Biocompatible surface functionalization architecture for a diamond quantum sensor

Quantum metrology enables some of the most precise measurements. In the life sciences, diamond-based quantum sensing has led to a new class of biophysical sensors and diagnostic devices that are being investigated as a platform for cancer screening and ultrasensitive immunoassays. However, a broader application in the life sciences based on nanoscale NMR spectroscopy has been hampered by the need to interface highly sensitive quantum bit (qubit) sensors with their biological targets. Here, we demonstrate an approach that combines quantum engineering with single-molecule biophysics to immobilize individual proteins and DNA molecules on the surface of a bulk diamond crystal that hosts coherent nitrogen vacancy qubit sensors. Our thin (sub–5 nm) functionalization architecture provides precise control over the biomolecule adsorption density and results in near-surface qubit coherence approaching 100 μs. The developed architecture remains chemically stable under physiological conditions for over 5 d, making our technique compatible with most biophysical and biomedical applications.

Recent developments in quantum engineering and diamond processing have brought us considerably closer to performing nanoscale NMR and electron paramagnetic resonance (EPR) spectroscopy of small ensembles and even individual biomolecules. Notably, these advances have enabled the detection of the nuclear spin noise from a single ubiquitin protein (1) and the probing of the EPR spectrum of an individual paramagnetic spin label conjugated to a protein (2) or DNA molecule (3). More recently, lock-in detection and signal reconstruction techniques (4, 5) have enabled one- and multidimensional NMR spectroscopy with 0.5-Hz spectral resolution (6–8). More advanced control sequences at cryogenic temperatures have further enabled mapping the precise location of up to 27 ¹³C nuclear spins inside of diamond (9). Yet biologically meaningful spectroscopy on intact biomolecules remains elusive. One of the main outstanding challenges, which is required to perform nanoscale magnetic resonance spectroscopy of biomolecules, is the need to immobilize the target molecules within the 10- to 30-nm sensing range (2, 3, 7) of a highly coherent nitrogen vacancy (NV) qubit sensor. Immobilization is necessary because an untethered molecule would otherwise diffuse out of the detection volume within a few tens of microseconds.Various avenues to the functionalization of high-quality, single-crystalline diamond chips have been pursued over the last decade (10–12). However, none of the currently known approaches has led to the desired results of interfacing a coherent quantum sensor with target biomolecules. For example, hydrogen-terminated diamond surfaces can be chemically modified and form biologically stable surfaces (10, 13); but near-surface NV centers are generally charge-unstable under hydrogen termination (14), posing open challenges for NV sensing. On the other hand, oxygen-terminated diamond surfaces have been used to create charge stable NV^– centers with exceptional coherence times within 10 nm from the diamond surface (15). However, perfectly arranged, ether-terminated diamond surfaces generally lack chemically functionalizable surface groups (such as carboxyl or hydroxyl groups), making it difficult to control immobilization density and surface passivation. Other platforms such as diamond nanocrystals can generally be functionalized (16, 17) because of their heterogeneous surface chemistry, but they do not possess the coherence times needed for nanoscale magnetic resonance spectroscopy. Our approach (Fig. 1A) overcomes these limitations by utilizing a 2-nm-thick Al₂O₃ layer deposited onto an oxygen-terminated diamond surface by atomic layer deposition (ALD). This Al₂O₃ “adhesion” layer is silanized by N-[3-(trimethoxysilyl)propyl]ethylenediamine to create an amine (–NH₂) -terminated surface, which in turn is then grafted with a monolayer of heterobifunctional polyethylene glycol (PEG) via an N-hydroxysuccinimide (NHS) reaction, a process also referred as PEGylation. The PEG layer serves two purposes. First, it passivates the diamond surface to prevent nonspecific adsorption of biomolecules. Second, by adjusting the density of PEG molecules with functional groups (e.g., biotin or azide), we can control the immobilization density of proteins or DNA target molecules on the diamond surface. Furthermore, the small persistence length of the PEG linker (∼0.35 nm) allows the immobilized biomolecules to undergo rotational diffusion (18). This tumbling motion is the basis for motional averaging of the NMR spectra and helps to prevent immobilization of molecules in biologically inactive orientations.Open in a separate windowFig. 1.Architecture and characterization of the diamond functionalization approach. (A) Schematic illustration of the functionalization process. A thin layer of Al₂O₃ (gray) was deposited to the pristine, oxygen-terminated diamond surfaces (blue), followed by silanization (purple) and PEGylation (green). Functional groups (biotin, yellow circle; azide, red triangle) allow for cross-linking with target biomolecules. AFM characterization of the surfaces (B) and XPS Al2p signal after each step of the functionalization (C). (D) Illustration of the overall chemical functionalization architecture (not to scale), with corresponding thicknesses. (E) Illustration of SPAAC reaction. (F) A lithographically fabricated Al₂O₃ pattern on the diamond surface by lift-off, with a thickness of ∼2.1 nm. The Al₂O₃ layer is uniform without the presence of pin holes. The elevated edges originate from lift-off combined with ALD deposition.     

Single-spin stochastic optical reconstruction microscopy

We experimentally demonstrate precision addressing of single-quantum emitters by combined optical microscopy and spin resonance techniques. To this end, we use nitrogen vacancy (NV) color centers in diamond confined within a few ten nanometers as individually resolvable quantum systems. By developing a stochastic optical reconstruction microscopy (STORM) technique for NV centers, we are able to simultaneously perform sub–diffraction-limit imaging and optically detected spin resonance (ODMR) measurements on NV spins. This allows the assignment of spin resonance spectra to individual NV center locations with nanometer-scale resolution and thus further improves spatial discrimination. For example, we resolved formerly indistinguishable emitters by their spectra. Furthermore, ODMR spectra contain metrology information allowing for sub–diffraction-limit sensing of, for instance, magnetic or electric fields with inherently parallel data acquisition. As an example, we have detected nuclear spins with nanometer-scale precision. Finally, we give prospects of how this technique can evolve into a fully parallel quantum sensor for nanometer resolution imaging of delocalized quantum correlations.Stochastic reconstruction microscopy (STORM) techniques have led to a wealth of applications in fluorescence imaging (1–3); for example, few ten-nanometers 3D spatial resolution (lateral 20 nm, axial 50 nm) has been achieved in cellular imaging. So far, STORM fluorophores have been used as markers to achieve nanoscale microscopy of specific targets (4). Here, we present a spin-based approach that promises to combine sub–diffraction-limit imaging via STORM and simultaneous sensing of various physical quantities.As a prominent multipurpose probe and highly photostable single emitter, we use the nitrogen vacancy (NV) spin defect in diamond. It can be applied for nanometer-scale scanning magnetometry (5–8) as well as magnetic imaging (9–14) (e.g., for imaging spin distributions, magnetic particles or organisms, or device intrinsic fields), the measurement of electric fields, and diamond lattice strain (15–18) (e.g., for imaging elementary charges or charge distributions, or for imaging strain fields induced by mechanical action on the diamond surface). Very recently, precise temperature measurements (19, 20) even in living cells (21) have been demonstrated.During the last decades, a variety of methods have been invented to circumvent the diffraction limit in farfield optical microscopy. One approach reduces the spatial region within a laser focus from which optical response of a single emitter is possible by exploiting optical nonlinearities. Examples are stimulated emission depletion (STED) and ground-state depletion (GSD) microscopy (22, 23). Another approach tailors the timing of optical response of several emitters from within a diffraction-limited spot to distinguish them in the time domain. One example is stochastic optical reconstruction microscopy (24–26). This latter technique is intrinsically parallel as it uses a CCD array for imaging and is therefore particularly suited for high-throughput imaging.STED and GSD microscopy, which are both scanning techniques, have been recently implemented for NV centers in diamond (27, 28) with resolutions down to a few nanometers (29). In addition, localization-based superresolution microscopy has been shown with NV centers in nanodiamonds (30).Here, we experimentally demonstrate STORM for NV centers in diamond as a new optical superresolution technique with wide-field parallel image acquisition for NV centers in bulk diamond. Our technique is based on recently gained profound knowledge about statistical charge state switching of single NV centers (31), and its scalability relies on the homogeneity of this charge state dynamics for NV centers in bulk diamond. Furthermore, we combine optical superresolution microscopy with high–spectral-resolution optically detected magnetic resonance (ODMR). On the one hand, we use the latter technique to assign magnetic resonance data to nanometer-scale locations, which is important for qubit or metrology applications (9–11, 32). On the other hand, different magnetic resonance fingerprints of closely spaced NV centers are used to further increase the already obtained superresolution, as demonstrated in refs. 32 and 33, which is important for emitter localization in imaging applications.     

Mantle–slab interaction and redox mechanism of diamond formation

Subduction tectonics imposes an important role in the evolution of the interior of the Earth and its global carbon cycle; however, the mechanism of the mantle–slab interaction remains unclear. Here, we demonstrate the results of high-pressure redox-gradient experiments on the interactions between Mg-Ca-carbonate and metallic iron, modeling the processes at the mantle–slab boundary; thereby, we present mechanisms of diamond formation both ahead of and behind the redox front. It is determined that, at oxidized conditions, a low-temperature Ca-rich carbonate melt is generated. This melt acts as both the carbon source and crystallization medium for diamond, whereas at reduced conditions, diamond crystallizes only from the Fe-C melt. The redox mechanism revealed in this study is used to explain the contrasting heterogeneity of natural diamonds, as seen in the composition of inclusions, carbon isotopic composition, and nitrogen impurity content.Subduction of crustal material plays an important role in the global carbon cycle (1–6). Depending on oxygen fugacity and pressure-temperature (P-T) conditions, carbon exists in the Earth''s interior in the form of carbides, diamond, graphite, hydrocarbons, carbonates, and CO₂ (7–11). In the upper mantle, the oxygen fugacity (fO₂) varies from one to five log units below the fayalite-magnetite-quartz (FMQ) buffer, with a trend of a decrease with depth (6, 12–15). At a depth of ∼250 km, mantle is reported to become metal saturated (16, 17), which holds true for all mantle regions below, including the transition zone and lower mantle. The subduction of the oxidized crustal material occurs to depths greater than 600 km (4–6). The main carbon-bearing minerals of the subducted materials are carbonates, which are thermodynamically stable up to P-T conditions of the lower mantle (10, 11, 18). As evidenced by the compositions of inclusions in diamond, which vary from strongly reduced, e.g., metallic iron and carbides (19–23), to oxidized, e.g., carbonates and CO₂ (6, 20, 24–28), carbonates may be involved in the reactions with reduced deep-seated rocks, including Fe⁰-bearing species (29–31). A scale of these reactions is determined mainly by the capacity of subducted carbonate-bearing domains. An important consequence of such an interaction is that it can produce diamond. However, studies on diamond synthesis via the reactions between oxidized and reduced phases are limited (32–35).To understand the mechanisms of the interaction of carbon-bearing oxidized- and reduced-mineral assemblages, we performed high-pressure experiments with an iron-carbonate system; an approach was used that enabled the creation of an oxygen fugacity gradient in the capsules (Materials and Methods and SI Materials and Methods).     

Immunomagnetic microscopy of tumor tissues using quantum sensors in diamond

Histological imaging is essential for the biomedical research and clinical diagnosis of human cancer. Although optical microscopy provides a standard method, it is a persistent goal to develop new imaging methods for more precise histological examination. Here, we use nitrogen-vacancy centers in diamond as quantum sensors and demonstrate micrometer-resolution immunomagnetic microscopy (IMM) for human tumor tissues. We immunomagnetically labeled cancer biomarkers in tumor tissues with magnetic nanoparticles and imaged them in a 400-nm resolution diamond-based magnetic microscope. There is barely magnetic background in tissues, and the IMM can resist the impact of a light background. The distribution of biomarkers in the high-contrast magnetic images was reconstructed as that of the magnetic moment of magnetic nanoparticles by employing deep-learning algorithms. In the reconstructed magnetic images, the expression intensity of the biomarkers was quantified with the absolute magnetic signal. The IMM has excellent signal stability, and the magnetic signal in our samples had not changed after more than 1.5 y under ambient conditions. Furthermore, we realized multimodal imaging of tumor tissues by combining IMM with hematoxylin-eosin staining, immunohistochemistry, or immunofluorescence microscopy in the same tissue section. Overall, our study provides a different histological method for both molecular mechanism research and accurate diagnosis of human cancer.

Cancer is one of the most common and most serious human diseases (1). Various medical imaging technologies have been widely used in the clinical examination of tumors (2), while histopathological examination is the gold standard for diagnosing human cancers (3). In addition, for targeted therapy, the screening of sensitive populations relies on quantitative histopathological analysis of tumor local biomarker levels (4). Optical microscopy is the most common method for histological examination. For instance, hematoxylin-eosin (HE) staining, immunohistochemistry (IHC), and immunofluorescence microscopy (IFM) have been developed for many years to obtain the morphological features and the distribution and expression level of biomarkers in tumor tissues (3, 5), respectively. There are many other tissue imaging technologies emerging, such as multispectral imaging based on tyramide signal amplification (6), highly multiplexed imaging based on mass cytometry (7), and label-free spectroscopic imaging (8–10). However, commonly used optical microscopic imaging cannot absolutely quantify the signal intensity and usually suffers from background signals in tissues (11, 12). Furthermore, it is difficult to correlate different optical imaging in the same tissue section, such as HE staining cannot be combined with other optical imaging. Each unique tissue section in imaging mass cytometry is a single-use sample due to its destructive nature. Therefore, developing a tissue-imaging method with outstanding properties remains a persistent pursuit and challenge for biologists and pathologists.MRI provides a powerful technique for tumor imaging. Conventional MRI has been widely used for in situ imaging from biological research to clinical diagnosis of cancer (2). However, its low spatial resolution, e.g., merely 60 μm resolution of anatomical imaging at 9.4 T (13), limits the applications in tissue-level imaging. Recently developed microscale magnetic-imaging technology based on nitrogen-vacancy (NV) centers in diamond (14) provides a way for breaking the spatial-resolution limitation. The NV center is a nanoscale point defect in diamond and has been proposed as an ultrasensitive quantum sensor to realize nanoscale magnetometry (15–17). Since then, magnetic resonance techniques based on NV centers have been greatly developed and applied in nanoscale magnetic resonance spectroscopy and imaging of biological samples (14, 18–23). Meanwhile, wide-field magnetic imaging using an NV ensemble with submicrometer resolution is more suitable for the detection of large samples (24–30). These previous works are the cornerstones for magnetic resonance researches of microscale biological samples, such as single molecules and cells. However, micrometer-resolution magnetic imaging (or MRI) in biological tissues has not been achieved because of many kinds of technical barriers, which buries the potentially powerful capabilities of NV center-based quantum sensing in histopathology. Combining NV-based wide-field magnetic imaging with an immunomagnetic labeling technique modified from conventional immunocytochemistry (ICC) and immunofluorescence (IF) techniques, here we establish immunomagnetic microscopy (IMM), an approach that is capable of imaging cancer biomarkers in tumor tissues at a cellular resolution of ∼1 μm and quantifying them with absolute magnetic signals.We here report four technical advancements to achieve an easy-to-use robust IMM of tumor tissues, including 1) quantifying the expression intensity of biomarkers as the absolute magnetic intensity, 2) reconstructing magnetic images using deep-learning algorithms, 3) immunomagnetically labeling tissue samples with magnetic nanoparticles (MNPs), and 4) attaching tissue sections over a large area close to the diamond to meet the limited detection range of NV centers. For the immunomagnetic examination, we developed a diamond magnetic microscope based on NV centers, combining magnetic imaging and multiplexed optical imaging. The wide-field magnetic imaging uses a layer of shallow NV centers below the diamond to detect weak magnetic fields in the biological sample close to the diamond surface.Applying this approach, we imaged magnetic-labeled human tumor tissues and magnetically quantified the expression of biomarkers, which exhibited unparalleled signal stability and clean magnetic background. Meanwhile, the NV center-based IMM can be combined with conventional optical imaging techniques, such as HE staining, IHC, and IFM, to realize multimodal imaging in the same tissue section and has the potential to influence both fundamental research and clinical diagnosis of human cancer.     

Multiplexed sensing of biomolecules with optically detected magnetic resonance of nitrogen-vacancy centers in diamond

In the past decade, a great effort has been devoted to develop new biosensor platforms for the detection of a wide range of analytes. Among the various approaches, magneto-DNA assay platforms have received extended interest for high sensitive and specific detection of targets with a simultaneous manipulation capacity. Here, using nitrogen-vacancy quantum centers in diamond as transducers for magnetic nanotags (MNTs), a hydrogel-based, multiplexed magneto-DNA assay is presented. Near–background-free sensing with diamond-based imaging combined with noninvasive control of chemically robust nanotags renders it a promising platform for applications in medical diagnostics, life science, and pharmaceutical drug research. To demonstrate its potential for practical applications, we employed the sensor platform in the sandwich DNA hybridization process and achieved a limit of detection in the attomolar range with single-base mismatch differentiation.

Thanks to their nanoscale size, MNTs exhibit a reversible, hysteresis-free magnetization and high magnetophoretic mobility, which make them attractive for biomedical applications (1). The extremely large surface-to-volume ratio allows efficient interaction with the surrounding medium. Their surface can be chemically modified to conjugate biomolecules of different sizes, including drugs, nucleic acids, and peptides (2, 3, 4). Compared to fluorescence-based labeling approaches, MNTs provide distinct advantages, such as lower background noise that is related to the negligible magnetic property of biological samples, stable detection without label-bleaching, and feasibility for integration to magnetophoresis-based external manipulation systems (5). Exploiting these properties, MNTs have already been used for a wide range of applications in biomedicine and the life sciences, including bioseparation (6), controlled drug delivery to living cells (7), hyperthermic cancer treatment (8), magnetogenetics (9), and NMR imaging (10).Although the size and morphology of magnetic nanotags (MNTs) can be characterized with existing analytical techniques such as dynamic light-scattering (11) and high-resolution transmission electron microscopy (12), the methods to probe their magnetic property in a physiological environment are limited. Superconducting quantum interference devices (SQUIDs) can achieve very high sensitivities but have limited spatial resolution, exhibit a size in the millimeter scale, and require cryogenic temperatures to operate (13), making them not appropriate for biologically relevant systems. While force microscopy with a magnetic tip allows to achieve high spatial resolution, it is impractical for the investigation of biological samples (14). First, physical contact with the sample can contaminate and damage the scanning probe, and incorporation in closed microfluidic platforms is difficult. Second, the measured signal is not only due to the magnetic field from the target but also includes other interactions such as Van der Waals and electrostatic forces, which undermine the sensitivity. Besides, as with SQUID magnetometers, magnetic force microscopy represents a time-consuming and low-throughput mechanical-scanning process. Similarly, various other methodologies such as tunneling magnetoresistance (15), Hall effect sensors (16), and atomic vapor cells (17) have been widely used for the detection of local magnetic fields, but complex experimental conditions, difficulties in miniaturization, and measurements with very slow readout limit their application in the biomedical field. An effective imaging technique for multiplex magneto-DNA assay sensors should not rely on a single point sensor or require precise positioning of the probe hardware that scans a local environment to extract the magnetic field distribution. Here, we use a thin layer of nitrogen-vacancy (NV) quantum centers in a diamond chip to map the magnetic profile of hydrogel microstructures that form a magneto-DNA assay platform. The platform exploits a nucleic acid sandwich hybridization technique in which the target nucleic acid binds to a capture-DNA immobilized within the hydrogel microstructure network and a complementary reporter DNA conjugated with an MNT.NV quantum centers, atomic-scale defects in diamond crystals, are another promising candidate for various applications in sensing, quantum optics, and metrology. Optically addressable spin states of NV quantum centers can be manipulated with an externally applied microwave signal (MW) and detected from the spin state–dependent photoluminescence (PL) signal. The NV centers are well suited for precise detection of physical quantities such as magnetic fields (18–20), electric fields (21), temperature (22), pressure (23), ion concentrations (24), and spin densities (25). The sensing capabilities of NV quantum centers under ambient conditions are extremely valuable for applications in biological systems.Bulk, diamond-based sensors employing an ensemble of NV quantum centers have been used to detect action potentials at the single-neuron level in whole organisms (26), image magnetically labeled cell lines (27) and localize magnetosomes generated in magnetotactic bacteria (28). Recently a chemically modified diamond pillar array has been introduced for the detection of individual, nitroxide spin-labeled DNA duplexes immobilized within ∼10-nm detection range of shallowly embedded NV quantum centers (29). Although this study did not focus on the detection of unmodified DNA duplex, it demonstrates the detection capacity of NV centers for individual spin labels in aqueous buffer solutions when immobilized in close proximity.In addition, in the form of single fluorescent nanodiamonds (FNDs), unique spin and optical properties of NV quantum centers have promoted the development of numerous fluorescent microscopy techniques such as superresolution microscopy (30), fluorescence resonance energy transfer experiments (31), long-term in vivo tracking (32), fluorescence lifetime imaging microscopy, and stimulated emission depletion microscopy (33). FNDs are also emerging as promising candidates for sensor applications operating at the nanoscale. They have been validated for nanoscale thermometry in living human cells with high resolution and precision (22). A recent study on a hybrid approach with magnetic nanoparticles and NV quantum centers in FNDs has shown that the sensitivity can be further enhanced with the critical magnetization near the ferromagnetic–paramagnetic transition temperature (34). It has been demonstrated that nanodiamonds with NV centers are nontoxic for cells and do not induce any detectable stress in organisms (35). They have been employed to investigate a wide range of biomedical processes, including early embryogenesis (36), organ development (37), and drug delivery (38). More recently, they have been used as ultra-sensitive fluorescent labels that can be manipulated by microwave irradiation for a better signal-to-background ratio and allow a detection limit of 8.2 × 10⁻¹⁹ molar for a biotin–avidin model (39).     

Fenton chemistry at aqueous interfaces

In a fundamental process throughout nature, reduced iron unleashes the oxidative power of hydrogen peroxide into reactive intermediates. However, notwithstanding much work, the mechanism by which Fe²⁺ catalyzes H₂O₂ oxidations and the identity of the participating intermediates remain controversial. Here we report the prompt formation of O=Fe^IVCl₃⁻ and chloride-bridged di-iron O=Fe^IV·Cl·Fe^IICl₄⁻ and O=Fe^IV·Cl·Fe^IIICl₅⁻ ferryl species, in addition to Fe^IIICl₄⁻, on the surface of aqueous FeCl₂ microjets exposed to gaseous H₂O₂ or O₃ beams for <50 μs. The unambiguous identification of such species in situ via online electrospray mass spectrometry let us investigate their individual dependences on Fe²⁺, H₂O₂, O₃, and H⁺ concentrations, and their responses to tert-butanol (an ·OH scavenger) and DMSO (an O-atom acceptor) cosolutes. We found that (i) mass spectra are not affected by excess tert-butanol, i.e., the detected species are primary products whose formation does not involve ·OH radicals, and (ii) the di-iron ferryls, but not O=Fe^IVCl₃⁻, can be fully quenched by DMSO under present conditions. We infer that interfacial Fe(H₂O)_n²⁺ ions react with H₂O₂ and O₃ >10³ times faster than Fe(H₂O)₆²⁺ in bulk water via a process that favors inner-sphere two-electron O-atom over outer-sphere one-electron transfers. The higher reactivity of di-iron ferryls vs. O=Fe^IVCl₃⁻ as O-atom donors implicates the electronic coupling of mixed-valence iron centers in the weakening of the Fe^IV–O bond in poly-iron ferryl species.High-valent Fe^IV=O (ferryl) species participate in a wide range of key chemical and biological oxidations (1–4). Such species, along with ·OH radicals, have long been deemed putative intermediates in the oxidation of Fe^II by H₂O₂ (Fenton’s reaction) (5, 6), O₃, or HOCl (7, 8). The widespread availability of Fe^II and peroxides in vivo (9–12), in natural waters and soils (13), and in the atmosphere (14–18) makes Fenton chemistry and Fe^IV=O groups ubiquitous features in diverse systems (19). A lingering issue regarding Fenton’s reaction is how the relative yields of ferryls vs. ·OH radicals depend on the medium. For example, by assuming unitary ·OH radical yields, some estimates suggest that Fenton’s reaction might account for ∼30% of the ·OH radical production in fog droplets (20). Conversely, if Fenton’s reaction mostly led to Fe^IV=O species, atmospheric chemistry models predict that their steady-state concentrations would be ∼10⁴ times larger than [·OH], thereby drastically affecting the rates and course of oxidative chemistry in such media (20). Fe^IV=O centers are responsible for the versatility of the family of cytochrome P450 enzymes in catalyzing the oxidative degradation of a vast range of xenobiotics in vivo (21–28), and the selective functionalization of saturated hydrocarbons (29). The bactericidal action of antibiotics has been linked to their ability to induce Fenton chemistry in vivo (9, 30–34). Oxidative damage from exogenous Fenton chemistry likely is responsible for acute and chronic pathologies of the respiratory tract (35–38).Despite its obvious importance, the mechanism of Fenton’s reaction is not fully understood. What is at stake is how the coordination sphere of Fe²⁺ (39–46) under specific conditions affects the competition between the one-electron transfer producing ·OH radicals (the Haber–Weiss mechanism) (47), reaction R1, and the two-electron oxidation via O-atom transfer (the Bray–Gorin mechanism) into Fe^IVO²⁺, reaction R2 (6, 23, 26, 27, 45, 48–51):Ozone reacts with Fe²⁺ via analogous pathways leading to (formally) the same intermediates, reactions R3a, R3b, and R4 (8, 49, 52, 53):At present, experimental evidence about these reactions is indirect, being largely based on the analysis of reaction products in bulk water in conjunction with various assumptions. Given the complex speciation of aqueous Fe²⁺/Fe³⁺ solutions, which includes diverse poly-iron species both as reagents and products, it is not surprising that classical studies based on the identification of reaction intermediates and products via UV-absorption spectra and the use of specific scavengers have fallen short of fully unraveling the mechanism of Fenton’s reaction. Herein we address these issues, focusing particularly on the critically important interfacial Fenton chemistry that takes place at boundaries between aqueous and hydrophobic media, such as those present in atmospheric clouds (16), living tissues, biomembranes, bio-microenvironments (38, 54, 55), and nanoparticles (56, 57).We exploited the high sensitivity, surface selectivity, and unambiguous identification capabilities of a newly developed instrument based on online electrospray mass spectrometry (ES-MS) (58–62) to identify the primary products of reactions R1–R4 on aqueous FeCl₂ microjets exposed to gaseous H₂O₂ and O₃ beams under ambient conditions [in N₂(g) at 1 atm at 293 ± 2 K]. Our experiments are conducted by intersecting the continuously refreshed, uncontaminated surfaces of free-flowing aqueous microjets with reactive gas beams for τ ∼10–50 μs, immediately followed (within 100 μs; see below) by in situ detection of primary interfacial anionic products and intermediates via ES-MS (Methods, SI Text, and Figs. S1 and S2). We have previously demonstrated that online mass spectrometric sampling of liquid microjets under ambient conditions is a surface-sensitive technique (58, 62–67).     

Thermodynamics of formation of coffinite,USiO4

Coffinite, USiO₄, is an important U(IV) mineral, but its thermodynamic properties are not well-constrained. In this work, two different coffinite samples were synthesized under hydrothermal conditions and purified from a mixture of products. The enthalpy of formation was obtained by high-temperature oxide melt solution calorimetry. Coffinite is energetically metastable with respect to a mixture of UO₂ (uraninite) and SiO₂ (quartz) by 25.6 ± 3.9 kJ/mol. Its standard enthalpy of formation from the elements at 25 °C is −1,970.0 ± 4.2 kJ/mol. Decomposition of the two samples was characterized by X-ray diffraction and by thermogravimetry and differential scanning calorimetry coupled with mass spectrometric analysis of evolved gases. Coffinite slowly decomposes to U₃O₈ and SiO₂ starting around 450 °C in air and thus has poor thermal stability in the ambient environment. The energetic metastability explains why coffinite cannot be synthesized directly from uraninite and quartz but can be made by low-temperature precipitation in aqueous and hydrothermal environments. These thermochemical constraints are in accord with observations of the occurrence of coffinite in nature and are relevant to spent nuclear fuel corrosion.In many countries with nuclear energy programs, spent nuclear fuel (SNF) and/or vitrified high-level radioactive waste will be disposed in an underground geological repository. Demonstrating the long-term (10⁶–10⁹ y) safety of such a repository system is a major challenge. The potential release of radionuclides into the environment strongly depends on the availability of water and the subsequent corrosion of the waste form as well as the formation of secondary phases, which control the radionuclide solubility. Coffinite (1), USiO₄, is expected to be an important alteration product of SNF in contact with silica-enriched groundwater under reducing conditions (2–8). It is also found, accompanied by thorium orthosilicate and uranothorite, in igneous and metamorphic rocks and ore minerals from uranium and thorium sedimentary deposits (2, 4, 5, 8–16). Under reducing conditions in the repository system, the uranium solubility (very low) in aqueous solutions is typically derived from the solubility product of UO₂. Stable U(IV) minerals, which could form as secondary phases, would impart lower uranium solubility to such systems. Thus, knowledge of coffinite thermodynamics is needed to constrain the solubility of U(IV) in natural environments and would be useful in repository assessment.In natural uranium deposits such as Oklo (Gabon) (4, 7, 11, 12, 14, 17, 18) and Cigar Lake (Canada) (5, 13, 15), coffinite has been suggested to coexist with uraninite, based on electron probe microanalysis (EPMA) (4, 5, 7, 11, 13, 17, 19, 20) and transmission electron microscopy (TEM) (8, 15). However, it is not clear whether such apparent replacement of uraninite by a coffinite-like phase is a direct solid-state process or occurs mediated by dissolution and reprecipitation.The precipitation of USiO₄ as a secondary phase should be favored in contact with silica-rich groundwater (21) [silica concentration >10⁻⁴ mol/L (22, 23)]. Natural coffinite samples are often fine-grained (4, 5, 8, 11, 13, 15, 24), due to the long exposure to alpha-decay event irradiation (4, 6, 25, 26) and are associated with other minerals and organic matter (6, 8, 12, 18, 27, 28). Hence the determination of accurate thermodynamic data from natural samples is not straightforward. However, the synthesis of pure coffinite also has challenges. It appears not to form by reacting the oxides under dry high-temperature conditions (24, 29). Synthesis from aqueous solutions usually produces UO₂ and amorphous SiO₂ impurities, with coffinite sometimes being only a minor phase (24, 30–35). It is not clear whether these difficulties arise from kinetic factors (slow reaction rates) or reflect intrinsic thermodynamic instability (33). Thus, there are only a few reported estimates of thermodynamic properties of coffinite (22, 36–40) and some of them are inconsistent. To resolve these uncertainties, we directly investigated the energetics of synthetic coffinite by high-temperature oxide melt solution calorimetry to obtain a reliable enthalpy of formation and explored its thermal decomposition.     

Global urban population exposure to extreme heat

Increased exposure to extreme heat from both climate change and the urban heat island effect—total urban warming—threatens the sustainability of rapidly growing urban settlements worldwide. Extreme heat exposure is highly unequal and severely impacts the urban poor. While previous studies have quantified global exposure to extreme heat, the lack of a globally accurate, fine-resolution temporal analysis of urban exposure crucially limits our ability to deploy adaptations. Here, we estimate daily urban population exposure to extreme heat for 13,115 urban settlements from 1983 to 2016. We harmonize global, fine-resolution (0.05°), daily temperature maxima and relative humidity estimates with geolocated and longitudinal global urban population data. We measure the average annual rate of increase in exposure (person-days/year⁻¹) at the global, regional, national, and municipality levels, separating the contribution to exposure trajectories from urban population growth versus total urban warming. Using a daily maximum wet bulb globe temperature threshold of 30 °C, global exposure increased nearly 200% from 1983 to 2016. Total urban warming elevated the annual increase in exposure by 52% compared to urban population growth alone. Exposure trajectories increased for 46% of urban settlements, which together in 2016 comprised 23% of the planet’s population (1.7 billion people). However, how total urban warming and population growth drove exposure trajectories is spatially heterogeneous. This study reinforces the importance of employing multiple extreme heat exposure metrics to identify local patterns and compare exposure trends across geographies. Our results suggest that previous research underestimates extreme heat exposure, highlighting the urgency for targeted adaptations and early warning systems to reduce harm from urban extreme heat exposure.

Increased exposure to extreme heat from both climate change (1–5) and the urban heat island (UHI) effect (6–9) threaten the sustainability of rapidly growing urban settlements worldwide. Exposure to dangerously high temperatures endangers urban health and development, driving reductions in labor productivity and economic output (10, 11) and increases in morbidity (1) and mortality (2, 3, 12). Within urban settlements, extreme heat exposure is highly unequal and most severely impacts the urban poor (13, 14). Despite the harmful and inequitable risks, we presently lack a globally comprehensive, fine-resolution understanding of where urban population growth intersects with increases in extreme heat (2, 6, 15). Without this knowledge, we have limited ability to tailor adaptations to reduce extreme heat exposure across the planet’s diverse urban settlements (6, 15, 16).Reducing the impacts of extreme heat exposure to urban populations requires globally consistent, accurate, and high-resolution measurement of both climate and demographic conditions that drive exposure (5, 15, 17). Such analysis provides decision makers with information to develop locally tailored interventions (7, 18, 19) and is also sufficiently broad in spatial coverage to transfer knowledge across urban geographies and climates (6). Information about exposures and interventions from diverse contexts is vital for the development of functional early warning systems (20) and can help guide risk assessments and inform future scenario planning (21). Existing global extreme heat exposure assessments (1, 2), however, do not meet these criteria (SI Appendix, Table S1) and are insufficient for decision makers. These studies are coarse grained (>0.5° spatial resolution), employ disparate or single metrics that do not capture the complexities of heat-health outcomes (22), do not separate urban from rural exposure (19), and rely on climate reanalysis products that can be substantially (∼1 to 3 °C) cooler than in situ data observations (5, 23, 24). In fact, widely cited benchmarks (25) that estimate extreme heat with the version 5 of the European Centre for Medium-Range Weather Forecasts Reanalysis (ERA5) (26) may greatly underestimate total global exposure to extreme heat (5, 23, 24). Using a 40.6 °C daily maximum 2-m air temperature threshold (T_max), recent analysis found that ERA5 T_max drastically underestimated the number of extreme heat days per year compared to in situ observations (23). Finally, few studies (2, 18) have assessed urban extreme heat exposure across data-sparse (23) rapidly urbanizing regions, such as sub-Saharan Africa, the Middle East, and Southern Asia (27), that may be most impacted by increased extreme heat events due to climate change (3, 5, 28).Here, we present a globally comprehensive, fine-resolution, and longitudinal estimate of urban population exposure to extreme heat––referred to henceforth as exposure––for 13,115 urban settlements from 1983 to 2016. To accomplish this, we harmonize global, fine-grained (0.05° spatial resolution) T_max estimates (23) with global urban population and spatial extent data (29). For each urban settlement, we calculate area-averaged daily wet bulb globe temperature (WBGT_max) (30) and heat index (HI_max) (31) maxima using Climate Hazards Center InfraRed Temperature with Stations Daily (CHIRTS-daily) T_max (23) and down-scaled daily minimum relative humidity (RH_min) estimates (32). CHIRTS-daily is better suited to measure urban extreme heat exposure than other gridded temperature datasets used in recent global extreme heat studies (SI Appendix, Table S1) for two reasons. First, it is more accurate, especially at long distances (refer to figure 3 in ref. 23), than widely used gridded temperature datasets to estimate urban temperature signals worldwide (SI Appendix, Figs. S1 and S2). Second, it better captures the spatial heterogeneity of T_max across diverse urban contexts (SI Appendix, Fig. S3). These factors are key for measuring extreme heat exposure in rapidly urbanizing, data-sparse regions.As discussed in refs. 23 and 24, the number of in situ temperature observations is far too low across rapidly urbanizing (27) regions to resolve spatial and temporal urban extreme heat fluctuations, which can vary dramatically over small distances and time periods. For example, of the more than 3,000 urban settlements in India (29), only 111 have reliable station observations (SI Appendix, Fig. S3). While climate reanalyses can help overcome these limitations, they are coarse grained (SI Appendix, Table S1) and suffer from mean bias, and, to a lesser degree, temporal fidelity. ERA5 has been shown to substantially underestimate the increasing frequencies of heat extremes (figure 4 in ref. 23), while Modern-Era Retrospective analysis for Research and Applications Version 2 (MERRA2) fails to represent the substantial increase in recent monthly T_max values (figure 8 in ref. 24). These datasets dramatically underestimate increases in warming. CHIRTS-daily overcomes these limitations by coherently stacking information from a high-resolution (0.05°) climatology-derived surface emission temperature (24), interpolated in situ observations, and ERA5 reanalysis to produce a product that has been explicitly developed to monitor and assess temperature related hazards (23). As such, CHIRTS-daily is best suited to capture variation in exposure across urban settlements in rapidly urbanizing (27), data-sparse regions such as sub-Saharan Africa, the Middle East, and Southern Asia (SI Appendix, Fig. S3) (24).We measure exposure in person-days/year⁻¹—the number of days per year that exceed a heat exposure threshold multiplied by the total urban population exposed (5). We then estimate annual rates of increase in exposure at the global (Fig. 1), regional (SI Appendix, Table S2), national (SI Appendix, Table S3), and municipality levels from 1983 to 2016 (SI Appendix, Table S4). At each spatial scale, we separate the contribution to exposure trajectories from total urban warming and population growth (5). For clarity, total urban warming refers to the combined increase of extreme heat in urban settlements from both the UHI effect and anthropogenic climate change. We do not decouple these two forcing agents (33, 34). However, we identify which urban settlements have warmed the fastest by measuring the rate of increase in the number of days per year that exceed the two extreme heat thresholds described below (15). Our main findings use an extreme heat exposure threshold defined as WBGT_max > 30 °C, the International Standards Organization (ISO) occupational heat stress threshold for risk of heat-related illness among acclimated persons at low metabolic rates (100 to 115 W) (30). WBGT_max is a widely used heat stress metric (35) that captures the biophysical response (36) of hot temperature–humidity combinations (3, 17) that reduce labor output (36), lead to heat-related illness (36), and can cause death (23). In using a threshold WBGT_max > 30 °C, which has been associated with higher mortality rates among vulnerable populations (37), we aim to identify truly extremely hot temperature–humidity combinations (17) that can harm human health and well-being. We recognize, however, that strict exposure thresholds do not account for individual-level risks and vulnerabilities related to acclimatization, socio-economic, or health status or local infrastructure (18, 19, 38). We also note that there are a range of definitions of exposure, and we provide further analysis identifying 2-d or longer periods during which the maximum heat index (HI_max) (31) exceeded 40.6 °C (SI Appendix, Figs. S4–S6) following the US National Weather Service’s definition for an excessive heat warning (39).Open in a separate windowFig. 1.Global urban population exposure to extreme heat, defined by 1-d or longer periods when WBGT_max > 30 °C, from 1983 to 2016 (A), with the contribution from population growth (B), and total urban warming (C) decoupled.     

Linking function to global and local dynamics in an elevator-type transporter

Transporters cycle through large structural changes to translocate molecules across biological membranes. The temporal relationships between these changes and function, and the molecular properties setting their rates, determine transport efficiency—yet remain mostly unknown. Using single-molecule fluorescence microscopy, we compare the timing of conformational transitions and substrate uptake in the elevator-type transporter Glt_Ph. We show that the elevator-like movements of the substrate-loaded transport domain across membranes and substrate release are kinetically heterogeneous, with rates varying by orders of magnitude between individual molecules. Mutations increasing the frequency of elevator transitions and reducing substrate affinity diminish transport rate heterogeneities and boost transport efficiency. Hydrogen deuterium exchange coupled to mass spectrometry reveals destabilization of secondary structure around the substrate-binding site, suggesting that increased local dynamics leads to faster rates of global conformational changes and confers gain-of-function properties that set transport rates.

Transporters are integral membrane proteins that move solutes across lipid bilayers. They undergo concerted conformational changes, allowing alternate exposure of their substrate-binding sites to external and internal solutions (1). In each of these so-called outward- and inward-facing states (OFS and IFS, respectively), further isomerizations accompany substrate binding and release. Transport efficiency depends on the rates of these rearrangements, but linking function and structural dynamics has presented methodological challenges. Single-molecule Forster resonance energy transfer (smFRET)-based total internal reflection fluorescence (TIRF) microscopy (2–4) has been used to monitor the dynamics of the OFS to IFS transitions (5–8) and single-transporter activity (9) in the elevator-type transporter Glt_Ph and other transporters (10–18). Hydrogen–deuterium exchange followed by mass spectrometry (HDX-MS) has been used to pinpoint local changes in structural dynamics in diverse biological systems (19–21). Here, we combine these approaches to link changes in local protein dynamics to the larger-scale conformational transitions and substrate transport in wild-type (WT) and gain-of-function mutants of Glt_Ph.Glt_Ph is an extensively studied archaeal aspartate transporter that is homologous to human excitatory amino acid transporters (EAATs). Structures of Glt_Ph (7, 22–30), and archaeal and mammalian homologs (31–37), show that the transporters assemble into homotrimers via scaffold domains. Each protomer features a mobile transport domain that binds l-Aspartate (l-Asp) and three Na⁺ ions (22, 23, 28, 31, 38) and symports the solutes by an elevator mechanism, moving ∼15 Å across the membrane from an OFS to an IFS (6, 8, 23, 24, 39). During the elevator transitions, two structurally symmetric helical hairpins (HPs) 1 and 2 form the cores of the domain interfaces in the OFS and IFS, respectively (SI Appendix, Fig. S1A) (23, 24, 40). Despite symmetry, they do not have the same function. HP1 is mostly rigid, while HP2 is a conformationally plastic “master regulator” of the transporter, gating substrate in the OFS and IFS and contributing to setting the elevator transition rates (5, 23, 24, 27, 29, 36, 41–47).In this study, we use three previously characterized mutants of Glt_Ph to pinpoint the rate-limiting steps of the transport cycle and probe the protein dynamic properties that correlate with increased transport rates. A K290A mutation at the base of HP1 disrupts a salt bridge with the scaffold domain in the OFS and dramatically increases the elevator dynamics (5, 6). A triple-mutant Y204L/A345V/V366A displays a more modest increase in elevator dynamics and substantially diminished l-Asp affinity (5). Finally, a Y204L/K290A/A345V/V366A mutant combines these substitutions and their effects (5). We compared our previously obtained smFRET data on the elevator dynamics of the WT transporter and the mutants (5) to single-transporter uptake measurements. For WT Glt_Ph, these dynamics and transport measurements established transporter subpopulations that move (5, 6) and work (9) with rates differing by orders of magnitude, with slow transporters dominating the ensemble. We now show that only mutations that both reduce the population of the slow-moving transporters and weaken substrate affinity, such as Y204L/A345V/V366A, reduce the population of the slow-working transporters and confer overall gain-of-function properties. The slow-working population comprises transporters with rare elevator transitions or slow substrate release. We then used HDX-MS to explore how the Y204L/A345V/V366A mutant differed from the WT protein. We found that the mutations decreased the stability of the secondary structure around the substrate-binding site, suggesting that the increased local dynamics underlie reduced kinetic heterogeneity within the mutant transporter ensemble.     

Structural basis for high-voltage activation and subtype-specific inhibition of human Nav1.8

The dorsal root ganglia–localized voltage-gated sodium (Na_v) channel Na_v1.8 represents a promising target for developing next-generation analgesics. A prominent characteristic of Na_v1.8 is the requirement of more depolarized membrane potential for activation. Here we present the cryogenic electron microscopy structures of human Na_v1.8 alone and bound to a selective pore blocker, A-803467, at overall resolutions of 2.7 to 3.2 Å. The first voltage-sensing domain (VSD_I) displays three different conformations. Structure-guided mutagenesis identified the extracellular interface between VSD_I and the pore domain (PD) to be a determinant for the high-voltage dependence of activation. A-803467 was clearly resolved in the central cavity of the PD, clenching S6_IV. Our structure-guided functional characterizations show that two nonligand binding residues, Thr397 on S6_I and Gly1406 on S6_III, allosterically modulate the channel’s sensitivity to A-803467. Comparison of available structures of human Na_v channels suggests the extracellular loop region to be a potential site for developing subtype-specific pore-blocking biologics.

Voltage-gated sodium (Na_v) channels govern membrane excitability in neurons and muscles (1, 2). Despite high-degree sequence and architectural similarity, different subtypes of Na_v channels have specific tissue distributions and distinct voltage dependence and kinetics for activation, inactivation, and recovery (3). Among the nine mammalian Na_v channels (SI Appendix, Fig. S1), Na_v1.8, a tetrodotoxin (TTX)-resistant subtype encoded by SCN10A, is primarily expressed in the sensory neurons, exemplified by the dorsal root ganglia (DRG) neurons (4–6). Compared with other Na_v subtypes, Na_v1.8 has several unique biophysical properties, such as activation at more depolarized voltage and slower inactivation with persistent current, which enable the hyperexcitability of the DRG neurons (4, 5, 7–11).Na_v1.8 functions in pain sensation (12–16). Proexcitatory mutations of Na_v1.8 were identified in patients with painful small fiber neuropathy (17–19). On the other hand, a natural variant, A1073V, that shifts the voltage dependence of activation to more depolarized direction appeared to ameliorate pain symptoms (20). Specific inhibition of the peripheral Na_v1.8 thus represents a potential strategy for developing nonaddictive pain killers (21, 22).Several Na_v1.8-selective blockers, such as VX-150 and PF-06305591, had been tested in clinical trials. However, most of the drug candidates failed to meet the endpoint(s) of phase II trials for various reasons, such as unsatisfactory efficacy or selectivity (21, 23–26). Structures of Na_v1.8 bound to lead compounds will shed light on drug optimization for improving potency and selectivity. We focused on A-803467, a Na_v1.8-selective blocker, for structural analysis. A-803467 was shown to inhibit Na_v1.8 in both the resting state and inactivated state. Despite a wide range of concentration that inhibits response by 50% (IC₅₀) from several nanomolar to 1 μM measured by different groups, A-803467 consistently shows a higher affinity for the inactivated channel (27–31).In this study, we report the structures of full-length human Na_v1.8 alone and bound to A-803467. The first voltage-sensing domain (VSD_I) was resolved in multiple conformations. Based on the structural and electrophysiological characterizations, we attempt to address two questions: What underlies the high-voltage activation of Na_v1.8, and what determines the subtype specificity of A-803467?     

Negative effects of nitrogen override positive effects of phosphorus on grassland legumes worldwide

Anthropogenic nutrient enrichment is driving global biodiversity decline and modifying ecosystem functions. Theory suggests that plant functional types that fix atmospheric nitrogen have a competitive advantage in nitrogen-poor soils, but lose this advantage with increasing nitrogen supply. By contrast, the addition of phosphorus, potassium, and other nutrients may benefit such species in low-nutrient environments by enhancing their nitrogen-fixing capacity. We present a global-scale experiment confirming these predictions for nitrogen-fixing legumes (Fabaceae) across 45 grasslands on six continents. Nitrogen addition reduced legume cover, richness, and biomass, particularly in nitrogen-poor soils, while cover of non–nitrogen-fixing plants increased. The addition of phosphorous, potassium, and other nutrients enhanced legume abundance, but did not mitigate the negative effects of nitrogen addition. Increasing nitrogen supply thus has the potential to decrease the diversity and abundance of grassland legumes worldwide regardless of the availability of other nutrients, with consequences for biodiversity, food webs, ecosystem resilience, and genetic improvement of protein-rich agricultural plant species.

Anthropogenic enrichment of nitrogen (N), phosphorus (P), and other nutrients from fertilizers and fossil fuel combustion is transforming natural ecosystems worldwide (1–5), leading to increased terrestrial plant productivity (6, 7) and loss of biodiversity (8, 9). Resource competition theory proposes that the capacity of species to persist at low levels of a limiting resource is a key mechanism underpinning competitive success. Consequently, plant functional types with specialized nutrient acquisition strategies are expected to have a competitive advantage in nutrient-limited environments but also to be especially vulnerable to nutrient enrichment (10–13).Legumes (Fabaceae) are one of the largest families of flowering plants, contributing over 650 genera and 19,000 taxa to global plant diversity (14). This diversity is important for biodiversity conservation and for genetic improvement of protein-rich crops and forage species for sustainable livestock production (15–17). Furthermore, the ability to fix atmospheric N₂ is one of the most important plant functional traits for influencing ecosystem processes, conferring N-fixing legumes with a disproportionately important role in ecosystem functioning (18, 19). For example, litter produced by legumes is nitrogen-rich and more easily decomposed by soil microorganisms, leading to flow on effects to higher trophic levels, including increased complexity of food webs and resistance of soil biophysical and chemical properties to ecosystem disturbance (20). As the success of legumes often arises from this capacity for symbiotic fixation of atmospheric N₂ in N-limited environments (21, 22), atmospheric N-deposition and other pathways of anthropogenic N supply are expected to drastically reduce their competitive advantage in plant communities (1, 5, 11, 23). This is especially the case for obligate-N-fixers that cannot down-regulate N-fixation (24, 25) and hence at higher soil N are disadvantaged by the high energetic cost of N-fixation (26).While concerns about global nutrient enrichment are focused on impacts of N on biodiversity and ecosystem productivity (1, 2, 27), changes in P and potassium (K) cycles (3, 4) or altered concentrations of other nutrients, can also influence the abundance and diversity of legumes in accordance with resource competition theory (10–13). Owing to the physiological demands of N-fixation, N-fixing legumes often have higher requirements for P, K, and other nutrients [e.g., molybdenum (Mo), iron (Fe), and calcium (Ca)] than non–N-fixing plants (28–31), and increases in these nutrients can favor N-fixing over non–N-fixing species, particularly in nutrient poor soils (21, 22). However, added nutrients may have synergistic effects (6, 32), leading to uncertainties in the expected net effect of P addition on the abundance of N-fixing legumes (26). For example, the phosphatases required for P acquisition from soils are rich in N; N addition may increase phosphatase investment, conferring legumes a superior phosphorus acquisition capacity in P- and N-limited environments (25, 29). Conversely, multiple nutrient addition is expected to allow nonlegumes to compete more effectively with legume species. Resulting light limitation may suppress legume growth and reduce the survival and establishment of new legume individuals (8, 9), especially of those legumes that are unable to reduce the costs of N fixation through down-regulation (10, 11, 15, 33–35).Despite these theoretical predictions, empirical evidence for the individual and interactive effects of changes in nutrient availability on legumes in natural ecosystems is limited (29, 36–39). Some experimental studies have shown decreased legume abundance with N addition and increased with P addition, but these studies are typically conducted at a single site and show both positive and negative interactive effects among nutrients (e.g., refs. 37, 40, and 41). Furthermore, minimal evidence is available regarding the influence of K or micronutrient enrichment on legume responses (29), and the underlying mechanisms of legume responses to nutrient addition, such as soil and climatic conditions, have not been investigated at global scales (but see ref. 26 for forest ecosystems).Using data from the Nutrient Network global collaborative experiment [https://nutnet.org/ (42)], we measured the cover, richness, and biomass responses of N-fixing legumes (hereafter legumes) to standardized experimental nutrient additions in 45 grasslands across six continents (SI Appendix, Fig. S1 and Table S1). Grasslands are a globally significant biome, covering more than one-third of the Earth’s ice-free land surface, accounting for a third of terrestrial net primary production (43), and supporting the livelihoods of more than 1.3 billion people. They are subject to chronic atmospheric nitrogen deposition due to fossil fuel combustion and are likely candidates for direct nitrogen fertilization (44). While N emissions in many regions of Europe have declined leading to plateaus or reductions in deposition (45), deposition in other world grasslands, such as the Mongolian Steppe, have increased in recent decades (e.g., ref. 46). Experimental sites included temperate and anthropic grasslands that spanned a broad range of geographical locations and ecological conditions, although were mostly from temperate latitudes (39) (SI Appendix, Table S1 and Fig. S1; see Methods for details).Three nutrients (N, P, K⁺) were applied in factorial combinations, resulting in eight treatments enabling evaluation of the interactive effects of N, P, and K addition (6, 8) on legumes. Over 3 to 6 y, 10 g⋅m⁻² N, P, and K were added annually to their respective treatment plots at the beginning of each site’s growing season; other nutrients in the K⁺ treatment [sulfur (S), magnesium (Mg), and micronutrients] were applied only in the first year to avoid toxicity (42). These nutrient levels were selected to ensure they were high enough to reduce nutrient limitation at a wide diversity of sites. They are at the higher end of the range for agricultural fertilizer application rates globally (5), and higher than atmospheric nutrient deposition rates (1, 3, 41, 43). In particular, our N-addition rate was about three times maximum current N-deposition rates in European grasslands and more generally across the globe (1, 47, 48).We used a standardized protocol (6, 42) to annually measure cover, richness, and biomass of legumes, forbs, and grasses in 1-m² permanent plots (Methods), starting in the year prior to the first nutrient application (Y_initial). Across all years and sites, we recorded 170 species of N-fixing grassland legumes, comprising 50 genera (SI Appendix, Table S2). The most species-rich genera were Trifolium (25 spp.), Astragalus (12 spp.), Vicia (11 spp.), and Lupinus (11 spp.). Vicia sativa, Trifolium repens, and Vicia hirsuta were the most frequent species across our sites (9.1%, 5.1%, and 4.9% of total occurrences, respectively). Each site contained one to eight legume species (Methods and SI Appendix, Table S1). Most legume species were perennials (∼60%), including 10 woody or shrub species (∼6% of species). On average, ∼3% and 4% of total live cover comprised annual and perennial legumes, respectively.We present results of nutrient addition for the third and the last available sampling year (years 3 to 6) after starting nutrient application in each site [noting sites started applying experimental treatments in different calendar years and ran for different lengths of time (SI Appendix, Table S1)]. To measure the relative impact of N, P, and K⁺ addition on legumes, we calculated the log ratio (LR) of legume abundance and richness in the third or last year in each plot versus the initial (pretreatment) value [LR = ln (Y_final/Y_initial)]. We used the pretreatment legume abundance in the LR instead of control plots (49) to control for initial legume abundance and spatial variability among plots (8, 50). We also calculated measures of legume colonization and extinction in each plot, and evaluated the effect of initial soil nutrient concentrations, community structure, and climatic conditions as contingencies for nutrient addition effects (see Methods for details). We analyzed the data using linear mixed-effects models (51–53), with nutrient treatments (i.e., N, P, K⁺, and their interactions) as fixed effects, and blocks nested within sites as random effects. Confidence intervals for model parameters were bootstrapped as a conservative method for hypothesis testing (51, 52) (see Methods for details).     

Complex agricultural landscapes host more biodiversity than simple ones: A global meta-analysis

Managing agricultural landscapes to support biodiversity conservation requires profound structural changes worldwide. Often, discussions are centered on management at the field level. However, a wide and growing body of evidence calls for zooming out and targeting agricultural policies, research, and interventions at the landscape level to halt and reverse the decline in biodiversity, increase biodiversity-mediated ecosystem services in agricultural landscapes, and improve the resilience and adaptability of these ecosystems. We conducted the most comprehensive assessment to date on landscape complexity effects on nondomesticated terrestrial biodiversity through a meta-analysis of 1,134 effect sizes from 157 peer-reviewed articles. Increasing landscape complexity through changes in composition, configuration, or heterogeneity significatively and positively affects biodiversity. More complex landscapes host more biodiversity (richness, abundance, and evenness) with potential benefits to sustainable agricultural production and conservation, and effects are likely underestimated. The few articles that assessed the combined contribution of linear (e.g., hedgerows) and areal (e.g., woodlots) elements resulted in a near-doubling of the effect sizes (i.e., biodiversity level) compared to the dominant number of studies measuring these elements separately. Similarly, positive effects on biodiversity are stronger in articles monitoring biodiversity for at least 2 y compared to the dominant 1-y monitoring efforts. Besides, positive and stronger effects exist when monitoring occurs in nonoverlapping landscapes, highlighting the need for long-term and robustly designed monitoring efforts. Living in harmony with nature will require shifting paradigms toward valuing and promoting multifunctional agriculture at the farm and landscape levels with a research agenda that untangles complex agricultural landscapes’ contributions to people and nature under current and future conditions.

Agriculture expansion, intensification, and simplification dramatically contribute to biodiversity collapse (1–3). This is fueled by the acute lack of recognition in development agendas of agriculture’s dependence on biodiversity (4) and agriculture’s siloed and field-level planning (1, 2, 5–10). Hence, agriculture is underplaying a pivotal role in actively contributing to biodiversity conservation for the ecosystem services it provides, as well as its intrinsic or bequest values (6, 10–16). Many researchers are calling for better consideration of the multifunctional role of agricultural landscapes, including supporting biodiversity (17–19) and improving ecosystem resilience (16) and human well-being (20). The largest managed terrestrial land cover can, as such, be actively managed to offer a high-quality matrix connecting remanent patches of habitat to benefit biodiversity and people (21, 22).While increasing agriculture’s contribution to biodiversity conservation will require field- and landscape-level planning to leverage synergies with production goals (23), cross-sector and multiobjective landscape planning in agriculture policies or interventions remains rare. For example, agrienvironmental schemes, the most common legal mechanism in the European Union to foster sustainable agriculture management, rarely considers interactions and impacts at the landscape level, despite the large body of evidence showing that planning agriculture interventions at the landscape level contributes more efficiently to preserving biodiversity (9, 24–39), including specialist species (40), and even multiple ecosystem services (12, 41). Increasing landscape-level management in agricultural lands requires a science of landscape agronomy yet to be fully developed (23).Planning and managing agriculture with a landscape perspective acknowledges landscape-level patterns supporting vital ecological processes that enable biodiversity to persist. For example, agricultural landscapes with complex patterns (i.e., complex landscapes) mitigate regional extinctions by providing complementary and accessible resources that enable species survival and interpatch migration (42). Here, we consider landscape complexity to have three broad dimensions with distinct contributions to ecological processes: composition, configuration, and heterogeneity (SI Appendix). Landscape composition (e.g., remanent patches of forest) serves as the pool of biodiversity, landscape heterogeneity (e.g., number of crops) offers year-round resources in dynamic landscapes, whereas landscape configuration (e.g., edge length) enables species movement across the landscape (13, 42, 43). Although these dimensions are clear and well documented, understanding landscape complexity–biodiversity interaction is not straightforward. Landscape complexity and biodiversity are measured through a plethora of methods and metrics which could explain, in some cases, biodiversity’s nonlinear and inconsistent response to landscape complexity across species, taxa, and functional groups (e.g., 2, 29, 44–47).Despite these methodological challenges, complex landscapes can host larger species diversity, cascading into increased resilience, stability, and capacity to recover from disturbances (16, 40, 48, 49). Similarly, production in complex agricultural landscapes benefits from more diverse (in terms of evolutionary history) pollinator populations, improving ecosystem functioning and services (50, 51), while yields (50, 52–57) and crop products’ market and quality value (56, 58) can improve due to the more diverse pollinators and natural pest control populations.Multiple efforts have synthesized the effect of landscape complexity on biodiversity for specific taxa or functional groups [e.g., arthropods (45, 59) and pollinators (14, 60, 61)] and cropping systems [orchard and vineyards (14)], often using different metrics for biodiversity (e.g., species richness, abundance, or evenness) and landscape complexity dimensions, indicators, or metrics (SI Appendix, Table S1). The differences in scope and metrics used in existing reviews hinders consolidating the evidence base to identify consistent versus context-specific results that can then be used for tailoring biodiversity conservation recommendations in agricultural landscapes (60).We contribute to closing this knowledge gap by creating the most comprehensive global evidence map and meta-analysis to date of field experiments exploring the landscape complexity–biodiversity relationship. We first identify how landscape complexity is currently being measured and assessed in the scientific literature. Then, we use meta-analysis to explore landscape complexity effects on biodiversity in different agronomic and environmental contexts. Finally, we identify critical knowledge gaps that remain (missing crops, biodiversity indicators, and regions). Our comprehensive analysis provides insights and offers tangible next steps to live in harmony with nature by managing and shaping the largest land cover altered by humans.     

A novel class of TMPRSS2 inhibitors potently block SARS-CoV-2 and MERS-CoV viral entry and protect human epithelial lung cells

The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered covalent small-molecule ketobenzothiazole (kbt) TMPRSS2 inhibitors which are structurally distinct from and have significantly improved activity over the existing known inhibitors Camostat and Nafamostat. Lead compound MM3122 (4) has an IC₅₀ (half-maximal inhibitory concentration) of 340 pM against recombinant full-length TMPRSS2 protein, an EC₅₀ (half-maximal effective concentration) of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV-SARS-CoV-2 chimeric virus, and an EC₅₀ of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East respiratory syndrome coronavirus (MERS-CoV) cell entry with an EC₅₀ of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice, with a half-life of 8.6 h in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the newly emerged, highly transmissible coronavirus responsible for the ongoing COVID-19 pandemic, which is associated with 136 million cases and almost 3 million deaths worldwide as of April 12, 2021 (https://coronavirus.jhu.edu/map.html). While three vaccines have recently been approved by the FDA, there are still no clinically approved small-molecule drugs available for the treatment of this disease except Remdesivir, and the effectiveness of the vaccines against immune escape variants might be reduced. Multiple therapeutic strategies have been proposed (1−2), including both viral and host proteins, but none have yet been fully validated for clinical application. One class of protein targets which have shown promising results are proteolytic enzymes including the viral proteases (1, 3–5), Papain-Like Protease (PLpro) and the 3C-like or “Main Protease” (3CL or MPro), and several host proteases involved in viral entry, replication, and effects on the immune system creating the life-threatening symptoms of COVID-19 infection (4–6). The latter include various members of the cathepsin family of cysteine proteases, including cathepsin L, furin, and the serine proteases factor Xa, plasmin, elastase, tryptase, TMPRSS2, and TMPRSS4.Coronavirus (SARS CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus [MERS]) entry is mediated by the viral spike protein, which must be cleaved by host proteases in order to trigger membrane fusion and entry into the host cell after binding to the host cell receptor Angiotensin Converting Enzyme-2 (ACE2) (7–10). This is mediated by initial cleavage at the S1/S2 junction of spike, which is thought to occur during processing in the producer cell, followed by cleavage at the S2′ site either by serine proteases at the cell surface or by cathepsin proteases in the late endosome or endolysosome (9, 10). Whether serine or cathepsin proteases are used for S2′ cleavage is cell type dependent. While entry into Calu-3 (human lung epithelial) or HAE (primarily human airway epithelial) cells is cathepsin independent, entry into Vero cells (African green monkey kidney epithelial), which do not express the required serine proteases, depends exclusively on cathepsins (7, 9–11).TMPRSS2 (12) is a type II transmembrane serine proteases (TTSP) (13) that has been shown to be crucial for host cell viral entry and spread of SARS-CoV-2 (7, 8, 14–16), as well as SARS-CoV (17, 18), MERS-CoV (19), and influenza A viruses (20–27). The Spike protein requires proteolytic processing/priming by TMPRSS2 to mediate entry into lung cells; thus, small-molecule inhibitors of this target offer much promise as new therapeutics for COVID-19 and other coronavirus diseases (7, 8). TMPRSS2 expression levels dictate the entry route used by SARS-CoV-2 to enter cells, as reported recently (28). In cells that express little or no TMPRSS2, cell entry occurs via the endosomal pathway, and cleavage of spike protein is performed by cathepsin L. It has been demonstrated that the TMPRSS2-expressing lung epithelial Calu-3 cells are highly permissive to SARS-CoV-2 infection. The irreversible serine protease inhibitors Camostat (7) and Nafamostat (29) are effective at preventing host cell entry and replication of SARS-CoV-2 in Calu-3 cells through a TMPRSS2-dependent mechanism (14, 15).Herein, we report on the discovery of a class of substrate-based ketobenzothiazole (kbt) inhibitors of TMPRSS2 with potent antiviral activity against SARS-CoV-2 which are significantly improved over Camostat and Nafamostat. Several compounds were found to be strong inhibitors of viral entry and replication, with EC₅₀ (half-maximal effective concentration) values exceeding the potency of Camostat and Nafamostat and without cytotoxicity. Newly developed compound MM3122 (4) has excellent pharmacokinetics (PK) and safety in mice and is thus a promising lead candidate drug for COVID-19 treatment.     

An ultrafast and facile nondestructive strategy to convert various inefficient commercial nanocarbons to highly active Fenton-like catalysts

The Fenton-like process catalyzed by metal-free materials presents one of the most promising strategies to deal with the ever-growing environmental pollution. However, to develop improved catalysts with adequate activity, complicated preparation/modification processes and harsh conditions are always needed. Herein, we proposed an ultrafast and facile strategy to convert various inefficient commercial nanocarbons into highly active catalysts by noncovalent functionalization with polyethylenimine (PEI). The modified catalysts could be in situ fabricated by direct addition of PEI aqueous solution into the nanocarbon suspensions within 30 s and without any tedious treatment. The unexpectedly high catalytic activity is even superior to that of the single-atom catalyst and could reach as high as 400 times higher than the pristine carbon material. Theoretical and experimental results reveal that PEI creates net negative charge via intermolecular charge transfer, rendering the catalyst higher persulfate activation efficiency.

Due to the rapid pace of urbanization and heavy industrialization, organic pollutants in the aquatic environment have become a serious and ubiquitous problem on a global scale. The Fenton or Fenton-like process, an effective approach to generate active species by activating oxidizing agents for the elimination of a wide range of organic pollutants, has been regarded as a promising strategy to deal with the ever-growing environmental pollution (1, 2). Among the various oxidants, persulfate (S₂O₈²⁻; PS), as an inexpensive, environmentally friendly, and easily handled strong oxidant (E⁰ = 2.1 V), has been widely utilized in various fields, including water oxidation (3, 4), chemical analysis (5, 6), microbial/microfluidic fuel cell (7, 8), organic (molecular/polymer) synthesis (9, 10), and environmental remediation (11, 12), ranging from bench-scale experiments to industrial processes. Since originally introduced for in situ soil and groundwater remediation in the late 1990s to overcome the technical limitations of hydrogen peroxide (H₂O₂) (13), the PS-based Fenton-like system has drawn significant attention as an alternative to the H₂O₂-based Fenton process in water/wastewater treatment, owing to the advantages including high oxidation capacity under circumstance conditions (at neutral pH or with background constituents), high-yield radical production, low cost of storage/transportation, and various activation strategies (11, 12). Over the past few years, various transition-metal-based materials have been widely investigated as Fenton-like catalysts for PS activation (14–16). In terms of sustainable development, metal-based catalysts suffer from prohibitive cost, scarcity in nature, and secondary pollution. In this regard, metal-free carbon materials such as carbon nanotubes (CNTs) and graphene are promising candidates, owing to their unique structures and properties (17–19).In general, the efficiencies of pristine nanocarbons in activation of PS are very low (19, 20). Researchers have successively explored different strategies to boost the PS activation by regulating the local electronic environment of carbocatalysts, such as covalent doping of suitable heteroatoms (e.g., N or S) (17, 21, 22), introduction of intrinsic defects on the edge (23, 24), and construction of specific structure (25). However, the existing strategies usually involve complicated preparation processes (the usage of templates), special equipment (plasma devices), and/or harsh reaction conditions (high-temperature annealing under special atmosphere), which are time-consuming and more laborious, greatly increasing the financial costs for large-scale production and industrialization (22, 25, 26).In principle, PS activation relies mainly on the cleavage of peroxyl bonds induced by electron transfer from catalysts, and the electron-transfer efficiency could be modulated by tuning the electron states (i.e., charge/spin density or density of state) of the catalysts (14, 18, 27). Recently, various polyelectrolytes, including polyethylenimine (PEI), have been utilized to tune the surface electronic structures of electrodes in electronic/electrochemical applications (28–31). Inspired by these observations, herein, we explored an ultrafast and facile approach to transform inefficient commercial nanocarbons into highly active, metal-free catalysts for PS activation by noncovalent functionalization with PEI (SI Appendix, Scheme S1). Density functional theory (DFT) calculations, X-ray photoelectron spectroscopy (XPS), Raman spectroscopy, and Kelvin probe force microscopy (KPFM) revealed that PEI could tune the local electronic environment of carbon atoms through the intermolecular electron donation from PEI to nanocarbons. As a result, the activities of the modified catalysts could be greatly enhanced and reach as high as 400 times higher than the pristine carbon material. Moreover, PEI-nanocarbon membranes were further in situ fabricated and applied to treat wastewater in a continuous-flow mode, revealing the feasibility of its practical application.     

Volcanically driven lacustrine ecosystem changes during the Carnian Pluvial Episode (Late Triassic)

The Late Triassic Carnian Pluvial Episode (CPE) saw a dramatic increase in global humidity and temperature that has been linked to the large-scale volcanism of the Wrangellia large igneous province. The climatic changes coincide with a major biological turnover on land that included the ascent of the dinosaurs and the origin of modern conifers. However, linking the disparate cause and effects of the CPE has yet to be achieved because of the lack of a detailed terrestrial record of these events. Here, we present a multidisciplinary record of volcanism and environmental change from an expanded Carnian lake succession of the Jiyuan Basin, North China. New U–Pb zircon dating, high-resolution chemostratigraphy, and palynological and sedimentological data reveal that terrestrial conditions in the region were in remarkable lockstep with the large-scale volcanism. Using the sedimentary mercury record as a proxy for eruptions reveals four discrete episodes during the CPE interval (ca. 234.0 to 232.4 Ma). Each eruptive phase correlated with large, negative C isotope excursions and major climatic changes to more humid conditions (marked by increased importance of hygrophytic plants), lake expansion, and eutrophication. Our results show that large igneous province eruptions can occur in multiple, discrete pulses, rather than showing a simple acme-and-decline history, and demonstrate their powerful ability to alter the global C cycle, cause climate change, and drive macroevolution, at least in the Triassic.

The Carnian Pluvial Episode (CPE; ca. 234 to ∼232 Ma; Late Triassic) was an interval of significant changes in global climate and biotas (1, 2). It was characterized by warming (3, 4) and enhancement of the hydrological cycle (5–7), linked to repeated C isotope fluctuations (8–11) and accompanied by increased rainfall (1), intensified continental weathering (9, 12), shutdown of carbonate platforms (13), widespread marine anoxia (4), and substantial biological turnover (1, 2, 10). Available stratigraphic data indicate that the Carnian climatic changes broadly coincide with, and could have been driven by, the emplacement of the Wrangellia large igneous province (LIP) (2, 4, 7, 8, 10, 14, 15) (Fig. 1A). It is postulated that the voluminous emission of volcanic CO₂, with consequent global warming and enhancement of a mega-monsoonal climate, was responsible for the CPE (9, 16), although the link is imprecise (2, 17) because the interval of Wrangellian eruptions have not yet been traced in the sedimentary records encompassing the CPE.Open in a separate windowFig. 1.Location and geological context for the study area. (A) Paleogeographic reconstruction for the Carnian (∼237 to 227 Ma) Stage (Late Triassic), showing locations of the study area and volcanic centers (revised after ref. 4, with volcanic data from refs. 4, 7, 49, and 50). (B) Tectono-paleogeographic map of the NCP during the Late Triassic (modified from ref. 21), showing the location of the study area. (C) Stratigraphic framework of the Upper Chunshuyao Formation (CSY) to the Lower Yangshuzhuang (YSZ) Formation from the Jiyuan Basin (modified from ref. 20). Abbreviations: LIP, Large Igneous Province; QDOB, Qingling-Dabie Orogenic Belt; S-NCP, southern NCP; SCP, South China Plate; Fm., Formation; m & s, coal, mudstone, and silty mudstone; s., sandstone; c, conglomerate; Dep. env., Depositional environment; and C.-P., Coniopteris-Phoenicopsis.The CPE was originally identified because of changes in terrestrial sedimentation, but most subsequent studies have been on marine strata (2, 4, 7–10). By contrast, much less is known about the effects of this climatic episode on terrestrial environments (2), although there were major extinctions and radiations among animals (including dinosaurs, crocodiles, turtles, and the first mammals and insects) and modern conifer families (2). Some of the new organisms may have flourished because of the spread of humid environments, such as the turtles and metoposaurids (18, 19).In this study, we have investigated terrestrial sediments from the Zuanjing-1 (ZJ-1) borehole in the Jiyuan Basin of the southern North China Plate (NCP) and use zircon U–Pb ages from two tuffaceous claystone horizons, fossil plant biostratigraphy, and organic C isotope (δ¹³C_org) and Hg chemostratigraphy to identify the CPE and volcanic activity.     

Recent Advances in Archaeological Science Techniques Special Feature: A Neandertal dietary conundrum: Insights provided by tooth enamel Zn isotopes from Gabasa,Spain

The characterization of Neandertals’ diets has mostly relied on nitrogen isotope analyses of bone and tooth collagen. However, few nitrogen isotope data have been recovered from bones or teeth from Iberia due to poor collagen preservation at Paleolithic sites in the region. Zinc isotopes have been shown to be a reliable method for reconstructing trophic levels in the absence of organic matter preservation. Here, we present the results of zinc (Zn), strontium (Sr), carbon (C), and oxygen (O) isotope and trace element ratio analysis measured in dental enamel on a Pleistocene food web in Gabasa, Spain, to characterize the diet and ecology of a Middle Paleolithic Neandertal individual. Based on the extremely low δ⁶⁶Zn value observed in the Neandertal’s tooth enamel, our results support the interpretation of Neandertals as carnivores as already suggested by δ¹⁵N isotope values of specimens from other regions. Further work could help identify if such isotopic peculiarities (lowest δ⁶⁶Zn and highest δ¹⁵N of the food web) are due to a metabolic and/or dietary specificity of the Neandertals.

Over the last 30 years, analyses of nitrogen isotopes in collagen (δ¹⁵N_collagen) have provided direct evidence for Neandertal diets across Europe and Asia. These studies all indicate a carnivorous (1–12), or at least a meat-heavy, diet for European Neandertals. However, one peculiarity of Neandertal δ¹⁵N_collagen remains the subject of an ongoing debate. From the one Siberian and eight western European sites, where both Neandertal and associated fauna have been analyzed, nitrogen isotope ratios in Neandertal collagen are systematically higher than that of other carnivores (3, 6–8, 10, 11, 13, 14). An explanation for such elevated values could be the consumption of herbivores, such as mammoths, which themselves exhibit elevated δ¹⁵N values due to the consumption of plants growing on arid soils (1, 2, 7). While mammoth remains are usually scarce at Neandertal fossil localities, they were nonetheless occasionally consumed, as suggested by remains with cut marks and other human butchery signatures (15). The absence of mammoth remains at Middle Paleolithic sites could be a result of 1) Neandertals chose to leave large bone elements at the kill site and transport other edible carcass products, mainly meat, back to the habitation site (15), or 2) mammoths were not frequently consumed, and the δ¹⁵N peculiarity consequently reflects the consumption of other resources enriched in ¹⁵N.Alongside this δ¹⁵N peculiarity, one major obstacle to our knowledge of Neandertals’ subsistence patterns is that the preservation of organic matter limits the application of collagen-bound nitrogen isotope analysis to fossil specimens. Collagen degrades over time at a varying speed depending on climatic and environmental conditions (16). The oldest hominin specimen in which bone protein is preserved is that of Scladina (Belgium), which dates to 90,000 cal BP (calibrated years before the present) (17), but most studied specimens are younger than 50,000 cal BP (1–3, 6–8, 10–13, 18). Furthermore, these specimens are only from sites in northwestern and central Europe and Siberia, where climatic conditions favored collagen preservation. As a result, the variability of Neandertals’ diet over time and between regions may not accurately be reflected by the currently available isotope data. In Iberia, where the latest surviving Neandertals have been discovered (19, 20), collagen was successfully extracted for only one site (21). Therefore, our knowledge of Iberian Neandertal diets mostly relies on zooarcheological and dental calculus data, which show some inconsistencies (21–25). For instance, similar to other western European sites, zooarcheological studies emphasize the consumption of terrestrial mammals and birds (21). In contrast, analysis of dental calculus for microremains and ancient DNA metagenomic analysis (26–28) highlight the frequent consumption of plants and mushrooms. Indeed, Weyrich et al. (26) even suggest that Neandertals at El Sidrón (Fig. 1) rarely consumed meat but often ate mushrooms, which would also result in elevated δ¹⁵N values (29). The consumption of marine foods is also attested for coastal Neandertals, but its frequency cannot be truly assessed in the absence of isotope studies (21, 23–25, 30). Finally, cannibalism has been documented at two Iberian sites (El Sidrón and Zafarraya) (22, 31) (Fig. 1), though such practices appear limited and most likely occurred only during periods of nutritional stress (32). Therefore, it is challenging to confirm the homogeneity of Neandertals’ diets across time and space, calling into question a direct link between their subsistence strategy and disappearance.Open in a separate windowFig. 1.(A) Location of the Gabasa site as well as other Neandertal sites mentioned in the text. (B) Detailed map of the Gabasa region. San Estaban de Litera and Benabarre are nearby modern cities.This study aims to investigate if the Zn isotope proxy could help elucidate the dietary behaviors of Neandertals and the source of their δ¹⁵N peculiarity, specifically by studying a Late Pleistocene Iberian food web where the presence of mammoth has not been documented (33). The development of Zn isotope analysis (⁶⁶Zn/⁶⁴Zn, expressed as δ⁶⁶Zn) has proven that trophic level information can be retrieved from mammalian tooth enamel (δ⁶⁶Zn_enamel) (34, 35), including fossil samples from Pleistocene food webs where organic matter is typically not preserved (36, 37). Previous studies have demonstrated that δ⁶⁶Zn_enamel decreases by ca. 0.30 to 0.60 ‰ with each step in archeological and modern food webs (34–38) and that δ⁶⁶Zn values associated with breastfeeding are higher than postweaning-associated values (39). While the main source of variation of δ⁶⁶Zn_enamel values is diet, local geology can also likely influence the isotope ratio of a given animal (36, 39). To date, three modern assemblages from Koobi Fora (Kenya), Kruger Park, and the western Cape (South Africa) (40), a few animals from a historical site (Rennes, France) (41), and three Late Pleistocene sites (Tam Hay Marklot, Nam Lot, and Tam Pa Ling, Laos) (36, 37) are the only terrestrial food webs for which Zn isotope data in teeth and/or bones have been published (SI Appendix, Fig. S14). In the modern Koobi Fora savannah food web, δ⁶⁶Zn_enamel differences have been observed between browsers and grazers (35), but this pattern was not seen in any of the three Pleistocene Asian forest food webs (36, 37). Among modern and historical human populations, historically documented diets relying on plants are associated with higher δ⁶⁶Zn values than those that include a substantial quantity of animal products (41–44). Zinc isotopes of ancient hominins have been analyzed only in one Pleistocene Homo sapiens individual (37) from Southeast Asia, but not yet in any Neandertal specimen.This current study contributes significantly to our understanding of Iberian Neandertal diets by providing information on their trophic ecology for a region where traditional nitrogen isotope analyses are unfeasible due to the poor preservation of organic matter. We use the Zn isotopic tool as well as other mobility, ecological, and dietary proxies applied on tooth enamel from hominin and animal remains from the cave site Cueva de los Moros 1 (Gabasa, Pyrenees, Spain; Fig. 1). The site, excavated in the 1980s, is very well documented [for stratigraphic context, see Montes and Utrilla (45) and SI Appendix, Section S1]. All remains come from layers e, f, and g of a single stratigraphic layer directly above layer h dated to 143 ± 43 ka. Numerous carnivore remains were recovered along with Neandertal remains (layers e and f), allowing for comparison of the different meat-eating taxa. Coexisting herbivores from three different types of environmental contexts are homogeneously represented in layers e, f, and g: mountains (Iberian ibex [Capra pyrenaica], chamois [Rupicapra rupicapra]), forest (cervids including red deer [Cervus elaphus]), and open environments (horses [Equus ferus], European wild asses [Equus hydruntinus]).     

C1q binding to surface-bound IgG is stabilized by C1r2s2 proteases

Complement is an important effector mechanism for antibody-mediated clearance of infections and tumor cells. Upon binding to target cells, the antibody’s constant (Fc) domain recruits complement component C1 to initiate a proteolytic cascade that generates lytic pores and stimulates phagocytosis. The C1 complex (C1qr₂s₂) consists of the large recognition protein C1q and a heterotetramer of proteases C1r and C1s (C1r₂s₂). While interactions between C1 and IgG-Fc are believed to be mediated by the globular heads of C1q, we here find that C1r₂s₂ proteases affect the capacity of C1q to form an avid complex with surface-bound IgG molecules (on various 2,4-dinitrophenol [DNP]-coated surfaces and pathogenic Staphylococcus aureus). The extent to which C1r₂s₂ contributes to C1q–IgG stability strongly differs between human IgG subclasses. Using antibody engineering of monoclonal IgG, we reveal that hexamer-enhancing mutations improve C1q–IgG stability, both in the absence and presence of C1r₂s₂. In addition, hexamer-enhanced IgGs targeting S. aureus mediate improved complement-dependent phagocytosis by human neutrophils. Altogether, these molecular insights into complement binding to surface-bound IgGs could be important for optimal design of antibody therapies.

Antibodies are important mediators of the human complement response, which offers critical protection against microbial infections and damaged host cells (1). In order to initiate a complement response, an antibody molecule first needs to bind antigens on the target cell via its antigen-binding (Fab) domains (2–5). Subsequently, the antibody’s constant (Fc) domain recruits the first complement protein complex, C1, to the cell surface (SI Appendix, Fig. S1A). The large C1 complex (also denoted as C1qr₂s₂, 766 kDa) consists of the recognition protein C1q (410 kDa) and a heterotetramer of serine proteases C1r and C1s (denoted C1r₂s₂, 356 kDa) (SI Appendix, Fig. S1B). While C1q is responsible for antibody recognition, its attached proteases C1r₂s₂ induce the activation of downstream enzymatic complexes (i.e., C3 convertases [C4b2b (6)]) that catalyze the covalent deposition of C3-derived molecules (e.g., C3b and its degradation product iC3b) onto the target cell surface (SI Appendix, Fig. S1A) (7, 8). C3b opsonizes the target cell surface and can induce formation of lytic pores (membrane attack complex [MAC]) in the target cell membrane (9–11). In contrast to human cells and gram-negative bacteria, gram-positive bacteria are not susceptible to the MAC due to their thick cell wall (12). On these bacteria, efficient decoration with C3b and iC3b is essential for triggering effective phagocytic uptake of target cells via complement receptors (CR) expressed on phagocytes of which the integrin CR3 (also denoted CD11b/CD18) is considered most important (13, 14).In recent years, our insights into IgG-dependent complement activation have increased significantly. A combination of structural, biophysical, and functional studies revealed that surface-bound IgG molecules (after Fab-mediated antigen binding) require organization into higher-ordered structures, namely hexamers, to induce complement activation most effectively (15–19). Hexamerized IgGs are being held together by noncovalent Fc–Fc interactions and form an optimal platform for C1q docking (SI Appendix, Fig. S1A). C1q has a “bunch of tulips–” like structure, consisting of six collagen arms that each end in a globular (gC1q) domain (SI Appendix, Fig. S1B) that binds the Fc region of an IgG. As the affinity of C1q for a single IgG is very weak (20, 21), avidity achieved through simultaneous binding of its globular domains to six oligomerized IgG molecules is paramount for a strong response (15, 17–19). Furthermore, it was found that IgG hexamerization could be manipulated by specific point mutations in the Fc–Fc contact region that enhance such oligomerization (15, 18, 22). While these hexamer-enhancing mutations in IgG potentiate the efficacy of MAC-dependent cytotoxicity on tumor cells and gram-negative bacteria (15, 23), their effect on complement-dependent phagocytosis is not known.Because complement is an important effector mechanism to kill bacteria and tumor cells, development of complement-enhancing antibodies represents an attractive strategy for immune therapies (1, 24). Immunotherapy based on human monoclonal antibodies is not yet available for bacterial infections (25–28). Such developments are mainly hampered by the fact that little is known about the basic mechanisms of complement activation on bacterial cells. For instance, we do not understand why certain antibodies induce complement activation on bacteria and others do not. In this study, we set out to investigate how antibacterial IgGs induce an effective complement response. By surprise, we noticed that C1q–IgG stability differs between human IgG subclasses. More detailed molecular investigations revealed that C1r₂s₂ proteases are important for generating stable C1q–IgG complexes on various target surfaces. Furthermore, we demonstrate that C1q–IgG stability is influenced by antibody oligomerization. These molecular insights into C1q binding to surface-bound IgGs may pave the way for optimal design of antibody therapies.     

From the Cover: Structural differences in amyloid-β fibrils from brains of nondemented elderly individuals and Alzheimer's disease patients

Although amyloid plaques composed of fibrillar amyloid-β (Aβ) assemblies are a diagnostic hallmark of Alzheimer''s disease (AD), quantities of amyloid similar to those in AD patients are observed in brain tissue of some nondemented elderly individuals. The relationship between amyloid deposition and neurodegeneration in AD has, therefore, been unclear. Here, we use solid-state NMR to investigate whether molecular structures of Aβ fibrils from brain tissue of nondemented elderly individuals with high amyloid loads differ from structures of Aβ fibrils from AD tissue. Two-dimensional solid-state NMR spectra of isotopically labeled Aβ fibrils, prepared by seeded growth from frontal lobe tissue extracts, are similar in the two cases but with statistically significant differences in intensity distributions of cross-peak signals. Differences in solid-state NMR data are greater for 42-residue amyloid-β (Aβ42) fibrils than for 40-residue amyloid-β (Aβ40) fibrils. These data suggest that similar sets of fibril polymorphs develop in nondemented elderly individuals and AD patients but with different relative populations on average.

Amyloid plaques in brain tissue, containing fibrils formed by amyloid-β (Aβ) peptides, are one of the diagnostic pathological signatures of Alzheimer''s disease (AD). Clear genetic and biomarker evidence indicates that Aβ is key to AD pathogenesis (1). However, Aβ is present as a diverse population of multimeric assemblies, ranging from soluble oligomers to insoluble fibrils and plaques, and may lead to neurodegeneration by a number of possible mechanisms (2–7).One argument against a direct neurotoxic role for Aβ plaques and fibrils in AD is the fact that plaques are not uncommon in the brains of nondemented elderly people, as shown both by traditional neuropathological studies (8, 9) and by positron emission tomography (10–13). On average, the quantity of amyloid is greater in AD patients (10) and (at least in some studies) increases with decreasing cognitive ability (12, 14, 15) or increasing rate of cognitive decline (16). However, a high amyloid load does not necessarily imply a high degree of neurodegeneration and cognitive impairment (11, 13, 17).A possible counterargument comes from studies of the molecular structures of Aβ fibrils, which show that Aβ peptides form multiple distinct fibril structures, called fibril polymorphs (18–20). Polymorphism has been demonstrated for fibrils formed by both 40-residue amyloid-β (Aβ40) (19, 21–24) and 42-residue amyloid-β (Aβ42) (22, 25–29) peptides, the two main Aβ isoforms. Among people with similar total amyloid loads, variations in neurodegeneration and cognitive impairment may conceivably arise from variations in the relative populations of different fibril polymorphs. As a hypothetical example, if polymorph A was neurotoxic but polymorph B was not, then people whose Aβ peptides happened to form polymorph A would develop AD, while people whose Aβ peptides happened to form polymorph B would remain cognitively normal. In practice, brains may contain a population of different propagating and/or neurotoxic Aβ species, akin to prion quasispecies or “clouds,” and the relative proportions of these and their dynamic interplay may affect clinical phenotype and rates of progression (30).Well-established connections between molecular structural polymorphism and variations in other neurodegenerative diseases lend credence to the hypothesis that Aβ fibril polymorphism plays a role in variations in the characteristics of AD. Distinct strains of prions causing the transmissible spongiform encephalopathies have been shown to involve different molecular structural states of the mammalian prion protein PrP (30–32). Distinct tauopathies involve different polymorphs of tau protein fibrils (33–37). In the case of synucleopathies, α-synuclein has been shown to be capable of forming polymorphic fibrils (38–40) with distinct biological effects (41–43).Experimental support for connections between Aβ polymorphism and variations in characteristics of AD comes from polymorph-dependent fibril toxicities in neuronal cell cultures (19), differences in neuropathology induced in transgenic mice by injection of amyloid-containing extracts from different sources (44–46), differences in conformation and stability with respect to chemical denaturation of Aβ assemblies prepared from brain tissue of rapidly or slowly progressing AD patients (47), and differences in fluorescence emission spectra of structure-sensitive dyes bound to amyloid plaques in tissue from sporadic or familial AD patients (48, 49).Solid-state NMR spectroscopy is a powerful method for investigating fibril polymorphism because even small, localized changes in molecular conformation or structural environment produce measurable changes in ¹³C and ¹⁵N NMR chemical shifts (i.e., in NMR frequencies of individual carbon and nitrogen sites). Full molecular structural models for amyloid fibrils can be developed from large sets of measurements on structurally homogeneous samples (21, 25, 26, 29, 38, 50). Alternatively, simple two-dimensional (2D) solid-state NMR spectra can serve as structural fingerprints, allowing assessments of polymorphism and comparisons between samples from different sources (22, 51).Solid-state NMR requires isotopic labeling and milligram-scale quantities of fibrils, ruling out direct measurements on amyloid fibrils extracted from brain tissue. However, Aβ fibril structures from autopsied brain tissue can be amplified and isotopically labeled by seeded fibril growth, in which fibril fragments (i.e., seeds) in a brain tissue extract are added to a solution of isotopically labeled peptide (21, 22, 52). Labeled “daughter” fibrils that grow from the seeds retain the molecular structures of the “parent” fibrils, as demonstrated for Aβ (19, 21, 24, 53) and other (54, 55) amyloid fibrils. Solid-state NMR measurements on the brain-seeded fibrils then provide information about molecular structures of fibrils that were present in the brain tissue at the time of autopsy. Using this approach, Lu et al. (21) developed a full molecular structure for Aβ40 fibrils derived from one AD patient with an atypical clinical history (patient 1), showed that Aβ40 fibrils from a second patient with a typical AD history (patient 2) were qualitatively different in structure, and showed that the predominant brain-derived Aβ40 polymorph was the same in multiple regions of the cerebral cortex from each patient. Subsequently, Qiang et al. (22) prepared isotopically labeled Aβ40 and Aβ42 fibrils from frontal, occipital, and parietal lobe tissue of 15 patients in three categories, namely typical long-duration Alzheimer''s disease (t-AD), the posterior cortical atrophy variant of Alzheimer''s disease (PCA-AD), and rapidly progressing Alzheimer''s disease (r-AD). Quantitative analyses of 2D solid-state NMR spectra led to the conclusions that Aβ40 fibrils derived from t-AD and PCA-AD tissue were indistinguishable, with both showing the same predominant polymorph; that Aβ40 fibrils derived from r-AD tissue were more structurally heterogeneous (i.e., more polymorphic); and that Aβ42 fibrils derived from all three categories were structurally heterogeneous, with at least two prevalent Aβ42 polymorphs (22).In this paper, we address the question of whether Aβ fibrils that develop in cortical tissue of nondemented elderly individuals with high amyloid loads are structurally distinguishable from fibrils that develop in cortical tissue of AD patients. As described below, quantitative analyses of 2D solid-state NMR spectra of brain-seeded samples indicate statistically significant differences for both Aβ40 and Aβ42 fibrils. Differences in the 2D spectra are subtle, however, indicating that nondemented individuals and AD patients do not develop entirely different Aβ fibril structures. Instead, data and analyses described below suggest overlapping distributions of fibril polymorphs, with different relative populations on average.     

Key role of organic carbon in the sunlight-enhanced atmospheric aging of soot by O2

Soot particles are ubiquitous in the atmosphere and have important climatic and health effects. The aging processes of soot during long-range transport result in variability in its morphology, microstructure, and hygroscopic and optical properties, subsequently leading to the modification of soot’s climatic and health effects. In the present study the aging process of soot by molecular O₂ under simulated sunlight irradiation is investigated. Organic carbon components on the surface of soot are found to play a key role in soot aging and are transformed into oxygen-containing organic species including quinones, ketones, aldehydes, lactones, and anhydrides. These oxygen-containing species may become adsorption centers of water and thus enhance the cloud condensation nuclei and ice nuclei activities of soot. Under irradiation of 25 mW·cm⁻², the apparent rate constants (k_1,obs) for loss or formation of species on soot aged by 20% O₂ were larger by factors of 1.5–3.5 than those on soot aged by 100 ppb O₃. Considering the abundance of O₂ in the troposphere and its higher photoreactivity rate, the photochemical oxidation by O₂ under sunlight irradiation should be a very important aging process for soot.Soot, which originates from incomplete combustion, is a mixture of elemental carbon and organic carbon (OC) compounds (1, 2). It has been widely recognized that soot particles in the atmosphere are partly responsible for global climate change. For instance, the contribution of soot to global warming from the direct absorption of solar radiation is estimated to be second only to that of CO₂ (3). Soot also contributes to regional climate change and air quality; it has been associated with the increase in droughts or floods in China over the past 20 y (4, 5) and with haze formation over South Asia (6). Soot also poses a health risk by causing and enhancing respiratory, cardiovascular, and allergic diseases (7).Once emitted into the atmosphere, soot undergoes aging processes through the uptake of reactive gases, such as OH, O₃, NO₂, NO₃, N₂O₅, HNO₃, and H₂SO₄ (8–17), and photochemical reactions (18). The aging processes of soot may not only affect the lifetime of some important gas-phase species, such as OH, O₃, and NO₃ (8–10, 14), but also modify the morphology, microstructure, and hygroscopic and optical properties of soot aggregates (19–22). At present, most studies have been focused on the kinetics of trace gas-phase species reactions with soot. Although large initial uptake coefficients for O₃ or NO₂ were measured in the dark, a rapid deactivation was usually observed because of the depletion of reactive sites (9, 11, 16, 23). Recently, it was found that UV light can persistently enhance the uptake reaction of NO₂ and O₃ on organic films or soot (24–28). This implies that photoreactions for soot may also be important in the troposphere. Compared with the uptake of gas-phase species by soot, however, the modification of soot during the aging processes remains unclear. A limited number of publications have studied changes of the surface composition and microstructure of soot during reaction with O₃, NO₂, and H₂SO₄ in the dark (2, 22, 29, 30). When soot was exposed to O₃ or NO₂, oxygen-related species or nitrogen-containing compounds were detected (2, 29, 30). Soot exposed to H₂SO₄ exhibited a marked change in morphology along with an increased fractal dimension and effective density (22). It should be pointed out that in all of these previous works, the aging processes focused only on the interactions between soot and gaseous pollutants in the dark. In this study, we investigated the aging process of soot resulting from photochemical oxidation by molecular O₂ and simulated sunlight. The photooxidation of surface OC is found to explain the aging process of soot.