Iron limitation and the role of siderophores in marine Synechococcus

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution June 2009Marine cyanobacteria in the genus Synechococcus are widely distributed and contribute significantly to global primary productivity. In many parts of the ocean their growth is limited by a lack of iron, an essential nutrient that is virtually insoluble in seawater. To overcome this, Synechococcus have evolved a number of strategies to acquire iron. Gene distribution, metagenomics and a novel immunological flow cytometry assay in the Costa Rica Upwelling Dome were used to estimate the importance of Fe stress. Genomic and metagenomic measures suggest that iron limitation is, paradoxically, more severe in coastal and upwelling areas than in the open ocean, where iron is less abundant. A serological assay found significant differences in the vertical distribution of the Fe stress protein IdiA over just a few meters. Despite average surface ocean iron concentrations of just 0.07 nM, most marine oligotrophic cyanobacteria lack iron-binding siderophores that are present in many heterotrophic marine bacteria. Siderophores are widely distributed in the surface ocean and compose an important portion of the pool of natural ligands that bind >99% of all soluble Fe. In bottle incubations from the Sargasso Sea we found the addition of Fe complexed to an excess of the siderophore desferrioxamine B (DFB) limited Synechococcus growth and stimulated the growth of heterotrophic bacteria in a concentration dependent manner. Laboratory work revealed that excess DFB decreased Synechococcus growth beyond Fe-limited controls at concentrations as low as 20-40 nM. The inhibition was aggravated by light but it could be reversed by the addition of Fe. The DFB inhibition could not be explained by thermodynamic or kinetic models of Fe’ or co-limitation with other metals. DFB may interact with some aspect of cellular physiology to directly inhibit cyanobacterial growth.Funding for this research was provided by Grant NSF-OCE0352241 from the National Science Foundation to Eric A. Webb and grants 495 and 495.01 from the Gordon and Betty Moore Foundation, grants DE-F602-07ER64506 and DE-F602- 08ER64516 from the Department of Energy, and grant Z792093-02 (C-MORE) from the University of Hawaii, to Sallie W. Chisholm

Iron limitation and the role of Siderophores in marine Synechococcus

Thesis (Ph. D.)--Joint Program in Oceanography (Massachusetts Institute of Technology, Dept. of Biology; and the Woods Hole Oceanographic Institution), 2009.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections."June 2009."Includes bibliographical references.Marine cyanobacteria in the genus Synechococcus are widely distributed and contribute significantly to global primary productivity. In many parts of the ocean their growth is limited by a lack of iron, an essential nutrient that is virtually insoluble in seawater. To overcome this, Synechococcus have evolved a number of strategies to acquire iron. Gene distribution, metagenomics and a novel immunological flow cytometry assay in the Costa Rica Upwelling Dome were used to estimate the importance of Fe stress. Genomic and metagenomic measures suggest that iron limitation is, paradoxically, more severe in coastal and upwelling areas than in the open ocean, where iron is less abundant. A serological assay found significant differences in the vertical distribution of the Fe stress protein IdiA over just a few meters. Despite average surface ocean iron concentrations of just 0.07 nM, most marine oligotrophic cyanobacteria lack iron-binding siderophores that are present in many heterotrophic marine bacteria. Siderophores are widely distributed in the surface ocean and compose an important portion of the pool of natural ligands that bind >99% of all soluble Fe. In bottle incubations from the Sargasso Sea we found the addition of Fe complexed to an excess of the siderophore desferrioxamine B (DFB) limited Synechococcus growth and stimulated the growth of heterotrophic bacteria in a concentration dependent manner.(cont.) Laboratory work revealed that excess DFB decreased Synechococcus growth beyond Fe-limited controls at concentrations as low as 20-40 nM. The inhibition was aggravated by light but it could be reversed by the addition of Fe. The DFB inhibition could not be explained by thermodynamic or kinetic models of Fe' or co-limitation with other metals. DFB may interact with some aspect of cellular physiology to directly inhibit cyanobacterial growth.by Adam R. Rivers.Ph.D

ITSxpress: Software to rapidly trim internally transcribed spacer sequences with quality scores for marker gene analysis [version 1; referees: 2 approved]

The internally transcribed spacer (ITS) region between the small subunit ribosomal RNA gene and large subunit ribosomal RNA gene is a widely used phylogenetic marker for fungi and other taxa. The eukaryotic ITS contains the conserved 5.8S rRNA and is divided into the ITS1 and ITS2 hypervariable regions. These regions are variable in length and are amplified using primers complementary to the conserved regions of their flanking genes. Previous work has shown that removing the conserved regions results in more accurate taxonomic classification. An existing software program, ITSx, is capable of trimming FASTA sequences by matching hidden Markov model profiles to the ends of the conserved genes using the software suite HMMER. ITSxpress was developed to extend this technique from marker gene studies using Operational Taxonomic Units (OTU’s) to studies using exact sequence variants; a method used by the software packages Dada2, Deblur, QIIME 2, and Unoise. The sequence variant approach uses the quality scores of each read to identify sequences that are statistically likely to represent real sequences. ITSxpress enables this by processing FASTQ rather than FASTA files. The software also speeds up the trimming of reads by a factor of 14-23 times on a 4-core computer by temporarily clustering highly similar sequences that are common in amplicon data and utilizing optimized parameters for Hmmsearch. ITSxpress is available as a QIIME 2 plugin and a stand-alone application installable from the Python package index, Bioconda, and Github

The role of functional single nucleotide polymorphisms of the human glucocorticoid receptor gene NR3C1 in Polish patients with bronchial asthma

N363S and ER22/23EK polymorphisms observed within glucocorticoid receptor gene (NR3C1) may play an important role in the development of bronchial asthma. NR3C1 gene is associated with an altered sensitivity to GCs. The aim of the research project was to study the correlation between this NR3C1 gene polymorphisms and occurrence of asthma in the population of Polish asthmatics. Peripheral blood was obtained from 207 healthy volunteers and 221 asthma patients. Genotyping was carried out with PCR-RFLP method. In the groups of patients with uncontrolled moderate asthma and uncontrolled severe disease, the genotype distribution for the investigated polymorphisms was as follows: N363S-AA, AG, GG occurring with 0.881/0.073/0.046 frequency and ER22/23EK-GG, GA, AA occurring with 0.963/0.037/0.000 frequency. Chi-square analysis revealed a significantly different (P < 0.05) distribution between cases and controls for the N363S polymorphisms. The N363S polymorphism of NR3C1 gene is significantly associated with bronchial asthma, susceptibility to the development of moderate to severe form of uncontrolled bronchial asthma

IgE and IgG4 epitopes of the peanut allergens shift following oral immunotherapy

Background Oral immunotherapy (OIT) with peanut (Arachis hypogaea) allergen powder-dnfp (PTAH; Aimmune Therapeutics) is an FDA-approved treatment to desensitize peanut allergic participants. Objective Here we assessed shifts in IgE and IgG4 binding to peanut allergens and their epitopes recognized by United States (US) peanut allergic participants (n = 20) enrolled in phase 3 PTAH OIT clinical trials. Methods Pre- and post- trial participant sera were collected approximately 12 months apart and tested for IgE binding to intact peanut proteins via ImmunoCAP ISAC immunoassays. IgE and IgG4 linear epitopes were identified based on binding to synthetic overlapping 15-mer linear peptides of 10 peanut allergens (Ara h 1-11) synthesized on microarray slides. Results Statistically significant decreases in IgE binding were identified for intact Ara h 2, 3, and 6, and known and newly identified IgE epitopes were shown to exhibit shifts towards IgG4 binding post-OIT, with most linear peptides having increased IgG4 binding after treatment with PTAH. While PTAH does not seem to alter the actual peptide binding patterns significantly after one year of treatment, the IgE and IgG4 binding ratios and intensity are altered. Conclusion At a population level, the linear IgE and IgG4 epitopes of 10 peanut allergens overlap and that increase in IgG4 with OIT results in displacement of IgE binding to both conformational and linear epitopes. Furthermore, it appears as though the increase in IgG4 is more important to achieve desensitization at the 12-month timepoint than the decrease in IgE. This type of knowledge can be useful in the identification of IgE and IgG4-binding allergen and peptide biomarkers that may indicate desensitization or sustained unresponsiveness of allergic individuals to peanut

Genetic mechanisms of critical illness in COVID-19.

Host-mediated lung inflammation is present1, and drives mortality2, in the critical illness caused by coronavirus disease 2019 (COVID-19). Host genetic variants associated with critical illness may identify mechanistic targets for therapeutic development3. Here we report the results of the GenOMICC (Genetics Of Mortality In Critical Care) genome-wide association study in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units. We have identified and replicated the following new genome-wide significant associations: on chromosome 12q24.13 (rs10735079, P = 1.65 × 10-8) in a gene cluster that encodes antiviral restriction enzyme activators (OAS1, OAS2 and OAS3); on chromosome 19p13.2 (rs74956615, P = 2.3 × 10-8) near the gene that encodes tyrosine kinase 2 (TYK2); on chromosome 19p13.3 (rs2109069, P = 3.98 ×  10-12) within the gene that encodes dipeptidyl peptidase 9 (DPP9); and on chromosome 21q22.1 (rs2236757, P = 4.99 × 10-8) in the interferon receptor gene IFNAR2. We identified potential targets for repurposing of licensed medications: using Mendelian randomization, we found evidence that low expression of IFNAR2, or high expression of TYK2, are associated with life-threatening disease; and transcriptome-wide association in lung tissue revealed that high expression of the monocyte-macrophage chemotactic receptor CCR2 is associated with severe COVID-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms and mediators of inflammatory organ damage in COVID-19. Both mechanisms may be amenable to targeted treatment with existing drugs. However, large-scale randomized clinical trials will be essential before any change to clinical practice

Protocol for the perfusion and angiography imaging sub-study of the Third International Stroke Trial (IST-3) of alteplase treatment within six-hours of acute ischemic stroke

RATIONALE: Intravenous thrombolysis with recombinant tissue Plasminogen Activator improves outcomes in patients treated early after stroke but at the risk of causing intracranial hemorrhage. Restricting recombinant tissue Plasminogen Activator use to patients with evidence of still salvageable tissue, or with definite arterial occlusion, might help reduce risk, increase benefit and identify patients for treatment at late time windows. AIMS: To determine if perfusion or angiographic imaging with computed tomography or magnetic resonance help identify patients who are more likely to benefit from recombinant tissue Plasminogen Activator in the context of a large multicenter randomized trial of recombinant tissue Plasminogen Activator given within six-hours of onset of acute ischemic stroke, the Third International Stroke Trial. DESIGN: Third International Stroke Trial is a prospective multicenter randomized controlled trial testing recombinant tissue Plasminogen Activator (0·9 mg/kg, maximum dose 90 mg) started up to six-hours after onset of acute ischemic stroke, in patients with no clear indication for or contraindication to recombinant tissue Plasminogen Activator. Brain imaging (computed tomography or magnetic resonance) was mandatory pre-randomization to exclude hemorrhage. Scans were read centrally, blinded to treatment and clinical information. In centers where perfusion and/or angiography imaging were used routinely in stroke, these images were also collected centrally, processed and assessed using validated visual scores and computational measures. STUDY OUTCOMES: The primary outcome in Third International Stroke Trial is alive and independent (Oxford Handicap Score 0-2) at 6 months; secondary outcomes are symptomatic and fatal intracranial hemorrhage, early and late death. The perfusion and angiography study additionally will examine interactions between recombinant tissue Plasminogen Activator and clinical outcomes, infarct growth and recanalization in the presence or absence of perfusion lesions and/or arterial occlusion at presentation. The study is registered ISRCTN25765518