Large enrichments in fatty acid 2H/1H ratios distinguish respiration from aerobic fermentation in yeast Saccharomyces cerevisiae.

Shifts in the hydrogen stable isotopic composition (2H/1H ratio) of lipids relative to water (lipid/water 2H-fractionation) at natural abundances reflect different sources of the central cellular reductant, NADPH, in bacteria. Here, we demonstrate that lipid/water 2H-fractionation (2εfattyacid/water) can also constrain the relative importance of key NADPH pathways in eukaryotes. We used the metabolically flexible yeast Saccharomyces cerevisiae, a microbial model for respiratory and fermentative metabolism in industry and medicine, to investigate 2εfattyacid/water. In chemostats, fatty acids from glycerol-respiring cells were >550‰ 2H-enriched compared to those from cells aerobically fermenting sugars via overflow metabolism, a hallmark feature in cancer. Faster growth decreased 2H/1H ratios, particularly in glycerol-respiring cells by 200‰. Variations in the activities and kinetic isotope effects among NADP+-reducing enzymes indicate cytosolic NADPH supply as the primary control on 2εfattyacid/water. Contributions of cytosolic isocitrate dehydrogenase (cIDH) to NAPDH production drive large 2H-enrichments with substrate metabolism (cIDH is absent during fermentation but contributes up to 20 percent NAPDH during respiration) and slower growth on glycerol (11 percent more NADPH from cIDH). Shifts in NADPH demand associated with cellular lipid abundance explain smaller 2εfattyacid/water variations (<30‰) with growth rate during fermentation. Consistent with these results, tests of murine liver cells had 2H-enriched lipids from slower-growing, healthy respiring cells relative to fast-growing, fermenting hepatocellular carcinoma. Our findings point to the broad potential of lipid 2H/1H ratios as a passive natural tracker of eukaryotic metabolism with applications to distinguish health and disease, complementing studies that rely on complex isotope-tracer addition methods.

Effects of increasing atmospheric CO2 on leaf water δ18O values are small and are attenuated in grasses and amplified in dicotyledonous herbs and legumes when transferred to cellulose δ18O values.

The oxygen isotope composition of cellulose (δ18O values) has been suggested to contain information on stomatal conductance (gs) responses to rising pCO2. The extent by which pCO2 affects leaf water and cellulose δ18O values (δ18OLW and δ18OC) and the isotope processes that determine pCO2 effects on δ18OLW and δ18OC are, however, unknown. We tested the effects of pCO2 on gs, δ18OLW and δ18OC in a glasshouse experiment, where six plant species were grown under pCO2 ranging from 200 to 500 ppm. Increasing pCO2 caused a decline in gs and an increase in δ18OLW, as expected. Importantly, the effects of pCO2 on gs and δ18OLW were small and pCO2 effects on δ18OLW were not directly transferred to δ18OC but were attenuated in grasses and amplified in dicotyledonous herbs and legumes. This is likely because of functional group-specific pCO2 effects on the model parameter pxpex. Our study highlights important uncertainties when using δ18OC as a proxy for gs. Specifically, pCO2-triggered gs effects on δ18OLW and δ18OC are possibly too small to be detected in natural settings and a pCO2 effect on pxpex may render the commonly assumed negative linkage between δ18OC and gs to be incorrect, potentially confounding δ18OC based gs reconstructions.

Covariation between oxygen and hydrogen stable isotopes declines along the path from xylem water to wood cellulose across an aridity gradient.

Oxygen and hydrogen isotopes of cellulose in plant biology are commonly used to infer environmental conditions, often from time series measurements of tree rings. However, the covariation (or the lack thereof) between δ18 O and δ2 H in plant cellulose is still poorly understood. We compared plant water, and leaf and branch cellulose from dominant tree species across an aridity gradient in Northern Australia, to examine how δ18 O and δ2 H relate to each other and to mean annual precipitation (MAP). We identified a decline in covariation from xylem to leaf water, and onwards from leaf to branch wood cellulose. Covariation in leaf water isotopic enrichment (Δ) was partially preserved in leaf cellulose but not branch wood cellulose. Furthermore, whilst δ2 H was well-correlated between leaf and branch, there was an offset in δ18 O between organs that increased with decreasing MAP. Our findings strongly suggest that postphotosynthetic isotope exchange with water is more apparent for oxygen isotopes, whereas variable kinetic and nonequilibrium isotope effects add complexity to interpreting metabolic-induced δ2 H patterns. Varying oxygen isotope exchange in wood and leaf cellulose must be accounted for when δ18 O is used to reconstruct climatic scenarios. Conversely, comparing δ2 H and δ18 O patterns may reveal environmentally induced shifts in metabolism.

Land-use-based freshwater sediment source fingerprinting using hydrogen isotope compositions of long-chain fatty acids.

Rapidly changing land use patterns and frequent extreme weather events have resulted in an increased sediment flux to freshwater systems globally, highlighting the need for land-use-based sediment source fingerprinting. Application of variability in hydrogen isotope compositions (δ2H values) of vegetation-specific biomarkers from soils and sediments is relatively underexplored for land-use-based freshwater suspended sediment (SS) source fingerprinting, but has the potential to complement the information from routinely applied carbon isotope analysis and provide new insights. We analysed δ2H values of long-chain fatty acids (LCFAs) as vegetation-specific biomarkers in source soils and SS collected from the mixed land use Tarland catchment (74 km2) in NE Scotland, to identify stream SS sources and quantify their contributions to SS. Plant growth form was the primary control on source soils LCFAs (n-C26:0, n-C28:0, n-C30:0) δ2H variability, while the isotopic composition of source water had no significant control. Forest and heather moorland soils covered with dicotyledonous and gymnosperm species were differentiated from arable land and grasslands soils covered with monocotyledonous species. SS samples collected for fourteen months from the Tarland catchment with a nested sampling approach showed monocot-based land use (cereal crops, grassland) to be the major source of SS with 71 ± 11% contribution on catchment-wide scale averaged throughout the sampling period. Storm events after a dry summer period and sustained high flow conditions in the streams during autumn and early winter suggested enhanced connectivity of more distant forest and heather moorland land uses covering relatively steep topography. This was shown by an increased contribution (44 ± 8%) on catchment-wide scale from dicot and gymnosperm-based land uses during the corresponding period. Our study demonstrated successful application of vegetation-specificity in δ2H values of LCFAs for land-use-based freshwater SS source fingerprinting in a mesoscale catchment where δ2H values of LCFAs were primarily controlled by plant growth forms.

The metabolic sensitivity of hydrogen isotope fractionation differs between plant compounds.

Hydrogen stable isotope analyses (δ2H) of plant derived organic compounds are a useful tool for ecological, environmental, and palaeoclimatological research. However, during organic compound synthesis, variable biosynthetic 2H-fractionation has been suggested to occur as a result of changes in plant carbon fluxes. So far, inference has been based on examining the δ2H patterns of plant compounds along environmental gradients, among plant species, and between plant organs. In an alternative approach, we used four plant species with four different types of mutations that cause impaired starch synthesis to assess whether variability in carbon metabolism affects the biosynthetic 2H-fractionation during cellulose, phytol, and acetogenic lipid synthesis. We found that mutants with impaired starch synthesis always had higher cellulose and phytol δ2H values compared to the wild type. By contrast, 2H-fractionation during acetogenic lipid biosynthesis generally did not show strong metabolic sensitivity. We rationalise these differences by considering the biosynthetic pathway of each compound and the likely source of the variable isotope fractionation. In different organic compounds, the sensitivity of variable biosynthetic 2H-fractionation to changes in C-metabolism depends on incorporation of specific H atoms from precursor molecules. As such, we determined that the similar increase in cellulose and phytol δ2H values as an effect of impaired starch synthesis most likely originates in triose-phosphates.

Continuous Medicaid coverage during the COVID-19 public health emergency reduced churning, but did not eliminate it.

Preserving insurance coverage in the wake of pandemic-related job loss was a priority in early 2020. To this end, the Families First Coronavirus Response Act implemented a continuous coverage policy in Medicaid to shore up access to health insurance. Prior to the pandemic, Medicaid enrollees experienced frequent coverage disruptions, known as "churning." The effect of the continuous coverage policy on churning during the COVID-19 public health emergency (PHE) is unknown. We performed a difference-in-differences analysis of nonelderly Medicaid enrollees using longitudinal national survey data to compare a 2019-2020 cohort exposed to the policy with a control cohort in 2018-2019. We found that the policy led to reduced transitions to uninsurance among adults, although not among children. The policy prevented over 300 000 transitions to uninsurance each month. However, disenrollment from Medicaid persisted at a low rate, despite the continuous coverage policy. As the PHE unwinds, policymakers should consider long-term continuous coverage policies to minimize churning in Medicaid.

Species variation in the hydrogen isotope composition of leaf cellulose is mostly driven by isotopic variation in leaf sucrose.

Experimental approaches to isolate drivers of variation in the carbon-bound hydrogen isotope composition (δ2 H) of plant cellulose are rare and current models are limited in their application. This is in part due to a lack in understanding of how 2 H-fractionations in carbohydrates differ between species. We analysed, for the first time, the δ2 H of leaf sucrose along with the δ2 H and δ18 O of leaf cellulose and leaf and xylem water across seven herbaceous species and a starchless mutant of tobacco. The δ2 H of sucrose explained 66% of the δ2 H variation in cellulose (R2 = 0.66), which was associated with species differences in the 2 H enrichment of sucrose above leaf water ( ε sucrose : -126% to -192‰) rather than by variation in leaf water δ2 H itself. ε sucrose was positively related to dark respiration (R2 = 0.27), and isotopic exchange of hydrogen in sugars was positively related to the turnover time of carbohydrates (R2 = 0.38), but only when ε sucrose was fixed to the literature accepted value of - 171 ‰. No relation was found between isotopic exchange of hydrogen and oxygen, suggesting large differences in the processes shaping post-photosynthetic fractionation between elements. Our results strongly advocate that for robust applications of the leaf cellulose hydrogen isotope model, parameterization utilizing δ2 H of sugars is needed.

Constraining parameter uncertainty for predicting oxygen and hydrogen isotope values in fruit.

Understanding δ18O and δ2H values of agricultural products like fruit is of particular scientific interest in plant physiology, ecology, and forensic studies. Applications of mechanistic stable isotope models to predict δ18O and δ2H values of water and organic compounds in fruit, however, are hindered by a lack of empirical parameterizations and validations. We addressed this lack of data by experimentally evaluating model parameter values required to model δ18O and δ2H values of water and organic compounds in berries and leaves from strawberry and raspberry plants grown at different relative humidities. Our study revealed substantial differences between leaf and berry isotope values, consistent across the different relative humidity treatments. We demonstrated that existing isotope models can reproduce water and organic δ18O and δ2H values for leaves and berries. Yet, these simulations require organ-specific model parameterization to accurately predict δ18O and δ2H values of leaf and berry tissue and water pools. We quantified these organ-specific model parameters for both species and relative humidity conditions. Depending on the required model accuracy, species- and environment-specific model parameters may be justified. The parameter values determined in this study thus facilitate applications of stable isotope models where understanding δ18O and δ2H values of fruit is of scientific interest.

Do 2 H and 18 O in leaf water reflect environmental drivers differently?

We compiled hydrogen and oxygen stable isotope compositions (δ2 H and δ18 O) of leaf water from multiple biomes to examine variations with environmental drivers. Leaf water δ2 H was more closely correlated with δ2 H of xylem water or atmospheric vapour, whereas leaf water δ18 O was more closely correlated with air relative humidity. This resulted from the larger proportional range for δ2 H of meteoric waters relative to the extent of leaf water evaporative enrichment compared with δ18 O. We next expressed leaf water as isotopic enrichment above xylem water (Δ2 H and Δ18 O) to remove the impact of xylem water isotopic variation. For Δ2 H, leaf water still correlated with atmospheric vapour, whereas Δ18 O showed no such correlation. This was explained by covariance between air relative humidity and the Δ18 O of atmospheric vapour. This is consistent with a previously observed diurnal correlation between air relative humidity and the deuterium excess of atmospheric vapour across a range of ecosystems. We conclude that 2 H and 18 O in leaf water do indeed reflect the balance of environmental drivers differently; our results have implications for understanding isotopic effects associated with water cycling in terrestrial ecosystems and for inferring environmental change from isotopic biomarkers that act as proxies for leaf water.

Effects of phenotypic variability on the oxygen and hydrogen isotope compositions of grains in different winter wheat varieties.

Stable isotope analyses are the leading method for geographic origin determination, especially of plant-based agricultural products. Origin analysis is typically done by comparing a suspicious sample to reference materials with known geographic origin. Reference materials are usually collected at the species level, assuming different varieties of a species to have comparable isotope compositions within a given location. We evaluated whether different phenotypes that are expressed in different varieties of winter wheat (Triticum aestivum L.) influence the oxygen (δ18O) and hydrogen (δ2H) isotope composition of plant tissue water and organic compounds. We found that mean δ18O and δ2H values among winter wheat varieties did not differ significantly in leaf water, however, differed significantly in bulk dried grain tissue. The differences in bulk dried grain δ18O and δ2H values among varieties can be related to differences in phenotypic trait expression among varieties. Despite this substantial phenotypic variability, the overall variability of bulk dried grain δ18O and δ2H values among varieties was small (SD 0.54 ‰ for oxygen, 3.60 ‰ for hydrogen). We thus conclude that reference materials collected at the species level should be sufficient for geographic origin analysis of winter wheat and possibly other cereals using δ18O and δ2H values.

Using plant physiological stable oxygen isotope models to counter food fraud.

Fraudulent food products, especially regarding false claims of geographic origin, impose economic damages of $30-$40 billion per year. Stable isotope methods, using oxygen isotopes (δ18O) in particular, are the leading forensic tools for identifying these crimes. Plant physiological stable oxygen isotope models simulate how precipitation δ18O values and climatic variables shape the δ18O values of water and organic compounds in plants. These models have the potential to simplify, speed up, and improve conventional stable isotope applications and produce temporally resolved, accurate, and precise region-of-origin assignments for agricultural food products. However, the validation of these models and thus the best choice of model parameters and input variables have limited the application of the models for the origin identification of food. In our study we test model predictions against a unique 11-year European strawberry δ18O reference dataset to evaluate how choices of input variable sources and model parameterization impact the prediction skill of the model. Our results show that modifying leaf-based model parameters specifically for fruit and with product-independent, but growth time specific environmental input data, plant physiological isotope models offer a new and dynamic method that can accurately predict the geographic origin of a plant product and can advance the field of stable isotope analysis to counter food fraud.

Precipitation isotope time series predictions from machine learning applied in Europe.

Hydrogen and oxygen isotope values of precipitation are critically important quantities for applications in Earth, environmental, and biological sciences. However, direct measurements are not available at every location and time, and existing precipitation isotope models are often not sufficiently accurate for examining features such as long-term trends or interannual variability. This can limit applications that seek to use these values to identify the source history of water or to understand the hydrological or meteorological processes that determine these values. We developed a framework using machine learning to calculate isotope time series at monthly resolution using available climate and location data in order to improve precipitation isotope model predictions. Predictions from this model are currently available for any location in Europe for the past 70 y (1950-2019), which is the period for which all climate data used as predictor variables are available. This approach facilitates simple, user-friendly predictions of precipitation isotope time series that can be generated on demand and are accurate enough to be used for exploration of interannual and long-term variability in both hydrogen and oxygen isotopic systems. These predictions provide important isotope input variables for ecological and hydrological applications, as well as powerful targets for paleoclimate proxy calibration, and they can serve as resources for probing historic patterns in the isotopic composition of precipitation with a high level of meteorological accuracy. Predictions from our modeling framework, Piso.AI, are available at https://isotope.bot.unibas.ch/PisoAI/.

Metabolic exchange between pathways for isoprenoid synthesis and implications for biosynthetic hydrogen isotope fractionation.

Hydrogen isotope ratios of plant lipids are used for paleoclimate reconstruction, but are influenced by both source water and biosynthetic processes. Measuring 2 H : 1 H ratios of multiple compounds produced by different pathways could allow these effects to be separated, but hydrogen isotope fractionations during isoprenoid biosynthesis remain poorly constrained. To investigate how hydrogen isotope fractionation during isoprenoid biosynthesis is influenced by molecular exchange between the cytosolic and plastidial production pathways, we paired position-specific 13 C-pyruvate labeling with hydrogen isotope measurements of lipids in Pachira aquatica saplings. We find that acetogenic compounds primarily incorporated carbon from 13 C2-pyruvate, whereas isoprenoids incorporated 13 C1- and 13 C2-pyruvate equally. This indicates that cytosolic pyruvate is primarily introduced into plastidial isoprenoids via glyceraldehyde 3-phosphate and that plastidial isoprenoid intermediates are incorporated into cytosolic isoprenoids. Probably as a result of the large differences in hydrogen isotope fractionation between plastidial and cytosolic isoprenoid pathways, sterols from P. aquatica are at least 50‰ less 2 H-enriched relative to phytol than sterols in other plants. These results provide the first experimental evidence that incorporation of plastidial intermediates reduces 2 H : 1 H ratios of sterols. This suggests that relative offsets between the 2 H : 1 H ratios of sterols and phytol can trace exchange between the two isoprenoid synthesis pathways.

Changes in Coverage, Access, and Health Following Implementation of Healthy Behavior Incentive Medicaid Expansions vs. Traditional Medicaid Expansions.

BACKGROUND: Several states expanded Medicaid under the Affordable Care Act using Section 1115 waivers to implement healthy behavior incentive (HBI) programs, but the impact of this type of expansion relative to traditional expansion is not well understood. OBJECTIVE: To examine whether Medicaid expansion with healthy behavior incentive programs and traditional Medicaid expansion were associated with differential changes in coverage, access, and self-rated health outcomes among low-income adults. DESIGN: Difference-in-differences analysis of American Community Survey and Behavioral Risk Factor Surveillance System data from 2011 to 2017. PARTICIPANTS: Low-income adults ages 19-64 in the Midwest Census region (American Community Survey, n = 665,653; Behavioral Risk Factor Surveillance System, n = 71,959). INTERVENTIONS: Exposure to either HBI waiver or traditional Medicaid expansion in the state of residence. MAIN MEASURES: Coverage: Medicaid, private, or any health insurance coverage; access: routine checkup, personal doctor, delaying care due to cost; health: cancer screening, preventive care, healthy behaviors, self-reported health. KEY RESULTS: Healthy behavior incentive (HBI) and traditional expansion (TE) states experienced reductions in uninsurance (- 5.6 [- 7.5, - 3.7] and - 6.2 [- 8.1, - 4.4] percentage points, respectively) and gains in Medicaid (HBI, + 7.6 [2.4, 12.8]; TE, + 9.7 [5.9, 13.4] percentage points) relative to non-expansion states. Both expansion types were associated with increases in rates of having a personal doctor (HBI, + 3.8 [2.0, 5.6]; TE, + 5.9 [2.2, 9.6] percentage points) and mammography (HBI, + 5.6 [0.6, 10.6]; TE, + 7.3 [0.7, 13.9] percentage points). Meanwhile, checkups increased more in HBI than in TE states (p < 0.01), but no other changes in health care services differed between expansion types. CONCLUSIONS: Medicaid expansion was associated with improvements in coverage and access to care with few differences between expansion types.

Expansion by Ballot Initiative: Challenges and Future Directions in Health Policy.

Since 2017, four states have successfully adopted the Medicaid expansion through ballot initiative. We analyze how states could potentially use ballot initiatives to implement these programs. We find there are serious legal and political challenges to expansion by initiative. Only six non-expansion states allow for a ballot initiative to pass and implement the Medicaid expansion. Amongst those states, there are challenges that limit the development, scope, and implementation of an initiative. Whether a state adopts the Medicaid expansion has important implications for health care providers.

Aging impairs mouse skeletal muscle macrophage polarization and muscle-specific abundance during recovery from disuse.

Impaired recovery of aged muscle following a disuse event is an unresolved issue facing the older adult population. Although investigations in young animals have suggested that rapid regrowth of skeletal muscle following a disuse event entails a coordinated involvement of skeletal muscle macrophages, this phenomenon has not yet been thoroughly tested as an explanation for impaired muscle recovery in aging. To examine this hypothesis, young (4-5 mo) and old (24-26 mo) male mice were examined as controls following 2 wk of hindlimb unloading (HU) and following 4 (RL4) and 7 (RL7) days of reloading after HU. Muscles were harvested to assess muscle weight, myofiber-specifc cross-sectional area, and skeletal muscle macrophages via immunofluorescence. Flow cytometry was used on gastrocnemius and soleus muscle (at RL4) single-cell suspensions to immunophenotype skeletal muscle macrophages. Our data demonstrated impaired muscle regrowth in aged compared with young mice following disuse, which was characterized by divergent muscle macrophage polarization patterns and muscle-specifc macrophage abundance. During reloading, young mice exhibited the classical increase in M1-like (MHC II+CD206-) macrophages that preceeded the increase in percentage of M2-like macrophages (MHC II-CD206+); however, old mice did not demonstrate this pattern. Also, at RL4, the soleus demonstrated reduced macrophage abundance with aging. Together, these data suggest that dysregulated macrophage phenotype patterns in aged muscle during recovery from disuse may be related to impaired muscle growth. Further investigation is needed to determine whether the dysregulated macrophage response in the old during regrowth from disuse is related to a reduced ability to recruit or activate specific immune cells.

Population-level factors associated with maternal mortality in the United States, 1997-2012.

BACKGROUND: In contrast to peer nations, the United States is experiencing rapid increases in maternal mortality. Trends in individual and population-level demographic factors and health trends may play a role in this change. METHODS: We analyzed state-level maternal mortality for the years 1997-2012 using multilevel mixed-effects regression grouped by state, using publicly available data including whether a state had adopted the 2003 U.S. Standard Certificate of Death, designed to simplify identification of pregnant and recently pregnant decedents. We calculated the proportion of the increase in maternal mortality attributable to specific factors during the study period. RESULTS: Maternal mortality was associated with higher population prevalence of obesity and high school non-completion among women of childbearing age; these factors explained 31.0% and 5.3% of the attributable increase in maternal mortality during the study period, respectively. Among delivering mothers, prevalence of diabetes (17.0%), attending fewer than 10 prenatal visits (4.9%), and African American race (2.0%) were also associated with higher maternal mortality, as was time-varying state adoption of the 2003 death certificate (31.1%). CONCLUSIONS: Our findings indicate that, in addition to better case ascertainment of maternal deaths, adverse changes in chronic diseases, insufficient healthcare access, and social determinants of health represent identifiable risks for maternal mortality that merit prompt attention in population-directed interventions and health policies.