Robust LC3B lipidation analysis by precisely adjusting autophagic flux

Autophagic flux can be quantified based on the accumulation of lipidated LC3B in the presence of late-stage autophagy inhibitors. This method has been widely applied to identify novel compounds that activate autophagy. Here we scrutinize this approach and show that bafilomycin A1 (BafA) but not chloroquine is suitable for flux quantification due to the stimulating effect of chloroquine on non-canonical LC3B-lipidation. Significant autophagic flux increase by rapamycin could only be observed when combining it with BafA concentrations not affecting basal flux, a condition which created a bottleneck, rather than fully blocking autophagosome-lysosome fusion, concomitant with autophagy stimulation. When rapamycin was combined with saturating concentrations of BafA, no significant further increase of LC3B lipidation could be detected over the levels induced by the late-stage inhibitor. The large assay window obtained by this approach enables an effective discrimination of autophagy activators based on their cellular potency. To demonstrate the validity of this approach, we show that a novel inhibitor of the acetyltransferase EP300 activates autophagy in a mTORC1-dependent manner. We propose that the creation of a sensitized background rather than a full block of autophagosome progression is required to quantitatively capture changes in autophagic flux.

Creating a bottleneck: Robust LC3B lipidation analysis by adjusting autophagic flux with low concentrations of Bafilomycin A1

Autophagic flux can be quantified based on the accumulation of lipidated LC3B in the presence of late-stage autophagy inhibitors. This method has been widely applied to identify novel compounds that activate autophagy. Here we scrutinize this approach and show that bafilomycin A1 (BafA) but not chloroquine is suitable for flux quantification due to the stimulating effect of chloroquine on non-canonical LC3B-lipidation. Significant autophagic flux increase by rapamycin could only be observed when combining it with BafA concentrations not affecting basal flux, a condition which created a bottleneck, rather than fully blocking autophagosome-lysosome fusion, concomitant with autophagy stimulation. When rapamycin was combined with saturating concentrations of BafA, no significant further increase of LC3B lipidation could be detected over the levels induced by the late-stage inhibitor. The large assay window obtained by this approach enables an effective discrimination of autophagy activators based on their cellular potency. To demonstrate the validity of this approach, we show that a novel inhibitor of the acetyltransferase EP300 activates autophagy in a mTORC1-dependent manner. We propose that the creation of a sensitized background rather than a full block of autophagosome progression is required to quantitatively capture changes in autophagic flux.

Precise multispecies agricultural gas flux determined using broadband open-path dual-comb spectroscopy

Advances in spectroscopy have the potential to improve our understanding of agricultural processes and associated trace gas emissions. We implement field-deployed, open-path dual-comb spectroscopy (DCS) for precise multispecies emissions estimation from livestock. With broad atmospheric dual-comb spectra, we interrogate upwind and downwind paths from pens containing approximately 300 head of cattle, providing time-resolved concentration enhancements and fluxes of CH4, NH3, CO2, and H2O. The methane fluxes determined from DCS data and fluxes obtained with a colocated closed-path cavity ring-down spectroscopy gas analyzer agree to within 6%. The NH3 concentration retrievals have sensitivity of 10 parts per billion and yield corresponding NH3 fluxes with a statistical precision of 8% and low systematic uncertainty. Open-path DCS offers accurate multispecies agricultural gas flux quantification without external calibration and is easily extended to larger agricultural systems where point-sampling-based approaches are insufficient, presenting opportunities for field-scale biogeochemical studies and ecological monitoring.

Testing the near-field Gaussian plume inversion flux quantification technique using unmanned aerial vehicle sampling

Methane emission fluxes from many facility-scale sources may be poorly quantified, potentially leading to uncertainties in the global methane budget. Accurate atmospheric measurement-based flux quantification is urgently required to address this. This paper describes the first test (using unbiased sampling) of a near-field Gaussian plume inversion (NGI) technique, suitable for facility-scale flux quantification, using a controlled release of methane gas. Two unmanned-aerial-vehicle (UAV) platforms were used to perform 22 flight surveys downwind of a point-source methane gas release from a regulated cylinder with a flowmeter. One UAV was tethered to an instrument on the ground, while the other UAV carried an on-board prototype instrument (both of which used the same near-infrared laser technology). Both instruments were calibrated using certified standards to account for variability in the instrumental gain factor, assuming fixed temperature and pressure. Furthermore, a water vapour correction factor, specifically calculated for the instrument, was applied and is described here in detail. We also provide guidance on potential systematic uncertainties associated with temperature and pressure, which may require further characterisation for improved measurement accuracy. The NGI technique was then used to derive emission fluxes for each UAV flight survey. We found good agreement of most NGI fluxes with the known controlled emission flux, within uncertainty, verifying the flux quantification methodology. The lower and upper NGI flux uncertainty bounds were, on average, 17 %±10(1σ) % and 227 %±98(1σ) % of the controlled emission flux, respectively. This range of conservative uncertainty bounds incorporate factors including the variability in the position of the time-invariant plume and potential for under-sampling. While these average uncertainties are large compared to methods such as tracer dispersion, we suggest that UAV sampling can be highly complementary to a toolkit of flux quantification approaches and may be a valuable alternative in situations where site access for tracer release is problematic. We see tracer release combined with UAV sampling as an effective approach in future flux quantification studies. Successful flux quantification using the UAV sampling methodology described here demonstrates its future utility in identifying and quantifying emissions from methane sources such as oil and gas extraction infrastructure facilities, livestock agriculture, and landfill sites, where site access may be difficult.

Methane emission detection and flux quantification from exploratory hydraulic fracturing in the United Kingdom, using unmanned aerial vehicle sampling

<p>Unmanned aerial vehicle (UAV) sampling was used to derive high-precision methane mole fraction measurements downwind of the United Kingdom’s first onshore exploratory operation to horizontally hydraulically fracture shale rock. Sampling took place using two UAVs on five intermittent sampling days between October 2018 and February 2019. One UAV carried an on-board prototype sensor while the other was connected to a sensor on the ground, using a tethered air inlet. Both instruments used near infrared spectroscopy. Methane emissions were observed on one sampling day (14<sup>th</sup> January 2019) over a 1.4-hour sampling window, due to cold venting of methane following a nitrogen lift. The nitrogen lift procedure was used to induce gas flow during liquid unloading. The near-field Gaussian plume inversion flux quantification method was used to derive four instantaneous flux ranges (within uncertainty) from the four UAV flight surveys conducted during the emission window.</p>

Quantitative fluxomics of circulating metabolites

SUMMARYMammalian organs are nourished by nutrients carried by the blood circulation. These nutrients originate from diet and internal stores, and can undergo various interconversions before their eventual use as tissue fuel. Here we develop isotope tracing, mass spectrometry, and mathematical analysis methods to determine the direct sources of circulating nutrients, their interconversion rates, and eventual tissue-specific contributions to TCA cycle metabolism. Experiments with fifteen nutrient tracers enabled extensive accounting both for circulatory metabolic cycles and tissue TCA inputs, across fed and fasted mice on either high carbohydrate or ketogenic diet. We find that a majority of circulating carbon flux is carried by two major cycles: glucose-lactate and triglyceride-glycerol-fatty acid. Futile cycling through these pathways is prominent when dietary content of the associated nutrients is low, rendering internal metabolic activity robust to food choice. The presented in vivo flux quantification methods are broadly applicable to different physiological and disease states.

Suitability of a Non-Dispersive Infrared Methane Sensor Package for Flux Quantification Using an Unmanned Aerial Vehicle

Point-source methane emission flux quantification is required to help constrain the global methane budget. Facility-scale fluxes can be derived using in situ methane mole fraction sampling, near-to-source, which may be acquired from an unmanned aerial vehicle (UAV) platform. We test a new non-dispersive infrared methane sensor by mounting it onto a small UAV, which flew downwind of a controlled methane release. Nine UAV flight surveys were conducted on a downwind vertical sampling plane, perpendicular to mean wind direction. The sensor was first packaged in an enclosure prior to sampling which contained a pump and a recording computer, with a total mass of 1.0 kg. The packaged sensor was then characterised to derive a gain factor of 0.92 ± 0.07, independent of water mole fraction, and an Allan deviation precision (at 1 Hz) of ±1.16 ppm. This poor instrumental precision and possible short-term drifts made it non-trivial to define a background mole fraction during UAV surveys, which may be important where any measured signal is small compared to sources of instrumental uncertainty and drift. This rendered the sensor incapable of deriving a meaningful flux from UAV sampling for emissions of the order of 1 g s−1. Nevertheless, the sensor may indeed be useful when sampling mole fraction enhancements of the order of at least 10 ppm (an order of magnitude above the 1 Hz Allan deviation), either from stationary ground-based sampling (in baseline studies) or from mobile sampling downwind of sources with greater source flux than those observed in this study. While many methods utilising low-cost sensors to determine methane flux are being developed, this study highlights the importance of adequately characterising and testing all new sensors before they are used in scientific research.