Superelastic oxide micropillars enabled by surface tension–modulated 90° domain switching with excellent fatigue resistance

Superelastic materials capable of recovering large nonlinear strains are ideal for a variety of applications in morphing structures, reconfigurable systems, and robots. However, making oxide materials superelastic has been a long-standing challenge due to their intrinsic brittleness. Here, we fabricate ferroelectric BaTiO₃ (BTO) micropillars that not only are superelastic but also possess excellent fatigue resistance, lasting over 1 million cycles without accumulating residual strains or noticeable variation in stress–strain curves. Phase field simulations reveal that the large recoverable strains of BTO micropillars arise from surface tension–modulated 90° domain switching and thus are size dependent, while the small energy barrier and ultralow energy dissipation are responsible for their unprecedented cyclic stability among superelastic materials. This work demonstrates a general strategy to realize superelastic and fatigue-resistant domain switching in ferroelectric oxides for many potential applications.

Superelastic materials are capable of recovering large amount of nonlinear “plastic” strains, way beyond their linear elastic regimes (1–4). They are ideal for a variety of applications from morphing structures, reconfigurable systems, to robots (5–8). The effects have traditionally been associated with macroscopically compliant/ductile rubbers (2) or microscopically phase-transforming shape memory alloys (SMAs) (7–11). The only macroscopically brittle oxide recently discovered to be superelastic is ZrO₂-based micropillars or particles (12–20), which is realized via austenite-martensite phase transformation similar to SMAs. Although ultimate strengths approaching the theoretical limit have been demonstrated in nanoscale samples (21, 22), long fatigue life is elusive, which is arguably more important for most applications. As a matter of fact, poor fatigue life has been a long-standing challenge for oxide ceramics in general (23, 24). Even for ductile SMAs that enjoy excellent fatigue life, irrecoverable residual strains gradually accumulate over cycling, leading to substantial variations in stress–strain curves at different cycles (9, 10, 25). We overcome these difficulties by reporting superelastic barium titanate (BaTiO₃ [BTO]) micropillars enabled by surface tension–modulated 90° domain switching, which exhibit excellent fatigue resistance, while bulk BTO crystals or ceramics are rather brittle. The demonstration of over one million cycles of loading and unloading without accumulating residual strains or noticeable variation in stress–strain curves is unprecedented among superelastic materials.BTO is a ferroelectric oxide exhibiting modest piezoelectric strains around 0.1 to 0.2% (26) and fracture toughness of ∼1 MPa ⋅ m^1/2, and thus it is quite brittle (27). Considerable research efforts have been devoted to enhancing its electric field–induced strain via 90° ferroelectric domain switching (28–30). However, the process is often irreversible, and external mechanisms such as restoring force (28, 29) and internal mechanisms such as defect pinning (30) have to be invoked to make the electrostrain recoverable. Nevertheless, it hints at the possibility of BTO being made superelastic by taking advantage of the stress-induced 90° domain switching (6). Earlier works suggest that surface tension induces an in-plane compressive stress that favors the axial polarization in one-dimensional ferroelectrics at small size (31, 32), which may provide the necessary restoring mechanism for the stress-switched domains. Thus, if a compressive axial force is applied, reversible domain switching may occur during unloading, leading to superelasticity. To verify this hypothesis, we fabricated single-crystalline BTO micropillars from [001]-oriented bulk crystals (SI Appendix, Fig. S1A) via focused ion beam (FIB), as detailed in Materials and Methods and SI Appendix, Fig. S1B. The diameters (Φ) of the micropillars range from 0.5 μm to 5 μm, with their height to diameter ratio fixed at 3. No visible defects can be seen from the scanning electron microscopy (SEM) images of these micropillars shown in Fig. 1 A–D, and their surfaces appear to be quite smooth, suggesting that no apparent damages are induced by FIB.Open in a separate windowFig. 1.Superelastic BTO micropillars below a critical size. (A–D) SEM images of the micropillars with Φ = 5, 3, 2, and 0.5 μm. (E–G) The first and second cycles of stress–strain curves for BTO micropillars with Φ = 5, 2, and 0.5 μm. (H) S_r/S_max and ΔW/W_max during the first cycle for BTO micropillars of different diameters. Here, S_r and S_max denote the residual strain and the maximum strain (SI Appendix, Fig. S6A), while ΔW and W_max are energy dissipated and stored in the first cycle, respectively (SI Appendix, Fig. S6F).     

Air quality–related health damages of food

Agriculture is a major contributor to air pollution, the largest environmental risk factor for mortality in the United States and worldwide. It is largely unknown, however, how individual foods or entire diets affect human health via poor air quality. We show how food production negatively impacts human health by increasing atmospheric fine particulate matter (PM_2.5), and we identify ways to reduce these negative impacts of agriculture. We quantify the air quality–related health damages attributable to 95 agricultural commodities and 67 final food products, which encompass >99% of agricultural production in the United States. Agricultural production in the United States results in 17,900 annual air quality–related deaths, 15,900 of which are from food production. Of those, 80% are attributable to animal-based foods, both directly from animal production and indirectly from growing animal feed. On-farm interventions can reduce PM_2.5-related mortality by 50%, including improved livestock waste management and fertilizer application practices that reduce emissions of ammonia, a secondary PM_2.5 precursor, and improved crop and animal production practices that reduce primary PM_2.5 emissions from tillage, field burning, livestock dust, and machinery. Dietary shifts toward more plant-based foods that maintain protein intake and other nutritional needs could reduce agricultural air quality–related mortality by 68 to 83%. In sum, improved livestock and fertilization practices, and dietary shifts could greatly decrease the health impacts of agriculture caused by its contribution to reduced air quality.

The health and environmental consequences of feeding the increasingly large and affluent global population are becoming increasingly apparent. These consequences have spurred interest in identifying food production practices and diets that improve human health and reduce environmental harm. Recent work has demonstrated that many of the opportunities for food producers and consumers to improve nutritional outcomes also have environmental benefits, such as reducing greenhouse gas emissions, land and water use, and eutrophication (1–6). It is largely unknown, however, how individual foods and diets affect air quality, even though air pollution is the largest environmental mortality risk factor in the United States and globally (7, 8), and agriculture is itself known to be a major contributor to reduced air quality (8, 9). In the United States alone, atmospheric fine particulate matter (PM_2.5) from anthropogenic sources is responsible for about 100,000 premature deaths each year, one-fifth of which are linked to agriculture (10, 11).Here, we show how different foods affect human health by reducing air quality. We consider the emission of pollutants that contribute to atmospheric PM_2.5, the chronic exposure to which increases the incidence of premature mortality from cardiovascular disease, cancer, and stroke (12, 13). These pollutants include directly emitted PM_2.5 (primary PM_2.5) and PM_2.5 formed in the atmosphere (secondary PM_2.5) from the precursors ammonia (NH₃), nitrogen oxides (NO_x), sulfur dioxide (SO₂), and nonmethane volatile organic compounds (NMVOCs). From a spatially explicit inventory of emissions of primary PM_2.5 and secondary PM_2.5 precursors from agricultural supply chain activities for commodities in the contiguous United States (SI Appendix, Figs. S1 and S2) (14, 15) (Materials and Methods), we estimate increases in atmospheric concentrations of total (primary + secondary) PM_2.5 attributable to agricultural emissions; total PM_2.5 transport, chemistry, and removal; and exposure of populations to total PM_2.5 using an ensemble of three independent air quality models (16–19). We describe damages attributable to 95 agricultural commodities and 67 final food products (full list in SI Appendix, Table S1), which cover >99% of US agricultural production (20).     

Crystal structure of a far-red–sensing cyanobacteriochrome reveals an atypical bilin conformation and spectral tuning mechanism

Cyanobacteriochromes (CBCRs) are small, linear tetrapyrrole (bilin)-binding photoreceptors in the phytochrome superfamily that regulate diverse light-mediated adaptive processes in cyanobacteria. More spectrally diverse than canonical red/far-red–sensing phytochromes, CBCRs were thought to be restricted to sensing visible and near UV light until recently when several subfamilies with far-red–sensing representatives (frCBCRs) were discovered. Two of these frCBCRs subfamilies have been shown to incorporate bilin precursors with larger pi-conjugated chromophores, while the third frCBCR subfamily uses the same phycocyanobilin precursor found in the bulk of the known CBCRs. To elucidate the molecular basis of far-red light perception by this third frCBCR subfamily, we determined the crystal structure of the far-red–absorbing dark state of one such frCBCR Anacy_2551g3 from Anabaena cylindrica PCC 7122 which exhibits a reversible far-red/orange photocycle. Determined by room temperature serial crystallography and cryocrystallography, the refined 2.7-Å structure reveals an unusual all-Z,syn configuration of the phycocyanobilin (PCB) chromophore that is considerably less extended than those of previously characterized red-light sensors in the phytochrome superfamily. Based on structural and spectroscopic comparisons with other bilin-binding proteins together with site-directed mutagenesis data, our studies reveal protein–chromophore interactions that are critical for the atypical bathochromic shift. Based on these analyses, we propose that far-red absorption in Anacy_2551g3 is the result of the additive effect of two distinct red-shift mechanisms involving cationic bilin lactim tautomers stabilized by a constrained all-Z,syn conformation and specific interactions with a highly conserved anionic residue.

Cyanobacteria have developed elaborate, spectrally tuned photoreceptors and light-harvesting systems for adaptation and survival in a wide range of ecological niches (1–5). Many photoreceptor systems are modular components of much larger signaling proteins that integrate different sensor and effector modules into a single protein molecule to interface with diverse signal transduction pathways. Photoreceptors in the phytochrome superfamily utilize a specific lineage of GAF (cGMP phosphodiesterase, adenylyl cyclase and FhlA) domain that binds a thioether-linked linear tetrapyrrole (bilin) chromophore for light perception (6–11). Bilin-based photoreceptors play critical roles in plant development as well as in regulating cyanobacterial phototaxis, development, and light harvesting (2, 3, 12–17). Protein structural changes following the primary photochemical event then alter the downstream enzymatic activities and/or protein–protein interactions via an interdomain allosteric mechanism (18).Phytochromes possess a tripartite photosensory region consisting of three N-terminal domains (PAS, GAF, and PHY), known as the photosensory core module, in which the PAS and GAF domains are tethered via a “figure-eight knot” (14, 19, 20). In prototypical phytochromes, the bilin chromophore embedded in the GAF domain adopts a protonated 5-Z,syn, 10-Z,syn, 15-Z,anti configuration in the dark-adapted state. Light absorption triggers photoisomerization of the 15,16 double bond to generate a 15E,anti photoproduct, which typically absorbs far-red light (9, 14, 21). A long extension from the adjacent PHY domain is responsible for stabilizing the far-red–absorbing Pfr state (14, 20). In cyanobacteria, the phytochrome superfamily has diversified to yield a large family of more streamlined sensors, designated cyanobacteriochromes (CBCRs) (2, 4, 22–26). Unlike canonical phytochromes, CBCR photosensory modules consist of one or more GAF domains that are sufficient for covalent attachment of bilin and photoconversion. These small CBCR domains have also been used as light-sensing modules in a variety of synthetic biology applications (27–32). In contrast to canonical red/far-red phytochromes, CBCRs are able to sense light from near UV to far-red, utilizing a common phycocyanobilin (PCB) chromophore precursor (22–24, 26).The remarkable spectral diversity of CBCRs (SI Appendix, Fig. S1A) arises from extensive molecular evolution of the GAF domain scaffold. Many CBCRs leverage two thioether linkages to sense blue, violet, or near-UV light (8, 22, 23, 25, 33–35). Such “two-Cys” CBCRs possess an additional thioether linkage to the C10 methine bridge of the bilin that splits the chromophore in half, significantly shortening the conjugated π-system. Rupture of this covalent bond can occur upon 15Z/15E photoisomerization, which restores bilin conjugation across C10 to generate a photostate absorbing at wavelengths from teal to red (8, 33, 36, 37). Dual cysteine CBCRs have evolved multiple times, yielding a wide range of photocycles with (ultra)violet, blue, teal, green, orange, and red states (22).Red/green CBCRs such as AnPixJg2 and NpR6012g4 have red-absorbing dark states similar to phytochromes that photoconvert to green-absorbing lit states. In this CBCR subfamily, the molecular mechanism responsible for photoproduct tuning relies on trapping the 15E bilin in a twisted geometry that results in blue-shifted absorption (10, 11). In contrast, green/red CBCRs exhibit a reversed photocycle: the green-absorbing 15Z dark state photoconverts to yield a red-absorbing 15E photoproduct. This subfamily uses a protochromic mechanism first reported for the light-regulated histidine kinase RcaE (SI Appendix, Fig. S1B) in which photoconversion triggers a proton transfer to an uncharged chromophore inducing a spectral red shift (2, 38).Until recently, the light-sensing range of CBCRs appeared limited to the visible spectrum, thereby implicating phytochromes to be exclusively responsible for far-red sensing in cyanobacteria. Indeed, far-red–dependent remodeling of the photosynthetic apparatus in multiple cyanobacterial species is mediated by the red/far-red phytochrome RfpA (3, 39). The discovery of two lineages of CBCRs with far-red-absorbing dark states (frCBCRs) was thus surprising (40). Upon far-red light absorption, these frCBCRs convert to either an orange- or red-absorbing photoproduct state. These frCBCRs evolved from green/red CBCRs as part of a greater green/red (GGR) lineage and independent from evolution of other frCBCRs within the XRG (extended red/green) lineage (35, 40, 41). Owing to their small size and spectral overlap with the therapeutic window of optimum tissue penetrance (700 to 800 nm) (42–46), frCBCRs represent tantalizing scaffolds for development of FR-responsive optogenetic reagents for biomedical research and imaging applications (45, 47–50).To understand the molecular basis of far-red spectral tuning of the frCBCR family that evolved within GGR lineage, we determined the crystal structures of the FR-absorbing dark state of the representative FR/O CBCR Anacy_2551g3 from Anabaena cylindrica PCC 7122 at both ambient and cryogenic temperatures. These structures revealed an all-Z,syn configuration of its PCB chromophore that differs from those found in all known CBCRs and phytochromes. Based upon these crystallographic results, spectra of site-directed mutants of Anacy_2551g3 and related frCBCRs in the GGR lineage, and comparisons with other bilin-binding proteins, we identify key protein–chromophore interactions that support two tuning mechanisms simultaneously at work for far-red light detection in this family of frCBCRs.     

High-resolution multicontrast tomography with an X-ray microarray anode–structured target source

Multicontrast X-ray imaging with high resolution and sensitivity using Talbot–Lau interferometry (TLI) offers unique imaging capabilities that are important to a wide range of applications, including the study of morphological features with different physical properties in biological specimens. The conventional X-ray TLI approach relies on an absorption grating to create an array of micrometer-sized X-ray sources, posing numerous limitations, including technical challenges associated with grating fabrication for high-energy operations. We overcome these limitations by developing a TLI system with a microarray anode–structured target (MAAST) source. The MAAST features an array of precisely controlled microstructured metal inserts embedded in a diamond substrate. Using this TLI system, tomography of a Drum fish tooth with high resolution and tri-contrast (absorption, phase, and scattering) reveals useful complementary structural information that is inaccessible otherwise. The results highlight the exceptional capability of high-resolution multicontrast X-ray tomography empowered by the MAAST-based TLI method in biomedical applications.

Adding phase and scattering/darkfield contrast to the conventional absorption contrast in X-ray microscopy is a rapidly expanding research field because it offers tremendous advantages in a wide range of applications. The different contrast mechanisms are highly complementary, as they feature different sample–beam interactions that fingerprint different material properties. For example, the real and imaginary parts of the refractive index exhibit very significant differences in their absolute values (SI Appendix, Fig. S1) and represent the phase shift and attenuation of the X-ray, respectively (1–7). This has major implications for biomedical imaging, e.g., mammography (6, 8, 9), lung pathology (10–13), and industrial applications such as the structural investigation of carbon-reinforced polymer composites (14, 15). With this motivation, a variety of X-ray phase-contrast imaging (XPCI) techniques have been developed (2, 5–7, 9, 16), and there is a strong emphasis on achieving the phase-contrast efficiently and quantitatively (17–20). Among all these methods, grating-based XPCI (GXPCI), especially the Talbot–Lau interferometry (TLI) (3), is a leading contender for bringing XPCI into widespread adoption. Its unique advantages include 1) the compatibility with conventional, low-brilliance laboratory X-ray sources (3), 2) high sensitivity at high X-ray energy (20), and 3) desirable spatial resolution down to the micrometer level (21). Furthermore, it simultaneously provides three different contrast mechanisms: attenuation, refraction (differential phase-shift), and scattering (dark-field) in a single GXPCI dataset. The multicontrast modalities of TLI can offer valuable and complementary information for better discrimination of different structural components with different physical properties (4, 19). The GXPCI-enabled scattering contrast corresponds to the ultra-small-angle scattering strength of a material and offers excellent sensitivity to the morphological features that are much finer than the nominal spatial resolution (22–24). The phase-contrast component, on the other hand, quantitatively reconstructs the spatial distribution of the electron density, which is a fundamental material property that has different implications in different applications (20, 25). For example, the electron density of a battery cathode material fingerprints its state of charge and evolves as the battery is charged and discharged (26). Therefore, the three-dimensional (3D) electron density map of a battery electrode can be used to quantify the reaction heterogeneity, which is critical to the battery performance. The extraordinary potential of TLI tomography is reflected by the broad interest in applying this method to biological and medical imaging (5, 6, 8, 9, 12, 13), nondestructive testing (14, 27), materials science (19), and security screening (28). There are, however, key limitations of this technique that have yet to be addressed.A main advantage of TLI is its compatibility with a high-power laboratory X-ray source, which could largely improve the throughput of the experiment (3). In a conventional TLI setup, an absorption source grating (G0) is utilized to formulate a structured illumination pattern (Fig. 1A). G0 is inserted near the exit window of the X-ray tube for improving the spatial coherence and for reinforcing a geometrical constraint that matches the configuration of the downstream optics (SI Appendix, Fig. S2). The drawback of using the source grating G0 is that more than half of the X-ray’s source flux is wasted, significantly jeopardizing the efficiency of the imaging system. To increase the imaging sensitivity, one needs to use G0 gratings with fine periods as small as a few micrometers (29). Meanwhile, to ensure a sufficient X-ray transmission contrast, the thickness of the G0 grating lines has to reach several tens of micrometers, manifesting a desire for a high aspect ratio (AR) that is technically very challenging (30). To overcome this issue, Thüring et al. tilted the gratings with respect to the beam to effectively increase the AR. This approach, however, significantly reduces the useful field of view (FOV), compromising the practicality of this technique (30). Additionally, a G0 with large AR collimates the X-ray beam, which is another reason for the diminished FOV (∝AR⁻¹) (SI Appendix, Fig. S3) (21). We acknowledge that curved and tiled gratings can potentially be fabricated to alleviate this issue; however, they are associated with great challenges in microfabrication, particularly when targeting high a AR and small radius (21, 31). Several approaches have been introduced to circumvent the use of G0 and its associated limitations. One demonstrated approach involves fabricating grooves on an anode target for structured illumination. However, this method has a rather limited FOV because the spatial coherence property changes as a function of the target position (32). Morimoto et al. developed a TLI with a transmission grating by using a structured X-ray source. They demonstrated two-dimensional imaging results with a rather low spatial resolution and at a low working energy (20 kV) (33, 34). Despite the tremendous research efforts devoted to this field, the aforementioned challenges have hindered the broad adoption of TLI as a standard tool for high-resolution structural investigations with high-energy X-rays.Open in a separate windowFig. 1.Schematic comparison of the conventional TLI setup and our approach with a MAAST source is shown in A. SEM images of the MAAST pattern with etched grooves (B) and with W-MMIs embedded in the polycrystalline diamond substrate (C).To tackle these challenges, we developed a TLI system with a microarray anode–structured target (MAAST) X-ray source. We overcome the limitations of the conventional configuration with an extended source and a G0 grating by designing and incorporating the illumination pattern into the MAAST source as a built-in feature. Our approach significantly improves the efficiency in the use of source X-rays for imaging at high resolution and with high sensitivity. We further present correlative tri-contrast tomography on a Drum fish tooth specimen and demonstrate a clear separation of biological features with different physical properties. Our results highlight the exceptional imaging capability empowered by the MAAST-based TLI method. Our approach also features a compact and robust setup that can potentially be made broadly available to academia research and industrial applications.     

Inhibition of 3-phosphoinositide–dependent protein kinase 1 (PDK1) can revert cellular senescence in human dermal fibroblasts

Cellular senescence is defined as a stable, persistent arrest of cell proliferation. Here, we examine whether senescent cells can lose senescence hallmarks and reenter a reversible state of cell-cycle arrest (quiescence). We constructed a molecular regulatory network of cellular senescence based on previous experimental evidence. To infer the regulatory logic of the network, we performed phosphoprotein array experiments with normal human dermal fibroblasts and used the data to optimize the regulatory relationships between molecules with an evolutionary algorithm. From ensemble analysis of network models, we identified 3-phosphoinositide–dependent protein kinase 1 (PDK1) as a promising target for inhibitors to convert the senescent state to the quiescent state. We showed that inhibition of PDK1 in senescent human dermal fibroblasts eradicates senescence hallmarks and restores entry into the cell cycle by suppressing both nuclear factor κB and mTOR signaling, resulting in restored skin regeneration capacity. Our findings provide insight into a potential therapeutic strategy to treat age-related diseases associated with the accumulation of senescent cells.

Cellular senescence is defined as a stable, persistent exit from the cell cycle in response to stresses such as telomere shortening, oxidative stress, oncogene activation, and DNA damage (1, 2). A benefit of cellular senescence is prevention of tumorigenesis by blocking proliferation of damaged cells that may undergo malignant transformation (2, 3). However, senescent cells accumulate in tissues during aging and secrete proinflammatory cytokines, which can contribute to aging and age-related diseases, including cancer (2, 3). In studies with animal models, elimination of senescent cells prevents, alleviates, or reverses symptoms of aging (4, 5) and various age-related diseases (6, 7), such as osteoarthritis and atherosclerosis.Cell-cycle arrest alone is not cellular senescence; cellular senescence requires additional signals that convert transient cell-cycle arrest into persistent exit from the cell cycle so that the cells fail to proliferate in response to growth signals, a process called geroconversion (8, 9). Terminally differentiated, nonmitotic cells can also undergo senescence; thus, cell-cycle exit is only one aspect of the senescent phenotype. Cellular senescence is a complex biological mechanism regulated by various signaling pathways (10, 11). Signaling pathways that mediate cellular senescence can be divided into three major categories. The first category includes the pathways that cause cell-cycle arrest in response to DNA damage, such as p53/p21^CIP1 and p16^INK4a/pRb pathways (11–14). The second category consists of the pathways mediating cell growth and energy metabolism, such as PI3K/AKT/mTOR and SIRT1/AMPK pathways (15–18). Activation of mTOR in cells arrested by persistent DNA damage represents a second stimulus that can convert transiently arrested cells into senescent cells that exhibit hypertrophy and an expanded lysosomal compartment (19). The last category consists of the pathways mediating the senescence-associated secretory phenotype (SASP) (3, 10, 20). The SASP is a characteristic feature of senescent cells and reflects their secretion of proinflammatory cytokines and chemokines. These cytokines and chemokines maintain cellular senescence through positive autoregulatory feedbacks, affect nonsenescent nearby cells, and promote aging and age-related diseases, including cancer (3, 21). Nuclear factor κB (NF-κB) activity is important for SASP, and suppression of NF-κB prevents age-related diseases and delays aging in mice (22, 23).Spontaneous reversion from senescence to proliferation is extremely rare, but the reversion through manipulations is not. Some studies reported that senescent cells can reenter the cell cycle (24). The current understanding of senescence is as a dynamic multistep process that is reversible under some conditions (25). About 70 to 90% of cells with low p16^INK4a levels in replicative senescence, which is senescence related to the finite number of divisions a cell can perform before telomeres become too short, resume proliferation following p53 inactivation (26). Inactivation of p53 also enables cells to escape from therapy-induced senescence, caused by the chemotherapeutic agent Adriamycin (27). Cells with oncogene-induced senescence can also escape from the senescent state. For instance, about 50% of mouse embryo fibroblasts with high Ras levels reenter the cell cycle upon inactivation of all three Rb family members (28), and about 70% of the fibroblasts reenter upon activation of H3K9 demethylases (29).Here, we applied a systems biology approach to identify mechanisms underlying cell-cycle arrest, cell growth, and the SASP with the goal of finding inhibitable targets to convert the senescent state to the quiescent state. We studied normal human dermal fibroblasts (NHDFs), which can be experimentally induced into the senescent state (8). We constructed a molecular signaling network of cellular senescence using information in the literature and network databases to identify the relevant molecules, experimental data from time series of phosphorylated proteins in NHDFs to define the input–output relationships that reflect cellular states upon each input condition, and an evolutionary algorithm to determine the regulatory logic of the network (SI Appendix, Fig. S1 A–C). By analyzing the regulatory signaling network, we predicted that PDK1 was an inhibitor target that can convert senescent fibroblasts to quiescent fibroblasts (SI Appendix, Fig. S1D). To validate this prediction, we conducted experiments with NHDFs exposed to PDK1 inhibitors (SI Appendix, Fig. S1E), which eliminated hallmarks of cellular senescence, restored the proliferation of the cells in response to growth factors, and restored skin regeneration capacity in two-dimensional (2D) culture and a three-dimensional (3D) skin equivalent model. Our findings provide insight into a potential therapeutic strategy to treat aging and age-related diseases.     

Legume–microbiome interactions unlock mineral nutrients in regrowing tropical forests

Legume trees form an abundant and functionally important component of tropical forests worldwide with N₂-fixing symbioses linked to enhanced growth and recruitment in early secondary succession. However, it remains unclear how N₂-fixers meet the high demands for inorganic nutrients imposed by rapid biomass accumulation on nutrient-poor tropical soils. Here, we show that N₂-fixing trees in secondary Neotropical forests triggered twofold higher in situ weathering of fresh primary silicates compared to non-N₂–fixing trees and induced locally enhanced nutrient cycling by the soil microbiome community. Shotgun metagenomic data from weathered minerals support the role of enhanced nitrogen and carbon cycling in increasing acidity and weathering. Metagenomic and marker gene analyses further revealed increased microbial potential beneath N₂-fixers for anaerobic iron reduction, a process regulating the pool of phosphorus bound to iron-bearing soil minerals. We find that the Fe(III)-reducing gene pool in soil is dominated by acidophilic Acidobacteria, including a highly abundant genus of previously undescribed bacteria, Candidatus Acidoferrum, genus novus. The resulting dependence of the Fe-cycling gene pool to pH determines the high iron-reducing potential encoded in the metagenome of the more acidic soils of N₂-fixers and their nonfixing neighbors. We infer that by promoting the activities of a specialized local microbiome through changes in soil pH and C:N ratios, N₂-fixing trees can influence the wider biogeochemical functioning of tropical forest ecosystems in a manner that enhances their ability to assimilate and store atmospheric carbon.

The legume family is the most diverse angiosperm family in the Neotropics (1–3), with dinitrogen (N₂)-fixing legume trees growing fast and supplying tropical forests with substantial quantities of nitrogen (N) during succession (4). This N₂-fixing strategy requires that trees can access scarce sources of inorganic nutrients, including bioavailable forms of phosphorus (P) for metabolites and growth and molybdenum (Mo) for nitrogenase functioning (5–7) (the enzyme that catalyzes conversion of atmospheric N₂ to bioavailable N).However, in highly weathered tropical soils, in addition to their pools in organic matter (8, 9), large amounts of P and Mo are often occluded in an inorganic form in insoluble iron (Fe) and aluminum (Al)-bearing minerals (10, 11) and thus not available for immediate biological uptake. For example, both P and Mo are scarce in the oxisols and inceptisols that have developed from Mo-poor basalt bedrock in Panamanian tropical forests (7, 12) (SI Appendix, Table S1) and that contain P-adsorbing kaolinite, goethite, and hematite secondary minerals (SI Appendix, Fig. S1). Our own observations from these Panamanian forests show significant differences in the chemistry of soils beneath N₂ fixing versus nonfixing trees, with significantly lower concentrations of nitric acid–extractable P, Fe, and Al and lower pH below N₂-fixing trees (SI Appendix, Table S2). Moreover, a strong association between extracted P and Fe plus Al, but not between P and soil organic carbon, implies that soil P is significantly influenced by mineral dynamics within these tropical soils (SI Appendix, Fig. S2, P < 0.001 for Fe and Al and P > 0.10 for carbon, Pearson test).These patterns raise the biogeochemical hypothesis that N₂-fixing legume trees may strategically employ specific mechanisms to enhance mineral weathering (5, 13, 14), resulting in improved access to occluded inorganic mineral nutrients, enhanced N₂-fixation, and enhanced carbon sequestration by forest biomass during succession. Here, we address this hypothesis by investigating 1) whether N₂-fixing trees induce locally elevated rates of silicate mineral (olivine) weathering (compared to nonfixing trees), causing the depletion of elements critical to N₂-fixation; 2) whether the altered soil beneath N₂-fixing trees is linked to compositional and functional differences in the microbiomes and metagenomes associated with soil minerals; and 3) whether the presence of N₂-fixers affects biogeochemical nutrient cycling in rooting zone soils beneath neighboring non-N₂–fixing forest trees.     

Thermal niches of planktonic foraminifera are static throughout glacial–interglacial climate change

Abiotic niche lability reduces extinction risk by allowing species to adapt to changing environmental conditions in situ. In contrast, species with static niches must keep pace with the velocity of climate change as they track suitable habitat. The rate and frequency of niche lability have been studied on human timescales (months to decades) and geological timescales (millions of years), but lability on intermediate timescales (millennia) remains largely uninvestigated. Here, we quantified abiotic niche lability at 8-ka resolution across the last 700 ka of glacial–interglacial climate fluctuations, using the exceptionally well-known fossil record of planktonic foraminifera coupled with Atmosphere–Ocean Global Climate Model reconstructions of paleoclimate. We tracked foraminiferal niches through time along the univariate axis of mean annual temperature, measured both at the sea surface and at species’ depth habitats. Species’ temperature preferences were uncoupled from the global temperature regime, undermining a hypothesis of local adaptation to changing environmental conditions. Furthermore, intraspecific niches were equally similar through time, regardless of climate change magnitude on short timescales (8 ka) and across contrasts of glacial and interglacial extremes. Evolutionary trait models fitted to time series of occupied temperature values supported widespread niche stasis above randomly wandering or directional change. Ecotype explained little variation in species-level differences in niche lability after accounting for evolutionary relatedness. Together, these results suggest that warming and ocean acidification over the next hundreds to thousands of years could redistribute and reduce populations of foraminifera and other calcifying plankton, which are primary components of marine food webs and biogeochemical cycles.

Abiotic niche dynamics determine patterns of community composition over space and regulate trajectories of diversity over time (1). Both niche lability (2, 3) and conservatism (1, 4) have been proposed to spur speciation, and abiotic niche lability has been associated with ecological invasions (5–7) and with reduced risk of extinction during times of climate change (8). Thus, a deeper understanding of species’ propensity for niche stasis versus lability could improve predictions of biodiversity restructuring in response to anthropogenic climate change (9).Stasis in species’ abiotic niches through time has been documented in empirical research, but most such studies have been limited to ecological niche modeling on decadal scales (reviewed in ref. 10) or paleoecological examination on 10⁶ to 10⁷ y scales (5, 11, 12). Since empirical rates of niche change are scarce and difficult to acquire, many studies merely assume that niche evolution occurs at a constant rate along branches of a phylogeny (2, 3, 6, 7). Niche dynamics at intermediate timescales of centuries to millennia are particularly poorly documented (10), and studies at this meso scale have been restricted to terrestrial systems (e.g., refs. 13–15) or to comparisons between the present day and the single historical time step of the Last Glacial Maximum, ∼21 ka (16–20). Quantifying the rate and relative frequency of niche change in marine species over timescales of 10² to 10⁵ years is important, however, because species will adapt or go extinct in response to anthropogenic ocean changes over this timescale (21).Here, we investigated climatic niche lability from the rich sedimentary archive of global planktonic foraminifera across the last 700 ka of glacial–interglacial cycles at 8-ka resolution. Planktonic foraminifera (Protista) construct “shells” (tests) of calcite, thereby sequestering carbon and recording an isotopic signature of past ocean conditions. Tests readily accumulate over large expanses of the seafloor. Consequently, the fossil record of foraminifera—arguably “the best fossil record on Earth” (22)—affords an exceptionally high-resolution view into past species distributions. This detailed record fuels studies of biostratigraphy, paleoclimatology, and paleoecology (20, 22–25). Moreover, the complete species diversity of planktonic foraminifera has been described for the Plio–Pleistocene, with good agreement between morphological and molecular phylogenies (22, 25–27). Although some have speculated that foraminifera competitively exclude each other (24), recent work found that planktonic foraminifera species seldom restrict each other’s distributions (28). Presumably, therefore, species occupy the full envelope of existing environmental conditions within their tolerance limits, and geographic distributions are determined almost entirely by physical ocean conditions.We developed five analyses to investigate the degree of abiotic niche lability in foraminifera. All methods examined the univariate niche axis of temperature, which is the single most important explanatory variable in regard to geographic distributions of foraminifera (20, 29–32) and is a climate-related stressor and extinction driver for diverse marine fauna across timescales (33, 34). The adaptive potential of thermal niches has been taken as a key determinant of global community structure and genetic connectance in plankton (35). Primary productivity and other environmental variables, however, may also structure abiotic niches of plankton (36). Our suite of analyses quantified whether and by how much planktonic foraminiferal niches shifted along a temperature axis. First, we correlated time series of species’ thermal optima with global temperature to determine whether species tracked suitable habitat or experienced environmental fluctuations in situ. We then quantified species’ niche dissimilarity between pairs of time bins—either tracking niches across bin boundaries or contrasting niches at climatic extremes of glacial maxima and interglacial thermal peaks. To characterize niche change we applied trait evolution models to time series of temperatures at occupied sites. Lastly, we explored variation in intraspecific niche lability among ecotypes while accounting for phylogenetic relatedness. SI Appendix, Table S1 lists the response variable and sample size for each analysis.     

LGI1–ADAM22–MAGUK configures transsynaptic nanoalignment for synaptic transmission and epilepsy prevention

Physiological functioning and homeostasis of the brain rely on finely tuned synaptic transmission, which involves nanoscale alignment between presynaptic neurotransmitter-release machinery and postsynaptic receptors. However, the molecular identity and physiological significance of transsynaptic nanoalignment remain incompletely understood. Here, we report that epilepsy gene products, a secreted protein LGI1 and its receptor ADAM22, govern transsynaptic nanoalignment to prevent epilepsy. We found that LGI1–ADAM22 instructs PSD-95 family membrane-associated guanylate kinases (MAGUKs) to organize transsynaptic protein networks, including NMDA/AMPA receptors, Kv₁ channels, and LRRTM4–Neurexin adhesion molecules. Adam22^ΔC5/ΔC5 knock-in mice devoid of the ADAM22–MAGUK interaction display lethal epilepsy of hippocampal origin, representing the mouse model for ADAM22-related epileptic encephalopathy. This model shows less-condensed PSD-95 nanodomains, disordered transsynaptic nanoalignment, and decreased excitatory synaptic transmission in the hippocampus. Strikingly, without ADAM22 binding, PSD-95 cannot potentiate AMPA receptor-mediated synaptic transmission. Furthermore, forced coexpression of ADAM22 and PSD-95 reconstitutes nano-condensates in nonneuronal cells. Collectively, this study reveals LGI1–ADAM22–MAGUK as an essential component of transsynaptic nanoarchitecture for precise synaptic transmission and epilepsy prevention.

Epilepsy, characterized by unprovoked, recurrent seizures, affects 1 to 2% of the population worldwide. Many genes that cause inherited epilepsy when mutated encode ion channels, and dysregulated synaptic transmission often causes epilepsy (1, 2). Although antiepileptic drugs have mainly targeted ion channels, they are not always effective and have adverse effects. It is therefore important to clarify the detailed processes for synaptic transmission and how they are affected in epilepsy.Recent superresolution imaging of the synapse reveals previously overlooked subsynaptic nano-organizations and pre- and postsynaptic nanodomains (3–6), and mathematical simulation suggests their nanometer-scale coordination in individual synapses for efficient synaptic transmission: presynaptic neurotransmitter release machinery and postsynaptic receptors precisely align across the synaptic cleft to make “transsynaptic nanocolumns” (7, 8).So far, numerous transsynaptic cell-adhesion molecules have been identified (9–12), including presynaptic Neurexins and type IIa receptor protein tyrosine phosphatases (PTPδ, PTPσ, and LAR) and postsynaptic Neuroligins, LRRTMs, NGL-3, IL1RAPL1, Slitrks, and SALMs. Neurexins–Neuroligins have attracted particular attention because of their synaptogenic activities when overexpressed and their genetic association with neuropsychiatric disorders (e.g., autism). Another type of transsynaptic adhesion complex mediated by synaptically secreted Cblns (e.g., Neurexin–Cbln1–GluD2) promotes synapse formation and maintenance (13–15). Genetic studies in Caenorhabditis elegans show that secreted Ce-Punctin, the ortholog of the mammalian ADAMTS-like family, specifies cholinergic versus GABAergic identity of postsynaptic domains and functions as an extracellular synaptic organizer (16). However, the molecular identity and in vivo physiological significance of transsynaptic nanocolumns remain incompletely understood.LGI1, a neuronal secreted protein, and its receptor ADAM22 have recently emerged as major determinants of brain excitability (17) as 1) mutations in the LGI1 gene cause autosomal dominant lateral temporal lobe epilepsy (18); 2) mutations in the ADAM22 gene cause infantile epileptic encephalopathy with intractable seizures and intellectual disability (19, 20); 3) Lgi1 or Adam22 knockout mice display lethal epilepsy (21–24); and 4) autoantibodies against LGI1 cause limbic encephalitis characterized by seizures and amnesia (25–28). Functionally, LGI1–ADAM22 regulates AMPA receptor (AMPAR) and NMDA receptor (NMDAR)-mediated synaptic transmission (17, 22, 29) and Kv₁ channel-mediated neuronal excitability (30, 31). Recent structural analysis shows that LGI1 and ADAM22 form a 2:2 heterotetrameric assembly (ADAM22–LGI1–LGI1–ADAM22) (32), suggesting the transsynaptic configuration.In this study, we identify ADAM22-mediated synaptic protein networks in the brain, including pre- and postsynaptic MAGUKs and their functional bindings to transmembrane proteins (NMDA/AMPA glutamate receptors, voltage-dependent ion channels, cell-adhesion molecules, and vesicle-fusion machinery). ADAM22 knock-in mice lacking the MAGUK-binding motif show lethal epilepsy of hippocampal origin. In this mouse, postsynaptic PSD-95 nano-assembly as well as nano-scale alignment between pre- and postsynaptic proteins are significantly impaired. Importantly, PSD-95 is no longer able to modulate AMPAR-mediated synaptic transmission without binding to ADAM22. These findings establish that LGI1–ADAM22 instructs MAGUKs to organize transsynaptic nanocolumns and guarantee the stable brain activity.     

Topological Weaire–Thorpe models of amorphous matter

Amorphous solids remain outside of the classification and systematic discovery of new topological materials, partially due to the lack of realistic models that are analytically tractable. Here we introduce the topological Weaire–Thorpe class of models, which are defined on amorphous lattices with fixed coordination number, a realistic feature of covalently bonded amorphous solids. Their short-range properties allow us to analytically predict spectral gaps. Their symmetry under permutation of orbitals allows us to analytically compute topological phase diagrams, which determine quantized observables like circular dichroism, by introducing symmetry indicators in amorphous systems. These models and our procedures to define invariants are generalizable to higher coordination number and dimensions, opening a route toward a complete classification of amorphous topological states in real space using quasilocal properties.

Although most solids can be grown amorphous, their lack of translational symmetries has kept amorphous solids outside the recently developed topological classifications of noninteracting matter (1–3), halting their discovery for robust applications. Amorphous Bi2Se3 was shown to be the sole exception recently, with spectral, spin, and transport data supporting a surface Dirac cone (4). Other condensed matter platforms based on nonstoichiometric growth of the same compound are promising alternatives (5, 6), and, as a proof of principle, amorphous topological states have been realized in two-dimensional (2D) systems of coupled gyroscopes (7). However, the challenge is to model realistic materials, and determine their topological phase diagram in a way that may establish a classification and aid their systematic discovery.Addressing this challenge seems possible, since the absence of amorphous topological solids is not fundamental; topological protection does not rely on translational invariance. This well-developed understanding dates back at least to studies of integer quantum Hall transitions (8–10). More recently, several classes of amorphous models have been shown to host integer quantum Hall (or Chern insulator) phases, as well as other topological states (7, 11–19), including numerical work that suggests differences compared to known quantum Hall transitions (19, 20). Although the corresponding topological phase diagrams can be computed numerically, by simulating responses to external fields (18) or through real space topological markers (7, 11), these methods are not generalizable to every discrete symmetry in every dimensionality. Crucially, a symmetry-based approach (21–24) for amorphous solids, which proved to be successful in high-throughput classifications of topological crystals (1–3), seems out of reach due to the absence of long-range atomic order.In this work, we find that an overlooked yet common property of covalently bonded amorphous solids, their fixed coordination number (25), can be exploited to overcome these problems. This property is rooted in the fact that the local chemical environment in an amorphous solid is similar to that of the crystalline phase of the same compound (4, 26, 27). The local environment determines the coarse properties of the density of states such as spectral gaps, while long-range correlations or periodicity determine the finer details. Such physical input has been a cornerstone in describing amorphous states (25, 28), allowing proving of the presence of spectral gaps in amorphous Si, eventually explaining why windows are transparent (26, 28, 29). Although topological properties are nonlocal in general, and quasi-local (30) at best, this useful chemical input remains unexploited in current models of amorphous topological states.The models we propose are an analytically tractable and generalizable set of topological amorphous models with fixed coordination. They generalize the Weaire–Thorpe Hamiltonian class (28) explicitly developed to respect the local environment across sites. We show, analytically, that they are generically gapped, and track the band crossings as a function of the parameters of the models. Remarkably, these models allow us to construct an amorphous version of symmetry indicators by exploiting the symmetry resulting from the equivalence between orbitals. We are able to map their topological phase diagram modulo an integer, without the need to compute local topological markers.For concreteness, in this paper, we exemplify our results using a triply coordinated 2D amorphous lattice without time-reversal symmetry (7), and consider a fourfold coordinated model in SI Appendix that emphasizes the generality of our results. We analytically compute the spectral gaps and numerically calculate the in-gap local density of states that shows topologically protected edge states. We numerically compute the local Chern marker (30), which we link to a quantized circular dichroism, mapping the topological phase diagram in parameter space. We then introduce the symmetry indicators for this model and combine them in a formula that delivers the Chern number modulo three (modulo four in the case of fourfold coordination), reproducing the topological phase diagram analytically. Finally, for the threefold coordinated model, we discuss to what extent an effective Hamiltonian approach (31), that projects the full Hamiltonian into a basis of plane waves, can also detect topological phase transitions.Due to their gapped structure and previous success in describing amorphous solids, the models we propose are natural candidates to describe realistic amorphous topological insulators and to track their topological phase transitions. Moreover, there is no fundamental restriction to extend our analytical arguments to different dimensionality, symmetry classes, and coordination number, hinting at a route to classify amorphous topological insulators.     

Experimental test of a predicted dynamics–structure–thermodynamics connection in molecularly complex glass-forming liquids

Understanding in a unified manner the generic and chemically specific aspects of activated dynamics in diverse glass-forming liquids over 14 or more decades in time is a grand challenge in condensed matter physics, physical chemistry, and materials science and engineering. Large families of conceptually distinct models have postulated a causal connection with qualitatively different “order parameters” including various measures of structure, free volume, thermodynamic properties, short or intermediate time dynamics, and mechanical properties. Construction of a predictive theory that covers both the noncooperative and cooperative activated relaxation regimes remains elusive. Here, we test using solely experimental data a recent microscopic dynamical theory prediction that although activated relaxation is a spatially coupled local–nonlocal event with barriers quantified by local pair structure, it can also be understood based on the dimensionless compressibility via an equilibrium statistical mechanics connection between thermodynamics and structure. This prediction is found to be consistent with observations on diverse fragile molecular liquids under isobaric and isochoric conditions and provides a different conceptual view of the global relaxation map. As a corollary, a theoretical basis is established for the structural relaxation time scale growing exponentially with inverse temperature to a high power, consistent with experiments in the deeply supercooled regime. A criterion for the irrelevance of collective elasticity effects is deduced and shown to be consistent with viscous flow in low-fragility inorganic network-forming melts. Finally, implications for relaxation in the equilibrated deep glass state are briefly considered.

An enormous number of seemingly orthogonal proposals exist for a fundamental connection between a (typically scalar) structural or excess (configurational) thermodynamic quantity and activated relaxation in supercooled liquids (1–12). High chemical complexity for fragile glass formers which exhibit strongly non-Arrhenius relaxation greatly complicates the formulation of predictive theories. A common generic view (1, 3, 8) is that the structural or alpha relaxation time (and viscosity, inverse diffusivity) evolves with cooling as shown in Fig. 1A. Different dynamical mechanisms in the high-, intermediate-, and low-temperature regimes are often envisioned: noncooperative Arrhenius (∼1 ps to 100 ps), critical power law (∼0.1 ns to 100 ns), and cooperative non-Arrhenius (∼0.1 μs to 100 s or beyond), respectively. Typically a causal connection is postulated between the logarithm of the alpha time (an effective barrier in thermal energy units) and a specific “order parameter”: 1) in the structural class (6, 7, 13–17), the intensity of the cage peak of the structure factor S(k), local aspect(s) of the radial distribution function g(r), or specific packing motifs; 2) in the thermodynamics class, various measures of free volume (18, 19), excess entropy (20), configurational entropy (21–25), internal energy and enthalpy (26), or with some arguing for an equilibrium phase transition at an inaccessibly low (high) temperature (density) (23, 27–29); 3) in the short time class, the high-frequency shear modulus (2, 30–32), Debye–Waller factor (33), or amplitude of special vibrational modes (33–35); and 4) in the intermediate time class, the concentration of dilute mobile excitations [e.g., strings (36, 37) or facilitating defects (38)]. Many of the proposed order parameters are hard or impossible to uniquely define and/or experimentally measure. The diverse models often claim to capture relaxation data over limited time windows typically based on fitting but usually fail at low and/or high enough temperature (5).Open in a separate windowFig. 1.Global relaxation map and theoretical picture and key predictions. (A) Three-regime relaxation map (curves) for the alpha time with Arrhenius and strongly non-Arrhenius behaviors separated by a crossover regime perhaps of a critical power law (6) form. The proposed two-regime scenario of ECNLE theory (39–42) is based solely on noncooperative and cooperative activated dynamics (slightly overlapping orange and green regions) with the inverse dimensionless compressibility (S0−1) as the relevant thermodynamic quantity. The approximately five to six decade range that simulations can probe is indicated. (B) Dynamic free energy for a metastable hard sphere (diameter σ) fluid (42) as a function of particle displacement at a high packing fraction of ϕ = 0.58. Relevant length and energy scales are indicated. (Inset) Schematic of the core physical idea for the alpha relaxation: hopping on the cage scale coupled with a collective elastic displacement of all particles outside the cage. (C) Main: local cage barrier as a function of inverse dimensionless compressibility for 0.44<ϕ <0.61 corresponding to a 16 decade increase of the alpha time (39, 41, 42). The metastable regime begins at ϕ ∼ 0.5 where the total barrier is ∼1.5 k_BT. (Inset) Total barrier as a function of S0−3 normalized by its ϕ = 0.5 value. The elastic barrier is 1 k_BT at ϕ ∼ 0.55. Packing fractions are given along the top x-axis.Here we present, using only experimental data, a test of a relationship between activated relaxation, local pair structure, and a specific thermodynamic property predicted by the Elastically Collective Nonlinear Langevin Equation (ECNLE) theory (39–41). The results provide support for the following: 1) the coupled local–nonlocal nature of relaxation deeply connected with collective elasticity, 2) the dimensionless amplitude of thermal density fluctuations, S₀, as the relevant (nonexcess) thermodynamic property, 3) a roadmap for organizing relaxation data in S₀, not in temperature, space, 4) irrelevance of collective elasticity as the origin for the crossover from fragile to strong glass formers, and 5) an explicit demonstration that a dynamics–thermodynamics correlation can be a noncausal consequence of the causal relation between local pair structure and S₀.     

Human-relevant near-organ neuromodulation of the immune system via the splenic nerve

Neuromodulation of immune function by stimulating the autonomic connections to the spleen has been demonstrated in rodent models. Consequently, neuroimmune modulation has been proposed as a new therapeutic strategy for the treatment of inflammatory conditions. However, demonstration of the translation of these immunomodulatory mechanisms in anatomically and physiologically relevant models is still lacking. Additionally, translational models are required to identify stimulation parameters that can be transferred to clinical applications of bioelectronic medicines. Here, we performed neuroanatomical and functional comparison of the mouse, rat, pig, and human splenic nerve using in vivo and ex vivo preparations. The pig was identified as a more suitable model of the human splenic innervation. Using functional electrophysiology, we developed a clinically relevant marker of splenic nerve engagement through stimulation-dependent reversible reduction in local blood flow. Translation of immunomodulatory mechanisms were then assessed using pig splenocytes and two models of acute inflammation in anesthetized pigs. The pig splenic nerve was shown to locally release noradrenaline upon stimulation, which was able to modulate cytokine production by pig splenocytes. Splenic nerve stimulation was found to promote cardiovascular protection as well as cytokine modulation in a high- and a low-dose lipopolysaccharide model, respectively. Importantly, splenic nerve–induced cytokine modulation was reproduced by stimulating the efferent trunk of the cervical vagus nerve. This work demonstrates that immune responses can be modulated by stimulation of spleen-targeted autonomic nerves in translational species and identifies splenic nerve stimulation parameters and biomarkers that are directly applicable to humans due to anatomical and electrophysiological similarities.

The inflammatory status of the body is monitored and regulated through the neuroimmune axis, connecting the brain to the immune system via both humoral and neural pathways (1–3). In particular, the inflammatory reflex (3) controls systemic immune responses; detection of inflammatory stimuli in the periphery is communicated to the brain that activates outflow of neural signals to promote peripheral immune responses proportional to the threat. Studies in rodent models have identified the cholinergic anti-inflammatory pathway (CAIP) as the brain’s efferent response to infection and inflammation through peripheral neurotransmitters released in lymphoid organs, mainly the spleen (4, 5). Within this pathway, the peripheral connection between the vagus nerve (VN), the splenic nerve (SpN), and its terminal release of noradrenaline (NA) into the spleen have been identified as crucial components of this neural circuit (6–8) (SI Appendix, Fig. S1A).Importantly, the CAIP can be harnessed to promote immune control. Activation of the cervical VN by electrical stimulation (vagus nerve stimulation—VNS; SI Appendix, Fig. S1A) has been shown to be effective in reducing lipopolysaccharide (LPS)-induced levels of tumor necrosis factor alpha (TNF-α) (4, 6, 7) and in preclinical rodent models of chronic inflammatory diseases (9, 10). Murine models have generally been used to demonstrate biological proof of concepts of novel neuromodulation therapies in this and other contexts. However, the development of clinical bioelectronic medicines requires the accurate estimation and validation of stimulation parameters in a histologically, surgically, and anatomically relevant model to define device and therapy requirements. The translation of stimulation parameters from rodent to human is hampered by anatomical (e.g., size of nerves), histological (e.g., number of axons, connective tissue thickness, proportion of adipose tissue), and physiological (e.g., immunological) differences. Therefore, it is suggested that the use of large animal models, human tissues, and in silico modeling are more appropriate for the optimization and scaling of human-relevant parameters (11, 12).Although early clinical feasibility studies have provided preliminary evidence of immunomodulatory effects of VNS in patients (13, 14), clear demonstration of the translation of the splenic anti-inflammatory pathway in clinically relevant species is currently lacking in the literature. The VN has a functionally and anatomically complex composition. In animals and humans, the VN contains both afferent and efferent axons of varying size (large, medium, and small) and degree of myelination (heavily myelinated, lightly myelinated, and unmyelinated axons) innervating multiple organs and muscles (15). As a consequence, currently used VNS results in activation of off-target circuits (SI Appendix, Fig. S1A) that can cause dysphonia, coughing, hoarseness, pain, and dyspnea (16–18); in some patients, these can be managed and can also improve over time (18). Further, it remains unclear which axons (efferent versus afferent, myelinated versus unmyelinated) within the VN relay immunomodulatory signals to peripheral organs (19, 20). As a result, it is difficult to optimize the stimulation parameters necessary to activate axons within the VN which carry signals to the spleen. Typically, clinical parameters are selected based on the individual patient’s tolerance of off-target effects (13, 21) without direct evidence of activation of the anti-inflammatory pathway because of a lack of an organ-specific biomarker. Since the SpN directly transmits neural signals to the spleen and is the fundamental nodal circuit in mediating the anti-inflammatory response (22), SpN stimulation (SpNS) may represent an alternative modality providing the opportunity for near-organ modulation of the immune system (SI Appendix, Fig. S1 B and C). Proof of concept experiments in rodents have shown that immune responses can indeed be modulated by stimulation of the SpN with comparable cytokine suppressive effects to VNS (7, 8, 23).Here, we anatomically, histologically, and functionally compared the mouse, rat, pig, and human SpN, demonstrating the superiority of the pig as a translational model of the human SpN. We then performed functional in vivo pig electrophysiological studies to identify organ-specific physiological biomarkers that can be used to assess nerve engagement and to refine stimulation parameters. Finally, we assessed the large animal translation of the spleen-dependent anti-inflammatory pathway in the pig using in vitro splenocyte preparations together with two in vivo models of acute inflammation.     

Characterization of the strain-rate–dependent mechanical response of single cell–cell junctions

Cell–cell adhesions are often subjected to mechanical strains of different rates and magnitudes in normal tissue function. However, the rate-dependent mechanical behavior of individual cell–cell adhesions has not been fully characterized due to the lack of proper experimental techniques and therefore remains elusive. This is particularly true under large strain conditions, which may potentially lead to cell–cell adhesion dissociation and ultimately tissue fracture. In this study, we designed and fabricated a single-cell adhesion micro tensile tester (SCAµTT) using two-photon polymerization and performed displacement-controlled tensile tests of individual pairs of adherent epithelial cells with a mature cell–cell adhesion. Straining the cytoskeleton–cell adhesion complex system reveals a passive shear-thinning viscoelastic behavior and a rate-dependent active stress-relaxation mechanism mediated by cytoskeleton growth. Under low strain rates, stress relaxation mediated by the cytoskeleton can effectively relax junctional stress buildup and prevent adhesion bond rupture. Cadherin bond dissociation also exhibits rate-dependent strengthening, in which increased strain rate results in elevated stress levels at which cadherin bonds fail. This bond dissociation becomes a synchronized catastrophic event that leads to junction fracture at high strain rates. Even at high strain rates, a single cell–cell junction displays a remarkable tensile strength to sustain a strain as much as 200% before complete junction rupture. Collectively, the platform and the biophysical understandings in this study are expected to build a foundation for the mechanistic investigation of the adaptive viscoelasticity of the cell–cell junction.

Adhesive organelles between neighboring epithelial cells form an integrated network as the foundation of complex tissues (1). As part of normal physiology, this integrated network is constantly exposed to mechanical stress and strain, which is essential to normal cellular activities, such as proliferation (2–4), migration (5, 6), differentiation (7), and gene regulation (7, 8) associated with a diverse set of functions in tissue morphogenesis (9–11) and wound healing (9). A host of developmental defects or clinical pathologies in the form of compromised cell–cell associations will arise when cells fail to withstand external mechanical stress due to genetic mutations or pathological perturbations (12, 13). Indeed, since the mechanical stresses are mainly sustained by the intercellular junctions, which may represent the weakest link and limit the stress tolerance within the cytoskeleton network of a cell sheet, mutations or disease-induced changes in junction molecules and components in adherens junctions and desmosomes lead to cell layer fracture and tissue fragility, which exacerbate the pathological conditions (14–17). This clinical relevance gives rise to the importance of understanding biophysical transformations of the cell–cell adhesion interface when cells are subjected to mechanical loads.As part of their normal functions, cells often experience strains of tens to a few hundred percent at strain rates of 10⁻⁴ to 1 s⁻¹ (18–21). For instance, embryonic epithelia are subjected to strain rates in the range of 10⁻⁴ to 10⁻³ s⁻¹ during normal embryogenesis (22). Strain rates higher than 0.1 s⁻¹ are often experienced by adult epithelia during various normal physiological functions (21, 23, 24), such as breathing motions in the lung (1 to 10 s⁻¹) (25), cardiac pulses in the heart (1 to 6.5 s⁻¹) (20), peristaltic movements in the gut (0.4 to 1.5 s⁻¹), and normal stretching of the skin (0.1 to 5 s⁻¹). Cells have different mechanisms to dissipate the internal stress produced by external strain to avoid fracture, often via cytoskeleton remodeling and cell–cell adhesion enhancement (26, 27). These coping mechanisms may have different characteristic timescales. Cytoskeleton remodeling can dissipate mechanical stress promptly due to its viscoelastic nature and the actomyosin-mediated cell contractility (17, 28–32). Adhesion enhancement at the cell–cell contact is more complex in terms of timescale. Load-induced cell–cell adhesion strengthening has been shown via the increase in the number of adhesion complexes (33–35) or by the clustering of adhesion complexes (36–39), which occurs on a timescale ranging from a few minutes up to a few hours after cells experience an initial load (28). External load on the cell–cell contact also results in a prolonged cell–cell adhesion dissociation time (40, 41), suggesting cadherin bonds may transition to catch bonds under certain loading conditions (42, 43), which can occur within seconds (44). With the increase in cellular tension, failure to dissipate the stress within the cell layer at a rate faster than the accumulation rate will inevitably lead to the fracture of the cell layer (45). Indeed, epithelial fracture often aggravates the pathological outcomes in several diseases, such as acute lung injuries (46), skin disorders (47), and development defects (48). It is generally accepted that stress accumulation in the cytoskeleton network (49, 50) and potentially in the cytoplasm is strain-rate–dependent (51). However, to date, there is a lack of understanding about the rate-dependent behavior of cell–cell adhesions, particularly about which of the stress-relaxation mechanisms are at play across the spectrum of strain rates. In addition, it remains unclear how the stress relaxation interplays with adhesion enhancement under large strains, especially at high strain rates which may lead to fracture, that is, a complete separation of mature cell–cell adhesions under a tensile load (45, 52, 53). Yet, currently, there is a lack of quantitative technology that enables the investigation of these mechanobiological processes in a precisely controlled manner. This is especially true at high strain rates.To delineate this mechanical behavior, the cleanest characterization method is to directly measure stress dynamics at a single mature cell–cell adhesion interface. Specifically, just as a monolayer cell sheet is a reduction from three-dimensional (3D) tissue, a single cell–cell adhesion interface, as a reduction from a monolayer system, represents the smallest unit to study the rheological behavior of cellular junctions. The mechanistic understanding uncovered with this single unit will inform cellular adaptations to a more complex stress microenvironment in vivo and in vitro, in healthy and diseased conditions. To this end, we developed a single-cell adhesion micro tensile tester (SCAµTT) platform based on nanofabricated polymeric structures using two-photon polymerization (TPP). This platform allows in situ investigation of stress–strain characteristics of a mature cell–cell junction through defined strains and strain rates. With SCAµTT, we reveal some interesting biophysical phenomena at the single cell–cell junction that were previously not possible to observe using existing techniques. We show that cytoskeleton growth can effectively relax intercellular stress between an adherent cell pair in a strain-rate–dependent manner. Along with cadherin-clustering–induced bond strengthening, it prevents failure to occur at low strain rates. At high strain rates, insufficient relaxation leads to stress accumulation, which results in cell–cell junction rupture. We show that a remarkably large strain can be sustained before junction rupture (>200%), even at a strain rate as high as 0.5 s⁻¹. Collectively, the rate-dependent mechanical characterization of the cell–cell junction builds the foundation for an improved mechanistic understanding of junction adaptation to an external load and potentially the spatiotemporal coordination of participating molecules at the cell–cell junction.     

From the Cover: Toward net-zero sustainable aviation fuel with wet waste–derived volatile fatty acids

With the increasing demand for net-zero sustainable aviation fuels (SAF), new conversion technologies are needed to process waste feedstocks and meet carbon reduction and cost targets. Wet waste is a low-cost, prevalent feedstock with the energy potential to displace over 20% of US jet fuel consumption; however, its complexity and high moisture typically relegates its use to methane production from anaerobic digestion. To overcome this, methanogenesis can be arrested during fermentation to instead produce C₂ to C₈ volatile fatty acids (VFA) for catalytic upgrading to SAF. Here, we evaluate the catalytic conversion of food waste–derived VFAs to produce n-paraffin SAF for near-term use as a 10 vol% blend for ASTM “Fast Track” qualification and produce a highly branched, isoparaffin VFA-SAF to increase the renewable blend limit. VFA ketonization models assessed the carbon chain length distributions suitable for each VFA-SAF conversion pathway, and food waste–derived VFA ketonization was demonstrated for >100 h of time on stream at approximately theoretical yield. Fuel property blending models and experimental testing determined normal paraffin VFA-SAF meets 10 vol% fuel specifications for “Fast Track.” Synergistic blending with isoparaffin VFA-SAF increased the blend limit to 70 vol% by addressing flashpoint and viscosity constraints, with sooting 34% lower than fossil jet. Techno-economic analysis evaluated the major catalytic process cost-drivers, determining the minimum fuel selling price as a function of VFA production costs. Life cycle analysis determined that if food waste is diverted from landfills to avoid methane emissions, VFA-SAF could enable up to 165% reduction in greenhouse gas emissions relative to fossil jet.

Over 21 billion gallons of jet fuel are consumed in the United States annually, with demand expected to double by 2050 (1). The aviation sector accounts for 2.5% of global greenhouse gas emissions, with airlines committing to reduce their carbon footprint by 50% before 2050 (2, 3). Sustainable aviation fuels (SAF) comprise a significant portion of the aviation sector’s strategy for CO₂ reductions given the limited near-term prospects for electrification (3–5). In addition, the low aromatic content of current SAF routes has been shown to reduce soot formation and aviation-related aerosol emissions by 50 to 70% (2, 6, 7), which can significantly impact the net global warming potential. Soot is the primary nucleator of aviation-induced contrails (8), which have a larger effective radiative forcing (57.4 mW/m²) than aviation-emitted CO₂ alone (34.3 mW/m²) (3).Commercial SAF production in the United States currently relies on the hydrotreating of esters and fatty acids (HEFA) using virgin vegetable oils as well as waste fats, oils, and greases. These feedstocks also serve the renewable diesel market, which in 2018, produced ∼300 million gallons of HEFA diesel compared to ∼2 million gallons of HEFA SAF (1). Global HEFA capacity is estimated at 1.1 billion gallons per year (BGPY) in 2017 (9). HEFA SAF competes with demand for HEFA diesel, with US fossil diesel consumption estimated at ∼47 BGPY (10). Producing HEFA SAF requires an additional catalytic cracking step to convert predominantly C₁₆ and C₁₈ long chain fatty acids into C₈ to C₁₈ hydrocarbons suitable for jet fuel. This consumes additional hydrogen and lowers the jet and diesel fuel yield, making HEFA SAF more expensive to produce than HEFA diesel (11). California’s Low Carbon Fuel Standard (LCFS) has provided significant economic incentive for producing HEFA from low carbon intensity feedstocks (12), with petroleum companies continuing to retrofit existing refineries (13). Although this expansion will significantly increase biofuel production, the US availability of fats, oils, and greases is capped at ∼1.7 BGPY of jet fuel equivalent (14, 15). As such, efforts are needed to develop alternative feedstocks and conversion routes for SAF that avoid direct competition with food resources.Wet waste is an underutilized feedstock in the United States, with an energy content equivalent to 10.5 BGPY of jet fuel equivalent (assumed 130.4 MJ/gallon). Wet waste includes food waste (2.5 BPGY), animal manure (4.4 BGPY), wastewater sludge (1.9 BPGY), and the abovementioned waste fats, oils, and grease (1.7 BGPY) (14, 15). While waste lipid feedstocks may be best suited for HEFA refining, valorization strategies are needed for the remaining wet waste feedstocks. Diverting food waste from landfills is of particular note for reducing greenhouse gas emissions, as landfilling one dry ton of food waste has been estimated to release as much as 1.8 tons of CO₂ equivalents, assuming landfill methane is collected and recovered for electricity generation (16, 17). Globally, food waste accounts for 6% of greenhouse emissions (18). The high moisture content of wet waste restricts the use of conventional thermochemical conversion approaches (e.g., pyrolysis and gasification) used to produce liquid biofuels from terrestrial biomass, directing technology development efforts toward hydrothermal liquefaction, biological conversion, and hybrid processes (19).Currently, anaerobic digestion to produce biogas is the leading technology to recover energy from wet waste (20). The high moisture content of wet waste limits its transport and necessitates local processing, with the majority of US anaerobic digestion facilities located near population-dense areas and airports (21). Biogas purification provides a route to pipeline quality renewable natural gas compatible with existing infrastructure. Life cycle analysis has shown that negative carbon intensity can be achieved when producing renewable natural gas from municipal solid waste (−23 g CO₂eq/MJ) and dairy waste (−276 g CO₂eq/MJ), providing a significant economic driver under the LCFS (12). While renewable natural gas targets an enormous US market (∼246 BGPY of jet fuel equivalent) (10), producing liquid hydrocarbon fuels from wet waste offers the potential to address the challenge of decarbonizing the aviation sector.Anaerobic digestion of wet waste can be arrested prior to methanogenesis to generate both short chain (C₂ to C₅) and medium chain length (C₆ to C₈) carboxylic acids as precursors for biofuels and biobased chemicals (14, 22–27), hereon collectively referred to as volatile fatty acids (VFAs). VFA production by arrested methanogenesis offers the potential to utilize existing biogas infrastructure and a wide variety of wet waste feedstocks (14, 22, 28) with ongoing research and development working to increase VFA titers, rates, and yields by tailoring feedstock composition, microbial consortia, fermentation parameters, and online separation technologies (14, 22, 29–31). Currently, C₂ to C₅ carboxylic acids are primarily produced from the oxidation of petroleum derivatives, while C₆ and C₈ carboxylic acids are primarily derived from coconut and palm oil (29). Propionic acid (C₃) and butyric acid (C₄) address chemical market volumes on the order of 0.1 to 0.2 BGPY (29), while medium chain length carboxylic acids target smaller specialty markets. Given the availability of wet waste and potential saturation of biobased chemical markets in the long term, VFAs provide a potential target intermediate for catalytic upgrading into low carbon intensity biofuel (23, 25, 26, 32–35).VFAs can be catalytically upgraded to SAF through carbon coupling and deoxygenation chemistries. Depending on their chain length, VFAs can be converted into normal paraffins identical to those found in petroleum or undergo an additional carbon coupling step to generate isoparaffin, cycloparaffin, and aromatic hydrocarbons with molecular structures distinct from fossil jet (Fig. 1).Open in a separate windowFig. 1.Overview scheme of the major oxygenate and hydrocarbon molecules produced when converting wet waste VFA into Fast Track VFA-SAF that is composed of normal paraffin-rich hydrocarbons (Top Right) and Aldol Condensation VFA-SAF composed of isoparaffin-rich hydrocarbons (Bottom Right).Ketonization is the first unit operation to elongate the carbon backbone of VFAs (14, 22). Ketonization reacts two VFAs to produce a single ketone that is one carbon shorter than the sum of both acids and removes oxygen in the form of water and carbon dioxide (36, 37). Ketonization of acetic acid to acetone has been commercialized (37), with longer chain acids actively researched for biofuel and biochemical applications. Following ketonization, ketones ≥C₈ can undergo direct hydrodeoxygenation (Fig. 1, Top) to produce normal paraffin-rich hydrocarbons. In comparison, ketones ≤C₇ require a second carbon coupling step prior to hydrodeoxygenation to fall within the C₈ to C₁₈ range of jet fuel (Fig. 1, Bottom). Ketone carbon coupling can take place by various pathways including aldol condensation chemistry (25) as well as ketone reduction to alcohols for further dehydration and oligomerization (32, 34). Aldol condensation of central ketones is an emerging bench-scale chemistry that can generate structurally unique isoparaffins (38) with significantly lower freezing points for jet fuel applications due to the high degree of branching as well as reduce the intrinsic sooting tendency relative to aromatic hydrocarbons by over twofold (25).Normal paraffins produced from VFAs (Fig. 1, Top) can provide fungible hydrocarbons identical to those in petroleum that offer a near-term path to SAF qualification and market entry. In the United States, new SAF conversion routes must complete a rigorous qualification process to ensure fuel safety and operability overseen by ASTM International, the Federal Aviation Administration, and aviation original equipment manufacturers (OEMs). There are currently seven ASTM-approved routes for SAF that are derived from Fischer–Tropsch processing of syngas, the abovementioned esters and fatty acids, farnesene, ethanol, isobutanol, and algal hydrocarbons (39). Further details summarizing current ASTM-qualified routes to SAF can be found in SI Appendix, Table S1. Historically, ASTM qualification can require jet fuel volumes on order of over 100,000 gallons in order to pass a four-tiered screening process and two OEM review stage gates that may take place over a period of 3 to 7 y (40). To help reduce this barrier, in January 2020, ASTM approved a new “Fast Track” qualification process for SAF routes that produce hydrocarbons structurally comparable to those in petroleum jet with a 10 vol% blend limit (41). “Fast Track” eliminates two tiers of testing and facilitates approval with under 1,000 gallons of fuel and within the timeframe of 1 to 2 y (42).In contrast, isoparaffins derived from aldol condensation can offer complementary fuel properties to increase the renewable blend content of VFA-SAF blends, but the unique chemical structures would not qualify for “Fast Track” approval. To accelerate the approval of SAF routes that produce molecules distinct from petroleum jet, small-volume fuel tests and predictive tools are being developed for the most critical bulk properties, which screen for potentially deleterious engine operability effects (i.e., lean blowout, cold ignition, and altitude relight) (43). These tests, referred to as Tier α and β prescreening (44), evaluate SAF candidates for established ASTM D7566 properties, as well as novel properties observed to be important through the National Jet Fuels Combustion Program (SI Appendix, Table S2) (43). New properties include surface tension and derived cetane number (CN), which impact ignition and lean blowout propensity, respectively. At less than one mL of test volume, Tier α can utilize gas chromatograph (GC) and GCxGC method data to predict all critical properties. With between 50 and 150 mL of neat material (depending on the CN measurement method used), Tier β test methods can measure the critical unblended operability properties. In terms of emissions, low-volume sooting tendency measurement methods have been developed that require <1 mL of fuel versus the 10 mL required to measure smoke point (45, 46). Combined, these new methods allow for rapid evaluation of developing SAF conversion routes.To advance the technology and fuel readiness level of VFA-SAF, this work evaluates the production of drop-in normal paraffins and structurally unique isoparaffins from food waste–derived VFAs. First, VFAs were biologically produced from food waste and recovered neat by an industry partner, Earth Energy Renewables. A simplified kinetic model was then developed for mixed VFA ketonization to determine the ketone carbon chain length distribution suitable for SAF production by each conversion route, with model results compared to experiments with biogenic VFAs. VFA ketonization was assessed for >100 h of continuous time-on-stream (TOS), with trace impurities characterized within the incoming biogenic VFA feed. Catalyst regeneration was evaluated for coke and impurity removal, as well as to compare fresh and regenerated catalyst activity. Following VFA ketonization, ≥C₈ ketones were processed by direct hydrodeoxygenation to generate predominantly normal paraffins suitable for 10% blend testing for ASTM Fast Track, hereon referred to as “Fast Track VFA-SAF.” In parallel, VFA-derived ketones ≤C₇ were processed via aldol condensation and hydrodeoxygenation to produce predominantly isoparaffin hydrocarbons for Tier α and Tier β prescreening, hereon referred to as “Aldol Condensation VFA-SAF.” Higher blends with both VFA-SAF fractions were examined to increase the renewable carbon content and reduce soot formation while still meeting fuel property specifications. Lastly, techno-economic and life cycle analysis was performed to evaluate the sensitivity of catalytic process parameters on VFA-SAF production costs as well as potential greenhouse gas reductions relative to fossil jet.     

Assessment of enzyme active site positioning and tests of catalytic mechanisms through X-ray–derived conformational ensembles

How enzymes achieve their enormous rate enhancements remains a central question in biology, and our understanding to date has impacted drug development, influenced enzyme design, and deepened our appreciation of evolutionary processes. While enzymes position catalytic and reactant groups in active sites, physics requires that atoms undergo constant motion. Numerous proposals have invoked positioning or motions as central for enzyme function, but a scarcity of experimental data has limited our understanding of positioning and motion, their relative importance, and their changes through the enzyme’s reaction cycle. To examine positioning and motions and test catalytic proposals, we collected “room temperature” X-ray crystallography data for Pseudomonas putida ketosteroid isomerase (KSI), and we obtained conformational ensembles for this and a homologous KSI from multiple PDB crystal structures. Ensemble analyses indicated limited change through KSI’s reaction cycle. Active site positioning was on the 1- to 1.5-Å scale, and was not exceptional compared to noncatalytic groups. The KSI ensembles provided evidence against catalytic proposals invoking oxyanion hole geometric discrimination between the ground state and transition state or highly precise general base positioning. Instead, increasing or decreasing positioning of KSI’s general base reduced catalysis, suggesting optimized Ångstrom-scale conformational heterogeneity that allows KSI to efficiently catalyze multiple reaction steps. Ensemble analyses of surrounding groups for WT and mutant KSIs provided insights into the forces and interactions that allow and limit active-site motions. Most generally, this ensemble perspective extends traditional structure–function relationships, providing the basis for a new era of “ensemble–function” interrogation of enzymes.

The central role of enzymes in biology is embodied in the decades of effort spent to deeply investigate the origins of their catalysis (e.g., refs. 1–6). Enzyme studies now routinely identify the active-site groups that interact with substrates and reveal their roles in binding and in facilitating chemical transformations. Nevertheless, these so-called “catalytic groups” alone, outside of the context of a folded enzyme, do not account for the enormous rate enhancements and exquisite specificities exhibited by enzymes (4). Classic proposals for enzyme catalysis have invoked the importance of positioning of active-site groups within a folded enzyme and of substrates localized and positioned by binding interactions (6–15). While these proposals universally invoke restricted motion of catalytic groups, the amount of restriction and the amount of catalysis provided by that restriction has been the subject of much discussion and debate (16–20). Conversely, it is also clear that motions are inherent to enzymes, and that conformational transitions and structural rearrangements are important for enzyme function (e.g., refs. 11 and 21–23). Considering both positioning and motions, it has been recognized that: “For catalysis, flexible but not too flexible, as well as rigid but not too rigid, is essential. Specifically, the protein must be rigid enough to maintain the required structure but flexible enough to permit atomic movements as the reaction proceeds” (3).The importance of both positioning and motions to enzyme function suggests a nuanced view of enzyme catalysis and underscores the need for direct experimental measurements of positioning and motions within enzymes.As Feynman noted, “Everything that living things do can be understood in terms of the jigglings and wigglings of atoms” (24). But simply observing motions of active-site residues does not tell us how enzymes achieve catalysis. To understand enzymes, we want to know how much an enzyme dampens and alters the motions of catalytic residues. We want to know which increases or decreases in motion increase or decrease the reaction rate and what interactions and forces are most responsible for dampening motions. With this information we may be able to better design new enzymes. Additionally, to what extent are active-site residues positioned upon folding of the enzyme, or adjusted as the reaction proceeds, and are active-site residues more precisely positioned than residues throughout an enzyme?To address fundamental questions about how enzymes function and evolve, and how to ultimately design highly efficient enzymes, we need to obtain experimental information about enzyme conformation ensembles: The distribution of enzyme states dictated by their highly complex multidimensional energy landscapes over which conformational rearrangements occur. Observations of well-resolved electron densities from X-ray diffraction data indicate positioning of residues in and around the active site, but do not provide information on the extent and nature of that positioning. Crystallographic B-factors of residues are sometimes used to infer motions, but are only indirectly related to intrinsic motion and contain contributions from additional factors, such as crystallographic order (25, 26). NMR experiments identify groups with greater motional freedom and can provide temporal information, but these experiments typically lack information about the directions and extent of these motions (27). Molecular dynamics simulations provide atomic-level models for entire systems, but we currently lack the rigorous experimental tests needed to determine whether or not computational outputs reflect actual physical behavior, which prevents firm mechanistic conclusions from being inferred (28, 29).Two X-ray crystallographic approaches have recently emerged that can provide experimentally-derived conformational ensemble information: High-sequence similarity Protein Data Bank (PDB) structural ensembles (referred to as “pseudoensembles” herein) (30, 31) and multiconformer models from X-ray data obtained at temperatures above the protein’s glass transition (referred to as “room temperature” or ”RT” X-ray diffraction in the literature and herein) (22, 32, 33). These approaches are complementary. Pseudoensembles provide information about residues that move in concert (i.e., coupled motions) but require dozens of structures (see also SI Appendix, Supplementary Text 1). RT X-ray data from single crystals can provide multiconformer models, so that ensemble information about new complexes and mutants can more readily be acquired, but do not provide direct information about coupled motions. Furthermore, RT X-ray studies provide direct information about equilibrium distributions without cryocooling, which can alter and quench motions, and without assuming that different cryocooled crystals reproduce an equilibrium distribution of states (32, 34–36).Here we demonstrate consistency between these approaches and take advantage of the strengths of each: The ability to evaluate correlated side-chain rearrangements in and near the active site via pseudoensembles, and the ability to obtain new ensemble-type information of new states from single X-ray datasets at temperatures above the glass transition. Importantly, these analyses report on conformational heterogeneity and cannot give information about the timescales of motions and interconversions between states. Additionally, each traditional model within the pseudoensemble represents predominantly a single rather than average state and combining these states captures an ensemble distribution. Similarly, the alternate conformations in multiconformer models explicitly reduce bias toward average structures of multistate systems. Focusing on a model enzyme with very high-resolution data and with ligands representing steps along its reaction path has allowed us to obtain insights that would not be possible from static structures, from either ensemble approach alone or from less-extensive or lower-resolution data.We chose to investigate the enzyme ketosteroid isomerase (KSI) (Fig. 1) because of our ability to obtain high-resolution diffraction data, because of the accumulated wealth of structural and mechanistic information, and because of KSI’s use of catalytic strategies common to many enzymes. As a single-substrate enzyme, KSI allows structural information to be obtained with a bonified reactant bound. Furthermore, we obtained ensemble data for KSI from two species, which gave consistent results and allowed us to address unresolved questions from decades of KSI studies. We also used our ensembles from these KSI homologs to ask—and answer—more general questions. Our in-depth analyses of KSI bring an ensemble perspective to bear on traditional structure–function studies and provide the basis for a new era of ensemble–function studies.Open in a separate windowFig. 1.The KSI reaction. Reaction mechanism and schematic depiction of the active site (A) and its 3D organization (B) [PDB ID code 1OH0 (87)]. KSI catalyzes double bond isomerization of steroid substrates (shown for the substrate 5-androstene-3,17-dione) utilizing a general acid/base D40 (which we refer to herein as a general base, for simplicity), and an oxyanion hole composed of the side chains of Y16 and D103 (protonated); general base and oxyanion hole residues are colored in red and orange, respectively. The product in A, 4-androstene-3,17-dione, is the substrate of the reverse reaction and was used for RT X-ray crystallography herein. (C) Examples of oxyanion KSI TSAs used for the KSI TSA ensembles: Equilenin (Left) and a substituted phenolate (Right).     

Trapping a cross-linked lysine–tryptophan radical in the catalytic cycle of the radical SAM enzyme SuiB

The radical S-adenosylmethionine (rSAM) enzyme SuiB catalyzes the formation of an unusual carbon–carbon bond between the sidechains of lysine (Lys) and tryptophan (Trp) in the biosynthesis of a ribosomal peptide natural product. Prior work on SuiB has suggested that the Lys–Trp cross-link is formed via radical electrophilic aromatic substitution (rEAS), in which an auxiliary [4Fe-4S] cluster (AuxI), bound in the SPASM domain of SuiB, carries out an essential oxidation reaction during turnover. Despite the prevalence of auxiliary clusters in over 165,000 rSAM enzymes, direct evidence for their catalytic role has not been reported. Here, we have used electron paramagnetic resonance (EPR) spectroscopy to dissect the SuiB mechanism. Our studies reveal substrate-dependent redox potential tuning of the AuxI cluster, constraining it to the oxidized [4Fe-4S]²⁺ state, which is active in catalysis. We further report the trapping and characterization of an unprecedented cross-linked Lys–Trp radical (Lys–Trp•) in addition to the organometallic Ω intermediate, providing compelling support for the proposed rEAS mechanism. Finally, we observe oxidation of the Lys–Trp• intermediate by the redox-tuned [4Fe-4S]²⁺ AuxI cluster by EPR spectroscopy. Our findings provide direct evidence for a role of a SPASM domain auxiliary cluster and consolidate rEAS as a mechanistic paradigm for rSAM enzyme-catalyzed carbon–carbon bond-forming reactions.

The radical S-adenosylmethionine (rSAM) enzyme superfamily is the largest known in nature, with over 570,000 annotated and predominantly uncharacterized members spanning all domains of life (1–4). The uniting feature of rSAM enzymes is a [4Fe-4S] cluster, usually bound by a CX₃CX₂C motif that catalyzes reductive cleavage of SAM to form L-Met and a strongly oxidizing 5′-deoxyadenosyl radical (5′-dA•) (5–7). Recent studies on a suite of rSAM enzymes have revealed the presence of a previously unknown organometallic intermediate in this process, termed Ω, in which the 5′-C of 5′-dA• is bound to the unique iron of the [4Fe-4S] cluster (Fig. 1A) (8, 9). Homolysis of the Fe–C bond ultimately liberates 5′-dA•, which abstracts a hydrogen atom from substrate to initiate a profoundly diverse set of chemical reactions in both primary and secondary metabolism, including DNA, cofactor, vitamin, and antibiotic biosynthesis (5, 10–13).Open in a separate windowFig. 1.(A) Accepted scheme for radical initiation in rSAM enzymes. (B) X-ray crystal structure of SuiB (PDB ID: 5V1T). The RS domain, SPASM domain, and RiPP recognition element are rendered blue, green, and pink, respectively. [4Fe-4S] clusters are shown as spheres with the distances separating them indicated. (C) Lys–Trp cross-link formation (20) catalyzed by SuiB. The carbon–carbon bond installed by SuiB is shown in red. (D and E) Previously proposed EAS (D) and rEAS (E) mechanisms for SuiB-catalyzed Lys–Trp cross-link formation.Of the 570,000 rSAM enzyme superfamily members, over a quarter (∼165,000 genes from the Enzyme Function Initiative-Enzyme Similarity Tool) possess C-terminal extensions, called SPASM and twitch domains, which bind auxiliary Fe-S clusters (4, 14–19). The SPASM domain typically binds two auxiliary Fe-S clusters and is named after the rSAM enzymes involved in the synthesis of subtilosin, pyrroloquinoline quinone, anaerobic sulfatase, and mycofactocin. The twitch domain is a truncated SPASM domain and only binds one auxiliary cluster (15). Despite the wide prevalence of these domains and the characterization of several different SPASM/twitch rSAM enzymes by spectroscopic and structural studies, direct evidence for their catalytic function(s) has remained elusive.We previously performed functional and structural characterization on the SPASM rSAM enzyme SuiB (Fig. 1B), which is involved in the biosynthesis of a ribosomal peptide natural product in human and mammalian microbiome streptococci (14, 20–22). SuiB introduces an unusual carbon–carbon bond onto its substrate peptide, SuiA, between the sidechains of Lys2 and Trp6 (Fig. 1C). The mechanism for this transformation is of broader relevance, as a number of enzymes, such as RrrB, PqqE, and MqnC (2, 23, 24), are known to join unactivated aliphatic and aromatic carbons to generate sp³-sp² cross-links. A general mechanistic paradigm for this class of transformations is not yet available. For SuiB, two pathways have been proposed (20), one through a typical electrophilic aromatic substitution (EAS) mechanism, which is involved in other enzyme-catalyzed indole modifications, such as indole prenylation or flavin adenine dinucleotide (FAD)-enzyme-dependent indole chlorination (25–27). In this pathway, the 5′-dA• generates an alkyl radical, which upon a second one-electron oxidation, creates an α,β-unsaturated amide electrophile with which the indole sidechain reacts via Michael addition (Fig. 1D). Lanthionine cross-links observed in diverse lanthipeptides are built by this general scheme, though via heterolytic chemistry, with Cys acting as the nucleophile (28, 29). Alternatively, a radical electrophilic aromatic substitution (rEAS) reaction has been proposed, wherein the alkyl radical, formed by 5′-dA•, would react with the indole sidechain to generate a radical σ complex, a cross-linked Lys–Trp radical (Lys–Trp•), which upon oxidation and rearomatization would yield product (Fig. 1E). In both mechanisms, AuxI is proposed as an oxidant. Although this role for an rSAM auxiliary cluster has been previously suggested (30, 31), it has yet to be directly demonstrated experimentally. Mechanistic studies have favored the rEAS pathway (20); however, intermediates in the reaction of SuiB and enzymes that catalyze similar reactions have not yet been detected (15).In the current work, we sought to differentiate between the proposed mechanisms by trapping intermediates in the catalytic cycle of SuiB and characterizing them using electron paramagnetic resonance (EPR) spectroscopy. We report observation of three transient reaction intermediates, most importantly the sought-after Lys–Trp•, which is fundamentally different from previously characterized Trp radicals, as it is cross-linked and carries an indole tetrahedral center. We also provide evidence for AuxI as the oxidant of the Lys–Trp• intermediate as well as insights into redox potential changes of Fe-S clusters in SuiB that accompany SuiA binding. Together, our findings support the rEAS pathway for formation of the sp³-sp² cross-link and carry important implications for other enzymes that catalyze related transformations.