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.     

Reductions in NO2 burden over north equatorial Africa from decline in biomass burning in spite of growing fossil fuel use, 2005 to 2017

Socioeconomic development in low- and middle-income countries has been accompanied by increased emissions of air pollutants, such as nitrogen oxides [NO_x: nitrogen dioxide (NO₂) + nitric oxide (NO)], which affect human health. In sub-Saharan Africa, fossil fuel combustion has nearly doubled since 2000. At the same time, landscape biomass burning—another important NO_x source—has declined in north equatorial Africa, attributed to changes in climate and anthropogenic fire management. Here, we use satellite observations of tropospheric NO₂ vertical column densities (VCDs) and burned area to identify NO₂ trends and drivers over Africa. Across the northern ecosystems where biomass burning occurs—home to hundreds of millions of people—mean annual tropospheric NO₂ VCDs decreased by 4.5% from 2005 through 2017 during the dry season of November through February. Reductions in burned area explained the majority of variation in NO₂ VCDs, though changes in fossil fuel emissions also explained some variation. Over Africa’s biomass burning regions, raising mean GDP density (USD⋅km⁻²) above its lowest levels is associated with lower NO₂ VCDs during the dry season, suggesting that economic development mitigates net NO₂ emissions during these highly polluted months. In contrast to the traditional notion that socioeconomic development increases air pollutant concentrations in low- and middle-income nations, our results suggest that countries in Africa’s northern biomass-burning region are following a different pathway during the fire season, resulting in potential air quality benefits. However, these benefits may be lost with increasing fossil fuel use and are absent during the rainy season.

Socioeconomic development and population growth in low- and middle-income countries have been widely associated with increased environmental degradation, including rapid increases in emissions of air pollutants (1–3). In contrast, in countries with a high per capita gross domestic product (GDP), various socioeconomic, institutional, and regulatory factors often cause economic growth to be accompanied by reductions of some pollutant emissions, though these emissions may simply be outsourced to lower income countries (4). The relationship between income level and environmental pressure—known as the Environmental Kuznets Curve—has often been conceptualized as an inverted U-shaped curve, but a wide array of functional relationships is possible (3). For emissions of air pollutants, the relationship has generally been described as an inverted U-shaped curve, though carbon dioxide generally does not follow such a curve (3, 5). Some researchers argue that low- and middle-income countries can mitigate or shorten the period of rapid emissions growth that tends to accompany socioeconomic development for at least some pollutants (4). Africa, and sub-Saharan Africa in particular, is characterized by countries with low but growing per capita GDP and rapid population growth, which have been linked to increases in emissions of carbon dioxide and particulate matter (6). As these countries continue their trajectories of economic development, emissions of air pollutants from fossil fuel and biofuel combustion are expected to experience explosive growth (7).Nitrogen dioxide (NO₂) is a reactive gas and air pollutant with a lifetime in the atmosphere on the order of hours (8). In the atmosphere, NO₂ interconverts rapidly with nitric oxide (NO), and the two species are collectively referred to as NO_x. NO₂ itself is toxic, is regulated by the US Environmental Protection Agency, and has been associated with premature mortality and asthma [though its direct effects on health are not clear (9) and it may instead function as a proxy for other pollutants, such as ozone and aerosols that have direct health and mortality impacts (10)]. NO_x is also a key precursor to the formation of tropospheric ozone (O₃), which is damaging to both crop productivity and human health; anthropogenic O₃ contributes to roughly half a million premature deaths annually, of which nearly 20,000 are in Africa (11). In addition, NO_x is involved in reactions with atmospheric ammonia (NH₃) to form nitrate aerosols, which contribute to particulate matter pollution (12) as well as in reactions with volatile organic compounds (VOCs), which form organic nitrates (13). Because of the short lifetime of NO₂, and because it can function as an indicator for other pollutants, it can serve as an indicator of overall changes in air quality.NO and NO₂ are emitted from a variety of natural and anthropogenic sources. Fossil fuel combustion and anthropogenic alterations to soils through fertilization or livestock management are the primary sources of NO_x in many parts of the world. In sub-Saharan Africa (excluding South Africa), fossil fuel combustion and fertilizer use has been considerably lower than elsewhere, and natural soils and biomass burning have historically been more important sources (14). This is true even in Nigeria (15), which experiences substantial emissions of VOCs from the oil and gas industry (16). NO_x emissions from Lagos have been shown to be either lower than (15) or comparable to other megacities (17), and NO₂ concentrations are generally low during the rainy season, but air quality can become heavily degraded during the biomass burning season (15, 18). However, fossil fuel combustion in the region nearly doubled between 2000 and 2016 (19) and associated emissions of NO_x are projected to increase sixfold by 2030 in the absence of regulation, as compared to 2005 levels (7).This increase in fossil fuel combustion is occurring against the backdrop of Africa’s unique, fire-prone savanna ecosystems, home to 70% of the global area burned each year (20). Biomass burning in Africa is estimated to be responsible for NO_x emissions of roughly 4 Tg N⋅yr⁻¹, equivalent to about half of all NO_x emissions for the continent (21), and one third to half of NO_x emissions from biomass burning globally (21–23). The majority of biomass burning in Africa occurs in northern and southern bands of savanna, savanna-forest mosaic, and woodland ecoregions, with a seasonality that follows the migration of the intertropical convergence zone.The early part of the 21st century has been accompanied by a global decline in burned area, with some of the largest declines occurring in Africa’s northern fire band (24). Some of the burned area decline in the northern fire band can be attributed to changes in precipitation that, in turn, affect the quantity and moisture content of available fuels (24–26). However, active anthropogenic suppression of fire has also played an important role (24, 25). Burning is thought to be used as a management strategy—among other uses, humans ignite fires to mineralize nutrients, improve grazing, and reduce fuel loads and the potential for large, uncontrolled fires (27). Increased population density and the introduction of agricultural land into African savanna landscapes—reflecting socioeconomic transitions from traditional nomadic pastoralist lifestyles (28)—have been associated with a sharp decrease in burned area as people either reduce ignition or suppress fires to protect villages and farms, with a reduction in the amount of pasture area to be maintained (25).Unfortunately, sub-Saharan Africa remains a severely understudied region—for example, agricultural soil NO fluxes have only been measured directly for two sites (29, 30), and surface air quality monitoring is extremely limited compared to other parts of the world (31). Remote sensing products provide an important tool for filling some of these data gaps. The short NO₂ lifetime in the planetary boundary layer makes it possible to use satellite observations to directly evaluate emissions sources, especially in regions with high temperatures, which tend to shorten the NO₂ lifetime, and in relatively polluted regions, where total column densities and surface emissions are highly correlated (ref. 8 and references therein). Although recent remote sensing work has evaluated long-term trends in NO₂ concentrations around the world, recent trends in the biomass burning region of northern Africa have not been explicitly evaluated, and the relative impacts of socioeconomic development—the possibility of reduced NO_x emissions because of anthropogenic fire suppression and of increasing NO_x emissions from growing fossil fuel use—remain unknown. In general, studies on global trends in NO₂ tend not to focus on Africa, likely because the regions with the highest NO₂ concentrations are in China, Europe, and the United States (e.g., refs. 1, 21). Some earlier studies observed a decline in NO₂ VCDs over north equatorial Africa (32, 33), but others did not (34). These and other large-scale studies (e.g., refs. 8, 34, 35) did not identify mechanisms for the observed NO₂ dynamics, but rather focused on understanding anthropogenic influences on trends in other regions.Indoor air pollution from biomass combustion for fuel is an important health concern (36). We do not focus on this source. Biofuel combustion is responsible for emissions of 0.6 Tg NO annually across all of Africa (37), which is less than 10% of the magnitude of landscape biomass burning emissions estimated by the Global Fire Emissions Database version 4s [GFED4s (38)] and represents a much smaller proportion of NO_x emissions from landscape biomass burning regions during the dry season.Here, we use observations of NO₂ by the Ozone Monitoring Instrument [OMI (39)] and burned area from the Moderate Resolution Imaging Spectroradiometer [MODIS (40)] to demonstrate that the recent decline in burned area in the productive savannas of north equatorial Africa—home to over 275 million people—is associated with large declines in tropospheric NO₂ VCDs during the biomass burning season from 2005 through 2017, though positive trends explained in part by increasing fossil fuel combustion were observed in other seasons, especially over Nigeria.     

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.     

First-in-class humanized FSH blocking antibody targets bone and fat

Blocking the action of FSH genetically or pharmacologically in mice reduces body fat, lowers serum cholesterol, and increases bone mass, making an anti-FSH agent a potential therapeutic for three global epidemics: obesity, osteoporosis, and hypercholesterolemia. Here, we report the generation, structure, and function of a first-in-class, fully humanized, epitope-specific FSH blocking antibody with a K_D of 7 nM. Protein thermal shift, molecular dynamics, and fine mapping of the FSH–FSH receptor interface confirm stable binding of the Fab domain to two of five receptor-interacting residues of the FSHβ subunit, which is sufficient to block its interaction with the FSH receptor. In doing so, the humanized antibody profoundly inhibited FSH action in cell-based assays, a prelude to further preclinical and clinical testing.

Obesity and osteoporosis affect nearly 650 million and 200 million people worldwide, respectively (1, 2). Yet the armamentarium for preventing and treating these disorders remains limited, particularly when compared with public health epidemics of a similar magnitude. It has also become increasingly clear that obesity and osteoporosis track together clinically. First, body mass does not protect against bone loss; instead, obesity can be permissive to osteoporosis and a high fracture risk (3, 4). Furthermore, the menopausal transition marks the onset not only of rapid bone loss, but also of visceral obesity and dysregulated energy balance (5–9). These physiologic aberrations have been attributed traditionally to a decline in serum estrogen, although, during the perimenopause—2 to 3 y prior to the last menstrual period—serum estrogen is within the normal range, while FSH levels rise to compensate for reduced ovarian reserve (10–12). In our view, therefore, the early skeletal and metabolic derangements cannot conceivably be explained solely by declining estrogen (13, 14).The past decade has shown that pituitary hormones can act directly on the skeleton and other tissues, a paradigm shift that is in stark contrast to previously held views on their sole regulation of endocrine targets (15–25). We and others have shown that FSH can bypass the ovary to act on G_i-coupled FSH receptors (FSHRs) on osteoclasts to stimulate bone resorption and inhibit bone formation (26, 27). This mechanism, which could underscore the bone loss during early menopause, is testified by the strong correlations between serum FSH, bone turnover, and bone mineral density (7–9, 14, 16, 26). Likewise, activating polymorphisms in the FSHR in postmenopausal women are linked to a high bone turnover and reduced bone mass (27). It therefore made biological and clinical sense to inhibit FSH action during this period to prevent bone loss.Toward this goal, we generated murine polyclonal and monoclonal antibodies to a 13-amino-acid–long binding epitope of FSHβ (28–31). The mouse and human FSHβ epitopes differ by just two amino acids; hence, blocking antibodies to the human epitope showed efficacy in mice (28). The antibodies displayed two sets of actions: they attenuated the loss of bone after ovariectomy by inhibiting bone resorption and stimulating bone formation and displayed profound effects on body composition and energy metabolism (28, 29, 31). Most notably, in a series of contemporaneously reproduced experiments, we (M.Z. and C.J.R.) found that FSH blockade reduced body fat, triggered adipocyte beiging, and increased thermogenesis in models of obesity, notably post ovariectomy and after high-fat diet (29). Our findings have been further confirmed independently by two groups who used a FSHβ–GST fusion protein or tandem repeats of the 13-amino-acid–long FSHβ epitope for studies on bone and fat, respectively (32, 33). Consistent with the mouse data, inhibiting FSH secretion using a GnRH agonist in prostate cancer patients resulted in low body fat compared with orchiectomy, wherein FSH levels are high (34). This interventional clinical trial provides evidence for a therapeutic benefit of reducing FSH levels on body fat in people. There is also new evidence that FSH blockade lowers serum cholesterol (35, 36).Thus, both emerging and validated datasets on the antiobesity, osteoprotective, and lipid-lowering actions of FSH blockade in mice and in humans prompted our current attempt to develop and characterize an array of fully humanized FSH-blocking antibodies for future testing in people. Here, we report that our lead first-in-class humanized antibody, Hu6, and two related molecules, Hu26 and Hu28, bind human FSH with a high affinity (K_Ds <10 nM), block the binding of FSH on the human FSHR, and inhibit FSH action in functional cell-based assays.     

Physical mixing in coastal waters controls and decouples nitrification via biomass dilution

Nitrification is a central process of the aquatic nitrogen cycle that controls the supply of nitrate used in other key processes, such as phytoplankton growth and denitrification. Through time series observation and modeling of a seasonally stratified, eutrophic coastal basin, we demonstrate that physical dilution of nitrifying microorganisms by water column mixing can delay and decouple nitrification. The findings are based on a 4-y, weekly time series in the subsurface water of Bedford Basin, Nova Scotia, Canada, that included measurement of functional (amoA) and phylogenetic (16S rRNA) marker genes. In years with colder winters, more intense winter mixing resulted in strong dilution of resident nitrifiers in subsurface water, delaying nitrification for weeks to months despite availability of ammonium and oxygen. Delayed regrowth of nitrifiers also led to transient accumulation of nitrite (3 to 8 μmol · kg_sw⁻¹) due to decoupling of ammonia and nitrite oxidation. Nitrite accumulation was enhanced by ammonia-oxidizing bacteria (Nitrosomonadaceae) with fast enzyme kinetics, which temporarily outcompeted the ammonia-oxidizing archaea (Nitrosopumilus) that dominated under more stable conditions. The study reveals how physical mixing can drive seasonal and interannual variations in nitrification through control of microbial biomass and diversity. Variable, mixing-induced effects on functionally specialized microbial communities are likely relevant to biogeochemical transformation rates in other seasonally stratified water columns. The detailed study reveals a complex mechanism through which weather and climate variability impacts nitrogen speciation, with implications for coastal ecosystem productivity. It also emphasizes the value of high-frequency, multiparameter time series for identifying complex controls of biogeochemical processes in aquatic systems.

Coastal waters worldwide are subject to inputs of anthropogenic nitrogen (N) which impact primary production and marine ecosystems through alteration of both the quantity and speciation (oxidized/reduced and inorganic/organic) of N (1, 2). These are key controls on phytoplankton growth and community composition, altering patterns and magnitude of primary production, causing eutrophication and harmful algae blooms and impacting carbon flux (2–4). For example, relative increase in ammonium over nitrate supply can shift phytoplankton community compositions toward smaller species with the potential to cause harmful algal blooms and reduced productivity (2). The speciation of N also exerts control on key microbial N cycling pathways, including the fixed N removal processes anammox and denitrification. These pathways depend on the availability of oxidized forms of N that can be converted to N₂ and thereby removed from the pool of readily bioavailable N within ocean waters.It is therefore essential to understand the processes and environmental factors that control the speciation of dissolved inorganic nitrogen (DIN = NO₃⁻ + NO₂⁻ + NH₃/NH₄⁺) between oxidized (nitrate and nitrite) and reduced (ammonium/ammonia) forms as well as between organic and inorganic forms (2, 5). A central process controlling DIN speciation is nitrification, the two-step oxidation of ammonia (NH₃) to nitrate (NO₃⁻) via nitrite (NO₂⁻). Ammonia-oxidizing organisms (AOO), either archaea (AOA) from the phylum Thaumarchaeota or bacteria (AOB), catalyze the oxidation of ammonia to nitrite:Ammonia oxidation AO: 2NH3+ 3O2→2NO2-+ 2H++ 2H2O.[1]AO kinetics differ between the two AOO groups, with higher maximum reaction velocities (V_max) and ammonium half-saturation constants (K_m) in AOB (6–8) compared to AOA (9–11). Nitrite-oxidizing bacteria (NOB) are responsible for the second step from nitrite to nitrate:Nitrite oxidation NO: 2NO2-+ O2→2NO3-.[2]Recently, an exception to the two-organism nitrification paradigm (12, 13) has been recognized through the discovery of complete ammonia oxidation to nitrate (“comammox”) by individual Nitrospirae bacteria (12, 13). This could play a role in coastal marine waters under some conditions (14).In the ocean, nitrification maxima typically occur at or below the base of the euphotic zone, spatially separated from photosynthetic primary production (10, 11, 15). However, vertical transport can supply products of nitrification to the euphotic zone (16, 17). Incomplete nitrification may therefore affect phototrophic communities, since the speciation of externally supplied N (e.g., ammonium versus nitrate) can significantly impact both the structure and productivity of phytoplankton (2).Accumulation of the intermediate product of nitrification, nitrite, has been documented in a wide range of marine systems, including at the base of the oceanic euphotic zone (18) and transiently in coastal bights, bays, and estuaries (19–23). In many cases, the presence of nitrite can be attributed to decoupling of AO and NO, which are usually tightly coupled despite ecophysiological differences between AOA and NOB (24). A large variety of environmental factors, including temperature and oxygen, have been associated with the decoupling of nitrification in marine systems (19, 20, 25, 26). Here, we describe nitrite accumulation arising from nitrifier regrowth following physical dilution, which might point to a role for nitrifier biomass in a more general mechanism for decoupling nitrification in seasonally stratified water columns.Physical transport is widely recognized to control phytoplankton growth in aquatic systems, for example through supply of nutrients to the euphotic zone from below (17, 27). Mixing also plays a role in the initiation of spring blooms according to the “dilution-recoupling” hypothesis (28), which posits that dilution of both phytoplankton and grazer biomass leads to fewer grazer–phytoplankton encounters. It has been shown that nitrification can be enhanced by the mixing of ammonium-rich waters into well-oxygenated waters (21, 29), whereas the mixing-induced transport of NOB biomass away from the depth of optimal growth at the base of the euphotic zone has recently been implicated as a factor explaining local nitrite accumulation (30). Here, we describe a different way in which mixing controls nitrification, whereby seasonal and interannual variations in mixing lead to temporally variable rates of nitrification as a consequence of nitrifier biomass dilution.High-frequency, long-term measurements of the physical and chemical environment along with the associated microorganisms have been shown to be a valuable tool set for determining environmental controls on microbial processes (17, 31–35). However, such time series are rare because of the sustained, multidisciplinary effort and teamwork they require.Here, we present results of such a time series–based study of nitrification within the bottom water (60 m) of Bedford Basin (BB), a eutrophic, anthropogenically impacted, fjord-like embayment located within the Halifax Regional Municipality on the Atlantic coast of Nova Scotia, Canada (see SI Appendix, SI Materials and Methods for more details). Restricted water exchange with the open ocean and annual cycles of stratification and winter mixing make BB a useful natural laboratory to study the relationship between microbial growth phases, geochemistry, and physical processes. Based on 4 y of weekly observations of ammonia monooxygenase subunit A (amoA) gene copy numbers (via qPCR), microbial community composition (16S ribosomal RNA [rRNA] gene amplicon sequencing), nutrient concentrations, and a biogeochemical model enhanced by functional gene modeling, we observed variable dilution of the nitrifier population following winter mixing events. We propose that intense winter mixing during cold winters flushes the resident nitrifier population from the basin bottom waters, resulting in a delay in nitrification and decoupling of AO and NO until the nitrifier community can reestablish. During warmer winters, when mixing is less intense, growth can keep pace with mixing and effectively prevent dilution.     

The evolution of red color vision is linked to coordinated rhodopsin tuning in lycaenid butterflies

Color vision has evolved multiple times in both vertebrates and invertebrates and is largely determined by the number and variation in spectral sensitivities of distinct opsin subclasses. However, because of the difficulty of expressing long-wavelength (LW) invertebrate opsins in vitro, our understanding of the molecular basis of functional shifts in opsin spectral sensitivities has been biased toward research primarily in vertebrates. This has restricted our ability to address whether invertebrate G_q protein-coupled opsins function in a novel or convergent way compared to vertebrate G_t opsins. Here we develop a robust heterologous expression system to purify invertebrate rhodopsins, identify specific amino acid changes responsible for adaptive spectral tuning, and pinpoint how molecular variation in invertebrate opsins underlie wavelength sensitivity shifts that enhance visual perception. By combining functional and optophysiological approaches, we disentangle the relative contributions of lateral filtering pigments from red-shifted LW and blue short-wavelength opsins expressed in distinct photoreceptor cells of individual ommatidia. We use in situ hybridization to visualize six ommatidial classes in the compound eye of a lycaenid butterfly with a four-opsin visual system. We show experimentally that certain key tuning residues underlying green spectral shifts in blue opsin paralogs have evolved repeatedly among short-wavelength opsin lineages. Taken together, our results demonstrate the interplay between regulatory and adaptive evolution at multiple G_q opsin loci, as well as how coordinated spectral shifts in LW and blue opsins can act together to enhance insect spectral sensitivity at blue and red wavelengths for visual performance adaptation.

Opsins belong to a diverse multigene family of G protein-coupled receptors that bind to a small nonprotein retinal moiety to form photosensitive rhodopsins and enable vision across animals (1–4). The tight relationship between opsin genotypes and spectral sensitivity phenotypes offers an ideal framework to analyze how specific molecular changes give rise to adaptations in visual behaviors (5). Notably, independent opsin gene gains and losses (6–13), genetic variation across opsins (14–16), spectral tuning mutations within opsins (17–21), and alterations in visual regulatory networks (22, 23) have contributed to opsin adaptation. Yet, the molecular and structural changes underlying the remarkable diversification of spectral sensitivity phenotypes identified in some invertebrates, including crustaceans and insects (24–27), are far less understood than those in vertebrate lineages (28–32).The diversity of opsin-based photoreceptors observed across animal visual systems is produced by distinct ciliary vertebrate c-opsin and invertebrate rhabdomeric based r-opsin subfamilies that mediate separate phototransduction cascades (31, 33–35). Vertebrate c-opsins function through the G protein transducing (G_t) signaling pathway, which activates cyclic nucleotide phosphodiesterase, ultimately resulting in a hyperpolarization response in photoreceptor cells through the opening of selective K⁺ channels (31, 36). By contrast, insect opsins transmit light stimuli through a G_q-type G protein (33, 37) with phosphoinositol (PLCβ) acting as an effector enzyme to achieve TRP channel depolarization in the invertebrate photoreceptor cell (34, 38).All vertebrate visual cone opsins derive from four gene families: short-wavelength-sensitive opsins SWS1 (or ultraviolet [UV]) with λ_max 344 to 445 nm and SWS2 with λ_max 400 to 470 nm, and longer-wavelength-sensitive opsins that specify the green MWS (or Rh2) pigments with λ_max 480 to 530 nm and red-sensitive LWS pigments with λ_max 500 to 570 nm (5, 30). Most birds and fish have retained the four ancestral opsin genes (39), with notable opsin expansions in cichlid fish opsins (23, 40), whereas SWS1 is extinct in monotremes, and SWS2 and M opsins are lost in marsupials and eutherian mammals (41). In primates, trichromatic vision is conferred through SWS1 (λ_max = 414 nm) and recent duplicate MWS (λ_max = 530 nm) and LWS opsins (λ_max = 560 nm) (42–44). In vertebrates, molecular evolutionary approaches and well-established in vitro opsin purification have identified the complex interplay between opsin duplications, regulatory and protein-coding mutations controlling opsin gene tuning, and spectral phenotypes notably in birds, fish, and mammals (45–47).Insect opsins are phylogenetically distinct but functionally analogous to those of vertebrates, and the ancestral opsin repertoire consists of three types of light-absorbing rhabdomeric G_q-type opsin specifying UV (350 nm), short-wavelength (blue, 440 nm) and long-wavelength pigments (LW, 530 nm) (48). Given the importance of color-guided behaviors and the remarkable photoreceptor spectral diversity observed in insects (26, 27), the dynamic opsin gene diversification found across lineages (Fig. 1) highlights their potentially central role in adaptation (27, 49, 50), yet the molecular basis of opsin functionality of rhabdomeric invertebrate G_q opsins remains understudied.Open in a separate windowFig. 1.Visual opsin gene evolution and spectral tuning mechanisms in insects. Visual opsin genes of the Atala hairstreak (E. atala, Lepidoptera, Lycaenidae) in comparison with those encoded in the genomes of diverse insects. The opsin types are highlighted in gray for UV, in blue for short wavelength (SW), and in green for long wavelength (LW). Numbers indicate multiple opsins, whereas no dot indicates gene loss. Colored circles indicate instances of shifted spectral sensitivities in at least one of the encoded opsins. The direction of shift is inferred from the opsin lambda max that departs from the typical range of absorbance in the opsin subfamily using wavelength boundaries for the various colors: UV <380 nm, violet 380 to 435 nm, blue 435 to 492 nm, green 492 to 530 nm, and red shifted >530 nm. Coleopteran lineages, and some hemipterans, lost the blue opsin locus and compensated for the loss of blue sensitivity via UV and/or LW gene duplications across lineages (11, 12). In butterflies, extended photosensitivity at short wavelengths is observed in Heliconius erato with two UV opsins at λ_max = 355 nm and 398 nm (10) and in P. rapae with two blue opsins with λ_max = 420 and 450 nm (17). A blue opsin duplication occurred independently in lycaenid butterflies (61). LW opsin duplications occurred independently in most major insect lineages (6, 16, 55) and confer a variable range of LW sensitivities with or without additional contributions from lateral filtering. In order to extend spectral sensitivity at longer wavelengths while sharpening blue acuity, some lycaenid butterflies have evolved a new color vision mechanism combining spectral shifts at a duplicate blue opsin and at the LW opsin. Images credit: Christopher Adams (illustrator).The recurrent evolution of red receptors in insects in particular suggests that perception of longer wavelengths can play an important role in the context of foraging, oviposition, and/or conspecific recognition (6, 27, 51–54). In butterflies, several mechanisms are likely to have provided extended spectral sensitivity to longer wavelengths. LW opsin duplications along with the evolution of lateral filtering between ommatidia has been demonstrated in two papilionids, Papilio xuthus (27) and Graphium sarpedon (55), as well as in a riodinid (Apodemia mormo) (6, 54). Lateral filtering pigments are relatively widespread across butterfly lineages, e.g., Heliconius (56), Pieris (57), Colias erate (58), and some moths [Adoxophyes orana (59) and Paysandisia archon (60)]. These pigments absorb short wavelengths and aid in shifting the sensitivity peak of green LW photoreceptors to longer wavelengths (27, 51, 56, 57, 61, 62). Despite creating distinct spectral types that can contribute to color vision, as identified in nymphalid (56), pierid (57), and lycaenid (62) species, all of which lack duplicated LW opsins (61, 63), lateral filtering alone cannot extend photoreceptor sensitivity toward the far red (700 to 750 nm) beyond the exponentially decaying long-wavelength rhodopsin absorbance spectrum (51). Thus, molecular variation of ancestral LW opsin genes is likely to have contributed an as yet underexplored mechanism to the diversification of long-wavelength photoreceptor spectral sensitivity. However, disentangling the relative contributions of lateral filtering and pure LW opsin properties has remained technically challenging using classical electrophysiological approaches (14, 64, although see, e.g., refs. 65, 66, 67) and has been limited by the lack of in vitro expression systems suitable for LW opsins.While opsin duplicates have been identified in numerous organisms, the spectral tuning mechanisms and interplay between new opsin photoreceptors in invertebrate visual system evolution are less well understood. Here we combine physiological, molecular, and heterologous approaches to start closing this gap in our knowledge of invertebrate G_q opsin evolution by investigating the functions, spectral tuning, and implications of evolving new combinations of short- and long-wavelength opsin types in lycaenid species. This butterfly group, comprising the famous blues, coppers, and hairstreaks, is the second largest family with about 5,200 (28%) of the some 18,770 described butterfly species (68). In light of their remarkable behavioral, ecological, and morphological diversity (69, 70), as well as pioneer studies in the Lycaena and Polyommatus genera supporting the rapid evolution of color vision in certain lineages (56, 61, 62), lycaenids provide an ideal candidate system for investigating opsin evolution and visual adaptations. Using the Atala hairstreak, Eumaeus atala, as a molecular and ecological model, we find coordinated spectral shifts at short- and long-wavelength G_q opsin loci and demonstrate that the combination of six ommatidial classes of photoreceptors in the compound eye uniquely extend spectral sensitivity at long wavelengths toward the far-red while concurrently sharpening acuity of multiple blue wavelengths. Together, these findings link the evolution of four-opsin visual systems to adaptation in the context of finely tuned color perception critical to the behavior of these butterflies.     

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.     

Chemokine CCL5 promotes robust optic nerve regeneration and mediates many of the effects of CNTF gene therapy

Ciliary neurotrophic factor (CNTF) is a leading therapeutic candidate for several ocular diseases and induces optic nerve regeneration in animal models. Paradoxically, however, although CNTF gene therapy promotes extensive regeneration, recombinant CNTF (rCNTF) has little effect. Because intraocular viral vectors induce inflammation, and because CNTF is an immune modulator, we investigated whether CNTF gene therapy acts indirectly through other immune mediators. The beneficial effects of CNTF gene therapy remained unchanged after deleting CNTF receptor alpha (CNTFRα) in retinal ganglion cells (RGCs), the projection neurons of the retina, but were diminished by depleting neutrophils or by genetically suppressing monocyte infiltration. CNTF gene therapy increased expression of C-C motif chemokine ligand 5 (CCL5) in immune cells and retinal glia, and recombinant CCL5 induced extensive axon regeneration. Conversely, CRISPR-mediated knockdown of the cognate receptor (CCR5) in RGCs or treating wild-type mice with a CCR5 antagonist repressed the effects of CNTF gene therapy. Thus, CCL5 is a previously unrecognized, potent activator of optic nerve regeneration and mediates many of the effects of CNTF gene therapy.

Like most pathways in the mature central nervous system (CNS), the optic nerve cannot regenerate once damaged due in part to cell-extrinsic suppressors of axon growth (1, 2) and the low intrinsic growth capacity of adult retinal ganglion cells (RGCs), the projection neurons of the eye (3–5). Consequently, traumatic or ischemic optic nerve injury or degenerative diseases such as glaucoma lead to irreversible visual losses. Experimentally, some degree of regeneration can be induced by intraocular inflammation or growth factors expressed by inflammatory cells (6–10), altering the cell-intrinsic growth potential of RGCs (3–5), enhancing physiological activity (11, 12), chelating free zinc (13, 14), and other manipulations (15–19). However, the extent of regeneration achieved to date remains modest, underlining the need for more effective therapies.Ciliary neurotrophic factor (CNTF) is a leading therapeutic candidate for glaucoma and other ocular diseases (20–23). Activation of the downstream signal transduction cascade requires CNTF to bind to CNTF receptor-α (CNTFRα) (24), which leads to recruitment of glycoprotein 130 (gp130) and leukemia inhibitory factor receptor-β (LIFRβ) to form a tripartite receptor complex (25). CNTFRα anchors to the plasma membrane through a glycosylphosphatidylinositol linkage (26) and can be released and become soluble through phospholipase C-mediated cleavage (27). CNTF has been reported to activate STAT3 phosphorylation in retinal neurons, including RGCs, and to promote survival, but it is unknown whether these effects are mediated by direct action of CNTF on RGCs via CNTFRα (28). Our previous studies showed that CNTF promotes axon outgrowth from neonate RGCs in culture (29) but fails to do so in cultured mature RGCs (8) or in vivo (6). Although some studies report that recombinant CNTF (rCNTF) can promote optic nerve regeneration (20, 30, 31), others find little or no effect unless SOCS3 (suppressor of cytokine signaling-3), an inhibitor of the Jak-STAT pathway, is deleted in RGCs (5, 6, 32). In contrast, multiple studies show that adeno-associated virus (AAV)-mediated expression of CNTF in RGCs induces strong regeneration (33–40). The basis for the discrepant effects of rCNTF and CNTF gene therapy is unknown but is of considerable interest in view of the many promising clinical and preclinical outcomes obtained with CNTF to date.Because intravitreal virus injections induce inflammation (41), we investigated the possibility that CNTF, a known immune modulator (42–44), might act by elevating expression of other immune-derived factors. We report here that the beneficial effects of CNTF gene therapy in fact require immune system activation and elevation of C-C motif chemokine ligand 5 (CCL5). Depletion of neutrophils, global knockout (KO) or RGC-selective deletion of the CCL5 receptor CCR5, or a CCR5 antagonist all suppress the effects of CNTF gene therapy, whereas recombinant CCL5 (rCCL5) promotes axon regeneration and increases RGC survival. These studies point to CCL5 as a potent monotherapy for optic nerve regeneration and to the possibility that other applications of CNTF and other forms of gene therapy might similarly act indirectly through other factors.     

A tandem activity-based sensing and labeling strategy enables imaging of transcellular hydrogen peroxide signaling

Reactive oxygen species (ROS) like hydrogen peroxide (H₂O₂) are transient species that have broad actions in signaling and stress, but spatioanatomical understanding of their biology remains insufficient. Here, we report a tandem activity-based sensing and labeling strategy for H₂O₂ imaging that enables capture and permanent recording of localized H₂O₂ fluxes. Peroxy Green-1 Fluoromethyl (PG1-FM) is a diffusible small-molecule probe that senses H₂O₂ by a boronate oxidation reaction to trigger dual release and covalent labeling of a fluorescent product, thus preserving spatial information on local H₂O₂ changes. This unique reagent enables visualization of transcellular redox signaling in a microglia–neuron coculture cell model, where selective activation of microglia for ROS production increases H₂O₂ in nearby neurons. In addition to identifying ROS-mediated cell-to-cell communication, this work provides a starting point for the design of chemical probes that can achieve high spatial fidelity by combining activity-based sensing and labeling strategies.

Reactive oxygen species (ROS) are a family of small molecules that play broad roles in physiology and pathology (1–6). In this context, hydrogen peroxide (H₂O₂) is an ROS that is both a source of oxidative stress and a potent signaling molecule. H₂O₂ has been shown to regulate cell growth, differentiation, migration, and death pathways. Indeed, beyond its classical roles in phagocytic killing of pathogens during immune response (7–9), production of H₂O₂ via superoxide by NADPH oxidase (Nox) enzymes in nonphagocytic cells (10) can trigger signaling events that contribute to a diverse array of physiological processes including neural activity and long-term potentiation (11–14) and depression, stem cell growth and proliferation (15–17), circadian rhythms (18–20), and wound healing (21, 22).Owing to its transient and reactive nature, the vast majority of studies on H₂O₂ signaling have focused on intracellular communication events. Indeed, despite its small size and relatively nonpolar nature, H₂O₂ is not freely diffusible through membranes, and its entry into cells is tightly regulated, as our laboratory (23) and others (24–27) have identified specific isoforms of aquaporin water channels as endogenous mediators of H₂O₂ transport. As such, roles for H₂O₂ in transcellular communication remain insufficiently understood. This gap in fundamental knowledge is due in part to limitations in chemical tools to visualize integrated H₂O₂ activity that can retain spatial information over larger and/or more complex cell populations. Indeed, there have been recent elegant developments in sensing platforms and probe design (28–31). In terms of H₂O₂ sensing, conventional small-molecule fluorescent probes for H₂O₂ can quickly access sites of local H₂O₂ elevations but can also diffuse away after ROS detection (32–43), diluting signal-to-noise responses. Likewise, traditional fluorescent protein–based indicators that reversibly respond to H₂O₂ can be localized by genetic encoding but are limited to studies within a microscope’s field of view (44–48) and do not provide a permanent signal.Here we report a dual activity-based sensing and labeling strategy for selective and sensitive fluorescence detection of H₂O₂ with the ability to capture and record spatial information over defined time scales. Peroxy Green-1 Fluoromethyl (PG1-FM, Scheme 1) promotes a tandem boronate oxidation sensing and quinone methide labeling sequence upon reaction with H₂O₂ to covalently trap the probe in cells and afford a permanent stain that preserves spatial information on localized H₂O₂ fluxes. PG1-FM is capable of monitoring elevations in endogenous H₂O₂ production in live cells and is useful for both microscopy and flow cytometry assays. As an example of its utility, we use this probe to visualize transcellular ROS signaling in a microglia–neuron coculture system. This approach presages further opportunities for combining chemical sensing and labeling strategies to decipher biology with improved spatial fidelity.Open in a separate windowScheme 1.Design and chemical structure of PG1-FM, a dual activity-based sensing and labeling probe for fluorescence H₂O₂ detection.     

Quantification of organic aerosol and brown carbon evolution in fresh wildfire plumes

The evolution of organic aerosol (OA) and brown carbon (BrC) in wildfire plumes, including the relative contributions of primary versus secondary sources, has been uncertain in part because of limited knowledge of the precursor emissions and the chemical environment of smoke plumes. We made airborne measurements of a suite of reactive trace gases, particle composition, and optical properties in fresh western US wildfire smoke in July through August 2018. We use these observations to quantify primary versus secondary sources of biomass-burning OA (BBPOA versus BBSOA) and BrC in wildfire plumes. When a daytime wildfire plume dilutes by a factor of 5 to 10, we estimate that up to one-third of the primary OA has evaporated and subsequently reacted to form BBSOA with near unit yield. The reactions of measured BBSOA precursors contribute only 13 ± 3% of the total BBSOA source, with evaporated BBPOA comprising the rest. We find that oxidation of phenolic compounds contributes the majority of BBSOA from emitted vapors. The corresponding particulate nitrophenolic compounds are estimated to explain 29 ± 15% of average BrC light absorption at 405 nm (BrC Abs₄₀₅) measured in the first few hours of plume evolution, despite accounting for just 4 ± 2% of average OA mass. These measurements provide quantitative constraints on the role of dilution-driven evaporation of OA and subsequent radical-driven oxidation on the fate of biomass-burning OA and BrC in daytime wildfire plumes and point to the need to understand how processing of nighttime emissions differs.

Biomass burning (BB) is a major global source of atmospheric trace gases (1, 2), fixed nitrogen (3–6), and primary organic carbonaceous fine particles, known as BB organic aerosol (BBOA) (7). These emissions and their subsequent atmospheric transformations play a major role in affecting air quality, atmospheric composition, and climate.Questions remain about the magnitude of BBOA emissions and evolution, particularly the relative contributions that are primary, i.e., directly emitted (BBPOA), versus secondary, i.e., formed from gas-to-particle conversion following the oxidation of emitted vapors (BBSOA). Measurements of laboratory burns of individual or ensemble biomass fuel types have suggested that the BBSOA source could be anywhere from negligible to twice as large as the BBPOA source (8–11). On the other hand, field measurements have generally suggested little to no net change in BBOA in wildfire plumes (12–17). A leading hypothesis is that any BBSOA formation is offset by evaporation of BBPOA due to dilution-driven repartitioning of semivolatile components to the gas phase, making the magnitude of each difficult to discern (e.g., refs. 12, 14, 16–19). Recent plume modeling has investigated this behavior in detail, although the magnitudes of evaporation and BBSOA formation remain unclear due to limited observational constraints (20, 21). This hypothesis is uncertain in part because previous wildfire studies of BBSOA often did not include all potentially important precursor gases such as phenolic compounds. Laboratory studies have indicated that oxygenated aromatic compounds (i.e., phenolic compounds) are a large and often dominant source of BBSOA in BB smoke, although other sources including reduced aromatic compounds, biogenic compounds, and heterocyclic compounds (e.g., furans) can also be major BBSOA sources (11, 22–25).BB is also considered a major contributor to atmospheric brown carbon aerosol (BrC), which absorbs solar radiation and thus acts similarly to black carbon (BC) to potentially warm and stabilize the atmosphere (26–28). The lifetime of BrC and the importance of secondary BrC (sBrC) formation both remain uncertain. Phenolic compound emissions from wildfires are thought to be potentially important precursors for sBrC formation given their propensity to form light-absorbing nitroaromatics upon oxidation in the nitrogen oxide-rich fire plumes (29, 30). Phenolic compound oxidation products, including nitrophenolic compounds, have been estimated to account for up to approximately one-third to one-half of BrC light absorption for regions impacted by residential wood smoke and agricultural BB (31–34). Similar to BBSOA, field measurements of BrC absorption have generally shown little or no secondary formation in wildfire plumes, instead showing mostly decay with lifetimes from 9 h up to more than 1 d (16, 35). Understanding the controls on BrC and its secondary source can enable better predictions of its contribution and fate as wildfire smoke ages.We introduce a method for quantifying the effects of secondary formation versus dilution-driven evaporation for both total organic aerosol (OA) and BrC evolution in wildfire plumes. OA was measured using a high-resolution time-of-flight aerosol mass spectrometer (AMS), and BrC was quantified using a photoacoustic absorption spectrometer (PAS). To address the relative contributions to BBSOA formation from either primary emitted precursors (e.g., phenolic compounds) versus evaporated BBPOA, we quantified all major secondary OA (SOA) precursor gases measured using an iodide-adduct, high-resolution time-of-flight chemical ionization mass spectrometer (I⁻ CIMS) and a proton-transfer reaction time-of-flight mass spectrometer (PTR-ToF-MS) in authentic wildfire smoke plumes during the airborne Western Wildfire Experiment for Cloud Chemistry, Aerosol Absorption, and Nitrogen (WE-CAN) field campaign. The WE-CAN project deployed a research aircraft across the western United States between 22 July and 13 September 2018 to sample wildfire smoke during the first several hours of atmospheric evolution. To investigate the contributions of particulate nitrophenolic compounds to BrC Abs₄₀₅, environmental chamber experiments were performed at the National Center for Atmospheric Research (NCAR) chamber facility in May/June 2019 as part of the Monoterpene and Oxygenated aromatic Oxidation at Night and under LIGHTs (MOONLIGHT) campaign. These experiments simulated phenolic compound oxidation chemistry in fresh wildfire plumes. Through a combined analysis of these data, we characterize the importance of phenolic compound emissions and nitrophenolic oxidation products as potential contributors to BBSOA and sBrC, compared to the sources from evaporated BBPOA vapors.     

Cholinergic suppression of hippocampal sharp-wave ripples impairs working memory

Learning and memory are assumed to be supported by mechanisms that involve cholinergic transmission and hippocampal theta. Using G protein–coupled receptor-activation–based acetylcholine sensor (GRAB_ACh3.0) with a fiber-photometric fluorescence readout in mice, we found that cholinergic signaling in the hippocampus increased in parallel with theta/gamma power during walking and REM sleep, while ACh3.0 signal reached a minimum during hippocampal sharp-wave ripples (SPW-R). Unexpectedly, memory performance was impaired in a hippocampus-dependent spontaneous alternation task by selective optogenetic stimulation of medial septal cholinergic neurons when the stimulation was applied in the delay area but not in the central (choice) arm of the maze. Parallel with the decreased performance, optogenetic stimulation decreased the incidence of SPW-Rs. These findings suggest that septo–hippocampal interactions play a task-phase–dependent dual role in the maintenance of memory performance, including not only theta mechanisms but also SPW-Rs.

The neurotransmitter acetylcholine is thought to be critical for hippocampus-dependent declarative memories (1, 2). Reduction in cholinergic neurotransmission, either in Alzheimer’s disease or in experiments with cholinergic antagonists, such as scopolamine, impairs memory function (3–8). Acetylcholine may bring about its beneficial effects on memory encoding by enhancing theta rhythm oscillations, decreasing recurrent excitation, and increasing synaptic plasticity (9–11). Conversely, drugs which activate cholinergic receptors enhance learning and, therefore, are a neuropharmacological target for the treatment of memory deficits in Alzheimer’s disease (5, 12, 13).The contribution of cholinergic mechanisms in the acquisition of long-term memories and the role of the hippocampal–entorhinal–cortical interactions are well supported by experimental data (5, 12, 13). In addition, working memory or “short-term” memory is also supported by the hippocampal–entorhinal–prefrontal cortex (14–16). Working memory in humans is postulated to be a conscious process to “keep things in mind” transiently (16). In rodents, matching to sample task, spontaneous alternation between reward locations, and the radial maze task have been suggested to function as a homolog of working memory [“working memory like” (17)].Cholinergic activity is a critical requirement for working memory (18, 19) and for sustaining theta oscillations (10, 20–22). In support of this contention, theta–gamma coupling and gamma power are significantly higher in the choice arm of the maze, compared with those in the side arms where working memory is no longer needed for correct performance (23–26). It has long been hypothesized that working memory is maintained by persistent firing of neurons, which keep the presented items in a transient store in the prefrontal cortex and hippocampal–entorhinal system (27–31), although the exact mechanisms are debated (32–37). An alternative hypothesis holds that items of working memory are stored in theta-nested gamma cycles (38). Common in these models of working memory is the need for an active, cholinergic system–dependent mechanism (39–41). However, in spontaneous alternation tasks, the animals are not moving continuously during the delay, and theta oscillations are not sustained either. During the immobility epochs, theta is replaced by intermittent sharp-wave ripples (SPW-R), yet memory performance does not deteriorate. On the contrary, artificial blockade of SPW-Rs can impair memory performance (42, 43), and prolongation of SPW-Rs improves performance (44). Under the cholinergic hypothesis of working memory, such a result is unexpected.To address the relationship between cholinergic/theta versus SPW-R mechanism in spontaneous alternation, we used a G protein–coupled receptor-activation–based acetylcholine sensor (GRAB_ACh3.0) (45) to monitor acetylcholine (ACh) activity during memory performance in mice. In addition, we optogenetically enhanced cholinergic tone, which suppresses SPW-Rs by a different mechanism than electrically or optogenetically induced silencing of neurons in the hippocampus (43, 44). We show that cholinergic signaling in the hippocampus increases in parallel with theta power/score during walking and rapid eye movement (REM) sleep and reaches a transient minimum during SPW-Rs. Selective optogenetic stimulation of medial septal cholinergic neurons decreased the incidence of SPW-Rs during non-REM sleep (46–48), as well as during the delay epoch of a working memory task and impaired memory performance. These findings demonstrate that memory performance is supported by complementary theta and SPW-R mechanisms.     

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.     

“Soft” oxidative coupling of methane to ethylene: Mechanistic insights from combined experiment and theory

The oxidative coupling of methane to ethylene using gaseous disulfur (2CH₄ + S₂ → C₂H₄ + 2H₂S) as an oxidant (SOCM) proceeds with promising selectivity. Here, we report detailed experimental and theoretical studies that examine the mechanism for the conversion of CH₄ to C₂H₄ over an Fe₃O₄-derived FeS₂ catalyst achieving a promising ethylene selectivity of 33%. We compare and contrast these results with those for the highly exothermic oxidative coupling of methane (OCM) using O₂ (2CH₄ + O₂ → C₂H₄ + 2H₂O). SOCM kinetic/mechanistic analysis, along with density functional theory results, indicate that ethylene is produced as a primary product of methane activation, proceeding predominantly via CH₂ coupling over dimeric S–S moieties that bridge Fe surface sites, and to a lesser degree, on heavily sulfided mononuclear sites. In contrast to and unlike OCM, the overoxidized CS₂ by-product forms predominantly via CH₄ oxidation, rather than from C₂ products, through a series of C–H activation and S-addition steps at adsorbed sulfur sites on the FeS₂ surface. The experimental rates for methane conversion are first order in both CH₄ and S₂, consistent with the involvement of two S sites in the rate-determining methane C–H activation step, with a CD₄/CH₄ kinetic isotope effect of 1.78. The experimental apparent activation energy for methane conversion is 66 ± 8 kJ/mol, significantly lower than for CH₄ oxidative coupling with O₂. The computed methane activation barrier, rate orders, and kinetic isotope values are consistent with experiment. All evidence indicates that SOCM proceeds via a very different pathway than that of OCM.

The oxidative coupling of methane (OCM) with O₂ would seem to be a concise, direct route to convert methane, one of the most Earth-abundant carbon sources (1), to ethylene (2CH₄ + O₂ → C₂H₄ + 2H₂O), a key chemical intermediate (2, 3), and this process has been extensively studied (1, 4–19) since 1982 (20). Nevertheless, the widespread use of OCM is challenged by methane overoxidation to CO₂ and other oxygenates. Furthermore, the severe reaction conditions of nonoxidative pathways (2, 21–28) typically risk carbon deposition and catalyst deactivation (2, 21–26). In preliminary studies, we reported a 2CH₄ + S₂ → C₂H₄ + 2H₂S coupling process that moderates the methane overoxidation driving force using gaseous disulfur (S₂) as a “soft” oxidant (SOCM; Fig. 1A) (29). S₂ is isoelectronic with O₂, the major sulfur vapor species at 700 to 925 °C (30–32), and is a less aggressive oxidant than O₂ (33). In this scenario, elemental sulfur is recovered from the H₂S coproduct via the known Claus process (Fig. 1B) (30), in a cycle where sulfur mediates/moderates the high nonselective O₂ reactivity. SOCM achieved promising ethylene selectivity, raising intriguing mechanistic questions and the possibility of higher selectivity. Methane + S_2(g) ethylene selectivities near ∼20% are achieved over a PdS/ZrO₂ catalyst (29), and oxide precatalysts give selectivities near 33% (34).Open in a separate windowFig. 1.Energetic comparison between the oxidative coupling of methane with O₂ (OCM) and with S₂ (SOCM) and the pathway to recover elemental sulfur from H₂S. (A) Gibbs free energy of desired and overoxidation processes in OCM and SOCM at 800 and 1,050 °C. (B) Industrialized catalytic Claus process used to recover elemental sulfur from H₂S.Nevertheless, in contrast to extensive OCM (17, 35–39) and nonoxidative CH₄ coupling studies (40), far less is known about the SOCM reaction pathway. Post-SOCM X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), and elemental analysis (29, 34) indicate that the oxide precatalysts are predominantly sulfided. Density functional theory (DFT) analyses of molybdenum sulfide catalysts suggest that methane is activated at M–S or S–S sites to form surface-bound CH₃* species which dehydrogenate to form CH₂* (methylidene) species, which then couple to produce C₂H₄. It was proposed that CH₃* species can also desorb as methyl radicals which couple to form ethane (29). The overoxidation product, CS₂, was suggested to form via sulfur addition to methylidene surface intermediates (29).Kinetic, mechanistic, and theoretical analyses are needed to better understand the CH₄ conversion pathways to C₂H₄ and other products. In principle, there are two plausible pathways following methane activation: 1) H abstraction from adsorbed methyl species forms methylidene (CH₂*) and methylidyne (CH*) species then couple to C₂ products or undergo oxidation to CS₂ or 2) coupling of surface or gas phase methyl species form ethane, which then dehydrogenates to form ethylene or oxidizes to CS₂. For further SOCM optimization it is important to determine which pathways are operative, their relative rates, and the C₂ and CS₂ formation sites.Here we investigate SOCM pathways over a sulfided Fe₃O₄ precatalyst which affords C₂H₄ selectivities near 33%, complete oxide to sulfide conversion, minimal carbon deposition (coking), and 48-h SOCM stability at 950 °C (34). We first summarize SOCM phenomenology, followed by analysis of the Fe phases during sulfurization and SOCM. Next, kinetic/mechanistic studies focus on the methane and S₂ reaction orders, activation energetics, and isotope effects and probe the pathways governing C₂ vs. CS₂ formation. Complementary DFT calculations focus on reaction mechanisms, the active sites, and their role in product formation. The results are used in a microkinetic model to simulate reaction rates, apparent activation barriers, and reaction rate orders and to compare with experiment. Finally, SOCM and OCM are compared, revealing that they follow distinctly different pathways.     

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.     

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.     

ICAM-1 induced rearrangements of capsid and genome prime rhinovirus 14 for activation and uncoating

Most rhinoviruses, which are the leading cause of the common cold, utilize intercellular adhesion molecule-1 (ICAM-1) as a receptor to infect cells. To release their genomes, rhinoviruses convert to activated particles that contain pores in the capsid, lack minor capsid protein VP4, and have an altered genome organization. The binding of rhinoviruses to ICAM-1 promotes virus activation; however, the molecular details of the process remain unknown. Here, we present the structures of virion of rhinovirus 14 and its complex with ICAM-1 determined to resolutions of 2.6 and 2.4 Å, respectively. The cryo-electron microscopy reconstruction of rhinovirus 14 virions contains the resolved density of octanucleotide segments from the RNA genome that interact with VP2 subunits. We show that the binding of ICAM-1 to rhinovirus 14 is required to prime the virus for activation and genome release at acidic pH. Formation of the rhinovirus 14–ICAM-1 complex induces conformational changes to the rhinovirus 14 capsid, including translocation of the C termini of VP4 subunits, which become poised for release through pores that open in the capsids of activated particles. VP4 subunits with altered conformation block the RNA–VP2 interactions and expose patches of positively charged residues. The conformational changes to the capsid induce the redistribution of the virus genome by altering the capsid–RNA interactions. The restructuring of the rhinovirus 14 capsid and genome prepares the virions for conversion to activated particles. The high-resolution structure of rhinovirus 14 in complex with ICAM-1 explains how the binding of uncoating receptors enables enterovirus genome release.

Human rhinoviruses are the cause of more than half of common colds (1). Medical visits and missed days of school and work cost tens of billions of US dollars annually (2, 3). There is currently no cure for rhinovirus infections, and the available treatments are only symptomatic. Rhinoviruses belong to the family Picornaviridae, genus Enterovirus, and are classified into species A, B, and C (4). Rhinoviruses A and B can belong to either “major” or “minor” groups, based on their utilization of intercellular adhesion molecule-1 (ICAM-1) or low-density lipoprotein receptor for cell entry (5–7). Type C rhinoviruses use CDHR3 as a receptor (8). Rhinovirus 14 belongs to the species rhinovirus B and uses ICAM-1 as a receptor. Receptors recognized by rhinoviruses and other enteroviruses can be divided into two groups based on their function in the infection process (9). Attachment receptors such as DAF, PSGL1, KREMEN1, CDHR3, and sialic acid enable the binding and endocytosis of virus particles into cells (10–13). In contrast, uncoating receptors including ICAM-1, CD155, CAR, and SCARB2 enable virus cell entry but also promote genome release from virus particles (5, 14–16).Virions of rhinoviruses are nonenveloped and have icosahedral capsids (17). Genomes of rhinoviruses are 7,000 to 9,000 nucleotide-long single-stranded positive-sense RNA molecules (1, 17). The rhinovirus genome encodes a single polyprotein that is co- and posttranslationally cleaved into functional protein subunits. Capsid proteins VP1, VP3, and VP0, originating from one polyprotein, form a protomer, 60 of which assemble into a pseudo-T = 3 icosahedral capsid. To render the virions mature and infectious, VP0 subunits are cleaved into VP2 and VP4 (18, 19). VP1 subunits form pentamers around fivefold symmetry axes, whereas subunits VP2 and VP3 form heterohexamers centered on threefold symmetry axes. The major capsid proteins VP1 through 3 have a jelly roll β-sandwich fold formed by two β-sheets, each containing four antiparallel β-strands, which are conventionally named B to I (20–22). The two β-sheets contain the strands BIDG and CHEF, respectively. The C termini of the capsid proteins are located at the virion surface, whereas the N termini mediate interactions between the capsid proteins and the RNA genome on the inner surface of the capsid. VP4 subunits are attached to the inner face of the capsid formed by the major capsid proteins. The surfaces of rhinovirus virions are characterized by circular depressions called canyons, which are centered around fivefold symmetry axes of the capsids (21).The VP1 subunits of most rhinoviruses, but not those of rhinovirus 14, contain hydrophobic pockets, which are filled by molecules called pocket factors (17, 21, 23, 24). It has been speculated that pocket factors are fatty acids or lipids (25). The pockets are positioned immediately below the canyons. The exposure of rhinoviruses to acidic pH induces expulsion of the pocket factors, which leads to the formation of activated particles and genome release (17, 26–32). The activated particles are characterized by capsid expansion, a reduction in interpentamer contacts, the release of VP4 subunits, externalization of N termini of VP1 subunits, and changes in the distribution of RNA genomes (17, 26–29, 33, 34). Artificial hydrophobic compounds that bind to VP1 pockets with high affinity inhibit infection by rhinoviruses (35, 36).ICAM-1 is an endothelial- and leukocyte-associated protein that stabilizes cell–cell interactions and facilitates the movement of leukocytes through endothelia (37). ICAM-1 can be divided into an extracellular amino-terminal part composed of five immunoglobulin domains, a single transmembrane helix, and a 29-residue–long carboxyl-terminal cytoplasmic domain. The immunoglobulin domains are characterized by a specific fold that consists of seven to eight β-strands, which form two antiparallel β-sheets in a sandwich arrangement (38–40). The immunoglobulin domains of ICAM-1 are stabilized by disulfide bonds and glycosylation (38–41). The connections between the immunoglobulin domains are formed by flexible linkers that enable bending of the extracellular part of ICAM-1. For example, the angle between domains 1 and 2 differs by 8° between molecules in distinct crystal forms (38, 42). As a virus receptor, ICAM-1 enables the virus particles to sequester at the cell surface and mediates their endocytosis (5). The structures of complexes of rhinoviruses 3, 14, and 16, and CVA21 with ICAM-1 have been determined to resolutions of 9 to 28 Å (42–46). It was shown that ICAM-1 molecules bind into the canyons at the rhinovirus surface, approximately between fivefold and twofold symmetry axes (42–46). ICAM-1 interacts with residues from all three major capsid proteins. It has been speculated that the binding of ICAM-1 triggers the transition of virions of rhinovirus 14 to activated particles and initiates genome release (45, 47). However, the limited resolution of the previous studies prevented characterization of the corresponding molecular mechanism.Here, we present the cryo-electron microscopy (cryo-EM) reconstruction of the rhinovirus 14 virion, which contains resolved density of octanucleotide segments of the RNA genome that interact with VP2 subunits. Furthermore, we show that the binding of ICAM-1 to rhinovirus 14 induces changes in its capsid and genome, which are required for subsequent virus activation and genome release at acidic pH.