Macrophage miR-210 induction and metabolic reprogramming in response to pathogen interaction boost life-threatening inflammation

Unbalanced immune responses to pathogens can be life-threatening although the underlying regulatory mechanisms remain unknown. Here, we show a hypoxia-inducible factor 1α–dependent microRNA (miR)–210 up-regulation in monocytes and macrophages upon pathogen interaction. MiR-210 knockout in the hematopoietic lineage or in monocytes/macrophages mitigated the symptoms of endotoxemia, bacteremia, sepsis, and parasitosis, limiting the cytokine storm, organ damage/dysfunction, pathogen spreading, and lethality. Similarly, pharmacologic miR-210 inhibition improved the survival of septic mice. Mechanistically, miR-210 induction in activated macrophages supported a switch toward a proinflammatory state by lessening mitochondria respiration in favor of glycolysis, partly achieved by downmodulating the iron-sulfur cluster assembly enzyme ISCU. In humans, augmented miR-210 levels in circulating monocytes correlated with the incidence of sepsis, while serum levels of monocyte/macrophage-derived miR-210 were associated with sepsis mortality. Together, our data identify miR-210 as a fine-tuning regulator of macrophage metabolism and inflammatory responses, suggesting miR-210–based therapeutic and diagnostic strategies

Upscaling Wetland Methane Emissions From the FLUXNET-CH4 Eddy Covariance Network (UpCH4 v1.0):Model Development, Network Assessment, and Budget Comparison

Wetlands are responsible for 20%–31% of global methane (CH4) emissions and account for a large source of uncertainty in the global CH4 budget. Data-driven upscaling of CH4 fluxes from eddy covariance measurements can provide new and independent bottom-up estimates of wetland CH4 emissions. Here, we develop a six-predictor random forest upscaling model (UpCH4), trained on 119 site-years of eddy covariance CH4 flux data from 43 freshwater wetland sites in the FLUXNET-CH4 Community Product. Network patterns in site-level annual means and mean seasonal cycles of CH4 fluxes were reproduced accurately in tundra, boreal, and temperate regions (Nash-Sutcliffe Efficiency ∼0.52–0.63 and 0.53). UpCH4 estimated annual global wetland CH4 emissions of 146 ± 43 TgCH4 y−1 for 2001–2018 which agrees closely with current bottom-up land surface models (102–181 TgCH4 y−1) and overlaps with top-down atmospheric inversion models (155–200 TgCH4 y−1). However, UpCH4 diverged from both types of models in the spatial pattern and seasonal dynamics of tropical wetland emissions. We conclude that upscaling of eddy covariance CH4 fluxes has the potential to produce realistic extra-tropical wetland CH4 emissions estimates which will improve with more flux data. To reduce uncertainty in upscaled estimates, researchers could prioritize new wetland flux sites along humid-to-arid tropical climate gradients, from major rainforest basins (Congo, Amazon, and SE Asia), into monsoon (Bangladesh and India) and savannah regions (African Sahel) and be paired with improved knowledge of wetland extent seasonal dynamics in these regions. The monthly wetland methane products gridded at 0.25° from UpCH4 are available via ORNL DAAC (https://doi.org/10.3334/ORNLDAAC/2253).</p

Upscaling Wetland Methane Emissions From the FLUXNET-CH4 Eddy Covariance Network (UpCH4 v1.0): Model Development, Network Assessment, and Budget Comparison

Wetlands are responsible for 20%-31% of global methane (CH4) emissions and account for a large source of uncertainty in the global CH4 budget. Data-driven upscaling of CH4 fluxes from eddy covariance measurements can provide new and independent bottom-up estimates of wetland CH4 emissions. Here, we develop a six-predictor random forest upscaling model (UpCH4), trained on 119 site-years of eddy covariance CH4 flux data from 43 freshwater wetland sites in the FLUXNET-CH4 Community Product. Network patterns in site-level annual means and mean seasonal cycles of CH4 fluxes were reproduced accurately in tundra, boreal, and temperate regions (Nash-Sutcliffe Efficiency similar to 0.52-0.63 and 0.53). UpCH(4) estimated annual global wetland CH4 emissions of 146 +/- 43 TgCH4 y(-1) for 2001-2018 which agrees closely with current bottom-up land surface models (102-181 TgCH4 y(-1)) and overlaps with top-down atmospheric inversion models (155-200 TgCH4 y -1). However, UpCH4 diverged from both types of models in the spatial pattern and seasonal dynamics of tropical wetland emissions. We conclude that upscaling of eddy covariance CH4 fluxes has the potential to produce realistic extra-tropical wetland CH4 emissions estimates which will improve with more flux data. To reduce uncertainty in upscaled estimates, researchers could prioritize new wetland flux sites along humid-to-arid tropical climate gradients, from major rainforest basins (Congo, Amazon, and SE Asia), into monsoon (Bangladesh and India) and savannah regions (African Sahel) and be paired with improved knowledge of wetland extent seasonal dynamics in these regions. The monthly wetland methane products gridded at 0.25 degrees from UpCH4 are available via ORNL DAAC (https://doi.org/10.3334/ ORNLDAAC/2253).Plain Language Summary Wetlands account for a large share of global methane emissions to the atmosphere, but current estimates vary widely in magnitude (similar to 30% uncertainty on annual global emissions) and spatial distribution, with diverging predictions for tropical rice growing (e.g., Bengal basin), rainforest (e.g., Amazon basin), and floodplain savannah (e.g., Sudd) regions. Wetland methane model estimates could be improved by increased use of land surface methane flux data. Upscaling approaches use flux data collected across globally distributed measurement networks in a machine learning framework to extrapolate fluxes in space and time. Here, we train and evaluate a methane upscaling model (UpCH4) and use it to generate monthly, globally gridded wetland methane emissions estimates for 2001-2018. The UpCH4 model uses only six predictor variables among which temperature is dominant. Global annual methane emissions estimates and associated uncertainty ranges from upscaling fall within state-of-the-art model ensemble estimates from the Global Carbon Project (GCP) methane budget. In some tropical regions, the spatial pattern of UpCH4 emissions diverged from GCP predictions, however, inclusion of flux measurements from additional ground-based sites, together with refined maps of tropical wetlands extent, could reduce these prediction uncertainties

Identifying dominant environmental predictors of freshwater wetland methane fluxes across diurnal to seasonal time scales

While wetlands are the largest natural source of methane (CH4) to the atmosphere, they represent a large source of uncertainty in the global CH4 budget due to the complex biogeochemical controls on CH4 dynamics. Here we present, to our knowledge, the first multi-site synthesis of how predictors of CH4 fluxes (FCH4) in freshwater wetlands vary across wetland types at diel, multiday (synoptic), and seasonal time scales. We used several statistical approaches (correlation analysis, generalized additive modeling, mutual information, and random forests) in a wavelet-based multi-resolution framework to assess the importance of environmental predictors, nonlinearities and lags on FCH4 across 23 eddy covariance sites. Seasonally, soil and air temperature were dominant predictors of FCH4 at sites with smaller seasonal variation in water table depth (WTD). In contrast, WTD was the dominant predictor for wetlands with smaller variations in temperature (e.g., seasonal tropical/subtropical wetlands). Changes in seasonal FCH4 lagged fluctuations in WTD by similar to 17 +/- 11 days, and lagged air and soil temperature by median values of 8 +/- 16 and 5 +/- 15 days, respectively. Temperature and WTD were also dominant predictors at the multiday scale. Atmospheric pressure (PA) was another important multiday scale predictor for peat-dominated sites, with drops in PA coinciding with synchronous releases of CH4. At the diel scale, synchronous relationships with latent heat flux and vapor pressure deficit suggest that physical processes controlling evaporation and boundary layer mixing exert similar controls on CH4 volatilization, and suggest the influence of pressurized ventilation in aerenchymatous vegetation. In addition, 1- to 4-h lagged relationships with ecosystem photosynthesis indicate recent carbon substrates, such as root exudates, may also control FCH4. By addressing issues of scale, asynchrony, and nonlinearity, this work improves understanding of the predictors and timing of wetland FCH4 that can inform future studies and models, and help constrain wetland CH4 emissions.Peer reviewe

Gap-filling eddy covariance methane fluxes : Comparison of machine learning model predictions and uncertainties at FLUXNET-CH4 wetlands

Time series of wetland methane fluxes measured by eddy covariance require gap-filling to estimate daily, seasonal, and annual emissions. Gap-filling methane fluxes is challenging because of high variability and complex responses to multiple drivers. To date, there is no widely established gap-filling standard for wetland methane fluxes, with regards both to the best model algorithms and predictors. This study synthesizes results of different gap-filling methods systematically applied at 17 wetland sites spanning boreal to tropical regions and including all major wetland classes and two rice paddies. Procedures are proposed for: 1) creating realistic artificial gap scenarios, 2) training and evaluating gap-filling models without overstating performance, and 3) predicting halfhourly methane fluxes and annual emissions with realistic uncertainty estimates. Performance is compared between a conventional method (marginal distribution sampling) and four machine learning algorithms. The conventional method achieved similar median performance as the machine learning models but was worse than the best machine learning models and relatively insensitive to predictor choices. Of the machine learning models, decision tree algorithms performed the best in cross-validation experiments, even with a baseline predictor set, and artificial neural networks showed comparable performance when using all predictors. Soil temperature was frequently the most important predictor whilst water table depth was important at sites with substantial water table fluctuations, highlighting the value of data on wetland soil conditions. Raw gap-filling uncertainties from the machine learning models were underestimated and we propose a method to calibrate uncertainties to observations. The python code for model development, evaluation, and uncertainty estimation is publicly available. This study outlines a modular and robust machine learning workflow and makes recommendations for, and evaluates an improved baseline of, methane gap-filling models that can be implemented in multi-site syntheses or standardized products from regional and global flux networks (e.g., FLUXNET).Peer reviewe

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

FLUXNET-CH4 : a global, multi-ecosystem dataset and analysis of methane seasonality from freshwater wetlands

Methane (CH4) emissions from natural landscapes constitute roughly half of global CH4 contributions to the atmosphere, yet large uncertainties remain in the absolute magnitude and the seasonality of emission quantities and drivers. Eddy covariance (EC) measurements of CH4 flux are ideal for constraining ecosystem-scale CH4 emissions due to quasi-continuous and high-temporal-resolution CH4 flux measurements, coincident carbon dioxide, water, and energy flux measurements, lack of ecosystem disturbance, and increased availability of datasets over the last decade. Here, we (1) describe the newly published dataset, FLUXNET-CH4 Version 1.0, the first open-source global dataset of CH4 EC measurements (available at https://fluxnet.org/data/fluxnet-ch4-community-product/, last access: 7 April 2021). FLUXNET-CH4 includes half-hourly and daily gap-filled and non-gap-filled aggregated CH4 fluxes and meteorological data from 79 sites globally: 42 freshwater wetlands, 6 brackish and saline wetlands, 7 formerly drained ecosystems, 7 rice paddy sites, 2 lakes, and 15 uplands. Then, we (2) evaluate FLUXNET-CH4 representativeness for freshwater wetland coverage globally because the majority of sites in FLUXNET-CH4 Version 1.0 are freshwater wetlands which are a substantial source of total atmospheric CH4 emissions; and (3) we provide the first global estimates of the seasonal variability and seasonality predictors of freshwater wetland CH4 fluxes. Our representativeness analysis suggests that the freshwater wetland sites in the dataset cover global wetland bioclimatic attributes (encompassing energy, moisture, and vegetation-related parameters) in arctic, boreal, and temperate regions but only sparsely cover humid tropical regions. Seasonality metrics of wetland CH4 emissions vary considerably across latitudinal bands. In freshwater wetlands (except those between 20 degrees S to 20 degrees N) the spring onset of elevated CH4 emissions starts 3 d earlier, and the CH4 emission season lasts 4 d longer, for each degree Celsius increase in mean annual air temperature. On average, the spring onset of increasing CH4 emissions lags behind soil warming by 1 month, with very few sites experiencing increased CH4 emissions prior to the onset of soil warming. In contrast, roughly half of these sites experience the spring onset of rising CH4 emissions prior to the spring increase in gross primary productivity (GPP). The timing of peak summer CH4 emissions does not correlate with the timing for either peak summer temperature or peak GPP. Our results provide seasonality parameters for CH4 modeling and highlight seasonality metrics that cannot be predicted by temperature or GPP (i.e., seasonality of CH4 peak). FLUXNET-CH4 is a powerful new resource for diagnosing and understanding the role of terrestrial ecosystems and climate drivers in the global CH4 cycle, and future additions of sites in tropical ecosystems and site years of data collection will provide added value to this database. All seasonality parameters are available at https://doi.org/10.5281/zenodo.4672601 (Delwiche et al., 2021). Additionally, raw FLUXNET-CH4 data used to extract seasonality parameters can be downloaded from https://fluxnet. org/data/fluxnet-ch4-community-product/ (last access: 7 April 2021), and a complete list of the 79 individual site data DOIs is provided in Table 2 of this paper.Peer reviewe

A roadmap for interpreting 13C metabolite labeling patterns from cells

Measuring intracellular metabolism has increasingly led to important insights in biomedical research. 13C tracer analysis, although less information-rich than quantitative 13C flux analysis that requires computational data integration, has been established as a time-efficient method to unravel relative pathway activities, qualitative changes in pathway contributions, and nutrient contributions. Here, we review selected key issues in interpreting 13C metabolite labeling patterns, with the goal of drawing accurate conclusions from steady state and dynamic stable isotopic tracer experiments