Pharmacologic inhibition of S-nitrosoglutathione reductase protects against experimental asthma in BALB/c mice through attenuation of both bronchoconstriction and inflammation

BACKGROUND: S-nitrosoglutathione (GSNO) serves as a reservoir for nitric oxide (NO) and thus is a key homeostatic regulator of airway smooth muscle tone and inflammation. Decreased levels of GSNO in the lungs of asthmatics have been attributed to increased GSNO catabolism via GSNO reductase (GSNOR) leading to loss of GSNO- and NO- mediated bronchodilatory and anti-inflammatory actions. GSNOR inhibition with the novel small molecule, N6022, was explored as a therapeutic approach in an experimental model of asthma. METHODS: Female BALB/c mice were sensitized and subsequently challenged with ovalbumin (OVA). Efficacy was determined by measuring both airway hyper-responsiveness (AHR) upon methacholine (MCh) challenge using whole body plethysmography and pulmonary eosinophilia by quantifying the numbers of these cells in the bronchoalveolar lavage fluid (BALF). Several other potential biomarkers of GSNOR inhibition were measured including levels of nitrite, cyclic guanosine monophosphate (cGMP), and inflammatory cytokines, as well as DNA binding activity of nuclear factor kappa B (NFκB). The dose response, onset of action, and duration of action of a single intravenous dose of N6022 given from 30 min to 48 h prior to MCh challenge were determined and compared to effects in mice not sensitized to OVA. The direct effect of N6022 on airway smooth muscle tone also was assessed in isolated rat tracheal rings. RESULTS: N6022 attenuated AHR (ED(50) of 0.015 ± 0.002 mg/kg; Mean ± SEM) and eosinophilia. Effects were observed from 30 min to 48 h after treatment and were comparable to those achieved with three inhaled doses of ipratropium plus albuterol used as the positive control. N6022 increased BALF nitrite and plasma cGMP, while restoring BALF and plasma inflammatory markers toward baseline values. N6022 treatment also attenuated the OVA-induced increase in NFκB activation. In rat tracheal rings, N6022 decreased contractile responses to MCh. CONCLUSIONS: The significant bronchodilatory and anti-inflammatory actions of N6022 in the airways are consistent with restoration of GSNO levels through GSNOR inhibition. GSNOR inhibition may offer a therapeutic approach for the treatment of asthma and other inflammatory lung diseases. N6022 is currently being evaluated in clinical trials for the treatment of inflammatory lung disease

Development of a sensitive trial-ready poly(GP) CSF biomarker assay for <i>C9orf72</i>-associated frontotemporal dementia and amyotrophic lateral sclerosis

Data availability statement: Data are available upon reasonable request.Supplementary Data: This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content. Data supplement 1 available at: https://jnnp.bmj.com/highwire/filestream/214878/field_highwire_adjunct_files/0/jnnp-2021-328710supp001_data_supplement.pdf .Copyright © Author(s) (or their employer(s)) 2022. Objective: A GGGGCC repeat expansion in the C9orf72 gene is the most common cause of genetic frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). As potential therapies targeting the repeat expansion are now entering clinical trials, sensitive biomarker assays of target engagement are urgently required. Our objective was to develop such an assay. Methods: We used the single molecule array (Simoa) platform to develop an immunoassay for measuring poly(GP) dipeptide repeat proteins (DPRs) generated by the C9orf72 repeat expansion in cerebrospinal fluid (CSF) of people with C9orf72-associated FTD/ALS. Results and conclusions: We show the assay to be highly sensitive and robust, passing extensive qualification criteria including low intraplate and interplate variability, a high precision and accuracy in measuring both calibrators and samples, dilutional parallelism, tolerance to sample and standard freeze–thaw and no haemoglobin interference. We used this assay to measure poly(GP) in CSF samples collected through the Genetic FTD Initiative (N=40 C9orf72 and 15 controls). We found it had 100% specificity and 100% sensitivity and a large window for detecting target engagement, as the C9orf72 CSF sample with the lowest poly(GP) signal had eightfold higher signal than controls and on average values from C9orf72 samples were 38-fold higher than controls, which all fell below the lower limit of quantification of the assay. These data indicate that a Simoa-based poly(GP) DPR assay is suitable for use in clinical trials to determine target engagement of therapeutics aimed at reducing C9orf72 repeat-containing transcripts.This work was funded by Wave Life Sciences, the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (648716 - C9ND) (AMI), the UK Dementia Research Institute, which receives its funding from UK DRI, funded by the UK Medical Research Council, Alzheimer's Society and Alzheimer's Research UK. The Dementia Research Centre is supported by Alzheimer's Research UK, Alzheimer's Society, Brain Research UK and The Wolfson Foundation. This work was supported by the NIHR UCL/H Biomedical Research Centre, the Leonard Wolfson Experimental Neurology Centre (LWENC) Clinical Research Facility and the NIHR Cambridge Biomedical Research Centre (BRC-1215-20014). The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care. AK is supported by a Weston Brain Institute and Selfridges Group Foundation award (UB170045). JMS is supported by Engineering and Physical Sciences Research Council (EP/J020990/1), British Heart Foundation (PG/17/90/33415), EU’s Horizon 2020 research and innovation programme (666992). HZ is a Wallenberg Scholar. Simoa instruments used were funded by Wellcome Trust, Fidelity International Foundation and UK DRI. JDR is supported by the Miriam Marks Brain Research UK Senior Fellowship and has received funding from an MRC Clinician Scientist Fellowship (MR/M008525/1) and the NIHR Rare Disease Translational Research Collaboration (BRC149/NS/MH). This work was also supported by the MRC UK GENFI grant (MR/M023664/1), the Bluefield Project and the JPND GENFI-PROX grant (2019-02248). Several authors of this publication are members of the European Reference Network for Rare Neurological Diseases - Project ID No 739510

Construction and Characterization of Cyanobacterial Bioreporters to Assess Nutrient (P, Fe) Availability in Marine Environments

Nutrient deficiency especially phosphorus (P) and iron (Fe), are well documented in world oceans, particularly associated with oligotrophic oceanic gyres and “high nutrient, low chlorophyll” (HNLC) regions. As a corresponding approach to identify bioavailable Fe in marine systems, my research has focused on the development, characterization and implementation of a whole-cell cyanobacterial Fe bioreporter to be used to assess the bioavailability of Fe in marine environments. A Fe responsive whole-cell marine bioreporter was developed by integrating a construct of the isiAB promoter fused to bacterial luciferase luxAB into the marine cyanobacterium Synechococcus PCC 7002. Dose-response characterization of the bioreporter was made in trace-metal buffered synthetic seawater medium containing Fe concentrations in the range of pFe [-log (Fe3+ free ferric)] 19.4 – 22.4. The luminescent response from the Fe bioreporter was best described by a sigmoidal dose-response curve. A comprehensive growth and physiological characterization of the bioreporter was conducted prior to implementing it to assess Fe bioavailability from various marine samples. The bioreporters response was optimum when it was incubated at 25 °C under an irradiance of 45 µmol photon m-2 s-1. Varying the amount of bioreporter inoculum or salinity of the test medium did not affect cellular luminescence over the 12 h assay period. Testing the ability of the bioreporter to acquire Fe3+ bound to ligands, the bioreporter was able to acquire bound Fe from rhodotorulic acid but, was unable to acquire Fe from ligands such as desferrioxamine B and N, N’-diethylenediamine-N,N’-diacetic acid. We assessed Fe availability in environmental samples from the Baltic Sea using the cyanobacterial bioreporter and demonstrated that the bioavailable Fe was one order of magnitude lower than the chemically-determined dissolved Fe and that samples from depth showed higher available Fe than at the surface. The Fe bioreporter was also implemented for assessing available Fe in samples collected during the mesoscale Fe fertilization experiment SERIES conducted in the HNLC eastern subarctic Pacific Ocean, and in samples collected from oligotrophic waters of the central north Pacific gyre during the ROMP study. The luminescent response from these open ocean samples was consistently higher than the corresponding calibration standard. As a result, the cyanobacterial Fe bioreporter can only be considered a qualitative tool with which to assess Fe availability in open ocean environments. Despite this constraint, we observed genuine differences in Fe availability from samples collected during each study

Cyanobacterial bioreporters as sensors of nutrient availability

Due to their ubiquity in aquatic environments and their contribution to total biomass, especially in oligotrophic systems, cyanobacteria can be viewed as a proxy for primary productivity in both marine and fresh waters. In this chapter we describe the development and use of picocyanobacterial bioreporters to measure the bioavailability of nutrients that may constrain total photosynthesis in both lacustrine and marine systems. Issues pertaining to bioreporter construction, performance and field applications are discussed. Specifically, luminescent Synechococcus spp. and Synechocystis spp. bioreporters are described that allow the bioavailability of phosphorus, nitrogen and iron to be accurately measured in environmental samples. © Springer-Verlag Berlin Heidelberg 2010

Luminescent Whole-Cell Cyanobacterial Bioreporter for Measuring Fe Availability in Diverse Marine Environments

A Synechococcus sp. strain PCC 7002 Fe bioreporter was constructed containing the isiAB promoter fused to the Vibrio harveyi luxAB genes. Bioreporter luminescence was characterized with respect to the free ferric ion concentration in trace metal-buffered synthetic medium. The applicability of the Fe bioreporter to assess Fe availability in the natural environment was tested by using samples collected from the Baltic Sea and from the high-nutrient, low-chlorophyll subarctic Pacific Ocean. Parallel assessment of dissolved Fe and bioreporter response confirmed that direct chemical measurements of dissolved Fe should not be considered alone when assessing Fe availability to phytoplankton

Development of a sensitive trial-ready poly(GP) CSF biomarker assay for C9orf72-associated frontotemporal dementia and amyotrophic lateral sclerosis.

Funder: Bluefield ProjectFunder: Fidelity Foundation; FundRef: http://dx.doi.org/10.13039/100005639Funder: European Reference Network for Rare Neurological DiseasesFunder: Wellcome Trust; FundRef: http://dx.doi.org/10.13039/100010269Funder: EU Joint Programme - Neurodegenerative Disease ResearchOBJECTIVE: A GGGGCC repeat expansion in the C9orf72 gene is the most common cause of genetic frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). As potential therapies targeting the repeat expansion are now entering clinical trials, sensitive biomarker assays of target engagement are urgently required. Our objective was to develop such an assay. METHODS: We used the single molecule array (Simoa) platform to develop an immunoassay for measuring poly(GP) dipeptide repeat proteins (DPRs) generated by the C9orf72 repeat expansion in cerebrospinal fluid (CSF) of people with C9orf72-associated FTD/ALS. RESULTS AND CONCLUSIONS: We show the assay to be highly sensitive and robust, passing extensive qualification criteria including low intraplate and interplate variability, a high precision and accuracy in measuring both calibrators and samples, dilutional parallelism, tolerance to sample and standard freeze-thaw and no haemoglobin interference. We used this assay to measure poly(GP) in CSF samples collected through the Genetic FTD Initiative (N=40 C9orf72 and 15 controls). We found it had 100% specificity and 100% sensitivity and a large window for detecting target engagement, as the C9orf72 CSF sample with the lowest poly(GP) signal had eightfold higher signal than controls and on average values from C9orf72 samples were 38-fold higher than controls, which all fell below the lower limit of quantification of the assay. These data indicate that a Simoa-based poly(GP) DPR assay is suitable for use in clinical trials to determine target engagement of therapeutics aimed at reducing C9orf72 repeat-containing transcripts