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.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:The effect of parathyroid hormone on osteogenesis is mediated partly by osteolectin

We previously described a new osteogenic growth factor, osteolectin/Clec11a, which is required for the maintenance of skeletal bone mass during adulthood. Osteolectin binds to Integrin α11 (Itga11), promoting Wnt pathway activation and osteogenic differentiation by leptin receptor⁺ (LepR⁺) stromal cells in the bone marrow. Parathyroid hormone (PTH) and sclerostin inhibitor (SOSTi) are bone anabolic agents that are administered to patients with osteoporosis. Here we tested whether osteolectin mediates the effects of PTH or SOSTi on bone formation. We discovered that PTH promoted Osteolectin expression by bone marrow stromal cells within hours of administration and that PTH treatment increased serum osteolectin levels in mice and humans. Osteolectin deficiency in mice attenuated Wnt pathway activation by PTH in bone marrow stromal cells and reduced the osteogenic response to PTH in vitro and in vivo. In contrast, SOSTi did not affect serum osteolectin levels and osteolectin was not required for SOSTi-induced bone formation. Combined administration of osteolectin and PTH, but not osteolectin and SOSTi, additively increased bone volume. PTH thus promotes osteolectin expression and osteolectin mediates part of the effect of PTH on bone formation.

The maintenance and repair of the skeleton require the generation of new bone cells throughout adult life. Osteoblasts are relatively short-lived cells that are constantly regenerated, partly by skeletal stem cells within the bone marrow (1). The main source of new osteoblasts in adult bone marrow is leptin receptor-expressing (LepR⁺) stromal cells (2–4). These cells include the multipotent skeletal stem cells that give rise to the fibroblast colony-forming cells (CFU-Fs) in the bone marrow (2), as well as restricted osteogenic progenitors (5) and adipocyte progenitors (6–8). LepR⁺ cells are a major source of osteoblasts for fracture repair (2) and growth factors for hematopoietic stem cell maintenance (9–11).One growth factor synthesized by LepR⁺ cells, as well as osteoblasts and osteocytes, is osteolectin/Clec11a, a secreted glycoprotein of the C-type lectin domain superfamily (5, 12, 13). Osteolectin is an osteogenic factor that promotes the maintenance of the adult skeleton by promoting the differentiation of LepR⁺ cells into osteoblasts. Osteolectin acts by binding to integrin α11β1, which is selectively expressed by LepR⁺ cells and osteoblasts, activating the Wnt pathway (12). Deficiency for either Osteolectin or Itga11 (the gene that encodes integrin α11) reduces osteogenesis during adulthood and causes early-onset osteoporosis in mice (12, 13). Recombinant osteolectin promotes osteogenic differentiation by bone marrow stromal cells in culture and daily injection of mice with osteolectin systemically promotes bone formation.Osteoporosis is a progressive condition characterized by reduced bone mass and increased fracture risk (14). Several factors contribute to osteoporosis development, including aging, estrogen insufficiency, mechanical unloading, and prolonged glucocorticoid use (14). Existing therapies include antiresorptive agents that slow bone loss, such as bisphosphonates (15, 16) and estrogens (17), and anabolic agents that increase bone formation, such as parathyroid hormone (PTH) (18), PTH-related protein (19), and sclerostin inhibitor (SOSTi) (20). While these therapies increase bone mass and reduce fracture risk, they are not a cure.PTH promotes both anabolic and catabolic bone remodeling (21–24). PTH is synthesized by the parathyroid gland and regulates serum calcium levels, partly by regulating bone formation and bone resorption (23–25). PTH1R is a PTH receptor (26, 27) that is strongly expressed by LepR⁺ bone marrow stromal cells (8, 28–30). Recombinant human PTH (Teriparatide; amino acids 1 to 34) and synthetic PTH-related protein (Abaloparatide) are approved by the US Food and Drug Administration (FDA) for the treatment of osteoporosis (19, 31). Daily (intermittent) administration of PTH increases bone mass by promoting the differentiation of osteoblast progenitors, inhibiting osteoblast and osteocyte apoptosis, and reducing sclerostin levels (32–35). PTH promotes osteoblast differentiation by activating Wnt and BMP signaling in bone marrow stromal cells (28, 36, 37), although the mechanisms by which it regulates Wnt pathway activation are complex and uncertain (38).Sclerostin is a secreted glycoprotein that inhibits Wnt pathway activation by binding to LRP5/6, a widely expressed Wnt receptor (7, 8), reducing bone formation (39, 40). Sclerostin is secreted by osteocytes (8, 41), negatively regulating bone formation by inhibiting the differentiation of osteoblasts (41, 42). SOSTi (Romosozumab) is a humanized monoclonal antibody that binds sclerostin, preventing binding to LRP5/6 and increasing Wnt pathway activation and bone formation (43). It is FDA-approved for the treatment of osteoporosis (20, 44) and has activity in rodents in addition to humans (45, 46).The discovery that osteolectin is a bone-forming growth factor raises the question of whether it mediates the effects of PTH or SOSTi on osteogenesis.     

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.     

Nitric oxide resets kisspeptin-excited GnRH neurons via PIP2 replenishment

Fertility relies upon pulsatile release of gonadotropin-releasing hormone (GnRH) that drives pulsatile luteinizing hormone secretion. Kisspeptin (KP) neurons in the arcuate nucleus are at the center of the GnRH pulse generation and the steroid feedback control of GnRH secretion. However, KP evokes a long-lasting response in GnRH neurons that is hard to reconcile with periodic GnRH activity required to drive GnRH pulses. Using calcium imaging, we show that 1) the tetrodotoxin-insensitive calcium response evoked by KP relies upon the ongoing activity of canonical transient receptor potential channels maintaining voltage-gated calcium channels in an activated state, 2) the duration of the calcium response is determined by the rate of resynthesis of phosphatidylinositol 4,5-bisphosphate (PIP₂), and 3) nitric oxide terminates the calcium response by facilitating the resynthesis of PIP₂ via the canonical pathway guanylyl cyclase/3′,5′-cyclic guanosine monophosphate/protein kinase G. In addition, our data indicate that exposure to nitric oxide after KP facilitates the calcium response to a subsequent KP application. This effect was replicated using electrophysiology on GnRH neurons in acute brain slices. The interplay between KP and nitric oxide signaling provides a mechanism for modulation of the refractory period of GnRH neurons after KP exposure and places nitric oxide as an important component for tonic GnRH neuronal pulses.

Gonadotropin-releasing hormone (GnRH)-secreting neurons integrate multiple physiological and environmental cues and translate them into GnRH secretory patterns, to drive gonadotrophs, which in turn control the gonads. In both sexes, tonic GnRH pulses regulate gametogenesis and steroidogenesis via luteinizing hormone (LH) and follicle-stimulating pulses. Females require a GnRH surge to trigger LH surge and ovulation [reviewed in (1, 2)]. However, disruption of the normal pulsatile GnRH secretion impairs fertility in both sexes (3). Although insufficient for optimal reproductive health (4), the direct action of kisspeptins (KP) on GnRH neurons, via the KP receptor Kiss1r, is required for fertility (4–6). KP neurons play a critical regulatory role in the hypothalamic–pituitary–gonadal axis, including puberty onset (7–9), the preovulatory GnRH surge (5, 9, 10), and tonic GnRH pulses (6, 11). In rodents, two KP neuronal subpopulations exist with distinct functions: the anteroventral periventricular nucleus (AVPV) subpopulation, linked to puberty onset [reviewed in (12)] and preovulatory surge [reviewed in (13)], and the arcuate nucleus (ARC) subpopulation, also known as Kisspeptin-Neurokinin B-Dynorphin (KNDy) neurons, linked to tonic pulses (reviewed in ref. 14).Exogenous KP at the GnRH cell body evokes a long-lasting increase in intracellular calcium levels ([Ca²⁺]_i) (15), often leading to the summation of individual oscillations into [Ca²⁺]_i plateaus (15, 16). This observation is in agreement with an increase in electrical activity where GnRH neurons in acute slices go from burst firing to tonic firing after KP application (17–19). One could argue that this prolonged response is an artifact of the exogenously applied KP. However, endogenously released KP by AVPV stimulation also evokes a long-lasting increase in firing rate (20). Under normal conditions, [Ca²⁺]_i oscillations are driven by bursts of action potentials (AP) (21, 22). Yet, AP are not necessary for the KP-evoked [Ca²⁺]_i response to occur, as it is driven by multiple effectors including transient receptor potential-canonical channels (TRPC), voltage-gated calcium channels (VGCC), and inositol 1,4,5-trisphosphate receptors (InsP3R) (15, 16, 19, 23). Thus, the versatility of Kiss1r signaling pathway underlies the functionality of KP projections along GnRH neuron processes (24), with KP locally applied on nerve terminals also evoking a long-lasting increase in [Ca²⁺]_i (16).While the long-lasting KP response is suitable to trigger the preovulatory surge, it seems incompatible with the KNDy model for tonic pulses. GnRH and LH pulses occur every ∼20 min in ovariectomized mice (25). Indeed, the KNDy model provides on-/off- signals for KNDy neurons, neurokinin B and dynorphin respectively, and an on-signal for GnRH neurons, KP. However, this model lacks an off-signal for GnRH neurons. KNDy neurons trigger GnRH/LH pulses via Kiss1r (26), but the “extinction” of KNDy neurons by dynorphin is not obligatory for the termination of GnRH/LH pulses in rodents (27, 28), and dynorphin is lacking in human KP-neurokinin neurons (29).In fact, how the response in firing and [Ca²⁺]_i to KP relate to GnRH secretion is unknown. In acute coronal brain slices, the KP-evoked increase in firing at the cell body cannot be repeated (19, 30). In contrast, in acute sagittal brain slices, using fast scan cyclic voltammetry, the KP-evoked GnRH secretion from cells in the preoptic area (POA) and fibers in the median eminence (ME) can be triggered repeatedly (31). The difference in repeatability at the cell body is puzzling. One explanation could be a technical consequence of brain slicing or conventional patch clamp. Another explanation could be a dissociation between firing and [Ca²⁺]_i during the second KP application. In fact, the common feature of the KP-evoked increase in [Ca²⁺]_i and GnRH secretion, at the GnRH neuron cell body and nerve terminal, is that it is AP-independent (15, 16, 31). The current study uses calcium imaging and electrophysiology to address the mechanisms that allow 1) [Ca²⁺]_i in GnRH neurons to return to baseline after KP stimulation, and 2) GnRH neurons to respond to a second KP stimulation (i.e., repeatability) and shows nitric oxide as an important component for tonic GnRH neuronal pulses.     

Impaired TRPV4-eNOS signaling in trabecular meshwork elevates intraocular pressure in glaucoma

Primary Open Angle Glaucoma (POAG) is the most common form of glaucoma that leads to irreversible vision loss. Dysfunction of trabecular meshwork (TM) tissue, a major regulator of aqueous humor (AH) outflow resistance, is associated with intraocular pressure (IOP) elevation in POAG. However, the underlying pathological mechanisms of TM dysfunction in POAG remain elusive. In this regard, transient receptor potential vanilloid 4 (TRPV4) cation channels are known to be important Ca²⁺ entry pathways in multiple cell types. Here, we provide direct evidence supporting Ca²⁺ entry through TRPV4 channels in human TM cells and show that TRPV4 channels in TM cells can be activated by increased fluid flow/shear stress. TM-specific TRPV4 channel knockout in mice elevated IOP, supporting a crucial role for TRPV4 channels in IOP regulation. Pharmacological activation of TRPV4 channels in mouse eyes also improved AH outflow facility and lowered IOP. Importantly, TRPV4 channels activated endothelial nitric oxide synthase (eNOS) in TM cells, and loss of eNOS abrogated TRPV4-induced lowering of IOP. Remarkably, TRPV4-eNOS signaling was significantly more pronounced in TM cells compared to Schlemm’s canal cells. Furthermore, glaucomatous human TM cells show impaired activity of TRPV4 channels and disrupted TRPV4-eNOS signaling. Flow/shear stress activation of TRPV4 channels and subsequent NO release were also impaired in glaucomatous primary human TM cells. Together, our studies demonstrate a central role for TRPV4-eNOS signaling in IOP regulation. Our results also provide evidence that impaired TRPV4 channel activity in TM cells contributes to TM dysfunction and elevated IOP in glaucoma.

Glaucoma is a heterogenic group of multifactorial neurodegenerative diseases characterized by progressive optic neuropathy. It is the leading cause of irreversible vision loss with more than 70 million people affected worldwide (1), and the prevalence is estimated to increase to 111.6 million by the year 2040 (2). Primary open angle glaucoma (POAG) is the most common form of glaucoma, accounting for ∼70% of all cases (1). POAG is characterized by progressive loss of retinal ganglion cell axons that leads to an irreversible loss of vision (1, 3). Elevated intraocular pressure (IOP) is a major, and the only treatable, risk factor associated with POAG (4). The trabecular meshwork (TM), a molecular sieve-like structure, maintains homeostatic control over IOP by constantly adjusting the resistance to aqueous humor (AH) outflow. In POAG, there is increased resistance to AH outflow, elevating IOP (5). This increase in AH outflow resistance is associated with dysfunction of the TM (6–8).The TM has an intrinsic ability to sense the AH flow and regulate outflow facility to maintain IOP homeostasis (6), although the precise flow-sensing mechanisms in TM cells are unclear. In this regard, transient receptor potential vanilloid 4 (TRPV4) cation channels have emerged as a major flow-activated Ca²⁺ entry pathway in multiple cell types (9–12). Upon activation, TRPV4 channels allow localized Ca²⁺ influx (termed as TRPV4 sparklets), which influences a variety of cellular homeostatic processes (13, 14). TRPV4 sparklets are spatially restricted signals with a spatial spread (maximum width at half maximal amplitude) of ∼11 microns (13). Treatment with a selective TRPV4 channel activator GSK1016790A (GSK101) lowered IOP in rats and mice (15). Furthermore, baseline IOP was higher in global TRPV4^−/− mice compared to their wild-type (WT) littermates (15). However, the exact cell type responsible for these IOP-lowering effects is not known. Previous studies have shown that TRPV4 channel protein is expressed in TM cells and tissues (15, 16). The physiological roles of TRPV4 channels in TM cells (TRPV4_TM) and downstream signaling mechanisms remain unknown. TM constitutively expresses Ca²⁺-sensitive endothelial nitric oxide synthase (eNOS) (17), a known regulator of outflow facility and IOP (18–22). In vascular endothelial cells, TRPV4 channels are important regulators of eNOS activity (23–26). We, therefore, hypothesized that TRPV4_TM-eNOS signaling promotes outflow facility and reduces IOP.Glaucoma-associated pathological changes are known to impair physiological function of TM (8). One of the hallmarks of the glaucomatous TM is its inability to maintain normal IOP and AH outflow resistance (6). Here, we postulated that impaired TRPV4_TM-eNOS signaling contributes to TM dysfunction and elevated IOP in glaucoma. In this report, our studies in human TM cells and TM tissue showed shear stress–mediated activation of TRPV4-eNOS signaling. Moreover, reduced AH outflow and elevated IOPs were observed in TM-specific TRPV4^−/− (TRPV4_TM^−/−) mice and eNOS^−/− mice. Importantly, TRPV4_TM activity and shear stress–mediated activation of TRPV4_TM-eNOS signaling are compromised in human glaucomatous TM cells. Our results provide direct evidence for a physiological role of TRPV4_TM-eNOS signaling and indicate that impaired TRPV4_TM-eNOS signaling may underlie TM dysfunction and IOP dysregulation in glaucoma.     

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.     

Neuromodulator release in neurons requires two functionally redundant calcium sensors

Neuropeptides and neurotrophic factors secreted from dense core vesicles (DCVs) control many brain functions, but the calcium sensors that trigger their secretion remain unknown. Here, we show that in mouse hippocampal neurons, DCV fusion is strongly and equally reduced in synaptotagmin-1 (Syt1)- or Syt7-deficient neurons, but combined Syt1/Syt7 deficiency did not reduce fusion further. Cross-rescue, expression of Syt1 in Syt7-deficient neurons, or vice versa, completely restored fusion. Hence, both sensors are rate limiting, operating in a single pathway. Overexpression of either sensor in wild-type neurons confirmed this and increased fusion. Syt1 traveled with DCVs and was present on fusing DCVs, but Syt7 supported fusion largely from other locations. Finally, the duration of single DCV fusion events was reduced in Syt1-deficient but not Syt7-deficient neurons. In conclusion, two functionally redundant calcium sensors drive neuromodulator secretion in an expression-dependent manner. In addition, Syt1 has a unique role in regulating fusion pore duration.

To date, over 100 genes encoding neuropeptides and neurotrophic factors, together referred to as neuromodulators, are identified, and most neurons express neuromodulators and neuromodulator receptors (1). Neuromodulators travel through neurons in dense core vesicles (DCVs) and, upon secretion, regulate neuronal excitability, synaptic plasticity, and neurite outgrowth (2–4). Dysregulation of DCV secretion is linked to many brain disorders (5–7). However, the molecular mechanisms that regulate neuromodulator secretion remain largely elusive.Neuromodulator secretion, like neurotransmitter secretion from synaptic vesicles (SVs), is tightly controlled by Ca²⁺. The Ca²⁺ sensors that regulate secretion have been described for other secretory pathways but not for DCV exocytosis in neurons. Synaptotagmin (Syt) and Doc2a/b are good candidate sensors due to their interaction with SNARE complexes, phospholipids, and Ca²⁺ (8–11). The Syt family consists of 17 paralogs (12, 13). Eight show Ca²⁺-dependent lipid binding: Syt1 to 3, Syt5 to 7, and Syt9 and 10 (14, 15). Syt1 mediates synchronous SV fusion (8), consistent with its low Ca²⁺-dependent lipid affinity (15, 16) and fast Ca²⁺/membrane dissociation kinetics (16, 17). Syt1 is also required for the fast fusion in chromaffin cells (18) and fast striatal dopamine release (19). Synaptotagmin-7 (Syt7), in contrast, drives asynchronous SV fusion (20), in line with its a higher Ca²⁺ affinity (15) and slower dissociation kinetics (16). Syt7 is also a major calcium sensor for neuroendocrine secretion (21) and secretion in pancreatic cells (22–24). Other sensors include Syt4, which negatively regulates brain-derived neurothropic factor (25) and oxytocin release (26), in line with its Ca²⁺ independency. Syt9 regulates hormone secretion in the anterior pituitary (27) and, together with Syt1, secretion from PC12 cells (28, 29). Syt10 controls growth factor secretion (30). However, Syt9 and Syt10 expression is highly restricted in the brain (31–33). Hence, the calcium sensors for neuronal DCV fusion remain largely elusive. Because DCVs are generally not located close to Ca²⁺ channels (34), we hypothesized that DCV fusion is triggered by high-affinity Ca²⁺ sensors. Because of their important roles in vesicle secretion, their Ca²⁺ binding ability, and their high expression levels in the brain (20, 31, 35–38), we addressed the roles of Doc2a/b, Syt1, and Syt7 in neuronal DCV fusion.In this study, we used primary Doc2a/b-, Syt1-, and Syt7-null (knockout, KO) neurons expressing DCV fusion reporters (34, 39–41) with single-vesicle resolution. We show that both Syt1 and Syt7, but not Doc2a/b, are required for ∼60 to 90% of DCV fusion events. Deficiency of both Syt1 and Syt7 did not produce an additive effect, suggesting they function in the same pathway. Syt1 overexpression (Syt1-OE) rescued DCV fusion in Syt7-null neurons, and vice versa, indicating that the two proteins compensate for each other in DCV secretion. Moreover, overexpression of Syt1 or Syt7 in wild-type (WT) neurons increased DCV fusion, suggesting they are both rate limiting for DCV secretion. We conclude that DCV fusion requires two calcium sensors, Syt1 and Syt7, that act in a single/serial pathway and that both sensors regulate fusion in a rate-limiting and dose-dependent manner.     

Elementary mechanisms of calmodulin regulation of NaV1.5 producing divergent arrhythmogenic phenotypes

In cardiomyocytes, Na_V1.5 channels mediate initiation and fast propagation of action potentials. The Ca²⁺-binding protein calmodulin (CaM) serves as a de facto subunit of Na_V1.5. Genetic studies and atomic structures suggest that this interaction is pathophysiologically critical, as human mutations within the Na_V1.5 carboxy-terminus that disrupt CaM binding are linked to distinct forms of life-threatening arrhythmias, including long QT syndrome 3, a “gain-of-function” defect, and Brugada syndrome, a “loss-of-function” phenotype. Yet, how a common disruption in CaM binding engenders divergent effects on Na_V1.5 gating is not fully understood, though vital for elucidating arrhythmogenic mechanisms and for developing new therapies. Here, using extensive single-channel analysis, we find that the disruption of Ca²⁺-free CaM preassociation with Na_V1.5 exerts two disparate effects: 1) a decrease in the peak open probability and 2) an increase in persistent Na_V openings. Mechanistically, these effects arise from a CaM-dependent switch in the Na_V inactivation mechanism. Specifically, CaM-bound channels preferentially inactivate from the open state, while those devoid of CaM exhibit enhanced closed-state inactivation. Further enriching this scheme, for certain mutant Na_V1.5, local Ca²⁺ fluctuations elicit a rapid recruitment of CaM that reverses the increase in persistent Na current, a factor that may promote beat-to-beat variability in late Na current. In all, these findings identify the elementary mechanism of CaM regulation of Na_V1.5 and, in so doing, unravel a noncanonical role for CaM in tuning ion channel gating. Furthermore, our results furnish an in-depth molecular framework for understanding complex arrhythmogenic phenotypes of Na_V1.5 channelopathies.

Voltage-gated sodium channels (Na_V) are responsible for the initiation and spatial propagation of action potentials (AP) in excitable cells (1, 2). Na_V channels undergo rapid activation that underlie the AP upstroke while ensuing inactivation permits AP repolarization. The Na_V1.5 channel constitutes the predominant isoform in cardiomyocytes, whose pore-forming α-subunit is encoded by the SCN5A gene. Na_V1.5 dysfunction underlies diverse forms of cardiac disease including cardiomyopathies, arrhythmias, and sudden death (3–6). Human mutations in Na_V1.5 are associated with two forms of inherited arrhythmias–congenital long QT syndrome 3 (LQTS3) and Brugada syndrome (BrS) (7). LQTS3 stems from delayed or incomplete inactivation of Na_V1.5 that causes persistent Na influx that prolongs AP repolarization—a “gain-of-function” phenotype (7–9). BrS predisposes patients to sudden death and is associated with a reduction in the peak Na current that may slow cardiac conduction or cause region-specific repolarization differences—a “loss-of-function” phenotype (10, 11). Genetic studies have identified an expanding array of mutations in multiple Na_V1.5 domains, including the channel carboxy-terminus (CT) that is a hotspot for mutations linked to both LQTS3 and BrS (12, 13). This domain interacts with the Ca²⁺-binding protein calmodulin (CaM), suggesting that altered CaM regulation of Na_V1.5 may be a common pathophysiological mechanism (12, 14–16). More broadly, human mutations in the homologous regions of neuronal Na_V1.1 (17, 18), Na_V1.2 (19, 20), and Na_V1.6 (21) as well as skeletal muscle Na_V1.4 (22) are linked to varied clinical phenotypes including epilepsy, autism spectrum disorder, neurodevelopmental delay, and myotonia (23). Taken together, a common Na_V mechanistic deficit—defective CaM regulation—may underlie these diverse diseases.CaM regulation of Na_V channels is complex, isoform specific, and mediated by multiple interfaces within the channel (14–16). The Na_V CT consists of a dual vestigial EF hand segment and a canonical CaM-binding “IQ” (isoleucine–glutamine) domain (24, 25) (Fig. 1A). The IQ domain of nearly all Na_V channels binds to both Ca²⁺-free CaM (apoCaM) and Ca²⁺/CaM, similar to Ca_V channels (26–31). As CaM is typically a Ca²⁺-dependent regulator, much attention has been focused on elucidating Ca²⁺-dependent changes in Na_V gating. For skeletal muscle Na_V1.4, transient elevation in cytosolic Ca²⁺ causes a dynamic reduction in the peak current, a process reminiscent of Ca²⁺/CaM-dependent inactivation of Ca_V channels (32). Cardiac Na_V1.5 by comparison exhibits no dynamic effect of Ca²⁺ on the peak current (32–34). Instead, sustained Ca²⁺ elevation has been shown to elicit a depolarizing shift in Na_V1.5 steady-state inactivation (SSI or h_∞), although the magnitude and the presence of a shift have been debated (32, 35). Additional CaM-binding sites have been identified in the channel amino terminus domain (36) and the III-IV linker near the isoleucine, phenylalanine, and methionine (IFM) motif that is well recognized for its role in fast inactivation (35, 37). However, recent cryogenic electron microscopy structures, biochemical, and functional analyses suggest that both the III-IV linker and the Domain IV voltage-sensing domain might instead interact with the channel CT in a state-dependent manner (38–43).Open in a separate windowFig. 1.Absence of dynamic Ca²⁺/CaM effects on WT Na_V1.5 SSI. (A, Left) Structure of Na_V1.5 transmembrane domain (6UZ3) (70) juxtaposed with that of Na_V1.5 CT–apoCaM complex (4OVN) (28). (Right) Arrhythmia-linked CT mutations highlighted in Na_V1.5 CT–apoCaM structure (LQTS3, blue; BrS, magenta; mixed syndrome, purple). (B) Dynamic Ca²⁺-dependent changes in Na_V1.5 SSI probed using Ca²⁺ photouncaging. Na currents specifying h_∞ at ∼100 nM (Left) and ∼4 μM Ca²⁺ step (Right). (C) Population data for Na_V1.5 SSI under low (black, Left) versus high (red, Right) intracellular Ca²⁺ reveal no differences (P = 0.55, paired t test). Dots and bars are mean ± SEM (n = 8 cells). (D) FRET two-hybrid analysis of Cerulean-tagged apoCaM interaction with various Venus-tagged Na_V1.5 CT (WT, black; IQ/AA, red; S[1904]L, blue). Each dot is FRET efficiency measured from a single cell. Solid line fits show 1:1 binding isotherm.Beyond Ca²⁺-dependent effects, the loss of apoCaM binding to the Na_V1.5 IQ domain increases persistent current (34, 44), suggesting that apoCaM itself may be pathophysiologically relevant. Indeed, Na_V1.5 mutations in the apoCaM-binding interface are associated with LQTS3 and atrial fibrillation (7), as well as a loss-of-function BrS phenotype and a mixed-syndrome phenotype whereby some patients present with BrS while others with LQTS3 (Fig. 1A) (13, 45). How alterations in CaM binding paradoxically elicits both gain-of-function and loss-of-function effects is not fully understood, though important to delineate pathophysiological mechanisms and for personalized therapies.Here, using single- and multichannel recordings, we show that apoCaM binding elicits two distinct effects on Na_V1.5 gating: 1) an increase in the peak channel open probability (P_O/peak) and 2) a reduction in the normalized persistent channel open probability (R_persist), consistent with previous studies (34, 44). The two effects may explain how mixed-syndrome mutations in the Na_V1.5 CT produce either BrS or LQTS3 phenotypes. On one hand, the loss of apoCaM association may diminish P_O/peak and induce BrS by shunting cardiac AP. On the other hand, increased R_persist may prevent normal AP repolarization, resulting in LQTS3. Analysis of elementary mechanisms suggests that these changes relate to a switch in the state dependence of channel inactivation. Furthermore, dynamic changes in Ca²⁺ can inhibit persistent current for certain mutant Na_V1.5 owing to enhanced Ca²⁺/CaM binding that occurs over the timescale of a cardiac AP. This effect may result in beat-to-beat variability in persistent Na current for some mutations. In all, these findings explain how a common deficit in CaM binding can contribute to distinct arrhythmogenic mechanisms.     

Cellular pathways of calcium transport and concentration toward mineral formation in sea urchin larvae

Sea urchin larvae have an endoskeleton consisting of two calcitic spicules. The primary mesenchyme cells (PMCs) are the cells that are responsible for spicule formation. PMCs endocytose sea water from the larval internal body cavity into a network of vacuoles and vesicles, where calcium ions are concentrated until they precipitate in the form of amorphous calcium carbonate (ACC). The mineral is subsequently transferred to the syncytium, where the spicule forms. Using cryo-soft X-ray microscopy we imaged intracellular calcium-containing particles in the PMCs and acquired Ca-L_2,3 X-ray absorption near-edge spectra of these Ca-rich particles. Using the prepeak/main peak (L₂′/ L₂) intensity ratio, which reflects the atomic order in the first Ca coordination shell, we determined the state of the calcium ions in each particle. The concentration of Ca in each of the particles was also determined by the integrated area in the main Ca absorption peak. We observed about 700 Ca-rich particles with order parameters, L₂′/ L₂, ranging from solution to hydrated and anhydrous ACC, and with concentrations ranging between 1 and 15 M. We conclude that in each cell the calcium ions exist in a continuum of states. This implies that most, but not all, water is expelled from the particles. This cellular process of calcium concentration may represent a widespread pathway in mineralizing organisms.

Calcium ions play a critical role in many cellular processes. Calcium ions are messengers for a wide range of cellular activities, including fertilization, cell differentiation, secretion, muscle contraction, and programmed cell death (1, 2). Therefore, the concentration of Ca²⁺ in the cytosol is highly regulated at around 100 to 200 nM during resting stages (3–5). Ca²⁺ homeostasis is mediated by Ca-binding proteins and different organelles in the cell, including the endoplasmic reticulum (ER) and the mitochondria that serve as significant Ca²⁺ stores and as signal generators (6–8).Calcium ions are an important component of many biominerals such as bones, teeth, shells, and spines (9). In comparison to Ca²⁺ signaling, which requires small amounts of Ca²⁺, biomineralization processes require massive sequestering and transport of ions from the environment and/or from the food to the site of mineralization. The sequestered ions can reach the mineralization site as solutes but can also concentrate intracellularly inside vesicles, where they precipitate to form highly disordered mineral phases (10–15). In the latter case, specialized cells take up the ions through ion pumps, ion channels, or by endocytosis of extracellular fluid and process the calcium ions until export to the final mineralization location (16–19).In this study, we evaluate the contents of calcium-containing vesicles in primary mesenchyme cells (PMCs), which are involved in the formation of the calcitic skeleton of the sea urchin larvae. In this way, we obtain insights into how calcium ions, extracted from the environment, are concentrated and stored for spicule formation.Paracentrotus lividus sea urchin embryos form an endoskeleton consisting of two calcitic spicules within 72 h after fertilization (20, 21). The source of the calcium ions is the surrounding sea water, whereas the carbonate ions are thought to originate from both sea water and metabolic processes in the embryo (22, 23). Sea water enters the embryonic body cavity (blastocoel) through the permeable ectoderm cell layer of the embryo (24). Endocytosis of sea water and blastocoel fluid into PMCs as well as endothelial and epithelial cells (25, 26) was tracked by labeling sea water with calcein, a fluorescent calcium-binding and membrane-impermeable dye (17, 26–28). The endocytosed fluid in the PMCs was observed to form a network of vacuoles and vesicles (26). Beniash et al. observed electron-dense granules of sizes 0.5 to 1.5 µm in PMCs, which are composed of amorphous calcium carbonate (ACC) (10). Intracellular vesicles of similar size were observed Vidavsky et al., using cryo-scanning electron microscopy (SEM) and air-SEM, containing calcium carbonate deposits composed of nanoparticles 20 to 30 nm in size (25). Ca deposits within the same range of sizes were also observed in the rough ER of PMCs (29).The intracellularly produced ACC is subsequently exported to the growing spicule, where it partially transforms into calcite through secondary nucleation (30–33). The location and distribution of ACC and calcite in the spicule were studied by using extended X-ray absorption fine structure, X-ray absorption near-edge spectroscopy (XANES), and photoelectron emission microscopy (PEEM) (30, 34, 35). Three distinct mineral phases were identified in the spicule: hydrated ACC (ACC*H₂O), anhydrous ACC, and crystalline calcite (34). According to theoretical simulations of Rez and Blackwell (36), the two different amorphous phases arise from different levels of ordering of the oxygen coordination polyhedron around calcium. The coordination polyhedron becomes more ordered as the transformation to the crystalline phase progresses. The phase information contained in the XANES spectra is exploited here to characterize the mineral phases in the intracellular vesicles.Cryo-soft X-ray transmission microscopy (cryo-SXM) is an attractive technique for tomography and spectromicroscopy of biological samples in the hydrated state (37–39). Imaging is performed in the “water window” interval of X-ray energies, namely between the carbon (C) K-edge (284 eV) and the oxygen (O) K-edge (543 eV). As a result, in the “water window” C is highly absorbing, whereas O, and thus H₂O, is almost transparent. Subsequently, carbon-rich moieties such as lipid bodies, proteins, and membranes appear dark in transmission, whereas the water rich cytosol appears lighter (40). The Ca L_2,3-absorption edge between ∼346 and 356 eV also resides in the “water window” (41). Therefore, imaging across this absorption edge enables the characterization of Ca-rich moieties in whole, hydrated cells. Each pixel of the same field of view, imaged as the energy is varied across the Ca L_2,3-edge, can be assigned an individual Ca L_2,3-edge XANES spectrum. This technique was applied to the calcifying coccolithophorid alga (42, 43). In this study, we use cryo-SXM and XANES to locate and characterize both the phases and the concentrations of Ca-rich bodies in sea urchin larval cells.     

The mitochondrial thioredoxin reductase system (TrxR2) in vascular endothelium controls peroxynitrite levels and tissue integrity

The mitochondrial thioredoxin/peroxiredoxin system encompasses NADPH, thioredoxin reductase 2 (TrxR2), thioredoxin 2, and peroxiredoxins 3 and 5 (Prx3 and Prx5) and is crucial to regulate cell redox homeostasis via the efficient catabolism of peroxides (TrxR2 and Trxrd2 refer to the mitochondrial thioredoxin reductase protein and gene, respectively). Here, we report that endothelial TrxR2 controls both the steady-state concentration of peroxynitrite, the product of the reaction of superoxide radical and nitric oxide, and the integrity of the vascular system. Mice with endothelial deletion of the Trxrd2 gene develop increased vascular stiffness and hypertrophy of the vascular wall. Furthermore, they suffer from renal abnormalities, including thickening of the Bowman’s capsule, glomerulosclerosis, and functional alterations. Mechanistically, we show that loss of Trxrd2 results in enhanced peroxynitrite steady-state levels in both vascular endothelial cells and vessels by using a highly sensitive redox probe, fluorescein-boronate. High steady-state peroxynitrite levels were further found to coincide with elevated protein tyrosine nitration in renal tissue and a substantial change of the redox state of Prx3 toward the oxidized protein, even though glutaredoxin 2 (Grx2) expression increased in parallel. Additional studies using a mitochondria-specific fluorescence probe (MitoPY1) in vessels revealed that enhanced peroxynitrite levels are indeed generated in mitochondria. Treatment with Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin [Mn(III)TMPyP], a peroxynitrite-decomposition catalyst, blunted intravascular formation of peroxynitrite. Our data provide compelling evidence for a yet-unrecognized role of TrxR2 in balancing the nitric oxide/peroxynitrite ratio in endothelial cells in vivo and thus establish a link between enhanced mitochondrial peroxynitrite and disruption of vascular integrity.

Selenocysteine-containing mitochondrial thioredoxin reductase (TrxR2) is the key regulator of the thioredoxin system and essential for mitochondrial redox homeostasis (1–3). This system constitutes a primary defense against peroxides produced in mitochondria, such as hydrogen peroxide and peroxynitrite (4). TrxR2 is necessary for maintaining thioredoxin 2 (Trx2) in its reduced state by using electrons from NADPH. In turn, Trx2 is a cofactor of mitochondrially localized peroxiredoxin 3 (Prx3) and peroxiredoxin 5 (Prx5) that reduce H₂O₂ and peroxynitrite^* generated by mitochondrial metabolism (1, 2, 4–7). The reaction rate constants of H₂O₂ and peroxynitrite with the fast reacting thiols of both Prx3 and Prx5 have been compiled recently (4). Notably, Prx3, the only peroxiredoxin which is exclusively localized in mitochondria (8, 9)^†, was reported to accept electrons also from glutaredoxin 2 (Grx2) (10). Among the biologically recognized functions, TrxR2 plays essential roles in hematopoiesis, heart development, and heart function (11, 12). In a previous study, we could show that loss of TrxR2 in endothelial cells (ECs) attenuated vascular remodeling processes following ischemic events and led to a prothrombotic and proinflammatory endothelium (13). While these studies pointed toward an important role of TrxR2 in the cardiovascular system, the underlying biochemical mechanisms, particularly in vivo, have remained largely unclear. Unlike many other cell types in the body, ECs are unique as they are constantly exposed to changing biochemical and mechanical stimuli. Furthermore, they do not just separate the circulating blood and the vascular smooth muscle cells but also have to fulfill a wide range of physiological tasks, including regulation of vascular tone, cellular adhesion, thromboresistance, smooth muscle cell proliferation, and inflammoresistance (14, 15). One of the most significant biomolecules that is involved in vascular endothelial function is the free radical nitric oxide (·NO) (16, 17). ·NO is not only an important vasodilator but also has antiinflammatory properties by inhibiting the synthesis and expression of cytokines and adhesion molecules that attract inflammatory cells and facilitate their entrance into the vessel wall (18, 19). Furthermore, ·NO suppresses platelet aggregation (20), vascular smooth muscle cell migration, and proliferation (21). Consequentially, a decreased synthesis of ·NO, as well as an enhanced inactivation, can result in endothelial dysfunction (22–24). Oxidative stress contributes to this phenomenon, starting with the diffusion-controlled reaction of ·NO with superoxide radical (O₂^·−), which shortens the biological half-life and compromises the signaling actions of ·NO (25–28). In addition, the oxidative inactivation of ·NO by O₂^·− yields a powerful oxidizing and nitrating species, peroxynitrite anion (28, 29). Moreover, peroxynitrite itself leads to uncoupling of endothelial nitric oxide synthase to become a dysfunctional O₂^·− and peroxynitrite-producing enzyme that additionally contributes to cellular oxidative stress (30, 31). A sustained overload of O₂^·− and, peroxynitrite combined with insufficient levels of ·NO may contribute to a switch of the endothelium from the quiescent stage toward an activated one, setting up a vicious cycle, causing endothelial dysfunction and inflammation. ECs are equipped with a number of antioxidant systems known to be potentially protective against vascular oxidative stress that, however, under persistent pathological stimuli, may become overwhelmed.In this context, it is increasingly recognized that mitochondrial-derived O₂^·− and the disruption of mitochondrial redox homeostasis contribute to alter the signaling actions of ·NO in vascular biology (32–34). Besides the mitochondrial thioredoxin system, a number of professional redox systems, including mitochondrial superoxide dismutase (MnSOD/SOD2), glutathione peroxidase, and glutathione reductase, are involved in maintaining mitochondrial redox homeostasis.However, the specific roles of these enzymes in the context of endothelial dysfunction are far from being understood. The fact that genetically modified mouse models revealed that many of the different antioxidant enzymes are indispensable for murine development (35–38) impedes further insights into their role for vascular homeostasis, and, to our knowledge, only a limited number of EC-specific transgenic mice have yet been described in this context (39–42). Interestingly, Trx2 transgenic mice that overexpress Trx2 specifically in the endothelium demonstrated an increased ·NO bioavailability and EC function, decreased oxidative stress, and reduced propensity to atheroma formation (3, 43).The aim of this study was to analyze the impact of endothelial TrxR2 on vascular homeostasis (in vitro, ex vivo, and in vivo), focusing on its role on mitochondrial peroxynitrite catabolism. The data support that enhanced mitochondrial steady-state levels of peroxynitrite in vascular ECs are connected with disruption of redox homeostasis and vascular structural and functional integrity.     

Coal fly ash is a major carbon flux in the Chang Jiang (Yangtze River) basin

Fly ash—the residuum of coal burning—contains a considerable amount of fossilized particulate organic carbon (FOC_ash) that remains after high-temperature combustion. Fly ash leaks into natural environments and participates in the contemporary carbon cycle, but its reactivity and flux remained poorly understood. We characterized FOC_ash in the Chang Jiang (Yangtze River) basin, China, and quantified the riverine FOC_ash fluxes. Using Raman spectral analysis, ramped pyrolysis oxidation, and chemical oxidation, we found that FOC_ash is highly recalcitrant and unreactive, whereas shale-derived FOC (FOC_rock) was much more labile and easily oxidized. By combining mass balance calculations and other estimates of fly ash input to rivers, we estimated that the flux of FOC_ash carried by the Chang Jiang was 0.21 to 0.42 Mt C⋅y⁻¹ in 2007 to 2008—an amount equivalent to 37 to 72% of the total riverine FOC export. We attributed such high flux to the combination of increasing coal combustion that enhances FOC_ash production and the massive construction of dams in the basin that reduces the flux of FOC_rock eroded from upstream mountainous areas. Using global ash data, a first-order estimate suggests that FOC_ash makes up to 16% of the present-day global riverine FOC flux to the oceans. This reflects a substantial impact of anthropogenic activities on the fluxes and burial of fossil organic carbon that has been made less reactive than the rocks from which it was derived.

Fossil particulate organic carbon (FOC) is a geologically stable form of carbon that was produced by the ancient biosphere and then buried and stored in the lithosphere; it is a key player in the geological carbon cycle (1–7). Uplift and erosion liberate FOC from bedrock, delivering it to the surficial carbon cycle. Some is oxidized in sediment routing systems, but a portion escapes and can be transported by rivers to the oceans (5, 8–10). Oxidation of FOC represents a long-term atmospheric carbon source and O₂ sink, whereas the reburial of FOC in sedimentary basins has no long-term net effect on atmospheric CO₂ and O₂ (1, 9, 11). Exhumation and erosion of bedrock provide a natural source of FOC (2, 8), which we refer to as FOC_rock. Human activities have introduced another form of FOC from the mining and combustion of coal. Burning coal emits CO₂ to the atmosphere but also leaves behind solid waste that contains substantial amounts of organic carbon (OC) that survives high-temperature combustion (12–14). This fossil-fuel-sourced carbon represents a poorly understood anthropogenic flux in the global carbon cycle; it also provides a major source of black carbon, which is a severe pollutant and climate-forcing agent (12–15).Previous studies sought to quantify black carbon in different terrestrial and marine environments and to distinguish fossil fuel versus forest fire sources (14–18). In this study, we focused on fly ash—the material left from incomplete coal combustion. As a major fossil fuel, coal supplies around 30% of global primary energy consumption (19, 20). Despite efforts to capture and utilize fly ash, a fraction enters soils and rivers; the resulting fossil OC from fly ash (FOC_ash) has become a measurable part of the contemporary carbon cycle (14). FOC_ash is also referred to as “unburned carbon” in fly ash (21–25); it provides a useful measure of combustion efficiency and the quality of fly ash as a building material (e.g., in concrete) (23–26). Industrial standards of FOC_ash content in fly ash have been established for material quality assurance (23, 24, 26, 27). However, the characteristics and fluxes of FOC_ash released to the environment, and how these compare to FOC_rock from bedrock erosion, remain less well understood.To fill this knowledge gap, we examined the Chang Jiang (Yangtze River) basin in China—a system that allowed us to evaluate the influence of FOC_ash on the carbon cycle at continental scales. In the 2000s, China became the largest coal-consuming country in the world, with an annual coal consumption of over 2,500 Mt, equating to ∼50% of worldwide consumption (19, 20, 28). Coal contributed over 60% of China’s national primary energy consumption through the 2000s. A significant portion of this coal (approximately one-third) was consumed in the Chang Jiang (CJ) basin, where China’s most populated and economically developed areas are located (29). Significant amounts of fly ash and FOC_ash continue to be produced and consumed in the CJ basin. To determine the human-induced FOC_ash flux, we investigated the FOC_ash cycle in the CJ basin. We characterized OC in a series of samples including fly ash, bedrock sedimentary shale, and river sediment through multiple geochemical analyses. We then estimated the CJ-exported FOC_ash flux and evaluated how human activities modulated FOC transfer at basin scales. We found that in the CJ basin, coal combustion and dam construction have conspired to boost the FOC_ash flux and reduce the FOC_rock flux carried by the CJ; as a result, these two fluxes converged over an interval of 60 y.     

Ammonium transporter expression in sperm of the disease vector Aedes aegypti mosquito influences male fertility

The ammonium transporter (AMT)/methylammonium permease (MEP)/Rhesus glycoprotein (Rh) family of ammonia (NH₃/NH₄⁺) transporters has been identified in organisms from all domains of life. In animals, fundamental roles for AMT and Rh proteins in the specific transport of ammonia across biological membranes to mitigate ammonia toxicity and aid in osmoregulation, acid–base balance, and excretion have been well documented. Here, we observed enriched Amt (AeAmt1) mRNA levels within reproductive organs of the arboviral vector mosquito, Aedes aegypti, prompting us to explore the role of AMTs in reproduction. We show that AeAmt1 is localized to sperm flagella during all stages of spermiogenesis and spermatogenesis in male testes. AeAmt1 expression in sperm flagella persists in spermatozoa that navigate the female reproductive tract following insemination and are stored within the spermathecae, as well as throughout sperm migration along the spermathecal ducts during ovulation to fertilize the descending egg. We demonstrate that RNA interference (RNAi)-mediated AeAmt1 protein knockdown leads to significant reductions (∼40%) of spermatozoa stored in seminal vesicles of males, resulting in decreased egg viability when these males inseminate nonmated females. We suggest that AeAmt1 function in spermatozoa is to protect against ammonia toxicity based on our observations of high NH₄⁺ levels in the densely packed spermathecae of mated females. The presence of AMT proteins, in addition to Rh proteins, across insect taxa may indicate a conserved function for AMTs in sperm viability and reproduction in general.

Ammonium transporters (AMTs), methylammonium permeases (MEPs), and Rhesus glycoproteins (Rh proteins) comprise a protein family with three clades, and homologs from each have been identified in virtually all domains of life (1). AMT proteins were first identified in plants (2) with the simultaneous discovery of MEP proteins in fungi (3), followed by Rh proteins in humans (4). Ammonia (NH₃/NH₄⁺) is vital for growth in plants and microorganisms and is retained in some animals for use as an osmolyte (5, 6), for buoyancy (7, 8), and for those lacking sufficient dietary nitrogen (9). In the majority of animals, however, ammonia is the toxic by-product of amino acid and nucleic acid metabolism and, accordingly, requires efficient mechanisms for its regulation, transport, and excretion (10–13). AMT, MEP, and Rh proteins are responsible for the selective movement of ammonia (NH₃) or ammonium (NH₄⁺) across biological membranes, a process that all organisms require. Unlike their vertebrate, bacterial, and fungal counterparts which function as putative NH₃ gas channels (14–18), a myriad of evidence suggests that plant AMT proteins and closely related members in some animals are functionally distinct and facilitate electrogenic ammonium (NH₄⁺) transport (17, 19–22). In contrast to vertebrates which only possess Rh proteins (23), many invertebrates are unique in that they express both AMT and Rh proteins, sometimes in the same cell (24–28). Among insects, the presence of both AMT and Rh proteins has been described in Drosophila melanogaster (29, 30) and mosquitoes that vector disease-causing pathogens, Anopheles gambiae (22, 31) and Aedes aegypti (32, 33). It is unclear whether, in these instances, AMT and Rh proteins can functionally substitute for one another, but in the anal papillae of A. aegypti larvae, knockdown of either Amt or Rh proteins causes decreases in ammonia transport, suggesting that they do not (32–34). To date, studies on ammonia transporter (AMT and Rh) function in insects have focused on ammonia sensing and tasting in sensory structures (22, 30, 31, 35), ammonia detoxification and acid–base balance in muscle, digestive, and excretory organs (15, 36), and ammonia excretion in a variety of organs involved in ion and water homeostasis (9, 24, 32–34).A. aegypti is the primary vector for the transmission of the human arboviral diseases Zika, yellow fever, chikungunya, and dengue virus, which are of global health concern due to rapid increases in the geographical distribution of this species, presently at its highest ever (37, 38). In light of the well-documented evolution of insecticide resistance in mosquitoes (39–42), more recent methods to control disease transmission such as the sterile insect technique (43), transinfection and sterilization of mosquitoes with the bacterium Wolbachia (44), and targeted genome editing rendering adult males sterile (45) have proven effective. These methods take advantage of various aspects of mosquito reproductive biology; however, an understanding of male reproductive biology and the male contributions to female reproductive processes is still in its infancy (46). Here, we describe the expression of an A. aegypti ammonium transporter (AeAmt1) in the sperm during all stages of spermatogenesis, spermiogenesis, and egg fertilization, which is critical for fertility.     

Structure and activity of SLAC1 channels for stomatal signaling in leaves

Stomata in leaves regulate gas exchange between the plant and its atmosphere. Various environmental stimuli elicit abscisic acid (ABA); ABA leads to phosphoactivation of slow anion channel 1 (SLAC1); SLAC1 activity reduces turgor pressure in aperture-defining guard cells; and stomatal closure ensues. We used electrophysiology for functional characterizations of Arabidopsis thaliana SLAC1 (AtSLAC1) and cryoelectron microscopy (cryo-EM) for structural analysis of Brachypodium distachyon SLAC1 (BdSLAC1), at 2.97-Å resolution. We identified 14 phosphorylation sites in AtSLAC1 and showed nearly 330-fold channel-activity enhancement with 4 to 6 of these phosphorylated. Seven SLAC1-conserved arginines are poised in BdSLAC1 for regulatory interaction with the N-terminal extension. This BdSLAC1 structure has its pores closed, in a basal state, spring loaded by phenylalanyl residues in high-energy conformations. SLAC1 phosphorylation fine-tunes an equilibrium between basal and activated SLAC1 trimers, thereby controlling the degree of stomatal opening.

The stomatal pore, formed by a pair of specialized guard cells in plant leaves, serves as the gateway for water transpiration and atmospheric CO₂ influx for photosynthesis (1, 2). These pores need to be tightly controlled, as inadequate CO₂ intake is harmful and excessive water loss is devastating for plants (3). The guard cells respond to a wide range of environmental stimuli, including high CO₂ levels, O₃, low air humidity, and drought, and transduce these signals into appropriate turgor pressure changes that can adjust stomatal pore aperture (4, 5). It is well established that turgor pressure of guard cells is regulated by ion transport across the membrane, notably anions and potassium ions (6–8).The phytohormone abscisic acid (ABA) plays a central role in controlling stomatal closure via activation of a complex signaling pathway that is mediated by receptors, kinase/phosphatases, and ion channels (5, 9). It has been known that the activation of a slow anion current in guard cells is a key event leading to stomatal closure (10–13). The corresponding gene, slow anion channel 1 (SLAC1), was discovered in genetic screens for O₃ or CO₂ sensitivity in Arabidopsis; when this particular channel was mutated out, the mutants showed impaired response to stimuli (14–16).Arabidopsis thaliana SLAC1 (AtSLAC1) contains 556 amino acid (aa) residues, with a conserved transmembrane domain (TMD) between hydrophilic tails at both the N and C termini (15). Within the ABA signaling pathway, SLAC1 channel activity is controlled by a protein kinase-phosphatase pair (OST1/ABI1) (17–19). In the absence of ABA, OST1 kinase is bound and inhibited by ABI1 phosphatase (20–22). Under drought condition, ABA is accumulated into guard cells and perceived by the PYR1 receptor (23, 24). This resulting hormone–receptor complex then binds to phosphatase ABI1 to form a ternary hormone–receptor–phosphatase complex (25–28), which activates OST1 kinase, and leads to phosphorylation of SLAC1 (17, 18). Subsequently, SLAC1 releases anions (Cl⁻ and NO₃⁻) from guard cells, leading to membrane depolarization. Depolarization in turn activates potassium channels to release K⁺ via K⁺ channels and drives water out of the cell, decreasing guard cell turgor to close down the stomatal pore (8).It has been shown that SLAC1 activation is due to phosphorylation at its N terminus (17, 18, 29–31). The phosphorylation of S120 by OST1 is essential but not sufficient for SLAC1 activation monitored in Xenopus oocytes (18). S59 has been shown to be the target for both OST1 and other kinases (29, 31). In another phosphoproteomic analysis, using the fragment SLAC1¹⁻¹⁸⁶ as substrate, S86 and S113 were also shown to be phosphorylated by OST1 (32). OST1 also phosphorylates SLAC1 at the C terminus (17), with T513 identified as a potential phosphorylation site for SLAC1 activation (31). Although multiple phosphorylation sites have been proposed, the underlying mechanism for SLAC1 activation remains elusive.Our previous study on a SLAC1 bacterial homolog from Haemophilus influenzae (HiTehA) revealed a superfamily of trimeric anion channels (33) and found each protomer gated by a highly conserved phenylalanine. We observed an unusual high-energy rotameric conformation for this gating phenylalanine (F262 in HiTehA), and further mutational study on the corresponding site in AtSLAC1 (F450) suggested a unique mechanism for channel activation (33). Nevertheless, since HiTehA shares a mere 19% in sequence identity with AtSLAC1 and lacks the substantial N and C termini that are essential for channel activation, questions remain about SLAC1 structure and its regulation by kinase phosphorylation (7).Here we describe the cryoelectron microscopy (cryo-EM) structure of SLAC1 from Brachypodium distachyon (BdSLAC1) at 2.97-Å resolution, and we further characterize the channel activity on Arabidopsis SLAC1. BdSLAC1 shares 63% overall sequence identity with AtSLAC1, with 73% for the pore-forming TMD and 48% for the regulatory N and C termini. As for the bacterial homolog, each BdSLAC1 TMD comprises five tandemly repeated helical hairpins that surround a transmembrane pore in each protomer of the trimeric assembly. The SLAC1 pores are electropositive, consistent with its function as an anion channel. The regulatory N-terminal domain (190 aa) and C-terminal domain (65 aa) are flexibly associated and not discernible in the structure.We employed a SLAC1::OST1 fusion design for examining SLAC1 phosphorylation, whereby we systematically characterized critical sites responsible for channel activation. Inspired by the structure, we also conducted mutagenesis on the highly conserved phenylalanine residues related to channel gating. Altogether, our structural and functional analyses provide insights into SLAC1 gating and activity modulation. These findings allow us to propose a mechanism for finely tuned SLAC1 control of the stomatal pore in response to environmental stimuli.     

Direct and continuous generation of pure acetic acid solutions via electrocatalytic carbon monoxide reduction

Electrochemical CO₂ or CO reduction to high-value C₂₊ liquid fuels is desirable, but its practical application is challenged by impurities from cogenerated liquid products and solutes in liquid electrolytes, which necessitates cost- and energy-intensive downstream separation processes. By coupling rational designs in a Cu catalyst and porous solid electrolyte (PSE) reactor, here we demonstrate a direct and continuous generation of pure acetic acid solutions via electrochemical CO reduction. With optimized edge-to-surface ratio, the Cu nanocube catalyst presents an unprecedented acetate performance in neutral pH with other liquid products greatly suppressed, delivering a maximal acetate Faradaic efficiency of 43%, partial current of 200 mA⋅cm⁻², ultrahigh relative purity of up to 98 wt%, and excellent stability of over 150 h continuous operation. Density functional theory simulations reveal the role of stepped sites along the cube edge in promoting the acetate pathway. Additionally, a PSE layer, other than a conventional liquid electrolyte, was designed to separate cathode and anode for efficient ion conductions, while not introducing any impurity ions into generated liquid fuels. Pure acetic acid solutions, with concentrations up to 2 wt% (0.33 M), can be continuously produced by employing the acetate-selective Cu catalyst in our PSE reactor.

Electrochemically reducing carbon dioxide (CO₂) or carbon monoxide (CO) to commodity fuels or chemicals, with the input of green and economical renewable electricity, has become an alternative route to traditional chemical engineering processes (1–7). To date, a variety of catalysts have been demonstrated to reduce CO₂ into C₁ products (CO, formate, etc.) with high Faradaic efficiencies (FEs) (6, 8–14); however, the critical step of C-C coupling toward high-value C₂₊ products was rarely observed on catalysts other than Cu (15–20). Due to its proper binding strength with *CO (19, 21, 22), Cu has shown the capability of catalyzing the coupling of two carbons or more in CO₂/CO reduction reactions (CO₂RR/CORR), which, however, typically suffers from large overpotential and low selectivity (23–25). In previous studies on Cu catalysts, tremendous efforts have been focused on how to promote C-C coupling toward high selectivity of C₂₊ products, including facet engineering (26–28), grain boundary engineering (29, 30), surface modifications (31–33), etc. For example, recent studies on facet dependence of C-C coupling have shown that the C₂₊ selectivity could be improved on Cu(100) compared to other facets due to more stabilized *OCCO intermediates (26, 34). While the overall C₂₊ selectivity is constantly improved by researchers in this field, the generated products are typically a mixture of different C₂₊ compounds, which subsequently necessitates energy- and cost-intensive downstream product separation and purification processes (35–37). A solution to continuously obtain a highly pure, single C₂₊ product stream, which can be directly fed into practical applications, will impose a significant scientific and economic impact but has been rarely studied before.Ethylene, ethanol, and acetate are the three major C₂ products that have been widely reported in CO₂RR/CORR on Cu materials (16–18, 38–44). Compared to gas-phase products, liquid products show significant advantages due to their high energy densities and ease of storage and distribution (45). Nevertheless, the generation of a high-purity C₂ liquid fuel via electrochemical CO₂RR/CORR is challenging due to the involvement of two types of impurities. First, in previous studies of Cu catalysts, while the C₂ product can be dramatically increased by strategies such as exposing (100) facets, ethanol and acetic acid usually share the same potential window and are cogenerated simultaneously, making it difficult to obtain a single C₂ liquid product (29, 46, 47); Second, in traditional H-cell or flow-cell reactors, the generated liquid products were mixed with solutes in liquid electrolytes such as KOH or KHCO₃, which requires extra separation and concentration processes to recover pure liquid fuel solutions in practical applications (37, 48, 49). An integration of rational design in both catalytic material and reactor, for high single-C₂ selectivity and electrolyte-free liquid fuel output, respectively, therefore becomes the key to achieve the goal of pure C₂ liquid fuels.Here we report the continuous generation of pure acetic acid solutions via CORR on Cu nanocube (NC) catalysts in a porous solid electrolyte (PSE) reactor. By flexibly tuning the edge to (100) surface ratio, the medium-size Cu NC catalyst exhibits a maximal acetate Faradaic efficiency of 43% with a partial current density of ∼200 mA⋅cm⁻² in neutral pH, setting up a different acetate performance benchmark (18, 40, 41). More importantly, the selectivity of other liquid products (ethanol and trace amount of n-propanol) was suppressed to below 2%, suggesting an ultrahigh acetic acid relative purity of up to 98 wt%. An impressive stability was demonstrated by a continuous operation of CORR under 150 mA⋅cm⁻² current for over 150 h with negligible degradation in selectivity and activity. This acetate-selective Cu NC catalyst was successfully employed into our PSE reactor for the continuous generation of electrolyte-free, pure acetic acid solutions, with overall current of up to 1 A⋅cm⁻² and acetic acid relative purity as high as 96 wt%. Different from the traditional liquid electrolyte, our PSE layer can efficiently conduct ions while it does not introduce any impurity ions into the generated liquid products. Density functional theory (DFT) calculations suggest that the stepped edge sites on the Cu NC readily dissociate *OCCOH into *CCO and *OH, which is a key step in promoting the formation of acetate. We propose that the large NC has a low edge-to-facet ratio and therefore cogenerates considerable ethanol and ethylene instead of acetate, while a small NC provides less active (100) area for C-C coupling and therefore predominantly catalyzes the hydrogen evolution reaction (HER) instead of yielding C₂ products.