A 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

A 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

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

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

Compositional and fluorescence characteristics of the giant diatom Ethmodiscus along a 3000 km transect (28°N) in the central North Pacific gyre

The giant diatom Ethmodiscus was examined along an east-west transect at 28-30°N during 2002 and 2003 to determine if abundance, chemical composition or physiological status of this largest of diatoms varied on the scale of 100\u27s-1000\u27s of km in North Pacific gyre. Abundance ranged from \u3c0.1-\u3e2.0 cells m-3 and supported the notion of an abundance mosaic reported previously. However, there was only minimal support for the relationship between abundance and nutrient concentration at 125 m reported previously. Cellular chlorophyll varied little along the transect (7.3-10.9 ng chl cell-1) except at the westernmost station. Cellular N and P quotas co-varied 3-4.5 fold (mean=50.8±3.7 and 3.7±0.8 nmol N and P cell-1) and yielded N:P ratios that closely clustered around the Redfield ratio (average=14.6±1.1). Only low levels of chlorophyll-normalized alkaline phosphatase (APase) activity were observed (0.4-2.5 nmol P μg chl-1 h-1) with APase activity lower than that in either the bulk water, or co-occurring Trichodesmium spp. and Pyrocystis noctiluca. The active fluorescence parameter Fv:Fm, a property sensitive to Fe stress, was uniformly high at all stations (average=0.73±0.04 for 2003, and 0.69±0.05 for 2002), indicating sufficient Fe for optimum photosynthetic competence. These results contrasted sharply with results from Rhizosolenia mats reported along the same transect where there was a significant decline westward in Fv:Fm. Both ferredoxin (Fd) and flavodoxin accumulated in cells of Ethmodiscus, resulting in Fd Index values of\u3c0.6. Iron cell quotas ranged from 0.7-5.1 pmol Fe cell-1. When normalized to cytoplasmic volume, the Fe μm-3 was comparable to that of Escherichia coli. We note that the disproportionate contribution of the vacuole (with its high organic content) to total volume typical of large diatoms is a potentially significant source of error in Fe:C ratios and suggest that Fe should be normalized to cytoplasmic volume whenever possible to permit valid intercomparisons between studies. The composition, Fv:Fm data and Fe:C ratio suggest a relatively uniform population experiencing little N, P or Fe stress. The uncoupling of the Fd Index from these measures is consistent with previous findings showing that the expression of flavodoxin can be characterized as an early stress response and that its accumulation is not necessarily correlated with physiological deficit. Ethmodiscus appears to be well adapted to some of the most oligotrophic waters in the ocean. Because it is an important sedimentary marker, the biology of living Ethmodiscus provides insights into the source of extensive Ethmodiscus oozes. Mass sedimentation after frontal accumulation has been suggested as a source for these oozes. Our data contain no evidence that the flux is linked directly to Fe, N or P stress. © 2007 Elsevier Ltd. All rights reserved

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