An isolated water droplet in the aqueous solution of a supramolecular tetrahedral cage

Water under nanoconfinement at ambient conditions has exhibited low-dimensional ice formation and liquid–solid phase transitions, but with structural and dynamical signatures that map onto known regions of water’s phase diagram. Using terahertz (THz) absorption spectroscopy and ab initio molecular dynamics, we have investigated the ambient water confined in a supramolecular tetrahedral assembly, and determined that a dynamically distinct network of 9 ± 1 water molecules is present within the nanocavity of the host. The low-frequency absorption spectrum and theoretical analysis of the water in the Ga₄L₆¹²⁻ host demonstrate that the structure and dynamics of the encapsulated droplet is distinct from any known phase of water. A further inference is that the release of the highly unusual encapsulated water droplet creates a strong thermodynamic driver for the high-affinity binding of guests in aqueous solution for the Ga₄L₆¹²⁻ supramolecular construct.

Supramolecular capsules create internal cavities that are thought to act like enzyme active sites (1). As aqueous enzymes provide inspiration for the design of supramolecular catalysts, one of the goals of supramolecular chemistry is the creation of synthetic “receptors” that have both a high affinity and a high selectivity for the binding of guests in water (2, 3). The Ga₄L₆¹²⁻ tetrahedral assembly formulated by Raymond and coworkers represents an excellent example of a water-soluble supramolecular cage that has provided host interactions that promotes guest encapsulation. Using steric interactions and electrostatic charge to chemically position the substrate while shielding the reaction from solvent, this host has been shown to provide enhanced reaction rates that approach the performance of natural biocatalysts (4–10). Moreover, aqueous solvation of the substrate, host, and encapsulated solvent also play an important role in the whole catalytic cycle. In particular, the driving forces that release water from the nanocage host to favor the direct binding with the substrate is thought to be a critical factor in successful catalysis, but is challenging to probe directly (7, 8, 11–14).In both natural and artificial nanometer-sized environments, confined water displays uniquely modified structure and dynamics with respect to the bulk liquid (15–18). Recently, these modified properties were also found to have significant implications for the mechanism and energetics of reactions taking place in confined water with respect to those observed in bulk aqueous solution (19–21). In a pioneering study on supramolecular assemblies, Cram and collaborators (22) concluded that the interior of those cages is a “new and unique phase of matter” for the incarcerated guests. In more recent studies, it was postulated that, similar to graphitic and zeolite nanopores (23, 24), confined water within supramolecular host cavities is organized in stable small clusters [(H₂O)_n, with n = 8 to 19] that are different from gas phase water clusters (25). In these studies, the hydrogen-bonded water clusters were reported to be mostly ice- or clathrate-like by X-ray and neutron diffraction in the solid state at both ambient and cryogenic temperatures (26–32). However, to the best of our knowledge, such investigations have not characterized the Ga₄L₆¹²⁻ supramolecular tetrahedral assembly in the liquid state near room temperature and pressure, where the [Ga₄L₆]¹²⁻ capsule can perform catalytic reactions (6, 8, 9).Here, we use terahertz (THz) absorption spectroscopy and ab initio molecular dynamics (AIMD) to characterize low-frequency vibrations and structural organization of water in the nanoconfined environment. THz is ideally suited to probe the intermolecular collective dynamics of the water hydrogen bond (HB) network with extremely high sensitivity, as illustrated for different phases of water (33–38), and for aqueous solutions of salts, osmolytes, alcohols, and amino acids (36, 39–42). The THz spectra of the water inside the nanocage has been quantitatively reproduced with AIMD, allowing us to confidently characterize the water network in the cage in order to provide a more complete dynamical, structural, and thermodynamic picture. We have determined that the spectroscopic signature of the confined water in the nanocage is a dynamically arrested state whose structure bears none of the features of water at any alternate thermodynamic state point such as pressurized liquid or ice. Our experimental and theoretical study provides insight into the role played by encapsulated water in supramolecular catalysis, creating a low entropy and low enthalpy water droplet readily displaced by a catalytic substrate.     

Vapor isotopic evidence for the worsening of winter air quality by anthropogenic combustion-derived water

Anthropogenic combustion-derived water (CDW) may accumulate in an airshed due to stagnant air, which may further enhance the formation of secondary aerosols and worsen air quality. Here we collected three-winter-season, hourly resolution, water-vapor stable H and O isotope compositions together with atmospheric physical and chemical data from the city of Xi’an, located in the Guanzhong Basin (GZB) in northwestern China, to elucidate the role of CDW in particulate pollution. Based on our experimentally determined water vapor isotope composition of the CDW for individual and weighted fuels in the basin, we found that CDW constitutes 6.2% of the atmospheric moisture on average and its fraction is positively correlated with [PM_2.5] (concentration of particulate matter with an aerodynamic diameter less than 2.5 μm) as well as relative humidity during the periods of rising [PM_2.5]. Our modeling results showed that CDW added additional average 4.6 μg m⁻³ PM_2.5 during severely polluted conditions in the GZB, which corresponded to an average 5.1% of local anthropogenic [PM_2.5] (average at ∼91.0 μg m⁻³). Our result is consistent with the proposed positive feedback between the relative humidity and a moisture sensitive air-pollution condition, alerting to the nontrivial role of CDW when considering change of energy structure such as a massive coal-to-gas switch in household heating in winter.

An estimated 3 million people are killed each year owing to outdoor air pollution (1). Overall mean mortality rate increases ∼1.2% with each 10 μg m⁻³ increase in [PM_2.5] (concentration of particulate matter with an aerodynamic diameter less than 2.5 μm) (2). Considering a close relationship between air pollution and energy structure (3, 4), countries facing severe air pollution have been adjusting their energy structure to improve air quality. In the past several years, China has invested heavily in reducing air pollution in major cities (5), and there has been a significant decrease in annual [PM_2.5] since 2013 (6). Despite many drastic efforts, haze events, correlated with high [PM_2.5], still occur frequently, especially in cities on the North China Plain (7, 8). In the heavily polluted Beijing-Tianjin-Hebei region, a series of regulatory policies has been implemented (5, 9), including using natural gas instead of coal (10, 11). Since 2015, a large-scale project of coal-to-gas switch has been deployed in urban and rural areas in China (12, 13).It has been proposed that in northern China, severe haze is the synergetic effect of the interactions between anthropogenic emissions and atmospheric processes (7). Among the many causes of haze events, atmospheric water vapor or specifically relative humidity (RH) enhances the rate of heterogeneous oxidation of SO₂ and NO_X and in turn exerts a positive feedback on the rising of [PM_2.5] (14–17). Water vapor in the planetary boundary layer (PBL) comes mostly from the oceans via evaporation and transport or from continental water via evapotranspiration (18, 19). Combustion-derived water (CDW), a source of water vapor coming from fossil fuel or biomass burning, is negligible in the global atmospheric water budget. However, Gorski et al. (20) reported up to 13% of the water vapor in PBL was from CDW during certain days in Salt Lake City, Utah, located in an enclosed basin in northern America. This is a significant water contribution and should be verified independently in similar urban environments. The increased CDW fraction in air moisture could simply be a passive result of multiday accumulation in a polluted/stagnant PBL. Or, the CDW added to an airshed during polluted days could accelerate the formation of secondary aerosols, further stabilize the PBL, and reinforce CDW accumulation (15, 20). Pinning the exact role of CDW is important to energy policy in mitigating air pollution in China and other developing nations. However, the nonlinearity of atmospheric processes renders any firm conclusion hard to come by. Here we report the results of a 3-y, multiparameter sampling campaign in combination with atmospheric chemical model to examine the role of CDW.     

Structural relaxation and crystallization in supercooled water from 170 to 260 K

The origin of water’s anomalous properties has been debated for decades. Resolution of the problem is hindered by a lack of experimental data in a crucial region of temperatures, T, and pressures where supercooled water rapidly crystallizes—a region often referred to as “no man’s land.” A recently developed technique where water is heated and cooled at rates greater than 10⁹ K/s now enables experiments in this region. Here, it is used to investigate the structural relaxation and crystallization of deeply supercooled water for 170 K < T < 260 K. Water’s relaxation toward a new equilibrium structure depends on its initial structure with hyperquenched glassy water (HQW) typically relaxing more quickly than low-density amorphous solid water (LDA). For HQW and T > 230 K, simple exponential relaxation kinetics is observed. For HQW at lower temperatures, increasingly nonexponential relaxation is observed, which is consistent with the dynamics expected on a rough potential energy landscape. For LDA, approximately exponential relaxation is observed for T > 230 K and T < 200 K, with nonexponential relaxation only at intermediate temperatures. At all temperatures, water’s structure can be reproduced by a linear combination of two, local structural motifs, and we show that a simple model accounts for the complex kinetics within this context. The relaxation time, τ_rel, is always shorter than the crystallization time, τ_xtal. For HQW, the ratio, τ_xtal/τ_rel, goes through a minimum at ∼198 K where the ratio is about 60.

Liquids below their melting point are, at best, metastable with respect to the crystalline phase. However, if the timescale for structural relaxation^* is short compared to the timescale for crystallization, then the liquid can exist in thermal equilibrium. Upon cooling, the equilibrium relaxation time increases, while the time to crystallize initially decreases. Continued cooling leads to either crystallization or glass formation, and the changes in the particle dynamics and kinetics upon cooling influence the eventual outcome (1–5). A common observation is that the Arrhenius temperature dependence and simple exponential relaxation kinetics seen at temperatures above the melting point are replaced by non-Arrhenius behavior and stretched exponential relaxation upon supercooling. It is widely believed that these and other changes are related to the development of dynamical heterogeneities in the liquids (1–12). Dynamical heterogeneities are probably connected to spatial heterogeneities in the liquid (4, 5, 9, 10), for example, as clusters of more mobile molecules that are commonly observed in molecular dynamics (MD) simulations (2, 3, 13–18). Whether the properties of supercooled liquids and glasses arise from purely dynamical effects or have thermodynamic origins is still debated, as is the precise role of the liquid’s structure (1, 3–5).Mildly supercooled water exhibits some of the properties seen in other liquids such as non-Arrhenius temperature dependence for the dielectric relaxation (19), self-diffusivity (20), and viscosity (21), and the dynamics of supercooled water in MD simulations are qualitatively similar to those found in “simple” liquids (17, 22). However, many of water’s properties are quite different from simple liquids (23). The leading theories to explain water’s many anomalies, such as the liquid–liquid critical point (LLCP) hypothesis and the singularity-free scenario, propose that it is a temperature- and pressure-dependent mixture of two local structures (18, 23–30). One structural motif is suggested to be ice-like with the four nearest neighbors in a nearly tetrahedral arrangement. The second motif is more disordered with a fifth nearest neighbor molecule that disrupts the hydrogen bonding network. The first structural motif, which has lower density and enthalpy, is often called the low-density liquid (LDL), while the second motif, which has higher density and entropy, is called the high-density liquid (HDL). These structural motifs, which correspond to “static heterogeneities” in the liquid (17), are distinct from the dynamical heterogeneities found in simple liquids. While connections between the static and dynamic heterogeneities have been observed in MD simulations (17, 18, 23, 31), rapid crystallization at temperatures between ∼160 K and ∼230 K has precluded dynamic and kinetic measurements on real water that are needed to test these proposed connections. Data in this temperature range are also needed to test the relevance of theories, such as Adam–Gibbs, which connect the thermodynamic and dynamic anomalies of water (15, 32).Techniques which limit the amount of time liquid water spends in no man’s land enable investigations of its properties prior to crystallization (33, 34). One approach is to heat and cool thin water films at high rates (>10⁹ K/s) with pulsed laser heating (35, 36). Recently, we used this technique to investigate the structure of nanoscale liquid water films as a function of the temperature, T_max, to which they were transiently heated for 170 K ≤ T_max < 260 K (37). The experiments showed that 1) at each temperature, the steady-state structure prior to the onset of appreciable crystallization could be reproduced as a linear combination of two local structural motifs—low-density amorphous ice (LDA) and hyperquenched water (HQW) and 2) at steady state, the mole fraction of the water corresponding to HQW, fHQWSS(Tmax), decreased rapidly with temperature below ∼235 K and was negligible below ∼180 K. The data could be fit with a logistic function, fHQWSS(T)=(1 + exp((T0−T)ΔT))−1, with T₀ = 210 ± 3 K and ΔT = 8.5 K. 3) Prior to the onset of crystallization, all the structural changes were “reversible” in the sense that by simply changing to a new T_max at some point in an experiment, the local structure of water evolved toward the structure appropriate for that new temperature. These results are consistent with the LLCP hypothesis and the singularity free scenario.Here, we investigate the kinetics of structural relaxation in transiently heated supercooled water films and its connections to the observed structural heterogeneity. Infrared (IR) spectroscopy was used to assess the structural evolution of the water as a function of the number of heat pulses applied to the samples for temperatures from 170 to 260 K. The results show that the relaxation kinetics depend on the initial conditions: For T_max > 200 K, water that was annealed at the glass transition temperature (T_g ∼136 K) prior to pulsed heating relaxed more slowly than water that was hyperquenched from 300 K. For T_max > 225 K, a simple exponential decay adequately describes the relaxation kinetics independent of the initial structure. At lower temperatures, increasingly nonexponential decay was observed for HQW. For LDA, exponential decay was also observed for T_max ≤ 195 K, but the relaxation kinetics were nonexponential at intermediate temperatures. We show that a simple kinetic model based on transitions between two local structural motifs for water in a rough energy landscape accounts for the observations. The structural relaxation times for HQW are similar to the characteristic diffusion times for water determined from transient-heating experiments (36) and equilibrium MD simulations (38). We also measured the crystallization times for the transiently heated water films and found that they were always long compared to the structural relaxation of the liquid. The ratio of the crystallization and relaxation times was minimized at about 196 K for the HQW and 216 K for the LDA. The measured crystallization times are consistent with results from MD simulations using the TIP4P/ICE model (38).Many experiments and simulations look at the relaxation of liquids at or near thermal equilibrium. In contrast, the experiments presented here follow the evolution of the system from one equilibrium configuration to another in conditions where the difference in equilibration temperatures, δT_max, is typically large. Two factors limit our ability to investigate supercooled water’s equilibrium response functions. First, the rapid crystallization of deeply supercooled water limits the amount of time available for experiments on equilibrated water, severely limiting the available experimental techniques. Second, because it is difficult to reliably identify small changes in the structure via changes in the IR spectra, larger perturbations are required. In contrast, experiments on good glass formers are not limited by rapid crystallization. As a result, many techniques have been used to investigate their structural relaxation both approaching and at equilibrium (39, 40). Below, we discuss our results in the context of prior research on relaxation toward and at equilibrium.     

Presynaptic α2δ subunits are key organizers of glutamatergic synapses

In nerve cells the genes encoding for α₂δ subunits of voltage-gated calcium channels have been linked to synaptic functions and neurological disease. Here we show that α₂δ subunits are essential for the formation and organization of glutamatergic synapses. Using a cellular α₂δ subunit triple-knockout/knockdown model, we demonstrate a failure in presynaptic differentiation evidenced by defective presynaptic calcium channel clustering and calcium influx, smaller presynaptic active zones, and a strongly reduced accumulation of presynaptic vesicle-associated proteins (synapsin and vGLUT). The presynaptic defect is associated with the downscaling of postsynaptic AMPA receptors and the postsynaptic density. The role of α₂δ isoforms as synaptic organizers is highly redundant, as each individual α₂δ isoform can rescue presynaptic calcium channel trafficking and expression of synaptic proteins. Moreover, α₂δ-2 and α₂δ-3 with mutated metal ion-dependent adhesion sites can fully rescue presynaptic synapsin expression but only partially calcium channel trafficking, suggesting that the regulatory role of α₂δ subunits is independent from its role as a calcium channel subunit. Our findings influence the current view on excitatory synapse formation. First, our study suggests that postsynaptic differentiation is secondary to presynaptic differentiation. Second, the dependence of presynaptic differentiation on α₂δ implicates α₂δ subunits as potential nucleation points for the organization of synapses. Finally, our results suggest that α₂δ subunits act as transsynaptic organizers of glutamatergic synapses, thereby aligning the synaptic active zone with the postsynaptic density.

In synapses neurotransmitter release is triggered by the entry of calcium through voltage-gated calcium channels (VGCCs). Neuronal VGCCs consist of an ion-conducting α₁ subunit and the auxiliary β and α₂δ subunits. α₂δ subunits, the targets of the widely prescribed antiepileptic and antiallodynic drugs gabapentin and pregabalin, are membrane-anchored extracellular glycoproteins, which modulate VGCC trafficking and calcium currents (1–5). In nerve cells α₂δ subunits have been linked to neuropathic pain and epilepsy (4) and they interact with mutant prion proteins (6) and regulate synaptic release probability (7). Importantly, all α₂δ isoforms are implicated in synaptic functions. Presynaptic effects of α₂δ-1, for example, may be mediated by an interaction with α-neurexins (8) or N-methyl-D-aspartate receptors (e.g., refs. 9 and 10). In contrast, postsynaptic α₂δ-1 acts as a receptor for thrombospondins (11) and promotes spinogenesis via postsynaptic Rac1 (12). α₂δ-2 is necessary for normal structure and function of auditory hair cell synapses (13); it has been identified as a regulator of axon growth and hence a suppressor of axonal regeneration (14) and was recently shown to control structure and function of cerebellar climbing fiber synapses (15). A splice variant of α₂δ-2 regulates postsynaptic GABA_A receptor (GABA_AR) abundance and axonal wiring (16). In invertebrates, α₂δ loss of function was associated with abnormal presynaptic development in motoneurons (17, 18) and in mice the loss of α₂δ-3 results in aberrant synapse formation of auditory nerve fibers (19). Finally, α₂δ-4 is required for the organization of rod and cone photoreceptor synapses (20, 21).Despite these important functions, knockout mice for α₂δ-1 and α₂δ-3 show only mild neurological phenotypes (5, 10, 22–25). In contrast, mutant mice for α₂δ-2 (ducky) display impaired gait, ataxia, and epileptic seizures (26), all phenotypes consistent with a cerebellar dysfunction due to the predominant expression of α₂δ-2 in the cerebellum (e.g., ref. 15). Hence, in contrast to the specific functions of α₂δ isoforms (discussed above) the phenotypes of the available knockout or mutant mouse models suggest a partial functional redundancy in central neurons. Moreover, detailed mechanistic insights into the putative synaptic functions of α₂δ subunits are complicated by the simultaneous and strong expression of three isoforms (α₂δ-1 to -3) in neurons of the central nervous system (27).In this study, by transfecting cultured hippocampal neurons from α₂δ-2/-3 double-knockout mice with short hairpin RNA (shRNA) against α₂δ-1, we developed a cellular α₂δ subunit triple-knockout/knockdown model. Excitatory synapses from these cultures show a severe failure of synaptic vesicle recycling associated with severely reduced presynaptic calcium transients, loss of presynaptic calcium channels and presynaptic vesicle-associated proteins, and a reduced size of the presynaptic active zone (AZ). Lack of presynaptic α₂δ subunits also induces a failure of postsynaptic PSD-95 and AMPA receptor (AMPAR) localization and a thinning of the postsynaptic density (PSD). Each individual α₂δ isoform (α₂δ-1 to -3) could rescue the severe phenotype, revealing the highly redundant role of presynaptic α₂δ isoforms in glutamatergic synapse formation and differentiation. Together our results show that α₂δ subunits regulate presynaptic differentiation as well as the transsynaptic alignment of postsynaptic receptors and are thus critical for the function of glutamatergic synapses.     

Pharmacological inhibition of PI5P4Kα/β disrupts cell energy metabolism and selectively kills p53-null tumor cells

Most human cancer cells harbor loss-of-function mutations in the p53 tumor suppressor gene. Genetic experiments have shown that phosphatidylinositol 5-phosphate 4-kinase α and β (PI5P4Kα and PI5P4Kβ) are essential for the development of late-onset tumors in mice with germline p53 deletion, but the mechanism underlying this acquired dependence remains unclear. PI5P4K has been previously implicated in metabolic regulation. Here, we show that inhibition of PI5P4Kα/β kinase activity by a potent and selective small-molecule probe disrupts cell energy homeostasis, causing AMPK activation and mTORC1 inhibition in a variety of cell types. Feedback through the S6K/insulin receptor substrate (IRS) loop contributes to insulin hypersensitivity and enhanced PI3K signaling in terminally differentiated myotubes. Most significantly, the energy stress induced by PI5P4Kαβ inhibition is selectively toxic toward p53-null tumor cells. The chemical probe, and the structural basis for its exquisite specificity, provide a promising platform for further development, which may lead to a novel class of diabetes and cancer drugs.

There are two synthetic routes for phosphatidylinositol 4,5-bisphosphate, or PI(4,5)P₂, a versatile phospholipid with both structural and signaling functions in most eukaryotic cells (1 –3). The bulk of PI(4,5)P₂ is found at the inner leaflet of the plasma membrane and is synthesized from phosphatidylinositol 4-phosphate, or PI(4)P, by type 1 phosphatidylinositol phosphate kinase PI4P5K (4, 5). A smaller fraction of PI(4,5)P₂ is generated from the much rarer phosphatidylinositol 5-phosphate, or PI(5)P, through the activity of type 2 phosphatidylinositol phosphate kinase PI5P4K (6, 7). Although PI5P4K is as abundantly expressed as PI4P5K (8), its function is less well understood (9). It has been proposed that PI5P4K may play a role in suppressing PI(5)P, which is often elevated by stress (10, 11), or produce local pools of PI(4,5)P₂ at subcellular compartments such as Golgi and nucleus (12).Higher animals have three PI5P4K isoforms, α, β, and γ, which are encoded by three different genes, PIP4K2A, PIP4K2B, and PIP4K2C. The three isoforms differ, at least in vitro, significantly in enzymatic activity: PI5P4Kα is two orders of magnitude more active than PI5P4Kβ, while PI5P4K-γ has very little activity (13). PI5P4Ks are dimeric proteins (14), and the possibility that they can form heterodimers may have important functional implications, especially for the lesser active isoforms (15, 16). PI5P4Kβ is the only isoform that preferentially localizes to the nucleus (17).Genetic studies have implicated PI5P4Kβ in metabolic regulation (18, 19). Mice with both PIP4K2B genes inactivated manifest hypersensitivity to insulin stimulation (adult males are also leaner). Although this is consistent with the observation that PI(5)P levels, which can be manipulated by overexpressing PI5P4K or a bacterial phosphatase that robustly produces PI(5)P from PI(4,5)P₂, correlate positively with PI3K/Akt signaling, the underlying molecular mechanisms remain undefined (20). Both male and female PIP4K2B ^−/− mice are mildly growth retarded. Inactivation of the only PI5P4K isoform in Drosophila also produced small and developmentally delayed animals (21). These phenotypes may be related to suppressed TOR signaling (22, 23), but again, the underlying mechanism is unclear since TORC1 is downstream of, and positively regulated by, PI3K/Akt. Knocking out the enzymatically more active PI5P4Kα, in contrast, did not produce any overt metabolic or developmental phenotypes (19).Malignant transformation is associated with profound changes in cell metabolism (24, 25). Although metabolic reprograming generally benefits tumor cells by increasing energy and material supplies, it can also, counterintuitively, generate unique dependencies (26, 27). Loss of p53, a tumor suppressor that is mutated in most human cancers, has been shown to render cells more susceptible to nutrient stress (28, 29) and to the antidiabetic drug metformin (30, 31). Although TP53 ^−/− and PIP4K2B ^−/− mice are themselves viable, combining the two is embryonically lethal (19). Knocking out three copies of PI5P4K (PIP4K2A ^−/− PIP4K2B ^+/− ) greatly reduces tumor formation and cancer-related death in TP53 ^−/− animals (19). The synthetic lethal interaction between p53 and PI5P4Kα/β was thought to result from suppressed glycolysis and increased reactive oxygen species (19), although how the lipid kinases impact glucose metabolism remains uncertain.Given the interest in the physiological function of this alternative synthetic route for PI(4,5)P₂, and the potential of PI5P4K inactivation in treating type 2 diabetes and cancer, several attempts have been made to identify chemical probes that target various PI5P4K isoforms, which yielded compounds with micromolar affinity and unknown selectivity (32 –35). Here, we report the development of a class of PI5P4Kα/β inhibitors that have much improved potency and better-defined selectivity. Using the chemical probe, we show that transient inhibition of the lipid kinases alters cell energy metabolism and induces different responses in muscle and cancer cells.     

Tropospheric carbonyl sulfide mass balance based on direct measurements of sulfur isotopes

Robust estimates for the rates and trends in terrestrial gross primary production (GPP; plant CO₂ uptake) are needed. Carbonyl sulfide (COS) is the major long-lived sulfur-bearing gas in the atmosphere and a promising proxy for GPP. Large uncertainties in estimating the relative magnitude of the COS sources and sinks limit this approach. Sulfur isotope measurements (³⁴S/³²S; δ³⁴S) have been suggested as a useful tool to constrain COS sources. Yet such measurements are currently scarce for the atmosphere and absent for the marine source and the plant sink, which are two main fluxes. Here we present sulfur isotopes measurements of marine and atmospheric COS, and of plant-uptake fractionation experiments. These measurements resulted in a complete data-based tropospheric COS isotopic mass balance, which allows improved partition of the sources. We found an isotopic (δ³⁴S ± SE) value of 13.9 ± 0.1‰ for the troposphere, with an isotopic seasonal cycle driven by plant uptake. This seasonality agrees with a fractionation of −1.9 ± 0.3‰ which we measured in plant-chamber experiments. Air samples with strong anthropogenic influence indicated an anthropogenic COS isotopic value of 8 ± 1‰. Samples of seawater-equilibrated-air indicate that the marine COS source has an isotopic value of 14.7 ± 1‰. Using our data-based mass balance, we constrained the relative contribution of the two main tropospheric COS sources resulting in 40 ± 17% for the anthropogenic source and 60 ± 20% for the oceanic source. This constraint is important for a better understanding of the global COS budget and its improved use for GPP determination.

The Earth system is going through rapid changes as the climate warms and CO₂ level rises. This rise in CO₂ is mitigated by plant uptake; hence, it is important to estimate global and regional photosynthesis rates and trends (1). Yet, robust tools for investigating these processes at a large scale are scarce (2). Recent studies suggest that carbonyl sulfide (COS) could provide an improved constraint on terrestrial photosynthesis (gross primary production, GPP) (2–12). COS is the major long-lived sulfur-bearing gas in the atmosphere and the main supplier of sulfur to the stratospheric sulfate aerosol layer (13), which exerts a cooling effect on the Earth’s surface and regulates stratospheric ozone chemistry (14).During terrestrial photosynthesis, COS diffuses into leaf stomata and is consumed by photosynthetic enzymes in a similar manner to CO₂ (3–5). Contrary to CO₂, COS undergoes rapid and irreversible hydrolysis mainly by the enzyme carbonic-anhydrase (6, 7). Thus, COS can be used as a proxy for the one-way flux of CO₂ removal from the atmosphere by terrestrial photosynthesis (2, 8–11). However, the large uncertainties in estimating the COS sources weaken this approach (10–12, 15). Tropospheric COS has two main sources: the oceans and anthropogenic emissions, and one main sink–terrestrial plant uptake (8, 10–13). Smaller sources include biomass burning, soil emissions, wetlands, volcanoes, and smaller sinks include OH destruction, stratospheric destruction, and soil uptake (12). The largest source of COS to the atmosphere is the ocean, both as direct COS emission, and as indirect carbon disulfide (CS₂) and dimethylsulfide (DMS) emissions that are rapidly oxidized to COS (10, 16–20). Recent studies suggest oceanic COS emissions are in the range of 200–4,000 GgS/y (19–22). The second major COS source is the anthropogenic source, which is dominated by indirect emissions derived from CS₂ oxidation, mainly from the use of CS₂ as an industrial solvent. Direct emissions of COS are mainly derived from coal and fuel combustion (17, 23, 24). Recent studies suggest that anthropogenic emissions are in the range of 150–585 GgS/y (23, 24). The terrestrial plant uptake is estimated to be in the range of 400–1,360 GgS/y (11). Measurements of sulfur isotope ratios (δ³⁴S) in COS may be used to track COS sources and thus reduce the uncertainties in their flux estimations (15, 25–27). However, the isotopic mass balance approach works best if the COS end members are directly measured and have a significantly different isotopic signature. Previous δ³⁴S measurements of atmospheric COS are scarce and there have been no direct measurements of two important components: the δ³⁴S of oceanic COS emissions, and the isotopic fractionation of COS during plant uptake (15, 25–27). In contrast to previous studies that used assessments for these isotopic values, our aim was to directly measure the isotopic values of these missing components, and to determine the tropospheric COS δ³⁴S variability over space and time.     

Redox control on the tungsten isotope composition of seawater

Free oxygen represents an essential basis for the evolution of complex life forms on a habitable Earth. The isotope composition of redox-sensitive trace elements such as tungsten (W) can possibly trace the earliest rise of oceanic oxygen in Earth’s history. However, the impact of redox changes on the W isotope composition of seawater is still unknown. Here, we report highly variable W isotope compositions in the water column of a redox-stratified basin (δ^186/184W between +0.347 and +0.810 ‰) that contrast with the homogenous W isotope composition of the open ocean (refined δ^186/184W of +0.543 ± 0.046 ‰). Consistent with experimental studies, the preferential scavenging of isotopically light W by Mn-oxides increases the δ^186/184W of surrounding seawater, whereas the redissolution of Mn-oxides causes decreasing seawater δ^186/184W. Overall, the distinctly heavy stable W isotopic signature of open ocean seawater mirrors predominantly fully oxic conditions in modern oceans. We expect, however, that the redox evolution from anoxic to hypoxic and finally oxic marine conditions in early Earth’s history would have continuously increased the seawater δ^186/184W. Stable W isotope compositions of chemical sediments that potentially preserve changing seawater W isotope signatures might therefore reflect global changes in marine redox conditions.

Tungsten (W) belongs to the transition group VI elements and is dissolved as the tetrahedral oxyanion tungstate (WO₄²⁻) in the modern oceans. Despite its relatively low concentration of 0.041 to 0.067 nM, a moderately long residence time between 14,000 to 61,000 y suggests a homogenous distribution of W in the oceans (1–3). Riverine and hydrothermal sources, assumed to be the main inputs for marine W, have dissolved W concentrations that may be orders of magnitudes higher (rivers: 0.02 to 1029 nM; hydrothermal fluids: 0.22 to 123 nM), arguing for an efficient scavenging of W in estuarine and marine environments (3–6). While the stable W isotope composition of rivers and hydrothermal fluids is still unknown, a pioneering study of Fujiwara et al. (7) indicates that the seawater stable W isotope composition of the Northern Pacific is homogeneous with δ^186/184W of +0.55 ± 0.12 ‰ (Eq. 1). Thus, the seawater dissolved W inventory is isotopically significantly heavier than modern igneous rocks [δ^186/184W of +0.096 ± 0.076 ‰ (8, 9)], the ultimate source for marine W. This suggests stable W isotope fractionation during processes such as weathering, hydrothermal alteration of the oceanic crust, or the scavenging of dissolved marine W.In euxinic environments, the sequestration of dissolved W is limited because of its increased solubility as thiotungstate species (WO_xS_4−x²⁻) that form when WO₄²⁻ is exposed to elevated H₂S_(aq) levels (e.g., ref. 10). However, particle shuttling by Mn-oxides and Fe-hydroxides acting at pelagic redoxclines efficiently scavenges dissolved WO₄²⁻ and may cause strong authigenic enrichments of W in modern chemical sediments (e.g., ref. 3). Adsorption of WO₄²⁻ onto these (hydr)oxides triggers a change in coordination from tetrahedral to octahedral (11). Because the bonding in octahedral coordination is longer and weaker, isotopically light W is preferentially adsorbed (12). Kashiwabara et al. (2017) experimentally determined similar stable W isotopic differences between dissolved and adsorbed W species for Mn-oxides and Fe-hydroxides, respectively, with Δ^186/184W_Mn-oxides = δ^186/184W_dissolved − δ^186/184W_adsorbed = 0.59 ± 0.14 ‰ and Δ^186/184W_{Fe-hydroxides} = δ^186/184W_dissolved − δ^186/184W_adsorbed = 0.51 ± 0.06 ‰ (12). Thus, adsorption processes in oxic marine environments may provide the key mechanism for the build-up of an isotopically heavy ocean.Molybdenum (Mo), the geochemical twin of W, also exists as a dissolved oxyanion (MoO₄²⁻) species and is homogeneously distributed in the modern ocean with a higher concentration of around 105 nM (13). As for W, the stable Mo isotope composition of seawater [δ^98/95Mo = 2.34 ± 0.10 ‰ (14, 15)] is distinctly heavy compared to the bulk crust [δ^98/95Mo = +0.47 ± 0.12 ‰ (16)]. In analogy to WO₄²⁻, adsorption of MoO₄²⁻ onto Mn-oxides also causes a change in coordination from tetrahedral to octahedral. However, there are some key differences in the redox sensitivity of W and Mo. During adsorption of MoO₄²⁻ onto Fe-hydroxides, the tetrahedral coordination is partially retained (11). The experimentally determined isotopic difference between dissolved MoO₄²⁻ and adsorbed Mo species is therefore significantly smaller for Fe-hydroxides [Δ^98/95Mo_{Fe-hydroxides} = δ^98/95Mo_dissolved − δ^98/95Mo_adsorbed = 1.11 ± 0.15 ‰ (17)] than for Mn-oxides [Δ^98/95Mo_Mn-oxides = δ^98/95Mo_dissolved − δ^98/95Mo_adsorbed = 2.67 ± 0.18 ‰ (18)]. In euxinic environments, MoO₄²⁻ is successively thiolated with increasing H₂S_(aq), forming predominantly thiomolybdate (MoS₄²⁻) at H₂S_(aq) > 11 µM (19). In contrast to WS₄²⁻, MoS₄²⁻ is particle reactive and readily removed from solution by forming Fe–Mo–S clusters (20). In restricted and euxinic basins such as the modern Black Sea, the near quantitative sequestration of MoS₄²⁻ results in the preservation of the seawater Mo isotopic signature in Black Sea sediments (21). However, incomplete scavenging of MoO_xS_4−x²⁻ species in less restricted and/or weakly euxinic environments with H₂S_(aq) < 11 µM leads to the preferential burial of isotopically light Mo (21, 22). Therefore, adsorption processes in oxic marine environments but also the incomplete sequestration of Mo in euxinic settings may increase the δ^98/95Mo of seawater. On the other hand, temporal variations in the δ^186/184W of seawater and authigenic sediments might be more intimately linked to adsorption and deposition of oxide minerals. The difference in sensitivity to changing redox environments suggests that combined W and Mo proxy data may be a powerful way to trace subtle changes in oxygenation levels of the early surface Earth, particularly if further combined with other redox-sensitive trace metal proxies. Increasingly, multiple-proxy approaches are being used to remove the ambiguities that may be associated with individual paleoredox proxies (e.g., refs. 23–26). Thus, stable W isotope analyses may represent a complementary tool for paleo-redox reconstructions that is particularly sensitive to oxide mineral formation in oxic marine environments. However, future paleo-redox applications require a better understanding of the processes that fractionate W isotopes and their definite implication on the seawater and sedimentary δ^186/184W as offered by modern systems characterized by changing redox conditions.In this study, we present W concentrations and stable W isotope compositions of oceanic water column profiles from the Southern Atlantic Ocean, the South China Sea in the Western Pacific Ocean, and from the Landsort Deep, a redox-stratified basin within the more restricted Baltic Sea (SI Appendix, Fig. S1). The dataset is complemented by stable W isotope compositions of suspended particles that form at the pelagic redoxcline of the Landsort Deep and euxinic porewaters from this site. Our results confirm the initial assumption of a deep ocean homogeneous in isotopically heavy W. Furthermore, we highlight the major redox-related processes that cause variations in the abundance and stable isotope composition of marine W. Overall, these findings provide the initial framework essential for the future application of stable W isotopes as a paleo-redox proxy.     

From the Cover: Condensing water vapor to droplets generates hydrogen peroxide

It was previously shown [J. K. Lee et al., Proc. Natl. Acad. Sci. U.S.A., 116, 19294–19298 (2019)] that hydrogen peroxide (H₂O₂) is spontaneously produced in micrometer-sized water droplets (microdroplets), which are generated by atomizing bulk water using nebulization without the application of an external electric field. Here we report that H₂O₂ is spontaneously produced in water microdroplets formed by dropwise condensation of water vapor on low-temperature substrates. Because peroxide formation is induced by a strong electric field formed at the water–air interface of microdroplets, no catalysts or external electrical bias, as well as precursor chemicals, are necessary. Time-course observations of the H₂O₂ production in condensate microdroplets showed that H₂O₂ was generated from microdroplets with sizes typically less than ∼10 µm. The spontaneous production of H₂O₂ was commonly observed on various different substrates, including silicon, plastic, glass, and metal. Studies with substrates with different surface conditions showed that the nucleation and the growth processes of condensate water microdroplets govern H₂O₂ generation. We also found that the H₂O₂ production yield strongly depends on environmental conditions, including relative humidity and substrate temperature. These results show that the production of H₂O₂ occurs in water microdroplets formed by not only atomizing bulk water but also condensing water vapor, suggesting that spontaneous water oxidation to form H₂O₂ from water microdroplets is a general phenomenon. These findings provide innovative opportunities for green chemistry at heterogeneous interfaces, self-cleaning of surfaces, and safe and effective disinfection. They also may have important implications for prebiotic chemistry.

Water molecules in liquid water are considered stable and inert. We and other investigators have reported that water molecules become electrochemically active and catalytic for various reactions when bulk water is formed into micrometer-sized droplets (microdroplets). Reaction rates for various chemical reactions are accelerated in microdroplets by factors of 10² or more compared to bulk solution (1). The microdroplet environment provides conditions for a lowered entropic barrier, which allows thermodynamically unfavorable reactions to proceed in microdroplets at room temperature (2, 3). We also have shown that water microdroplets induce spontaneous charge exchanges between solutes and water molecules to induce the spontaneous reduction of organic molecules and metal ions as well as the formation of nanostructures without any added reducing agent or template (4, 5). Moreover, we have reported that water molecules undergo spontaneous oxidation to form reactive oxygen species, including hydroxyl radicals (OH) and hydrogen peroxide (H₂O₂) (6–8). Recent investigations attributed the origin of these unique physicochemical properties observed in microdroplets to the enrichment of reactants at the interface (9–11), restricted molecular rotations (12), partial solvation at the water surface (1, 13), and a strong interfacial electric field at the surface of the water microdroplet (14).Microdroplets can be formed either by atomizing bulk water (top down) with various methods such as high-pressure gas nebulization (15), ultrasonic nebulization (16), vibrating micromesh nebulization (17), and piezoelectric nebulization (18), or by condensing vapor-phase molecules (bottom up) (19). A question may be asked whether those unique properties of microdroplets arise only in microdroplets formed by atomization of bulk water. In addition, it may be wondered whether the spontaneous oxidation of water to form H₂O₂ in microdroplets (6) was caused by the atomizing process involving friction or vibration. These questions motivated us to investigate whether H₂O₂ becomes spontaneously generated in water microdroplets formed by the condensation of water vapor in air on cold surfaces, and how universal might this process be. We have paid special attention to the influence of different surface properties, including hydrophilicity and surface roughness, as well as environmental factors, including relative humidity and surface temperature.     

Reconstitution of β-adrenergic regulation of CaV1.2: Rad-dependent and Rad-independent protein kinase A mechanisms

L-type voltage-gated Ca_V1.2 channels crucially regulate cardiac muscle contraction. Activation of β-adrenergic receptors (β-AR) augments contraction via protein kinase A (PKA)–induced increase of calcium influx through Ca_V1.2 channels. To date, the full β-AR cascade has never been heterologously reconstituted. A recent study identified Rad, a Ca_V1.2 inhibitory protein, as essential for PKA regulation of Ca_V1.2. We corroborated this finding and reconstituted the complete pathway with agonist activation of β1-AR or β2-AR in Xenopus oocytes. We found, and distinguished between, two distinct pathways of PKA modulation of Ca_V1.2: Rad dependent (∼80% of total) and Rad independent. The reconstituted system reproduces the known features of β-AR regulation in cardiomyocytes and reveals several aspects: the differential regulation of posttranslationally modified Ca_V1.2 variants and the distinct features of β1-AR versus β2-AR activity. This system allows for the addressing of central unresolved issues in the β-AR–Ca_V1.2 cascade and will facilitate the development of therapies for catecholamine-induced cardiac pathologies.

Cardiac excitation–contraction coupling crucially depends on the L-type voltage-dependent Ca²⁺ channel, Ca_V1.2. Influx of extracellular Ca²⁺ via Ca_V1.2 triggers Ca²⁺ release from the sarcoplasmic reticulum via the Ca²⁺ release channel (1). Activation of the sympathetic nervous system increases heart rate, relaxation rate and contraction force. The latter is largely due to increased Ca²⁺ influx via Ca_V1.2 (2, 3). Pathological prolonged sympathetic activation progressively impairs cardiac function, causing heart failure, partly due to misregulation of Ca_V1.2 (4, 5).Cardiac Ca_V1.2 is a heterotrimer comprising the pore-forming subunit α_1C (∼240 kDa), the intracellular Ca_Vβ₂ (∼68 kDa) and the extracellular α2δ (∼170 kDa) (Fig. 1A) (6, 7). The N and C termini (NT, CT respectively) of α_1C are cytosolic and vary among Ca_V1.2 isoforms. Further, most of the cardiac α_1C protein is posttranslationally cleaved at the CT, around amino acid (a.a.) 1800, to produce the truncated ∼210-kDa α_1C protein and the ∼35-kDa cleaved distal CT (dCT); however, the full-length protein is also present (8–11).Open in a separate windowFig. 1.cAMP regulation of Ca_V1.2 is enhanced by coexpression of Rad. (A) Ca_V1.2 and Rad. α_1C and α2δ subunits are shown schematically, with structures of β_2b (38) and Rad (74). The truncation in α_1CΔ1821 was at a.a. 1,821 (red cross mark) similar to naturally truncated cardiac α_1C, ∼a.a. 1800 (9). Ca_Vβ binds to the cytosolic loop I, L1, that connects repeat domains I and II. Rad exerts inhibitory action on the channel, in part through an interaction with Ca_Vβ. (B) Rad reduces the Ba²⁺ current of Ca_V1.2-α_1CΔ1821 (α_1CΔ1821, β_2b and α2δ; 1.5 ng RNA of each subunit) in a dose-dependent manner. Pearson correlation, r = −0.82, P = 0.023. Each point represents mean ± SEM from 7 to 10 oocytes recorded during 1 d. The linear regression line was drawn for nonzero doses of Rad. (C) Rad enhances the cAMP-induced increase in I_Ba. Diary plots of the time course of change in I_Ba (normalized to initial I_Ba) are shown before and after intracellular injection of cAMP in representative cells. No Rad: Upper; with Rad: Lower. (Insets) Currents at +20 mV before (black trace) and 10 min after cAMP injection (red trace). (D) “before–after” plots of cAMP-induced changes in I_Ba in individual cells injected Rad RNA while varying Rad:β_2b RNA ratio (by weight, wt/wt). Empty symbols–before cAMP; red-filled–after cAMP. n = 3 experiments; statistics: paired t test. (E) cAMP-induced increase in I_Ba at different Rad/β_2b RNA levels (summary of data from D). Each symbol represents fold increase in I_Ba induced by cAMP injection in one cell. Here and in the following figures, box plots show 25 to 75 percentiles, whiskers show the 5/95 percentiles, and black and red horizontal lines within the boxes are the median and mean, respectively. At all Rad:β_2b RNA ratios except 1:20, the cAMP-induced increase in I_Ba was significantly greater than without Rad (Kruskal–Wallis test; H = 36.1, 6 degrees of freedom, P < 0.001). (F) Summary of cAMP effects in 10 experiments without and with Rad at 1:2 and 1:1 Rad:β_2b RNA ratios (pooled). Number of cells: within the bars. Statistics: Mann–Whitney U test; U = 19.0, P < 0.001.The sympathetic nervous system activates cardiac β-adrenergic receptors (β-AR), primarily β1-AR (which is coupled to G_s, is globally distributed in cardiomyocytes, and mediates most of the β-AR-enhancement of contraction and Ca_V1.2 activity) and β2-AR, which can couple to both G_s and G_i (12). The cascade of adrenergic modulation of Ca_V1.2 comprises agonist binding to β-ARs, activation of G_s and adenylyl cyclase, elevated intracellular cAMP levels, and activation of protein kinase A (PKA) by cAMP-induced dissociation of its catalytic subunit (PKA-CS) from the regulatory subunit. However, the final step, how PKA-CS enhances Ca_V1.2 activity, remained enigmatic. A long-standing paradigm was a direct phosphorylation by PKA-CS of α_1C and/or Ca_Vβ subunits (3, 13–16). However, numerous studies critically challenged this theory. In particular, mutated Ca_V1.2 channels in genetically engineered mice lacking putative PKA phosphorylation sites on α_1C and/or β_2b, were still up-regulated by PKA (9, 17–21) (reviewed in refs. 6 and 22).One significant obstacle in deciphering the mechanism of PKA regulation of Ca_V1.2 was a recurrent lack of success in reconstituting the regulation in heterologous systems, which proved challenging and controversial (23). Studies in heterologous cellular models, including Xenopus oocytes, demonstrated that cAMP failed to up-regulate Ca_V1.2 containing the full-length α_1C, Ca_V1.2-α_1C (24–26). However, robust β-AR–induced up-regulation of Ca²⁺ currents was observed in oocytes injected with total heart RNA (27, 28), suggesting the necessity of an auxiliary protein, the “missing link” (24, 25). Interestingly, partial regulation was observed with dCT-truncated α_1C (16, 29). Intracellular injection of cAMP or PKA-CS in Xenopus oocytes caused a modest (30 to 40%) up-regulation of Ca_V1.2, containing a dCT-truncated α_1C, Ca_V1.2-α_1CΔ1821 (29). This regulation required the presence of the initial segment of the long-NT of α_1C but did not involve Ca_Vβ subunit. We proposed that this mechanism might account for part of the adrenergic regulation of Ca_V1.2 in the heart (29). Normally adrenergic stimulation in cardiomyocytes increases the Ca²⁺ current two- to threefold; thus, a major part of the regulation has remained unexplained.Recently, Liu et al. identified Rad as the “missing link” in PKA regulation of Ca_V1.2 (20). Rad is a member of the Ras-related GTP-binding protein subfamily (RGK) that inhibit high voltage-gated calcium channels Ca_V1 and Ca_V2 (30). Rad tonically inhibits Ca_V1.2, largely via an interaction with Ca_Vβ (31, 32). Ablation of Rad in murine heart was shown to increase basal Ca_V1.2 activity and rendered the channel insensitive to β-AR regulation, probably through a “ceiling” effect (33, 34). Liu et al. (20) reconstituted a major part of the Ca_V1.2 regulation cascade, initiated by forskolin-activated adenylyl cyclase in mammalian cells, ultimately attaining an approximately twofold increase in Ca²⁺ current. The regulation required phosphorylation of Rad, the presence of Ca_Vβ, and the interaction of Ca_Vβ with the cytosolic loop I of α_1C, suggesting that PKA phosphorylation of Rad reduces its interaction with Ca_Vβ and relieves the tonic inhibition of Ca_V1.2 (20, 35).Importantly, the complete adrenergic cascade, starting with β-AR activation, has not yet been heterologously reconstituted for Ca_V1.2. Also, the relation between the Rad-dependent regulation and the regulation reported in our previous study (29) is not clear. Here, we utilized the Xenopus oocyte heterologous expression system and successfully reconstituted the entire β-AR cascade. We demonstrate two distinct pathways of PKA modulation of Ca_V1.2 (Rad dependent and Rad independent) and characterize the roles of NT and CT of α_1C, β_2b, and Rad in the adrenergic modulation of cardiac Ca_V1.2 channels. Reproducing the complete β-AR cascade in a heterologous expression system will promote the identification and characterization of intracellular proteins that regulate the cascade, eventually assisting efforts to develop therapies to treat heart failure and other catecholamine-induced cardiac pathologies.     

Structural insights into α-synuclein monomer–fibril interactions

Protein aggregation into amyloid fibrils is associated with multiple neurodegenerative diseases, including Parkinson’s disease. Kinetic data and biophysical characterization have shown that the secondary nucleation pathway highly accelerates aggregation via the absorption of monomeric protein on the surface of amyloid fibrils. Here, we used NMR and electron paramagnetic resonance spectroscopy to investigate the interaction of monomeric α-synuclein (α-Syn) with its fibrillar form. We demonstrate that α-Syn monomers interact transiently via their positively charged N terminus with the negatively charged flexible C-terminal ends of the fibrils. These intermolecular interactions reduce intramolecular contacts in monomeric α-Syn, yielding further unfolding of the partially collapsed intrinsically disordered states of α-Syn along with a possible increase in the local concentration of soluble α-Syn and alignment of individual monomers on the fibril surface. Our data indicate that intramolecular unfolding critically contributes to the aggregation kinetics of α-Syn during secondary nucleation.

Synucleinopathies, including Parkinson’s disease (PD), are associated with the accumulation of intracellular neuronal aggregates termed as Lewy bodies and Lewy neuritis, which contain high concentration of the protein α-synuclein (α-Syn) in an aggregated state (1, 2). The disease-relevant role of α-Syn is further highlighted by mutations in the α-Syn gene (SNCA) causing familial PD [i.e., A30P (3), E46K (4), H50Q (5), G51D (6), A53E (7), and A53T (8)] and the duplication or triplication of the SNCA leading to early-onset PD in affected families (9, 10). α-Syn is a 140-residue intrinsically disordered protein (IDP) in solution (11) but adopts a helical structure in the presence of acidic lipid surfaces (12, 13). The positively charged N terminus (residues 1 to 60) is rich in lysine residues and contains KTKEGV binding repeats associated with vesicle binding (14). Moreover, the N-terminal domain includes all known SNCA familial PD mutations. The central region (residues 61 to 95) defines the non-amyloid-β component (NAC) (15), which is essential for α-Syn aggregation (16), while the C terminus (residues 96 to 140) is highly negatively charged.In vitro, α-Syn forms polymorphic amyloid fibrils (17–19) with unique arrangements of cross-β-sheet motifs (20–22). When injected into model animals, these fibrils induce a PD-like pathology (23) where the aggregation pathway of α-Syn plays a key role in the development of the disease (24). A detailed analysis of the aggregation kinetics of α-Syn into amyloids is therefore important toward understanding the toxic mechanisms relevant for synucleinopathies.Amyloid formation of α-Syn is very sensitive to solution conditions, including pH (25), temperature (26), and salt concentration (27). It further requires the presence of an air–water interface (28) or negatively charged lipid membranes (29) for which α-Syn has a high affinity. Previous studies suggest that amyloid fibril growth of α-Syn occurs via a nucleation-dependent polymerization reaction (30). Following a fairly slow primary nucleus formation, α-Syn fibrils are elongated by addition of single monomers. In a next step, the amyloid fibrils multiply by fragmentation or can catalyze the formation of new amyloids from monomers on their surface—a process known as secondary nucleation that was first described for sickle cell anemia 40 y ago (31). Fragmentation and secondary nucleation critically depend on the fibril mass and accelerate the aggregation kinetics (30). In the case of α-Syn aggregation under quiescent condition fragmentation does not exist and only the described secondary nucleation process occurs. While detailed kinetic experiments showed no significant secondary nucleation at pH 7, it strongly contributes at pH values lower than 6 (25, 30). However, mechanistic or structural information of the secondary nucleation process in α-Syn aggregation has been lacking so far.In this study we investigated the structural properties of α-Syn monomer–fibril interactions by NMR and electron paramagnetic resonance (EPR) spectroscopy. Our results provide insights into how monomeric α-Syn transiently interacts in vitro via its positively charged N terminus with the negatively charged C-terminal residues of the α-Syn fibrils, giving detailed insights into the mechanism of the secondary nucleation process.     

Wild-type α-synuclein inherits the structure and exacerbated neuropathology of E46K mutant fibril strain by cross-seeding

Heterozygous point mutations of α-synuclein (α-syn) have been linked to the early onset and rapid progression of familial Parkinson’s diseases (fPD). However, the interplay between hereditary mutant and wild-type (WT) α-syn and its role in the exacerbated pathology of α-syn in fPD progression are poorly understood. Here, we find that WT mice inoculated with the human E46K mutant α-syn fibril (hE46K) strain develop early-onset motor deficit and morphologically different α-syn aggregation compared with those inoculated with the human WT fibril (hWT) strain. By using cryo-electron microscopy, we reveal at the near-atomic level that the hE46K strain induces both human and mouse WT α-syn monomers to form the fibril structure of the hE46K strain. Moreover, the induced hWT strain inherits most of the pathological traits of the hE46K strain as well. Our work suggests that the structural and pathological features of mutant strains could be propagated by the WT α-syn in such a way that the mutant pathology would be amplified in fPD.

α-Synuclein (α-Syn) is the main component of Lewy bodies, which serve as the common histological hallmark of Parkinson’s disease (PD) and other synucleinopathies (1, 2). α-Syn fibrillation and cell-to-cell transmission in the brain play essential roles in disease progression (3–5). Interestingly, WT α-syn could form fibrils with distinct polymorphs, which exhibit disparate seeding capability in vitro and induce distinct neuropathologies in mouse models (6–10). Therefore, it is proposed that α-syn fibril polymorphism may underlie clinicopathological variability of synucleinopathies (6, 9). In fPD, several single-point mutations of SNCA have been identified, which are linked to early-onset, severe, and highly heterogeneous clinical symptoms (11–13). These mutations have been reported to influence either the physiological or pathological function of α-syn (14). For instance, A30P weakens while E46K strengthens α-syn membrane binding affinity that may affect its function in synaptic vesicle trafficking (14, 15). E46K, A53T, G51D, and H50Q have been found to alter the aggregation kinetics of α-syn in different manners (15–17). Recently, several cryogenic electron microscopy (cryo-EM) studies revealed that α-syn with these mutations forms diverse fibril structures that are distinct from the WT α-syn fibrils (18–26). Whether and how hereditary mutations induced fibril polymorphism contributes to the early-onset and exacerbated pathology in fPD remains to be elucidated. More importantly, most fPD patients are heterozygous for SNCA mutations (12, 13, 27, 28), which leads to another critical question: could mutant fibrils cross-seed WT α-syn to orchestrate neuropathology in fPD patients?E46K mutation is one of the eight disease-causing mutations on SNCA originally identified from a Spanish family with autosomal-dominant PD (11). E46K-associated fPD features early-onset motor symptoms and rapid progression of dementia with Lewy bodies (11). Studies have shown that E46K mutant has higher neurotoxicity than WT α-syn in neurons and mouse models overexpressing α-syn (29–32). The underlying mechanism is debatable. Some reported that E46K promotes the formation of soluble species of α-syn without affecting the insoluble fraction (29, 30), while others suggested that E46K mutation may destabilize α-syn tetramer and induce aggregation (31, 32). Our previous study showed that E46K mutation disrupts the salt bridge between E46 and K80 in the WT fibril strain and rearranges α-syn into a different polymorph (33). Compared with the WT strain, the E46K fibril strain is prone to be fragmented due to its smaller and less stable fibril core (33). Intriguingly, the E46K strain exhibits higher seeding ability in vitro, suggesting that it might induce neuropathology different from the WT strain in vivo (33).In this study, we found that human E46K and WT fibril strains (referred to as hE46K and hWT strains) induced α-syn aggregates with distinct morphologies in mice. Mice injected with the hE46K strain developed more α-syn aggregation and early-onset motor deficits compared with the mice injected with the hWT strain. Notably, the hE46K strain was capable of cross-seeding both human and mouse WT (mWT) α-syn to form fibrils (named as hWT_cs and mWT_cs). The cross-seeded fibrils replicated the structure and seeding capability of the hE46K template both in vitro and in vivo. Our results suggest that the hE46K strain could propagate its structure as well as the seeding properties to the WT monomer so as to amplify the α-syn pathology in fPD.     

Enhancement and maximum in the isobaric specific-heat capacity measurements of deeply supercooled water using ultrafast calorimetry

Knowledge of the temperature dependence of the isobaric specific heat (C_p) upon deep supercooling can give insights regarding the anomalous properties of water. If a maximum in C_p exists at a specific temperature, as in the isothermal compressibility, it would further validate the liquid–liquid critical point model that can explain the anomalous increase in thermodynamic response functions. The challenge is that the relevant temperature range falls in the region where ice crystallization becomes rapid, which has previously excluded experiments. Here, we have utilized a methodology of ultrafast calorimetry by determining the temperature jump from femtosecond X-ray pulses after heating with an infrared laser pulse and with a sufficiently long time delay between the pulses to allow measurements at constant pressure. Evaporative cooling of ∼15-µm diameter droplets in vacuum enabled us to reach a temperature down to ∼228 K with a small fraction of the droplets remaining unfrozen. We observed a sharp increase in C_p, from 88 J/mol/K at 244 K to about 218 J/mol/K at 229 K where a maximum is seen. The C_p maximum is at a similar temperature as the maxima of the isothermal compressibility and correlation length. From the C_p measurement, we estimated the excess entropy and self-diffusion coefficient of water and these properties decrease rapidly below 235 K.

Water is one of the most exceptional liquids due to its importance, abundance, and many properties that are anomalous with respect to a normal liquid (1–3). This anomalous behavior is already evident at ambient conditions and is enhanced when water is supercooled below the freezing point into the metastable regime (2, 4, 5). In particular, the observation that the isothermal compressibility (κ_T), heat capacity (C_p), thermal expansion coefficient (α_P), and correlation length (ξ) appear to diverge toward a singular temperature (T_s) of about 228 K at 1 bar, as estimated by power-law fits (6, 7), has led to several hypotheses about the origin of water’s anomalous properties (2, 3, 8). One of the hypotheses proposes the existence of a liquid–liquid transition in supercooled water between high-density (HDL) and low-density (LDL) liquids, separated by a phase-coexistence line (8, 9) and terminating at a liquid–liquid critical point (LLCP) at positive pressure (8). Beyond the LLCP, at lower pressures, water is characterized by fluctuations between local structures of HDL and LDL (10). The locus of maxima in ξ of these fluctuations defines the Widom line in the pressure–temperature phase diagram, which emanates from the LLCP as an extension of the phase-coexistence line (11). Near the ξWidom line, the other thermodynamic response functions could also have maxima defining κ_T and C_p Widom lines, merging with the ξ Widom line in close proximity to the critical point. Such a merging was observed for the maxima in κ_T and C_p, and for the minimum in α_P at the liquid–gas critical point (LGCP), based on molecular-dynamic (MD) simulations (12).It has been challenging to experimentally determine the existence of a Widom line in supercooled water due to the extremely fast ice-forming crystallization at temperatures below 235 K. Nevertheless, rapid evaporative cooling of micrometer-sized droplets followed by ultrafast interrogation with an X-ray laser have allowed us to probe water at temperatures down to 227 K (13, 14). Recently, using this approach, maxima in ξ and κ_T were observed at 229 K, coinciding with the temperature of the most rapid change of the local tetrahedral structure in the liquid (13). Other experiments using sound velocity in stretched liquid water (15) also predict a maxima in κ_T and C_p. Based on a combination of MD simulations and temperature-dependent structure factor measurements, a consistency was derived with which α_P may also exhibit a minimum at 229 K (16). If all thermodynamic response functions showed evidence of a Widom line with maxima or minima, this would validate the LLCP scenario, more so if they were in close proximity in temperature. Currently, no measurements exist below 236 K (17) for C_p and it is necessary to develop experimental techniques to study water upon deep supercooling where rapid ice crystallization occurs. Measurements of the value of the C_p maximum also allow us to derive to which extent the excess entropy has decreased upon supercooling and compare this to the entropy of low-density amorphous ice (LDA) at the glass transition temperature (18–20). Interest in excess entropy was one of the original motivations in 1969 behind the study of supercooled water (18). Based on the expectation that C_p should decrease upon cooling, the excess entropy was expected to rapidly decrease, as C_p approaches that of LDA. Surprisingly, though, an accelerated increase was observed instead (21–23).Here, we show that the C_p can be measured down to 228 K using a method based on ultrafast calorimetry. The data are consistent with the existence of a maximum of C_p at 229 K, as well as a rapid decrease of the excess entropy at temperatures beyond the Widom line. Fig. 1 shows the experimental setup of our ultrafast calorimetry approach. The droplets are cooled by evaporation and the temperature is calculated using Knudsen’s theory of evaporation and Fourier’s law of heat conduction (24, 25). This approach to determining droplet temperatures has been proven to be successful in various experimental setups (13, 14, 26) and has been validated using ME simulations (25). A 2.05-μm infrared (IR) pulse heats the sample, increasing the temperature of the droplets by 0.5–1 K. The droplets are then probed by a femtosecond X-ray pulse after a 1-µs delay time, allowing the liquid to expand. The difference in the X-ray scattering patterns between IR laser on and off is used as a thermometer. The pattern from each X-ray shot is also used to detect whether Bragg peaks appear from small ice crystals so that crystallized droplets can be excluded from the analysis. Using a calibration curve of the scattering signal versus temperature, we estimate the increase of temperature from the heating pulse and derive the heat capacity at constant pressure, C_p. We observe a rapid increase in C_p at temperatures below 235 K with a maximum appearing at 229 K, followed by a suggested decrease toward lower temperatures. The rise and maximum of C_p is consistent with the existence of a Widom line for C_p as previously observed for κ_T and ξ (13).Open in a separate windowFig. 1.(A) Schematic of the experimental setup (Left) and (B) angularly integrated scattering intensity (Right). The time delay (∆t) between the IR laser and the X-rays is 1 µs. IR laser is ON for every alternate X-ray pulse. The difference in the scattering profile of the laser ON and laser OFF shots is ∼2% of the signal.     

Coupled impacts of sea ice variability and North Pacific atmospheric circulation on Holocene hydroclimate in Arctic Alaska

Arctic Alaska lies at a climatological crossroads between the Arctic and North Pacific Oceans. The modern hydroclimate of the region is responding to rapidly diminishing sea ice, driven in part by changes in heat flux from the North Pacific. Paleoclimate reconstructions have improved our knowledge of Alaska’s hydroclimate, but no studies have examined Holocene sea ice, moisture, and ocean−atmosphere circulation in Arctic Alaska, limiting our understanding of the relationship between these phenomena in the past. Here we present a sedimentary diatom assemblage and diatom isotope dataset from Schrader Pond, located ∼80 km from the Arctic Ocean, which we interpret alongside synthesized regional records of Holocene hydroclimate and sea ice reduction scenarios modeled by the Hadley Centre Coupled Model Version 3 (HadCM3). The paleodata synthesis and model simulations suggest the Early and Middle Holocene in Arctic Alaska were characterized by less sea ice, a greater contribution of isotopically heavy Arctic-derived moisture, and wetter climate. In the Late Holocene, sea ice expanded and regional climate became drier. This climatic transition is coincident with a documented shift in North Pacific circulation involving the Aleutian Low at ∼4 ka, suggesting a Holocene teleconnection between the North Pacific and Arctic. The HadCM3 simulations reveal that reduced sea ice leads to a strengthened Aleutian Low shifted west, potentially increasing transport of warm North Pacific water to the Arctic through the Bering Strait. Our findings demonstrate the interconnectedness of the Arctic and North Pacific on multimillennial timescales, and are consistent with future projections of less sea ice and more precipitation in Arctic Alaska.

Rapidly rising Arctic air and sea surface temperatures have resulted in the reduced annual duration and extent of Arctic sea ice (1), which in turn drives the ice−albedo feedback leading to amplified warming in the Arctic (2). These reductions in sea ice are projected to continue in future decades (3) and have important implications for Arctic terrestrial hydroclimate, as sea ice extent and duration impact the seasonality, type, and amount of precipitation in this region (4). Recent studies have also suggested teleconnections between the extent and duration of Arctic sea ice and midlatitudinal storm tracks (5, 6), as well as synoptic-scale processes involving the Aleutian Low atmospheric pressure cell (AL) (7, 8) and ocean−atmosphere circulation in the Bering Strait (9–11), which might link North Pacific hydroclimate directly to changes in Arctic sea ice. While recent observations show the influence of North Pacific climate on Arctic sea ice, little is known about their long-term dynamics or their coupled influence on hydroclimate in the western Arctic.Our understanding of past hydroclimate in Arctic Alaska is based in part on stable isotope reconstructions that reflect changes in the oxygen (δ¹⁸O) and hydrogen (δD) isotope composition of water. δ¹⁸O has proven particularly useful for studying both current (12, 13) and past (14–20) hydroclimate in the region, because it is sensitive to climate and environmental variables. As a result, δ¹⁸O has been used as a paleoclimate proxy for precipitation source (16), effective moisture (14), and temperature (20) in Arctic Alaska. Interpretations of these paleoclimate datasets have considered the impact of Holocene changes in AL variability (15, 16, 18), but they have not been used to examine the influence of Holocene Arctic sea ice variability on western Arctic climate, despite well-established sea ice conditions for this time period (e.g., ref. 21). The influence of sea ice extent on δ¹⁸O in various climate archives has been demonstrated in Arctic Alaska during the Pleistocene−Holocene transition (19), as well as in Greenland during the Holocene (22) and the Last Interglacial period (LIG) (23), suggesting that sites adjacent to seasonally ice-free Arctic waters can be sensitive recorders of sea ice conditions.In light of increasing evidence from both data and models for a modern connection between North Pacific circulation and Arctic sea ice (5–8), as well as the demonstrated influence of North Pacific (15, 16, 18) and Arctic (13, 19) ocean−atmosphere systems on past and present terrestrial hydroclimate conditions, it appears that northern Alaska lies at a climatological crossroads within the western Arctic. This means that paleoclimate records from Arctic Alaska are especially well situated for studying the effects of both changing Arctic sea ice and North Pacific circulation. However, existing paleoclimate datasets from this region have not been interpreted in the context of such a coupled system, and little has been done to synthesize possible multimillennial patterns among these and other datasets. Potential teleconnections during the Holocene must be explored, because this paleoclimate context is important for understanding the coevolution of Arctic and Pacific hydroclimate systems on longer timescales, which could help clarify predictions of their continued coevolution in the future.Here we present Holocene diatom assemblage and oxygen isotope (δ¹⁸O_diatom) datasets from Arctic Alaska, which we interpret in terms of past hydroclimatic change. Our results show that Holocene variability in δ¹⁸O_diatom at Schrader Pond (SP) in the northeastern Brooks Range was driven by changes in moisture source associated with fluctuating Arctic sea ice extent. We also present a data−model comparison, featuring a synthesis of Holocene hydroclimate and sea ice reconstructions from regional terrestrial and marine sites, together with coupled atmosphere−ocean model simulations, which supports our interpretation of δ¹⁸O_diatom variability. Our data highlight a prominent shift in terrestrial hydroclimate and sea ice in the region, concomitant with a well-documented shift in North Pacific hydroclimate at ∼4 ka (24). The timing of these near-synchronous shifts suggests an Arctic−Pacific teleconnection has been present over the Middle to Late Holocene, emphasizing the important role of both sea ice and lower-latitude ocean−atmosphere dynamics in the past and future of the Arctic.     

Evidence for root adaptation to a spatially discontinuous water availability in the absence of external water potential gradients

We hereby show that root systems adapt to a spatially discontinuous pattern of water availability even when the gradients of water potential across them are vanishingly small. A paper microfluidic approach allowed us to expose the entire root system of Brassica rapa plants to a square array of water sources, separated by dry areas. Gradients in the concentration of water vapor across the root system were as small as 10⁻⁴⋅mM⋅m⁻¹ (∼4 orders of magnitude smaller than in conventional hydrotropism assays). Despite such minuscule gradients (which greatly limit the possible influence of the well-understood gradient-driven hydrotropic response), our results show that 1) individual roots as well as the root system as a whole adapt to the pattern of water availability to maximize access to water, and that 2) this adaptation increases as water sources become more rare. These results suggest that either plant roots are more sensitive to water gradients than humanmade water sensors by 3–5 orders of magnitude, or they might have developed, like other organisms, mechanisms for water foraging that allow them to find water in the absence of an external gradient in water potential.

A secure water supply is the strongest predictor of survival in crops (1, 2) and most plants [not all (3)] uptake water mostly from their roots. Therefore, harvesting water is one of the most important, and yet still poorly understood functions of the root system. For example, despite great progress (4–11), we still do not fully understand how the architecture of the root system develops to optimize its access to a water supply that is inhomogeneously distributed. Therefore, we have limited information on how to design genomes or select phenotypes that promote, for example, tolerance to drought (4).The availability of water to plants is usually determined by the water potential (WP), and the hydraulic conductivity (HC) (12). Intuitively, these parameters help quantify, respectively, how easy it is to pull the water (i.e., the lower the WP, the more thermodynamically stable the water is, and the more difficult it is, in general, to change its state), and how rapidly it can be pulled (i.e., the flow of water under a certain pressure differential). These physical parameters can have biological consequences and induce a response: e.g., low water availability can limit the rate of water uptake by the plant and therefore induce water stress. Such limitation on water uptake can be due to the water being too hard to pull, too slow to obtain, and/or too limited in quantity.Plants can adapt to water scarcity by collecting information about the distribution and availability of water in the surrounding volume of soil and develop the structure of their root system accordingly (13).Organisms generally “collect information” about their environment by sensing some external potential gradient (e.g., gravitational potential in gravitropism, chemical potential in chemotropism). Therefore, the study of the adaptation of roots to an inhomogeneous water supply has historically focused on understanding how roots grow toward higher WP [i.e., hydrotropism, first reported in 1811 (13)]. Since 1872, hydrotropism was further investigated by Sachs (14), Molisch (15), Darwin (16), and, more recently, by others (17–21). These recent studies have focused on observing deflections of single roots (22) when exposed to gradients in the potential of water vapor (23) or of the water in the nutrient solution (18).Nonetheless we were prompted by the observation that in the animal kingdom, foraging is not always guided by sensing of the food source (olfactory, auditory, vision, tactile). Forage can be collected by trapping (24), harvesting (25), luring (26), symbiosis (27), parasitism (28), or its location can be encoded in memory (29) or into a chemical trail (30, 31). These distinct mechanisms allow animals [and some plants (32)] to forage for food sources that cannot be sensed due to their distance, or that move too rapidly to be caught. It is therefore conceivable that plants might have developed one or more mechanisms to seek water in the absence of external gradients of water potential. Therefore we set out to find out.     

Autoregulation of insulin receptor signaling through MFGE8 and the αvβ5 integrin

The role of integrins, in particular αv integrins, in regulating insulin resistance is incompletely understood. We have previously shown that the αvβ5 integrin ligand milk fat globule epidermal growth factor like 8 (MFGE8) regulates cellular uptake of fatty acids. In this work, we evaluated the impact of MFGE8 on glucose homeostasis. We show that acute blockade of the MFGE8/β5 pathway enhances while acute augmentation dampens insulin-stimulated glucose uptake. Moreover, we find that insulin itself induces cell-surface enrichment of MFGE8 in skeletal muscle, which then promotes interaction between the αvβ5 integrin and the insulin receptor leading to dampening of skeletal-muscle insulin receptor signaling. Blockade of the MFGE8/β5 pathway also enhances hepatic insulin sensitivity. Our work identifies an autoregulatory mechanism by which insulin-stimulated signaling through its cognate receptor is terminated through up-regulation of MFGE8 and its consequent interaction with the αvβ5 integrin, thereby establishing a pathway that can potentially be targeted to improve insulin sensitivity.

Acute insulin resistance can be viewed as a protective response under specific physiological conditions that necessitate increased insulin secretion. Nevertheless, the increasing prevalence of chronic insulin resistance (1) in the current obesity epidemic hastens the development of type 2 diabetes (T2D) and induces compensatory hyperinsulinemia. Hyperinsulinemia can produce potentially maladaptive consequences at least in part, due to the mitogenic roles of insulin (2–4). As such, there remains a critical need for new therapies to improve insulin sensitivity in order to prevent T2D, avoid the need for insulin treatment in patients with T2D, or reduce the insulin dose required to normalize blood glucose in such individuals.Insulin binding to the alpha subunit of the insulin receptor induces a conformational change that triggers activation of insulin receptor beta subunit (IRβ) tyrosine kinase activity (5–7). The activated insulin receptor phosphorylates target molecules that mediate downstream signaling leading to glucose uptake and other metabolic effects (8, 9). Dephosphorylation of IRβ and insulin receptor substrate-1 (IRS-1) aids in termination of insulin signaling pathways (10, 11) and is the basis of clinical trials targeting putative phosphatases to treat diabetes (12). Despite their potential therapeutic relevance, there is a relative paucity of knowledge regarding molecular mechanisms that lead to termination of insulin receptor signaling.The integrin families of cell surface receptors mediate bidirectional signaling between the cell and its external environment. Previous work has identified interactions between integrin receptors and other growth factor receptor tyrosine kinases (13–16) that lead to modulation of downstream signaling (17–19). For example, the αvβ3 and α6β4 integrins function as coreceptors for insulin-like growth factor-1 and 2 (IGF1 and 2) and potentiate IGF1 receptor (IGF1R)-mediated signaling (19–23). Immunoprecipitation studies have demonstrated a physical association between the αv integrins and IRβ (24, 25). The impact of these associations on glucose homeostasis has not been evaluated. A role for β1 integrins in the regulation of glucose homeostasis is well established. This class of integrins appears to be particularly important in regulating insulin-mediated glucose homeostasis in the obese state. The effect of β1 integrins on glucose homeostasis appears to be primarily due to obesity-associated matrix remodeling (26–30) rather than a direct effect secondary to a physical association between β1 integrins and the insulin receptor.Milk fat globule epidermal growth factor like 8 (MFGE8) is a secreted integrin ligand which binds the αvβ3, αvβ5, and α8β1 integrins (31, 32). Several recent observations suggest a role for MFGE8 in modulating insulin resistance. In humans, serum MFGE8 levels are increased in the context of diabetes and correlate positively with the extent of hemoglobin glycosylation (33, 34). Indeed, serum MFGE8 levels correlate with indices of insulin resistance in two independent cohorts of patients with T2D or gestational diabetes from China (35, 36). A missense variation in the gene encoding MFGE8, present in South Asian Punjabi Sikhs, is associated with increased circulating MFGE8 levels and increased risk of developing T2D (37). Increased circulating levels of MFGE8 in diabetic patients may impact T2D through effects on inflammation and cardiovascular disease. Humans with increased MFGE8 expression have a greater risk of developing coronary artery disease (38). In contrast, in murine models, MFGE8 deficiency exacerbates cardiac hypertrophy and atherosclerosis (39, 40). MFGE8 also improves wound healing responses in diabetic foot ulcers (41, 42) by triggering apoptotic cell clearance and promoting resolution of inflammation (43–45).Despite the notable links between MFGE8, insulin resistance, and T2D pathology, the biology underlying these associations has not been investigated. We therefore evaluated the effect of acute antibody-mediated disruption of the MFGE8/β5 pathway on glucose homeostasis in wild-type (WT) mice. We report here that MFGE8 markedly attenuates the effect of insulin on skeletal muscle glucose uptake. Antibody-mediated blockade of MFGE8 or αvβ5 enhances while recombinant MFGE8 (rMFGE8) reduces insulin-stimulated glucose uptake in vitro and in vivo. Mechanistically, insulin acts to promotes cell-surface enrichment of skeletal muscle MFGE8, which then binds to cell surface αvβ5 and increases the interaction between the integrin and the insulin receptor. This interaction subsequently aids in terminating insulin receptor signaling.     

The intrinsic instability of the hydrolase domain of lipoprotein lipase facilitates its inactivation by ANGPTL4-catalyzed unfolding

The complex between lipoprotein lipase (LPL) and its endothelial receptor (GPIHBP1) is responsible for the lipolytic processing of triglyceride-rich lipoproteins (TRLs) along the capillary lumen, a physiologic process that releases lipid nutrients for vital organs such as heart and skeletal muscle. LPL activity is regulated in a tissue-specific manner by endogenous inhibitors (angiopoietin-like [ANGPTL] proteins 3, 4, and 8), but the molecular mechanisms are incompletely understood. ANGPTL4 catalyzes the inactivation of LPL monomers by triggering the irreversible unfolding of LPL’s α/β-hydrolase domain. Here, we show that this unfolding is initiated by the binding of ANGPTL4 to sequences near LPL’s catalytic site, including β2, β3–α3, and the lid. Using pulse-labeling hydrogen‒deuterium exchange mass spectrometry, we found that ANGPTL4 binding initiates conformational changes that are nucleated on β3–α3 and progress to β5 and β4–α4, ultimately leading to the irreversible unfolding of regions that form LPL’s catalytic pocket. LPL unfolding is context dependent and varies with the thermal stability of LPL’s α/β-hydrolase domain (T_m of 34.8 °C). GPIHBP1 binding dramatically increases LPL stability (T_m of 57.6 °C), while ANGPTL4 lowers the onset of LPL unfolding by ∼20 °C, both for LPL and LPL•GPIHBP1 complexes. These observations explain why the binding of GPIHBP1 to LPL retards the kinetics of ANGPTL4-mediated LPL inactivation at 37 °C but does not fully suppress inactivation. The allosteric mechanism by which ANGPTL4 catalyzes the irreversible unfolding and inactivation of LPL is an unprecedented pathway for regulating intravascular lipid metabolism.

The lipolytic processing of triglyceride-rich lipoproteins (TRLs) along the luminal surface of capillaries plays an important role in the delivery of lipid nutrients to vital tissues (e.g., heart, skeletal muscle, adipose tissue). A complex of lipoprotein lipase (LPL) and its endothelial cell transporter, glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1), is responsible for the margination of TRLs and their lipolytic processing (1–3). The importance of the LPL•GPIHBP1 complex for TRL processing is underscored by the development of severe hypertriglyceridemia (chylomicronemia) with loss-of-function mutations in LPL or GPIHBP1 or with GPIHBP1 autoantibodies that disrupt GPIHBP1•LPL interactions (4–7). Chylomicronemia is associated with a high risk for acute pancreatitis, which is debilitating and often life threatening (8, 9). Interestingly, increased efficiency of plasma triglyceride processing appears to be beneficial, reducing both plasma triglyceride levels and the risk for coronary heart disease (CHD). For example, genome-wide population studies have revealed that single-nucleotide polymorphisms that limit the ability of angiopoietin-like proteins 3 or 4 (ANGPTLs) to inhibit LPL are associated with lower plasma triglyceride levels and a reduced risk of CHD (10–14).GPIHBP1 is an atypical member of the LU domain superfamily because it contains a long intrinsically disordered and highly acidic N-terminal extension in addition to a canonical disulfide-rich three-fingered LU domain (15). At the abluminal surface of capillaries, GPIHBP1 is responsible for capturing LPL from heparan sulfate proteoglycans (HSPGs) in the subendothelial spaces and shuttling it to its site of action in the capillary lumen (3, 16). The capture of LPL from subendothelial HSPGs depends on electrostatic interactions with GPIHBP1’s intrinsically disordered acidic domain and stable hydrophobic interactions with GPIHBP1’s LU domain (15, 17, 18). In the setting of GPIHBP1 deficiency, LPL never reaches the capillary lumen and remains mislocalized, bound to HSPGs, in the subendothelial spaces. Aside from promoting the formation of GPIHBP1•LPL complexes, the acidic domain plays an important role in preserving LPL activity. The acidic domain is positioned to form a fuzzy complex with a large basic patch on the surface of LPL, which is formed by the confluence of several heparin-binding motifs. This electrostatic interaction stabilizes LPL structure and activity, even in the face of physiologic inhibitors of LPL (e.g., ANGPTL4) (19–22).Distinct expression profiles for ANGPTL-3, -4, and -8 underlie the tissue-specific regulation of LPL and serve to match the supply of lipoprotein-derived lipid nutrients to the metabolic demands of nearby tissues (21, 23–29). In the fasted state, ANGPTL4 inhibits LPL activity in adipose tissue, resulting in increased delivery of lipid nutrients to oxidative tissues. In the fed state, ANGPTL3•ANGPTL8 complexes inhibit LPL activity in oxidative tissues and thereby channel lipid delivery to adipocytes. While the physiologic relevance of tissue-specific LPL regulation is clear, the mechanisms by which ANGPTL proteins inhibit LPL activity remain both incompletely understood and controversial. One view holds that ANGPTL4 inhibits LPL activity by a reversible mechanism (30, 31). An opposing view, formulated early on by the laboratory of Gunilla Olivecrona, is that ANGPTL4 irreversibly inhibits LPL by a “molecular unfolding chaperone-like mechanism” (32). Hydrogen–deuterium exchange mass spectrometry (HDX-MS) studies have supported the latter view. Recent studies by our group revealed that ANGPTL4 catalyzes the irreversible unfolding (and inactivation) of LPL’s α/β-hydrolase domain and that the unfolding is substantially mitigated by the binding of GPIHBP1 to LPL (17, 19, 22). We further showed that ANGPTL4 functions by unfolding catalytically active LPL monomers rather than by promoting the dissociation of catalytically active LPL homodimers (33, 34). Despite these newer findings, a host of issues remains unresolved. For example, the binding site for ANGPTL4 on LPL has been controversial (31, 35); the initial conformational changes induced by ANGPTL4 binding have not been delineated, and how the early conformational changes in LPL progress to irreversible inactivation is unknown. In the current studies, we show, using time-resolved HDX-MS, that ANGPTL4 binds to LPL sequences proximal to the entrance of LPL’s catalytic pocket. That binding event triggers alterations in the dynamics of LPL secondary structure elements that are central to the architecture of the catalytic triad. Progression of those conformational changes leads to irreversible unfolding and collapse of LPL’s catalytic pocket. The binding of GPIHBP1 to LPL limits the progression of these allosteric changes, explaining why GPIHBP1 protects LPL from ANGPTL4-induced inhibition.     

In situ organic Fenton-like catalysis triggered by anodic polymeric intermediates for electrochemical water purification

Organic Fenton-like catalysis has been recently developed for water purification, but redox-active compounds have to be ex situ added as oxidant activators, causing secondary pollution problem. Electrochemical oxidation is widely used for pollutant degradation, but suffers from severe electrode fouling caused by high-resistance polymeric intermediates. Herein, we develop an in situ organic Fenton-like catalysis by using the redox-active polymeric intermediates, e.g., benzoquinone, hydroquinone, and quinhydrone, generated in electrochemical pollutant oxidation as H₂O₂ activators. By taking phenol as a target pollutant, we demonstrate that the in situ organic Fenton-like catalysis not only improves pollutant degradation, but also refreshes working electrode with a better catalytic stability. Both ¹O₂ nonradical and ·OH radical are generated in the anodic phenol conversion in the in situ organic Fenton-like catalysis. Our findings might provide a new opportunity to develop a simple, efficient, and cost-effective strategy for electrochemical water purification.

The efficient generation of reactive oxygen species is essential for pollutant degradation in water purification. The metal-mediated Fenton catalysis has been widely used for several decades owning to its high efficiency, low cost, and easy operation (1). However, it has several technical drawbacks to largely limit further applications, e.g., harsh pH, metal-rich sludge, secondary pollution, and poor stability (1). Alternatively, the metal-free Fenton catalysis has recently attracted increasing interests. Redox-active compounds serve as the oxidant activator to decompose pollutants via radical and/or nonradical pathways (2–5). These pathways depend highly on the atomic and electronic structures and molecular configurations of compounds and their molecular interactions with oxidants (6–18). So far organic activators are ex situ introduced and cause secondary pollution, although the performance can be largely improved (2–18). Such an intrinsic drawback greatly restricts its practical applications. Thus, in situ organic Fenton-like catalysis without secondary pollution is greatly desired for clean and safe water purification.Electrochemical oxidation (EO) at low bias is widely used for pollutant degradation owning to its high current efficiency and low energy consumption, but largely suffers from electrode fouling (19, 20). Such fouling is mainly caused by anodic polymeric intermediates with large molecular size, low geometric polarity, and high structural stability, thus anodic oxidation is thermodynamically terminated at this stage (19, 20). How to remove polymeric intermediates is essential for electrochemical water purification. It is interesting to note that anodic polymeric intermediates usually contain quinonelike moieties (C = O) and persistent organic radicals, as the electrons in nucleophilic C-OH can be readily transferred to generate C-O· and C = O (19, 20). Quinonelike moieties are redox-active because of their high electron density and strong electron-donating properties, thus can serve as the metal ligand and reductant to enhance transition-metal redox cycling, and also be involved in the environmental geochemistry of natural organic matters (21–30). Moreover, quinonelike moieties and persistent organic radicals can directly serve as an organic activator to initiate organic Fenton-like catalysis for environmental remediation (31–40). Thus, these redox-active anodic polymeric intermediates are likely to trigger organic Fenton-like catalysis.Inspired by above analyses, we constructed and validated in situ organic Fenton-like catalysis for electrochemical water purification at low bias before oxygen evolution (Scheme 1). Phenol, a model chemical widely present in environments, and other typical halogenated and nonhalogenated aromatic compounds were selected as target pollutants. Carbon felt (CF), a model material with high activity and low cost, and other typical dimensionally stable anodes were selected as target electrodes. Reaction systems were named in the form of “EO + ex situ added reagent + cathode,” as their anodes were identical. Pollutant degradation and electrode antifouling performances were evaluated under various conditions. After the major reactive oxygen species were identified using a suite of testing methods, and the potential role of trace transition metals, especially iron and copper, was examined, the possible molecular mechanism of the in situ organic Fenton-like catalysis was proposed.Open in a separate windowScheme 1.Scheme diagrams of the EO-Ti, EO/H₂O₂-Ti, and EO/O₂-CF systems.