低分子依存性緑色蛍光タンパク質UnaGの基礎的研究とその応用

早大学位記番号:新7827早稲田大

Detection of CO\u3csub\u3e2\u3c/sub\u3e leakage from a simulated sub-seabed storage site using three different types of pCO\u3csub\u3e2\u3c/sub\u3e sensors

© 2015 Elsevier Ltd. All rights reserved. This work is focused on results from a recent controlled sub-seabed in situ carbon dioxide (CO2) release experiment carried out during May-October 2012 in Ardmucknish Bay on the Scottish west coast. Three types of pCO2 sensors (fluorescence, NDIR and ISFET-based technologies) were used in combination with multiparameter instruments measuring oxygen, temperature, salinity and currents in the water column at the epicentre of release and further away. It was shown that distribution of seafloor CO2 emissions features high spatial and temporal heterogeneity. The highest pCO2 values (~1250 μatm) were detected at low tide around a bubble stream and within centimetres distance from the seafloor. Further up in the water column, 30-100 cm above the seabed, the gradients decreased, but continued to indicate elevated pCO2 at the epicentre of release throughout the injection campaign with the peak values between 400 and 740 μatm. High-frequency parallel measurements from two instruments placed within 1 m from each other, relocation of one of the instruments at the release site and 2D horizontal mapping of the release and control sites confirmed a localized impact from CO2 emissions. Observed effects on the water column were temporary and post-injection recovery took O2, and when it was influenced by purposefully released CO2. Use of a hydrodynamic circulation model, calibrated with in situ data, was crucial to establishing background conditions in this complex and dynamic shallow water system

Measuring pH variability using an experimental sensor on an underwater glider

Autonomous underwater gliders offer the capability of measuring oceanic parameters continuously at high resolution in both vertical and horizontal planes, with timescales that can extend to many months. An experimental ion sensitive field effect transistor (ISFET) sensor measuring pH on the total scale was attached to a glider during the REP14 – MED experiment in June 2014 in the northwestern Mediterranean Sea. During the deployment, pH was sampled at depths of up to 1000 m, along an 80 km transect over a period of 12 days. Water samples were collected from a nearby ship and analysed for dissolved inorganic carbon concentration and total alkalinity to derive pH for validating the ISFET measurements. The vertical resolution of the pH sensor was good (1 to 2 m), but stability was poor, and the sensor drifted in a non-monotonous fashion. In order to remove the sensor drift, a time-dependent, depth-invariant offset was applied throughout the water column for each dive, reducing the spread of the data by approximately two thirds. Furthermore, the ISFET sensor required temperature and pressure-based corrections, which were achieved using linear regression. Correcting for this decreased the apparent sensor pH variability by a further 13 to 31 %. Sunlight caused an apparent sensor pH decrease of up to 0.1 in surface waters around local noon, highlighting the importance of shielding the sensor away from light in future deployments. The corrected pH from the ISFET sensor is presented along with potential temperature, salinity, potential density anomalies (σθ), and dissolved oxygen concentrations (c(O2)) measured by the glider, providing insights into physical and biogeochemical variability in this region. pH maxima were identified at the depth of the summer chlorophyll maximum, where high c(O2) values were also found. Longitudinal pH variations at depth (σθ > 28.8 kg m−3) highlighted variability of water masses in this region. Higher pH was observed where salinity was > 38.65, and lower pH was found where salinity ranged between 38.3 and 38.65. It seemed that the higher pH was associated with saltier Levantine Intermediate Water. Furthermore, shoaling isopycnals closer to shore coinciding with low pH, high salinity, low c(O2) waters may be indicative of upwelling

Bright ligand-activatable fluorescent protein for high-quality multicolor live-cell super-resolution microscopy

We introduce UnaG as a green-to-dark photoswitching fluorescent protein capable of high-quality super-resolution imaging with photon numbers equivalent to the brightest photoswitchable red protein. UnaG only fluoresces upon binding of a fluorogenic metabolite, bilirubin, enabling UV-free reversible photoswitching with easily controllable kinetics and low background under Epi illumination. The on- and off-switching rates are controlled by the concentration of the ligand and the excitation light intensity, respectively, where the dissolved oxygen also promotes the off-switching. The photo-oxidation reaction mechanism of bilirubin in UnaG suggests that the lack of ligand-protein covalent bond allows the oxidized ligand to detach from the protein, emptying the binding cavity for rebinding to a fresh ligand molecule. We demonstrate super-resolution single-molecule localization imaging of various subcellular structures genetically encoded with UnaG, which enables facile labeling and simultaneous multicolor imaging of live cells. UnaG has the promise of becoming a default protein for high-performance super-resolution imaging. Photoconvertible proteins occupy two color channels thereby limiting multicolour localisation microscopy applications. Here the authors present UnaG, a new green-to-dark photoswitching fluorescent protein for super-resolution imaging, whose activation is based on a noncovalent binding with bilirubin

High-resolution observations in the Western Mediterranean Sea: The REP14-MED experiment

The observational part of the REP14-MED experiment was conducted in June 2014 in the Sardo-Balearic Sea west of Sardinia Island (Western Mediterranean Sea). Two research vessels collected high-resolution oceanographic data by means of hydrographic casts, towed systems, and underway measurements. In addition, a vast amount of data was provided by a fleet of 11 gliders, time series were available from moored instruments, and information on Lagrangian flow patterns were obtained from surface drifters and one profiling float. The spatial resolution of the observations encompasses a spectrum over four orders of magnitude from O(101 m) to O(105 m), and the time series from the moored instruments cover a spectral range of five orders from O(101 s) to O(106 s). The objective of this article is to provide an overview of the huge data set which is utilized by various ongoing studies, focusing on (i) sub-mesoscale and mesoscale pattern analyses, (ii) operational forecasting in terms of the development and assessment of sampling strategies, assimilation methods, and model validation, (iii) modeling the variability of the ocean, and (iv) testing of new payloads for gliders

Detection of CO2 leakage from a simulated sub-seabed storage site using three different types of pCO2 sensors

This work is focused on results from a recent controlled sub-seabed in situ carbon dioxide (CO2) release experiment carried out during May–October 2012 in Ardmucknish Bay on the Scottish west coast. Three types of pCO2 sensors (fluorescence, NDIR and ISFET-based technologies) were used in combination with multiparameter instruments measuring oxygen, temperature, salinity and currents in the water column at the epicentre of release and further away. It was shown that distribution of seafloor CO2 emissions features high spatial and temporal heterogeneity. The highest pCO2 values (∼1250 μatm) were detected at low tide around a bubble stream and within centimetres distance from the seafloor. Further up in the water column, 30–100 cm above the seabed, the gradients decreased, but continued to indicate elevated pCO2 at the epicentre of release throughout the injection campaign with the peak values between 400 and 740 μatm. High-frequency parallel measurements from two instruments placed within 1 m from each other, relocation of one of the instruments at the release site and 2D horizontal mapping of the release and control sites confirmed a localized impact from CO2 emissions. Observed effects on the water column were temporary and post-injection recovery took <7 days. A multivariate statistical approach was used to recognize the periods when the system was dominated by natural forcing with strong correlation between variation in pCO2 and O2, and when it was influenced by purposefully released CO2. Use of a hydrodynamic circulation model, calibrated with in situ data, was crucial to establishing background conditions in this complex and dynamic shallow water system

High-resolution observations in the western Mediterranean Sea: the REP14-MED experiment

The observational part of the REP14-MED experiment was conducted in June 2014 in the Sardo-Balearic Basin west of Sardinia (western Mediterranean Sea). Two research vessels collected high-resolution oceanographic data by means of hydrographic casts, towed systems, and underway measurements. In addition, a vast amount of data was provided by a fleet of 11 ocean gliders, time series were available from moored instruments, and information on Lagrangian flow patterns was obtained from surface drifters and one profiling float. The spatial resolution of the observations encompasses a spectrum over 4 orders of magnitude from (10¹ m) to (10⁵ m), and the time series from the moored instruments cover a spectral range of 5 orders from (10¹ s) to (10⁶ s). The objective of this article is to provide an overview of the huge data set which has been utilised by various studies, focusing on (i) water masses and circulation, (ii) operational forecasting, (iii) data assimilation, (iv) variability of the ocean, and (v) new payloads for gliders

Net community production in the northwestern Mediterranean Sea from glider and buoy measurements

The Mediterranean Sea comprises just 0.8 % of the global oceanic surface, yet considering its size, it is regarded as a disproportionately large sink for anthropogenic carbon due to its physical and biogeochemical characteristics. An underwater glider mission was carried out in March–April 2016 close to the BOUSSOLE and DyFAMed time series moorings in the northwestern Mediterranean Sea. The glider deployment served as a test of a prototype ion-sensitive field-effect transistor pH sensor. Dissolved oxygen (O2) concentrations and optical backscatter were also observed by the glider and increased between 19 March and 1 April, along with pH. These changes indicated the start of a phytoplankton spring bloom, following a period of intense mixing. Concurrent measurements of CO2 fugacity and O2 concentrations at the BOUSSOLE mooring buoy showed fluctuations, in qualitative agreement with the pattern of glider measurements. Mean net community production rates (N) were estimated from glider and buoy measurements of dissolved O2 and inorganic carbon (DIC) concentrations, based on their mass budgets. Glider and buoy DIC concentrations were derived from a salinity-based total alkalinity parameterisation, glider pH and buoy CO2 fugacity. The spatial coverage of glider data allowed the calculation of advective O2 and DIC fluxes. Mean N estimates for the euphotic zone between 10 March and 3 April were (-17±36) for glider O2, (44±94) for glider DIC, (17±37) for buoy O2 and (49±86)  mmolm-2d-1 for buoy DIC, all indicating net metabolic balance over these 25 d. However, these 25 d were actually split into a period of net DIC increase and O2 decrease between 10 and 19 March and a period of net DIC decrease and O2 increase between 19 March and 3 April. The latter period is interpreted as the onset of the spring bloom. The regression coefficients between O2 and DIC-based N estimates were 0.25 ± 0.08 for the glider data and 0.54 ± 0.06 for the buoy, significantly lower than the canonical metabolic quotient of 1.45±0.15. This study shows the added value of co-locating a profiling glider with moored time series buoys, but also demonstrates the difficulty in estimating N, and the limitations in achievable precision

Metabolically active microbial communities in marine sediment under high-CO2 and low-pH extremes

Sediment-hosting hydrothermal systems in the Okinawa Trough maintain a large amount of liquid, supercritical and hydrate phases of CO2 in the seabed. The emission of CO2 may critically impact the geochemical, geophysical and ecological characteristics of the deep-sea sedimentary environment. So far it remains unclear whether microbial communities that have been detected in such high-CO2 and low-pH habitats are metabolically active, and if so, what the biogeochemical and ecological consequences for the environment are. In this study, RNA-based molecular approaches and radioactive tracer-based respiration rate assays were combined to study the density, diversity and metabolic activity of microbial communities in CO2-seep sediment at the Yonaguni Knoll IV hydrothermal field of the southern Okinawa Trough. In general, the number of microbes decreased sharply with increasing sediment depth and CO2 concentration. Phylogenetic analyses of community structure using reverse-transcribed 16S ribosomal RNA showed that the active microbial community became less diverse with increasing sediment depth and CO2 concentration, indicating that microbial activity and community structure are sensitive to CO2 venting. Analyses of RNA-based pyrosequences and catalyzed reporter deposition-fluorescence in situ hybridization data revealed that members of the SEEP-SRB2 group within the Deltaproteobacteria and anaerobic methanotrophic archaea (ANME-2a and -2c) were confined to the top seafloor, and active archaea were not detected in deeper sediments (13–30 cm in depth) characterized by high CO2. Measurement of the potential sulfate reduction rate at pH conditions of 3–9 with and without methane in the headspace indicated that acidophilic sulfate reduction possibly occurs in the presence of methane, even at very low pH of 3. These results suggest that some members of the anaerobic methanotrophs and sulfate reducers can adapt to the CO2-seep sedimentary environment; however, CO2 and pH in the deep-sea sediment were found to severely impact the activity and structure of the microbial community

Detection and impacts of leakage from sub-seafloor deep geological carbon dioxide storage

Fossil fuel power generation and other industrial emissions of carbon dioxide are a threat to global climate1, yet many economies will remain reliant on these technologies for several decades2. Carbon dioxide capture and storage (CCS) in deep geological formations provides an effective option to remove these emissions from the climate system3. In many regions storage reservoirs are located offshore4, 5, over a kilometre or more below societally important shelf seas6. Therefore, concerns about the possibility of leakage7, 8 and potential environmental impacts, along with economics, have contributed to delaying development of operational CCS. Here we investigate the detectability and environmental impact of leakage from a controlled sub-seabed release of CO2. We show that the biological impact and footprint of this small leak analogue (&lt;1 tonne CO2 d?1) is confined to a few tens of metres. Migration of CO2 through the shallow seabed is influenced by near-surface sediment structure, and by dissolution and re-precipitation of calcium carbonate naturally present in sediments. Results reported here advance the understanding of environmental sensitivity to leakage and identify appropriate monitoring strategies for full-scale carbon storage operations