an index of toxicity are investigated as a function of time, DNA dose, and presence of helper and stealth lipids. Introduction of a biodegradable linker between the phosphatidylcholine and detergent moieties significantly attenuates the severity of inflammatory response that characterizes cationic lipid-mediated gene transfer. Concurrent introduction of polyunsaturated fatty acid chains in the carrier scaffold improves transgene expression and further reduces airway inflammation. Finally, the biodegradable phosphatidylcholine-detergent conjugates favorably compare to GL67A, the gold standard for DNA delivery to the airway that is currently under clinical evaluation. Our findings indicate that the lipid formulations described herein may have great potential as nucleic acid carriers for gene therapy. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification of airway mucosal type 2 inflammation by using clinical biomarkers in asthmatic patients.

PubMed

Silkoff, Philip E; Laviolette, Michel; Singh, Dave; FitzGerald, J Mark; Kelsen, Steven; Backer, Vibeke; Porsbjerg, Celeste M; Girodet, Pierre-Olivier; Berger, Patrick; Kline, Joel N; Chupp, Geoffrey; Susulic, Vedrana S; Barnathan, Elliot S; Baribaud, Frédéric; Loza, Matthew J

2017-09-01

The Airways Disease Endotyping for Personalized Therapeutics (ADEPT) study profiled patients with mild, moderate, and severe asthma and nonatopic healthy control subjects. We explored this data set to define type 2 inflammation based on airway mucosal IL-13-driven gene expression and how this related to clinically accessible biomarkers. IL-13-driven gene expression was evaluated in several human cell lines. We then defined type 2 status in 25 healthy subjects, 28 patients with mild asthma, 29 patients with moderate asthma, and 26 patients with severe asthma based on airway mucosal expression of (1) CCL26 (the most differentially expressed gene), (2) periostin, or (3) a multigene IL-13 in vitro signature (IVS). Clinically accessible biomarkers included fraction of exhaled nitric oxide (Feno) values, blood eosinophil (bEOS) counts, serum CCL26 expression, and serum CCL17 expression. Expression of airway mucosal CCL26, periostin, and IL-13-IVS all facilitated segregation of subjects into type 2-high and type 2-low asthmatic groups, but in the ADEPT study population CCL26 expression was optimal. All subjects with high airway mucosal CCL26 expression and moderate-to-severe asthma had Feno values (≥35 ppb) and/or high bEOS counts (≥300 cells/mm 3 ) compared with a minority (36%) of subjects with low airway mucosal CCL26 expression. A combination of Feno values, bEOS counts, and serum CCL17 and CCL26 expression had 100% positive predictive value and 87% negative predictive value for airway mucosal CCL26-high status. Clinical variables did not differ between subjects with type 2-high and type 2-low status. Eosinophilic inflammation was associated with but not limited to airway mucosal type 2 gene expression. A panel of clinical biomarkers accurately classified type 2 status based on airway mucosal CCL26, periostin, or IL-13-IVS gene expression. Use of Feno values, bEOS counts, and serum marker levels (eg, CCL26 and CCL17) in combination might allow patient

Lateral gene transfer and the origins of prokaryotic groups.

PubMed

Boucher, Yan; Douady, Christophe J; Papke, R Thane; Walsh, David A; Boudreau, Mary Ellen R; Nesbø, Camilla L; Case, Rebecca J; Doolittle, W Ford

2003-01-01

Lateral gene transfer (LGT) is now known to be a major force in the evolution of prokaryotic genomes. To date, most analyses have focused on either (a) verifying phylogenies of individual genes thought to have been transferred, or (b) estimating the fraction of individual genomes likely to have been introduced by transfer. Neither approach does justice to the ability of LGT to effect massive and complex transformations in basic biology. In some cases, such transformation will be manifested as the patchy distribution of a seemingly fundamental property (such as aerobiosis or nitrogen fixation) among the members of a group classically defined by the sharing of other properties (metabolic, morphological, or molecular, such as small subunit ribosomal RNA sequence). In other cases, the lineage of recipients so transformed may be seen to comprise a new group of high taxonomic rank ("class" or even "phylum"). Here we review evidence for an important role of LGT in the evolution of photosynthesis, aerobic respiration, nitrogen fixation, sulfate reduction, methylotrophy, isoprenoid biosynthesis, quorum sensing, flotation (gas vesicles), thermophily, and halophily. Sometimes transfer of complex gene clusters may have been involved, whereas other times separate exchanges of many genes must be invoked.

Gene Transfer in Eukaryotic Cells Using Activated Dendrimers

NASA Astrophysics Data System (ADS)

Dennig, Jörg

Gene transfer into eukaryotic cells plays an important role in cell biology. Over the last 30 years a number of transfection methods have been developed to mediate gene transfer into eukaryotic cells. Classical methods include co-precipitation of DNA with calcium phosphate, charge-dependent precipitation of DNA with DEAE-dextran, electroporation of nucleic acids, and formation of transfection complexes between DNA and cationic liposomes. Gene transfer technologies based on activated PAMAM-dendrimers provide another class of transfection reagents. PAMAM-dendrimers are highly branched, spherical molecules. Activation of newly synthesized dendrimers involves hydrolytic removal of some of the branches, and results in a molecule with a higher degree of flexibility. Activated dendrimers assemble DNA into compact structures via charge interactions. Activated dendrimer - DNA complexes bind to the cell membrane of eukaryotic cells, and are transported into the cell by non-specific endocytosis. A structural model of the activated dendrimer - DNA complex and a potential mechanism for its uptake into cells will be discussed.

On the need for widespread horizontal gene transfers under genome size constraint.

PubMed

Isambert, Hervé; Stein, Richard R

2009-08-25

While eukaryotes primarily evolve by duplication-divergence expansion (and reduction) of their own gene repertoire with only rare horizontal gene transfers, prokaryotes appear to evolve under both gene duplications and widespread horizontal gene transfers over long evolutionary time scales. But, the evolutionary origin of this striking difference in the importance of horizontal gene transfers remains by and large a mystery. We propose that the abundance of horizontal gene transfers in free-living prokaryotes is a simple but necessary consequence of two opposite effects: i) their apparent genome size constraint compared to typical eukaryote genomes and ii) their underlying genome expansion dynamics through gene duplication-divergence evolution, as demonstrated by the presence of many tandem and block repeated genes. In principle, this combination of genome size constraint and underlying duplication expansion should lead to a coalescent-like process with extensive turnover of functional genes. This would, however, imply the unlikely, systematic reinvention of functions from discarded genes within independent phylogenetic lineages. Instead, we propose that the long-term evolutionary adaptation of free-living prokaryotes must have resulted in the emergence of efficient non-phylogenetic pathways to circumvent gene loss. This need for widespread horizontal gene transfers due to genome size constraint implies, in particular, that prokaryotes must remain under strong selection pressure in order to maintain the long-term evolutionary adaptation of their "mutualized" gene pool, beyond the inevitable turnover of individual prokaryote species. By contrast, the absence of genome size constraint for typical eukaryotes has presumably relaxed their need for widespread horizontal gene transfers and strong selection pressure. Yet, the resulting loss of genetic functions, due to weak selection pressure and inefficient gene recovery mechanisms, must have ultimately favored the

Horizontal Gene Transfer is a Significant Driver of Gene Innovation in Dinoflagellates

PubMed Central

Wisecaver, Jennifer H.; Brosnahan, Michael L.; Hackett, Jeremiah D.

2013-01-01

The dinoflagellates are an evolutionarily and ecologically important group of microbial eukaryotes. Previous work suggests that horizontal gene transfer (HGT) is an important source of gene innovation in these organisms. However, dinoflagellate genomes are notoriously large and complex, making genomic investigation of this phenomenon impractical with currently available sequencing technology. Fortunately, de novo transcriptome sequencing and assembly provides an alternative approach for investigating HGT. We sequenced the transcriptome of the dinoflagellate Alexandrium tamarense Group IV to investigate how HGT has contributed to gene innovation in this group. Our comprehensive A. tamarense Group IV gene set was compared with those of 16 other eukaryotic genomes. Ancestral gene content reconstruction of ortholog groups shows that A. tamarense Group IV has the largest number of gene families gained (314–1,563 depending on inference method) relative to all other organisms in the analysis (0–782). Phylogenomic analysis indicates that genes horizontally acquired from bacteria are a significant proportion of this gene influx, as are genes transferred from other eukaryotes either through HGT or endosymbiosis. The dinoflagellates also display curious cases of gene loss associated with mitochondrial metabolism including the entire Complex I of oxidative phosphorylation. Some of these missing genes have been functionally replaced by bacterial and eukaryotic xenologs. The transcriptome of A. tamarense Group IV lends strong support to a growing body of evidence that dinoflagellate genomes are extraordinarily impacted by HGT. PMID:24259313

Ultrasound -Assisted Gene Transfer to Adipose Tissue-Derived Stem/Progenitor Cells (ASCs)

NASA Astrophysics Data System (ADS)

Miyamoto, Yoshitaka; Ueno, Hitomi; Hokari, Rei; Yuan, Wenji; Kuno, Shuichi; Kakimoto, Takashi; Enosawa, Shin; Negishi, Yoichi; Yoshinaka, Kiyoshi; Matsumoto, Yoichiro; Chiba, Toshio; Hayashi, Shuji

2011-09-01

In recent years, multilineage adipose tissue-derived stem cells (ASCs) have become increasingly attractive as a promising source for cell transplantation and regenerative medicine. Particular interest has been expressed in the potential to make tissue stem cells, such as ASCs and marrow stromal cells (MSCs), differentiate by gene transfection. Gene transfection using highly efficient viral vectors such as adeno- and sendai viruses have been developed for this purpose. Sonoporation, or ultrasound (US)-assisted gene transfer, is an alternative gene manipulation technique which employs the creation of a jet stream by ultrasonic microbubble cavitation. Sonoporation using non-viral vectors is expected to be a much safer, although less efficient, tool for prospective clinical gene therapy. In this report, we assessed the efficacy of the sonoporation technique for gene transfer to ASCs. We isolated and cultured adipocyets from mouse adipose tissue. ASCs that have the potential to differentiate with transformation into adipocytes or osteoblasts were obtained. Using the US-assisted system, plasmid DNA containing beta-galactosidase (beta-Gal) and green fluorescent protein (GFP) genes were transferred to the ASCs. For this purpose, a Sonopore 4000 (NEPAGENE Co.) and a Sonazoid (Daiichi Sankyo Co.) instrument were used in combination. ASCs were subjected to US (3.1 MHz, 50% duty cycle, burst rate 2.0 Hz, intensity 1.2 W/cm2, exposure time 30 sec). We observed that the gene was more efficiently transferred with increased concentrations of plasmid DNA (5-150 μg/mL). However, further optimization of the US parameters is required, as the gene transfer efficiency was still relatively low. In conclusion, we herein demonstrate that a gene can be transferred to ASCs using our US-assisted system. In regenerative medicine, this system might resolve the current issues surrounding the use of viral vectors for gene transfer.

Plant transformation via pollen tube-mediated gene transfer

USDA-ARS?s Scientific Manuscript database

Genetic transformation using foreign genes and the subsequent development of transgenic plants has been employed to develop enhanced elite germplasm. Although some skepticism exits regarding pollen tube-mediated gene transfer (PTT), reports demonstrating improved transformation efficiency with PTT ...

Genomic Data Quality Impacts Automated Detection of Lateral Gene Transfer in Fungi

PubMed Central

Dupont, Pierre-Yves; Cox, Murray P.

2017-01-01

Lateral gene transfer (LGT, also known as horizontal gene transfer), an atypical mechanism of transferring genes between species, has almost become the default explanation for genes that display an unexpected composition or phylogeny. Numerous methods of detecting LGT events all rely on two fundamental strategies: primary structure composition or gene tree/species tree comparisons. Discouragingly, the results of these different approaches rarely coincide. With the wealth of genome data now available, detection of laterally transferred genes is increasingly being attempted in large uncurated eukaryotic datasets. However, detection methods depend greatly on the quality of the underlying genomic data, which are typically complex for eukaryotes. Furthermore, given the automated nature of genomic data collection, it is typically impractical to manually verify all protein or gene models, orthology predictions, and multiple sequence alignments, requiring researchers to accept a substantial margin of error in their datasets. Using a test case comprising plant-associated genomes across the fungal kingdom, this study reveals that composition- and phylogeny-based methods have little statistical power to detect laterally transferred genes. In particular, phylogenetic methods reveal extreme levels of topological variation in fungal gene trees, the vast majority of which show departures from the canonical species tree. Therefore, it is inherently challenging to detect LGT events in typical eukaryotic genomes. This finding is in striking contrast to the large number of claims for laterally transferred genes in eukaryotic species that routinely appear in the literature, and questions how many of these proposed examples are statistically well supported. PMID:28235827

Gene transfer mediated by alpha2-macroglobulin.

PubMed Central

Schneider, H; Huse, K; Birkenmeier, G; Otto, A; Scholz, G H

1996-01-01

alpha2-Macroglobulin covalently linked to poly(L)-lysine can be used as a vehicle for receptor-mediated gene transfer. This modified alpha2-macroglobulin maintains its ability to bind to the alpha2-macroglobulin receptor, and was shown to introduce a luciferase reporter gene plasmid into HepG2 human hepatoma cells in vitro. The alpha2-macroglobulin receptor is a very large and multifunctional cell surface receptor, whose rapid and efficient internalization rate makes it attractive for gene therapy, e.g. for hepatic gene targeting via injection into the portal vein. PMID:8871570

Early growth response gene 1 is essential for urban particulate matter-induced inflammation and mucus hyperproduction in airway epithelium.

PubMed

Xu, Feng; Cao, Jiaofei; Luo, Man; Che, Luanqing; Li, Wen; Ying, Songmin; Chen, Zhihua; Shen, Huahao

2018-05-19

Particulate matter (PM) has been implicated as a risk factor for human airway disorders. However, the biological mechanisms underlying the correlation between PM exposure and adverse airway effects have not yet been fully clarified. The objective of this study was to explore the possible role of early growth response gene 1 (Egr-1) in PM-induced toxic effects in pulmonary inflammation and mucus hyperproduction in vitro and in vivo. Particulate matter exposure induced a rapid Egr-1 expression in human bronchial epithelial (HBE) cells and in mouse lungs. Genetic blockage of Egr-1 markedly reduced PM-induced inflammatory cytokines, e.g., IL6 and IL8, and MUC5AC in HBE cells, and these effects were mechanistically mediated by the nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) pathways, respectively. Egr-1-knockout mice displayed significantly reduced airway inflammation and mucus hyperproduction in response to PM exposure in vivo. Moreover, polycyclic aromatic hydrocarbons (PAHs) contained in the PM also induced Egr-1 expression, and also played a role in the inflammatory responses and mucus production. Taken together, our data reveal novel Egr-1 signaling that mediates the NF-κB and AP-1 pathways to orchestrate PM-induced pulmonary inflammation and mucus hyperproduction, suggesting that Egr-1 inhibition could be an effective therapeutic approach for airway disorders or disease exacerbations induced by airborne particulate pollution. Copyright © 2018. Published by Elsevier B.V.

Differential Roles of Hydrogen Peroxide in Adaptive and Inflammatory Gene Expression Induced by Exposure of Human Airway Epithelial Cells to Zn2+

EPA Science Inventory

Oxidant stress is believed to play an important role in particulate matter (PM)–mediated toxicity in the respiratory tract. Zinc (Zn2+) is a ubiquitous component of PM that has been shown to induce adverse responses such as inflammatory and adaptive gene expression in airway epit...

Gene transfer agents: phage-like elements of genetic exchange

PubMed Central

Lang, Andrew S.; Zhaxybayeva, Olga; Beatty, J. Thomas

2013-01-01

Horizontal gene transfer is important in the evolution of bacterial and archaeal genomes. An interesting genetic exchange process is carried out by diverse phage-like gene transfer agents (GTAs) that are found in a wide range of prokaryotes. Although GTAs resemble phages, they lack the hallmark capabilities that define typical phages, and they package random pieces of the producing cell’s genome. In this Review, we discuss the defining characteristics of the GTAs that have been identified to date, along with potential functions for these agents and the possible evolutionary forces that act on the genes involved in their production. PMID:22683880

UCP2 muscle gene transfer modifies mitochondrial membrane potential.

PubMed

Marti, A; Larrarte, E; Novo, F J; Garcia, M; Martinez, J A

2001-01-01

The aim of this work was to evaluate the effect of uncoupling protein 2 (UCP2) muscle gene transfer on mitochondrial activity. Five week-old male Wistar rats received an intramuscular injection of plasmid pXU1 containing UCP2 cDNA in the right tibialis anterior muscles. Left tibialis anterior muscles were injected with vehicle as control. Ten days after DNA injection, tibialis anterior muscles were dissected and muscle mitochondria isolated and analyzed. There were two mitochondrial populations in the muscle after UCP2 gene transfer, one of low fluorescence and complexity and the other, showing high fluorescence and complexity. UCP2 gene transfer resulted in a 3.6 fold increase in muscle UCP2 protein levels compared to control muscles assessed by Western blotting. Furthermore, a significant reduction in mitochondria membrane potential assessed by spectrofluorometry and flow cytometry was observed. The mitochondria membrane potential reduction might account for a decrease in fluorescence of the low fluorescence mitochondrial subpopulation. It has been demonstrated that UCP2 muscle gene transfer in vivo is associated with a lower mitochondria membrane potential. Our results suggest the potential involvement of UCP2 in uncoupling respiration. International Journal of Obesity (2001) 25, 68-74

Transferred interbacterial antagonism genes augment eukaryotic innate immune function.

PubMed

Chou, Seemay; Daugherty, Matthew D; Peterson, S Brook; Biboy, Jacob; Yang, Youyun; Jutras, Brandon L; Fritz-Laylin, Lillian K; Ferrin, Michael A; Harding, Brittany N; Jacobs-Wagner, Christine; Yang, X Frank; Vollmer, Waldemar; Malik, Harmit S; Mougous, Joseph D

2015-02-05

Horizontal gene transfer allows organisms to rapidly acquire adaptive traits. Although documented instances of horizontal gene transfer from bacteria to eukaryotes remain rare, bacteria represent a rich source of new functions potentially available for co-option. One benefit that genes of bacterial origin could provide to eukaryotes is the capacity to produce antibacterials, which have evolved in prokaryotes as the result of eons of interbacterial competition. The type VI secretion amidase effector (Tae) proteins are potent bacteriocidal enzymes that degrade the cell wall when delivered into competing bacterial cells by the type VI secretion system. Here we show that tae genes have been transferred to eukaryotes on at least six occasions, and that the resulting domesticated amidase effector (dae) genes have been preserved for hundreds of millions of years through purifying selection. We show that the dae genes acquired eukaryotic secretion signals, are expressed within recipient organisms, and encode active antibacterial toxins that possess substrate specificity matching extant Tae proteins of the same lineage. Finally, we show that a dae gene in the deer tick Ixodes scapularis limits proliferation of Borrelia burgdorferi, the aetiologic agent of Lyme disease. Our work demonstrates that a family of horizontally acquired toxins honed to mediate interbacterial antagonism confers previously undescribed antibacterial capacity to eukaryotes. We speculate that the selective pressure imposed by competition between bacteria has produced a reservoir of genes encoding diverse antimicrobial functions that are tailored for co-option by eukaryotic innate immune systems.

Staphylococci on ICE: Overlooked agents of horizontal gene transfer.

PubMed

Sansevere, Emily A; Robinson, D Ashley

2017-01-01

Horizontal gene transfer plays a significant role in spreading antimicrobial resistance and virulence genes throughout the genus Staphylococcus , which includes species of clinical relevance to humans and animals. While phages and plasmids are the most well-studied agents of horizontal gene transfer in staphylococci, the contribution of integrative conjugative elements (ICEs) has been mostly overlooked. Experimental work demonstrating the activity of ICEs in staphylococci remained frozen for years after initial work in the 1980s that showed Tn 916 was capable of transfer from Enterococcus to Staphylococcus . However, recent work has begun to thaw this field. To date, 2 families of ICEs have been identified among staphylococci - Tn 916 that includes the Tn 5801 subfamily, and ICE 6013 that includes at least 7 subfamilies. Both Tn 5801 and ICE 6013 commonly occur in clinical strains of S. aureus . Tn 5801 is the most studied of the Tn 916 family elements in staphylococci and encodes tetracycline resistance and a protein that, when expressed in Escherichia coli , inhibits restriction barriers to incoming DNA. ICE 6013 is among the shortest known ICEs, but it still includes many uncharacterized open reading frames. This element uses an IS 30 -like transposase as its recombinase, providing some versatility in integration sites. ICE 6013 also conjugatively transfers among receptive S. aureus strains at relatively higher frequency than Tn 5801 . Continued study of these mobile genetic elements may reveal the full extent to which ICEs impact horizontal gene transfer and the evolution of staphylococci.

Myb permits multilineage airway epithelial cell differentiation

PubMed Central

Pan, Jie-hong; Adair-Kirk, Tracy L.; Patel, Anand C.; Huang, Tao; Yozamp, Nicholas S.; Xu, Jian; Reddy, E. Premkumar; Byers, Derek E.; Pierce, Richard A.; Holtzman, Michael J.; Brody, Steven L.

2014-01-01

The epithelium of the pulmonary airway is specially differentiated to provide defense against environmental insults, but also subject to dysregulated differentiation that results in lung disease. The current paradigm for airway epithelial differentiation is a one-step program whereby a p63+ basal epithelial progenitor cell generates a ciliated or secretory cell lineage, but the cue for this transition and whether there are intermediate steps is poorly defined. Here we identify transcription factor Myb as a key regulator that permits early multilineage differentiation of airway epithelial cells. Myb+ cells were identified as p63− and therefore distinct from basal progenitor cells, but were still negative for markers of differentiation. Myb RNAi treatment of primary-culture airway epithelial cells and Myb gene deletion in mice resulted in a p63− population with failed maturation of Foxj1+ ciliated cells, as well as Scbg1a1+ and Muc5ac+ secretory cells. Consistent with these findings, analysis of whole genome expression of Myb-deficient cells identified Myb-dependent programs for ciliated and secretory cell differentiation. Myb+ cells were rare in human airways but were increased in regions of ciliated cells and mucous cell hyperplasia in samples from subjects with chronic obstructive pulmonary disease. Together, the results show that a p63− Myb+ population of airway epithelial cells represents a distinct intermediate stage of differentiation that is required under normal conditions and may be heightened in airway disease. PMID:25103188

Role of OSGIN1 in mediating smoking-induced autophagy in the human airway epithelium.

PubMed

Wang, Guoqing; Zhou, Haixia; Strulovici-Barel, Yael; Al-Hijji, Mohammed; Ou, Xuemei; Salit, Jacqueline; Walters, Matthew S; Staudt, Michelle R; Kaner, Robert J; Crystal, Ronald G

2017-07-03

Enhanced macroautophagy/autophagy is recognized as a component of the pathogenesis of smoking-induced airway disease. Based on the knowledge that enhanced autophagy is linked to oxidative stress and the DNA damage response, both of which are linked to smoking, we used microarray analysis of the airway epithelium to identify smoking upregulated genes known to respond to oxidative stress and the DNA damage response. This analysis identified OSGIN1 (oxidative stress induced growth inhibitor 1) as significantly upregulated by smoking, in both the large and small airway epithelium, an observation confirmed by an independent small airway microarray cohort, TaqMan PCR of large and small airway samples and RNA-Seq of small airway samples. High and low OSGIN1 expressors have different autophagy gene expression patterns in vivo. Genome-wide correlation of RNAseq analysis of airway basal/progenitor cells showed a direct correlation of OSGIN1 mRNA levels to multiple classic autophagy genes. In vitro cigarette smoke extract exposure of primary airway basal/progenitor cells was accompanied by a dose-dependent upregulation of OSGIN1 and autophagy induction. Lentivirus-mediated expression of OSGIN1 in human primary basal/progenitor cells induced puncta-like staining of MAP1LC3B and upregulation of MAP1LC3B mRNA and protein and SQSTM1 mRNA expression level in a dose and time-dependent manner. OSGIN1-induction of autophagosome, amphisome and autolysosome formation was confirmed by colocalization of MAP1LC3B with SQSTM1 or CD63 (endosome marker) and LAMP1 (lysosome marker). Both OSGIN1 overexpression and knockdown enhanced the smoking-evoked autophagic response. Together, these observations support the concept that smoking-induced upregulation of OSGIN1 is one link between smoking-induced stress and enhanced-autophagy in the human airway epithelium.

Regulation of allergic airway inflammation by adoptive transfer of CD4+ T cells preferentially producing IL-10.

PubMed

Matsuda, Masaya; Doi, Kana; Tsutsumi, Tatsuya; Fujii, Shinya; Kishima, Maki; Nishimura, Kazuma; Kuroda, Ikue; Tanahashi, Yu; Yuasa, Rino; Kinjo, Toshihiko; Kuramoto, Nobuyuki; Mizutani, Nobuaki; Nabe, Takeshi

2017-10-05

Anti-inflammatory pharmacotherapy for asthma has mainly depended on the inhalation of glucocorticoids, which non-specifically suppress immune responses. If the anti-inflammatory cytokine interleukin (IL)-10 can be induced by a specific antigen, asthmatic airway inflammation could be suppressed when individuals are exposed to the antigen. The purpose of this study was to develop cellular immunotherapeutics for atopic diseases using IL-10-producing CD4 + T cells. Spleen cells isolated from ovalbumin (OVA)-sensitized mice were cultured with the antigen, OVA and growth factors, IL-21, IL-27 and TGF-β for 7 days. After the 7-day culture, the CD4 + T cells were purified using a murine CD4 magnetic beads system. When the induced CD4 + T cells were stimulated by OVA in the presence of antigen-presenting cells, IL-10 was preferentially produced in vitro. When CD4 + T cells were adoptively transferred to OVA-sensitized mice followed by intratracheal OVA challenges, IL-10 was preferentially produced in the serum and bronchoalveolar lavage fluid in vivo. IL-10 production coincided with the inhibition of eosinophilic airway inflammation and epithelial mucus plugging. Most of the IL-10-producing CD4 + T cells were negative for Foxp3 and GATA-3, transcription factors of naturally occurring regulatory T cells and Th2 cells, respectively, but double positive for LAG-3 and CD49b, surface markers of inducible regulatory T cells, Tr1 cells. Collectively, most of the induced IL-10-producing CD4 + T cells could be Tr1 cells, which respond to the antigen to produce IL-10, and effectively suppressed allergic airway inflammation. The induced Tr1 cells may be useful for antigen-specific cellular immunotherapy for atopic diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression Profiling Identifies Klf15 as a Glucocorticoid Target That Regulates Airway Hyperresponsiveness

PubMed Central

Masuno, Kiriko; Haldar, Saptarsi M.; Jeyaraj, Darwin; Mailloux, Christina M.; Huang, Xiaozhu; Panettieri, Rey A.; Jain, Mukesh K.

2011-01-01

Glucocorticoids (GCs), which activate GC receptor (GR) signaling and thus modulate gene expression, are widely used to treat asthma. GCs exert their therapeutic effects in part through modulating airway smooth muscle (ASM) structure and function. However, the effects of genes that are regulated by GCs on airway function are not fully understood. We therefore used transcription profiling to study the effects of a potent GC, dexamethasone, on human ASM (HASM) gene expression at 4 and 24 hours. After 24 hours of dexamethasone treatment, nearly 7,500 genes had statistically distinguishable changes in expression; quantitative PCR validation of a 40-gene subset of putative GR-regulated genes in 6 HASM cell lines suggested that the early transcriptional targets of GR signaling are similar in independent HASM lines. Gene ontology analysis implicated GR targets in controlling multiple aspects of ASM function. One GR-regulated gene, the transcription factor, Kruppel-like factor 15 (Klf15), was already known to modulate vascular smooth and cardiac muscle function, but had no known role in the lung. We therefore analyzed the pulmonary phenotype of Klf15−/− mice after ovalbumin sensitization and challenge. We found diminished airway responses to acetylcholine in ovalbumin-challenged Klf15−/− mice without a significant change in the induction of asthmatic inflammation. In cultured cells, overexpression of Klf15 reduced proliferation of HASM cells, whereas apoptosis in Klf15−/− murine ASM cells was increased. Together, these results further characterize the GR-regulated gene network in ASM and establish a novel role for the GR target, Klf15, in modulating airway function. PMID:21257922

Transfer of skills and comparison of performance between king vision® video laryngoscope and macintosh blade following an AHA airway management course.

PubMed

Wolf, Lukas E; Aguirre, José A; Vogt, Christian; Keller, Christian; Borgeat, Alain; Bruppacher, Heinz R

2017-01-10

To potentially optimize intubation skill teaching in an American Heart Association® Airway Management Course® for novices, we investigated the transfer of skills from video laryngoscopy to direct laryngoscopy and vice versa using King Vision® and Macintosh blade laryngoscopes respectively. Ninety volunteers (medical students, residents and staff physicians) without prior intubation experience were randomized into three groups to receive intubation training with either King Vision® or Macintosh blade or both. Afterwards they attempted intubation on two human cadavers with both tools. The primary outcome was skill transfer from video laryngoscopy to direct laryngoscopy assessed by first attempt success rates within 60 s. Secondary outcomes were skill transfer in the opposite direction, the efficacy of teaching both tools, and the success rates and esophageal intubation rates of Macintosh blade versus King Vision®. Performance with the Macintosh blade was identical following training with either Macintosh blade or King Vision® (unadjusted odds ratio [OR] 1.09, 95% confidence interval [95% CI] 0.5-2.6). Performance with the King Vision® was significantly better in the group that was trained on it (OR 2.7, 95% CI 1.2-5.9). Success rate within 60 s with Macintosh blade was 48% compared to 52% with King Vision® (OR 0.85, 95% CI 0.4-2.0). Rate of esophageal intubations with Macintosh blade was significantly higher (17% versus 4%, OR 5.0, 95% CI 1.1-23). We found better skill transfer from King Vision® to Macintosh blade than vice versa and fewer esophageal intubations with video laryngoscopy. For global skill improvement in an airway management course for novices, teaching only video laryngoscopy may be sufficient. However, success rates were low for both devices.

Gene Transfers Shaped the Evolution of De Novo NAD+ Biosynthesis in Eukaryotes

PubMed Central

Ternes, Chad M.; Schönknecht, Gerald

2014-01-01

NAD+ is an essential molecule for life, present in each living cell. It can function as an electron carrier or cofactor in redox biochemistry and energetics, and serves as substrate to generate the secondary messenger cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate. Although de novo NAD+ biosynthesis is essential, different metabolic pathways exist in different eukaryotic clades. The kynurenine pathway starting with tryptophan was most likely present in the last common ancestor of all eukaryotes, and is active in fungi and animals. The aspartate pathway, detected in most photosynthetic eukaryotes, was probably acquired from the cyanobacterial endosymbiont that gave rise to chloroplasts. An evolutionary analysis of enzymes catalyzing de novo NAD+ biosynthesis resulted in evolutionary trees incongruent with established organismal phylogeny, indicating numerous gene transfers. Endosymbiotic gene transfers probably introduced the aspartate pathway into eukaryotes and may have distributed it among different photosynthetic clades. In addition, several horizontal gene transfers substituted eukaryotic genes with bacterial orthologs. Although horizontal gene transfer is accepted as a key mechanism in prokaryotic evolution, it is supposed to be rare in eukaryotic evolution. The essential metabolic pathway of de novo NAD+ biosynthesis in eukaryotes was shaped by numerous gene transfers. PMID:25169983

Nasal continuous positive airway pressure therapy in a non-tertiary neonatal unit: reduced need for up-transfers.

PubMed

Kiran, Sai; Murki, Srinivas; Pratap, Oleti Tejo; Kandraju, Hemasree; Reddy, Anupama

2015-02-01

To evaluate the need for up-transfer after starting of nasal continuous positive airway pressure (n-CPAP) services in a Level II special newborn care unit (SNCU). Five hundred fifty infants admitted to Level II SNCU, 252 infants during one year prior to introduction of n-CPAP (retrospective data from case records and electronic data base) and 298 infants during one year after introduction of n-CPAP services (prospective data in predefined case reporting form) were evaluated in this before and after intervention trial. The primary outcome was proportion of infants needing up-transfers from Level II SNCU for any indication. Baseline demographic data like birth weight, gestation and other perinatal factors were similar between the two epochs. Among the infants admitted to Level II SNCU, up-transfer for any reason was significantly higher in the pre-CPAP epoch compared with CPAP epoch (n = 93, 36 % vs. n = 74, 24.8 %, p = 0.002, OR 0.56, 95 % CI 0.38 to 0.83). However parent desired up-transfers were similar between the two epochs (n = 9, 3 % vs. n = 16, 5 %, p = 0.40). Introduction of n-CPAP treatment modality reduced up-transfers in subgroups of very low birth weight infants (VLBW) (n = 20, 74 % vs. n = 15, 37 %, p = 0.003) and also in preterm infants (n = 50, 54 % vs. n = 34, 32 %, p = 0.002). Introduction of n-CPAP services in a non-tertiary care neonatal unit, significantly reduced the need for up-transfers, especially in VLBW and preterm infants.

A new computational method for the detection of horizontal gene transfer events.

PubMed

Tsirigos, Aristotelis; Rigoutsos, Isidore

2005-01-01

In recent years, the increase in the amounts of available genomic data has made it easier to appreciate the extent by which organisms increase their genetic diversity through horizontally transferred genetic material. Such transfers have the potential to give rise to extremely dynamic genomes where a significant proportion of their coding DNA has been contributed by external sources. Because of the impact of these horizontal transfers on the ecological and pathogenic character of the recipient organisms, methods are continuously sought that are able to computationally determine which of the genes of a given genome are products of transfer events. In this paper, we introduce and discuss a novel computational method for identifying horizontal transfers that relies on a gene's nucleotide composition and obviates the need for knowledge of codon boundaries. In addition to being applicable to individual genes, the method can be easily extended to the case of clusters of horizontally transferred genes. With the help of an extensive and carefully designed set of experiments on 123 archaeal and bacterial genomes, we demonstrate that the new method exhibits significant improvement in sensitivity when compared to previously published approaches. In fact, it achieves an average relative improvement across genomes of between 11 and 41% compared to the Codon Adaptation Index method in distinguishing native from foreign genes. Our method's horizontal gene transfer predictions for 123 microbial genomes are available online at http://cbcsrv.watson.ibm.com/HGT/.

Environmental factors influencing gene transfer agent (GTA) mediated transduction in the subtropical ocean.

PubMed

McDaniel, Lauren D; Young, Elizabeth C; Ritchie, Kimberly B; Paul, John H

2012-01-01

Microbial genomic sequence analyses have indicated widespread horizontal gene transfer (HGT). However, an adequate mechanism accounting for the ubiquity of HGT has been lacking. Recently, high frequencies of interspecific gene transfer have been documented, catalyzed by Gene Transfer Agents (GTAs) of marine α-Proteobacteria. It has been proposed that the presence of bacterial genes in highly purified viral metagenomes may be due to GTAs. However, factors influencing GTA-mediated gene transfer in the environment have not yet been determined. Several genomically sequenced strains containing complete GTA sequences similar to Rhodobacter capsulatus (RcGTA, type strain) were screened to ascertain if they produced putative GTAs, and at what abundance. Five of nine marine strains screened to date spontaneously produced virus-like particles (VLP's) in stationary phase. Three of these strains have demonstrated gene transfer activity, two of which were documented by this lab. These two strains Roseovarius nubinhibens ISM and Nitratireductor 44B9s, were utilized to produce GTAs designated RnGTA and NrGTA and gene transfer activity was verified in culture. Cell-free preparations of purified RnGTA and NrGTA particles from marked donor strains were incubated with natural microbial assemblages to determine the level of GTA-mediated gene transfer. In conjunction, several ambient environmental parameters were measured including lysogeny indicated by prophage induction. GTA production in culture systems indicated that approximately half of the strains produced GTA-like particles and maximal GTA counts ranged from 10-30% of host abundance. Modeling of GTA-mediated gene transfer frequencies in natural samples, along with other measured environmental variables, indicated a strong relationship between GTA mediated gene transfer and the combined factors of salinity, multiplicity of infection (MOI) and ambient bacterial abundance. These results indicate that GTA-mediated HGT in the

Environmental Factors Influencing Gene Transfer Agent (GTA) Mediated Transduction in the Subtropical Ocean

PubMed Central

McDaniel, Lauren D.; Young, Elizabeth C.; Ritchie, Kimberly B.; Paul, John H.

2012-01-01

Microbial genomic sequence analyses have indicated widespread horizontal gene transfer (HGT). However, an adequate mechanism accounting for the ubiquity of HGT has been lacking. Recently, high frequencies of interspecific gene transfer have been documented, catalyzed by Gene Transfer Agents (GTAs) of marine α-Proteobacteria. It has been proposed that the presence of bacterial genes in highly purified viral metagenomes may be due to GTAs. However, factors influencing GTA-mediated gene transfer in the environment have not yet been determined. Several genomically sequenced strains containing complete GTA sequences similar to Rhodobacter capsulatus (RcGTA, type strain) were screened to ascertain if they produced putative GTAs, and at what abundance. Five of nine marine strains screened to date spontaneously produced virus-like particles (VLP's) in stationary phase. Three of these strains have demonstrated gene transfer activity, two of which were documented by this lab. These two strains Roseovarius nubinhibens ISM and Nitratireductor 44B9s, were utilized to produce GTAs designated RnGTA and NrGTA and gene transfer activity was verified in culture. Cell-free preparations of purified RnGTA and NrGTA particles from marked donor strains were incubated with natural microbial assemblages to determine the level of GTA-mediated gene transfer. In conjunction, several ambient environmental parameters were measured including lysogeny indicated by prophage induction. GTA production in culture systems indicated that approximately half of the strains produced GTA-like particles and maximal GTA counts ranged from 10–30% of host abundance. Modeling of GTA-mediated gene transfer frequencies in natural samples, along with other measured environmental variables, indicated a strong relationship between GTA mediated gene transfer and the combined factors of salinity, multiplicity of infection (MOI) and ambient bacterial abundance. These results indicate that GTA-mediated HGT in the

Nociceptin effects in the airways.

PubMed

Peiser, C; Undem, B J; Fischer, A

2000-07-01

The opioid-like heptadecapeptide nociceptin (NC) has the following effects in the airways (investigated in isolated tracheae and bronchi from guinea pig or rat): the electric field stimulation (EFS)-induces release of acetylcholine (ACh), the tachykinin substance P (SP) and calcitonin gene-related peptide (CGRP) is reduced after pretreatment with NC, and EFS-induced tachykinergic nonadrenergic-noncholinergic (NANC) bronchoconstriction is inhibited by NC. Both the NC-mediated inhibition of neurotransmission and of smooth muscle contraction occurred in a concentration-dependent manner. Because these effects were naloxone-insensitive, were blocked by the NC receptor antagonist [F/G]NC(1-13)NH(2), and could be mimicked by the NC analogs, NCNH(2) and NC(1-13)NH(2), it is thought that they are distinct from the classic opioid receptors. That these pharmacological actions of NC are of relevance for airway physiology is highly probable given the presence of NC-immunoreactivity in the nerve fibers of the airways and of opioid-like receptor (ORL-1) transcripts in the jugular ganglia, from where the tachykinin-containing afferents arise.

Lipopolysaccharide does not alter small airway reactivity in mouse lung slices.

PubMed

Donovan, Chantal; Royce, Simon G; Vlahos, Ross; Bourke, Jane E

2015-01-01

The bacterial endotoxin, lipopolysaccharide (LPS) has been associated with occupational airway diseases with asthma-like symptoms and in acute exacerbations of COPD. The direct and indirect effects of LPS on small airway reactivity have not been fully elucidated. We tested the hypothesis that both in vitro and in vivo LPS treatment would increase contraction and impair relaxation of mouse small airways. Lung slices were prepared from naïve Balb/C mice and cultured in the absence or presence of LPS (10 μg/ml) for up to 48 h for measurement of TNFα levels in conditioned media. Alternatively, mice were challenged with PBS or LPS in vivo once a day for 4 days for preparation of lung slices or for harvest of lungs for Q-PCR analysis of gene expression of pro-inflammatory cytokines and receptors involved in airway contraction. Reactivity of small airways to contractile agonists, methacholine and serotonin, and bronchodilator agents, salbutamol, isoprenaline and rosiglitazone, were assessed using phase-contrast microscopy. In vitro LPS treatment of slices increased TNFα release 6-fold but did not alter contraction or relaxation to any agonists tested. In vivo LPS treatment increased lung gene expression of TNFα, IL-1β and ryanodine receptor isoform 2 more than 5-fold. However there were no changes in reactivity in lung slices from these mice, even when also incubated with LPS ex vivo. Despite evidence of LPS-induced inflammation, neither airway hyperresponsiveness or impaired dilator reactivity were evident. The increase in ryanodine receptor isoform 2, known to regulate calcium signaling in vascular smooth muscle, warrants investigation. Since LPS failed to elicit changes in small airway reactivity in mouse lung slices following in vitro or in vivo treatment, alternative approaches are required to define the potential contribution of this endotoxin to altered small airway reactivity in human lung diseases.

Lipopolysaccharide Does Not Alter Small Airway Reactivity in Mouse Lung Slices

PubMed Central

Donovan, Chantal; Royce, Simon G.; Vlahos, Ross; Bourke, Jane E.

2015-01-01

The bacterial endotoxin, lipopolysaccharide (LPS) has been associated with occupational airway diseases with asthma-like symptoms and in acute exacerbations of COPD. The direct and indirect effects of LPS on small airway reactivity have not been fully elucidated. We tested the hypothesis that both in vitro and in vivo LPS treatment would increase contraction and impair relaxation of mouse small airways. Lung slices were prepared from naïve Balb/C mice and cultured in the absence or presence of LPS (10 μg/ml) for up to 48 h for measurement of TNFα levels in conditioned media. Alternatively, mice were challenged with PBS or LPS in vivo once a day for 4 days for preparation of lung slices or for harvest of lungs for Q-PCR analysis of gene expression of pro-inflammatory cytokines and receptors involved in airway contraction. Reactivity of small airways to contractile agonists, methacholine and serotonin, and bronchodilator agents, salbutamol, isoprenaline and rosiglitazone, were assessed using phase-contrast microscopy. In vitro LPS treatment of slices increased TNFα release 6-fold but did not alter contraction or relaxation to any agonists tested. In vivo LPS treatment increased lung gene expression of TNFα, IL-1β and ryanodine receptor isoform 2 more than 5-fold. However there were no changes in reactivity in lung slices from these mice, even when also incubated with LPS ex vivo. Despite evidence of LPS-induced inflammation, neither airway hyperresponsiveness or impaired dilator reactivity were evident. The increase in ryanodine receptor isoform 2, known to regulate calcium signaling in vascular smooth muscle, warrants investigation. Since LPS failed to elicit changes in small airway reactivity in mouse lung slices following in vitro or in vivo treatment, alternative approaches are required to define the potential contribution of this endotoxin to altered small airway reactivity in human lung diseases. PMID:25822969

Downregulation of p300 gene expression in airway mesenchyme of nitrofen-induced hypoplastic lungs.

PubMed

Takahashi, Hiromizu; Friedmacher, Florian; Fujiwara, Naho; Hofmann, Alejandro; Takahashi, Toshiaki; Puri, Prem

2014-04-01

Congenital diaphragmatic hernia (CDH) is a relatively common developmental abnormality causing life-threatening respiratory distress at birth. The nitrofen model has been widely used to investigate the pathogenesis of hypoplastic lungs associated with CDH. Embryos lacking p300 and CBP genes are significantly smaller in lung formation. We hypothesized that pulmonary gene expression of p300 and CBP is downregulated during late gestation in the nitrofen-induced CDH model. Time-pregnant rats were treated with either nitrofen or vehicle on gestational day 9 (D9). Fetal lungs were harvested on D18 and D21 (n = 8 at each time point). Pulmonary gene expression of p300 and CBP was analyzed by quantitative real-time PCR. Immunohistochemistry was performed to investigate expression and localization of pulmonary p300 and CBP proteins. Relative mRNA expression levels of p300 were significantly decreased in nitrofen-induced hypoplastic lungs on D18 compared to controls (3.00 ± 0.20 vs. 3.76 ± 0.14; p = 0.0039), while CBP levels were not altered. p300 immunoreactivity was markedly diminished in surrounding mesenchymal compartments and nuclei of proximal and distal airway cells, while CBP expression was not altered. Downregulation of p300 gene expression during the early canalicular stage may disrupt epithelial-mesenchymal signaling interactions, contributing to the development of hypoplastic lungs in the nitrofen-induced CDH model.

Risks from GMOs due to horizontal gene transfer.

PubMed

Keese, Paul

2008-01-01

Horizontal gene transfer (HGT) is the stable transfer of genetic material from one organism to another without reproduction or human intervention. Transfer occurs by the passage of donor genetic material across cellular boundaries, followed by heritable incorporation to the genome of the recipient organism. In addition to conjugation, transformation and transduction, other diverse mechanisms of DNA and RNA uptake occur in nature. The genome of almost every organism reveals the footprint of many ancient HGT events. Most commonly, HGT involves the transmission of genes on viruses or mobile genetic elements. HGT first became an issue of public concern in the 1970s through the natural spread of antibiotic resistance genes amongst pathogenic bacteria, and more recently with commercial production of genetically modified (GM) crops. However, the frequency of HGT from plants to other eukaryotes or prokaryotes is extremely low. The frequency of HGT to viruses is potentially greater, but is restricted by stringent selection pressures. In most cases the occurrence of HGT from GM crops to other organisms is expected to be lower than background rates. Therefore, HGT from GM plants poses negligible risks to human health or the environment.

Aircraft transfer of pediatric patients with intractable cardiac or airway problems – single-institutional experience of a specialty hospital.

PubMed

Ando, Makoto; Takahashi, Yukihiro; Park, In-Sam; Tomoike, Hitonobu

2015-01-01

There is currently a well-established network for the allocation of donor organs for transplantation in Japan, and emergency patients are often transported by the "Doctor Helicopter". However, interhospital transfer of patients, which can require aircraft with specialized equipment, depends on arrangement by each responsible hospital. Since 2009 there were 41 interhospital aviation transfers of pediatric patients with intractable cardiac or airway diseases seeking surgical treatment at Sakakibara Heart Institute. Of these, 22 were newborns, 21 were on continuous drip infusion and 14 on mechanical ventilator support. In 15 cases (36.6%), a commercial airliner was used, with the remaining using chartered emergency aircraft (eg, local fire department helicopter, Self-Defense-Forces of Japan and the Doctor Helicopter). The median transfer time was 239 min for commercial airliners, 51 min for chartered aircraft departing directly from the referring hospital and 120.5 min for chartered aircraft departing from a nearby location. The efficiency of the transfer exemplified by the percentage of the time on board the aircraft was significantly lower for commercial airliners compared with chartered emergency aircraft. Further efforts and cooperation with government are required to obtain geographically uniform availability of carriers with optimal medical equipment to improve pediatric patient outcomes.

Center for Fetal Monkey Gene Transfer for Heart, Lung, and Blood Diseases: An NHLBI Resource for the Gene Therapy Community

PubMed Central

Skarlatos, Sonia I.

2012-01-01

Abstract The goals of the National Heart, Lung, and Blood Institute (NHLBI) Center for Fetal Monkey Gene Transfer for Heart, Lung, and Blood Diseases are to conduct gene transfer studies in monkeys to evaluate safety and efficiency; and to provide NHLBI-supported investigators with expertise, resources, and services to actively pursue gene transfer approaches in monkeys in their research programs. NHLBI-supported projects span investigators throughout the United States and have addressed novel approaches to gene delivery; “proof-of-principle”; assessed whether findings in small-animal models could be demonstrated in a primate species; or were conducted to enable new grant or IND submissions. The Center for Fetal Monkey Gene Transfer for Heart, Lung, and Blood Diseases successfully aids the gene therapy community in addressing regulatory barriers, and serves as an effective vehicle for advancing the field. PMID:22974119

Clinical Applications Involving CNS Gene Transfer

PubMed Central

Kantor, Boris; McCown, Thomas; Leone, Paola; Gray, Steven J.

2015-01-01

Diseases of the central nervous system (CNS) have traditionally been the most difficult to treat by traditional pharmacological methods, due mostly to the blood–brain barrier and the difficulties associated with repeated drug administration targeting the CNS. Viral vector gene transfer represents a way to permanently provide a therapeutic protein within the nervous system after a single administration, whether this be a gene replacement strategy for an inherited disorder or a disease-modifying protein for a disease such as Parkinson's. Gene therapy approaches for CNS disorders has evolved considerably over the last two decades. Although a breakthrough treatment has remained elusive, current strategies are now considerably safer and potentially much more effective. This chapter will explore the past, current, and future status of CNS gene therapy, focusing on clinical trials utilizing adeno-associated virus and lentiviral vectors. PMID:25311921

Multimodality Imaging of Gene Transfer with a Receptor-Based Reporter Gene

PubMed Central

Chen, Ron; Parry, Jesse J.; Akers, Walter J.; Berezin, Mikhail Y.; El Naqa, Issam M.; Achilefu, Samuel; Edwards, W. Barry; Rogers, Buck E.

2010-01-01

Gene therapy trials have traditionally used tumor and tissue biopsies for assessing the efficacy of gene transfer. Non-invasive imaging techniques offer a distinct advantage over tissue biopsies in that the magnitude and duration of gene transfer can be monitored repeatedly. Human somatostatin receptor subtype 2 (SSTR2) has been used for the nuclear imaging of gene transfer. To extend this concept, we have developed a somatostatin receptor–enhanced green fluorescent protein fusion construct (SSTR2-EGFP) for nuclear and fluorescent multimodality imaging. Methods An adenovirus containing SSTR2-EGFP (AdSSTR2-EGFP) was constructed and evaluated in vitro and in vivo. SCC-9 human squamous cell carcinoma cells were infected with AdEGFP, AdSSTR2, or AdSSTR2-EGFP for in vitro evaluation by saturation binding, internalization, and fluorescence spectroscopy assays. In vivo biodistribution and nano-SPECT imaging studies were conducted with mice bearing SCC-9 tumor xenografts directly injected with AdSSTR2-EGFP or AdSSTR2 to determine the tumor localization of 111In-diethylenetriaminepentaacetic acid (DTPA)-Tyr3-octreotate. Fluorescence imaging was conducted in vivo with mice receiving intratumoral injections of AdSSTR2, AdSSTR2-EGFP, or AdEGFP as well as ex vivo with tissues extracted from mice. Results The similarity between AdSSTR2-EGFP and wild-type AdSSTR2 was demonstrated in vitro by the saturation binding and internalization assays, and the fluorescence emission spectra of cells infected with AdSSTR2-EGFP was almost identical to the spectra of cells infected with wild-type AdEGFP. Biodistribution studies demonstrated that the tumor uptake of 111In-DTPA-Tyr3-octreotate was not significantly different (P > 0.05) when tumors (n = 5) were injected with AdSSTR2 or AdSSTR2-EGFP but was significantly greater than the uptake in control tumors. Fluorescence was observed in tumors injected with AdSSTR2-EGFP and AdEGFP in vivo and ex vivo but not in tumors injected with AdSSTR2

Cystic fibrosis gene modifier SLC26A9 modulates airway response to CFTR-directed therapeutics.

PubMed

Strug, Lisa J; Gonska, Tanja; He, Gengming; Keenan, Katherine; Ip, Wan; Boëlle, Pierre-Yves; Lin, Fan; Panjwani, Naim; Gong, Jiafen; Li, Weili; Soave, David; Xiao, Bowei; Tullis, Elizabeth; Rabin, Harvey; Parkins, Michael D; Price, April; Zuberbuhler, Peter C; Corvol, Harriet; Ratjen, Felix; Sun, Lei; Bear, Christine E; Rommens, Johanna M

2016-10-15

Cystic fibrosis is realizing the promise of personalized medicine. Recent advances in drug development that target the causal CFTR directly result in lung function improvement, but variability in response is demanding better prediction of outcomes to improve management decisions. The genetic modifier SLC26A9 contributes to disease severity in the CF pancreas and intestine at birth and here we assess its relationship with disease severity and therapeutic response in the airways. SLC26A9 association with lung disease was assessed in individuals from the Canadian and French CF Gene Modifier consortia with CFTR-gating mutations and in those homozygous for the common Phe508del mutation. Variability in response to a CFTR-directed therapy attributed to SLC26A9 genotype was assessed in Canadian patients with gating mutations. A primary airway model system determined if SLC26A9 shows modification of Phe508del CFTR function upon treatment with a CFTR corrector. In those with gating mutations that retain cell surface-localized CFTR we show that SLC26A9 modifies lung function while this is not the case in individuals homozygous for Phe508del where cell surface expression is lacking. Treatment response to ivacaftor, which aims to improve CFTR-channel opening probability in patients with gating mutations, shows substantial variability in response, 28% of which can be explained by rs7512462 in SLC26A9 (P = 0.0006). When homozygous Phe508del primary bronchial cells are treated to restore surface CFTR, SLC26A9 likewise modifies treatment response (P = 0.02). Our findings indicate that SLC26A9 airway modification requires CFTR at the cell surface, and that a common variant in SLC26A9 may predict response to CFTR-directed therapeutics.

The contribution of airway smooth muscle to airway narrowing and airway hyperresponsiveness in disease.

PubMed

Martin, J G; Duguet, A; Eidelman, D H

2000-08-01

Airway hyperresponsiveness (AHR), the exaggerated response to constrictor agonists in asthmatic subjects, is incompletely understood. Changes in either the quantity or properties of airway smooth muscle (ASM) are possible explanations for AHR. Morphometric analyses demonstrate structural changes in asthmatic airways, including subepithelial fibrosis, gland hyperplasia/hypertrophy, neovascularization and an increase in ASM mass. Mathematical modelling of airway narrowing suggests that, of all the changes in structure, the increase in ASM mass is the most probable cause of AHR. An increase in ASM mass in the large airways is more closely associated with a greater likelihood of dying from asthma than increases in ASM mass in other locations within the airway tree. ASM contraction is opposed by the elastic recoil of the lungs and airways, which appears to limit the degree of bronchoconstriction in vivo. The cyclical nature of tidal breathing applies stresses to the airway wall that enhance the bronchodilating influence of the lung tissues on the contracting ASM, in all probability by disrupting cross-bridges. However, the increase in ASM mass in asthma may overcome the limitation resulting from the impedances to ASM shortening imposed by the lung parenchyma and airway wall tissues. Additionally, ASM with the capacity to shorten rapidly may achieve shorter lengths and cause a greater degree of bronchoconstriction when stimulated to contract than slower ASM. Changes in ASM properties are induced by the process of sensitization and allergen-exposure such as enhancement of phospholipase C activity and inositol phosphate turnover, and increases in myosin light chain kinase activity. Whether changes in ASM mass or biochemical/biomechanical properties form the basis for asthma remains to be determined.

Generation of SV40-transformed rabbit tracheal-epithelial-cell-derived blastocyst by somatic cell nuclear transfer

PubMed Central

de Semir, D.; Maurisse, R.; Du, F.; Xu, J.; Yang, X.; Illek, B.; Gruenert, D. C.

2013-01-01

The prospect of developing large animal models for the study of inherited diseases, such as cystic fibrosis (CF), through somatic cell nuclear transfer (SCNT) has opened up new opportunities for enhancing our understanding of disease pathology and for identifying new therapies. Thus, the development of species-specific in vitro cell systems that will provide broader insight into organ- and cell-type-specific functions relevant to the pathology of the disease is crucial. Studies have been undertaken to establish transformed rabbit airway epithelial cell lines that display differentiated features characteristic of the primary airway epithelium. This study describes the successful establishment and characterization of two SV40-transformed rabbit tracheal epithelial cell lines. These cell lines, 5RTEo- and 9RTEo-, express the CF transmembrane conductance regulator (CFTR) gene, retain epithelial-specific differentiated morphology and show CFTR-based cAMP-dependent Cl− ion transport across the apical membrane of a confluent monolayer. Immunocytochemical analysis indicates the presence of airway cytokeratins and tight-junction proteins in the 9RTEo- cell line after multiple generations. However, the tight junctions appear to diminish in their efficacy in both cell lines after at least 100 generations. Initial SCNT studies with the 9RTEo- cells have revealed that SV40-transformed rabbit airway epithelial donor cells can be used to generate blastocysts. These cell systems provide valuable models for studying the developmental and metabolic modulation of CFTR gene expression and rabbit airway epithelial cell biology. PMID:22234514

Exact Algorithms for Duplication-Transfer-Loss Reconciliation with Non-Binary Gene Trees.

PubMed

Kordi, Misagh; Bansal, Mukul S

2017-06-01

Duplication-Transfer-Loss (DTL) reconciliation is a powerful method for studying gene family evolution in the presence of horizontal gene transfer. DTL reconciliation seeks to reconcile gene trees with species trees by postulating speciation, duplication, transfer, and loss events. Efficient algorithms exist for finding optimal DTL reconciliations when the gene tree is binary. In practice, however, gene trees are often non-binary due to uncertainty in the gene tree topologies, and DTL reconciliation with non-binary gene trees is known to be NP-hard. In this paper, we present the first exact algorithms for DTL reconciliation with non-binary gene trees. Specifically, we (i) show that the DTL reconciliation problem for non-binary gene trees is fixed-parameter tractable in the maximum degree of the gene tree, (ii) present an exponential-time, but in-practice efficient, algorithm to track and enumerate all optimal binary resolutions of a non-binary input gene tree, and (iii) apply our algorithms to a large empirical data set of over 4700 gene trees from 100 species to study the impact of gene tree uncertainty on DTL-reconciliation and to demonstrate the applicability and utility of our algorithms. The new techniques and algorithms introduced in this paper will help biologists avoid incorrect evolutionary inferences caused by gene tree uncertainty.

Airway-parenchymal interdependence

PubMed Central

Paré, Peter D; Mitzner, Wayne

2015-01-01

In this manuscript we discuss the interaction of the lung parenchyma and the airways as well as the physiological and pathophysiological significance of this interaction. These two components of the respiratory organ can be thought of as two independent elastic structures but in fact the mechanical properties of one influence the behavior of the other. Traditionally the interaction has focused on the effects of the lung on the airways but there is good evidence that the opposite is also true, i.e., that the mechanical properties of the airways influence the elastic properties of the parenchyma. The interplay between components of the respiratory system including the airways, parenchyma and vasculature is often referred to as “interdependence.” This interdependence transmits the elastic recoil of the lung to create an effective pressure that dilates the airways as transpulmonary pressure and lung volume increase. By using a continuum mechanics analysis of the lung parenchyma, it is possible to predict the effective pressure between the airways and parenchyma, and these predictions can be empirically evaluated. Normal airway caliber is maintained by this pressure in the adventitial interstitium of the airway, and it counteracts airway compression during forced expiration as well as the ability of airway smooth muscle to narrow airways. Interdependence has physiological and pathophysiological significance. Weakening of the forces of interdependence contributes to airway dysfunction and gas exchange impairment in acute and chronic airway diseases including asthma and emphysema. PMID:23723029

A theoretical model of the application of RF energy to the airway wall and its experimental validation.

PubMed

Jarrard, Jerry; Wizeman, Bill; Brown, Robert H; Mitzner, Wayne

2010-11-27

Bronchial thermoplasty is a novel technique designed to reduce an airway's ability to contract by reducing the amount of airway smooth muscle through controlled heating of the airway wall. This method has been examined in animal models and as a treatment for asthma in human subjects. At the present time, there has been little research published about how radiofrequency (RF) energy and heat is transferred to the airways of the lung during bronchial thermoplasty procedures. In this manuscript we describe a computational, theoretical model of the delivery of RF energy to the airway wall. An electro-thermal finite-element-analysis model was designed to simulate the delivery of temperature controlled RF energy to airway walls of the in vivo lung. The model includes predictions of heat generation due to RF joule heating and transfer of heat within an airway wall due to thermal conduction. To implement the model, we use known physical characteristics and dimensions of the airway and lung tissues. The model predictions were tested with measurements of temperature, impedance, energy, and power in an experimental canine model. Model predictions of electrode temperature, voltage, and current, along with tissue impedance and delivered energy were compared to experiment measurements and were within ± 5% of experimental averages taken over 157 sample activations.The experimental results show remarkable agreement with the model predictions, and thus validate the use of this model to predict the heat generation and transfer within the airway wall following bronchial thermoplasty. The model also demonstrated the importance of evaporation as a loss term that affected both electrical measurements and heat distribution. The model predictions showed excellent agreement with the empirical results, and thus support using the model to develop the next generation of devices for bronchial thermoplasty. Our results suggest that comparing model results to RF generator electrical measurements

Gene doping detection: evaluation of approach for direct detection of gene transfer using erythropoietin as a model system.

PubMed

Baoutina, A; Coldham, T; Bains, G S; Emslie, K R

2010-08-01

As clinical gene therapy has progressed toward realizing its potential, concern over misuse of the technology to enhance performance in athletes is growing. Although 'gene doping' is banned by the World Anti-Doping Agency, its detection remains a major challenge. In this study, we developed a methodology for direct detection of the transferred genetic material and evaluated its feasibility for gene doping detection in blood samples from athletes. Using erythropoietin (EPO) as a model gene and a simple in vitro system, we developed real-time PCR assays that target sequences within the transgene complementary DNA corresponding to exon/exon junctions. As these junctions are absent in the endogenous gene due to their interruption by introns, the approach allows detection of trace amounts of a transgene in a large background of the endogenous gene. Two developed assays and one commercial gene expression assay for EPO were validated. On the basis of ability of these assays to selectively amplify transgenic DNA and analysis of literature on testing of gene transfer in preclinical and clinical gene therapy, it is concluded that the developed approach would potentially be suitable to detect gene doping through gene transfer by analysis of small volumes of blood using regular out-of-competition testing.

Incorporation of a horizontally transferred gene into an operon during cnidarian evolution.

PubMed

Dana, Catherine E; Glauber, Kristine M; Chan, Titus A; Bridge, Diane M; Steele, Robert E

2012-01-01

Genome sequencing has revealed examples of horizontally transferred genes, but we still know little about how such genes are incorporated into their host genomes. We have previously reported the identification of a gene (flp) that appears to have entered the Hydra genome through horizontal transfer. Here we provide additional evidence in support of our original hypothesis that the transfer was from a unicellular organism, and we show that the transfer occurred in an ancestor of two medusozoan cnidarian species. In addition we show that the gene is part of a bicistronic operon in the Hydra genome. These findings identify a new animal phylum in which trans-spliced leader addition has led to the formation of operons, and define the requirements for evolution of an operon in Hydra. The identification of operons in Hydra also provides a tool that can be exploited in the construction of transgenic Hydra strains.

The transfer and transformation of collective network information in gene-matched networks.

PubMed

Kitsukawa, Takashi; Yagi, Takeshi

2015-10-09

Networks, such as the human society network, social and professional networks, and biological system networks, contain vast amounts of information. Information signals in networks are distributed over nodes and transmitted through intricately wired links, making the transfer and transformation of such information difficult to follow. Here we introduce a novel method for describing network information and its transfer using a model network, the Gene-matched network (GMN), in which nodes (neurons) possess attributes (genes). In the GMN, nodes are connected according to their expression of common genes. Because neurons have multiple genes, the GMN is cluster-rich. We show that, in the GMN, information transfer and transformation were controlled systematically, according to the activity level of the network. Furthermore, information transfer and transformation could be traced numerically with a vector using genes expressed in the activated neurons, the active-gene array, which was used to assess the relative activity among overlapping neuronal groups. Interestingly, this coding style closely resembles the cell-assembly neural coding theory. The method introduced here could be applied to many real-world networks, since many systems, including human society and various biological systems, can be represented as a network of this type.

Gene transfer into the kidney: current status and limitations.

PubMed

Moullier, P; Salvetti, A; Champion-Arnaud, P; Ronco, P M

1997-01-01

Gene therapy is obviously a controversial issue and a wave of suspicion has dampened the initial enthusiasm raised by this new therapeutic approach. It has now become fashionable to downplay the potential for gene therapy in most fields including kidney-related diseases. In our opinion, this is an unfair and unrealistic view of the future. In fact, gene therapy of well-selected kidney diseases will certainly become feasible, but a large data base on vectors and transfer methods both in the normal kidney and in disease models has first to be collected. Any significant progress in the biology of the vectors, in the cellular interactions of the newly introduced DNA, and in the regulation and persistency of the transgene should be rapidly translated to the kidney in relevant experimental models. Herein, we present the use and current limitations of gene transfer to the kidney and the potential therapeutic perspectives.

Gene Transfer in Mycobacterium tuberculosis: Shuttle Phasmids to Enlightenment

PubMed Central

JACOBS, WILLIAM R.

2016-01-01

Infectious diseases have plagued humankind throughout history and have posed serious public health problems. Yet vaccines have eradicated smallpox and antibiotics have drastically decreased the mortality rate of many infectious agents. These remarkable successes in the control of infections came from knowing the causative agents of the diseases, followed by serendipitous discoveries of attenuated viruses and antibiotics. The discovery of DNA as genetic material and the understanding of how this information translates into specific phenotypes have changed the paradigm for developing new vaccines, drugs, and diagnostic tests. Knowledge of the mechanisms of immunity and mechanisms of action of drugs has led to new vaccines and new antimicrobial agents. The key to the acquisition of the knowledge of these mechanisms has been identifying the elemental causes (i.e., genes and their products) that mediate immunity and drug resistance. The identification of these genes is made possible by being able to transfer the genes or mutated forms of the genes into causative agents or surrogate hosts. Such an approach was limited in Mycobacterium tuberculosis by the difficulty of transferring genes or alleles into M. tuberculosis or a suitable surrogate mycobacterial host. The construction of shuttle phasmids—chimeric molecules that replicate in Escherichia coli as plasmids and in mycobacteria as mycobacteriophages—was instrumental in developing gene transfer systems for M. tuberculosis. This review will discuss M. tuberculosis genetic systems and their impact on tuberculosis research. “I had to know my enemy in order to prevail against him.”Nelson Mandela PMID:26105819

Kinetics of conjugative gene transfer on surfaces in granular porous media

NASA Astrophysics Data System (ADS)

Massoudieh, A.; Crain, C.; Lambertini, E.; Nelson, K. E.; Barkouki, T.; L'Amoreaux, P.; Loge, F. J.; Ginn, T. R.

2010-03-01

The transfer of genetic material among bacteria in the environment can occur both in the planktonic and attached state. Given the propensity of organisms to exist in sessile microbial communities in oligotrophic subsurface conditions, and that such conditions typify the subsurface, this study focuses on exploratory modeling of horizontal gene transfer among surface-associated Escherichiacoli in the subsurface. The mathematics so far used to describe the kinetics of conjugation in biofilms are developed largely from experimental observations of planktonic gene transfer, and are absent of lags or plasmid stability that appear experimentally. We develop a model and experimental system to quantify bacterial filtration and gene transfer in the attached state, on granular porous media. We include attachment kinetics described in Nelson et al. (2007) using the filtration theory approach of Nelson and Ginn (2001, 2005) with motility of E. coli described according to Biondi et al. (1998).

Kinetics of conjugative gene transfer on surfaces in granular porous media

NASA Astrophysics Data System (ADS)

Ginn, T.; Massoudieh, A.; Nelson, K.; Mathew, A.; Lambertini, E.

2005-12-01

The transfer of genetic material among bacteria in the environment can occur both in the planktonic and attached state. Given the propensity of organisms to exist in sessile microbial communities in oligotrophic conditions, and that such conditions typify the subsurface, this study focuses on exploratory modeling of horizontal gene transfer among surface-associated E. coli in the subsurface. The mathematics so far used to describe the kinetics of conjugation in biofilms are developed largely from experimental observations of planktonic gene transfer, and are absent of lags or plasmid stability that appear experimentally. We develop a model for bacterial filtration and gene transfer in the attached state, for the early stages of biofilm formation using a recently revised filtration theory approach (Nelson and Ginn, 2005) with motility of E. coli described as a continuous time random walk according to data from microflow chamber experiments (Biondi et al., 2002).

Gene transfer and expression in plants.

PubMed

Lorence, Argelia; Verpoorte, Robert

2004-01-01

Until recently, agriculture and plant breeding relied solely on the accumulated experience of generations of farmers and breeders that is, on sexual transfer of genes between plant species. However, recent developments in plant molecular biology and genomics now give us access to knowledge and understanding of plant genomes and the possibility of modifying them. This chapter presents an updated overview of the two most powerful technologies for transferring genetic material (DNA) into plants: Agrobacterium-mediated transformation and microparticle bombardment (biolistics). Some of the topics that are discussed in detail are the main variables controlling the transformation efficiency that can be achieved using each one of these approaches; the advantages and limitations of each methodology; transient versus stable transformation approaches; the potential of some in planta transformation systems; alternatives to developing transgenic plants without selection markers; the availability of diverse genetic tools generated as part of the genome sequencing of different plant species; transgene expression, gene silencing, and their association with regulatory elements; and prospects and ways to possibly overcome some transgene expression difficulties, in particular the use of matrix-attachment regions (MARs).

Timothy grass pollen extract-induced gene expression and signalling pathways in airway epithelial cells.

PubMed

Röschmann, K I L; Luiten, S; Jonker, M J; Breit, T M; Fokkens, W J; Petersen, A; van Drunen, C M

2011-06-01

Grass pollen allergy is one of the most common allergies worldwide and airborne allergens are the major cause of allergic rhinitis. Airway epithelial cells (AECs) are the first to encounter and respond to aeroallergens and are therefore interesting targets for the development of new therapeutics. Our understanding of the epithelial contribution to immune responses is limited as most studies focus on only a few individual genes or proteins. To describe in detail the Timothy grass pollen extract (GPE)-induced gene expression in AECs. NCI-H292 cells were exposed to GPE for 24 h, and isolated RNA and cell culture supernatants were used for microarray analysis and multiplex ELISA, respectively. Eleven thousand and seven hundred fifty-eight transcripts were affected after exposure to GPE, with 141 genes up-regulated and 121 genes down-regulated by more than threefold. The gene ontology group cell communication was among the most prominent categories. Network analysis revealed that a substantial part of regulated genes are related to the cytokines IL-6, IL-8, IL-1A, and the transcription factor FOS. After analysing significantly regulated signalling pathways, we found, among others, epidermal growth factor receptor 1, IL-1, Notch-, and Wnt-related signalling members. Unexpectedly, we found Jagged to be down-regulated and an increased release of IL-12, in line with a more Th1-biased response induced by GPE. Our data show that the stimulation of AECs with GPE results in the induction of a broad response on RNA and protein level by which they are able to affect the initiation and regulation of local immune responses. Detailed understanding of GPE-induced genes and signalling pathways will allow us to better define the pathogenesis of the allergic response and to identify new targets for treatment. © 2011 Blackwell Publishing Ltd.

Trefoil factor-2 reverses airway remodeling changes in allergic airways disease.

PubMed

Royce, Simon G; Lim, Clarice; Muljadi, Ruth C; Samuel, Chrishan S; Ververis, Katherine; Karagiannis, Tom C; Giraud, Andrew S; Tang, Mimi L K

2013-01-01

Trefoil factor 2 (TFF2) is a small peptide with an important role in mucosal repair. TFF2 is up-regulated in asthma, suggesting a role in asthma pathogenesis. Given its known biological role in promoting epithelial repair, TFF2 might be expected to exert a protective function in limiting the progression of airway remodeling in asthma. The contribution of TFF2 to airway remodeling in asthma was investigated by examining the expression of TFF2 in the airway and lung, and evaluating the effects of recombinant TFF2 treatment on established airway remodeling in a murine model of chronic allergic airways disease (AAD). BALB/c mice were sensitized and challenged with ovalbumin (OVA) or saline for 9 weeks, whereas mice with established OVA-induced AAD were treated with TFF2 or vehicle control (intranasally for 14 d). Effects on airway remodeling, airway inflammation, and airway hyperresponsiveness were then assessed, whereas TFF2 expression was determined by immunohistochemistry. TFF2 expression was significantly increased in the airways of mice with AAD, compared with expression levels in control mice. TFF2 treatment resulted in reduced epithelial thickening, subepithelial collagen deposition, goblet-cell metaplasia, bronchial epithelium apoptosis, and airway hyperresponsiveness (all P < 0.05, versus vehicle control), but TFF2 treatment did not influence airway inflammation. The increased expression of endogenous TFF2 in response to chronic allergic inflammation is insufficient to prevent the progression of airway inflammation and remodeling in a murine model of chronic AAD. However, exogenous TFF2 treatment is effective in reversing aspects of established airway remodeling. TFF2 has potential as a novel treatment for airway remodeling in asthma.

Endosymbiotic gene transfer in tertiary plastid-containing dinoflagellates.

PubMed

Burki, Fabien; Imanian, Behzad; Hehenberger, Elisabeth; Hirakawa, Yoshihisa; Maruyama, Shinichiro; Keeling, Patrick J

2014-02-01

Plastid establishment involves the transfer of endosymbiotic genes to the host nucleus, a process known as endosymbiotic gene transfer (EGT). Large amounts of EGT have been shown in several photosynthetic lineages but also in present-day plastid-lacking organisms, supporting the notion that endosymbiotic genes leave a substantial genetic footprint in the host nucleus. Yet the extent of this genetic relocation remains debated, largely because the long period that has passed since most plastids originated has erased many of the clues to how this process unfolded. Among the dinoflagellates, however, the ancestral peridinin-containing plastid has been replaced by tertiary plastids on several more recent occasions, giving us a less ancient window to examine plastid origins. In this study, we evaluated the endosymbiotic contribution to the host genome in two dinoflagellate lineages with tertiary plastids. We generated the first nuclear transcriptome data sets for the "dinotoms," which harbor diatom-derived plastids, and analyzed these data in combination with the available transcriptomes for kareniaceans, which harbor haptophyte-derived plastids. We found low level of detectable EGT in both dinoflagellate lineages, with only 9 genes and 90 genes of possible tertiary endosymbiotic origin in dinotoms and kareniaceans, respectively, suggesting that tertiary endosymbioses did not heavily impact the host dinoflagellate genomes.

The lower airway microbiota in early cystic fibrosis lung disease: a longitudinal analysis.

PubMed

Frayman, Katherine B; Armstrong, David S; Carzino, Rosemary; Ferkol, Thomas W; Grimwood, Keith; Storch, Gregory A; Teo, Shu Mei; Wylie, Kristine M; Ranganathan, Sarath C

2017-12-01

In infants and young children with cystic fibrosis, lower airway infection and inflammation are associated with adverse respiratory outcomes. However, the role of lower airway microbiota in the pathogenesis of early cystic fibrosis lung disease remains uncertain. To assess the development of the lower airway microbiota over time in infants and young children with cystic fibrosis, and to explore its association with airway inflammation and pulmonary function at age 6 years. Serial, semi-annual bronchoscopies and bronchoalveolar lavage (BAL) procedures were performed in infants newly diagnosed with cystic fibrosis following newborn screening. Quantitative microbiological cultures and inflammatory marker (interleukin 8 and neutrophil elastase) measurements were undertaken contemporaneously. 16S ribosomal RNA gene sequencing was conducted on stored BAL samples. Spirometry results recorded at 6 years of age were extracted from medical records. Ninety-five BAL samples provided 16S ribosomal RNA gene data. These were collected from 48 subjects aged 1.2-78.3 months, including longitudinal samples from 27 subjects and 13 before age 6 months. The lower airway microbiota varied, but diversity decreased with advancing age. Detection of recognised cystic fibrosis bacterial pathogens was associated with reduced microbial diversity and greater lower airway inflammation. There was no association between the lower airway microbiota and pulmonary function at age 6 years. In infants with cystic fibrosis, the lower airway microbiota is dynamic. Dominance of the microbiota by recognised cystic fibrosis bacterial pathogens is associated with increased lower airway inflammation, however early microbial diversity is not associated with pulmonary function at 6 years of age. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Comparative biology of rAAV transduction in ferret, pig and human airway epithelia.

PubMed

Liu, X; Luo, M; Guo, C; Yan, Z; Wang, Y; Engelhardt, J F

2007-11-01

Differences between rodent and human airway cell biology have made it difficult to translate recombinant adeno-associated virus (rAAV)-mediated gene therapies to the lung for cystic fibrosis (CF). As new ferret and pig models for CF become available, knowledge about host cell/vector interactions in these species will become increasingly important for testing potential gene therapies. To this end, we have compared the transduction biology of three rAAV serotypes (AAV1, 2 and 5) in human, ferret, pig and mouse-polarized airway epithelia. Our results indicate that apical transduction of ferret and pig airway epithelia with these rAAV serotypes closely mirrors that observed in human epithelia (rAAV1>rAAV2 congruent withrAAV5), while transduction of mouse epithelia was significantly different (rAAV1>rAAV5>rAAV2). Similarly, ferret, pig and human epithelia also shared serotype-specific differences in the polarity (apical vs basolateral) and proteasome dependence of rAAV transduction. Despite these parallels, N-linked sialic acid receptors were required for rAAV1 and rAAV5 transduction of human and mouse airway epithelia, but not ferret or pig airway epithelia. Hence, although the airway tropisms of rAAV serotypes 1, 2 and 5 are conserved better among ferret, pig and human as compared to mouse, viral receptors/co-receptors appear to maintain considerable species diversity.

Alignment-free detection of horizontal gene transfer between closely related bacterial genomes.

PubMed

Domazet-Lošo, Mirjana; Haubold, Bernhard

2011-09-01

Bacterial epidemics are often caused by strains that have acquired their increased virulence through horizontal gene transfer. Due to this association with disease, the detection of horizontal gene transfer continues to receive attention from microbiologists and bioinformaticians alike. Most software for detecting transfer events is based on alignments of sets of genes or of entire genomes. But despite great advances in the design of algorithms and computer programs, genome alignment remains computationally challenging. We have therefore developed an alignment-free algorithm for rapidly detecting horizontal gene transfer between closely related bacterial genomes. Our implementation of this algorithm is called alfy for "ALignment Free local homologY" and is freely available from http://guanine.evolbio.mpg.de/alfy/. In this comment we demonstrate the application of alfy to the genomes of Staphylococcus aureus. We also argue that-contrary to popular belief and in spite of increasing computer speed-algorithmic optimization is becoming more, not less, important if genome data continues to accumulate at the present rate.

Amoebozoa Possess Lineage-Specific Globin Gene Repertoires Gained by Individual Horizontal Gene Transfers

PubMed Central

Dröge, Jasmin; Buczek, Dorota; Suzuki, Yutaka; Makałowski, Wojciech

2014-01-01

The Amoebozoa represent a clade of unicellular amoeboid organisms that display a wide variety of lifestyles, including free-living and parasitic species. For example, the social amoeba Dictyostelium discoideum has the ability to aggregate into a multicellular fruiting body upon starvation, while the pathogenic amoeba Entamoeba histolytica is a parasite of humans. Globins are small heme proteins that are present in almost all extant organisms. Although several genomes of amoebozoan species have been sequenced, little is known about the phyletic distribution of globin genes within this phylum. Only two flavohemoglobins (FHbs) of D. discoideum have been reported and characterized previously while the genomes of Entamoeba species are apparently devoid of globin genes. We investigated eleven amoebozoan species for the presence of globin genes by genomic and phylogenetic in silico analyses. Additional FHb genes were identified in the genomes of four social amoebas and the true slime mold Physarum polycephalum. Moreover, a single-domain globin (SDFgb) of Hartmannella vermiformis, as well as two truncated hemoglobins (trHbs) of Acanthamoeba castellanii were identified. Phylogenetic evidence suggests that these globin genes were independently acquired via horizontal gene transfer from some ancestral bacteria. Furthermore, the phylogenetic tree of amoebozoan FHbs indicates that they do not share a common ancestry and that a transfer of FHbs from bacteria to amoeba occurred multiple times. PMID:25013378

Cox4i2, Ifit2, and Prdm11 Mutant Mice: Effective Selection of Genes Predisposing to an Altered Airway Inflammatory Response from a Large Compendium of Mutant Mouse Lines.

PubMed

Horsch, Marion; Aguilar-Pimentel, Juan Antonio; Bönisch, Clemens; Côme, Christophe; Kolster-Fog, Cathrine; Jensen, Klaus T; Lund, Anders H; Lee, Icksoo; Grossman, Lawrence I; Sinkler, Christopher; Hüttemann, Maik; Bohn, Erwin; Fuchs, Helmut; Ollert, Markus; Gailus-Durner, Valérie; de Angelis, Martin Hrabĕ; Beckers, Johannes

2015-01-01

We established a selection strategy to identify new models for an altered airway inflammatory response from a large compendium of mutant mouse lines that were systemically phenotyped in the German Mouse Clinic (GMC). As selection criteria we included published gene functional data, as well as immunological and transcriptome data from GMC phenotyping screens under standard conditions. Applying these criteria we identified a few from several hundred mutant mouse lines and further characterized the Cox4i2tm1Hutt, Ifit2tm1.1Ebsb, and Prdm11tm1.1ahl lines following ovalbumin (OVA) sensitization and repeated OVA airway challenge. Challenged Prdm11tm1.1ahl mice exhibited changes in B cell counts, CD4+ T cell counts, and in the number of neutrophils in bronchoalveolar lavages, whereas challenged Ifit2tm1.1Ebsb mice displayed alterations in plasma IgE, IgG1, IgG3, and IgM levels compared to the challenged wild type littermates. In contrast, challenged Cox4i2tm1Hutt mutant mice did not show alterations in the humoral or cellular immune response compared to challenged wild type mice. Transcriptome analyses from lungs of the challenged mutant mouse lines showed extensive changes in gene expression in Prdm11tm1.1ahl mice. Functional annotations of regulated genes of all three mutant mouse lines were primarily related to inflammation and airway smooth muscle (ASM) remodeling. We were thus able to define an effective selection strategy to identify new candidate genes for the predisposition to an altered airway inflammatory response under OVA challenge conditions. Similar selection strategies may be used for the analysis of additional genotype-envirotype interactions for other diseases.

Cox4i2, Ifit2, and Prdm11 Mutant Mice: Effective Selection of Genes Predisposing to an Altered Airway Inflammatory Response from a Large Compendium of Mutant Mouse Lines

PubMed Central

Bönisch, Clemens; Côme, Christophe; Kolster-Fog, Cathrine; Jensen, Klaus T.; Lund, Anders H.; Lee, Icksoo; Grossman, Lawrence I.; Sinkler, Christopher; Hüttemann, Maik; Bohn, Erwin; Fuchs, Helmut; Ollert, Markus; Gailus-Durner, Valérie; Hrabĕ de Angelis, Martin; Beckers, Johannes

2015-01-01

We established a selection strategy to identify new models for an altered airway inflammatory response from a large compendium of mutant mouse lines that were systemically phenotyped in the German Mouse Clinic (GMC). As selection criteria we included published gene functional data, as well as immunological and transcriptome data from GMC phenotyping screens under standard conditions. Applying these criteria we identified a few from several hundred mutant mouse lines and further characterized the Cox4i2tm1Hutt, Ifit2tm1.1Ebsb, and Prdm11tm1.1ahl lines following ovalbumin (OVA) sensitization and repeated OVA airway challenge. Challenged Prdm11tm1.1ahl mice exhibited changes in B cell counts, CD4+ T cell counts, and in the number of neutrophils in bronchoalveolar lavages, whereas challenged Ifit2tm1.1Ebsb mice displayed alterations in plasma IgE, IgG1, IgG3, and IgM levels compared to the challenged wild type littermates. In contrast, challenged Cox4i2tm1Hutt mutant mice did not show alterations in the humoral or cellular immune response compared to challenged wild type mice. Transcriptome analyses from lungs of the challenged mutant mouse lines showed extensive changes in gene expression in Prdm11tm1.1ahl mice. Functional annotations of regulated genes of all three mutant mouse lines were primarily related to inflammation and airway smooth muscle (ASM) remodeling. We were thus able to define an effective selection strategy to identify new candidate genes for the predisposition to an altered airway inflammatory response under OVA challenge conditions. Similar selection strategies may be used for the analysis of additional genotype – envirotype interactions for other diseases. PMID:26263558

Highly efficient gene transfer into adult ventricular myocytes by recombinant adenovirus.

PubMed Central

Kirshenbaum, L A; MacLellan, W R; Mazur, W; French, B A; Schneider, M D

1993-01-01

Molecular dissection of mechanisms that govern the differentiated cardiac phenotype has, for cogent technical reasons, largely been undertaken to date in neonatal ventricular myocytes. To circumvent expected limitations of other methods, the present study was initiated to determine whether replication-deficient adenovirus would enable efficient gene transfer to adult cardiac cells in culture. Adult rat ventricular myocytes were infected, 24 h after plating, with adenovirus type 5 containing a cytomegalovirus immediate-early promoter-driven lacZ reporter gene and were assayed for the presence of beta-galactosidase 48 h after infection. The frequency of lacZ+ rod-shaped myocytes was half-maximal at 4 x 10(5) plaque-forming units (PFU) and approached 90% at 1 x 10(8) PFU. Uninfected cells and cells infected with lacZ- virus remained colorless. Beta-galactosidase activity concurred with the proportion of lacZ+ cells and was contingent on the exogenous lacZ gene. At 10(8) PFU/dish, cell number, morphology, and viability each were comparable to uninfected cells. Thus, adult ventricular myocytes are amenable to efficient gene transfer with recombinant adenovirus. The relative uniformity for gene transfer by adenovirus should facilitate tests to determine the impact of putative regulators upon the endogenous genes and gene products of virally modified adult ventricular muscle cells. Images PMID:8326005

Microbial Evolution Is in the Cards: Horizontal Gene Transfer in the Classroom

ERIC Educational Resources Information Center

Kagle, Jeanne; Hay, Anthony G.

2007-01-01

Horizontal gene transfer, the exchange of genetic material between bacteria, is a potentially important factor in the degradation of synthetic compounds introduced to the environment and in the acquisition of other characteristics including antibiotic resistance. This game-based activity illustrates the role of horizontal gene transfer in the…

CD133-targeted gene transfer into long-term repopulating hematopoietic stem cells.

PubMed

Brendel, Christian; Goebel, Benjamin; Daniela, Abriss; Brugman, Martijn; Kneissl, Sabrina; Schwäble, Joachim; Kaufmann, Kerstin B; Müller-Kuller, Uta; Kunkel, Hana; Chen-Wichmann, Linping; Abel, Tobias; Serve, Hubert; Bystrykh, Leonid; Buchholz, Christian J; Grez, Manuel

2015-01-01

Gene therapy for hematological disorders relies on the genetic modification of CD34(+) cells, a heterogeneous cell population containing about 0.01% long-term repopulating cells. Here, we show that the lentiviral vector CD133-LV, which uses a surface marker on human primitive hematopoietic stem cells (HSCs) as entry receptor, transfers genes preferentially into cells with high engraftment capability. Transduction of unstimulated CD34(+) cells with CD133-LV resulted in gene marking of cells with competitive proliferative advantage in vitro and in immunodeficient mice. The CD133-LV-transduced population contained significantly more cells with repopulating capacity than cells transduced with vesicular stomatitis virus (VSV)-LV, a lentiviral vector pseudotyped with the vesicular stomatitis virus G protein. Upon transfer of a barcode library, CD133-LV-transduced cells sustained gene marking in vivo for a prolonged period of time with a 6.7-fold higher recovery of barcodes compared to transduced control cells. Moreover, CD133-LV-transduced cells were capable of repopulating secondary recipients. Lastly, we show that this targeting strategy can be used for transfer of a therapeutic gene into CD34(+) cells obtained from patients suffering of X-linked chronic granulomatous disease. In conclusion, direct gene transfer into CD133(+) cells allows for sustained long-term engraftment of gene corrected cells.

Dynamic monitoring of horizontal gene transfer in soil

NASA Astrophysics Data System (ADS)

Cheng, H. Y.; Masiello, C. A.; Silberg, J. J.; Bennett, G. N.

2015-12-01

Soil microbial gene expression underlies microbial behaviors (phenotypes) central to many aspects of C, N, and H2O cycling. However, continuous monitoring of microbial gene expression in soils is challenging because genetically-encoded reporter proteins widely used in the lab are difficult to deploy in soil matrices: for example, green fluorescent protein cannot be easily visualized in soils, even in the lab. To address this problem we have developed a reporter protein that releases small volatile gases. Here, we applied this gas reporter in a proof-of-concept soil experiment, monitoring horizontal gene transfer, a microbial activity that alters microbial genotypes and phenotypes. Horizontal gene transfer is central to bacterial evolution and adaptation and is relevant to problems such as the spread of antibiotic resistance, increasing metal tolerance in superfund sites, and bioremediation capability of bacterial consortia. This process is likely to be impacted by a number of matrix properties not well-represented in the petri dish, such as microscale variations in water, nutrients, and O2, making petri-dish experiments a poor proxy for environmental processes. We built a conjugation system using synthetic biology to demonstrate the use of gas-reporting biosensors in safe, lab-based biogeochemistry experiments, and here we report the use of these sensors to monitor horizontal gene transfer in soils. Our system is based on the F-plasmid conjugation in Escherichia coli. We have found that the gas signal reports on the number of cells that acquire F-plasmids (transconjugants) in a loamy Alfisol collected from Kellogg Biological Station. We will report how a gas signal generated by transconjugants varies with the number of F-plasmid donor and acceptor cells seeded in a soil, soil moisture, and soil O2 levels.

The Effects of Airway Microbiome on Corticosteroid Responsiveness in Asthma

PubMed Central

Goleva, Elena; Jackson, Leisa P.; Harris, J. Kirk; Robertson, Charles E.; Sutherland, E. Rand; Hall, Clifton F.; Good, James T.; Gelfand, Erwin W.; Martin, Richard J.

2013-01-01

Rationale: The role of airway microbiome in corticosteroid response in asthma is unknown. Objectives: To examine airway microbiome composition in patients with corticosteroid-resistant (CR) asthma and compare it with patients with corticosteroid-sensitive (CS) asthma and normal control subjects and explore whether bacteria in the airways of subjects with asthma may direct alterations in cellular responses to corticosteroids. Methods: 16S rRNA gene sequencing was performed on bronchoalveolar lavage (BAL) samples of 39 subjects with asthma and 12 healthy control subjects. In subjects with asthma, corticosteroid responsiveness was characterized, BAL macrophages were stimulated with pathogenic versus commensal microorganisms, and analyzed by real-time polymerase chain reaction for the expression of corticosteroid-regulated genes and cellular p38 mitogen-activated protein kinase (MAPK) activation. Measurements and Main Results: Of the 39 subjects with asthma, 29 were CR and 10 were CS. BAL microbiome from subjects with CR and CS asthma did not differ in richness, evenness, diversity, and community composition at the phylum level, but did differ at the genus level, with distinct genus expansions in 14 subjects with CR asthma. Preincubation of asthmatic airway macrophages with Haemophilus parainfluenzae, a uniquely expanded potential pathogen found only in CR asthma airways, resulted in p38 MAPK activation, increased IL-8 (P < 0.01), mitogen-activated kinase phosphatase 1 mRNA (P < 0.01) expression, and inhibition of corticosteroid responses (P < 0.05). This was not observed after exposure to commensal bacterium Prevotella melaninogenica. Inhibition of transforming growth factor-β–associated kinase-1 (TAK1), upstream activator of MAPK, but not p38 MAPK restored cellular sensitivity to corticosteroids. Conclusions: A subset of subjects with CR asthma demonstrates airway expansion of specific gram-negative bacteria, which trigger TAK1/MAPK activation and induce

Postentry processing of recombinant adeno-associated virus type 1 and transduction of the ferret lung are altered by a factor in airway secretions.

PubMed

Yan, Ziying; Sun, Xingshen; Evans, Idil A; Tyler, Scott R; Song, Yi; Liu, Xiaoming; Sui, Hongshu; Engelhardt, John F

2013-09-01

We recently created a cystic fibrosis ferret model that acquires neonatal lung infection. To develop lung gene therapies for this model, we evaluated recombinant adeno-associated virus (rAAV)-mediated gene transfer to the neonatal ferret lung. Unlike in vitro ferret airway epithelial (FAE) cells, in vivo infection of the ferret lung with rAAV1 required proteasome inhibitors to achieve efficient airway transduction. We hypothesized that differences in transduction between these two systems were because of an in vivo secreted factor that alter the transduction biology of rAAV1. Indeed, treatment of rAAV1 with ferret airway secretory fluid (ASF) strongly inhibited rAAV1, but not rAAV2, transduction of primary FAE and HeLa cells. Properties of the ASF inhibitory factor included a strong affinity for the AAV1 capsid, heat-stability, negative charge, and sensitivity to endoproteinase Glu-C. ASF-treated rAAV1 dramatically inhibited apical transduction of FAE ALI cultures (512-fold), while only reducing viral entry by 55-fold, suggesting that postentry processing of virus was influenced by the inhibitor factor. Proteasome inhibitors rescued transduction in the presence of ASF (~1600-fold) without effecting virus internalization, while proteasome inhibitors only enhanced transduction 45-fold in the absence of ASF. These findings demonstrate that a factor in lung secretions can influence intracellular processing of rAAV1 in a proteasome-dependent fashion.

Bacteriophage Mediates Efficient Gene Transfer in Combination with Conventional Transfection Reagents

PubMed Central

Donnelly, Amanda; Yata, Teerapong; Bentayebi, Kaoutar; Suwan, Keittisak; Hajitou, Amin

2015-01-01

The development of commercially available transfection reagents for gene transfer applications has revolutionized the field of molecular biology and scientific research. However, the challenge remains in ensuring that they are efficient, safe, reproducible and cost effective. Bacteriophage (phage)-based viral vectors have the potential to be utilized for general gene transfer applications within research and industry. Yet, they require adaptations in order to enable them to efficiently enter cells and overcome mammalian cellular barriers, as they infect bacteria only; furthermore, limited progress has been made at increasing their efficiency. The production of a novel hybrid nanocomplex system consisting of two different nanomaterial systems, phage vectors and conventional transfection reagents, could overcome these limitations. Here we demonstrate that the combination of cationic lipids, cationic polymers or calcium phosphate with M13 bacteriophage-derived vectors, engineered to carry a mammalian transgene cassette, resulted in increased cellular attachment, entry and improved transgene expression in human cells. Moreover, addition of a targeting ligand into the nanocomplex system, through genetic engineering of the phage capsid further increased gene expression and was effective in a stable cell line generation application. Overall, this new hybrid nanocomplex system (i) provides enhanced phage-mediated gene transfer; (ii) is applicable for laboratory transfection processes and (iii) shows promise within industry for large-scale gene transfer applications. PMID:26670247

Bacteriophage Mediates Efficient Gene Transfer in Combination with Conventional Transfection Reagents.

PubMed

Donnelly, Amanda; Yata, Teerapong; Bentayebi, Kaoutar; Suwan, Keittisak; Hajitou, Amin

2015-12-08

The development of commercially available transfection reagents for gene transfer applications has revolutionized the field of molecular biology and scientific research. However, the challenge remains in ensuring that they are efficient, safe, reproducible and cost effective. Bacteriophage (phage)-based viral vectors have the potential to be utilized for general gene transfer applications within research and industry. Yet, they require adaptations in order to enable them to efficiently enter cells and overcome mammalian cellular barriers, as they infect bacteria only; furthermore, limited progress has been made at increasing their efficiency. The production of a novel hybrid nanocomplex system consisting of two different nanomaterial systems, phage vectors and conventional transfection reagents, could overcome these limitations. Here we demonstrate that the combination of cationic lipids, cationic polymers or calcium phosphate with M13 bacteriophage-derived vectors, engineered to carry a mammalian transgene cassette, resulted in increased cellular attachment, entry and improved transgene expression in human cells. Moreover, addition of a targeting ligand into the nanocomplex system, through genetic engineering of the phage capsid further increased gene expression and was effective in a stable cell line generation application. Overall, this new hybrid nanocomplex system (i) provides enhanced phage-mediated gene transfer; (ii) is applicable for laboratory transfection processes and (iii) shows promise within industry for large-scale gene transfer applications.

Plant nodulation inducers enhance horizontal gene transfer of Azorhizobium caulinodans symbiosis island

PubMed Central

Ling, Jun; Wang, Hui; Wu, Ping; Li, Tao; Tang, Yu; Naseer, Nawar; Zheng, Huiming; Masson-Boivin, Catherine; Zhong, Zengtao

2016-01-01

Horizontal gene transfer (HGT) of genomic islands is a driving force of bacterial evolution. Many pathogens and symbionts use this mechanism to spread mobile genetic elements that carry genes important for interaction with their eukaryotic hosts. However, the role of the host in this process remains unclear. Here, we show that plant compounds inducing the nodulation process in the rhizobium-legume mutualistic symbiosis also enhance the transfer of symbiosis islands. We demonstrate that the symbiosis island of the Sesbania rostrata symbiont, Azorhizobium caulinodans, is an 87.6-kb integrative and conjugative element (ICEAc) that is able to excise, form a circular DNA, and conjugatively transfer to a specific site of gly-tRNA gene of other rhizobial genera, expanding their host range. The HGT frequency was significantly increased in the rhizosphere. An ICEAc-located LysR-family transcriptional regulatory protein AhaR triggered the HGT process in response to plant flavonoids that induce the expression of nodulation genes through another LysR-type protein, NodD. Our study suggests that rhizobia may sense rhizosphere environments and transfer their symbiosis gene contents to other genera of rhizobia, thereby broadening rhizobial host-range specificity. PMID:27849579

Plant nodulation inducers enhance horizontal gene transfer of Azorhizobium caulinodans symbiosis island.

PubMed

Ling, Jun; Wang, Hui; Wu, Ping; Li, Tao; Tang, Yu; Naseer, Nawar; Zheng, Huiming; Masson-Boivin, Catherine; Zhong, Zengtao; Zhu, Jun

2016-11-29

Horizontal gene transfer (HGT) of genomic islands is a driving force of bacterial evolution. Many pathogens and symbionts use this mechanism to spread mobile genetic elements that carry genes important for interaction with their eukaryotic hosts. However, the role of the host in this process remains unclear. Here, we show that plant compounds inducing the nodulation process in the rhizobium-legume mutualistic symbiosis also enhance the transfer of symbiosis islands. We demonstrate that the symbiosis island of the Sesbania rostrata symbiont, Azorhizobium caulinodans, is an 87.6-kb integrative and conjugative element (ICE Ac ) that is able to excise, form a circular DNA, and conjugatively transfer to a specific site of gly-tRNA gene of other rhizobial genera, expanding their host range. The HGT frequency was significantly increased in the rhizosphere. An ICE Ac -located LysR-family transcriptional regulatory protein AhaR triggered the HGT process in response to plant flavonoids that induce the expression of nodulation genes through another LysR-type protein, NodD. Our study suggests that rhizobia may sense rhizosphere environments and transfer their symbiosis gene contents to other genera of rhizobia, thereby broadening rhizobial host-range specificity.

Evolution of Antifreeze Protein Genes in the Diatom Genus Fragilariopsis: Evidence for Horizontal Gene Transfer, Gene Duplication and Episodic Diversifying Selection

PubMed Central

Sorhannus, Ulf

2011-01-01

Hypotheses about horizontal transfer of antifreeze protein genes to ice-living diatoms were addressed using two different statistical methods available in the program Prunier. The role of diversifying selection in driving the differentiation of a set of antifreeze protein genes in the diatom genus Fragilariopsis was also investigated. Four horizontal gene transfer events were identified. Two of these took place between two major eukaryote lineages, that is from the diatom Chaetoceros neogracile to the copepod Stephos longipes and from a basidiomycete clade to a monophyletic group, consisting of the diatom species Fragilariopsis curta and Fragilariopsis cylindrus. The remaining two events included transfers from an ascomycete lineage to the proteobacterium Stigmatella aurantiaca and from the proteobacterium Polaribacter irgensii to a group composed of 4 proteobacterium species. After the Fragilariopsis lineage acquired the antifreeze protein gene from the basidiomycetes, it duplicated and went through episodic evolution, characterized by strong positive selection acting on short segments of the branches in the tree. This selection pattern suggests that the paralogs differentiated functionally over relatively short time periods. Taken together, the results obtained here indicate that the group of antifreeze protein genes considered here have a complex evolutionary history. PMID:22253534

Intracellular gene transfer in action: Dual transcription and multiple silencings of nuclear and mitochondrial cox2 genes in legumes

PubMed Central

Adams, Keith L.; Song, Keming; Roessler, Philip G.; Nugent, Jacqueline M.; Doyle, Jane L.; Doyle, Jeff J.; Palmer, Jeffrey D.

1999-01-01

The respiratory gene cox2, normally present in the mitochondrion, was previously shown to have been functionally transferred to the nucleus during flowering plant evolution, possibly during the diversification of legumes. To search for novel intermediate stages in the process of intracellular gene transfer and to assess the evolutionary timing and frequency of cox2 transfer, activation, and inactivation, we examined nuclear and mitochondrial (mt) cox2 presence and expression in over 25 legume genera and mt cox2 presence in 392 genera. Transfer and activation of cox2 appear to have occurred during recent legume evolution, more recently than previously inferred. Many intermediate stages of the gene transfer process are represented by cox2 genes in the studied legumes. Nine legumes contain intact copies of both nuclear and mt cox2, although transcripts could not be detected for some of these genes. Both cox2 genes are transcribed in seven legumes that are phylogenetically interspersed with species displaying only nuclear or mt cox2 expression. Inactivation of cox2 in each genome has taken place multiple times and in a variety of ways, including loss of detectable transcripts or transcript editing and partial to complete gene loss. Phylogenetic evidence shows about the same number (3–5) of separate inactivations of nuclear and mt cox2, suggesting that there is no selective advantage for a mt vs. nuclear location of cox2 in plants. The current distribution of cox2 presence and expression between the nucleus and mitochondrion in the studied legumes is probably the result of chance mutations silencing either cox2 gene. PMID:10570164

Cell-to-Cell Contact and Nectin-4 Govern Spread of Measles Virus from Primary Human Myeloid Cells to Primary Human Airway Epithelial Cells.

PubMed

Singh, Brajesh K; Li, Ni; Mark, Anna C; Mateo, Mathieu; Cattaneo, Roberto; Sinn, Patrick L

2016-08-01

Measles is a highly contagious, acute viral illness. Immune cells within the airways are likely first targets of infection, and these cells traffic measles virus (MeV) to lymph nodes for amplification and subsequent systemic dissemination. Infected immune cells are thought to return MeV to the airways; however, the mechanisms responsible for virus transfer to pulmonary epithelial cells are poorly understood. To investigate this process, we collected blood from human donors and generated primary myeloid cells, specifically, monocyte-derived macrophages (MDMs) and dendritic cells (DCs). MDMs and DCs were infected with MeV and then applied to primary cultures of well-differentiated airway epithelial cells from human donors (HAE). Consistent with previous results obtained with free virus, infected MDMs or DCs were incapable of transferring MeV to HAE when applied to the apical surface. Likewise, infected MDMs or DCs applied to the basolateral surface of HAE grown on small-pore (0.4-μm) support membranes did not transfer virus. In contrast, infected MDMs and DCs applied to the basolateral surface of HAE grown on large-pore (3.0-μm) membranes successfully transferred MeV. Confocal microscopy demonstrated that MDMs and DCs are capable of penetrating large-pore membranes but not small-pore membranes. Further, by using a nectin-4 blocking antibody or recombinant MeV unable to enter cells through nectin-4, we demonstrated formally that transfer from immune cells to HAE occurs in a nectin-4-dependent manner. Thus, both infected MDMs and DCs rely on cell-to-cell contacts and nectin-4 to efficiently deliver MeV to the basolateral surface of HAE. Measles virus spreads rapidly and efficiently in human airway epithelial cells. This rapid spread is based on cell-to-cell contact rather than on particle release and reentry. Here we posit that MeV transfer from infected immune cells to epithelial cells also occurs by cell-to-cell contact rather than through cell-free particles. In

Evolutionary change and phylogenetic relationships in light of horizontal gene transfer.

PubMed

Boto, Luis

2015-06-01

Horizontal gene transfer has, over the past 25 years, become a part of evolutionary thinking. In the present paper I discuss horizontal gene transfer (HGT) in relation to contingency, natural selection, evolutionary change speed and the Tree-of-Life endeavour, with the aim of contributing to the understanding of the role of HGT in evolutionary processes. In addition, the challenges that HGT imposes on the current view of evolution are emphasized.

Horizontal Gene Transfers in Mycoplasmas (Mollicutes).

PubMed

Citti, C; Dordet-Frisoni, E; Nouvel, L X; Kuo, C H; Baranowski, E

2018-04-12

The class Mollicutes (trivial name "mycoplasma") is composed of wall-less bacteria with reduced genomes whose evolution was long thought to be only driven by gene losses. Recent evidences of massive horizontal gene transfer (HGT) within and across species provided a new frame to understand the successful adaptation of these minimal bacteria to a broad range of hosts. Mobile genetic elements are being identified in a growing number of mycoplasma species, but integrative and conjugative elements (ICEs) are emerging as pivotal in HGT. While sharing common traits with other bacterial ICEs, such as their chromosomal integration and the use of a type IV secretion system to mediate horizontal dissemination, mycoplasma ICEs (MICEs) revealed unique features: their chromosomal integration is totally random and driven by a DDE recombinase related to the Mutator-like superfamily. Mycoplasma conjugation is not restricted to ICE transmission, but also involves the transfer of large chromosomal fragments that generates progenies with mosaic genomes, nearly every position of chromosome being mobile. Mycoplasmas have thus developed efficient ways to gain access to a considerable reservoir of genetic resources distributed among a vast number of species expanding the concept of minimal cell to the broader context of flowing information.

Quantitative analysis of recombination between YFP and CFP genes of FRET biosensors introduced by lentiviral or retroviral gene transfer

PubMed Central

Komatsubara, Akira T.; Matsuda, Michiyuki; Aoki, Kazuhiro

2015-01-01

Biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) have been developed to visualize spatio-temporal dynamics of signalling molecules in living cells. Many of them adopt a backbone of intramolecular FRET biosensor with a cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) as donor and acceptor, respectively. However, there remains the difficulty of establishing cells stably expressing FRET biosensors with a YFP and CFP pair by lentiviral or retroviral gene transfer, due to the high incidence of recombination between YFP and CFP genes. To address this, we examined the effects of codon-diversification of YFP on the recombination of FRET biosensors introduced by lentivirus or retrovirus. The YFP gene that was fully codon-optimized to E.coli evaded the recombination in lentiviral or retroviral gene transfer, but the partially codon-diversified YFP did not. Further, the length of spacer between YFP and CFP genes clearly affected recombination efficiency, suggesting that the intramolecular template switching occurred in the reverse-transcription process. The simple mathematical model reproduced the experimental data sufficiently, yielding a recombination rate of 0.002–0.005 per base. Together, these results show that the codon-diversified YFP is a useful tool for expressing FRET biosensors by lentiviral or retroviral gene transfer. PMID:26290434

Effect of MUC8 on Airway Inflammation: A Friend or a Foe?

PubMed

Cha, Hee-Jae; Song, Kyoung Seob

2018-02-06

In this review, we compile identifying molecular mechanisms of MUC8 gene expression and studies characterizing the physiological functions of MUC8 in the airway and analyzing how altered MUC8 gene expression in the lung is affected by negative regulators.

Complexity of genetic sequences modified by horizontal gene transfer and degraded-DNA uptake

NASA Astrophysics Data System (ADS)

Tremberger, George; Dehipawala, S.; Nguyen, A.; Cheung, E.; Sullivan, R.; Holden, T.; Lieberman, D.; Cheung, T.

2015-09-01

Horizontal gene transfer has been a major vehicle for efficient transfer of genetic materials among living species and could be one of the sources for noncoding DNA incorporation into a genome. Our previous study of lnc- RNA sequence complexity in terms of fractal dimension and information entropy shows a tight regulation among the studied genes in numerous diseases. The role of sequence complexity in horizontal transferred genes was investigated with Mealybug in symbiotic relation with a 139K genome microbe and Deinococcus radiodurans as examples. The fractal dimension and entropy showed correlation R-sq of 0.82 (N = 6) for the studied Deinococcus radiodurans sequences. For comparison the Deinococcus radiodurans oxidative stress tolerant catalase and superoxide dismutase genes under extracellular dGMP growth condition showed R-sq ~ 0.42 (N = 6); and the studied arsenate reductase horizontal transferred genes for toxicity survival in several microorganisms showed no correlation. Simulation results showed that R-sq < 0.4 would be improbable at less than one percent chance, suggestive of additional selection pressure when compared to the R-sq ~ 0.29 (N = 21) in the studied transferred genes in Mealybug. The mild correlation of R-sq ~ 0.5 for fractal dimension versus transcription level in the studied Deinococcus radiodurans sequences upon extracellular dGMP growth condition would suggest that lower fractal dimension with less electron density fluctuation favors higher transcription level.

Status of Therapeutic Gene Transfer to Treat Cardiovascular Disease in Dogs and Cats.

PubMed

Sleeper, Meg M

2017-09-01

Gene therapy is a procedure resulting in the transfer of a gene into an individual's cells to treat a disease. One goal of gene transfer is to express a functional gene when the endogenous gene is inactive. However, because heart failure is a complex disease characterized by multiple abnormalities at the cellular level, an alternate gene delivery approach is to alter myocardial protein levels to improve function. This article discusses background information on gene delivery, including packaging, administration, and a brief discussion of some of the candidate transgenes likely to alter the progression of naturally occurring heart disease in dogs and cats. Copyright © 2017 Elsevier Inc. All rights reserved.

Automated airway evaluation system for multi-slice computed tomography using airway lumen diameter, airway wall thickness and broncho-arterial ratio

NASA Astrophysics Data System (ADS)

Odry, Benjamin L.; Kiraly, Atilla P.; Novak, Carol L.; Naidich, David P.; Lerallut, Jean-Francois

2006-03-01

Pulmonary diseases such as bronchiectasis, asthma, and emphysema are characterized by abnormalities in airway dimensions. Multi-slice computed tomography (MSCT) has become one of the primary means to depict these abnormalities, as the availability of high-resolution near-isotropic data makes it possible to evaluate airways at oblique angles to the scanner plane. However, currently, clinical evaluation of airways is typically limited to subjective visual inspection only: systematic evaluation of the airways to take advantage of high-resolution data has not proved practical without automation. We present an automated method to quantitatively evaluate airway lumen diameter, wall thickness and broncho-arterial ratios. In addition, our method provides 3D visualization of these values, graphically illustrating the location and extent of disease. Our algorithm begins by automatic airway segmentation to extract paths to the distal airways, and to create a map of airway diameters. Normally, airway diameters decrease as paths progress distally; failure to taper indicates abnormal dilatation. Our approach monitors airway lumen diameters along each airway path in order to detect abnormal profiles, allowing even subtle degrees of pathologic dilatation to be identified. Our method also systematically computes the broncho-arterial ratio at every terminal branch of the tree model, as a ratio above 1 indicates potentially abnormal bronchial dilatation. Finally, the airway wall thickness is computed at corresponding locations. These measurements are used to highlight abnormal branches for closer inspection, and can be summed to compute a quantitative global score for the entire airway tree, allowing reproducible longitudinal assessment of disease severity. Preliminary tests on patients diagnosed with bronchiectasis demonstrated rapid identification of lack of tapering, which also was confirmed by corresponding demonstration of elevated broncho-arterial ratios.

GFP as a marker for transient gene transfer and expression in Mycoplasma hyorhinis.

PubMed

Ishag, Hassan Z A; Liu, Maojun; Yang, Ruosong; Xiong, Qiyan; Feng, Zhixin; Shao, Guoqing

2016-01-01

Mycoplasma hyorhinis (M. hyorhinis) is an opportunistic pathogen of pigs and has been shown to transform cell cultures, which has increased the interest of researchers. The green florescence proteins (GFP) gene of Aquorea victoria, proved to be a vital marker to identify transformed cells in mixed populations. Use of GFP to observe gene transfer and expression in M. hyorhinis (strain HUB-1) has not been described. We have constructed a pMD18-O/MHRgfp plasmid containing the p97 gene promoter, origin of replication, tetracycline resistance marker and GFP gene controlled by the p97 gene promoter. The plasmid transformed into M. hyorhinis with a frequency of ~4 × 10(-3) cfu/µg plasmid DNA and could be detected by PCR amplification of the GFP gene from the total DNA of the transformant mycoplasmas. Analysis of a single clone grown on KM2-Agar containing tetracycline, showed a green fluorescence color. Conclusively, this report suggests the usefulness of GFP to monitor transient gene transfer and expression in M. hyorhinis, eventually minimizing screening procedures for gene transfer and expression.

On the Complexity of Duplication-Transfer-Loss Reconciliation with Non-Binary Gene Trees.

PubMed

Kordi, Misagh; Bansal, Mukul S

2017-01-01

Duplication-Transfer-Loss (DTL) reconciliation has emerged as a powerful technique for studying gene family evolution in the presence of horizontal gene transfer. DTL reconciliation takes as input a gene family phylogeny and the corresponding species phylogeny, and reconciles the two by postulating speciation, gene duplication, horizontal gene transfer, and gene loss events. Efficient algorithms exist for finding optimal DTL reconciliations when the gene tree is binary. However, gene trees are frequently non-binary. With such non-binary gene trees, the reconciliation problem seeks to find a binary resolution of the gene tree that minimizes the reconciliation cost. Given the prevalence of non-binary gene trees, many efficient algorithms have been developed for this problem in the context of the simpler Duplication-Loss (DL) reconciliation model. Yet, no efficient algorithms exist for DTL reconciliation with non-binary gene trees and the complexity of the problem remains unknown. In this work, we resolve this open question by showing that the problem is, in fact, NP-hard. Our reduction applies to both the dated and undated formulations of DTL reconciliation. By resolving this long-standing open problem, this work will spur the development of both exact and heuristic algorithms for this important problem.

Adeno-Associated Virus Type 6 (AAV6) Vectors Mediate Efficient Transduction of Airway Epithelial Cells in Mouse Lungs Compared to That of AAV2 Vectors

PubMed Central

Halbert, Christine L.; Allen, James M.; Miller, A. Dusty

2001-01-01

Although vectors derived from adeno-associated virus type 2 (AAV2) promote gene transfer and expression in many somatic tissues, studies with animal models and cultured cells show that the apical surface of airway epithelia is resistant to transduction by AAV2 vectors. Approaches to increase transduction rates include increasing the amount of vector and perturbing the integrity of the epithelia. In this study, we explored the use of vectors based on AAV6 to increase transduction rates in airways. AAV vectors were made using combinations of rep, cap, and packaged genomes from AAV2 or AAV6. The packaged genomes encoded human placental alkaline phosphatase and contained terminal repeat sequences from AAV2 or AAV6. We found that transduction efficiency was primarily dependent on the source of Cap protein, defined here as the vector pseudotype. The AAV6 and AAV2 pseudotype vectors exhibited different tropisms in tissue-cultured cells, and cell transduction by AAV6 vectors was not inhibited by heparin, nor did they compete for entry in a transduction assay, indicating that AAV6 and AAV2 capsid bind different receptors. In vivo analysis of vectors showed that AAV2 pseudotype vectors gave high transduction rates in alveolar cells but much lower rates in the airway epithelium. In contrast, the AAV6 pseudotype vectors exhibited much more efficient transduction of epithelial cells in large and small airways, showing up to 80% transduction in some airways. These results, combined with our previous results showing lower immunogenicity of AAV6 than of AAV2 vectors, indicate that AAV6 vectors may provide significant advantages over AAV2 for gene therapy of lung diseases like cystic fibrosis. PMID:11413329

Analysis of gene transfer rate with immobilized retroviral vectors.

PubMed

Peng, Ching-An

2009-04-01

Efficient delivery of transgenes into the cell nucleus by retroviral vectors in a static culture system is limited by the intrinsic features of incompetent retroviruses (i.e., thermodynamically unstable envelope proteins and low titers). Although several physicochemical approaches (e.g., adding polycationic polymer and applying magnetic force) have been reported to augment the retroviral gene transfer rate, none are suitable for scaling up to a setting for clinical use. The study of using acoustic fields with the form of standing waves has recently been reported to be a feasible way to enhance retroviral gene delivery efficiency in large-scale settings. The concept of using ultrasound standing-wave fields to increase retrovirus-mediated gene transfer is based on quickly established cell bands on acoustic nodal planes as nucleating sites to capture unstable colloidlike retroviruses. In this study, instead of having retroviral nanoparticles circulated between nodal planes, we proposed to immobilize retroviruses onto acoustic transparent films arranged in an acoustic chamber. Then, cells inoculated into the acoustic chamber can be driven by the primary radiation forces to the retrovirus-coated films that are constructed on the nodal planes. To obtain the optimal time of immobilizing retroviruses onto the acoustic transparent film prior to the inception of acoustic fields, we developed a retroviral diffusion-reaction model to describe such a static retroviral system. Analysis of viral transport model has its merit to guide experimental design for attaining high gene transfer efficiency.

Oral Gene Application Using Chitosan-DNA Nanoparticles Induces Transferable Tolerance

PubMed Central

Ensminger, Stephan M.; Spriewald, Bernd M.

2012-01-01

Oral tolerance is a promising approach to induce unresponsiveness to various antigens. The development of tolerogenic vaccines could be exploited in modulating the immune response in autoimmune disease and allograft rejection. In this study, we investigated a nonviral gene transfer strategy for inducing oral tolerance via antigen-encoding chitosan-DNA nanoparticles (NP). Oral application of ovalbumin (OVA)-encoding chitosan-DNA NP (OVA-NP) suppressed the OVA-specific delayed-type hypersensitivity (DTH) response and anti-OVA antibody formation, as well as spleen cell proliferation following OVA stimulation. Cytokine expression patterns following OVA stimulation in vitro showed a shift from a Th1 toward a Th2/Th3 response. The OVA-NP-induced tolerance was transferable from donor to naïve recipient mice via adoptive spleen cell transfer and was mediated by CD4+CD25+ T cells. These findings indicate that nonviral oral gene transfer can induce regulatory T cells for antigen-specific immune modulation. PMID:22933401

Protective effects of valproic acid against airway hyperresponsiveness and airway remodeling in a mouse model of allergic airways disease.

PubMed

Royce, Simon G; Dang, William; Ververis, Katherine; De Sampayo, Nishika; El-Osta, Assam; Tang, Mimi L K; Karagiannis, Tom C

2011-12-01

Airway remodeling and airway hyperresponsiveness are major aspects of asthma pathology that are not targeted optimally by existing anti-inflammatory drugs. Histone deacetylase inhibitors have a wide range of effects that may potentially abrogate aspects of remodeling. One such histone deacetylase inhibitor is valproic acid (2-propylvaleric acid). Valproic acid is used clinically as an anti-epileptic drug and is a potent inhibitor of class I histone deacetylases but also inhibits class II histone deacetylases. We used valproic acid as a molecular model of histone deacetylase inhibition in vivo in chronic allergic airways disease mice with airway remodeling and airway hyperresponsiveness. Wild-type Balb/c mice with allergic airways disease were treated with valproic acid or vehicle control. Airway inflammation was assessed by bronchoalveolar lavage fluid cell counts and examination of lung tissue sections. Remodeling was assessed by morphometric analysis of histochemically stained slides and lung function was assessed by invasive plethysmography measurement of airway resistance. Valproic acid treatment did not affect inflammation parameters; however, valproic acid treatment resulted in reduced epithelial thickness as compared to vehicle treated mice (p < 0.01), reduced subepithelial collagen deposition (p < 0.05) and attenuated airway hyperresponsiveness (p < 0.05 and p < 0.01 for the two highest doses of methacholine, respectively). These findings show that treatment with valproic acid can reduce structural airway remodeling changes and hyperresponsiveness, providing further evidence for the potential use of histone deacetylase inhibitors for the treatment of asthma.

Horizontal transfer of a eukaryotic plastid-targeted protein gene to cyanobacteria

PubMed Central

Rogers, Matthew B; Patron, Nicola J; Keeling, Patrick J

2007-01-01

Background Horizontal or lateral transfer of genetic material between distantly related prokaryotes has been shown to play a major role in the evolution of bacterial and archaeal genomes, but exchange of genes between prokaryotes and eukaryotes is not as well understood. In particular, gene flow from eukaryotes to prokaryotes is rarely documented with strong support, which is unusual since prokaryotic genomes appear to readily accept foreign genes. Results Here, we show that abundant marine cyanobacteria in the related genera Synechococcus and Prochlorococcus acquired a key Calvin cycle/glycolytic enzyme from a eukaryote. Two non-homologous forms of fructose bisphosphate aldolase (FBA) are characteristic of eukaryotes and prokaryotes respectively. However, a eukaryotic gene has been inserted immediately upstream of the ancestral prokaryotic gene in several strains (ecotypes) of Synechococcus and Prochlorococcus. In one lineage this new gene has replaced the ancestral gene altogether. The eukaryotic gene is most closely related to the plastid-targeted FBA from red algae. This eukaryotic-type FBA once replaced the plastid/cyanobacterial type in photosynthetic eukaryotes, hinting at a possible functional advantage in Calvin cycle reactions. The strains that now possess this eukaryotic FBA are scattered across the tree of Synechococcus and Prochlorococcus, perhaps because the gene has been transferred multiple times among cyanobacteria, or more likely because it has been selectively retained only in certain lineages. Conclusion A gene for plastid-targeted FBA has been transferred from red algae to cyanobacteria, where it has inserted itself beside its non-homologous, functional analogue. Its current distribution in Prochlorococcus and Synechococcus is punctate, suggesting a complex history since its introduction to this group. PMID:17584924

Airway stents

PubMed Central

Keyes, Colleen

2018-01-01

Stents and tubes to maintain the patency of the airways are commonly used for malignant obstruction and are occasionally employed in benign disease. Malignant airway obstruction usually results from direct involvement of bronchogenic carcinoma, or by extension of carcinomas occurring in the esophagus or the thyroid. External compression from lymph nodes or metastatic disease from other organs can also cause central airway obstruction. Most malignant airway lesions are surgically inoperable due to advanced disease stage and require multimodality palliation, including stent placement. As with any other medical device, stents have significantly evolved over the last 50 years and deserve an in-depth understanding of their true capabilities and complications. Not every silicone stent is created equal and the same holds for metallic stents. Herein, we present an overview of the topic as well as some of the more practical and controversial issues surrounding airway stents. We also try to dispel the myths surrounding stent removal and their supposed use only in central airways. At the end, we come to the long-held conclusion that stents should not be used as first line treatment of choice, but after ruling out the possibility of curative surgical resection or repair. PMID:29707506

Horizontal gene transfer in parasitic plants.

PubMed

Davis, Charles C; Xi, Zhenxiang

2015-08-01

Horizontal gene transfer (HGT) between species has been a major focus of plant evolutionary research during the past decade. Parasitic plants, which establish a direct connection with their hosts, have provided excellent examples of how these transfers are facilitated via the intimacy of this symbiosis. In particular, phylogenetic studies from diverse clades indicate that parasitic plants represent a rich system for studying this phenomenon. Here, HGT has been shown to be astonishingly high in the mitochondrial genome, and appreciable in the nuclear genome. Although explicit tests remain to be performed, some transgenes have been hypothesized to be functional in their recipient species, thus providing a new perspective on the evolution of novelty in parasitic plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

Gene Transfer Efficiency in Gonococcal Biofilms: Role of Biofilm Age, Architecture, and Pilin Antigenic Variation.

PubMed

Kouzel, Nadzeya; Oldewurtel, Enno R; Maier, Berenike

2015-07-01

Extracellular DNA is an important structural component of many bacterial biofilms. It is unknown, however, to which extent external DNA is used to transfer genes by means of transformation. Here, we quantified the acquisition of multidrug resistance and visualized its spread under selective and nonselective conditions in biofilms formed by Neisseria gonorrhoeae. The density and architecture of the biofilms were controlled by microstructuring the substratum for bacterial adhesion. Horizontal transfer of antibiotic resistance genes between cocultured strains, each carrying a single resistance, occurred efficiently in early biofilms. The efficiency of gene transfer was higher in early biofilms than between planktonic cells. It was strongly reduced after 24 h and independent of biofilm density. Pilin antigenic variation caused a high fraction of nonpiliated bacteria but was not responsible for the reduced gene transfer at later stages. When selective pressure was applied to dense biofilms using antibiotics at their MIC, the double-resistant bacteria did not show a significant growth advantage. In loosely connected biofilms, the spreading of double-resistant clones was prominent. We conclude that multidrug resistance readily develops in early gonococcal biofilms through horizontal gene transfer. However, selection and spreading of the multiresistant clones are heavily suppressed in dense biofilms. Biofilms are considered ideal reaction chambers for horizontal gene transfer and development of multidrug resistances. The rate at which genes are exchanged within biofilms is unknown. Here, we quantified the acquisition of double-drug resistance by gene transfer between gonococci with single resistances. At early biofilm stages, the transfer efficiency was higher than for planktonic cells but then decreased with biofilm age. The surface topography affected the architecture of the biofilm. While the efficiency of gene transfer was independent of the architecture, spreading of

Interfamily Transfer of Dual NB-LRR Genes Confers Resistance to Multiple Pathogens

PubMed Central

Narusaka, Mari; Kubo, Yasuyuki; Hatakeyama, Katsunori; Imamura, Jun; Ezura, Hiroshi; Nanasato, Yoshihiko; Tabei, Yutaka; Takano, Yoshitaka; Shirasu, Ken; Narusaka, Yoshihiro

2013-01-01

A major class of disease resistance (R) genes which encode nucleotide binding and leucine rich repeat (NB-LRR) proteins have been used in traditional breeding programs for crop protection. However, it has been difficult to functionally transfer NB-LRR-type R genes in taxonomically distinct families. Here we demonstrate that a pair of Arabidopsis (Brassicaceae) NB-LRR-type R genes, RPS4 and RRS1, properly function in two other Brassicaceae, Brassica rapa and Brassica napus, but also in two Solanaceae, Nicotiana benthamiana and tomato (Solanum lycopersicum). The solanaceous plants transformed with RPS4/RRS1 confer bacterial effector-specific immunity responses. Furthermore, RPS4 and RRS1, which confer resistance to a fungal pathogen Colletotrichum higginsianum in Brassicaceae, also protect against Colletotrichum orbiculare in cucumber (Cucurbitaceae). Importantly, RPS4/RRS1 transgenic plants show no autoimmune phenotypes, indicating that the NB-LRR proteins are tightly regulated. The successful transfer of two R genes at the family level implies that the downstream components of R genes are highly conserved. The functional interfamily transfer of R genes can be a powerful strategy for providing resistance to a broad range of pathogens. PMID:23437080

Local small airway epithelial injury induces global smooth muscle contraction and airway constriction.

PubMed

Zhou, Jian; Alvarez-Elizondo, Martha B; Botvinick, Elliot; George, Steven C

2012-02-01

Small airway epithelial cells form a continuous sheet lining the conducting airways, which serves many functions including a physical barrier to protect the underlying tissue. In asthma, injury to epithelial cells can occur during bronchoconstriction, which may exacerbate airway hyperreactivity. To investigate the role of epithelial cell rupture in airway constriction, laser ablation was used to precisely rupture individual airway epithelial cells of small airways (<300-μm diameter) in rat lung slices (∼250-μm thick). Laser ablation of single epithelial cells using a femtosecond laser reproducibly induced airway contraction to ∼70% of the original cross-sectional area within several seconds, and the contraction lasted for up to 40 s. The airway constriction could be mimicked by mechanical rupture of a single epithelial cell using a sharp glass micropipette but not with a blunt glass pipette. These results suggest that soluble mediators released from the wounded epithelial cell induce global airway contraction. To confirm this hypothesis, the lysate of primary human small airway epithelial cells stimulated a similar airway contraction. Laser ablation of single epithelial cells triggered a single instantaneous Ca(2+) wave in the epithelium, and multiple Ca(2+) waves in smooth muscle cells, which were delayed by several seconds. Removal of extracellular Ca(2+) or decreasing intracellular Ca(2+) both blocked laser-induced airway contraction. We conclude that local epithelial cell rupture induces rapid and global airway constriction through release of soluble mediators and subsequent Ca(2+)-dependent smooth muscle shortening.

Toll-like receptor-2 agonist-allergen coupling efficiently redirects Th2 cell responses and inhibits allergic airway eosinophilia.

PubMed

Krishnaswamy, Jayendra Kumar; Jirmo, Adan Chari; Baru, Abdul Mannan; Ebensen, Thomas; Guzmán, Carlos A; Sparwasser, Tim; Behrens, Georg M N

2012-12-01

Toll-like receptor (TLR) agonists beneficially modulate allergic airway inflammation. However, the efficiency of TLR agonists varies considerably, and their exact cellular mechanisms (especially of TLR 2/6 agonists) are incompletely understood. We investigated at a cellular level whether the administration of the pharmacologically improved TLR2/6 agonist S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxy polyethylene glycol (BPP) conjugated to antigenic peptide (BPP-OVA) could divert an existing Th2 response and influence airway eosinophilia. The effects of BPP-OVA on airway inflammation were assessed in a classic murine sensitization/challenge model and an adoptive transfer model, which involved the adoptive transfer of in vitro differentiated ovalbumin (OVA)-specific Th2 cells. Functional T-cell stimulation by lung dendritic cells (DCs) was determined both in vitro and in vivo, combined with a cytokine secretion analysis. A single mucosal application of BPP-OVA efficiently delivered antigen, led to TLR2-mediated DC activation, and resulted in OVA-specific T-cell proliferation via lung DCs in vivo. In alternative models of allergic airway disease, a single administration of BPP-OVA before OVA challenge (but not BPP alone) significantly reduced airway eosinophilia, most likely through altered antigen-specific T-cell stimulation via DCs. Analyses of adoptively transferred Th2-biased cells after BPP-OVA administration in vivo suggested that BPP-OVA guides antigen-specific Th2 cells to produce significantly higher amounts of IFN-γ upon allergen challenge. In conclusion, our data show for the first time that a single mucosal administration of a TLR 2/6 agonist-allergen conjugate can provoke IFN-γ responses in Th2-biased cells and alleviate allergic airway inflammation.

Atopic asthmatic immune phenotypes associated with airway microbiota and airway obstruction.

PubMed

Turturice, Benjamin A; McGee, Halvor S; Oliver, Brian; Baraket, Melissa; Nguyen, Brian T; Ascoli, Christian; Ranjan, Ravi; Rani, Asha; Perkins, David L; Finn, Patricia W

2017-01-01

Differences in asthma severity may be related to inflammation in the airways. The lower airway microbiota has been associated with clinical features such as airway obstruction, symptom control, and response to corticosteroids. To assess the relationship between local airway inflammation, severity of disease, and the lower airway microbiota in atopic asthmatics. A cohort of young adult, atopic asthmatics with intermittent or mild/moderate persistent symptoms (n = 13) were assessed via bronchoscopy, lavage, and spirometry. These individuals were compared to age matched non-asthmatic controls (n = 6) and to themselves after six weeks of treatment with fluticasone propionate (FP). Inflammation of the airways was assessed via a cytokine and chemokine panel. Lower airway microbiota composition was determined by metagenomic shotgun sequencing. Unsupervised clustering of cytokines and chemokines prior to treatment with FP identified two asthmatic phenotypes (AP), termed AP1 and AP2, with distinct bronchoalveolar lavage inflammatory profiles. AP2 was associated with more obstruction, compared to AP1. After treatment with FP reduced MIP-1β and TNF-α and increased IL-2 was observed. A module of highly correlated cytokines that include MIP-1β and TNF-α was identified that negatively correlated with pulmonary function. Independently, IL-2 was positively correlated with pulmonary function. The airway microbiome composition correlated with asthmatic phenotypes. AP2, prior to FP treatment, was enriched with Streptococcus pneumoniae. Unique associations between IL-2 or the cytokine module and the microbiota composition of the airways were observed in asthmatics subjects prior to treatment but not after or in controls. The underlying inflammation in atopic asthma is related to the composition of microbiota and is associated with severity of airway obstruction. Treatment with inhaled corticosteroids was associated with changes in the airway inflammatory response to microbiota.

Targeting a Newly Established Spontaneous Feline Fibrosarcoma Cell Line by Gene Transfer

PubMed Central

Nande, Rounak; De Carlo, Flavia; Carper, Miranda; Claudio, Charlene D.; Denvir, Jim; Valluri, Jagan; Duncan, Gary C.; Claudio, Pier Paolo

2012-01-01

Fibrosarcoma is a deadly disease in cats and is significantly more often located at classical vaccine injections sites. More rare forms of spontaneous non-vaccination site (NSV) fibrosarcomas have been described and have been found associated to genetic alterations. Purpose of this study was to compare the efficacy of adenoviral gene transfer in NVS fibrosarcoma. We isolated and characterized a NVS fibrosarcoma cell line (Cocca-6A) from a spontaneous fibrosarcoma that occurred in a domestic calico cat. The feline cells were karyotyped and their chromosome number was counted using a Giemsa staining. Adenoviral gene transfer was verified by western blot analysis. Flow cytometry assay and Annexin-V were used to study cell-cycle changes and cell death of transduced cells. Cocca-6A fibrosarcoma cells were morphologically and cytogenetically characterized. Giemsa block staining of metaphase spreads of the Cocca-6A cells showed deletion of one of the E1 chromosomes, where feline p53 maps. Semi-quantitative PCR demonstrated reduction of p53 genomic DNA in the Cocca-6A cells. Adenoviral gene transfer determined a remarkable effect on the viability and growth of the Cocca-6A cells following single transduction with adenoviruses carrying Mda-7/IL-24 or IFN-γ or various combination of RB/p105, Ras-DN, IFN-γ, and Mda-7 gene transfer. Therapy for feline fibrosarcomas is often insufficient for long lasting tumor eradication. More gene transfer studies should be conducted in order to understand if these viral vectors could be applicable regardless the origin (spontaneous vs. vaccine induced) of feline fibrosarcomas. PMID:22666387

Targeting a newly established spontaneous feline fibrosarcoma cell line by gene transfer.

PubMed

Nande, Rounak; Di Benedetto, Altomare; Aimola, Pierpaolo; De Carlo, Flavia; Carper, Miranda; Claudio, Charlene D; Denvir, Jim; Valluri, Jagan; Duncan, Gary C; Claudio, Pier Paolo

2012-01-01

Fibrosarcoma is a deadly disease in cats and is significantly more often located at classical vaccine injections sites. More rare forms of spontaneous non-vaccination site (NSV) fibrosarcomas have been described and have been found associated to genetic alterations. Purpose of this study was to compare the efficacy of adenoviral gene transfer in NVS fibrosarcoma. We isolated and characterized a NVS fibrosarcoma cell line (Cocca-6A) from a spontaneous fibrosarcoma that occurred in a domestic calico cat. The feline cells were karyotyped and their chromosome number was counted using a Giemsa staining. Adenoviral gene transfer was verified by western blot analysis. Flow cytometry assay and Annexin-V were used to study cell-cycle changes and cell death of transduced cells. Cocca-6A fibrosarcoma cells were morphologically and cytogenetically characterized. Giemsa block staining of metaphase spreads of the Cocca-6A cells showed deletion of one of the E1 chromosomes, where feline p53 maps. Semi-quantitative PCR demonstrated reduction of p53 genomic DNA in the Cocca-6A cells. Adenoviral gene transfer determined a remarkable effect on the viability and growth of the Cocca-6A cells following single transduction with adenoviruses carrying Mda-7/IL-24 or IFN-γ or various combination of RB/p105, Ras-DN, IFN-γ, and Mda-7 gene transfer. Therapy for feline fibrosarcomas is often insufficient for long lasting tumor eradication. More gene transfer studies should be conducted in order to understand if these viral vectors could be applicable regardless the origin (spontaneous vs. vaccine induced) of feline fibrosarcomas.

Degrees of reality: airway anatomy of high-fidelity human patient simulators and airway trainers.

PubMed

Schebesta, Karl; Hüpfl, Michael; Rössler, Bernhard; Ringl, Helmut; Müller, Michael P; Kimberger, Oliver

2012-06-01

Human patient simulators and airway training manikins are widely used to train airway management skills to medical professionals. Furthermore, these patient simulators are employed as standardized "patients" to evaluate airway devices. However, little is known about how realistic these patient simulators and airway-training manikins really are. This trial aimed to evaluate the upper airway anatomy of four high-fidelity patient simulators and two airway trainers in comparison with actual patients by means of radiographic measurements. The volume of the pharyngeal airspace was the primary outcome parameter. Computed tomography scans of 20 adult trauma patients without head or neck injuries were compared with computed tomography scans of four high-fidelity patient simulators and two airway trainers. By using 14 predefined distances, two cross-sectional areas and three volume parameters of the upper airway, the manikins' similarity to a human patient was assessed. The pharyngeal airspace of all manikins differed significantly from the patients' pharyngeal airspace. The HPS Human Patient Simulator (METI®, Sarasota, FL) was the most realistic high-fidelity patient simulator (6/19 [32%] of all parameters were within the 95% CI of human airway measurements). The airway anatomy of four high-fidelity patient simulators and two airway trainers does not reflect the upper airway anatomy of actual patients. This finding may impact airway training and confound comparative airway device studies.

Local small airway epithelial injury induces global smooth muscle contraction and airway constriction

PubMed Central

Zhou, Jian; Alvarez-Elizondo, Martha B.; Botvinick, Elliot

2012-01-01

Small airway epithelial cells form a continuous sheet lining the conducting airways, which serves many functions including a physical barrier to protect the underlying tissue. In asthma, injury to epithelial cells can occur during bronchoconstriction, which may exacerbate airway hyperreactivity. To investigate the role of epithelial cell rupture in airway constriction, laser ablation was used to precisely rupture individual airway epithelial cells of small airways (<300-μm diameter) in rat lung slices (∼250-μm thick). Laser ablation of single epithelial cells using a femtosecond laser reproducibly induced airway contraction to ∼70% of the original cross-sectional area within several seconds, and the contraction lasted for up to 40 s. The airway constriction could be mimicked by mechanical rupture of a single epithelial cell using a sharp glass micropipette but not with a blunt glass pipette. These results suggest that soluble mediators released from the wounded epithelial cell induce global airway contraction. To confirm this hypothesis, the lysate of primary human small airway epithelial cells stimulated a similar airway contraction. Laser ablation of single epithelial cells triggered a single instantaneous Ca2+ wave in the epithelium, and multiple Ca2+ waves in smooth muscle cells, which were delayed by several seconds. Removal of extracellular Ca2+ or decreasing intracellular Ca2+ both blocked laser-induced airway contraction. We conclude that local epithelial cell rupture induces rapid and global airway constriction through release of soluble mediators and subsequent Ca2+-dependent smooth muscle shortening. PMID:22114176

Innate Functions of Immunoglobulin M Lessen Liver Gene Transfer with Helper-Dependent Adenovirus

PubMed Central

Unzu, Carmen; Morales-Kastresana, Aizea; Sampedro, Ana; Serrano-Mendioroz, Irantzu; Azpilikueta, Arantza; Ochoa, María Carmen; Dubrot, Juan; Martínez-Ansó, Eduardo

2014-01-01

The immune system poses obstacles to viral vectors, even in the first administration to preimmunized hosts. We have observed that the livers of B cell-deficient mice were more effectively transduced by a helper-dependent adenovirus serotype-5 (HDA) vector than those of WT mice. This effect was T-cell independent as shown in athymic mice. Passive transfer of the serum from adenovirus-naïve WT to Rag1KO mice resulted in a reduction in gene transfer that was traced to IgM purified from serum of adenovirus-naïve mice. To ascribe the gene transfer inhibition activity to either adenoviral antigen-specific or antigen-unspecific functions of IgM, we used a monoclonal IgM antibody of unrelated specificity. Both the polyclonal and the irrelevant monoclonal IgM inhibited gene transfer by the HDA vector to either cultured hepatocellular carcinoma cells or to the liver of mice in vivo. Adsorption of polyclonal or monoclonal IgMs to viral capsids was revealed by ELISAs on adenovirus-coated plates. These observations indicate the existence of an inborn IgM mechanism deployed against a prevalent virus to reduce early post-infection viremia. In conclusion, innate IgM binding to adenovirus serotype-5 capsids restrains gene-transfer and offers a mechanism to be targeted for optimization of vector dosage in gene therapy with HDA vectors. PMID:24465560

Receptor for advanced glycation end products and its ligand high-mobility group box-1 mediate allergic airway sensitization and airway inflammation.

PubMed

Ullah, Md Ashik; Loh, Zhixuan; Gan, Wan Jun; Zhang, Vivian; Yang, Huan; Li, Jian Hua; Yamamoto, Yasuhiko; Schmidt, Ann Marie; Armour, Carol L; Hughes, J Margaret; Phipps, Simon; Sukkar, Maria B

2014-08-01

The receptor for advanced glycation end products (RAGE) shares common ligands and signaling pathways with TLR4, a key mediator of house dust mite (Dermatophagoides pteronyssinus) (HDM) sensitization. We hypothesized that RAGE and its ligand high-mobility group box-1 (HMGB1) cooperate with TLR4 to mediate HDM sensitization. To determine the requirement for HMGB1 and RAGE, and their relationship with TLR4, in airway sensitization. TLR4(-/-), RAGE(-/-), and RAGE-TLR4(-/-) mice were intranasally exposed to HDM or cockroach (Blatella germanica) extracts, and features of allergic inflammation were measured during the sensitization or challenge phase. Anti-HMGB1 antibody and the IL-1 receptor antagonist Anakinra were used to inhibit HMGB1 and the IL-1 receptor, respectively. The magnitude of allergic airway inflammation in response to either HDM or cockroach sensitization and/or challenge was significantly reduced in the absence of RAGE but not further diminished in the absence of both RAGE and TLR4. HDM sensitization induced the release of HMGB1 from the airway epithelium in a biphasic manner, which corresponded to the sequential activation of TLR4 then RAGE. Release of HMGB1 in response to cockroach sensitization also was RAGE dependent. Significantly, HMGB1 release occurred downstream of TLR4-induced IL-1α, and upstream of IL-25 and IL-33 production. Adoptive transfer of HDM-pulsed RAGE(+/+)dendritic cells to RAGE(-/-) mice recapitulated the allergic responses after HDM challenge. Immunoneutralization of HMGB1 attenuated HDM-induced allergic airway inflammation. The HMGB1-RAGE axis mediates allergic airway sensitization and airway inflammation. Activation of this axis in response to different allergens acts to amplify the allergic inflammatory response, which exposes it as an attractive target for therapeutic intervention. Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Status of therapeutic gene transfer to treat cardiovascular disease in dogs and cats.

PubMed

Sleeper, Meg; Bish, Lawrence T; Haskins, Mark; Ponder, Katherine P; Sweeney, H Lee

2011-06-01

Gene therapy is a procedure resulting in the transfer of a gene(s) into an individual's cells to treat a disease, which is designed to produce a protein or functional RNA (the gene product). Although most current gene therapy clinical trials focus on cancer and inherited diseases, multiple studies have evaluated the efficacy of gene therapy to abrogate various forms of heart disease. Indeed, human clinical trials are currently underway. One goal of gene transfer may be to express a functional gene when the endogenous gene is inactive. Alternatively, complex diseases such as end stage heart failure are characterized by a number of abnormalities at the cellular level, many of which can be targeted using gene delivery to alter myocardial protein levels. This review will discuss issues related to gene vector systems, gene delivery strategies and two cardiovascular diseases in dogs successfully treated with therapeutic gene delivery. Copyright © 2011 Elsevier B.V. All rights reserved.

Parasympathetic Control of Airway Submucosal Glands: Central Reflexes and the Airway Intrinsic Nervous System

PubMed Central

Wine, Jeffrey J.

2007-01-01

Airway submucosal glands produce the mucus that lines the upper airways to protect them against insults. This review summarizes evidence for two forms of gland secretion, and hypothesizes that each is mediated by different but partially overlapping neural pathways. Airway innate defense comprises low level gland secretion, mucociliary clearance and surveillance by airway-resident phagocytes to keep the airways sterile in spite of nearly continuous inhalation of low levels of pathogens. Gland secretion serving innate defense is hypothesized to be under the control of intrinsic (peripheral) airway neurons and local reflexes, and these may depend disproportionately on non-cholinergic mechanisms, with most secretion being produced by VIP and tachykinins. In the genetic disease cystic fibrosis, airway glands no longer secrete in response to VIP alone and fail to show the synergy between VIP, tachykinins and ACh that is observed in normal glands. The consequent crippling of the submucosal gland contribution to innate defense may be one reason that cystic fibrosis airways are infected by mucus-resident bacteria and fungi that are routinely cleared from normal airways. By contrast, the acute (emergency) airway defense reflex is centrally mediated by vagal pathways, is primarily cholinergic, and stimulates copious volumes of gland mucus in response to acute, intense challenges to the airways, such as those produced by very vigorous exercise or aspiration of foreign material. In cystic fibrosis, the acute airway defense reflex can still stimulate the glands to secrete large amounts of mucus, although its properties are altered. Importantly, treatments that recruit components of the acute reflex, such as inhalation of hypertonic saline, are beneficial in treating cystic fibrosis airway disease. The situation for recipients of lung transplants is the reverse; transplanted airways retain the airway intrinsic nervous system but lose centrally mediated reflexes. The consequences

Oxytetracycline Inhibits Mucus Secretion and Inflammation in Human Airway Epithelial Cells.

PubMed

Shah, Said Ahmad; Ishinaga, Hajime; Takeuchi, Kazuhiko

2017-01-01

Oxytetracycline is a broad-spectrum antibiotic, but its nonantibacterial effects in the human respiratory tract are unknown. In this study, the effects of oxytetracycline on mucus secretion and inflammation were examined by PCR and ELISA in the human airway epithelial cell line NCI-H292. Oxytetracycline (10 μg/mL) significantly inhibited TNF-α-induced MUC5AC gene expression and MUC5AC protein levels in NCI-H292 cells. It also downregulated IL-8 and IL-1β gene expression and IL-1β protein levels. Our findings demonstrated that oxytetracycline suppressed mucus production and inflammation in human respiratory epithelial cells, providing further evidence for the usefulness of oxytetracycline for human airway inflammatory diseases. © 2017 S. Karger AG, Basel.

Horizontal gene transfer from Agrobacterium to plants.

PubMed

Matveeva, Tatiana V; Lutova, Ludmila A

2014-01-01

Most genetic engineering of plants uses Agrobacterium mediated transformation to introduce novel gene content. In nature, insertion of T-DNA in the plant genome and its subsequent transfer via sexual reproduction has been shown in several species in the genera Nicotiana and Linaria. In these natural examples of horizontal gene transfer from Agrobacterium to plants, the T-DNA donor is assumed to be a mikimopine strain of A. rhizogenes. A sequence homologous to the T-DNA of the Ri plasmid of Agrobacterium rhizogenes was found in the genome of untransformed Nicotiana glauca about 30 years ago, and was named "cellular T-DNA" (cT-DNA). It represents an imperfect inverted repeat and contains homologs of several T-DNA oncogenes (NgrolB, NgrolC, NgORF13, NgORF14) and an opine synthesis gene (Ngmis). A similar cT-DNA has also been found in other species of the genus Nicotiana. These presumably ancient homologs of T-DNA genes are still expressed, indicating that they may play a role in the evolution of these plants. Recently T-DNA has been detected and characterized in Linaria vulgaris and L. dalmatica. In Linaria vulgaris the cT-DNA is present in two copies and organized as a tandem imperfect direct repeat, containing LvORF2, LvORF3, LvORF8, LvrolA, LvrolB, LvrolC, LvORF13, LvORF14, and the Lvmis genes. All L. vulgaris and L. dalmatica plants screened contained the same T-DNA oncogenes and the mis gene. Evidence suggests that there were several independent T-DNA integration events into the genomes of these plant genera. We speculate that ancient plants transformed by A. rhizogenes might have acquired a selective advantage in competition with the parental species. Thus, the events of T-DNA insertion in the plant genome might have affected their evolution, resulting in the creation of new plant species. In this review we focus on the structure and functions of cT-DNA in Linaria and Nicotiana and discuss their possible evolutionary role.

Postentry Processing of Recombinant Adeno-Associated Virus Type 1 and Transduction of the Ferret Lung Are Altered by a Factor in Airway Secretions

PubMed Central

Yan, Ziying; Sun, Xingshen; Evans, Idil A.; Tyler, Scott R.; Song, Yi; Liu, Xiaoming; Sui, Hongshu

2013-01-01

Abstract We recently created a cystic fibrosis ferret model that acquires neonatal lung infection. To develop lung gene therapies for this model, we evaluated recombinant adeno-associated virus (rAAV)-mediated gene transfer to the neonatal ferret lung. Unlike in vitro ferret airway epithelial (FAE) cells, in vivo infection of the ferret lung with rAAV1 required proteasome inhibitors to achieve efficient airway transduction. We hypothesized that differences in transduction between these two systems were because of an in vivo secreted factor that alter the transduction biology of rAAV1. Indeed, treatment of rAAV1 with ferret airway secretory fluid (ASF) strongly inhibited rAAV1, but not rAAV2, transduction of primary FAE and HeLa cells. Properties of the ASF inhibitory factor included a strong affinity for the AAV1 capsid, heat-stability, negative charge, and sensitivity to endoproteinase Glu-C. ASF-treated rAAV1 dramatically inhibited apical transduction of FAE ALI cultures (512-fold), while only reducing viral entry by 55-fold, suggesting that postentry processing of virus was influenced by the inhibitor factor. Proteasome inhibitors rescued transduction in the presence of ASF (∼1600-fold) without effecting virus internalization, while proteasome inhibitors only enhanced transduction 45-fold in the absence of ASF. These findings demonstrate that a factor in lung secretions can influence intracellular processing of rAAV1 in a proteasome-dependent fashion. PMID:23948055

Horizontal gene transfer of the algal nuclear gene psbO to the photosynthetic sea slug Elysia chlorotica

PubMed Central

Rumpho, Mary E.; Worful, Jared M.; Lee, Jungho; Kannan, Krishna; Tyler, Mary S.; Bhattacharya, Debashish; Moustafa, Ahmed; Manhart, James R.

2008-01-01

The sea slug Elysia chlorotica acquires plastids by ingestion of its algal food source Vaucheria litorea. Organelles are sequestered in the mollusc's digestive epithelium, where they photosynthesize for months in the absence of algal nucleocytoplasm. This is perplexing because plastid metabolism depends on the nuclear genome for >90% of the needed proteins. Two possible explanations for the persistence of photosynthesis in the sea slug are (i) the ability of V. litorea plastids to retain genetic autonomy and/or (ii) more likely, the mollusc provides the essential plastid proteins. Under the latter scenario, genes supporting photosynthesis have been acquired by the animal via horizontal gene transfer and the encoded proteins are retargeted to the plastid. We sequenced the plastid genome and confirmed that it lacks the full complement of genes required for photosynthesis. In support of the second scenario, we demonstrated that a nuclear gene of oxygenic photosynthesis, psbO, is expressed in the sea slug and has integrated into the germline. The source of psbO in the sea slug is V. litorea because this sequence is identical from the predator and prey genomes. Evidence that the transferred gene has integrated into sea slug nuclear DNA comes from the finding of a highly diverged psbO 3′ flanking sequence in the algal and mollusc nuclear homologues and gene absence from the mitochondrial genome of E. chlorotica. We demonstrate that foreign organelle retention generates metabolic novelty (“green animals”) and is explained by anastomosis of distinct branches of the tree of life driven by predation and horizontal gene transfer. PMID:19004808

Ventilation heterogeneity is a major determinant of airway hyperresponsiveness in asthma, independent of airway inflammation

PubMed Central

Downie, Sue R; Salome, Cheryl M; Verbanck, Sylvia; Thompson, Bruce; Berend, Norbert; King, Gregory G

2007-01-01

Background Airway hyperresponsiveness is the ability of airways to narrow excessively in response to inhaled stimuli and is a key feature of asthma. Airway inflammation and ventilation heterogeneity have been separately shown to be associated with airway hyperresponsiveness. A study was undertaken to establish whether ventilation heterogeneity is associated with airway hyperresponsiveness independently of airway inflammation in subjects with asthma and to determine the effect of inhaled corticosteroids on this relationship. Methods Airway inflammation was measured in 40 subjects with asthma by exhaled nitric oxide, ventilation heterogeneity by multiple breath nitrogen washout and airway hyperresponsiveness by methacholine challenge. In 18 of these subjects with uncontrolled symptoms, measurements were repeated after 3 months of treatment with inhaled beclomethasone dipropionate. Results At baseline, airway hyperresponsiveness was independently predicted by airway inflammation (partial r2 = 0.20, p<0.001) and ventilation heterogeneity (partial r2 = 0.39, p<0.001). Inhaled corticosteroid treatment decreased airway inflammation (p = 0.002), ventilation heterogeneity (p = 0.009) and airway hyperresponsiveness (p<0.001). After treatment, ventilation heterogeneity was the sole predictor of airway hyperresponsiveness (r2 = 0.64, p<0.001). Conclusions Baseline ventilation heterogeneity is a strong predictor of airway hyperresponsiveness, independent of airway inflammation in subjects with asthma. Its persistent relationship with airway hyperresponsiveness following anti‐inflammatory treatment suggests that it is an important independent determinant of airway hyperresponsiveness. Normalisation of ventilation heterogeneity is therefore a potential goal of treatment that may lead to improved long‐term outcomes. PMID:17311839

Protease-activated receptor 2 activation of myeloid dendritic cells regulates allergic airway inflammation

PubMed Central

2011-01-01

Background A common characteristic of allergens is that they contain proteases that can activate protease-activated receptor (PAR-2); however the mechanism by which PAR-2 regulates allergic airway inflammation is unclear. Methods Mice (wild type and PAR-2-deficient) were sensitized using German cockroach (GC) feces (frass), the isolated protease from GC frass, or through adoptive transfer of GC frass-treated bone marrow-derived dendritic cells (BMDC) and measurements of airway inflammation (cellular infiltration, cytokine expression, and mucin production), serum IgE levels and airway hyperresponsiveness (AHR) were assessed. BMDC were cultured, treated with GC frass and assessed for cytokine production. PAR-2 expression on pulmonary mDCs was determined by flow cytometry. Results Exposure to GC frass induced AHR and airway inflammation in wild type mice; however PAR-2-deficient mice had significantly attenuated responses. To directly investigate the role of the protease, we isolated the protease from GC frass and administered the endotoxin-free protease into the airways of mice in the presence of OVA. GC frass proteases were sufficient to promote the development of AHR, serum IgE, and Th2 cytokine production. PAR-2 expression on mDC was upregulated following GC frass exposure, but the presence of a functional PAR-2 did not alter antigen uptake. To determine if PAR-2 activation led to differential cytokine production, we cultured BMDC in the presence of GM-CSF and treated these cells ex vivo with GC frass. PAR-2-deficient BMDC released significantly less IL-6, IL-23 and TNFα compared to BMDC from wild type mice, suggesting PAR-2 activation was important in Th2/Th17 skewing cytokine production. To determine the role for PAR-2 on mDCs on the initiation of allergic airway inflammation, BMDCs from wild type and PAR-2-deficient mice were treated in the presence or absence of GC frass and then adoptively transferred into the airway of wild type mice. Importantly, GC frass

Functional and Evolutionary Characterization of a Gene Transfer Agent’s Multilocus “Genome”

PubMed Central

Hynes, Alexander P.; Shakya, Migun; Mercer, Ryan G.; Grüll, Marc P.; Bown, Luke; Davidson, Fraser; Steffen, Ekaterina; Matchem, Heidi; Peach, Mandy E.; Berger, Tim; Grebe, Katherine; Zhaxybayeva, Olga; Lang, Andrew S.

2016-01-01

Gene transfer agents (GTAs) are phage-like particles that can package and transfer a random piece of the producing cell’s genome, but are unable to transfer all the genes required for their own production. As such, GTAs represent an evolutionary conundrum: are they selfish genetic elements propagating through an unknown mechanism, defective viruses, or viral structures “repurposed” by cells for gene exchange, as their name implies? In Rhodobacter capsulatus, production of the R. capsulatus GTA (RcGTA) particles is associated with a cluster of genes resembling a small prophage. Utilizing transcriptomic, genetic and biochemical approaches, we report that the RcGTA “genome” consists of at least 24 genes distributed across five distinct loci. We demonstrate that, of these additional loci, two are involved in cell recognition and binding and one in the production and maturation of RcGTA particles. The five RcGTA “genome” loci are widespread within Rhodobacterales, but not all loci have the same evolutionary histories. Specifically, two of the loci have been subject to frequent, probably virus-mediated, gene transfer events. We argue that it is unlikely that RcGTA is a selfish genetic element. Instead, our findings are compatible with the scenario that RcGTA is a virus-derived element maintained by the producing organism due to a selective advantage of within-population gene exchange. The modularity of the RcGTA “genome” is presumably a result of selection on the host organism to retain GTA functionality. PMID:27343288

Airway-Specific Inducible Transgene Expression Using Aerosolized Doxycycline

PubMed Central

Tata, Purushothama Rao; Pardo-Saganta, Ana; Prabhu, Mythili; Vinarsky, Vladimir; Law, Brandon M.; Fontaine, Benjamin A.; Tager, Andrew M.

2013-01-01

Tissue-specific transgene expression using tetracycline (tet)-regulated promoter/operator elements has been used to revolutionize our understanding of cellular and molecular processes. However, because most tet-regulated mouse strains use promoters of genes expressed in multiple tissues, to achieve exclusive expression in an organ of interest is often impossible. Indeed, in the extreme case, unwanted transgene expression in other organ systems causes lethality and precludes the study of the transgene in the actual organ of interest. Here, we describe a novel approach to activating tet-inducible transgene expression solely in the airway by administering aerosolized doxycycline. By optimizing the dose and duration of aerosolized doxycycline exposure in mice possessing a ubiquitously expressed Rosa26 promoter–driven reverse tet-controlled transcriptional activator (rtTA) element, we induce transgene expression exclusively in the airways. We detect no changes in the cellular composition or proliferative behavior of airway cells. We used this newly developed method to achieve airway basal stem cell–specific transgene expression using a cytokeratin 5 (also known as keratin 5)–driven rtTA driver line to induce Notch pathway activation. We observed a more robust mucous metaplasia phenotype than in mice receiving doxycycline systemically. In addition, unwanted phenotypes outside of the lung that were evident when doxycycline was received systemically were now absent. Thus, our approach allows for rapid and efficient airway-specific transgene expression. After the careful strain by strain titration of the dose and timing of doxycycline inhalation, a suite of preexisting transgenic mice can now be used to study airway biology specifically in cases where transient transgene expression is sufficient to induce a phenotype. PMID:23848320

Collective evolution of cyanobacteria and cyanophages mediated by horizontal gene transfer

NASA Astrophysics Data System (ADS)

Shih, Hong-Yan; Rogers, Tim; Goldenfeld, Nigel

We describe a model for how antagonistic predator-prey coevolution can lead to mutualistic adaptation to an environment, as a result of horizontal gene transfer. Our model is a simple description of ecosystems such as marine cyanobacteria and their predator cyanophages, which carry photosynthesis genes. These genes evolve more rapidly in the virosphere than the bacterial pan-genome, and thus the bacterial population could potentially benefit from phage predation. By modeling both the barrier to predation and horizontal gene transfer, we study this balance between individual sacrifice and collective benefits. The outcome is an emergent mutualistic coevolution of improved photosynthesis capability, benefiting both bacteria and phage. This form of multi-level selection can contribute to niche stratification in the cyanobacteria-phage ecosystem. This work is supported in part by a cooperative agreement with NASA, Grant NNA13AA91A/A0018.

CRISPR-Cas-Mediated Phage Resistance Enhances Horizontal Gene Transfer by Transduction.

PubMed

Watson, Bridget N J; Staals, Raymond H J; Fineran, Peter C

2018-02-13

A powerful contributor to prokaryotic evolution is horizontal gene transfer (HGT) through transformation, conjugation, and transduction, which can be advantageous, neutral, or detrimental to fitness. Bacteria and archaea control HGT and phage infection through CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) adaptive immunity. Although the benefits of resisting phage infection are evident, this can come at a cost of inhibiting the acquisition of other beneficial genes through HGT. Despite the ability of CRISPR-Cas to limit HGT through conjugation and transformation, its role in transduction is largely overlooked. Transduction is the phage-mediated transfer of bacterial DNA between cells and arguably has the greatest impact on HGT. We demonstrate that in Pectobacterium atrosepticum , CRISPR-Cas can inhibit the transduction of plasmids and chromosomal loci. In addition, we detected phage-mediated transfer of a large plant pathogenicity genomic island and show that CRISPR-Cas can inhibit its transduction. Despite these inhibitory effects of CRISPR-Cas on transduction, its more common role in phage resistance promotes rather than diminishes HGT via transduction by protecting bacteria from phage infection. This protective effect can also increase transduction of phage-sensitive members of mixed populations. CRISPR-Cas systems themselves display evidence of HGT, but little is known about their lateral dissemination between bacteria and whether transduction can contribute. We show that, through transduction, bacteria can acquire an entire chromosomal CRISPR-Cas system, including cas genes and phage-targeting spacers. We propose that the positive effect of CRISPR-Cas phage immunity on enhancing transduction surpasses the rarer cases where gene flow by transduction is restricted. IMPORTANCE The generation of genetic diversity through acquisition of DNA is a powerful contributor to microbial evolution and occurs through

Horizontal Gene Transfer of Pectinases from Bacteria Preceded the Diversification of Stick and Leaf Insects

PubMed Central

Shelomi, Matan; Danchin, Etienne G. J.; Heckel, David; Wipfler, Benjamin; Bradler, Sven; Zhou, Xin; Pauchet, Yannick

2016-01-01

Genes acquired by horizontal transfer are increasingly being found in animal genomes. Understanding their origin and evolution requires knowledge about the phylogenetic relationships from both source and recipient organisms. We used RNASeq data and respective assembled transcript libraries to trace the evolutionary history of polygalacturonase (pectinase) genes in stick insects (Phasmatodea). By mapping the distribution of pectinase genes on a Polyneoptera phylogeny, we identified the transfer of pectinase genes from known phasmatodean gut microbes into the genome of an early euphasmatodean ancestor that took place between 60 and 100 million years ago. This transfer preceded the rapid diversification of the suborder, enabling symbiont-free pectinase production that would increase the insects’ digestive efficiency and reduce dependence on microbes. Bacteria-to-insect gene transfer was thought to be uncommon, however the increasing availability of large-scale genomic data may change this prevailing notion. PMID:27210832

Preliminary Study on Gene Expression of Chitinase-Like Cytokines in Human Airway Epithelial Cell Under Chitin and Chitosan Microparticles Treatment.

PubMed

Alimohammadi, Masumeh; Yeganeh, Farshid; Haji Molla Hoseini, Mostafa

2016-06-01

Small-sized chitin and chitosan microparticles (MPs) reduce allergic inflammation. We examined the capacity of these glycans to stimulate A549 human airway epithelial cells to determine the feasibility of using of these glycans as allergic therapeutic modality. A549 cells were treated with MPs and then expressions levels of chitinase domain-containing 1 (CHID1) and chitinase 3-like 1 (CHI3L1) genes were determined by quantitative real-time PCR. IL-6 production was measured by ELISA. Chitin MPs resulted in upregulation of CHI3L1 expression by 35.7-fold while mRNA expression did not change with chitosan MPs. Compared to the untreated group, production of IL-6 was significantly decreased in the chitosan MPs-treated group, but chitin MPs treatment cause elevation of IL-6 level. This study demonstrates that chitin potently induces CHI3L1 expression, but chitosan is relatively inert. This effect and inhibition of pro-inflammatory cytokine (IL-6) suggest that chitosan MPs may possess more potential for therapeutic uses in human airway allergic inflammation.

Endosymbiotic gene transfer from prokaryotic pangenomes: Inherited chimerism in eukaryotes.

PubMed

Ku, Chuan; Nelson-Sathi, Shijulal; Roettger, Mayo; Garg, Sriram; Hazkani-Covo, Einat; Martin, William F

2015-08-18

Endosymbiotic theory in eukaryotic-cell evolution rests upon a foundation of three cornerstone partners--the plastid (a cyanobacterium), the mitochondrion (a proteobacterium), and its host (an archaeon)--and carries a corollary that, over time, the majority of genes once present in the organelle genomes were relinquished to the chromosomes of the host (endosymbiotic gene transfer). However, notwithstanding eukaryote-specific gene inventions, single-gene phylogenies have never traced eukaryotic genes to three single prokaryotic sources, an issue that hinges crucially upon factors influencing phylogenetic inference. In the age of genomes, single-gene trees, once used to test the predictions of endosymbiotic theory, now spawn new theories that stand to eventually replace endosymbiotic theory with descriptive, gene tree-based variants featuring supernumerary symbionts: prokaryotic partners distinct from the cornerstone trio and whose existence is inferred solely from single-gene trees. We reason that the endosymbiotic ancestors of mitochondria and chloroplasts brought into the eukaryotic--and plant and algal--lineage a genome-sized sample of genes from the proteobacterial and cyanobacterial pangenomes of their respective day and that, even if molecular phylogeny were artifact-free, sampling prokaryotic pangenomes through endosymbiotic gene transfer would lead to inherited chimerism. Recombination in prokaryotes (transduction, conjugation, transformation) differs from recombination in eukaryotes (sex). Prokaryotic recombination leads to pangenomes, and eukaryotic recombination leads to vertical inheritance. Viewed from the perspective of endosymbiotic theory, the critical transition at the eukaryote origin that allowed escape from Muller's ratchet--the origin of eukaryotic recombination, or sex--might have required surprisingly little evolutionary innovation.

Effects of maternal folic acid supplementation on airway remodeling and allergic airway disease development.

PubMed

İscan, Burcin; Tuzun, Funda; Eroglu Filibeli, Berna; Cilekar Micili, Serap; Ergur, Bekir Ugur; Duman, Nuray; Ozkan, Hasan; Kumral, Abdullah

2018-03-27

Maternal folic acid supplementation has been recommended prior to and during the first trimester of pregnancy to reduce the risk of infant neural tube defects. However, an uncertain relationship between folic acid supplementation during pregnancy and development of childhood asthma exists. Recent data show a methyl donor-rich diet could increase the risk of developing allergic airway disease through DNA methylation and aberrant gene transcription. This study evaluated the effect of folic acid supplementation during pregnancy on airway remodeling and allergic airway disease vulnerability in a mouse asthma model. BALB/c mice were divided into four groups according to gestational folic acid supplementation and postnatal ovalbumin (OVA) exposure: Group 1 (whole pregnancy folic acid supplementation + OVA-exposed group), Group 2 (first gestational week folic acid supplementation + OVA-exposed group), Group 3 (no folic acid supplementation + OVA-exposed group), and Group 4 (control group). Offspring were sacrificed on day 45 for immunohistological and ultrastructural tests. In OVA challenged groups, folic acid supplementation led to a thicker epithelial and subepithelial smooth muscle layer than in the unsupplemented group. Moreover, folic acid supplementation during whole pregnancy (Group 1) was associated with a thicker epithelial and subepithelial smooth muscle layer than folic acid supplementation during the first week of pregnancy (Group 2), suggesting a duration-response relationship. Electron microscopic imaging revealed that structural changes including the loss of epithelial integrity, thickening of basement membrane, and subepithelial fibrosis were more prominent in the folic acid supplementation groups. This study suggested that maternal folic acid supplementation during pregnancy affects airway remodeling and increases the allergic responses induced by ovalbumin challenge in offspring. In addition, the effect size increased as the duration and

Parasympathetic control of airway submucosal glands: central reflexes and the airway intrinsic nervous system.

PubMed

Wine, Jeffrey J

2007-04-30

Airway submucosal glands produce the mucus that lines the upper airways to protect them against insults. This review summarizes evidence for two forms of gland secretion, and hypothesizes that each is mediated by different but partially overlapping neural pathways. Airway innate defense comprises low level gland secretion, mucociliary clearance and surveillance by airway-resident phagocytes to keep the airways sterile in spite of nearly continuous inhalation of low levels of pathogens. Gland secretion serving innate defense is hypothesized to be under the control of intrinsic (peripheral) airway neurons and local reflexes, and these may depend disproportionately on non-cholinergic mechanisms, with most secretion being produced by VIP and tachykinins. In the genetic disease cystic fibrosis, airway glands no longer secrete in response to VIP alone and fail to show the synergy between VIP, tachykinins and ACh that is observed in normal glands. The consequent crippling of the submucosal gland contribution to innate defense may be one reason that cystic fibrosis airways are infected by mucus-resident bacteria and fungi that are routinely cleared from normal airways. By contrast, the acute (emergency) airway defense reflex is centrally mediated by vagal pathways, is primarily cholinergic, and stimulates copious volumes of gland mucus in response to acute, intense challenges to the airways, such as those produced by very vigorous exercise or aspiration of foreign material. In cystic fibrosis, the acute airway defense reflex can still stimulate the glands to secrete large amounts of mucus, although its properties are altered. Importantly, treatments that recruit components of the acute reflex, such as inhalation of hypertonic saline, are beneficial in treating cystic fibrosis airway disease. The situation for recipients of lung transplants is the reverse; transplanted airways retain the airway intrinsic nervous system but lose centrally mediated reflexes. The consequences

Evidence and Role for Bacterial Mucin Degradation in Cystic Fibrosis Airway Disease

PubMed Central

Flynn, Jeffrey M.; Niccum, David; Dunitz, Jordan M.

2016-01-01

Chronic lung infections in cystic fibrosis (CF) patients are composed of complex microbial communities that incite persistent inflammation and airway damage. Despite the high density of bacteria that colonize the lower airways, nutrient sources that sustain bacterial growth in vivo, and how those nutrients are derived, are not well characterized. In this study, we examined the possibility that mucins serve as an important carbon reservoir for the CF lung microbiota. While Pseudomonas aeruginosa was unable to efficiently utilize mucins in isolation, we found that anaerobic, mucin-fermenting bacteria could stimulate the robust growth of CF pathogens when provided intact mucins as a sole carbon source. 16S rRNA sequencing and enrichment culturing of sputum also identified that mucin-degrading anaerobes are ubiquitous in the airways of CF patients. The collective fermentative metabolism of these mucin-degrading communities in vitro generated amino acids and short chain fatty acids (propionate and acetate) during growth on mucin, and the same metabolites were also found in abundance within expectorated sputum. The significance of these findings was supported by in vivo P. aeruginosa gene expression, which revealed a heightened expression of genes required for the catabolism of propionate. Given that propionate is exclusively derived from bacterial fermentation, these data provide evidence for an important role of mucin fermenting bacteria in the carbon flux of the lower airways. More specifically, microorganisms typically defined as commensals may contribute to airway disease by degrading mucins, in turn providing nutrients for pathogens otherwise unable to efficiently obtain carbon in the lung. PMID:27548479

Involvement of β-carbonic anhydrase (β-CA) genes in bacterial genomic islands and horizontal transfer to protists.

PubMed

Zolfaghari Emameh, Reza; Barker, Harlan R; Hytönen, Vesa P; Parkkila, Seppo

2018-05-25

Genomic islands (GIs) are a type of mobile genetic element (MGE) that are present in bacterial chromosomes. They consist of a cluster of genes which produce proteins that contribute to a variety of functions, including, but not limited to, regulation of cell metabolism, anti-microbial resistance, pathogenicity, virulence, and resistance to heavy metals. The genes carried in MGEs can be used as a trait reservoir in times of adversity. Transfer of genes using MGEs, occurring outside of reproduction, is called horizontal gene transfer (HGT). Previous literature has shown that numerous HGT events have occurred through endosymbiosis between prokaryotes and eukaryotes.Beta carbonic anhydrase (β-CA) enzymes play a critical role in the biochemical pathways of many prokaryotes and eukaryotes. We have previously suggested horizontal transfer of β-CA genes from plasmids of some prokaryotic endosymbionts to their protozoan hosts. In this study, we set out to identify β-CA genes that might have transferred between prokaryotic and protist species through HGT in GIs. Therefore, we investigated prokaryotic chromosomes containing β-CA-encoding GIs and utilized multiple bioinformatics tools to reveal the distinct movements of β-CA genes among a wide variety of organisms. Our results identify the presence of β-CA genes in GIs of several medically and industrially relevant bacterial species, and phylogenetic analyses reveal multiple cases of likely horizontal transfer of β-CA genes from GIs of ancestral prokaryotes to protists. IMPORTANCE The evolutionary process is mediated by mobile genetic elements (MGEs), such as genomic islands (GIs). A gene or set of genes in the GIs are exchanged between and within various species through horizontal gene transfer (HGT). Based on the crucial role that GIs can play in bacterial survival and proliferation, they were introduced as the environmental- and pathogen-associated factors. Carbonic anhydrases (CAs) are involved in many critical

Phylogenetic Analysis of the Incidence of lux Gene Horizontal Transfer in Vibrionaceae▿ †

PubMed Central

Urbanczyk, Henryk; Ast, Jennifer C.; Kaeding, Allison J.; Oliver, James D.; Dunlap, Paul V.

2008-01-01

Horizontal gene transfer (HGT) is thought to occur frequently in bacteria in nature and to play an important role in bacterial evolution, contributing to the formation of new species. To gain insight into the frequency of HGT in Vibrionaceae and its possible impact on speciation, we assessed the incidence of interspecies transfer of the lux genes (luxCDABEG), which encode proteins involved in luminescence, a distinctive phenotype. Three hundred three luminous strains, most of which were recently isolated from nature and which represent 11 Aliivibrio, Photobacterium, and Vibrio species, were screened for incongruence of phylogenies based on a representative housekeeping gene (gyrB or pyrH) and a representative lux gene (luxA). Strains exhibiting incongruence were then subjected to detailed phylogenetic analysis of horizontal transfer by using multiple housekeeping genes (gyrB, recA, and pyrH) and multiple lux genes (luxCDABEG). In nearly all cases, housekeeping gene and lux gene phylogenies were congruent, and there was no instance in which the lux genes of one luminous species had replaced the lux genes of another luminous species. Therefore, the lux genes are predominantly vertically inherited in Vibrionaceae. The few exceptions to this pattern of congruence were as follows: (i) the lux genes of the only known luminous strain of Vibrio vulnificus, VVL1 (ATCC 43382), were evolutionarily closely related to the lux genes of Vibrio harveyi; (ii) the lux genes of two luminous strains of Vibrio chagasii, 21N-12 and SB-52, were closely related to those of V. harveyi and Vibrio splendidus, respectively; (iii) the lux genes of a luminous strain of Photobacterium damselae, BT-6, were closely related to the lux genes of the lux-rib2 operon of Photobacterium leiognathi; and (iv) a strain of the luminous bacterium Photobacterium mandapamensis was found to be merodiploid for the lux genes, and the second set of lux genes was closely related to the lux genes of the lux-rib2

Stable and Efficient Gene Transfer into the Retina Using an HIV-Based Lentiviral Vector

NASA Astrophysics Data System (ADS)

Miyoshi, Hiroyuki; Takahashi, Masayo; Gage, Fred H.; Verma, Inder M.

1997-09-01

The development of methods for efficient gene transfer to terminally differentiated retinal cells is important to study the function of the retina as well as for gene therapy of retinal diseases. We have developed a lentiviral vector system based on the HIV that can transduce terminally differentiated neurons of the brain in vivo. In this study, we have evaluated the ability of HIV vectors to transfer genes into retinal cells. An HIV vector containing a gene encoding the green fluorescent protein (GFP) was injected into the subretinal space of rat eyes. The GFP gene under the control of the cytomegalovirus promoter was efficiently expressed in both photoreceptor cells and retinal pigment epithelium. However, the use of the rhodopsin promoter resulted in expression predominantly in photoreceptor cells. Most successfully transduced eyes showed that photoreceptor cells in >80% of the area of whole retina expressed the GFP. The GFP expression persisted for at least 12 weeks with no apparent decrease. The efficient gene transfer into photoreceptor cells by HIV vectors will be useful for gene therapy of retinal diseases such as retinitis pigmentosa.

Beyond Agrobacterium-Mediated Transformation: Horizontal Gene Transfer from Bacteria to Eukaryotes.

PubMed

Lacroix, Benoît; Citovsky, Vitaly

2018-03-03

Besides the massive gene transfer from organelles to the nuclear genomes, which occurred during the early evolution of eukaryote lineages, the importance of horizontal gene transfer (HGT) in eukaryotes remains controversial. Yet, increasing amounts of genomic data reveal many cases of bacterium-to-eukaryote HGT that likely represent a significant force in adaptive evolution of eukaryotic species. However, DNA transfer involved in genetic transformation of plants by Agrobacterium species has traditionally been considered as the unique example of natural DNA transfer and integration into eukaryotic genomes. Recent discoveries indicate that the repertoire of donor bacterial species and of recipient eukaryotic hosts potentially are much wider than previously thought, including donor bacterial species, such as plant symbiotic nitrogen-fixing bacteria (e.g., Rhizobium etli) and animal bacterial pathogens (e.g., Bartonella henselae, Helicobacter pylori), and recipient species from virtually all eukaryotic clades. Here, we review the molecular pathways and potential mechanisms of these trans-kingdom HGT events and discuss their utilization in biotechnology and research.

Immune Recognition of Gene Transfer Vectors: Focus on Adenovirus as a Paradigm

PubMed Central

Aldhamen, Yasser Ali; Seregin, Sergey S.; Amalfitano, Andrea

2011-01-01

Recombinant Adenovirus (Ad) based vectors have been utilized extensively as a gene transfer platform in multiple pre-clinical and clinical applications. These applications are numerous, and inclusive of both gene therapy and vaccine based approaches to human or animal diseases. The widespread utilization of these vectors in both animal models, as well as numerous human clinical trials (Ad-based vectors surpass all other gene transfer vectors relative to numbers of patients treated, as well as number of clinical trials overall), has shed light on how this virus vector interacts with both the innate and adaptive immune systems. The ability to generate and administer large amounts of this vector likely contributes not only to their ability to allow for highly efficient gene transfer, but also their elicitation of host immune responses to the vector and/or the transgene the vector expresses in vivo. These facts, coupled with utilization of several models that allow for full detection of these responses has predicted several observations made in human trials, an important point as lack of similar capabilities by other vector systems may prevent detection of such responses until only after human trials are initiated. Finally, induction of innate or adaptive immune responses by Ad vectors may be detrimental in one setting (i.e., gene therapy) and be entirely beneficial in another (i.e., prophylactic or therapeutic vaccine based applications). Herein, we review the current understanding of innate and adaptive immune responses to Ad vectors, as well some recent advances that attempt to capitalize on this understanding so as to further broaden the safe and efficient use of Ad-based gene transfer therapies in general. PMID:22566830

Host-microbe interactions in distal airways: relevance to chronic airway diseases.

PubMed

Martin, Clémence; Burgel, Pierre-Régis; Lepage, Patricia; Andréjak, Claire; de Blic, Jacques; Bourdin, Arnaud; Brouard, Jacques; Chanez, Pascal; Dalphin, Jean-Charles; Deslée, Gaetan; Deschildre, Antoine; Gosset, Philippe; Touqui, Lhousseine; Dusser, Daniel

2015-03-01

This article is the summary of a workshop, which took place in November 2013, on the roles of microorganisms in chronic respiratory diseases. Until recently, it was assumed that lower airways were sterile in healthy individuals. However, it has long been acknowledged that microorganisms could be identified in distal airway secretions from patients with various respiratory diseases, including cystic fibrosis (CF) and non-CF bronchiectasis, chronic obstructive pulmonary disease, asthma and other chronic airway diseases (e.g. post-transplantation bronchiolitis obliterans). These microorganisms were sometimes considered as infectious agents that triggered host immune responses and contributed to disease onset and/or progression; alternatively, microorganisms were often considered as colonisers, which were considered unlikely to play roles in disease pathophysiology. These concepts were developed at a time when the identification of microorganisms relied on culture-based methods. Importantly, the majority of microorganisms cannot be cultured using conventional methods, and the use of novel culture-independent methods that rely on the identification of microorganism genomes has revealed that healthy distal airways display a complex flora called the airway microbiota. The present article reviews some aspects of current literature on host-microbe (mostly bacteria and viruses) interactions in healthy and diseased airways, with a special focus on distal airways. Copyright ©ERS 2015.

The New Perilaryngeal Airway (CobraPLA™)1 Is as Efficient as the Laryngeal Mask Airway (LMA™)2, But Provides Better Airway Sealing Pressures

PubMed Central

Akça, Ozan; Wadhwa, Anupama; Sengupta, Papiya; Durrani, Jaleel; Hanni, Keith; Wenke, Mary; Yücel, Yüksel; Lenhardt, Rainer; Doufas, Anthony G.; Sessler, Daniel I.

2006-01-01

The Laryngeal Mask Airway (LMA) is a frequently-used efficient airway device, yet it sometimes seals poorly, thus reducing the efficacy of positive-pressure ventilation. The Perilaryngeal Airway (CobraPLA) is a novel airway device with a larger pharyngeal cuff (when inflated). We tested the hypothesis that the CobraPLA was superior to LMA with regard to insertion time and airway sealing pressure and comparable to LMA in airway adequacy and recovery characteristics. After midazolam and fentanyl, 81 ASA I-II outpatients having elective surgery were randomized to receive an LMA or CobraPLA. Anesthesia was induced with propofol (2.5 mg/kg, IV), and the airway inserted. We measured 1) insertion time; 2) adequacy of the airway (no leak at 15-cm-H2O peak pressure or tidal volume of 5 ml/kg); 3) airway sealing pressure; 4) number of repositioning attempts; and 5) sealing quality (no leak at tidal volume of 8 ml/kg). At the end of surgery, gastric insufflation, postoperative sore throat, dysphonia, and dysphagia were evaluated. Data were compared with unpaired t-tests, chi-square tests, or Fisher’s Exact tests; P<0.05 was significant. Patient characteristics, insertion times, airway adequacy, number of repositioning attempts, and recovery were similar in each group. Airway sealing pressure was significantly greater with CobraPLA (23±6 cm H2O) than LMA (18±5 cm H2O, P<0.001). The CobraPLA has insertion characteristics similar to LMA, but better airway sealing capabilities. PMID:15281543

Horizontal Transfer of Plasmid-Mediated Cephalosporin Resistance Genes in the Intestine of Houseflies (Musca domestica).

PubMed

Fukuda, Akira; Usui, Masaru; Okubo, Torahiko; Tamura, Yutaka

2016-06-01

Houseflies are a mechanical vector for various types of bacteria, including antimicrobial-resistant bacteria (ARB). If the intestine of houseflies is a suitable site for the transfer of antimicrobial resistance genes (ARGs), houseflies could also serve as a biological vector for ARB. To clarify whether cephalosporin resistance genes are transferred efficiently in the housefly intestine, we compared with conjugation experiments in vivo (in the intestine) and in vitro by using Escherichia coli with eight combinations of four donor and two recipient strains harboring plasmid-mediated cephalosporin resistance genes and chromosomal-encoded rifampicin resistance genes, respectively. In the in vivo conjugation experiment, houseflies ingested donor strains for 6 hr and then recipient strains for 3 hr, and 24 hr later, the houseflies were surface sterilized and analyzed. In vitro conjugation experiments were conducted using the broth-mating method. In 3/8 combinations, the in vitro transfer frequency (Transconjugants/Donor) was ≥1.3 × 10(-4); the in vivo transfer rates of cephalosporin resistance genes ranged from 2.0 × 10(-4) to 5.7 × 10(-5). Moreover, cephalosporin resistance genes were transferred to other species of enteric bacteria of houseflies such as Achromobacter sp. and Pseudomonas fluorescens. These results suggest that houseflies are not only a mechanical vector for ARB but also a biological vector for the occurrence of new ARB through the horizontal transfer of ARGs in their intestine.

Airway surface mycosis in chronic TH2-associated airway disease.

PubMed

Porter, Paul C; Lim, Dae Jun; Maskatia, Zahida Khan; Mak, Garbo; Tsai, Chu-Lin; Citardi, Martin J; Fakhri, Samer; Shaw, Joanne L; Fothergil, Annette; Kheradmand, Farrah; Corry, David B; Luong, Amber

2014-08-01

Environmental fungi have been linked to TH2 cell-related airway inflammation and the TH2-associated chronic airway diseases asthma, chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP), and allergic fungal rhinosinusitis (AFRS), but whether these organisms participate directly or indirectly in disease pathology remains unknown. To determine the frequency of fungus isolation and fungus-specific immunity in patients with TH2-associated and non-TH2-associated airway disease. Sinus lavage fluid and blood were collected from sinus surgery patients (n = 118) including patients with CRSwNP, patients with CRS without nasal polyps, patients with AFRS, and non-CRS/nonasthmatic control patients. Asthma status was determined from medical history. Sinus lavage fluids were cultured and directly examined for evidence of viable fungi. PBMCs were restimulated with fungal antigens in an enzyme-linked immunocell spot assay to determine total memory fungus-specific IL-4-secreting cells. These data were compared with fungus-specific IgE levels measured from plasma by ELISA. Filamentous fungi were significantly more commonly cultured in patients with TH2-associated airway disease (asthma, CRSwNP, or AFRS: n = 68) than in control patients with non-TH2-associated disease (n = 31): 74% vs 16%, respectively (P < .001). Both fungus-specific IL-4 enzyme-linked immunocell spot (n = 48) and specific IgE (n = 70) data correlated with TH2-associated diseases (sensitivity 73% and specificity 100% vs 50% and 77%, respectively). The frequent isolation of fungi growing directly within the airways accompanied by specific immunity to these organisms only in patients with TH2-associated chronic airway diseases suggests that fungi participate directly in the pathogenesis of these conditions. Efforts to eradicate airway fungi from the airways should be considered in selected patients. Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Toward Gene Therapy for Cystic Fibrosis Using a Lentivirus Pseudotyped With Sendai Virus Envelopes

PubMed Central

Mitomo, Katsuyuki; Griesenbach, Uta; Inoue, Makoto; Somerton, Lucinda; Meng, Cuixiang; Akiba, Eiji; Tabata, Toshiaki; Ueda, Yasuji; Frankel, Gad M; Farley, Raymond; Singh, Charanjit; Chan, Mario; Munkonge, Felix; Brum, Andrea; Xenariou, Stefania; Escudero-Garcia, Sara; Hasegawa, Mamoru; Alton, Eric WFW

2010-01-01

Gene therapy for cystic fibrosis (CF) is making encouraging progress into clinical trials. However, further improvements in transduction efficiency are desired. To develop a novel gene transfer vector that is improved and truly effective for CF gene therapy, a simian immunodeficiency virus (SIV) was pseudotyped with envelope proteins from Sendai virus (SeV), which is known to efficiently transduce unconditioned airway epithelial cells from the apical side. This novel vector was evaluated in mice in vivo and in vitro directed toward CF gene therapy. Here, we show that (i) we can produce relevant titers of an SIV vector pseudotyped with SeV envelope proteins for in vivo use, (ii) this vector can transduce the respiratory epithelium of the murine nose in vivo at levels that may be relevant for clinical benefit in CF, (iii) this can be achieved in a single formulation, and without the need for preconditioning, (iv) expression can last for 15 months, (v) readministration is feasible, (vi) the vector can transduce human air–liquid interface (ALI) cultures, and (vii) functional CF transmembrane conductance regulator (CFTR) chloride channels can be generated in vitro. Our data suggest that this lentiviral vector may provide a step change in airway transduction efficiency relevant to a clinical programme of gene therapy for CF. PMID:20332767

Cystic Fibrosis Transmembrane Conductance Regulator in Sarcoplasmic Reticulum of Airway Smooth Muscle. Implications for Airway Contractility

PubMed Central

Cook, Daniel P.; Rector, Michael V.; Bouzek, Drake C.; Michalski, Andrew S.; Gansemer, Nicholas D.; Reznikov, Leah R.; Li, Xiaopeng; Stroik, Mallory R.; Ostedgaard, Lynda S.; Abou Alaiwa, Mahmoud H.; Thompson, Michael A.; Prakash, Y. S.; Krishnan, Ramaswamy; Meyerholz, David K.; Seow, Chun Y.

2016-01-01

Rationale: An asthma-like airway phenotype has been described in people with cystic fibrosis (CF). Whether these findings are directly caused by loss of CF transmembrane conductance regulator (CFTR) function or secondary to chronic airway infection and/or inflammation has been difficult to determine. Objectives: Airway contractility is primarily determined by airway smooth muscle. We tested the hypothesis that CFTR is expressed in airway smooth muscle and directly affects airway smooth muscle contractility. Methods: Newborn pigs, both wild type and with CF (before the onset of airway infection and inflammation), were used in this study. High-resolution immunofluorescence was used to identify the subcellular localization of CFTR in airway smooth muscle. Airway smooth muscle function was determined with tissue myography, intracellular calcium measurements, and regulatory myosin light chain phosphorylation status. Precision-cut lung slices were used to investigate the therapeutic potential of CFTR modulation on airway reactivity. Measurements and Main Results: We found that CFTR localizes to the sarcoplasmic reticulum compartment of airway smooth muscle and regulates airway smooth muscle tone. Loss of CFTR function led to delayed calcium reuptake following cholinergic stimulation and increased myosin light chain phosphorylation. CFTR potentiation with ivacaftor decreased airway reactivity in precision-cut lung slices following cholinergic stimulation. Conclusions: Loss of CFTR alters porcine airway smooth muscle function and may contribute to the airflow obstruction phenotype observed in human CF. Airway smooth muscle CFTR may represent a therapeutic target in CF and other diseases of airway narrowing. PMID:26488271

Chloroplast genes transferred to the nuclear plant genome have adjusted to nuclear base composition and codon usage.

PubMed Central

Oliver, J L; Marín, A; Martínez-Zapater, J M

1990-01-01

During plant evolution, some plastid genes have been moved to the nuclear genome. These transferred genes are now correctly expressed in the nucleus, their products being transported into the chloroplast. We compared the base compositions, the distributions of some dinucleotides and codon usages of transferred, nuclear and chloroplast genes in two dicots and two monocots plant species. Our results indicate that transferred genes have adjusted to nuclear base composition and codon usage, being now more similar to the nuclear genes than to the chloroplast ones in every species analyzed. PMID:2308837

Design of retrovirus vectors for transfer and expression of the human. beta. -globin gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miller, A.D.; Bender, M.A.; Harris, E.A.S.

1988-11-01

Regulated expression of the human ..beta..-globin gene has been demonstrated in cultured murine erythroleukemia cells and in mice after retrovirus-mediated gene transfer. However, the low titer of recombinant viruses described to date results in relatively inefficient gene transfer, which limits their usefulness for animal studies and for potential gene therapy in humans for diseases involving defective ..beta..-globin genes. The authors found regions that interfered with virus production within intron 2 of the ..beta..-globin gene and on both sides of the gene. The flanking regions could be removed, but intron 2 was required for ..beta..-globin expression. Inclusion of ..beta..-globin introns necessitatesmore » an antisense orientation of the gene within the retrovirus vector. However, they found no effect of the antisense ..beta..-globin transcription on virus production. A region downstream of the ..beta..-globin gene that stimulates expression of the gene in transgenic mice was included in the viruses without detrimental effects on virus titer. Virus titers of over 10/sup 6/ CFU/ml were obtained with the final vector design, which retained the ability to direct regulated expression of human ..beta..-globin in murine erythroleukemia cells. The vector also allowed transfer and expression of the human ..beta..-globin gene in hematopoietic cells (CFU-S cells) in mice.« less

Airway smooth muscle in airway reactivity and remodeling: what have we learned?

PubMed Central

2013-01-01

It is now established that airway smooth muscle (ASM) has roles in determining airway structure and function, well beyond that as the major contractile element. Indeed, changes in ASM function are central to the manifestation of allergic, inflammatory, and fibrotic airway diseases in both children and adults, as well as to airway responses to local and environmental exposures. Emerging evidence points to novel signaling mechanisms within ASM cells of different species that serve to control diverse features, including 1) [Ca2+]i contractility and relaxation, 2) cell proliferation and apoptosis, 3) production and modulation of extracellular components, and 4) release of pro- vs. anti-inflammatory mediators and factors that regulate immunity as well as the function of other airway cell types, such as epithelium, fibroblasts, and nerves. These diverse effects of ASM “activity” result in modulation of bronchoconstriction vs. bronchodilation relevant to airway hyperresponsiveness, airway thickening, and fibrosis that influence compliance. This perspective highlights recent discoveries that reveal the central role of ASM in this regard and helps set the stage for future research toward understanding the pathways regulating ASM and, in turn, the influence of ASM on airway structure and function. Such exploration is key to development of novel therapeutic strategies that influence the pathophysiology of diseases such as asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis. PMID:24142517

In vitro study for laser gene transfer in BHK-21 fibroblast cell line

NASA Astrophysics Data System (ADS)

Abdel Aziz, M.; Salem, D. S.; Salama, M. S.; Badr, Y.

2009-02-01

Modifications to our previously introduced system for laser microbeam cell surgery were carried out in the present work to match animal cells. These modifications included: 1- Using other laser system that used before, Excimer laser with 193 and 308 nm wavelengths. The used laser here, is He-Cd with low power and 441.5 nm wavelength in the visible region. 2- Instead of using pulsed laser, we used here CW He-Cd chopped by electrical chopper, which is synchronized with the mechanical motion of the mobile stage with step 40 microns, according to cell dimensions to avoid puncturing the same cell twice. The advantages of the modified here laser setup for gene transfer is: it is less damaging to the sensitive animal cell which has thin cell membrane. The present work aimed to: 1- Design a modified laser microbeam cell surgery, applicable to animal cells, such as fibroblast cells 2- To examine the efficiency of such system. 3- To assure gene transfer and its expression in the used cells. 4- To evaluate the ultra damages produced from using the laser beam as a modality for gene transfer. On the other wards, to introduce: safe, efficient and less damaging modality for gene transfer in animal cells. To achieve these goals, we applied the introduced here home-made laser setup with its synchronized parameters to introduce pBK-CMV phagemid, containing LacZ and neomycin resistance (neor )genes into BHK-21 fibroblast cell line. The results of the present work showed that: 1- Our modified laser microbeam cell surgery setup proved to be useful and efficient tool for gene transfer into fibroblast cells. 2- The presence and expression of LacZ gene was achieved using histochemical LacZ assay. 3- Selection of G418 antibiotic sensitivity assay confirmed the presence and expression towards stability of neor gene with time. 4- Presence of LacZ and neor genes in the genomic DNA of transfected fibroblast cells was indicated using PCR analysis. 5- Transmission electron microscopy indicated

Difficult airway response team: a novel quality improvement program for managing hospital-wide airway emergencies.

PubMed

Mark, Lynette J; Herzer, Kurt R; Cover, Renee; Pandian, Vinciya; Bhatti, Nasir I; Berkow, Lauren C; Haut, Elliott R; Hillel, Alexander T; Miller, Christina R; Feller-Kopman, David J; Schiavi, Adam J; Xie, Yanjun J; Lim, Christine; Holzmueller, Christine; Ahmad, Mueen; Thomas, Pradeep; Flint, Paul W; Mirski, Marek A

2015-07-01

Difficult airway cases can quickly become emergencies, increasing the risk of life-threatening complications or death. Emergency airway management outside the operating room is particularly challenging. We developed a quality improvement program-the Difficult Airway Response Team (DART)-to improve emergency airway management outside the operating room. DART was implemented by a team of anesthesiologists, otolaryngologists, trauma surgeons, emergency medicine physicians, and risk managers in 2005 at The Johns Hopkins Hospital in Baltimore, Maryland. The DART program had 3 core components: operations, safety, and education. The operations component focused on developing a multidisciplinary difficult airway response team, standardizing the emergency response process, and deploying difficult airway equipment carts throughout the hospital. The safety component focused on real-time monitoring of DART activations and learning from past DART events to continuously improve system-level performance. This objective entailed monitoring the paging system, reporting difficult airway events and DART activations to a Web-based registry, and using in situ simulations to identify and mitigate defects in the emergency airway management process. The educational component included development of a multispecialty difficult airway curriculum encompassing case-based lectures, simulation, and team building/communication to ensure consistency of care. Educational materials were also developed for non-DART staff and patients to inform them about the needs of patients with difficult airways and ensure continuity of care with other providers after discharge. Between July 2008 and June 2013, DART managed 360 adult difficult airway events comprising 8% of all code activations. Predisposing patient factors included body mass index >40, history of head and neck tumor, prior difficult intubation, cervical spine injury, airway edema, airway bleeding, and previous or current tracheostomy. Twenty

Difficult Airway Response Team: A Novel Quality Improvement Program for Managing Hospital-Wide Airway Emergencies

PubMed Central

Mark, Lynette J.; Herzer, Kurt R.; Cover, Renee; Pandian, Vinciya; Bhatti, Nasir I.; Berkow, Lauren C.; Haut, Elliott R.; Hillel, Alexander T.; Miller, Christina R.; Feller-Kopman, David J.; Schiavi, Adam J.; Xie, Yanjun J.; Lim, Christine; Holzmueller, Christine; Ahmad, Mueen; Thomas, Pradeep; Flint, Paul W.; Mirski, Marek A.

2015-01-01

Background Difficult airway cases can quickly become emergencies, increasing the risk of life-threatening complications or death. Emergency airway management outside the operating room is particularly challenging. Methods We developed a quality improvement program—the Difficult Airway Response Team (DART)—to improve emergency airway management outside the operating room. DART was implemented by a team of anesthesiologists, otolaryngologists, trauma surgeons, emergency medicine physicians, and risk managers in 2005 at The Johns Hopkins Hospital in Baltimore, Maryland. The DART program had three core components: operations, safety, and education. The operations component focused on developing a multidisciplinary difficult airway response team, standardizing the emergency response process, and deploying difficult airway equipment carts throughout the hospital. The safety component focused on real-time monitoring of DART activations and learning from past DART events to continuously improve system-level performance. This objective entailed monitoring the paging system, reporting difficult airway events and DART activations to a web-based registry, and using in situ simulations to identify and mitigate defects in the emergency airway management process. The educational component included development of a multispecialty difficult airway curriculum encompassing case-based lectures, simulation, and team building/communication to ensure consistency of care. Educational materials were also developed for non-DART staff and patients to inform them about the needs of patients with difficult airways and ensure continuity of care with other providers after discharge. Results Between July 2008 and June 2013, DART managed 360 adult difficult airway events comprising 8% of all code activations. Predisposing patient factors included body mass index > 40, history of head and neck tumor, prior difficult intubation, cervical spine injury, airway edema, airway bleeding, and previous

Horizontal gene transfer of an entire metabolic pathway between a eukaryotic alga and its DNA virus

PubMed Central

Monier, Adam; Pagarete, António; de Vargas, Colomban; Allen, Michael J.; Read, Betsy; Claverie, Jean-Michel; Ogata, Hiroyuki

2009-01-01

Interactions between viruses and phytoplankton, the main primary producers in the oceans, affect global biogeochemical cycles and climate. Recent studies are increasingly revealing possible cases of gene transfers between cyanobacteria and phages, which might have played significant roles in the evolution of cyanobacteria/phage systems. However, little has been documented about the occurrence of horizontal gene transfer in eukaryotic phytoplankton/virus systems. Here we report phylogenetic evidence for the transfer of seven genes involved in the sphingolipid biosynthesis pathway between the cosmopolitan eukaryotic microalga Emiliania huxleyi and its large DNA virus EhV. PCR assays indicate that these genes are prevalent in E. huxleyi and EhV strains isolated from different geographic locations. Patterns of protein and gene sequence conservation support that these genes are functional in both E. huxleyi and EhV. This is the first clear case of horizontal gene transfer of multiple functionally linked enzymes in a eukaryotic phytoplankton–virus system. We examine arguments for the possible direction of the gene transfer. The virus-to-host direction suggests the existence of ancient viruses that controlled the complex metabolic pathway in order to infect primitive eukaryotic cells. In contrast, the host-to-virus direction suggests that the serial acquisition of genes involved in the same metabolic pathway might have been a strategy for the ancestor of EhVs to stay ahead of their closest relatives in the great evolutionary race for survival. PMID:19451591

Laryngeal mask airway for airway control during percutaneous dilatational tracheostomy.

PubMed

Pratt, T; Bromilow, J

2011-11-01

Percutaneous dilatational tracheostomy is a common bedside procedure in critical care for patients requiring prolonged mechanical ventilation. The traditional technique requires withdrawal of the endotracheal tube to a proximal position to facilitate tracheostomy insertion, but this carries the risk of inadvertent extubation and does not prevent cuff rupture. Use of a supraglottic airway such as the laryngeal mask airway may avoid these risks and could provide a safe alternative to the endotracheal tube. We present an appraisal of the literature to date. We found reasonable evidence to show improved ventilation and bronchoscopic visualisation with the laryngeal mask airway, but this has not been translated into improved outcome. There is currently insufficient evidence to draw conclusions about the safety of the laryngeal mask airway during percutaneous dilatational tracheostomy.

“Is a cure in my sight?” Multi-stakeholder perspectives on phase I choroideremia gene transfer clinical trials

PubMed Central

Benjaminy, Shelly; MacDonald, Ian; Bubela, Tania

2014-01-01

Purpose: Ocular gene transfer clinical trials are raising patient hopes for the treatment of choroideremia – a blinding degenerative retinopathy. Phase I choroideremia gene transfer trials necessitate communicating about the risks of harm and potential benefits with patients while avoiding the sensationalism that has historically undermined this field of translational medicine. Methods: We conducted interviews between June 2011 and June 2012 with 6 choroideremia patient advocates, 20 patients, and 15 clinicians about their hopes for benefits, perceived risks of harm, and hopes for the time frame of clinical implementation of choroideremia gene transfer. Results: Despite the safety focus of phase I trials, participants hoped for direct visual benefits with evident discrepancies between stakeholder perspectives about the degree of visual benefit. Clinicians and patient advocates were concerned by limited patient attention to risks of harm. Interviews revealed confusion about the time frames for the clinical implementation of choroideremia gene transfer and patient urgency to access gene transfer within a limited therapeutic window. Conclusion: Differences in stakeholder perspectives about choroideremia gene transfer necessitate strategies that promote responsible communications about choroideremia gene transfer and aid in its translation. Strategies should counter historical sensationalism associated with gene transfer, promote informed consent, and honor patient hope while grounding communications in current clinical realities. PMID:24071795

The gene transfer agent-like particle of the marine phototrophic bacterium Rhodovulum sulfidophilum.

PubMed

Nagao, Nobuyoshi; Yamamoto, Junya; Komatsu, Hiroyuki; Suzuki, Hiromichi; Hirose, Yuu; Umekage, So; Ohyama, Takashi; Kikuchi, Yo

2015-12-01

Gene transfer agents (GTAs) are shaped like bacteriophage particles but have many properties that distinguish them from bacteriophages. GTAs play a role in horizontal gene transfer in nature and thus affect the evolution of prokaryotic genomes. In the course of studies on the extracellular production of designed RNAs using the marine bacterium Rhodovulum sulfidophilum , we found that this bacterium produces a GTA-like particle. The particle contains DNA fragments of 4.5 kb, which consist of randomly fragmented genomic DNA from the bacterium. This 4.5-kb DNA production was prevented while quorum sensing was inhibited. Direct observation of the particle by transmission electron microscopy revealed that the particle resembles a tailed phage and has a head diameter of about 40 nm and a tail length of about 60 nm. We also identified the structural genes for the GTA in the genome. Translated amino acid sequences and gene positions are closely related to those of the genes that encode the Rhodobacter capsulatus GTA. This is the first report of a GTA-like particle from the genus Rhodovulum . However, gene transfer activity of this particle has not yet been confirmed. The differences between this particle and other GTAs are discussed.

Use of a Novel Airway Kit and Simulation in Resident Training on Emergent Pediatric Airways.

PubMed

Melzer, Jonathan M; Hamersley, Erin R S; Gallagher, Thomas Q

2017-06-01

Objective Development of a novel pediatric airway kit and implementation with simulation to improve resident response to emergencies with the goal of improving patient safety. Methods Prospective study with 9 otolaryngology residents (postgraduate years 1-5) from our tertiary care institution. Nine simulated pediatric emergency airway drills were carried out with the existing system and a novel portable airway kit. Response times and time to successful airway control were noted with both the extant airway system and the new handheld kit. Results were analyzed to ensure parametric data and compared with t tests. A Bonferroni adjustment indicated that an alpha of 0.025 was needed for significance. Results Use of the airway kit significantly reduced the mean time of resident arrival by 47% ( P = .013) and mean time of successful intubation by 50% ( P = .007). Survey data indicated 100% improved resident comfort with emergent airway scenarios with use of the kit. Discussion Times to response and meaningful intervention were significantly reduced with implementation of the handheld airway kit. Use of simulation training to implement the new kit improved residents' comfort and airway skills. This study describes an affordable novel mobile airway kit and demonstrates its ability to improve response times. Implications for Practice The low cost of this airway kit makes it a tenable option even for smaller hospitals. Simulation provides a safe and effective way to familiarize oneself with novel equipment, and, when possible, realistic emergent airway simulations should be used to improve provider performance.

Intravascular local gene transfer mediated by protein-coated metallic stent.

PubMed

Yuan, J; Gao, R; Shi, R; Song, L; Tang, J; Li, Y; Tang, C; Meng, L; Yuan, W; Chen, Z

2001-10-01

To assess the feasibility, efficiency and selectivity of adenovirus-mediated gene transfer to local arterial wall by protein-coated metallic stent. A replication-defective recombinant adenovirus carrying the Lac Z reporter gene for nuclear-specific beta-galactosidase (Ad-beta gal) was used in this study. The coating for metallic stent was made by immersing it in a gelatin solution containing crosslinker. The coated stents were mounted on a 4.0 or 3.0 mm percutaneous transluminal coronary angioplasty (PTCA) balloon and submersed into a high-titer Ad-beta gal viral stock (2 x 10(10) pfu/ml) for 3 min, and then implanted into the carotid arteries in 4 mini-swines and into the left anterior descending branch of the coronary artery in 2 mini-swines via 8F large lumen guiding catheters. The animals were sacrificed 7 (n = 4), 14 (n = 1) and 21 (n = 1) days after implantation, respectively. The beta-galactosidase expression was assessed by X-gal staining. The results showed that the expression of transgene was detected in all animal. In 1 of carotid artery with an intact intima, the beta-gal expression was limited to endothelial cells. In vessels with denuded endothelium, gene expression was found in the sub-intima, media and adventitia. The transfection efficiency of medial smooth muscle cells was 38.6%. In 2 animals sacrificed 7 days after transfection, a microscopic examination of X-gal-stained samples did not show evidence of transfection in remote organs and arterial segments adjacent to the treated arterial site. Adenovirus-mediated arterial gene transfer to endothelial, smooth muscle cells and adventitia by protein-coated metallic stent is feasible. The transfection efficiency is higher. The coated stent may act as a good carrier of adenovirus-mediated gene transfer and have a potential to prevent restenosis following PTCA.

A proof-of-concept clinical study examining the NRF2 activator sulforaphane against neutrophilic airway inflammation.

PubMed

Duran, Charity G; Burbank, Allison J; Mills, Katherine H; Duckworth, Heather R; Aleman, Maria M; Kesic, Matthew J; Peden, David B; Pan, Yinghao; Zhou, Haibo; Hernandez, Michelle L

2016-07-22

Sulforaphane (SFN), a naturally occurring isothiocyanate found in cruciferous vegetables, is implicated as a possible therapy for airway inflammation via induction of the transcription factor NF-E2-related factor 2 (NRF2). In this proof-of-concept clinical study, we show that supplementation of SFN with broccoli sprout homogenate in healthy human subjects did not induce expression of antioxidant genes or protect against neutrophilic airway inflammation in an ozone-exposure model. Therefore, dietary sulforaphane supplementation is not a promising candidate for larger scale clinical trials targeting airway inflammation. NCT01625130 . Registered 19 June, 2012.

Efficacy of Surgical Airway Plasty for Benign Airway Stenosis.

PubMed

Tsukioka, Takuma; Takahama, Makoto; Nakajima, Ryu; Kimura, Michitaka; Inoue, Hidetoshi; Yamamoto, Ryoji

2016-01-01

Long-term patency is required during treatment for benign airway stenosis. This study investigated the effectiveness of surgical airway plasty for benign airway stenosis. Clinical courses of 20 patients, who were treated with surgical plasty for their benign airway stenosis, were retrospectively investigated. Causes of stenosis were tracheobronchial tuberculosis in 12 patients, post-intubation stenosis in five patients, malacia in two patients, and others in one patient. 28 interventional pulmonology procedures and 20 surgical plasty were performed. Five patients with post-intubation stenosis and four patients with tuberculous stenosis were treated with tracheoplasty. Eight patients with tuberculous stenosis were treated with bronchoplasty, and two patients with malacia were treated with stabilization of the membranous portion. Anastomotic stenosis was observed in four patients, and one to four additional treatments were required. Performance status, Hugh-Jones classification, and ventilatory functions were improved after surgical plasty. Outcomes were fair in patients with tuberculous stenosis and malacia. However, efficacy of surgical plasty for post-intubation stenosis was not observed. Surgical airway plasty may be an acceptable treatment for tuberculous stenosis. Patients with malacia recover well after surgical plasty. There may be untreated patients with malacia who have the potential to benefit from surgical plasty.

Efficacy of Surgical Airway Plasty for Benign Airway Stenosis

PubMed Central

Takahama, Makoto; Nakajima, Ryu; Kimura, Michitaka; Inoue, Hidetoshi; Yamamoto, Ryoji

2015-01-01

Background: Long-term patency is required during treatment for benign airway stenosis. This study investigated the effectiveness of surgical airway plasty for benign airway stenosis. Methods: Clinical courses of 20 patients, who were treated with surgical plasty for their benign airway stenosis, were retrospectively investigated. Results: Causes of stenosis were tracheobronchial tuberculosis in 12 patients, post-intubation stenosis in five patients, malacia in two patients, and others in one patient. 28 interventional pulmonology procedures and 20 surgical plasty were performed. Five patients with post-intubation stenosis and four patients with tuberculous stenosis were treated with tracheoplasty. Eight patients with tuberculous stenosis were treated with bronchoplasty, and two patients with malacia were treated with stabilization of the membranous portion. Anastomotic stenosis was observed in four patients, and one to four additional treatments were required. Performance status, Hugh–Jones classification, and ventilatory functions were improved after surgical plasty. Outcomes were fair in patients with tuberculous stenosis and malacia. However, efficacy of surgical plasty for post-intubation stenosis was not observed. Conclusion: Surgical airway plasty may be an acceptable treatment for tuberculous stenosis. Patients with malacia recover well after surgical plasty. There may be untreated patients with malacia who have the potential to benefit from surgical plasty. PMID:26567879

Insights on persistent airway infection by non-typeable Haemophilus influenzae in chronic obstructive pulmonary disease

PubMed Central

Ahearn, Christian P.; Gallo, Mary C.

2017-01-01

Abstract Non-typeable Haemophilus influenzae (NTHi) is the most common bacterial cause of infection of the lower airways in adults with chronic obstructive pulmonary disease (COPD). Infection of the COPD airways causes acute exacerbations, resulting in substantial morbidity and mortality. NTHi has evolved multiple mechanisms to establish infection in the hostile environment of the COPD airways, allowing the pathogen to persist in the airways for months to years. Persistent infection of the COPD airways contributes to chronic airway inflammation that increases symptoms and accelerates the progressive loss of pulmonary function, which is a hallmark of the disease. Persistence mechanisms of NTHi include the expression of multiple redundant adhesins that mediate binding to host cellular and extracellular matrix components. NTHi evades host immune recognition and clearance by invading host epithelial cells, forming biofilms, altering gene expression and displaying surface antigenic variation. NTHi also binds host serum factors that confer serum resistance. Here we discuss the burden of COPD and the role of NTHi infections in the course of the disease. We provide an overview of NTHi mechanisms of persistence that allow the pathogen to establish a niche in the hostile COPD airways. PMID:28449098

Volunteering for early phase gene transfer research in Parkinson disease.

PubMed

Kim, S Y H; Holloway, R G; Frank, S; Beck, C A; Zimmerman, C; Wilson, R; Kieburtz, K

2006-04-11

For early phase trials of novel interventions-such as gene transfer for Parkinson disease (PD)--whose focus is primarily on safety and tolerability, it is important that participants have a realistic understanding of the goals of such research. Recently, some have expressed concern that patients with PD may have unrealistic expectations. The authors examined why patients with PD might volunteer for invasive early phase research by interviewing 92 patients with PD and comparing those who would (n = 46) and those who would not (n = 46) participate in a hypothetical phase I gene-transfer study. The two groups' demographic, clinical, functional, and quality of life measures, as well as their understanding of the research protocol, were similar. The groups did not differ on their perception of potential for personal benefit nor on the level of likelihood of benefit they saw as a precondition for volunteering. However, those willing to participate tended to perceive lower probability of risk, were tolerant of greater probability of risk, and were more optimistic about the phase I study's potential benefits to society. They also appeared more decisive and action-oriented than the unwilling group. It is likely that the decision whether to participate in early phase PD gene transfer studies will depend mostly on patients' attitudes regarding risk, optimism about science, and an action orientation, rather than on their clinical, functional, or demographic characteristics.

Staphylococcus aureus genomics and the impact of horizontal gene transfer.

PubMed

Lindsay, Jodi A

2014-03-01

Whole genome sequencing and microarrays have revealed the population structure of Staphylococcus aureus, and identified epidemiological shifts, transmission routes, and adaptation of major clones. S. aureus genomes are highly diverse. This is partly due to a population structure of conserved lineages, each with unique combinations of genes encoding surface proteins, regulators, immune evasion and virulence pathways. Even more variable are the mobile genetic elements (MGE), which encode key proteins for antibiotic resistance, virulence and host-adaptation. MGEs can transfer at high frequency between isolates of the same lineage by horizontal gene transfer (HGT). There is increasing evidence that HGT is key to bacterial adaptation and success. Recent studies have shed light on new mechanisms of DNA transfer such as transformation, the identification of receptors for transduction, on integration of DNA pathways, mechanisms blocking transfer including CRISPR and new restriction systems, strategies for evasion of restriction barriers, as well as factors influencing MGE selection and stability. These studies have also lead to new tools enabling construction of genetically modified clinical S. aureus isolates. This review will focus on HGT mechanisms and their importance in shaping the evolution of new clones adapted to antibiotic resistance, healthcare, communities and livestock. Copyright © 2013 Elsevier GmbH. All rights reserved.

Prehospital removal improves neurological outcomes in elderly patient with foreign body airway obstruction.

PubMed

Igarashi, Yutaka; Yokobori, Shoji; Yoshino, Yudai; Masuno, Tomohiko; Miyauchi, Masato; Yokota, Hiroyuki

2017-10-01

In Japan, the number of patients with foreign body airway obstruction by food is rapidly increasing with the increase in the population of the elderly and a leading cause of unexpected death. This study aimed to determine the factors that influence prognosis of these patients. This is a retrospective single institutional study. A total of 155 patients were included. We collected the variables from the medical records and analyzed them to determine the factors associated with patient outcome. Patient outcomes were evaluated using cerebral performance categories (CPCs) when patients were discharged or transferred to other hospitals. A favorable outcome was defined as CPC 1 or 2, and an unfavorable outcome was defined as CPC 3, 4, or 5. A higher proportion of patients with favorable outcomes than unfavorable outcomes had a witness present at the accident scene (68.8% vs. 44.7%, P=0.0154). Patients whose foreign body were removed by a bystander at the accident scene had a significantly high rate of favorable outcome than those whose foreign body were removed by emergency medical technicians or emergency physician at the scene (73.7% vs. 31.8%, P<0.0075) and at the hospital after transfer (73.7% vs. 9.6%, P<0.0001). The presence of a witness to the aspiration and removal of the airway obstruction of patients by bystanders at the accident scene improves outcomes in patients with foreign body airway obstruction. When airway obstruction occurs, bystanders should remove foreign bodies immediately. Copyright © 2017 Elsevier Inc. All rights reserved.

Horizontal transfer of archaeal genes into the deinococcaceae: detection by molecular and computer-based approaches

NASA Technical Reports Server (NTRS)

Olendzenski, L.; Liu, L.; Zhaxybayeva, O.; Murphey, R.; Shin, D. G.; Gogarten, J. P.

2000-01-01

Members of the Deinococcaceae (e.g., Thermus, Meiothermus, Deinococcus) contain A/V-ATPases typically found in Archaea or Eukaryotes which were probably acquired by horizontal gene transfer. Two methods were used to quantify the extent to which archaeal or eukaryotic genes have been acquired by this lineage. Screening of a Meiothermus ruber library with probes made against Thermoplasma acidophilum DNA yielded a number of clones which hybridized more strongly than background. One of these contained the prolyl tRNA synthetase (RS) gene. Phylogenetic analysis shows the M. ruber and D. radiodurans prolyl RS to be more closely related to archaeal and eukaryal forms of this gene than to the typical bacterial type. Using a bioinformatics approach, putative open reading frames (ORFs) from the prerelease version of the D. radiodurans genome were screened for genes more closely related to archaeal or eukaryotic genes. Putative ORFs were searched against representative genomes from each of the three domains using automated BLAST. ORFs showing the highest matches against archaeal and eukaryotic genes were collected and ranked. Among the top-ranked hits were the A/V-ATPase catalytic and noncatalytic subunits and the prolyl RS genes. Using phylogenetic methods, ORFs were analyzed and trees assessed for evidence of horizontal gene transfer. Of the 45 genes examined, 20 showed topologies in which D. radiodurans homologues clearly group with eukaryotic or archaeal homologues, and 17 additional trees were found to show probable evidence of horizontal gene transfer. Compared to the total number of ORFs in the genome, those that can be identified as having been acquired from Archaea or Eukaryotes are relatively few (approximately 1%), suggesting that interdomain transfer is rare.

Two Horizontally Transferred Xenobiotic Resistance Gene Clusters Associated with Detoxification of Benzoxazolinones by Fusarium Species

PubMed Central

Glenn, Anthony E.; Davis, C. Britton; Gao, Minglu; Gold, Scott E.; Mitchell, Trevor R.; Proctor, Robert H.; Stewart, Jane E.; Snook, Maurice E.

2016-01-01

Microbes encounter a broad spectrum of antimicrobial compounds in their environments and often possess metabolic strategies to detoxify such xenobiotics. We have previously shown that Fusarium verticillioides, a fungal pathogen of maize known for its production of fumonisin mycotoxins, possesses two unlinked loci, FDB1 and FDB2, necessary for detoxification of antimicrobial compounds produced by maize, including the γ-lactam 2-benzoxazolinone (BOA). In support of these earlier studies, microarray analysis of F. verticillioides exposed to BOA identified the induction of multiple genes at FDB1 and FDB2, indicating the loci consist of gene clusters. One of the FDB1 cluster genes encoded a protein having domain homology to the metallo-β-lactamase (MBL) superfamily. Deletion of this gene (MBL1) rendered F. verticillioides incapable of metabolizing BOA and thus unable to grow on BOA-amended media. Deletion of other FDB1 cluster genes, in particular AMD1 and DLH1, did not affect BOA degradation. Phylogenetic analyses and topology testing of the FDB1 and FDB2 cluster genes suggested two horizontal transfer events among fungi, one being transfer of FDB1 from Fusarium to Colletotrichum, and the second being transfer of the FDB2 cluster from Fusarium to Aspergillus. Together, the results suggest that plant-derived xenobiotics have exerted evolutionary pressure on these fungi, leading to horizontal transfer of genes that enhance fitness or virulence. PMID:26808652

Phylogenetic modeling of lateral gene transfer reconstructs the pattern and relative timing of speciations

PubMed Central

Szöllősi, Gergely J.; Boussau, Bastien; Abby, Sophie S.; Tannier, Eric; Daubin, Vincent

2012-01-01

The timing of the evolution of microbial life has largely remained elusive due to the scarcity of prokaryotic fossil record and the confounding effects of the exchange of genes among possibly distant species. The history of gene transfer events, however, is not a series of individual oddities; it records which lineages were concurrent and thus provides information on the timing of species diversification. Here, we use a probabilistic model of genome evolution that accounts for differences between gene phylogenies and the species tree as series of duplication, transfer, and loss events to reconstruct chronologically ordered species phylogenies. Using simulations we show that we can robustly recover accurate chronologically ordered species phylogenies in the presence of gene tree reconstruction errors and realistic rates of duplication, transfer, and loss. Using genomic data we demonstrate that we can infer rooted species phylogenies using homologous gene families from complete genomes of 10 bacterial and archaeal groups. Focusing on cyanobacteria, distinguished among prokaryotes by a relative abundance of fossils, we infer the maximum likelihood chronologically ordered species phylogeny based on 36 genomes with 8,332 homologous gene families. We find the order of speciation events to be in full agreement with the fossil record and the inferred phylogeny of cyanobacteria to be consistent with the phylogeny recovered from established phylogenomics methods. Our results demonstrate that lateral gene transfers, detected by probabilistic models of genome evolution, can be used as a source of information on the timing of evolution, providing a valuable complement to the limited prokaryotic fossil record. PMID:23043116

In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment.

PubMed Central

Yang, N S; Burkholder, J; Roberts, B; Martinell, B; McCabe, D

1990-01-01

Chimeric chloramphenicol acetyltransferase and beta-galactosidase marker genes were coated onto fine gold particles and used to bombard a variety of mammalian tissues and cells. Transient expression of the genes was obtained in liver, skin, and muscle tissues of rat and mouse bombarded in vivo. Similar results were obtained with freshly isolated ductal segments of rat and human mammary glands and primary cultures derived from these explants. Gene transfer and transient expression were also observed in eight human cell culture lines, including cells of epithelial, endothelial, fibroblast, and lymphocyte origin. Using CHO and MCF-7 cell cultures as models, we obtained stable gene transfer at frequencies of 1.7 x 10(-3) and 6 x 10(-4), respectively. The particle bombardment technology thus provides a useful means to transfer foreign genes into a variety of mammalian somatic cell systems. The method is applicable to tissues in vivo as well as to isolated cells in culture and has proven effective with all cell or tissue types tested thus far. This technology may therefore prove to be applicable in various aspects of gene therapy. Images PMID:2175906

The effects of exogenous lipid on THP-1 cells: an in vitro model of airway aspiration?

PubMed

Hayman, Yvette A; Sadofsky, Laura R; Williamson, James D; Hart, Simon P; Morice, Alyn H

2017-01-01

Chronic inflammatory diseases of the airways are associated with gastro-oesophageal reflux (GOR) and aspiration events. The observation of lipid-laden macrophages (LLMs) within the airway may indicate aspiration secondary to GOR. The proposed mechanism, that lipid droplets from undigested or partially digested food are aspirated leading to accumulation in scavenging macrophages, led us to hypothesise that an activated population of LLMs could interact with other immune cells to induce bronchial inflammation. To test this, we generated an in vitro model using differentiated THP-1 cells, which were treated with a high-fat liquid feed. Here, we show that THP-1 cells can take up lipid from the high-fat feed independent of actin polymerisation or CD36-dependent phagocytosis. These cells did not exhibit M1 or M2 polarisation. Gene array analysis confirmed over 8000 genes were upregulated by at least twofold following high fat exposure, and IL-8 was the most upregulated gene. Pathway analysis revealed upregulation of genes known to be involved in chronic obstructive pulmonary disease (COPD) pathophysiology. We suggest that aspiration and macrophage phagocytosis may be important mechanisms in the aetiology of diseases such as COPD and cystic fibrosis that are characterised by high levels of IL-8 within the airways.

Airway management in neuroanesthesiology.

PubMed

Aziz, Michael

2012-06-01

Airway management for neuroanesthesiology brings together some key principles that are shared throughout neuroanesthesiology. This article appropriately targets the cervical spine with associated injury and the challenges surrounding airway management. The primary focus of this article is on the unique airway management obstacles encountered with cervical spine injury or cervical spine surgery, and unique considerations regarding functional neurosurgery are addressed. Furthermore, topics related to difficult airway management for those with rheumatoid arthritis or pituitary surgery are reviewed. Copyright © 2012 Elsevier Inc. All rights reserved.

Mesenchymal stem cells alleviate oxidative stress-induced mitochondrial dysfunction in the airways.

PubMed

Li, Xiang; Michaeloudes, Charalambos; Zhang, Yuelin; Wiegman, Coen H; Adcock, Ian M; Lian, Qizhou; Mak, Judith C W; Bhavsar, Pankaj K; Chung, Kian Fan

2018-05-01

Oxidative stress-induced mitochondrial dysfunction can contribute to inflammation and remodeling in patients with chronic obstructive pulmonary disease (COPD). Mesenchymal stem cells protect against lung damage in animal models of COPD. It is unknown whether these effects occur through attenuating mitochondrial dysfunction in airway cells. We sought to examine the effect of induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) on oxidative stress-induce mitochondrial dysfunction in human airway smooth muscle cells (ASMCs) in vitro and in mouse lungs in vivo. ASMCs were cocultured with iPSC-MSCs in the presence of cigarette smoke medium (CSM), and mitochondrial reactive oxygen species (ROS) levels, mitochondrial membrane potential (ΔΨm), and apoptosis were measured. Conditioned medium from iPSC-MSCs and transwell cocultures were used to detect any paracrine effects. The effect of systemic injection of iPSC-MSCs on airway inflammation and hyperresponsiveness in ozone-exposed mice was also investigated. Coculture of iPSC-MSCs with ASMCs attenuated CSM-induced mitochondrial ROS, apoptosis, and ΔΨm loss in ASMCs. iPSC-MSC-conditioned medium or transwell cocultures with iPSC-MSCs reduced CSM-induced mitochondrial ROS but not ΔΨm or apoptosis in ASMCs. Mitochondrial transfer from iPSC-MSCs to ASMCs was observed after direct coculture and was enhanced by CSM. iPSC-MSCs attenuated ozone-induced mitochondrial dysfunction, airway hyperresponsiveness, and inflammation in mouse lungs. iPSC-MSCs offered protection against oxidative stress-induced mitochondrial dysfunction in human ASMCs and in mouse lungs while reducing airway inflammation and hyperresponsiveness. These effects are, at least in part, dependent on cell-cell contact, which allows for mitochondrial transfer, and paracrine regulation. Therefore iPSC-MSCs show promise as a therapy for oxidative stress-dependent lung diseases, such as COPD. Copyright © 2017 American Academy of Allergy

Gene transfer of Alcaligenes eutrophus JMP134 plasmid pJP4 to indigenous soil recipients.

PubMed Central

DiGiovanni, G D; Neilson, J W; Pepper, I L; Sinclair, N A

1996-01-01

This study evaluated the potential for gene transfer of a large catabolic plasmid from an introduced organism to indigenous soil recipients. The donor organism Alcaligenes eutrophus JMP134 contained the 80-kb plasmid pJP4, which contains genes that code for mercury resistance. Genes on this plasmid plus chromosomal genes also allow degradation of 2,4-dichloruphenoxyacetic acid (2,4-D). When JMP134 was inoculated into a nonsterile soil microcosm amended with 1,000 micrograms of 2,4-D g-1, significant (10(6) g of soil-1) populations of indigenous recipients or transconjugants arose. These transconjugants all contained an 80-kb plasmid similar in size to pJP4, and all degraded 2,4-D. In addition, all transconjugants were resistant to mercury and contained the tfdB gene of pJP4 as detected by PCR. No mercury-resistant, 2,4-D-degrading organisms with large plasmids or the tfdB gene were found in the 2,4-D-amended but uninoculated control microcosm. These data clearly show that the plasmid pJP4 was transferred to indigenous soil recipients. Even more striking is the fact that not only did the indigenous transconjugant population survive and proliferate but also enhanced rates of 2,4-D degradation occurred relative to microcosms in which no such gene transfer occurred. Overall, these data indicate that gene transfer from introduced organisms is an effective means of bioaugmentation and that survival of the introduced organism is not a prerequisite for biodegradation that utilizes introduced biodegradative genes. PMID:8779592

[The research on the airway hyperresponsiveness and IOS airway resistance index of industrial area resident].

PubMed

Xu, Jin; Wang, Zhen; Sun, Hongcun

2015-09-01

To study airway reactivity and impulse oscillation (IOS)-measured airway resistance indicators of residents of Zhenhai industrial area in Ningbo city. In the form of follow-up, both. airway reactivity and respiratory functions of populations in Zhenhai industrial zone (n = 215) and urban (n = 203) were measured, comparing difference degree between different regions. Ninty-five of 215 cases in industrial area were identified as suspected airway hyperresponsiveness, but only 43 of 203 cases were in urban areas. Forty-seven of 95 cases (49.5%) in industrial zone were positive, while only 14 cases (32.6%) in urban. The proportions of people in the two regions on different types of airway hyperresponsiveness were significantly different (P < 0.01). All airway resistance indexes of urban populations were significantly lower than that of industrial zone (P < 0.05). The prevalence of airway hyperresponsiveness and IOS airway resistance aspects of industrial area residents was higher than that of urban residents. Monitoring and evaluating the airway diseases, inflammatory lesions and respiratory function in the region were good for understanding the severe pollution in the local area in certain significance.

Emergency airway puncture

MedlinePlus

... support for only a very short period of time. Alternative Names Needle cricothyrotomy Images Emergency airway puncture Cricoid cartilage Emergency airway puncture - series References Hebert RB, Bose S, Mace SE. Cricothyrotomy and ...

Maternal immune response to helminth infection during pregnancy determines offspring susceptibility to allergic airway inflammation.

PubMed

Straubinger, Kathrin; Paul, Sabine; Prazeres da Costa, Olivia; Ritter, Manuel; Buch, Thorsten; Busch, Dirk H; Layland, Laura E; Prazeres da Costa, Clarissa U

2014-12-01

Schistosomiasis, a chronic helminth infection, elicits distinct immune responses within the host, ranging from an initial TH1 and subsequent TH2 phase to a regulatory state, and is associated with dampened allergic reactions within the host. We sought to evaluate whether non-transplacental helminth infection during pregnancy alters the offspring's susceptibility to allergy. Ovalbumin-induced allergic airway inflammation was analyzed in offspring from Schistosoma mansoni-infected mothers mated during the TH1, TH2, or regulatory phase of infection. Embryos derived from in vitro fertilized oocytes of acutely infected females were transferred into uninfected foster mice to determine the role of placental environment. The fetomaternal unit was further characterized by helminth-specific immune responses and microarray analyses. Eventually, IFN-γ-deficient mice were infected to evaluate the role of this predominant cytokine on the offspring's allergy phenotype. We demonstrate that offspring from schistosome-infected mothers that were mated in the TH1 and regulatory phases, but not the TH2 immune phase, are protected against the onset of allergic airway inflammation. Interestingly, these effects were associated with distinctly altered schistosome-specific cytokine and gene expression profiles within the fetomaternal interface. Furthermore, we identified that it is not the transfer of helminth antigens but rather maternally derived IFN-γ during the acute phase of infection that is essential for the progeny's protective immune phenotype. Overall, we present a novel immune phase-dependent coherency between the maternal immune responses during schistosomiasis and the progeny's predisposition to allergy. Therefore, we propose to include helminth-mediated transmaternal immune modulation into the expanded hygiene hypothesis. Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Dynamic evolution of Geranium mitochondrial genomes through multiple horizontal and intracellular gene transfers.

PubMed

Park, Seongjun; Grewe, Felix; Zhu, Andan; Ruhlman, Tracey A; Sabir, Jamal; Mower, Jeffrey P; Jansen, Robert K

2015-10-01

The exchange of genetic material between cellular organelles through intracellular gene transfer (IGT) or between species by horizontal gene transfer (HGT) has played an important role in plant mitochondrial genome evolution. The mitochondrial genomes of Geraniaceae display a number of unusual phenomena including highly accelerated rates of synonymous substitutions, extensive gene loss and reduction in RNA editing. Mitochondrial DNA sequences assembled for 17 species of Geranium revealed substantial reduction in gene and intron content relative to the ancestor of the Geranium lineage. Comparative analyses of nuclear transcriptome data suggest that a number of these sequences have been functionally relocated to the nucleus via IGT. Evidence for rampant HGT was detected in several Geranium species containing foreign organellar DNA from diverse eudicots, including many transfers from parasitic plants. One lineage has experienced multiple, independent HGT episodes, many of which occurred within the past 5.5 Myr. Both duplicative and recapture HGT were documented in Geranium lineages. The mitochondrial genome of Geranium brycei contains at least four independent HGT tracts that are absent in its nearest relative. Furthermore, G. brycei mitochondria carry two copies of the cox1 gene that differ in intron content, providing insight into contrasting hypotheses on cox1 intron evolution. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Horizontal gene transfer is more frequent with increased heterotrophy and contributes to parasite adaptation.

PubMed

Yang, Zhenzhen; Zhang, Yeting; Wafula, Eric K; Honaas, Loren A; Ralph, Paula E; Jones, Sam; Clarke, Christopher R; Liu, Siming; Su, Chun; Zhang, Huiting; Altman, Naomi S; Schuster, Stephan C; Timko, Michael P; Yoder, John I; Westwood, James H; dePamphilis, Claude W

2016-10-24

Horizontal gene transfer (HGT) is the transfer of genetic material across species boundaries and has been a driving force in prokaryotic evolution. HGT involving eukaryotes appears to be much less frequent, and the functional implications of HGT in eukaryotes are poorly understood. We test the hypothesis that parasitic plants, because of their intimate feeding contacts with host plant tissues, are especially prone to horizontal gene acquisition. We sought evidence of HGTs in transcriptomes of three parasitic members of Orobanchaceae, a plant family containing species spanning the full spectrum of parasitic capabilities, plus the free-living Lindenbergia Following initial phylogenetic detection and an extensive validation procedure, 52 high-confidence horizontal transfer events were detected, often from lineages of known host plants and with an increasing number of HGT events in species with the greatest parasitic dependence. Analyses of intron sequences in putative donor and recipient lineages provide evidence for integration of genomic fragments far more often than retro-processed RNA sequences. Purifying selection predominates in functionally transferred sequences, with a small fraction of adaptively evolving sites. HGT-acquired genes are preferentially expressed in the haustorium-the organ of parasitic plants-and are strongly biased in predicted gene functions, suggesting that expression products of horizontally acquired genes are contributing to the unique adaptive feeding structure of parasitic plants.

Horizontal gene transfer is more frequent with increased heterotrophy and contributes to parasite adaptation

PubMed Central

Yang, Zhenzhen; Zhang, Yeting; Wafula, Eric K.; Honaas, Loren A.; Ralph, Paula E.; Jones, Sam; Clarke, Christopher R.; Liu, Siming; Su, Chun; Zhang, Huiting; Altman, Naomi S.; Schuster, Stephan C.; Timko, Michael P.; Yoder, John I.; dePamphilis, Claude W.

2016-01-01

Horizontal gene transfer (HGT) is the transfer of genetic material across species boundaries and has been a driving force in prokaryotic evolution. HGT involving eukaryotes appears to be much less frequent, and the functional implications of HGT in eukaryotes are poorly understood. We test the hypothesis that parasitic plants, because of their intimate feeding contacts with host plant tissues, are especially prone to horizontal gene acquisition. We sought evidence of HGTs in transcriptomes of three parasitic members of Orobanchaceae, a plant family containing species spanning the full spectrum of parasitic capabilities, plus the free-living Lindenbergia. Following initial phylogenetic detection and an extensive validation procedure, 52 high-confidence horizontal transfer events were detected, often from lineages of known host plants and with an increasing number of HGT events in species with the greatest parasitic dependence. Analyses of intron sequences in putative donor and recipient lineages provide evidence for integration of genomic fragments far more often than retro-processed RNA sequences. Purifying selection predominates in functionally transferred sequences, with a small fraction of adaptively evolving sites. HGT-acquired genes are preferentially expressed in the haustorium—the organ of parasitic plants—and are strongly biased in predicted gene functions, suggesting that expression products of horizontally acquired genes are contributing to the unique adaptive feeding structure of parasitic plants. PMID:27791104

Allergic Sensitization through the Airway Primes Th17-dependent Neutrophilia and Airway Hyperresponsiveness

PubMed Central

Wilson, Rhonda H.; Whitehead, Gregory S.; Nakano, Hideki; Free, Meghan E.; Kolls, Jay K.; Cook, Donald N.

2009-01-01

Rationale: In humans, immune responses to inhaled aeroallergens develop in the lung and draining lymph nodes. Many animal models of asthma bypass this route and instead use intraperitoneal injections of allergen using aluminum hydroxide as an adjuvant. Objectives: We investigated whether allergic sensitization through the airway elicits immune responses qualitatively different than those arising in the peritoneum. Methods: Mice were sensitized to allergen through the airway using low-dose LPS as an adjuvant, or through the peritoneum using aluminum hydroxide as an adjuvant. After a single allergen challenge, ELISA and flow cytometry were used to measure cytokines and leukocyte subsets. Invasive measurements of airway resistance were used to measure allergen-induced airway hyperreactivity (AHR). Measurements and Main Results: Sensitization through the peritoneum primed strong Th2 responses and eosinophilia, but not AHR, after a single allergen challenge. By contrast, allergic sensitization through the airway primed only modest Th2 responses, but strong Th17 responses. Th17 cells homed to the lung and released IL-17 into the airway on subsequent encounter with inhaled allergen. As a result, these mice developed IL-17–dependent airway neutrophilia and AHR. This AHR was neutrophil-dependent because it was abrogated in CXCR2-deficient mice and also in wild-type mice receiving a neutrophil-depleting antibody. Individually, neither IL-17 nor ongoing Th2 responses were sufficient to confer AHR, but together they acted synergistically to promote neutrophil recruitment, eosinophil recruitment and AHR. Conclusions: Allergic sensitization through the airway primes modest Th2 responses but strong Th17 responses that promote airway neutrophilia and acute AHR. These findings support a causal role for neutrophils in severe asthma. PMID:19661246

Careers in Airway Science.

ERIC Educational Resources Information Center

Federal Aviation Administration (DOT), Washington, DC.

The Federal Aviation Administration (FAA) has initiated the Airway Science curriculum as a method of preparing the next generation of aviation technicians and managers. This document: (1) discusses the FAA's role in the Airway Science program; (2) describes some of the career fields that FAA offers to Airway Science graduates (air traffic control…

Heat and water rate transfer processes in the human respiratory tract at various altitudes.

PubMed

Kandjov, I M

2001-02-01

The process of the respiratory air conditioning as a process of heat and mass exchange at the interface inspired air-airways surface was studied. Using a model of airways (Olson et al., 1970) where the segments of the respiratory tract are like cylinders with a fixed length and diameter, the corresponding heat transfer equations, in the paper are founded basic rate exchange parameters-convective heat transfer coefficient h(c)(W m(-2) degrees C(-1)) and evaporative heat transfer coefficient h(e)(W m(-2)hPa(-1)). The rate transfer parameters assumed as sources with known heat power are connected to airflow rate in different airways segments. Relationships expressing warming rate of inspired air due to convection, warming rate of inspired air due to evaporation, water diffused in the inspired air from the airways wall, i.e. a system of air conditioning parameters, was composed. The altitude dynamics of the relations is studied. Every rate conditioning parameter is an increasing function of altitude. The process of diffusion in the peripheral bronchial generations as a basic transfer process is analysed. The following phenomenon is in effect: the diffusion coefficient increases with altitude and causes a compensation of simultaneous decreasing of O(2)and CO(2)densities in atmospheric air. Due to this compensation, the diffusion in the peripheral generations with altitude is approximately constant. The elements of the human anatomy optimality as well as the established dynamics are discussed and assumed. The square form of the airways after the trachea expressed in terms of transfer supposes (in view of maximum contact surface), that a maximum heat and water exchange is achieved, i.e. high degree of air condition at fixed environmental parameters and respiration regime. Copyright 2001 Academic Press.

Phylogeographic reconstruction of a bacterial species with high levels of lateral gene transfer

USGS Publications Warehouse

Pearson, T.; Giffard, P.; Beckstrom-Sternberg, S.; Auerbach, R.; Hornstra, H.; Tuanyok, A.; Price, E.P.; Glass, M.B.; Leadem, B.; Beckstrom-Sternberg, J. S.; Allan, G.J.; Foster, J.T.; Wagner, D.M.; Okinaka, R.T.; Sim, S.H.; Pearson, O.; Wu, Z.; Chang, J.; Kaul, R.; Hoffmaster, A.R.; Brettin, T.S.; Robison, R.A.; Mayo, M.; Gee, J.E.; Tan, P.; Currie, B.J.; Keim, P.

2009-01-01

Background: Phylogeographic reconstruction of some bacterial populations is hindered by low diversity coupled with high levels of lateral gene transfer. A comparison of recombination levels and diversity at seven housekeeping genes for eleven bacterial species, most of which are commonly cited as having high levels of lateral gene transfer shows that the relative contributions of homologous recombination versus mutation for Burkholderia pseudomallei is over two times higher than for Streptococcus pneumoniae and is thus the highest value yet reported in bacteria. Despite the potential for homologous recombination to increase diversity, B. pseudomallei exhibits a relative lack of diversity at these loci. In these situations, whole genome genotyping of orthologous shared single nucleotide polymorphism loci, discovered using next generation sequencing technologies, can provide very large data sets capable of estimating core phylogenetic relationships. We compared and searched 43 whole genome sequences of B. pseudomallei and its closest relatives for single nucleotide polymorphisms in orthologous shared regions to use in phylogenetic reconstruction. Results: Bayesian phylogenetic analyses of >14,000 single nucleotide polymorphisms yielded completely resolved trees for these 43 strains with high levels of statistical support. These results enable a better understanding of a separate analysis of population differentiation among >1,700 B. pseudomallei isolates as defined by sequence data from seven housekeeping genes. We analyzed this larger data set for population structure and allele sharing that can be attributed to lateral gene transfer. Our results suggest that despite an almost panmictic population, we can detect two distinct populations of B. pseudomallei that conform to biogeographic patterns found in many plant and animal species. That is, separation along Wallace's Line, a biogeographic boundary between Southeast Asia and Australia. Conclusion: We describe an

Massive Gene Transfer and Extensive RNA Editing of a Symbiotic Dinoflagellate Plastid Genome

PubMed Central

Mungpakdee, Sutada; Shinzato, Chuya; Takeuchi, Takeshi; Kawashima, Takeshi; Koyanagi, Ryo; Hisata, Kanako; Tanaka, Makiko; Goto, Hiroki; Fujie, Manabu; Lin, Senjie; Satoh, Nori; Shoguchi, Eiichi

2014-01-01

Genome sequencing of Symbiodinium minutum revealed that 95 of 109 plastid-associated genes have been transferred to the nuclear genome and subsequently expanded by gene duplication. Only 14 genes remain in plastids and occur as DNA minicircles. Each minicircle (1.8–3.3 kb) contains one gene and a conserved noncoding region containing putative promoters and RNA-binding sites. Nine types of RNA editing, including a novel G/U type, were discovered in minicircle transcripts but not in genes transferred to the nucleus. In contrast to DNA editing sites in dinoflagellate mitochondria, which tend to be highly conserved across all taxa, editing sites employed in DNA minicircles are highly variable from species to species. Editing is crucial for core photosystem protein function. It restores evolutionarily conserved amino acids and increases peptidyl hydropathy. It also increases protein plasticity necessary to initiate photosystem complex assembly. PMID:24881086

RAGE: a new frontier in chronic airways disease

PubMed Central

Sukkar, Maria B; Ullah, Md Ashik; Gan, Wan Jun; Wark, Peter AB; Chung, Kian Fan; Hughes, J Margaret; Armour, Carol L; Phipps, Simon

2012-01-01

Asthma and chronic obstructive pulmonary disease (COPD) are heterogeneous inflammatory disorders of the respiratory tract characterized by airflow obstruction. It is now clear that the environmental factors that drive airway pathology in asthma and COPD, including allergens, viruses, ozone and cigarette smoke, activate innate immune receptors known as pattern-recognition receptors, either directly or indirectly by causing the release of endogenous ligands. Thus, there is now intense research activity focused around understanding the mechanisms by which pattern-recognition receptors sustain the airway inflammatory response, and how these mechanisms might be targeted therapeutically. One pattern-recognition receptor that has recently come to attention in chronic airways disease is the receptor for advanced glycation end products (RAGE). RAGE is a member of the immunoglobulin superfamily of cell surface receptors that recognizes pathogen- and host-derived endogenous ligands to initiate the immune response to tissue injury, infection and inflammation. Although the role of RAGE in lung physiology and pathophysiology is not well understood, recent genome-wide association studies have linked RAGE gene polymorphisms with airflow obstruction. In addition, accumulating data from animal and clinical investigations reveal increased expression of RAGE and its ligands, together with reduced expression of soluble RAGE, an endogenous inhibitor of RAGE signalling, in chronic airways disease. In this review, we discuss recent studies of the ligand–RAGE axis in asthma and COPD, highlight important areas for future research and discuss how this axis might potentially be harnessed for therapeutic benefit in these conditions. PMID:22506507

Targeted and efficient transfer of multiple value-added genes into wheat varieties

USDA-ARS?s Scientific Manuscript database

With an objective to optimize an approach to transfer multiple value added genes to a wheat variety while maintaining and improving agronomic performance, two alleles with mutations in the acetolactate synthase (ALS) gene located on wheat chromosomes 6B and 6D providing tolerance to imidazolinone (I...

The type VI secretion system of Vibrio cholerae fosters horizontal gene transfer.

PubMed

Borgeaud, Sandrine; Metzger, Lisa C; Scrignari, Tiziana; Blokesch, Melanie

2015-01-02

Natural competence for transformation is a common mode of horizontal gene transfer and contributes to bacterial evolution. Transformation occurs through the uptake of external DNA and its integration into the genome. Here we show that the type VI secretion system (T6SS), which serves as a predatory killing device, is part of the competence regulon in the naturally transformable pathogen Vibrio cholerae. The T6SS-encoding gene cluster is under the positive control of the competence regulators TfoX and QstR and is induced by growth on chitinous surfaces. Live-cell imaging revealed that deliberate killing of nonimmune cells via competence-mediated induction of T6SS releases DNA and makes it accessible for horizontal gene transfer in V. cholerae. Copyright © 2015, American Association for the Advancement of Science.

Phylogenetic Evidence for Lateral Gene Transfer in the Intestine of Marine Iguanas

PubMed Central

Nelson, David M.; Cann, Isaac K. O.; Altermann, Eric; Mackie, Roderick I.

2010-01-01

Background Lateral gene transfer (LGT) appears to promote genotypic and phenotypic variation in microbial communities in a range of environments, including the mammalian intestine. However, the extent and mechanisms of LGT in intestinal microbial communities of non-mammalian hosts remains poorly understood. Methodology/Principal Findings We sequenced two fosmid inserts obtained from a genomic DNA library derived from an agar-degrading enrichment culture of marine iguana fecal material. The inserts harbored 16S rRNA genes that place the organism from which they originated within Clostridium cluster IV, a well documented group that habitats the mammalian intestinal tract. However, sequence analysis indicates that 52% of the protein-coding genes on the fosmids have top BLASTX hits to bacterial species that are not members of Clostridium cluster IV, and phylogenetic analysis suggests that at least 10 of 44 coding genes on the fosmids may have been transferred from Clostridium cluster XIVa to cluster IV. The fosmids encoded four transposase-encoding genes and an integrase-encoding gene, suggesting their involvement in LGT. In addition, several coding genes likely involved in sugar transport were probably acquired through LGT. Conclusion Our phylogenetic evidence suggests that LGT may be common among phylogenetically distinct members of the phylum Firmicutes inhabiting the intestinal tract of marine iguanas. PMID:20520734

Herpes simplex virus-mediated human hypoxanthine-guanine phosphoribosyltransferase gene transfer into neuronal cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Palella, T.D.; Silverman, L.J.; Schroll, C.T.

1988-01-01

The virtually complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, Lesch-Nyhan syndrome. Transfer of the HPRT gene into fibroblasts and lymphoblasts in vitro and into hematopoietic cells in vivo has been accomplished by other groups with retroviral-derived vectors. It appears to be necessary, however, to transfer the HPRT gene into neuronal cells to correct the neurological dysfunction of this disorder. The neurotropic virus herpes simplex virus type 1 has features that make it suitable for use as a vector to transfer the HPRT gene into neuronal tissue. This report describes the isolationmore » of an HPRT-deficient rat neuroma cell line, designated B103-4C, and the construction of a recombinant herpes simplex virus type 1 that contained human HPRT cDNA. These recombinant viruses were used to infect B103-4C cells. Infected cells expressed HPRT activity which was human in origin.« less

In vivo exposure to nitrogen dioxide (NO2) induces a decrease in calcitonin gene-related peptide (CGRP) and tachykinin immunoreactivity in guinea-pig peripheral airways.

PubMed

Lucchini, R E; Springall, D R; Chitano, P; Fabbri, L M; Polak, J M; Mapp, C E

1996-09-01

The mammalian respiratory tract is densely innervated by sensory and autonomic fibres. Subsets of the nerves contain bioactive regulatory peptides, such as substance P, calcitonin gene-related peptide (CGRP), and neurokinins. The sensory nervous system responds to inhaled irritants, resulting in a release of neuropeptides and, thus, a decrease in the peptide immunoreactivity of the fibres. We examined the effects of inhaled nitrogen dioxide (NO2), a well-known indoor and outdoor air pollutant, on pulmonary sensory neuropeptides. Guinea-pigs were exposed for 4 h to 18 parts per million (ppm) NO2 or to air (n = 5 each). At the end of the exposure, they were killed with urethane and their lungs were fixed in 1% paraformaldehyde in phosphate-buffered saline. Cryostat sections were stained with antisera to an anatomical nerve marker, protein gene product (PGP) 9.5, and to CGRP and tachykinins, utilizing the avidin-biotinylated peroxidase method. In the noncartilaginous airways (diameter < 250 microns) of NO2-exposed animals, less tachykinin- and CGRP-immunoreactive nerve fibres were found compared with controls. No change was seen in the total nerve fibre distribution (PGP 9.5). It is concluded that the peptidergic nerves of guinea-pig peripheral airways are a sensitive indicator of exposure to nitrogen dioxide.

Winter flounder antifreeze protein genes: demonstration of a cold-inducible promoter and gene transfer to other species

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, R.C.; Gourlie, B.; Price, J.

1987-05-01

During the late fall and winter, the winter flounder produces a family of unique antifreeze proteins (AFP) to prevent the lethal formation of ice crystals in its blood. They have been able to induce winter flounder AFP mRNA synthesis in vivo by lowering the ambient temperature of the fish from 18/sup 0/C in the summer months when AFP synthesis is at a minimum to 4/sup 0/C. Furthermore, they have demonstrated and thoroughly investigated this cold induction of AFP mRNA synthesis in vitro in isolated liver tissue and in nuclear preparations isolated from liver tissue. A drug selection vector (pRSV/sub gpt/)more » which uses RSV promoter for the expression of xanthine-guanine phosphoribosyltransferase (gpt) gene and contains an AFP gene and 1.7 kb of its 5' upstream control region has been constructed for studies of gene transfer into cells of other fish species. These studies were made using a variety of gene transfer techniques into tissue culture cell lines derived from rainbow trout, bluegill, and salmon. Drug resistant colonies from all three species have been obtained and the presence of AFP DNA has been positively identified by Southern analysis. In addition, Northern blot analysis has shown that both gpt gene and AFP gene are active in these cells since mRNA/sub gpt/ and mRNA/sub AFP/ can be detected by probing with the respective gene sequences.« less

Alteration of Airway Reactivity and Reduction of Ryanodine Receptor Expression by Cigarette Smoke in Mice.

PubMed

Donovan, Chantal; Seow, Huei Jiunn; Royce, Simon G; Bourke, Jane E; Vlahos, Ross

2015-10-01

Small airways are a major site of airflow limitation in chronic obstructive pulmonary disease (COPD). Despite the detrimental effects of long-term smoking in COPD, the effects of acute cigarette smoke (CS) exposure on small airway reactivity have not been fully elucidated. Balb/C mice were exposed to room air (sham) or CS for 4 days to cause airway inflammation. Changes in small airway lumen area in response to contractile agents were measured in lung slices in situ using phase-contrast microscopy. Separate slices were pharmacologically maintained at constant intracellular Ca(2+) using caffeine/ryanodine before contractile measurements. Gene and protein analysis of contractile signaling pathways were performed on separate lungs. Monophasic contraction to serotonin became biphasic after CS exposure, whereas contraction to methacholine was unaltered. This altered pattern of contraction was normalized by caffeine/ryanodine. Expression of contractile agonist-specific receptors was unaltered; however, all isoforms of the ryanodine receptor were down-regulated. This is the first study to show that acute CS exposure selectively alters small airway contraction to serotonin and down-regulates ryanodine receptors involved in maintaining Ca(2+) oscillations in airway smooth muscle. Understanding the contribution of ryanodine receptors to altered airway reactivity may inform the development of novel treatment strategies for COPD.

Gene transfer from a parasitic flowering plant to a fern

PubMed Central

Davis, Charles C; Anderson, William R; Wurdack, Kenneth J

2005-01-01

The rattlesnake fern (Botrychium virginianum (L.) Sw.) is obligately mycotrophic and widely distributed across the northern hemisphere. Three mitochondrial gene regions place this species with other ferns in Ophioglossaceae, while two regions place it as a member of the largely parasitic angiosperm order Santalales (sandalwoods and mistletoes). These discordant phylogenetic placements suggest that part of the genome in B. virginianum was acquired by horizontal gene transfer (HGT), perhaps from root-parasitic Loranthaceae. These transgenes are restricted to B. virginianum and occur across the range of the species. Molecular and life-history traits indicate that the transfer preceded the global expansion of B. virginianum, and that the latter may have happened very rapidly. This is the first report of HGT from an angiosperm to a fern, through either direct parasitism or the mediation of interconnecting fungal symbionts. PMID:16191635

Association of IL-13 gene polymorphisms with airway hyperresponsiveness in a Japanese adult asthmatic population.

PubMed

Utsumi, Yu; Sasaki, Nobuhito; Nagashima, Hiromi; Suzuki, Naomi; Nakamura, Yutaka; Yamashita, Masahiro; Kobayashi, Hitoshi; Yamauchi, Kohei

2013-09-01

A single nucleotide polymorphism (SNP; rs20541) in the IL-13 gene has been recognized as a risk factor for asthma. This SNP causes Arg to Gln (Q) substitution at position 110 in the mature IL-13 protein. We have recently showed that FEV1 in asthmatics with the Q110 variant of IL-13 declined faster, and progressive airway remodeling was observed in these subjects (Wynn, 2003 [1]). However, the effects of the IL-13 variant on airway hyperresponsiveness (AHR) remain to be elucidated. We analyzed the relationship between SNP rs20541 in IL-13 and AHR in asthmatics. We recruited 182 asthmatics who visited the asthma outpatient clinic at Iwate Medical University Hospital from 2006 to 2011. Subjects were genotyped for rs20541. Asthma severity, atopic status, age of asthma onset, serum IgE concentration, AHR, and pulmonary function were studied in these subjects. AHR was measured using the continuous methacholine inhalation method (Astograph; Chest; Tokyo, Japan). Genotyping of rs20541 revealed 26 A/A, 77 A/G, and 79 G/G patient genotypes. The D min (U) of the 3 genotypes was 1.17±0.300 in A/A, 1.99±0.35 in A/G, and 2.85±0.39 in G/G. The D min in the 3 genotypes was significantly different. Spirometric data revealed that % FEV1 and % FEF75 were significantly different among the 3 groups of IL-13 genotypes, whereas no significant differences were observed in therapeutic steps, atopic status, house dust mite sensitization, or serum IgE concentration. The SNP rs20541 in IL-13 was associated with AHR in Japanese adult asthmatics. Copyright © 2013 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.

Relapsing polychondritis and airway involvement.

PubMed

Ernst, Armin; Rafeq, Samaan; Boiselle, Phillip; Sung, Arthur; Reddy, Chakravarthy; Michaud, Gaetane; Majid, Adnan; Herth, Felix J F; Trentham, David

2009-04-01

To assess the prevalence and characteristics of airway involvement in relapsing polychondritis (RP). Retrospective chart review and data analysis of RP patients seen in the Rheumatology Clinic and the Complex Airway Center at Beth Israel Deaconess Medical Center from January 2004 through February 2008. RP was diagnosed in 145 patients. Thirty-one patients had airway involvement, a prevalence of 21%. Twenty-two patients were women (70%), and they were between 11 and 61 years of age (median age, 42 years) at the time of first symptoms. Airway symptoms were the first manifestation of disease in 17 patients (54%). Dyspnea was the most common symptom in 20 patients (64%), followed by cough, stridor, and hoarseness. Airway problems included the following: subglottic stenosis (n = 8; 26%); focal and diffuse malacia (n = 15; 48%); and focal stenosis in different areas of the bronchial tree in the rest of the patients. Twelve patients (40%) required and underwent intervention including balloon dilatation, stent placement, tracheotomy, or a combination of the above with good success. The majority of patients experienced improvement in airway symptoms after intervention. One patient died during the follow-up period from the progression of airway disease. The rest of the patients continue to undergo periodic evaluation and intervention. In this largest cohort described in the English language literature, we found symptomatic airway involvement in RP to be common and at times severe. The nature of airway problems is diverse, with tracheomalacia being the most common. Airway intervention is frequently required and in experienced hands results in symptom improvement.

Airway recovery after face transplantation.

PubMed

Fischer, Sebastian; Wallins, Joe S; Bueno, Ericka M; Kueckelhaus, Maximilian; Chandawarkar, Akash; Diaz-Siso, J Rodrigo; Larson, Allison; Murphy, George F; Annino, Donald J; Caterson, Edward J; Pomahac, Bohdan

2014-12-01

Severe facial injuries can compromise the upper airway by reducing airway volume, obstructing or obliterating the nasal passage, and interfering with oral airflow. Besides the significant impact on quality of life, upper airway impairments can have life-threatening or life-altering consequences. The authors evaluated improvements in functional airway after face transplantation. Between 2009 and 2011, four patients underwent face transplantation at the authors' institution, the Brigham and Women's Hospital. Patients were examined preoperatively and postoperatively and their records reviewed for upper airway infections and sleeping disorders. The nasal mucosa was biopsied after face transplantation and analyzed using scanning electron microscopy. Volumetric imaging software was used to evaluate computed tomographic scans of the upper airway and assess airway volume changes before and after transplantation. Before transplantation, two patients presented an exposed naked nasal cavity and two suffered from occlusion of the nasal passage. Two patients required tracheostomy tubes and one had a prosthetic nose. Sleeping disorders were seen in three patients, and chronic cough was diagnosed in one. After transplantation, there was no significant improvement in sleeping disorders. The incidence of sinusitis increased because of mechanical interference of the donor septum and disappeared after surgical correction. All patients were decannulated after transplantation and were capable of nose breathing. Scanning electron micrographs of the respiratory mucosa revealed viable tissue capable of mucin production. Airway volume significantly increased in all patients. Face transplantation successfully restored the upper airway in four patients. Unhindered nasal breathing, viable respiratory mucosa, and a significant increase in airway volume contributed to tracheostomy decannulation.

TRPA1 is a major oxidant sensor in murine airway sensory neurons

PubMed Central

Bessac, Bret F.; Sivula, Michael; von Hehn, Christian A.; Escalera, Jasmine; Cohn, Lauren; Jordt, Sven-Eric

2008-01-01

Sensory neurons in the airways are finely tuned to respond to reactive chemicals threatening airway function and integrity. Nasal trigeminal nerve endings are particularly sensitive to oxidants formed in polluted air and during oxidative stress as well as to chlorine, which is frequently released in industrial and domestic accidents. Oxidant activation of airway neurons induces respiratory depression, nasal obstruction, sneezing, cough, and pain. While normally protective, chemosensory airway reflexes can provoke severe complications in patients affected by inflammatory airway conditions like rhinitis and asthma. Here, we showed that both hypochlorite, the oxidizing mediator of chlorine, and hydrogen peroxide, a reactive oxygen species, activated Ca2+ influx and membrane currents in an oxidant-sensitive subpopulation of chemosensory neurons. These responses were absent in neurons from mice lacking TRPA1, an ion channel of the transient receptor potential (TRP) gene family. TRPA1 channels were strongly activated by hypochlorite and hydrogen peroxide in primary sensory neurons and heterologous cells. In tests of respiratory function, Trpa1–/– mice displayed profound deficiencies in hypochlorite- and hydrogen peroxide–induced respiratory depression as well as decreased oxidant-induced pain behavior. Our results indicate that TRPA1 is an oxidant sensor in sensory neurons, initiating neuronal excitation and subsequent physiological responses in vitro and in vivo. PMID:18398506

Histone deacetylase inhibitors up-regulate LL-37 expression independent of toll-like receptor mediated signalling in airway epithelial cells.

PubMed

Liu, Quan; Liu, Juan; Roschmann, Kristina Irene Lisolette; van Egmond, Danielle; Golebski, Korneliusz; Fokkens, Wytske Johanna; Wang, Dehui; van Drunen, Cornelis Maria

2013-04-11

HDAC inhibitors have been proposed as anticancer agents. However, their roles in innate genes expression remain not well known. Cathelicidin LL-37 is one of the few human bactericidal peptides, but the regulation of histone acetylation on LL-37 expression in airway epithelium remains largely unknown. Therefore, we investigated the effects of two non-selective HDACi, trichostatin A (TSA) and sodium butyrate (SB), on the expression of the cathelicidin LL-37 in human airway epithelial cells. LL37 in human NCI-H292 airway epithelial cells and the primary cultures of normal nasal epithelial cells(PNEC) in response to HDAC inhibitors with or without poly (I:C) stimulation was assessed using real-time PCR and western blot. In parallel, IL-6 expression was evaluated by ELISA. Our results showed that HDAC inhibitors up-regulated LL-37 gene expression independent of poly (I:C) stimulation in PNEC as well as in NCI-H292 cells. HDAC inhibitors increased LL37 protein expression in NCI-H292 cells but not in PNEC. In addition, HDAC inhibitors significantly inhibited poly (I:C)-induced IL-6 production in both of the epithelial cells. In conclusion, HDAC inhibitors directly up-regulated LL-37 gene expression in human airway epithelial cells.

Histone deacetylase inhibitors up-regulate LL-37 expression independent of toll-like receptor mediated signalling in airway epithelial cells

PubMed Central

2013-01-01

HDAC inhibitors have been proposed as anticancer agents. However, their roles in innate genes expression remain not well known. Cathelicidin LL-37 is one of the few human bactericidal peptides, but the regulation of histone acetylation on LL-37 expression in airway epithelium remains largely unknown. Therefore, we investigated the effects of two non-selective HDACi, trichostatin A (TSA) and sodium butyrate (SB), on the expression of the cathelicidin LL-37 in human airway epithelial cells. LL37 in human NCI-H292 airway epithelial cells and the primary cultures of normal nasal epithelial cells(PNEC) in response to HDAC inhibitors with or without poly (I:C) stimulation was assessed using real-time PCR and western blot. In parallel, IL-6 expression was evaluated by ELISA. Our results showed that HDAC inhibitors up-regulated LL-37 gene expression independent of poly (I:C) stimulation in PNEC as well as in NCI-H292 cells. HDAC inhibitors increased LL37 protein expression in NCI-H292 cells but not in PNEC. In addition, HDAC inhibitors significantly inhibited poly (I:C)-induced IL-6 production in both of the epithelial cells. In conclusion, HDAC inhibitors directly up-regulated LL-37 gene expression in human airway epithelial cells. PMID:23577829

Histidine-rich stabilized polyplexes for cMet-directed tumor-targeted gene transfer

NASA Astrophysics Data System (ADS)

Kos, Petra; Lächelt, Ulrich; Herrmann, Annika; Mickler, Frauke Martina; Döblinger, Markus; He, Dongsheng; Krhač Levačić, Ana; Morys, Stephan; Bräuchle, Christoph; Wagner, Ernst

2015-03-01

Overexpression of the hepatocyte growth factor receptor/c-Met proto oncogene on the surface of a variety of tumor cells gives an opportunity to specifically target cancerous tissues. Herein, we report the first use of c-Met as receptor for non-viral tumor-targeted gene delivery. Sequence-defined oligomers comprising the c-Met binding peptide ligand cMBP2 for targeting, a monodisperse polyethylene glycol (PEG) for polyplex surface shielding, and various cationic (oligoethanamino) amide cores containing terminal cysteines for redox-sensitive polyplex stabilization, were assembled by solid-phase supported syntheses. The resulting oligomers exhibited a greatly enhanced cellular uptake and gene transfer over non-targeted control sequences, confirming the efficacy and target-specificity of the formed polyplexes. Implementation of endosomal escape-promoting histidines in the cationic core was required for gene expression without additional endosomolytic agent. The histidine-enriched polyplexes demonstrated stability in serum as well as receptor-specific gene transfer in vivo upon intratumoral injection. The co-formulation with an analogous PEG-free cationic oligomer led to a further compaction of pDNA polyplexes with an obvious change of shape as demonstrated by transmission electron microscopy. Such compaction was critically required for efficient intravenous gene delivery which resulted in greatly enhanced, cMBP2 ligand-dependent gene expression in the distant tumor.Overexpression of the hepatocyte growth factor receptor/c-Met proto oncogene on the surface of a variety of tumor cells gives an opportunity to specifically target cancerous tissues. Herein, we report the first use of c-Met as receptor for non-viral tumor-targeted gene delivery. Sequence-defined oligomers comprising the c-Met binding peptide ligand cMBP2 for targeting, a monodisperse polyethylene glycol (PEG) for polyplex surface shielding, and various cationic (oligoethanamino) amide cores containing

Massive gene transfer and extensive RNA editing of a symbiotic dinoflagellate plastid genome.

PubMed

Mungpakdee, Sutada; Shinzato, Chuya; Takeuchi, Takeshi; Kawashima, Takeshi; Koyanagi, Ryo; Hisata, Kanako; Tanaka, Makiko; Goto, Hiroki; Fujie, Manabu; Lin, Senjie; Satoh, Nori; Shoguchi, Eiichi

2014-05-31

Genome sequencing of Symbiodinium minutum revealed that 95 of 109 plastid-associated genes have been transferred to the nuclear genome and subsequently expanded by gene duplication. Only 14 genes remain in plastids and occur as DNA minicircles. Each minicircle (1.8-3.3 kb) contains one gene and a conserved noncoding region containing putative promoters and RNA-binding sites. Nine types of RNA editing, including a novel G/U type, were discovered in minicircle transcripts but not in genes transferred to the nucleus. In contrast to DNA editing sites in dinoflagellate mitochondria, which tend to be highly conserved across all taxa, editing sites employed in DNA minicircles are highly variable from species to species. Editing is crucial for core photosystem protein function. It restores evolutionarily conserved amino acids and increases peptidyl hydropathy. It also increases protein plasticity necessary to initiate photosystem complex assembly. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

41 CFR 102-75.435 - Does the Airport and Airway Development Act of 1970, as amended (Airport Act of 1970), apply to...

Code of Federal Regulations, 2011 CFR

2011-01-01

... airports to State and local agencies? No, the Airport and Airway Development Act of 1970, as amended (49 U... Airway Development Act of 1970, as amended (Airport Act of 1970), apply to the transfer of airports to State and local agencies? 102-75.435 Section 102-75.435 Public Contracts and Property Management Federal...

41 CFR 102-75.435 - Does the Airport and Airway Development Act of 1970, as amended (Airport Act of 1970), apply to...

Code of Federal Regulations, 2013 CFR

2013-07-01

... airports to State and local agencies? No, the Airport and Airway Development Act of 1970, as amended (49 U... Airway Development Act of 1970, as amended (Airport Act of 1970), apply to the transfer of airports to State and local agencies? 102-75.435 Section 102-75.435 Public Contracts and Property Management Federal...

41 CFR 102-75.435 - Does the Airport and Airway Development Act of 1970, as amended (Airport Act of 1970), apply to...

Code of Federal Regulations, 2012 CFR

2012-01-01

... airports to State and local agencies? No, the Airport and Airway Development Act of 1970, as amended (49 U... Airway Development Act of 1970, as amended (Airport Act of 1970), apply to the transfer of airports to State and local agencies? 102-75.435 Section 102-75.435 Public Contracts and Property Management Federal...

41 CFR 102-75.435 - Does the Airport and Airway Development Act of 1970, as amended (Airport Act of 1970), apply to...

Code of Federal Regulations, 2010 CFR

2010-07-01

... airports to State and local agencies? No, the Airport and Airway Development Act of 1970, as amended (49 U... Airway Development Act of 1970, as amended (Airport Act of 1970), apply to the transfer of airports to State and local agencies? 102-75.435 Section 102-75.435 Public Contracts and Property Management Federal...

41 CFR 102-75.435 - Does the Airport and Airway Development Act of 1970, as amended (Airport Act of 1970), apply to...

Code of Federal Regulations, 2014 CFR

2014-01-01

... airports to State and local agencies? No, the Airport and Airway Development Act of 1970, as amended (49 U... Airway Development Act of 1970, as amended (Airport Act of 1970), apply to the transfer of airports to State and local agencies? 102-75.435 Section 102-75.435 Public Contracts and Property Management Federal...

Bacterial gene transfer by natural genetic transformation in the environment.

PubMed Central

Lorenz, M G; Wackernagel, W

1994-01-01

Natural genetic transformation is the active uptake of free DNA by bacterial cells and the heritable incorporation of its genetic information. Since the famous discovery of transformation in Streptococcus pneumoniae by Griffith in 1928 and the demonstration of DNA as the transforming principle by Avery and coworkers in 1944, cellular processes involved in transformation have been studied extensively by in vitro experimentation with a few transformable species. Only more recently has it been considered that transformation may be a powerful mechanism of horizontal gene transfer in natural bacterial populations. In this review the current understanding of the biology of transformation is summarized to provide the platform on which aspects of bacterial transformation in water, soil, and sediments and the habitat of pathogens are discussed. Direct and indirect evidence for gene transfer routes by transformation within species and between different species will be presented, along with data suggesting that plasmids as well as chromosomal DNA are subject to genetic exchange via transformation. Experiments exploring the prerequisites for transformation in the environment, including the production and persistence of free DNA and factors important for the uptake of DNA by cells, will be compiled, as well as possible natural barriers to transformation. The efficiency of gene transfer by transformation in bacterial habitats is possibly genetically adjusted to submaximal levels. The fact that natural transformation has been detected among bacteria from all trophic and taxonomic groups including archaebacteria suggests that transformability evolved early in phylogeny. Probable functions of DNA uptake other than gene acquisition will be discussed. The body of information presently available suggests that transformation has a great impact on bacterial population dynamics as well as on bacterial evolution and speciation. PMID:7968924

Investigation of mucus transport in an idealized lung airway model using multiphase CFD analysis

NASA Astrophysics Data System (ADS)

Rajendran, Rahul; Banerjee, Arindam

2015-11-01

Mucus, a Bingham fluid is transported in the pulmonary airways by consistent beating of the cilia and exhibits a wide range of physical properties in response to the core air flow and various pathological conditions. A better understanding of the interfacial instability is required as it plays a crucial role in gas transport, mixing, mucus clearance and drug delivery. In the current study, mucus is modelled as a Newtonian fluid and the two phase gas-liquid flow in the airways is investigated using an inhomogeneous Eulerian-Eulerian approach. The complex interface between the phases is tracked using the conventional VOF (Volume of Fluid) method. Results from our CFD simulations which are performed in idealized single and double bifurcation geometries will be presented and the influence of airflow rate, mucus layer thickness, mucus viscosity, airway geometry (branching & diameter) and surface tension on mucus flow behavior will be discussed. Mean mucus layer thickness, pressure drop due to momentum transfer & increased airway resistance, mucus transport speed and the flow morphology will be compared to existing experimental and theoretical data.

Genome-wide identification of horizontal gene transfer in Fusarium verticillioides

USDA-ARS?s Scientific Manuscript database

Horizontal gene transfer (HGT), the exchange and stable integration of genetic material between different lineages, breaks species boundaries and generates new biological diversity. In eukaryotes, despite potential barriers, like the nuclear envelope and multicellularity, HGT may be facilitated by t...

[Quality assurance in airway management: education and training for difficult airway management].

PubMed

Kaminoh, Yoshiroh

2006-01-01

Respiratory problem is one of the main causes of death or severe brain damage in perioperative period. Three major factors of respiratory problem are esophageal intubation, inadequate ventilation, and difficult airway. The wide spread of pulse oximeter and capnograph reduced the incidences of esophageal intubation and inadequate ventilation, but the difficult airway still occupies the large portion in the causes of adverse events during anesthesia. "Practice guideline for management of the difficult airway" was proposed by American Society of Anesthesiologists (ASA) in 1992 and 2002. Improvement of knowledge, technical skills, and cognitive skills are necessary for the education and training of the difficult airway management. "The practical seminar of difficult airway management (DAM practical seminar)" has been cosponsored by the Japanese Association of Medical Simulation (JAMS) in the 51 st and 52 nd annual meetings of Japanese Society of Anesthesiologists and the 24th annual meeting of Japanese Society for Clinical Anesthesia. The DAM practical seminar is composed of the lecture session for ASA difficult airway algorithm, the hands-on training session for technical skills, and the scenario-based training session for cognitive skills. Ninty six Japanese anesthesiologists have completed the DAM practical seminar in one year. "The DAM instructor course" should be immediately prepared to organize the seminar more frequently.

Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors.

PubMed Central

Podsakoff, G; Wong, K K; Chatterjee, S

1994-01-01

Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthesis inhibitors fluorodeoxyuridine and aphidicolin or by contact inhibition induced by confluence and serum starvation. Cells in logarithmic growth or DNA synthesis arrest were transduced with vCWR:beta gal, an AAV-based vector encoding beta-galactosidase under Rous sarcoma virus long terminal repeat promoter control. Under each condition tested, vCWR:beta Gal expression in nondividing cells was at least equivalent to that in actively proliferating cells, suggesting that mechanisms for virus attachment, nuclear transport, virion uncoating, and perhaps some limited second-strand synthesis of AAV vectors were present in nondividing cells. Southern hybridization analysis of vector sequences from cells transduced while in DNA synthetic arrest and expanded after release of the block confirmed ultimate integration of the vector genome into cellular chromosomal DNA. These findings may provide the basis for the use of AAV-based vectors for gene transfer into quiescent cell populations such as totipotent hematopoietic stem cells. Images PMID:8057446

Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors.

PubMed

Podsakoff, G; Wong, K K; Chatterjee, S

1994-09-01

Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthesis inhibitors fluorodeoxyuridine and aphidicolin or by contact inhibition induced by confluence and serum starvation. Cells in logarithmic growth or DNA synthesis arrest were transduced with vCWR:beta gal, an AAV-based vector encoding beta-galactosidase under Rous sarcoma virus long terminal repeat promoter control. Under each condition tested, vCWR:beta Gal expression in nondividing cells was at least equivalent to that in actively proliferating cells, suggesting that mechanisms for virus attachment, nuclear transport, virion uncoating, and perhaps some limited second-strand synthesis of AAV vectors were present in nondividing cells. Southern hybridization analysis of vector sequences from cells transduced while in DNA synthetic arrest and expanded after release of the block confirmed ultimate integration of the vector genome into cellular chromosomal DNA. These findings may provide the basis for the use of AAV-based vectors for gene transfer into quiescent cell populations such as totipotent hematopoietic stem cells.

Evidence for extensive horizontal gene transfer from the draft genome of a tardigrade

PubMed Central

Boothby, Thomas C.; Tenlen, Jennifer R.; Smith, Frank W.; Wang, Jeremy R.; Patanella, Kiera A.; Osborne Nishimura, Erin; Tintori, Sophia C.; Li, Qing; Jones, Corbin D.; Yandell, Mark; Glasscock, Jarret; Goldstein, Bob

2015-01-01

Horizontal gene transfer (HGT), or the transfer of genes between species, has been recognized recently as more pervasive than previously suspected. Here, we report evidence for an unprecedented degree of HGT into an animal genome, based on a draft genome of a tardigrade, Hypsibius dujardini. Tardigrades are microscopic eight-legged animals that are famous for their ability to survive extreme conditions. Genome sequencing, direct confirmation of physical linkage, and phylogenetic analysis revealed that a large fraction of the H. dujardini genome is derived from diverse bacteria as well as plants, fungi, and Archaea. We estimate that approximately one-sixth of tardigrade genes entered by HGT, nearly double the fraction found in the most extreme cases of HGT into animals known to date. Foreign genes have supplemented, expanded, and even replaced some metazoan gene families within the tardigrade genome. Our results demonstrate that an unexpectedly large fraction of an animal genome can be derived from foreign sources. We speculate that animals that can survive extremes may be particularly prone to acquiring foreign genes. PMID:26598659

Evidence for extensive horizontal gene transfer from the draft genome of a tardigrade.

PubMed

Boothby, Thomas C; Tenlen, Jennifer R; Smith, Frank W; Wang, Jeremy R; Patanella, Kiera A; Nishimura, Erin Osborne; Tintori, Sophia C; Li, Qing; Jones, Corbin D; Yandell, Mark; Messina, David N; Glasscock, Jarret; Goldstein, Bob

2015-12-29

Horizontal gene transfer (HGT), or the transfer of genes between species, has been recognized recently as more pervasive than previously suspected. Here, we report evidence for an unprecedented degree of HGT into an animal genome, based on a draft genome of a tardigrade, Hypsibius dujardini. Tardigrades are microscopic eight-legged animals that are famous for their ability to survive extreme conditions. Genome sequencing, direct confirmation of physical linkage, and phylogenetic analysis revealed that a large fraction of the H. dujardini genome is derived from diverse bacteria as well as plants, fungi, and Archaea. We estimate that approximately one-sixth of tardigrade genes entered by HGT, nearly double the fraction found in the most extreme cases of HGT into animals known to date. Foreign genes have supplemented, expanded, and even replaced some metazoan gene families within the tardigrade genome. Our results demonstrate that an unexpectedly large fraction of an animal genome can be derived from foreign sources. We speculate that animals that can survive extremes may be particularly prone to acquiring foreign genes.

Use of electroporation for high-molecular-weight DNA-mediated gene transfer.

PubMed

Jastreboff, M M; Ito, E; Bertino, J R; Narayanan, R

1987-08-01

Electroporation was used to introduce high-molecular-weight DNA into murine hematopoietic cells and NIH3T3 cells. CCRF-CEM cells were stably transfected with SV2NEO plasmid and the genomic DNA from G-418-resistant clones (greater than 65 kb) was introduced into mouse bone marrow and NIH3T3 cells by electroporation. NEO sequences and expression were detected in the hematopoietic tissues of lethally irradiated mice, with 24% of individual spleen colonies expressing NEO. The frequency of genomic DNA transfer into NIH3T3 cells was 0.25 X 10(-3). Electroporation thus offers a powerful mode of gene transfer not only of cloned genes but also of high-molecular-weight DNA into cells.

Inhibition of TRPC3 downregulates airway hyperresponsiveness, remodeling of OVA-sensitized mouse

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Lingwei; Li, Jie; Integrated Chinese and Western Medicine Postdoctoral Research Station, Jinan University, Guangzhou

Airway hyperresponsiveness (AHR), airway remodeling and inflammation are the fundamental pathological alterations that occur in asthma. Transient receptor potential canonical 3 (TRPC3) has been implicated in diverse functions of airway smooth muscle cells (ASMCs) in asthma. However, the underlying mechanisms remain incompletely understood. We investigated the mRNA and protein expression of TRPC3 in ASMCs from normal and OVA-sensitized mouse. And the effects of inhibition or knockdown of TRPC3 with Ethyl-1- (4- (2,3,3-trichloroacrylamide) phenyl) −5 - (trifluoromethyl) -1H -pyrazole -4-carboxylate (Pyr3) and lentiviral shRNA on OVA-sensitized mouse AHR, airway remodeling, circulating inflammatory cytokines, cell proliferation and migration. We found that TRPC3 mRNAmore » and protein expression levels were significantly increased in ASMCs from OVA-sensitized mouse. Inhibiting TRPC3 with continuous subcutaneous administration of Pyr3 decreased enhanced pause (Penh) of OVA-sensitized mouse. Meanwhile, both Pyr3 and lentiviral shRNA treatment of ASMCs in OVA-sensitized mouse significantly decreased their proliferation and migration. These results suggest that TRPC3 plays a critical role in asthma and represents a promising new target for asthma treatment. - Highlights: • Penh, airway remodeling and the gene expression and protein of TRPC3 are increased in OVA-sensitized mice. • Inhibition of TRPC3 suppresses the OVA-sensitized mice Penh and airway remodeling. • Inhibition of TRPC3 decreases OVA-sensitized mice ASMC proliferation and migration.« less

Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

PubMed Central

Rodriguez-Osorio, Nelida; Wang, Zhongde; Kasinathan, Poothappillai; Page, Grier P; Robl, James M; Memili, Erdogan

2009-01-01

Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT) is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT). Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively) have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF) than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively). However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping. Conclusion The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research. PMID:19393066

Root parasitic plant Orobanche aegyptiaca and shoot parasitic plant Cuscuta australis obtained Brassicaceae-specific strictosidine synthase-like genes by horizontal gene transfer

PubMed Central

2014-01-01

Background Besides gene duplication and de novo gene generation, horizontal gene transfer (HGT) is another important way of acquiring new genes. HGT may endow the recipients with novel phenotypic traits that are important for species evolution and adaption to new ecological niches. Parasitic systems expectedly allow the occurrence of HGT at relatively high frequencies due to their long-term physical contact. In plants, a number of HGT events have been reported between the organelles of parasites and the hosts, but HGT between host and parasite nuclear genomes has rarely been found. Results A thorough transcriptome screening revealed that a strictosidine synthase-like (SSL) gene in the root parasitic plant Orobanche aegyptiaca and the shoot parasitic plant Cuscuta australis showed much higher sequence similarities with those in Brassicaceae than with those in their close relatives, suggesting independent gene horizontal transfer events from Brassicaceae to these parasites. These findings were strongly supported by phylogenetic analysis and their identical unique amino acid residues and deletions. Intriguingly, the nucleus-located SSL genes in Brassicaceae belonged to a new member of SSL gene family, which were originated from gene duplication. The presence of introns indicated that the transfer occurred directly by DNA integration in both parasites. Furthermore, positive selection was detected in the foreign SSL gene in O. aegyptiaca but not in C. australis. The expression of the foreign SSL genes in these two parasitic plants was detected in multiple development stages and tissues, and the foreign SSL gene was induced after wounding treatment in C. australis stems. These data imply that the foreign genes may still retain certain functions in the recipient species. Conclusions Our study strongly supports that parasitic plants can gain novel nuclear genes from distantly related host species by HGT and the foreign genes may execute certain functions in the new hosts

Root parasitic plant Orobanche aegyptiaca and shoot parasitic plant Cuscuta australis obtained Brassicaceae-specific strictosidine synthase-like genes by horizontal gene transfer.

PubMed

Zhang, Dale; Qi, Jinfeng; Yue, Jipei; Huang, Jinling; Sun, Ting; Li, Suoping; Wen, Jian-Fan; Hettenhausen, Christian; Wu, Jinsong; Wang, Lei; Zhuang, Huifu; Wu, Jianqiang; Sun, Guiling

2014-01-13

Besides gene duplication and de novo gene generation, horizontal gene transfer (HGT) is another important way of acquiring new genes. HGT may endow the recipients with novel phenotypic traits that are important for species evolution and adaption to new ecological niches. Parasitic systems expectedly allow the occurrence of HGT at relatively high frequencies due to their long-term physical contact. In plants, a number of HGT events have been reported between the organelles of parasites and the hosts, but HGT between host and parasite nuclear genomes has rarely been found. A thorough transcriptome screening revealed that a strictosidine synthase-like (SSL) gene in the root parasitic plant Orobanche aegyptiaca and the shoot parasitic plant Cuscuta australis showed much higher sequence similarities with those in Brassicaceae than with those in their close relatives, suggesting independent gene horizontal transfer events from Brassicaceae to these parasites. These findings were strongly supported by phylogenetic analysis and their identical unique amino acid residues and deletions. Intriguingly, the nucleus-located SSL genes in Brassicaceae belonged to a new member of SSL gene family, which were originated from gene duplication. The presence of introns indicated that the transfer occurred directly by DNA integration in both parasites. Furthermore, positive selection was detected in the foreign SSL gene in O. aegyptiaca but not in C. australis. The expression of the foreign SSL genes in these two parasitic plants was detected in multiple development stages and tissues, and the foreign SSL gene was induced after wounding treatment in C. australis stems. These data imply that the foreign genes may still retain certain functions in the recipient species. Our study strongly supports that parasitic plants can gain novel nuclear genes from distantly related host species by HGT and the foreign genes may execute certain functions in the new hosts.

Airway malacia in children with achondroplasia.

PubMed

Dessoffy, Kimberly E; Modaff, Peggy; Pauli, Richard M

2014-02-01

This study was undertaken to assess the frequency of airway malacia in infants and young children with achondroplasia, a population well known to be at risk for a variety of respiratory problems. We also wished to evaluate what, if any, contribution airway malacia makes to the complex respiratory issues that may be present in those with achondroplasia. Retrospective chart review of all infants and young children with achondroplasia who were assessed through the Midwest Regional Bone Dysplasia Clinics from 1985 through 2012 (n = 236) was completed. Records of comprehensive clinical examinations, polysomnographic assessments, and airway visualization were reviewed and abstracted using a data collection form. Analyses were completed comparing the group with and those without evidence for airway malacia. Thirteen of 236 patients (5.5%) were found to have airway malacia. Most of those affected had lower airway involvement (9/13). The presence of airway malacia was correlated with an increased occurrence of obstructive sleep apnea as well as need for oxygen supplementation, airway surgeries and tracheostomy placement. Although estimates of the frequency of airway malacia in the general population are limited, its frequency in children with achondroplasia appears to be much higher than any published general population estimate. The presence of airway malacia appears to confound other breathing abnormalities in this population and results in the need for more invasive airway treatments. © 2013 Wiley Periodicals, Inc.

Investigating rate-limiting barriers to nanoscale nonviral gene transfer with nanobiophotonics

NASA Astrophysics Data System (ADS)

Chen, Hunter H.

Nucleic acids are a novel class of therapeutics poised to address many unmet clinical needs. Safe and efficient delivery remains a significant challenge that has delayed the realization of the full therapeutic potential of nucleic acids. Nanoscale nonviral vectors offer an attractive alternative to viral vectors as natural and synthetic polymers or polypeptides may be rationally designed to meet the unique demands of individual applications. A mechanistic understanding of cellular barriers is necessary to develop guidelines for designing custom gene carriers which are expected to greatly impact this delivery challenge. The work herein focused on the relationships among nanocomplex stability, intracellular trafficking and unpacking kinetics, and DNA degradation. Ultrasensitive nanosensors based on QD-FRET were developed to characterize the biophysical properties of nanocomplexes and study these rate-limiting steps. Quantitative image analysis enabled the distributions of the subpopulation of condensed or released DNA to be determined within the major cellular compartments encountered during gene transfer. The steady state stability and unpacking kinetics within these compartments were found to impact transgene expression, elucidating multiple design strategies to achieve efficient gene transfer. To address enzymatic barriers, a novel two-step QD-FRET nanosensor was developed to analyze unpacking and DNA degradation simultaneously, which has not been accomplished previously. Bioresponsive strategies such as disulfide crosslinking and thermosensitivity were evaluated by QD-FRET and quantitative compartmental analysis as case studies to determine appropriate design specifications for thiolated polymers and thermoresponsive polypeptides. Relevant nanobiophotonic tools were developed as a platform to study major rate-limiting barriers to nanomedicine and demonstrated the feasibility of using mechanistic information gained from these tools to guide the rational design of

Horizontal gene transfer and gene dosage drives adaptation to wood colonization in a tree pathogen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dhillon, Braham; Feau, Nicolas; Aerts, Andrea L.

Some of the most damaging tree diseases are caused by pathogens that induce cankers, a stem deformation often lethal. To investigate the cause of this adaptation, we sequenced the genomes of poplar pathogens that do and do not cause cankers. We found a unique cluster of genes that produce secondary metabolites and are co-activated when the canker pathogen is grown on poplar wood and leaves. The gene genealogy is discordant with the species phylogeny, showing a signature of horizontal transfer from fungi associated with wood decay. Furthermore, genes encoding hemicellulose-degrading enzymes are up-regulated on poplar wood chips, with some havingmore » been acquired horizontally. In conclusion, we propose that adaptation to colonize poplar woody stems is the result of acquisition of these genes.« less

Horizontal gene transfer and gene dosage drives adaptation to wood colonization in a tree pathogen

DOE PAGES

Dhillon, Braham; Feau, Nicolas; Aerts, Andrea L.; ...

2015-03-02

Some of the most damaging tree diseases are caused by pathogens that induce cankers, a stem deformation often lethal. To investigate the cause of this adaptation, we sequenced the genomes of poplar pathogens that do and do not cause cankers. We found a unique cluster of genes that produce secondary metabolites and are co-activated when the canker pathogen is grown on poplar wood and leaves. The gene genealogy is discordant with the species phylogeny, showing a signature of horizontal transfer from fungi associated with wood decay. Furthermore, genes encoding hemicellulose-degrading enzymes are up-regulated on poplar wood chips, with some havingmore » been acquired horizontally. In conclusion, we propose that adaptation to colonize poplar woody stems is the result of acquisition of these genes.« less

Extraglottic airway devices: technology update.

PubMed

Sharma, Bimla; Sahai, Chand; Sood, Jayashree

2017-01-01

Extraglottic airway devices (EADs) have revolutionized the field of airway management. The invention of the laryngeal mask airway was a game changer, and since then, there have been several innovations to improve the EADs in design, functionality, safety and construction material. These have ranged from changes in the shape of the mask, number of cuffs and material used, like rubber, polyvinylchloride and latex. Phthalates, which were added to the construction material in order to increase device flexibility, were later omitted when this chemical was found to have serious adverse reproductive outcomes. The various designs brought out by numerous companies manufacturing EADs resulted in the addition of several devices to the airway market. These airway devices were put to use, many of them with inadequate or no evidence base regarding their efficacy and safety. To reduce the possibility of compromising the safety of the patient, the Difficult Airway Society (DAS) formed the Airway Device Evaluation Project Team (ADEPT) to strengthen the evidence base for airway equipment and vet the new extraglottic devices. A preuse careful analysis of the design and structure may help in better understanding of the functionality of a particular device. In the meantime, the search for the ideal EAD continues.

Persistent activation of interlinked type 2 airway epithelial gene networks in sputum-derived cells from aeroallergen-sensitized symptomatic asthmatics.

PubMed

Jones, Anya C; Troy, Niamh M; White, Elisha; Hollams, Elysia M; Gout, Alexander M; Ling, Kak-Ming; Kicic, Anthony; Stick, Stephen M; Sly, Peter D; Holt, Patrick G; Hall, Graham L; Bosco, Anthony

2018-01-24

Atopic asthma is a persistent disease characterized by intermittent wheeze and progressive loss of lung function. The disease is thought to be driven primarily by chronic aeroallergen-induced type 2-associated inflammation. However, the vast majority of atopics do not develop asthma despite ongoing aeroallergen exposure, suggesting additional mechanisms operate in conjunction with type 2 immunity to drive asthma pathogenesis. We employed RNA-Seq profiling of sputum-derived cells to identify gene networks operative at baseline in house dust mite-sensitized (HDM S ) subjects with/without wheezing history that are characteristic of the ongoing asthmatic state. The expression of type 2 effectors (IL-5, IL-13) was equivalent in both cohorts of subjects. However, in HDM S -wheezers they were associated with upregulation of two coexpression modules comprising multiple type 2- and epithelial-associated genes. The first module was interlinked by the hubs EGFR, ERBB2, CDH1 and IL-13. The second module was associated with CDHR3 and mucociliary clearance genes. Our findings provide new insight into the molecular mechanisms operative at baseline in the airway mucosa in atopic asthmatics undergoing natural aeroallergen exposure, and suggest that susceptibility to asthma amongst these subjects involves complex interactions between type 2- and epithelial-associated gene networks, which are not operative in equivalently sensitized/exposed atopic non-asthmatics.

Airway Protective Mechanisms

PubMed Central

Pitts, Teresa

2014-01-01

Cough and swallow are highly coordinated reflex behaviors whose common purpose is to protect the airway. The pharynx is the common tube for air and food/liquid movement from the mouth into the thorax, has been largely overlooked, and is potentially seen as just a passive space. The thyropharyngeus muscle responds to cough inducing stimuli to prepare a transient holding area for material that has been removed from the subglottic airway. The cricopharyngeus muscle participates with the larynx to ensure regulation of pressure when a bolus/air is moving from the upper airway through to the thorax (i.e inspiration or swallow) or the reverse (i.e expiration reflex or vomiting).These vital mechanisms have not been evaluated in clinical conditions, but could be impaired in many neurodegenerative diseases leading to aspiration pneumonia. These newly described airway protective mechanisms need further study, especially in healthy and pathologic human populations. PMID:24297325

Resistance gene transfer: induction of transducing phage by sub-inhibitory concentrations of antimicrobials is not correlated to induction of lytic phage

PubMed Central

Stanczak-Mrozek, Kinga I.; Laing, Ken G.

2017-01-01

Objectives: Horizontal gene transfer of antimicrobial resistance (AMR) genes between clinical isolates via transduction is poorly understood. MRSA are opportunistic pathogens resistant to all classes of antimicrobial agents but currently no strains are fully drug resistant. AMR gene transfer between Staphylococcus aureus isolates is predominantly due to generalized transduction via endogenous bacteriophage, and recent studies have suggested transfer is elevated during host colonization. The aim was to investigate whether exposure to sub-MIC concentrations of antimicrobials triggers bacteriophage induction and/or increased efficiency of AMR gene transfer. Methods: Isolates from MRSA carriers were exposed to nine antimicrobials and supernatants were compared for lytic phage particles and ability to transfer an AMR gene. A new technology, droplet digital PCR, was used to measure the concentration of genes in phage particles. Results: All antibiotics tested induced lytic phage and AMR gene transduction, although the ratio of transducing particles to lytic particles differed substantially for each antibiotic. Mupirocin induced the highest ratio of transducing versus lytic particles. Gentamicin and novobiocin reduced UV-induced AMR transduction. The genes carried in phage particles correlated with AMR transfer or lytic particle activity, suggesting antimicrobials influence which DNA sequences are packaged into phage particles. Conclusions: Sub-inhibitory antibiotics induce AMR gene transfer between clinical MRSA, while combination therapy with an inhibiting antibiotic could potentially alter AMR gene packaging into phage particles, reducing AMR transfer. In a continually evolving environment, pathogens have an advantage if they can transfer DNA while lowering the risk of lytic death. PMID:28369562

The effect of body weight on distal airway function and airway inflammation.

PubMed

van de Kant, Kim D G; Paredi, Paolo; Meah, Sally; Kalsi, Harpal S; Barnes, Peter J; Usmani, Omar S

Obesity is a global health problem that adversely influences the respiratory system. We assessed the effects of body mass index (BMI) on distal airway function and airway inflammation. Impulse oscillometry (IOS) as a measure of distal airway function, together with spirometry, were assessed in adults with a range of different BMIs. Airway inflammation was assessed with the fraction of exhaled nitric oxide (FeNO) and participants exhaled at various exhalation flows to determine alveolar and bronchial NO. In total 34 subjects were enrolled in the study; 19 subjects had a normal BMI (18.50-24.99), whilst 15 subjects were overweight (BMI 25.00-29.99), or obese (BMI ≥30). All subjects had normal spirometry. However, IOS measures of airway resistance (R) at 5Hz, 20Hz and frequency dependence (R 5-20 ) were elevated in overweight/obese individuals, compared to subjects with a normal BMI (median (interquartile range)); 5Hz: 0.41 (0.37, 0.45) vs. 0.32 (0.30, 0.37)kPa/l/s; 20Hz: 0.34 (0.30, 0.37) vs. 0.30 (0.26, 0.33)kPa/l/s; R 5-20 : 0.06 (0.04, 0.11) vs. 0.03 (0.01, 0.05)kPa/l/s; p<0.05), whereas airway reactance at 20Hz was decreased in overweight/obese individuals (20Hz: 0.07 (0.03, 0.09) vs. 0.10 (0.07, 0.13)kPa/l/s, p=0.009; 5Hz: -0.12 (-0.15, -0.10) vs. -0.10 (-0.13, -0.09)kPa/l/s, p=0.07). In contrast, within-breath IOS measures (a sign of expiratory flow limitation) and FeNO inflammatory measures, did not differ between groups (p>0.05). Being overweight has significant effects on distal and central airway function as determined by IOS, which is not detected by spirometry. Obesity does not influence airway inflammation as measured by FeNO. IOS is a reliable technique to identify airway abnormalities in the presence of normal spirometry in overweight people. Copyright © 2015 Asia Oceania Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.

Hydrodynamic liver gene transfer mechanism involves transient sinusoidal blood stasis and massive hepatocyte endocytic vesicles.

PubMed

Crespo, A; Peydró, A; Dasí, F; Benet, M; Calvete, J J; Revert, F; Aliño, S F

2005-06-01

The present study contributes to clarify the mechanism underlying the high efficacy of hepatocyte gene transfer mediated by hydrodynamic injection. Gene transfer experiments were performed employing the hAAT gene, and the efficacy and differential identification in mouse plasma of human transgene versus mouse gene was assessed by ELISA and proteomic procedures, respectively. By applying different experimental strategies such as cumulative dose-response efficacy, hemodynamic changes reflected by venous pressures, intravital microscopy, and morphological changes established by transmission electron microscopy, we found that: (a) cumulative multiple doses of transgene by hydrodynamic injection are efficient and well tolerated, resulting in therapeutic plasma levels of hAAT; (b) hydrodynamic injection mediates a transient inversion of intrahepatic blood flow, with circulatory stasis for a few minutes mainly in pericentral vein sinusoids; (c) transmission electron microscopy shows hydrodynamic injection to promote massive megafluid endocytic vesicles among hepatocytes around the central vein but not in hepatocytes around the periportal vein. We suggest that the mechanism of hydrodynamic liver gene transfer involves transient inversion of intrahepatic flow, sinusoidal blood stasis, and massive fluid endocytic vesicles in pericentral vein hepatocytes.

Difficult airway in a pediatric case of pycodysostosis

PubMed Central

Alkhalaf, Maizar M.; Ali, Hassan Mohamed; Al Otaibi, Rashed

2015-01-01

Pycodysostosis is a genetic autosomal rare disease with an incidence of 1:1.7 million births; the pathophysiology of the disease is related to mutation of cathepsin K gene. Sleep apnea, respiratory difficulties because of chest and oral abnormalities may cause a challenge to the anesthetist during intubation and/or mechanical ventilation. In this case report we will discuss a case of pycodysostosis with a difficult airway. PMID:25886438

Spontaneous Chitin Accumulation in Airways and Age-Related Fibrotic Lung Disease.

PubMed

Van Dyken, Steven J; Liang, Hong-Erh; Naikawadi, Ram P; Woodruff, Prescott G; Wolters, Paul J; Erle, David J; Locksley, Richard M

2017-04-20

The environmentally widespread polysaccharide chitin is degraded and recycled by ubiquitous bacterial and fungal chitinases. Although vertebrates express active chitinases from evolutionarily conserved loci, their role in mammalian physiology is unclear. We show that distinct lung epithelial cells secrete acidic mammalian chitinase (AMCase), which is required for airway chitinase activity. AMCase-deficient mice exhibit premature morbidity and mortality, concomitant with accumulation of environmentally derived chitin polymers in the airways and expression of pro-fibrotic cytokines. Over time, these mice develop spontaneous pulmonary fibrosis, which is ameliorated by restoration of lung chitinase activity by genetic or therapeutic approaches. AMCase-deficient epithelial cells express fibrosis-associated gene sets linked with cell stress pathways. Mice with lung fibrosis due to telomere dysfunction and humans with interstitial lung disease also accumulate excess chitin polymers in their airways. These data suggest that altered chitin clearance could exacerbate fibrogenic pathways in the setting of lung diseases characterized by epithelial cell dysfunction. Copyright © 2017 Elsevier Inc. All rights reserved.

Definitive airway management after pre-hospital supraglottic airway insertion: Outcomes and a management algorithm for trauma patients.

PubMed

Hernandez, Matthew C; Aho, Johnathon M; Zielinski, Martin D; Zietlow, Scott P; Kim, Brian D; Morris, David S

2018-01-01

Prehospital airway management increasingly involves supraglottic airway insertion and a paucity of data evaluates outcomes in trauma populations. We aim to describe definitive airway management in traumatically injured patients who necessitated prehospital supraglottic airway insertion. We performed a single institution retrospective review of multisystem injured patients (≥15years) that received prehospital supraglottic airway insertion during 2009 to 2016. Baseline demographics, number and type of: supraglottic airway insertion attempts, definitive airway and complications were recorded. Primary outcome was need for tracheostomy. Univariate and multivariable statistics were performed. 56 patients met inclusion criteria and were reviewed, 78% were male. Median age [IQR] was 36 [24-56] years. Injuries comprised blunt (94%), penetrating (4%) and burns (2%). Median ISS was 26 [22-41]. Median number of prehospital endotracheal intubation (PETI) attempts was 2 [1-3]. Definitive airway management included: (n=20, 36%, tracheostomy), (n=10, 18%, direct laryngoscopy), (n=6, 11%, bougie), (n=9, 15%, Glidescope), (n=11, 20%, bronchoscopic assistance). 24-hour mortality was 41%. Increasing number of PETI was associated with increasing facial injury. On regression, increasing cervical and facial injury patterns as well as number of PETI were associated with definitive airway control via surgical tracheostomy. After supraglottic airway insertion, operative or non-operative approaches can be utilized to obtain a definitive airway. Patients with increased craniofacial injuries have an increased risk for airway complications and need for tracheostomy. We used these factors to generate an evidence based algorithm that requires prospective validation. Level IV - Retrospective study. Retrospective single institution study. Copyright © 2017 Elsevier Inc. All rights reserved.

Airway compliance and dynamics explain the apparent discrepancy in length adaptation between intact airways and smooth muscle strips.

PubMed

Dowie, Jackson; Ansell, Thomas K; Noble, Peter B; Donovan, Graham M

2016-01-01

Length adaptation is a phenomenon observed in airway smooth muscle (ASM) wherein over time there is a shift in the length-tension curve. There is potential for length adaptation to play an important role in airway constriction and airway hyper-responsiveness in asthma. Recent results by Ansell et al., 2015 (JAP 2014 10.1152/japplphysiol.00724.2014) have cast doubt on this role by testing for length adaptation using an intact airway preparation, rather than strips of ASM. Using this technique they found no evidence for length adaptation in intact airways. Here we attempt to resolve this apparent discrepancy by constructing a minimal mathematical model of the intact airway, including ASM which follows the classic length-tension curve and undergoes length adaptation. This allows us to show that (1) no evidence of length adaptation should be expected in large, cartilaginous, intact airways; (2) even in highly compliant peripheral airways, or at more compliant regions of the pressure-volume curve of large airways, the effect of length adaptation would be modest and at best marginally detectable in intact airways; (3) the key parameters which control the appearance of length adaptation in intact airways are airway compliance and the relaxation timescale. The results of this mathematical simulation suggest that length adaptation observed at the level of the isolated ASM may not clearly manifest in the normal intact airway. Copyright © 2015 Elsevier B.V. All rights reserved.

Airway Microbiota Is Associated with Up-Regulation of the PI3K Pathway in Lung Cancer.

PubMed

Tsay, Jun-Chieh J; Wu, Benjamin G; Badri, Michelle H; Clemente, Jose C; Shen, Nan; Meyn, Peter; Li, Yonghua; Yie, Ting-An; Lhakhang, Tenzin; Olsen, Evan; Murthy, Vivek; Michaud, Gaetane; Sulaiman, Imran; Tsirigos, Aristotelis; Heguy, Adriana; Pass, Harvey; Weiden, Michael D; Rom, William N; Sterman, Daniel H; Bonneau, Richard; Blaser, Martin J; Segal, Leopoldo N

2018-06-04

In lung cancer, upregulation of the PI3K pathway is an early event that contributes to cell proliferation, survival, and tissue invasion. Upregulation of this pathway was recently described as associated with enrichment of the lower airways with bacteria identified as oral commensals. We hypothesize that host-microbe interactions in the lower airways of subjects with lung cancer affect known cancer pathways. Airway brushes were collected prospectively from subjects with lung nodules at time of diagnostic bronchoscopy, including 39 subjects with final lung cancer diagnoses and 36 subjects with non-cancer diagnosis. Additionally, samples from 10 healthy control subjects were included. 16S rRNA gene amplicon sequencing and paired transcriptome sequencing (RNAseq) were performed on all airway samples. In addition, an in vitro model with airway epithelial cells exposed to bacteria/bacterial products was performed. The composition of the lower airway transcriptome in the cancer patients was significantly different from the controls, which included upregulation of ERK and PI3K signaling pathways. The lower airways of lung cancer patients were enriched for oral taxa (Streptococcus and Veillonella), which was associated with upregulation of the ERK and PI3K signaling pathways. In vitro exposure of airway epithelial cells to Veillonella, Prevotella, and Streptococcus led to upregulation of these same signaling pathways. The data presented here shows that several transcriptomic signatures previously identified as relevant to lung cancer pathogenesis are associated with enrichment of the lower airway microbiota with oral commensals.

Adenoviral-Mediated Imaging of Gene Transfer Using a Somatostatin Receptor-Cytosine Deaminase Fusion Protein

PubMed Central

Lears, Kimberly A.; Parry, Jesse J.; Andrews, Rebecca; Nguyen, Kim; Wadas, Thaddeus J.; Rogers, Buck E.

2015-01-01

Suicide gene therapy is a process by which cells are administered a gene that encodes a protein capable of converting a nontoxic prodrug into an active toxin. Cytosine deaminase (CD) has been widely investigated as a means of suicide gene therapy due to the enzyme’s ability to convert the prodrug 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil (5-FU). However, the extent of gene transfer is a limiting factor in predicting therapeutic outcome. The ability to monitor gene transfer, non-invasively, would strengthen the efficiency of therapy. In this regard, we have constructed and evaluated a replication-deficient adenovirus (Ad) containing the human somatostatin receptor subtype 2 (SSTR2) fused with a C-terminal yeast CD gene for the non-invasive monitoring of gene transfer and therapy. The resulting Ad (AdSSTR2-yCD) was evaluated in vitro in breast cancer cells to determine the function of the fusion protein. These studies demonstrated that the both the SSTR2 and yCD were functional in binding assays, conversion assays, and cytotoxicity assays. In vivo studies similarly demonstrated the functionality using conversion assays, biodistribution studies, and small animal positron-emission tomography (PET) imaging studies. In conclusion, the fusion protein has been validated as useful for the non-invasive imaging of yCD expression and will be evaluated in the future for monitoring yCD-based therapy. PMID:25837665

[Cloning, mutagenesis and symbiotic phenotype of three lipid transfer protein encoding genes from Mesorhizobium huakuii 7653R].

PubMed

Li, Yanan; Zeng, Xiaobo; Zhou, Xuejuan; Li, Youguo

2016-12-04

Lipid transfer protein superfamily is involved in lipid transport and metabolism. This study aimed to construct mutants of three lipid transfer protein encoding genes in Mesorhizobium huakuii 7653R, and to study the phenotypes and function of mutations during symbiosis with Astragalus sinicus. We used bioinformatics to predict structure characteristics and biological functions of lipid transfer proteins, and conducted semi-quantitative and fluorescent quantitative real-time PCR to analyze the expression levels of target genes in free-living and symbiotic conditions. Using pK19mob insertion mutagenesis to construct mutants, we carried out pot plant experiments to observe symbiotic phenotypes. MCHK-5577, MCHK-2172 and MCHK-2779 genes encoding proteins belonged to START/RHO alpha_C/PITP/Bet_v1/CoxG/CalC (SRPBCC) superfamily, involved in lipid transport or metabolism, and were identical to M. loti at 95% level. Gene relative transcription level of the three genes all increased compared to free-living condition. We obtained three mutants. Compared with wild-type 7653R, above-ground biomass of plants and nodulenitrogenase activity induced by the three mutants significantly decreased. Results indicated that lipid transfer protein encoding genes of Mesorhizobium huakuii 7653R may play important roles in symbiotic nitrogen fixation, and the mutations significantly affected the symbiotic phenotypes. The present work provided a basis to study further symbiotic function mechanism associated with lipid transfer proteins from rhizobia.

Asialofetuin liposome-mediated human alpha1-antitrypsin gene transfer in vivo results in stationary long-term gene expression.

PubMed

Dasí, F; Benet, M; Crespo, J; Crespo, A; Aliño, S F

2001-05-01

The development of nonviral vectors for in vivo gene delivery to hepatocytes is an interesting topic in view of their safety and tremendous gene therapy potential. Since cationic liposomes and liposome uptake by receptor-mediated mechanisms could offer advantages in the efficacy of liposome-mediated gene transfer, we studied the effect of liposome charge (anionic vs. cationic) and the covalently coupled asialofetuin ligand on the liposome surface in mediating human alpha1-antitrypsin (hAAT) gene transfer to mice in vivo. The changes in liposome charge were made by adding the following lipids to the backbone liposomes: anionic phosphatidylserine, cationic N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate or a lipopeptide synthesized from dipalmitoylphosphatidylethanolamine and covalently coupled to the cationic nuclear localization signal peptide. Two plasmids containing the hAAT gene were used: pTG7101, containing the complete genomic sequence of the human gene driven by the natural promoter, and p216, containing the human hAAT cDNA under the control of the CMV promoter. The results indicate that both untargeted anionic and cationic liposomes mediate plasma levels of hAAT that decline over time. However, asialofetuin liposomes increase the plasma levels of hAAT and can mediate long-term gene expression (>12 months) with stationary plasma levels of protein. Results from quantitative and qualitative reverse transcriptase polymerase chain reaction match those from protein plasma levels and confirm both the human origin of the message and the liver as source of the protein. The use of asialofetuin liposomes in hepatic gene therapy may both increase and prolong in vivo gene expression of hAAT and other clinically important genes.

Topical cystic fibrosis transmembrane conductance regulator gene replacement for cystic fibrosis-related lung disease.

PubMed

Lee, T; Southern, K W

2007-04-18

measured ion transport in the lower airways and demonstrated significant changes toward normal values in the participants who received gene transfer agents (P < 0.0001), weighted mean difference 6.86 (95% CI of 3.77 to 9.95). In these participants there was also evidence of increased salt transport in cells obtained by brushing the lower airway. These outcomes, whilst important, are not of direct clinical relevance. There is currently no evidence to support the use of CFTR gene transfer reagents as a treatment for lung disease in people with cystic fibrosis. Future studies need to investigate clinically important outcome measures.

Signal Transducer and Activator of Transcription 1 (STAT1) is Essential for Chromium Silencing of Gene Induction in Human Airway Epithelial Cells

PubMed Central

Nemec, Antonia A.; Barchowsky, Aaron

2009-01-01

Hexavalent chromium (Cr(VI)) promotes lung injury and pulmonary diseases through poorly defined mechanisms that may involve the silencing of inducible protective genes. The current study investigated the hypothesis that Cr(VI) actively signals through a signal transducer and activator of transcription 1 (STAT1)–dependent pathway to silence nickel (Ni)–induced expression of vascular endothelial cell growth factor A (VEGFA), an important mediator of lung injury and repair. In human bronchial airway epithelial (BEAS-2B) cells, Ni-induced VEGFA transcription by stimulating an extracellular regulated kinase (ERK) signaling cascade that involved Src kinase–activated Sp1 transactivation, as well as increased hypoxia-inducible factor-1α (HIF-1α) stabilization and DNA binding. Ni-stimulated ERK, Src, and HIF-1α activities, as well as Ni-induced VEGFA transcript levels were inhibited in Cr(VI)-exposed cells. We previously demonstrated that Cr(VI) stimulates STAT1 to suppress VEGFA expression. In BEAS-2B cells stably expressing STAT1 short hairpin RNA, Cr(VI) increased VEGFA transcript levels and Sp1 transactivation. Moreover, in the absence of STAT1, Cr(VI), and Ni coexposures positively interacted to further increase VEGFA transcripts. This study demonstrates that metal-stimulated signaling cascades interact to regulate transcription and induction of adaptive or repair responses in airway cells. In addition, the data implicate STAT1 as a rate limiting mediator of Cr(VI)-stimulated gene regulation and suggest that cells lacking STAT1, such as many tumor cell lines, have opposite responses to Cr(VI) relative to normal cells. PMID:19403854

Psoriatic inflammation enhances allergic airway inflammation through IL-23/STAT3 signaling in a murine model.

PubMed

Nadeem, Ahmed; Al-Harbi, Naif O; Ansari, Mushtaq A; Al-Harbi, Mohammed M; El-Sherbeeny, Ahmed M; Zoheir, Khairy M A; Attia, Sabry M; Hafez, Mohamed M; Al-Shabanah, Othman A; Ahmad, Sheikh F

2017-01-15

Psoriasis is an autoimmune inflammatory skin disease characterized by activated IL-23/STAT3/Th17 axis. Recently psoriatic inflammation has been shown to be associated with asthma. However, no study has previously explored how psoriatic inflammation affects airway inflammation. Therefore, this study investigated the effect of imiquimod (IMQ)-induced psoriatic inflammation on cockroach extract (CE)-induced airway inflammation in murine models. Mice were subjected to topical and intranasal administration of IMQ and CE to develop psoriatic and airway inflammation respectively. Various analyses in lung/spleen related to inflammation, Th17/Th2/Th1 cell immune responses, and their signature cytokines/transcription factors were carried out. Psoriatic inflammation in allergic mice was associated with increased airway inflammation with concurrent increase in Th2/Th17 cells/signature cytokines/transcription factors. Splenic CD4+ T and CD11c+ dendritic cells in psoriatic mice had increased STAT3/RORC and IL-23 mRNA expression respectively. This led us to explore the effect of systemic IL-23/STAT3 signaling on airway inflammation. Topical application of STA-21, a small molecule STAT3 inhibitor significantly reduced airway inflammation in allergic mice having psoriatic inflammation. On the other hand, adoptive transfer of IL-23-treated splenic CD4+ T cells from allergic mice into naive recipient mice produced mixed neutrophilic/eosinophilic airway inflammation similar to allergic mice with psoriatic inflammation. Our data suggest that systemic IL-23/STAT3 axis is responsible for enhanced airway inflammation during psoriasis. The current study also suggests that only anti-asthma therapy may not be sufficient to alleviate airway inflammatory burden in asthmatics with psoriasis. Copyright © 2016 Elsevier Inc. All rights reserved.

Adaptive Evolution of Extreme Acidophile Sulfobacillus thermosulfidooxidans Potentially Driven by Horizontal Gene Transfer and Gene Loss

PubMed Central

Zhang, Xian; Liu, Xueduan; Liang, Yili; Guo, Xue; Xiao, Yunhua; Ma, Liyuan; Miao, Bo; Liu, Hongwei; Peng, Deliang; Huang, Wenkun; Zhang, Yuguang

2017-01-01

ABSTRACT Recent phylogenomic analysis has suggested that three strains isolated from different copper mine tailings around the world were taxonomically affiliated with Sulfobacillus thermosulfidooxidans. Here, we present a detailed investigation of their genomic features, particularly with respect to metabolic potentials and stress tolerance mechanisms. Comprehensive analysis of the Sulfobacillus genomes identified a core set of essential genes with specialized biological functions in the survival of acidophiles in their habitats, despite differences in their metabolic pathways. The Sulfobacillus strains also showed evidence for stress management, thereby enabling them to efficiently respond to harsh environments. Further analysis of metabolic profiles provided novel insights into the presence of genomic streamlining, highlighting the importance of gene loss as a main mechanism that potentially contributes to cellular economization. Another important evolutionary force, especially in larger genomes, is gene acquisition via horizontal gene transfer (HGT), which might play a crucial role in the recruitment of novel functionalities. Also, a successful integration of genes acquired from archaeal donors appears to be an effective way of enhancing the adaptive capacity to cope with environmental changes. Taken together, the findings of this study significantly expand the spectrum of HGT and genome reduction in shaping the evolutionary history of Sulfobacillus strains. IMPORTANCE Horizontal gene transfer (HGT) and gene loss are recognized as major driving forces that contribute to the adaptive evolution of microbial genomes, although their relative importance remains elusive. The findings of this study suggest that highly frequent gene turnovers within microorganisms via HGT were necessary to incur additional novel functionalities to increase the capacity of acidophiles to adapt to changing environments. Evidence also reveals a fascinating phenomenon of potential cross

Adaptive Evolution of Extreme Acidophile Sulfobacillus thermosulfidooxidans Potentially Driven by Horizontal Gene Transfer and Gene Loss.

PubMed

Zhang, Xian; Liu, Xueduan; Liang, Yili; Guo, Xue; Xiao, Yunhua; Ma, Liyuan; Miao, Bo; Liu, Hongwei; Peng, Deliang; Huang, Wenkun; Zhang, Yuguang; Yin, Huaqun

2017-04-01

Recent phylogenomic analysis has suggested that three strains isolated from different copper mine tailings around the world were taxonomically affiliated with Sulfobacillus thermosulfidooxidans Here, we present a detailed investigation of their genomic features, particularly with respect to metabolic potentials and stress tolerance mechanisms. Comprehensive analysis of the Sulfobacillus genomes identified a core set of essential genes with specialized biological functions in the survival of acidophiles in their habitats, despite differences in their metabolic pathways. The Sulfobacillus strains also showed evidence for stress management, thereby enabling them to efficiently respond to harsh environments. Further analysis of metabolic profiles provided novel insights into the presence of genomic streamlining, highlighting the importance of gene loss as a main mechanism that potentially contributes to cellular economization. Another important evolutionary force, especially in larger genomes, is gene acquisition via horizontal gene transfer (HGT), which might play a crucial role in the recruitment of novel functionalities. Also, a successful integration of genes acquired from archaeal donors appears to be an effective way of enhancing the adaptive capacity to cope with environmental changes. Taken together, the findings of this study significantly expand the spectrum of HGT and genome reduction in shaping the evolutionary history of Sulfobacillus strains. IMPORTANCE Horizontal gene transfer (HGT) and gene loss are recognized as major driving forces that contribute to the adaptive evolution of microbial genomes, although their relative importance remains elusive. The findings of this study suggest that highly frequent gene turnovers within microorganisms via HGT were necessary to incur additional novel functionalities to increase the capacity of acidophiles to adapt to changing environments. Evidence also reveals a fascinating phenomenon of potential cross-kingdom HGT

Rapid Neurofibrillary Tangle Formation after Localized Gene Transfer of Mutated Tau

PubMed Central

Klein, Ronald L.; Lin, Wen-Lang; Dickson, Dennis W.; Lewis, Jada; Hutton, Michael; Duff, Karen; Meyer, Edwin M.; King, Michael A.

2004-01-01

Neurofibrillary pathology was produced in the brains of adult rats after localized gene transfer of human tau carrying the P301L mutation, which is associated with frontotemporal dementia with parkinsonism. Within 1 month of in situ transfection of the basal forebrain region of normal rats, tau-immunoreactive and argyrophilic neuronal lesions formed. The fibrillar lesions had features of neurofibrillary tangles and tau immunoreactivity at light and electron microscopic levels. In addition to neurofibrillary tangles, other tau pathology, including pretangles and neuropil threads, was abundant and widespread. Tau gene transfer to the hippocampal region of amyloid-depositing transgenic mice produced pretangles and threads, as well as intensely tau-immunoreactive neurites in amyloid plaques. The ability to produce neurofibrillary pathology in adult rodents makes this a useful method to study tau-related neurodegeneration. PMID:14695347

Neutrophil elastase-mediated increase in airway temperature during inflammation.

PubMed

Schmidt, Annika; Belaaouaj, Azzaq; Bissinger, Rosi; Koller, Garrit; Malleret, Laurette; D'Orazio, Ciro; Facchinelli, Martino; Schulte-Hubbert, Bernhard; Molinaro, Antonio; Holst, Otto; Hammermann, Jutta; Schniederjans, Monika; Meyer, Keith C; Damkiaer, Soeren; Piacentini, Giorgio; Assael, Baroukh; Bruce, Kenneth; Häußler, Susanne; LiPuma, John J; Seelig, Joachim; Worlitzsch, Dieter; Döring, Gerd

2014-12-01

How elevated temperature is generated during airway infections represents a hitherto unresolved physiological question. We hypothesized that innate immune defence mechanisms would increase luminal airway temperature during pulmonary infection. We determined the temperature in the exhaled air of cystic fibrosis (CF) patients. To further test our hypothesis, a pouch inflammatory model using neutrophil elastase-deficient mice was employed. Next, the impact of temperature changes on the dominant CF pathogen Pseudomonas aeruginosa growth was tested by plating method and RNAseq. Here we show a temperature of ~38°C in neutrophil-dominated mucus plugs of chronically infected CF patients and implicate neutrophil elastase:α1-proteinase inhibitor complex formation as a relevant mechanism for the local temperature rise. Gene expression of the main pathogen in CF, P. aeruginosa, under anaerobic conditions at 38°C vs 30°C revealed increased virulence traits and characteristic cell wall changes. Neutrophil elastase mediates increase in airway temperature, which may contribute to P. aeruginosa selection during the course of chronic infection in CF. Copyright © 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Critical Airway Team: A Retrospective Study of an Airway Response System in a Pediatric Hospital.

PubMed

Sterrett, Emily C; Myer, Charles M; Oehler, Jennifer; Das, Bobby; Kerrey, Benjamin T

2017-12-01

Objective Study the performance of a pediatric critical airway response team. Study Design Case series with chart review. Setting Freestanding academic children's hospital. Subjects and Methods A structured review of the electronic medical record was conducted for all activations of the critical airway team. Characteristics of the activations and patients are reported using descriptive statistics. Activation of the critical airway team occurred 196 times in 46 months (March 2012 to December 2015); complete data were available for 162 activations (83%). For 49 activations (30%), patients had diagnoses associated with difficult intubation; 45 (28%) had a history of difficult laryngoscopy. Results Activation occurred at least 4 times per month on average (vs 3 per month for hospital-wide codes). The most common reasons for team activation were anticipated difficult intubation (45%) or failed intubation attempt (20%). For 79% of activations, the team performed an airway procedure, most commonly direct laryngoscopy and tracheal intubation. Bronchoscopy was performed in 47% of activations. Surgical airway rescue was attempted 4 times. Cardiopulmonary resuscitation occurred in 41 activations (25%). Twenty-nine patients died during or following team activation (18%), including 10 deaths associated with the critical airway event. Conclusion Critical airway team activation occurred at least once per week on average. Direct laryngoscopy, tracheal intubation, and bronchoscopic procedures were performed frequently; surgical airway rescue was rare. Most patients had existing risk factors for difficult intubation. Given our rate of serious morbidity and mortality, primary prevention of critical airway events will be a focus of future efforts.

A 4-Week Model of House Dust Mite (HDM) Induced Allergic Airways Inflammation with Airway Remodeling.

PubMed

Woo, L N; Guo, W Y; Wang, X; Young, A; Salehi, S; Hin, A; Zhang, Y; Scott, J A; Chow, C W

2018-05-02

Animal models of allergic airways inflammation are useful tools in studying the pathogenesis of asthma and potential therapeutic interventions. The different allergic airways inflammation models available to date employ varying doses, frequency, duration and types of allergen, which lead to the development of different features of asthma; showing varying degrees of airways inflammation and hyper-responsiveness (AHR) and airways remodeling. Models that also exhibit airway remodeling, a key feature of asthma, in addition to AHR and airway inflammation typically require 5-12 weeks to develop. In this report, we describe a 4-week mouse model of house dust mite (HDM)-induced allergic airways inflammation, and compare the phenotypic features of two different doses of HDM exposures (10 µg and 25 µg) for 5 days/week with a well-characterized 8-week chronic HDM model. We found that 4 weeks of intranasal HDM (25 µg in 35 µl saline; 5 days/week) resulted in AHR, airway inflammation and airway remodeling that were comparable to the 8-week model. We conclude that this new 4-week HDM model is another useful tool in studies of human asthma that offers advantages of shorter duration for development and decreased costs when compared to other models that require longer durations of exposure (5-12 weeks) to develop.

Horizontal transfer of the msp130 gene supported the evolution of metazoan biomineralization.

PubMed

Ettensohn, Charles A

2014-05-01

It is widely accepted that biomineralized structures appeared independently in many metazoan clades during the Cambrian. How this occurred, and whether it involved the parallel co-option of a common set of biochemical and developmental pathways (i.e., a shared biomineralization "toolkit"), are questions that remain unanswered. Here, I provide evidence that horizontal gene transfer supported the evolution of biomineralization in some metazoans. I show that Msp130 proteins, first described as proteins expressed selectively by the biomineral-forming primary mesenchyme cells of the sea urchin embryo, have a much wider taxonomic distribution than was previously appreciated. Msp130 proteins are present in several invertebrate deuterostomes and in one protostome clade (molluscs). Surprisingly, closely related proteins are also present in many bacteria and several algae, and I propose that msp130 genes were introduced into metazoan lineages via multiple, independent horizontal gene transfer events. Phylogenetic analysis shows that the introduction of an ancestral msp130 gene occurred in the sea urchin lineage more than 250 million years ago and that msp130 genes underwent independent, parallel duplications in each of the metazoan phyla in which these genes are found. © 2014 Wiley Periodicals, Inc.

Operative endoscopy of the airway

PubMed Central

Walters, Dustin M.

2016-01-01

Airway endoscopy has long been an important and useful tool in the management of thoracic diseases. As thoracic specialists have gained experience with both flexible and rigid bronchoscopic techniques, the technology has continued to evolve so that bronchoscopy is currently the foundation for diagnosis and treatment of many thoracic ailments. Airway endoscopy plays a significant role in the biopsy of tumors within the airways, mediastinum, and lung parenchyma. Endoscopic methods have been developed to treat benign and malignant airway stenoses and tracheomalacia. And more recently, techniques have been conceived to treat end-stage emphysema and prolonged air leaks in select patients. This review describes the abundant uses of airway endoscopy, as well as technical considerations and limitations of the current technologies. PMID:26981263

Light-controlled inhibition of malignant glioma by opsin gene transfer

PubMed Central

Yang, F; Tu, J; Pan, J-Q; Luo, H-L; Liu, Y-H; Wan, J; Zhang, J; Wei, P-F; Jiang, T; Chen, Y-H; Wang, L-P

2013-01-01

Glioblastomas are aggressive cancers with low survival rates and poor prognosis because of their highly proliferative and invasive capacity. In the current study, we describe a new optogenetic strategy that selectively inhibits glioma cells through light-controlled membrane depolarization and cell death. Transfer of the engineered opsin ChETA (engineered Channelrhodopsin-2 variant) gene into primary human glioma cells or cell lines, but not normal astrocytes, unexpectedly decreased cell proliferation and increased mitochondria-dependent apoptosis, upon light stimulation. These optogenetic effects were mediated by membrane depolarization-induced reductions in cyclin expression and mitochondrial transmembrane potential. Importantly, the ChETA gene transfer and light illumination in mice significantly inhibited subcutaneous and intracranial glioma growth and increased the survival of the animals bearing the glioma. These results uncover an unexpected effect of opsin ion channels on glioma cells and offer the opportunity for the first time to treat glioma using a light-controllable optogenetic approach. PMID:24176851

Light-controlled inhibition of malignant glioma by opsin gene transfer.

PubMed

Yang, F; Tu, J; Pan, J-Q; Luo, H-L; Liu, Y-H; Wan, J; Zhang, J; Wei, P-F; Jiang, T; Chen, Y-H; Wang, L-P

2013-10-31

Glioblastomas are aggressive cancers with low survival rates and poor prognosis because of their highly proliferative and invasive capacity. In the current study, we describe a new optogenetic strategy that selectively inhibits glioma cells through light-controlled membrane depolarization and cell death. Transfer of the engineered opsin ChETA (engineered Channelrhodopsin-2 variant) gene into primary human glioma cells or cell lines, but not normal astrocytes, unexpectedly decreased cell proliferation and increased mitochondria-dependent apoptosis, upon light stimulation. These optogenetic effects were mediated by membrane depolarization-induced reductions in cyclin expression and mitochondrial transmembrane potential. Importantly, the ChETA gene transfer and light illumination in mice significantly inhibited subcutaneous and intracranial glioma growth and increased the survival of the animals bearing the glioma. These results uncover an unexpected effect of opsin ion channels on glioma cells and offer the opportunity for the first time to treat glioma using a light-controllable optogenetic approach.

Specific gene transfer mediated by galactosylated poly-L-lysine into hepatoma cells.

PubMed

Han, J; Il Yeom, Y

2000-07-20

Plasmid DNA/galactosylated poly-L-lysine(GalPLL) complex was used to transfer luciferase reporter gene in vitro into human hepatoma cells by a receptor-mediated endocytosis process. DNA was combined with galPLL via charge interaction (DNA:GalPLL:fusogenic peptide, 1:0.4:5, w/w/w) and the resulting complex was characterized by dynamic light scattering, gel retardation assay and zeta potential analyzer to determine the particle size, electrostatic charge interaction, and apparent surface charge. The complex was tested for the efficiency of gene transfer in cultured human hepatoblastoma cell line Hep G2 and fibroblast cells NIH/3T3 in vitro. The mean diameter of the complex (DNA:GalPLL=1:0.4, w/w) was 256+/-34.8 nm, and at this ratio, it was positively charged (zeta potential of this complex was 10.1 mV). Hep G2 cells, which express a galactose specific membrane lectin, were efficiently and selectively transfected with the RSV Luc/GalPLL complex in a sugar-dependent manner. NIH/3T3 cells, which do not express the galactose-specific membrane lectin, showed only a marginal level of gene expression. The transfection efficiency of GalPLL-conjugated DNA complex into Hep G2 cells was greatly enhanced in the presence of fusogenic peptide that can disrupt endosomes, where the GalPLL-DNA complex is entrapped with the fusogenic peptide. With the fusogenic peptide KALA, the luciferase activity in Hep G2 cells was ten-fold higher than that of cells transfected in the absence of the fusogenic peptide. Our gene transfer formulation may find potential application for the gene therapy of liver diseases.

Gene transfer device utilizing micron-spiked electrodes produced by the self-organization phenomenon of Fe-alloy.

PubMed

Miyano, Naoki; Inoue, Yuuki; Teramura, Yuji; Fujii, Keisuke; Tsumori, Fujio; Iwata, Hiroo; Kotera, Hidetoshi

2008-07-01

In the diffusional phase transformation of two-phase alloys, the new phase precipitates form the matrix phase at specific temperatures, followed by the formation of a mixed microstructure comprising the precipitate and the matrix. It has been found that by specific chemical-etching treatment, the precipitate in Fe-25Cr-6Ni alloy projects substantially and clusters at the surface. The configuration of the precipitate has an extremely high aspect ratio: it is several microns in width and several tens of microns in length (known as micron-spiked). This study targets the development of a gene transfer device with a micro-spike produced based on the self-organization phenomenon of the Fe-25Cr-6Ni alloy. With this spike-projected device, we tried to efficiently transfer plasmid DNA into adherent cells by electric pulse-triggered gene transfer using a plasmid-loaded electrode (electroporation-based reverse transfection). The spiked structure was applied to a substrate of the device to allow efficient gene transfer into adherent cells, although the general substrate was flat and had a smooth surface. The results suggest that this unique spike-projected device has potential applications in gene transfer devices for the analysis of the human genome in the post-genome period.

Endogenous gamma-aminobutyric acid modulates tonic guinea pig airway tone and propofol-induced airway smooth muscle relaxation.

PubMed

Gallos, George; Gleason, Neil R; Virag, Laszlo; Zhang, Yi; Mizuta, Kentaro; Whittington, Robert A; Emala, Charles W

2009-04-01

Emerging evidence indicates that an endogenous autocrine/paracrine system involving gamma-aminobutyric acid (GABA) is present in airways. GABAA channels, GABAB receptors, and the enzyme that synthesizes GABA have been identified in airway epithelium and smooth muscle. However, the endogenous ligand itself, GABA, has not been measured in airway tissues. The authors sought to demonstrate that GABA is released in response to contractile agonists and tonically contributes a prorelaxant component to contracted airway smooth muscle. The amount and cellular localization of GABA in upper guinea pig airways under resting and contracted tone was determined by high pressure liquid chromatography and immunohistochemistry, respectively. The contribution that endogenous GABA imparts on the maintenance of airway smooth muscle acetylcholine-induced contraction was assessed in intact guinea pig airway tracheal rings using selective GABAA antagonism (gabazine) under resting or acetylcholine-contracted conditions. The ability of an allosteric agent (propofol) to relax a substance P-induced relaxation in an endogenous GABA-dependent manner was assessed. GABA levels increased and localized to airway smooth muscle after contractile stimuli in guinea pig upper airways. Acetylcholine-contracted guinea pig tracheal rings exhibited an increase in contracted force upon addition of the GABAA antagonist gabazine that was subsequently reversed by the addition of the GABAA agonist muscimol. Propofol dose-dependently relaxed a substance P contraction that was blocked by gabazine. These studies demonstrate that GABA is endogenously present and increases after contractile stimuli in guinea pig upper airways and that endogenous GABA contributes a tonic prorelaxant component in the maintenance of airway smooth muscle tone.

Endogenous γ-aminobutyric Acid Modulates Tonic Guinea Pig Airway Tone and Propofol-induced Airway Smooth Muscle Relaxation

PubMed Central

Gallos, George; Gleason, Neil R.; Virag, Laszlo; Zhang, Yi; Mizuta, Kentauro; Whittington, Robert A.; Emala, Charles W.

2009-01-01

Background Emerging evidence indicates that an endogenous autocrine/paracrine system involving γ-aminobutyric acid (GABA) is present in airways. GABAA channels, GABAB receptors and the enzyme that synthesizes GABA have been identified in airway epithelium and smooth muscle. However, the endogenous ligand itself, GABA, has not been measured in airway tissues. We sought to demonstrate that GABA is released in response to contractile agonists and tonically contributes a pro-relaxant component to contracted airway smooth muscle. Methods The amount and cellular localization of GABA in upper guinea pig airways under resting and contracted tone was determined by high pressure liquid chromatography and immunohistochemistry, respectively. The contribution that endogenous GABA imparts on the maintenance of airway smooth muscle acetylcholine-induced contraction was assessed in intact guinea pig airway tracheal rings using selective GABAA antagonism (gabazine) under resting or acetylcholine-contracted conditions. The ability of an allosteric agent (propofol) to relax a substance P-induced relaxation in an endogenous GABA-dependent manner was assessed. Results GABA levels increased and localized to airway smooth muscle following contractile stimuli in guinea pig upper airways. Acetylcholine-contracted guinea pig tracheal rings exhibited an increase in contracted force upon addition of the GABAA antagonist gabazine which was subsequently reversed by the addition of the GABAA agonist muscimol. Propofol dose-dependently relaxed a substance P contraction that was blocked by gabazine. Conclusion These studies demonstrate that GABA is endogenously present and increases following contractile stimuli in guinea pig upper airways and that endogenous GABA contributes a tonic pro-relaxant component in the maintenance of airway smooth muscle tone. PMID:19322939

Neurokinin-1 receptor mediates stress-exacerbated allergic airway inflammation and airway hyperresponsiveness in mice.

PubMed

Joachim, Ricarda A; Sagach, Viktoriya; Quarcoo, David; Dinh, Q Thai; Arck, Petra C; Klapp, Burghard F

2004-01-01

A wealth of clinical observation has suggested that stress and asthma morbidity are associated. We have previously established a mouse model of stress-exacerbated allergic airway inflammation, which reflects major clinical findings. The aim of the current study was to investigate the role of the neurokinin- (NK-)1 receptor in the mediation of stress effects in allergic airway inflammation. BALB/c mice were systemically sensitized with ovalbumin (OVA) on assay days 1, 14, and 21 and repeatedly challenged with OVA aerosol on days 26 and 27. Sound stress was applied to the animals for 24 hours, starting with the first airway challenge. Additionally, one group of stressed and one group of nonstressed mice received the highly specific NK-1 receptor antagonist RP 67580. Bronchoalveolar lavage fluid was obtained, and cell numbers and differentiation were determined. Airway hyperreactivity was measured in vitro by electrical field stimulation of tracheal smooth-muscle elements. Application of stress in sensitized and challenged animals resulted in a significant increase in leukocyte number in the bronchoalveolar lavage fluid. Furthermore, stressed animals showed enhanced airway reactivity. The increase of inflammatory cells and airway reactivity was blocked by treatment of animals with the NK-1 receptor antagonist. These data indicate that the NK-1 receptor plays an important role in mediating stress effects in allergen-induced airway inflammation.

A pathophysiological role of PDE3 in allergic airway inflammation

PubMed Central

Beute, Jan; Lukkes, Melanie; Koekoek, Ewout P.; Nastiti, Hedwika; Ganesh, Keerthana; de Bruijn, Marjolein J.W.; Hockman, Steve; van Nimwegen, Menno; Braunstahl, Gert-Jan; Boon, Louis; Lambrecht, Bart N.; Manganiello, Vince C.; Hendriks, Rudi W.

2018-01-01

Phosphodiesterase 3 (PDE3) and PDE4 regulate levels of cyclic AMP, which are critical in various cell types involved in allergic airway inflammation. Although PDE4 inhibition attenuates allergic airway inflammation, reported side effects preclude its application as an antiasthma drug in humans. Case reports showed that enoximone, which is a smooth muscle relaxant that inhibits PDE3, is beneficial and lifesaving in status asthmaticus and is well tolerated. However, clinical observations also showed antiinflammatory effects of PDE3 inhibition. In this study, we investigated the role of PDE3 in a house dust mite–driven (HDM-driven) allergic airway inflammation (AAI) model that is characterized by T helper 2 cell activation, eosinophilia, and reduced mucosal barrier function. Compared with wild-type (WT) littermates, mice with a targeted deletion of the PDE3A or PDE3B gene showed significantly reduced HDM-driven AAI. Therapeutic intervention in WT mice showed that all hallmarks of HDM-driven AAI were abrogated by the PDE3 inhibitors enoximone and milrinone. Importantly, we found that enoximone also reduced the upregulation of the CD11b integrin on mouse and human eosinophils in vitro, which is crucial for their recruitment during allergic inflammation. This study provides evidence for a hitherto unknown antiinflammatory role of PDE3 inhibition in allergic airway inflammation and offers a potentially novel treatment approach. PMID:29367458

Novel recA-Independent Horizontal Gene Transfer in Escherichia coli K-12.

PubMed

Kingston, Anthony W; Roussel-Rossin, Chloé; Dupont, Claire; Raleigh, Elisabeth A

2015-01-01

In bacteria, mechanisms that incorporate DNA into a genome without strand-transfer proteins such as RecA play a major role in generating novelty by horizontal gene transfer. We describe a new illegitimate recombination event in Escherichia coli K-12: RecA-independent homologous replacements, with very large (megabase-length) donor patches replacing recipient DNA. A previously uncharacterized gene (yjiP) increases the frequency of RecA-independent replacement recombination. To show this, we used conjugal DNA transfer, combining a classical conjugation donor, HfrH, with modern genome engineering methods and whole genome sequencing analysis to enable interrogation of genetic dependence of integration mechanisms and characterization of recombination products. As in classical experiments, genomic DNA transfer begins at a unique position in the donor, entering the recipient via conjugation; antibiotic resistance markers are then used to select recombinant progeny. Different configurations of this system were used to compare known mechanisms for stable DNA incorporation, including homologous recombination, F'-plasmid formation, and genome duplication. A genome island of interest known as the immigration control region was specifically replaced in a minority of recombinants, at a frequency of 3 X 10(-12) CFU/recipient per hour.

Plasmid transfer by conjugation as a possible route of horizontal gene transfer and recombination in Xylella fastidiosa

USDA-ARS?s Scientific Manuscript database

Horizontal gene transfer is an important component of evolution and adaptation of bacterial species. Xylella fastidiosa has the ability to incorporate exogenous DNA into its genome by homologous recombination at relatively high rates. This genetic recombination is believed to play a role in adaptati...

Gene transfer agent (GTA) genes reveal diverse and dynamic Roseobacter and Rhodobacter populations in the Chesapeake Bay.

PubMed

Zhao, Yanlin; Wang, Kui; Budinoff, Charles; Buchan, Alison; Lang, Andrew; Jiao, Nianzhi; Chen, Feng

2009-03-01

Within the bacterial class Alphaproteobacteria, the order Rhodobacterales contains the Roseobacter and Rhodobacter clades. Roseobacters are abundant and play important biogeochemical roles in marine environments. Roseobacter and Rhodobacter genomes contain a conserved gene transfer agent (GTA) gene cluster, and GTA-mediated gene transfer has been observed in these groups of bacteria. In this study, we investigated the genetic diversity of these two groups in Chesapeake Bay surface waters using a specific PCR primer set targeting the conserved Rhodobacterales GTA major capsid protein gene (g5). The g5 gene was successfully amplified from 26 Rhodobacterales isolates and the bay microbial communities using this primer set. Four g5 clone libraries were constructed from microbial assemblages representing different regions and seasons of the bay and yielded diverse sequences. In total, 12 distinct g5 clusters could be identified among 158 Chesapeake Bay clones, 11 fall within the Roseobacter clade, and one falls in the Rhodobacter clade. The vast majority of the clusters (10 out of 12) lack cultivated representatives. The composition of g5 sequences varied dramatically along the bay during the wintertime, and a distinct Roseobacter population composition between winter and summer was observed. The congruence between g5 and 16S rRNA gene phylogenies indicates that g5 may serve as a useful genetic marker to investigate diversity and abundance of Roseobacter and Rhodobacter in natural environments. The presence of the g5 gene in the natural populations of Roseobacter and Rhodobacter implies that genetic exchange through GTA transduction could be an important mechanism for maintaining the metabolic flexibility of these groups of bacteria.

A Jagged 1-Notch 4 molecular switch mediates airway inflammation induced by ultrafine particles.

PubMed

Xia, Mingcan; Harb, Hani; Saffari, Arian; Sioutas, Constantinos; Chatila, Talal A

2018-04-05

Exposure to traffic-related particulate matter promotes asthma and allergic diseases. However, the precise cellular and molecular mechanisms by which particulate matter exposure acts to mediate these effects remain unclear. We sought to elucidate the cellular targets and signaling pathways critical for augmentation of allergic airway inflammation induced by ambient ultrafine particles (UFP). We used in vitro cell-culture assays with lung-derived antigen-presenting cells and allergen-specific T cells and in vivo mouse models of allergic airway inflammation with myeloid lineage-specific gene deletions, cellular reconstitution approaches, and antibody inhibition studies. We identified lung alveolar macrophages (AM) as the key cellular target of UFP in promoting airway inflammation. Aryl hydrocarbon receptor-dependent induction of Jagged 1 (Jag1) expression in AM was necessary and sufficient for augmentation of allergic airway inflammation by UFP. UFP promoted T H 2 and T H 17 cell differentiation of allergen-specific T cells in a Jag1- and Notch 4-dependent manner. Treatment of mice with an anti-Notch 4 antibody abrogated exacerbation of allergic airway inflammation induced by UFP. UFP exacerbate allergic airway inflammation by promoting a Jag1-Notch 4-dependent interaction between AM and allergen-specific T cells, leading to augmented T H cell differentiation. Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Microbial evolution of sulphate reduction when lateral gene transfer is geographically restricted.

PubMed

Chi Fru, E

2011-07-01

Lateral gene transfer (LGT) is an important mechanism by which micro-organisms acquire new functions. This process has been suggested to be central to prokaryotic evolution in various environments. However, the influence of geographical constraints on the evolution of laterally acquired genes in microbial metabolic evolution is not yet well understood. In this study, the influence of geographical isolation on the evolution of laterally acquired dissimilatory sulphite reductase (dsr) gene sequences in the sulphate-reducing micro-organisms (SRM) was investigated. Sequences on four continental blocks related to SRM known to have received dsr by LGT were analysed using standard phylogenetic and multidimensional statistical methods. Sequences related to lineages with large genetic diversity correlated positively with habitat divergence. Those affiliated to Thermodesulfobacterium indicated strong biogeographical delineation; hydrothermal-vent sequences clustered independently from hot-spring sequences. Some of the hydrothermal-vent and hot-spring sequences suggested to have been acquired from a common ancestral source may have diverged upon isolation within distinct habitats. In contrast, analysis of some Desulfotomaculum sequences indicated they could have been transferred from different ancestral sources but converged upon isolation within the same niche. These results hint that, after lateral acquisition of dsr genes, barriers to gene flow probably play a strong role in their subsequent evolution.

Airway management in cervical spine injury

PubMed Central

Austin, Naola; Krishnamoorthy, Vijay; Dagal, Arman

2014-01-01

To minimize risk of spinal cord injury, airway management providers must understand the anatomic and functional relationship between the airway, cervical column, and spinal cord. Patients with known or suspected cervical spine injury may require emergent intubation for airway protection and ventilatory support or elective intubation for surgery with or without rigid neck stabilization (i.e., halo). To provide safe and efficient care in these patients, practitioners must identify high-risk patients, be comfortable with available methods of airway adjuncts, and know how airway maneuvers, neck stabilization, and positioning affect the cervical spine. This review discusses the risks and benefits of various airway management strategies as well as specific concerns that affect patients with known or suspected cervical spine injury. PMID:24741498

Group 2 Innate Lymphoid Cells Exhibit a Dynamic Phenotype in Allergic Airway Inflammation

PubMed Central

Li, Bobby W. S.; Stadhouders, Ralph; de Bruijn, Marjolein J. W.; Lukkes, Melanie; Beerens, Dior M. J. M.; Brem, Maarten D.; KleinJan, Alex; Bergen, Ingrid; Vroman, Heleen; Kool, Mirjam; van IJcken, Wilfred F. J.; Rao, Tata Nageswara; Fehling, Hans Jörg; Hendriks, Rudi W.

2017-01-01

Group 2 innate lymphoid cells (ILC2) are implicated in allergic asthma as an early innate source of the type 2 cytokines IL-5 and IL-13. However, their induction in house dust mite (HDM)-mediated airway inflammation additionally requires T cell activation. It is currently unknown whether phenotypic differences exist between ILC2s that are activated in a T cell-dependent or T cell-independent fashion. Here, we compared ILC2s in IL-33- and HDM-driven airway inflammation. Using flow cytometry, we found that surface expression levels of various markers frequently used to identify ILC2s were dependent on their mode of activation, highly variable over time, and differed between tissue compartments, including bronchoalveolar lavage (BAL) fluid, lung, draining lymph nodes, and spleen. Whereas in vivo IL-33-activated BAL fluid ILC2s exhibited an almost uniform CD25+CD127+T1/ST2+ICOS+KLRG1+ phenotype, at a comparable time point after HDM exposure BAL fluid ILC2s had a very heterogeneous surface marker phenotype. A major fraction of HDM-activated ILC2s were CD25lowCD127+T1/ST2low ICOSlowKLRG1low, but nevertheless had the capacity to produce large amounts of type 2 cytokines. HDM-activated CD25low ILC2s in BAL fluid and lung rapidly reverted to CD25high ILC2s upon in vivo stimulation with IL-33. Genome-wide transcriptional profiling of BAL ILC2s revealed ~1,600 differentially expressed genes: HDM-stimulated ILC2s specifically expressed genes involved in the regulation of adaptive immunity through B and T cell interactions, whereas IL-33-stimulated ILC2s expressed high levels of proliferation-related and cytokine genes. In both airway inflammation models ILC2s were present in the lung submucosa close to epithelial cells, as identified by confocal microscopy. In chronic HDM-driven airway inflammation ILC2s were also found inside organized cellular infiltrates near T cells. Collectively, our findings show that ILC2s are phenotypically more heterogeneous than previously thought

Relationships between equine airway reactivity measured by flowmetric plethysmography and specific indicators of airway inflammation in horses with suspected inflammatory airway disease.

PubMed

Wichtel, M; Gomez, D; Burton, S; Wichtel, J; Hoffman, A

2016-07-01

Agreement between airway reactivity measured by flowmetric plethysmography and histamine bronchoprovocation, and lower airway inflammation measured by bronchoalveolar lavage (BAL) cytology, has not been studied in horses with suspected inflammatory airway disease (IAD). We tested the hypothesis that airway reactivity is associated with BAL cytology in horses presenting for unexplained poor performance and/or chronic cough. Prospective clinical study. Forty-five horses, predominantly young Standardbred racehorses, presenting for unexplained poor performance or chronic cough, underwent endoscopic evaluation, tracheal wash, flowmetric plethysmography with histamine bronchoprovocation and BAL. Histamine response was measured by calculating PC35, the concentration of nebulised histamine eliciting an increase in Δflow of 35%. In this population, there was no significant correlation between histamine response and cell populations in BAL cytology. When airway hyperreactivity (AHR) was defined as ≥35% increase in Δflow at a histamine concentration of <6 mg/ml, 24 of the 45 horses (53%) were determined to have AHR. Thirty-three (73%) had either abnormal BAL cytology or AHR, and were diagnosed with IAD on this basis. Of horses diagnosed with IAD, 9 (27%) had an abnormal BAL, 11 (33%) had AHR and 13 (39%) had both. Airway reactivity and BAL cytology did not show concordance in this population of horses presenting for unexplained poor performance and/or chronic cough. Failure to include tests of airway reactivity may lead to underdiagnosis of IAD in young Standardbred racehorses that present with clinical signs suggestive of IAD. © 2015 EVJ Ltd.

Anatomic Optical Coherence Tomography of Upper Airways

NASA Astrophysics Data System (ADS)

Chin Loy, Anthony; Jing, Joseph; Zhang, Jun; Wang, Yong; Elghobashi, Said; Chen, Zhongping; Wong, Brian J. F.

The upper airway is a complex and intricate system responsible for respiration, phonation, and deglutition. Obstruction of the upper airways afflicts an estimated 12-18 million Americans. Pharyngeal size and shape are important factors in the pathogenesis of airway obstructions. In addition, nocturnal loss in pharyngeal muscular tone combined with high pharyngeal resistance can lead to collapse of the airway and periodic partial or complete upper airway obstruction. Anatomical optical coherence tomography (OCT) has the potential to provide high-speed three-dimensional tomographic images of the airway lumen without the use of ionizing radiation. In this chapter we describe the methods behind endoscopic OCT imaging and processing to generate full three dimensional anatomical models of the human airway which can be used in conjunction with numerical simulation methods to assess areas of airway obstruction. Combining this structural information with flow dynamic simulations, we can better estimate the site and causes of airway obstruction and better select and design surgery for patients with obstructive sleep apnea.

Radiology-guided forceps biopsy and airway stenting in severe airway stenosis.

PubMed

Li, Zong Ming; Wu, Gang; Han, Xin Wei; Ren, Ke Wei; Zhu, Ming

2014-01-01

We aimed to determine the feasibility, safety, and effectiveness of radiology-guided forceps biopsy and airway stenting in patients with severe airway stenosis. This study involved 28 patients with severe airway stenosis who underwent forceps biopsy between October 2006 and September 2011. Chest multislice computed tomography was used to determine the location and extent of stenosis. Sixteen patients had tracheal stenosis, two patients had stenosis of the tracheal carina, six patients had stenosis of the left main bronchus, and four patients had stenosis of the right main bronchus. Forceps biopsy and stenting of the stenosed area were performed under fluoroscopic guidance in digital subtraction angiography and the biopsy specimens were analyzed histopathologically. We contacted the patients via phone call and utilized a standardized questionnaire to determine their medical condition during a postoperative three-month follow-up. The technical success rate of radiology-guided forceps biopsy was 100%. Biopsy specimens were obtained in all patients. Dyspnea was relieved immediately after stent placement. No serious complications, such as tracheal hemorrhage or perforation, mediastinal emphysema, or asphyxia, occurred. Radiology-guided forceps biopsy and airway stenting can be used for the emergency treatment of severe airway stenosis. This method appears to be safe and effective, and it may be an alternative therapeutic option in patients who cannot tolerate fiberoptic bronchoscopy.

Radiology-guided forceps biopsy and airway stenting in severe airway stenosis

PubMed Central

Li, Zong-Ming; Wu, Gang; Han, Xin-Wei; Ren, Ke-Wei; Zhu, Ming

2014-01-01

PURPOSE We aimed to determine the feasibility, safety, and effectiveness of radiology-guided forceps biopsy and airway stenting in patients with severe airway stenosis. MATERIALS AND METHODS This study involved 28 patients with severe airway stenosis who underwent forceps biopsy between October 2006 and September 2011. Chest multislice computed tomography was used to determine the location and extent of stenosis. Sixteen patients had tracheal stenosis, two patients had stenosis of the tracheal carina, six patients had stenosis of the left main bronchus, and four patients had stenosis of the right main bronchus. Forceps biopsy and stenting of the stenosed area were performed under fluoroscopic guidance in digital subtraction angiography and the biopsy specimens were analyzed histopathologically. We contacted the patients via phone call and utilized a standardized questionnaire to determine their medical condition during a postoperative three-month follow-up. RESULTS The technical success rate of radiology-guided forceps biopsy was 100%. Biopsy specimens were obtained in all patients. Dyspnea was relieved immediately after stent placement. No serious complications, such as tracheal hemorrhage or perforation, mediastinal emphysema, or asphyxia, occurred. CONCLUSION Radiology-guided forceps biopsy and airway stenting can be used for the emergency treatment of severe airway stenosis. This method appears to be safe and effective, and it may be an alternative therapeutic option in patients who cannot tolerate fiberoptic bronchoscopy. PMID:24808434

Receptor-mediated gene transfer vectors: progress towards genetic pharmaceuticals.

PubMed

Molas, M; Gómez-Valadés, A G; Vidal-Alabró, A; Miguel-Turu, M; Bermudez, J; Bartrons, R; Perales, J C

2003-10-01

Although specific delivery to tissues and unique cell types in vivo has been demonstrated for many non-viral vectors, current methods are still inadequate for human applications, mainly because of limitations on their efficiencies. All the steps required for an efficient receptor-mediated gene transfer process may in principle be exploited to enhance targeted gene delivery. These steps are: DNA/vector binding, internalization, subcellular trafficking, vesicular escape, nuclear import, and unpacking either for transcription or other functions (i.e., antisense, RNA interference, etc.). The large variety of vector designs that are currently available, usually aimed at improving the efficiency of these steps, has complicated the evaluation of data obtained from specific derivatives of such vectors. The importance of the structure of the final vector and the consequences of design decisions at specific steps on the overall efficiency of the vector will be discussed in detail. We emphasize in this review that stability in serum and thus, proper bioavailability of vectors to their specific receptors may be the single greatest limiting factor on the overall gene transfer efficiency in vivo. We discuss current approaches to overcome the intrinsic instability of synthetic vectors in the blood. In this regard, a summary of the structural features of the vectors obtained from current protocols will be presented and their functional characteristics evaluated. Dissecting information on molecular conjugates obtained by such methodologies, when carefully evaluated, should provide important guidelines for the creation of effective, targeted and safe DNA therapeutics.

Differences in lateral gene transfer in hypersaline versus thermal environments.

PubMed

Rhodes, Matthew E; Spear, John R; Oren, Aharon; House, Christopher H

2011-07-08

The role of lateral gene transfer (LGT) in the evolution of microorganisms is only beginning to be understood. While most LGT events occur between closely related individuals, inter-phylum and inter-domain LGT events are not uncommon. These distant transfer events offer potentially greater fitness advantages and it is for this reason that these "long distance" LGT events may have significantly impacted the evolution of microbes. One mechanism driving distant LGT events is microbial transformation. Theoretically, transformative events can occur between any two species provided that the DNA of one enters the habitat of the other. Two categories of microorganisms that are well-known for LGT are the thermophiles and halophiles. We identified potential inter-class LGT events into both a thermophilic class of Archaea (Thermoprotei) and a halophilic class of Archaea (Halobacteria). We then categorized these LGT genes as originating in thermophiles and halophiles respectively. While more than 68% of transfer events into Thermoprotei taxa originated in other thermophiles, less than 11% of transfer events into Halobacteria taxa originated in other halophiles. Our results suggest that there is a fundamental difference between LGT in thermophiles and halophiles. We theorize that the difference lies in the different natures of the environments. While DNA degrades rapidly in thermal environments due to temperature-driven denaturization, hypersaline environments are adept at preserving DNA. Furthermore, most hypersaline environments, as topographical minima, are natural collectors of cellular debris. Thus halophiles would in theory be exposed to a greater diversity and quantity of extracellular DNA than thermophiles.

Temperature-dependent innate defense against the common cold virus limits viral replication at warm temperature in mouse airway cells.

PubMed

Foxman, Ellen F; Storer, James A; Fitzgerald, Megan E; Wasik, Bethany R; Hou, Lin; Zhao, Hongyu; Turner, Paul E; Pyle, Anna Marie; Iwasaki, Akiko

2015-01-20

Most isolates of human rhinovirus, the common cold virus, replicate more robustly at the cool temperatures found in the nasal cavity (33-35 °C) than at core body temperature (37 °C). To gain insight into the mechanism of temperature-dependent growth, we compared the transcriptional response of primary mouse airway epithelial cells infected with rhinovirus at 33 °C vs. 37 °C. Mouse airway cells infected with mouse-adapted rhinovirus 1B exhibited a striking enrichment in expression of antiviral defense response genes at 37 °C relative to 33 °C, which correlated with significantly higher expression levels of type I and type III IFN genes and IFN-stimulated genes (ISGs) at 37 °C. Temperature-dependent IFN induction in response to rhinovirus was dependent on the MAVS protein, a key signaling adaptor of the RIG-I-like receptors (RLRs). Stimulation of primary airway cells with the synthetic RLR ligand poly I:C led to greater IFN induction at 37 °C relative to 33 °C at early time points poststimulation and to a sustained increase in the induction of ISGs at 37 °C relative to 33 °C. Recombinant type I IFN also stimulated more robust induction of ISGs at 37 °C than at 33 °C. Genetic deficiency of MAVS or the type I IFN receptor in infected airway cells permitted higher levels of viral replication, particularly at 37 °C, and partially rescued the temperature-dependent growth phenotype. These findings demonstrate that in mouse airway cells, rhinovirus replicates preferentially at nasal cavity temperature due, in part, to a less efficient antiviral defense response of infected cells at cool temperature.

Synthetic Fatty Acids Prevent Plasmid-Mediated Horizontal Gene Transfer

PubMed Central

Getino, María; Sanabria-Ríos, David J.; Fernández-López, Raúl; Campos-Gómez, Javier; Sánchez-López, José M.; Fernández, Antonio; Carballeira, Néstor M.

2015-01-01

ABSTRACT Bacterial conjugation constitutes a major horizontal gene transfer mechanism for the dissemination of antibiotic resistance genes among human pathogens. Antibiotic resistance spread could be halted or diminished by molecules that interfere with the conjugation process. In this work, synthetic 2-alkynoic fatty acids were identified as a novel class of conjugation inhibitors. Their chemical properties were investigated by using the prototype 2-hexadecynoic acid and its derivatives. Essential features of effective inhibitors were the carboxylic group, an optimal long aliphatic chain of 16 carbon atoms, and one unsaturation. Chemical modification of these groups led to inactive or less-active derivatives. Conjugation inhibitors were found to act on the donor cell, affecting a wide number of pathogenic bacterial hosts, including Escherichia, Salmonella, Pseudomonas, and Acinetobacter spp. Conjugation inhibitors were active in inhibiting transfer of IncF, IncW, and IncH plasmids, moderately active against IncI, IncL/M, and IncX plasmids, and inactive against IncP and IncN plasmids. Importantly, the use of 2-hexadecynoic acid avoided the spread of a derepressed IncF plasmid into a recipient population, demonstrating the feasibility of abolishing the dissemination of antimicrobial resistances by blocking bacterial conjugation. PMID:26330514

[Retroviral-mediated transfer of a hygromycin phosphotransferase-thymidine kinase fusion gene into human bladder carcinoma cell].

PubMed

Ye, C; Chen, S; Pei, X; Li, L; Feng, K

1999-08-01

To evaluate the therapeutic efficacy of retroviral-mediated hygromycin phosphotransferase-thymidine kinase fusion gene (HyTK)/GCV on human bladder carcinoma cell. A retroviral expression vector pL (HyTK) SN was constructed. By using FuGENE 6-mediated transfection and "ping-pong effect" technique, high-titer of retroviral supernatant was obtained and HyTK gene was transferred into EJ cells. A retroviral vector encoding, enhanced green fluorescent protein, EGFP was used to rapidly detect the transduction efficiency. Antitumor effects were observed after GCV treatment. In vitro experiments demonstrated the EJ cells transferred by HyTK gene were killed in the GCV treatment. Non-transduced parental cells were not sensitive to GCV, but they were dead by the bystander killing of neighboring cells when mixed with EJ/HyTK cells at various ratios. In addition, this not only affect wild-type EJ cells but also cells from different bladder carcinoma cell lines. Retroviral-mediated HyTK/GCV systems were a promising suicide gene therapy for bladder carcinoma. EGFP may act as a convenient and rapid reporter to monitor retroviral-mediated gene transfer and expression in bladder carcinoma cells.

Using complementary approaches to identify trans-domain nuclear gene transfers in the extremophile Galdieria sulphuraria (Rhodophyta).

PubMed

Pandey, Ravi S; Saxena, Garima; Bhattacharya, Debashish; Qiu, Huan; Azad, Rajeev K

2017-02-01

Identification of horizontal gene transfers (HGTs) has primarily relied on phylogenetic tree based methods, which require a rich sampling of sequenced genomes to ensure a reliable inference. Because the success of phylogenetic approaches depends on the breadth and depth of the database, researchers usually apply stringent filters to detect only the most likely gene transfers in the genomes of interest. One such study focused on a highly conservative estimate of trans-domain gene transfers in the extremophile eukaryote, Galdieria sulphuraria (Galdieri) Merola (Rhodophyta), by applying multiple filters in their phylogenetic pipeline. This led to the identification of 75 inter-domain acquisitions from Bacteria or Archaea. Because of the evolutionary, ecological, and potential biotechnological significance of foreign genes in algae, alternative approaches and pipelines complementing phylogenetics are needed for a more comprehensive assessment of HGT. We present here a novel pipeline that uncovered 17 novel foreign genes of prokaryotic origin in G. sulphuraria, results that are supported by multiple lines of evidence including composition-based, comparative data, and phylogenetics. These genes encode a variety of potentially adaptive functions, from metabolite transport to DNA repair. © 2016 Phycological Society of America.

Ocular gene transfer in the spotlight: implications of newspaper content for clinical communications

PubMed Central

2014-01-01

Background Ocular gene transfer clinical trials are raising hopes for blindness treatments and attracting media attention. News media provide an accessible health information source for patients and the public, but are often criticized for overemphasizing benefits and underplaying risks of novel biomedical interventions. Overly optimistic portrayals of unproven interventions may influence public and patient expectations; the latter may cause patients to downplay risks and over-emphasize benefits, with implications for informed consent for clinical trials. We analyze the news media communications landscape about ocular gene transfer and make recommendations for improving communications between clinicians and potential trial participants in light of media coverage. Methods We analyzed leading newspaper articles about ocular gene transfer (1990-2012) from United States (n = 55), Canada (n = 26), and United Kingdom (n = 77) from Factiva and Canadian Newsstand databases using pre-defined coding categories. We evaluated the content of newspaper articles about ocular gene transfer for hereditary retinopathies, exploring representations of framing techniques, research design, risks/benefits, and translational timelines. Results The dominant frame in 61% of stories was a celebration of progress, followed by human-interest in 30% of stories. Missing from the positive frames were explanations of research design; articles conflated clinical research with treatment. Conflicts-of-interest and funding sources were similarly omitted. Attention was directed to the benefits of gene transfer, while risks were only reported in 43% of articles. A range of visual outcomes was described from slowing vision loss to cure, but the latter was the most frequently represented even though it is clinically infeasible. Despite the prominence of visual benefit portrayals, 87% of the articles failed to provide timelines for the commencement of clinical trials or for clinical

Ocular gene transfer in the spotlight: implications of newspaper content for clinical communications.

PubMed

Benjaminy, Shelly; Bubela, Tania

2014-07-16

Ocular gene transfer clinical trials are raising hopes for blindness treatments and attracting media attention. News media provide an accessible health information source for patients and the public, but are often criticized for overemphasizing benefits and underplaying risks of novel biomedical interventions. Overly optimistic portrayals of unproven interventions may influence public and patient expectations; the latter may cause patients to downplay risks and over-emphasize benefits, with implications for informed consent for clinical trials. We analyze the news media communications landscape about ocular gene transfer and make recommendations for improving communications between clinicians and potential trial participants in light of media coverage. We analyzed leading newspaper articles about ocular gene transfer (1990-2012) from United States (n = 55), Canada (n = 26), and United Kingdom (n = 77) from Factiva and Canadian Newsstand databases using pre-defined coding categories. We evaluated the content of newspaper articles about ocular gene transfer for hereditary retinopathies, exploring representations of framing techniques, research design, risks/benefits, and translational timelines. The dominant frame in 61% of stories was a celebration of progress, followed by human-interest in 30% of stories. Missing from the positive frames were explanations of research design; articles conflated clinical research with treatment. Conflicts-of-interest and funding sources were similarly omitted. Attention was directed to the benefits of gene transfer, while risks were only reported in 43% of articles. A range of visual outcomes was described from slowing vision loss to cure, but the latter was the most frequently represented even though it is clinically infeasible. Despite the prominence of visual benefit portrayals, 87% of the articles failed to provide timelines for the commencement of clinical trials or for clinical implementation. Our analysis confirms

Parallel Histories of Horizontal Gene Transfer Facilitated Extreme Reduction of Endosymbiont Genomes in Sap-Feeding Insects

PubMed Central

Sloan, Daniel B.; Nakabachi, Atsushi; Richards, Stephen; Qu, Jiaxin; Murali, Shwetha Canchi; Gibbs, Richard A.; Moran, Nancy A.

2014-01-01

Bacteria confined to intracellular environments experience extensive genome reduction. In extreme cases, insect endosymbionts have evolved genomes that are so gene-poor that they blur the distinction between bacteria and endosymbiotically derived organelles such as mitochondria and plastids. To understand the host’s role in this extreme gene loss, we analyzed gene content and expression in the nuclear genome of the psyllid Pachypsylla venusta, a sap-feeding insect that harbors an ancient endosymbiont (Carsonella) with one of the most reduced bacterial genomes ever identified. Carsonella retains many genes required for synthesis of essential amino acids that are scarce in plant sap, but most of these biosynthetic pathways have been disrupted by gene loss. Host genes that are upregulated in psyllid cells housing Carsonella appear to compensate for endosymbiont gene losses, resulting in highly integrated metabolic pathways that mirror those observed in other sap-feeding insects. The host contribution to these pathways is mediated by a combination of native eukaryotic genes and bacterial genes that were horizontally transferred from multiple donor lineages early in the evolution of psyllids, including one gene that appears to have been directly acquired from Carsonella. By comparing the psyllid genome to a recent analysis of mealybugs, we found that a remarkably similar set of functional pathways have been shaped by independent transfers of bacterial genes to the two hosts. These results show that horizontal gene transfer is an important and recurring mechanism driving coevolution between insects and their bacterial endosymbionts and highlight interesting similarities and contrasts with the evolutionary history of mitochondria and plastids. PMID:24398322

AAV-Mediated Gene Transfer to Dorsal Root Ganglion.

PubMed

Yu, Hongwei; Fischer, Gregory; Hogan, Quinn H

2016-01-01

Transferring genetic molecules into the peripheral sensory nervous system to manipulate nociceptive pathophysiology is a powerful approach for experimental modulation of sensory signaling and potentially for translation into therapy for chronic pain. This can be efficiently achieved by the use of recombinant adeno-associated virus (rAAV) in conjunction with nociceptor-specific regulatory transgene cassettes. Among different routes of delivery, direct injection into the dorsal root ganglia (DRGs) offers the most efficient AAV-mediated gene transfer selectively into the peripheral sensory nervous system. Here, we briefly discuss the advantages and applications of intraganglionic microinjection, and then provide a detailed approach for DRG injection, including a list of the necessary materials and description of a method for performing DRG microinjection experiments. We also discuss our experience with several adeno-associated virus (AAV) options for in vivo transgene expression in DRG neurons.

Inflammatory bowel disease and airway diseases.

PubMed