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).     

The evolution and changing ecology of the African hominid oral microbiome

The oral microbiome plays key roles in human biology, health, and disease, but little is known about the global diversity, variation, or evolution of this microbial community. To better understand the evolution and changing ecology of the human oral microbiome, we analyzed 124 dental biofilm metagenomes from humans, including Neanderthals and Late Pleistocene to present-day modern humans, chimpanzees, and gorillas, as well as New World howler monkeys for comparison. We find that a core microbiome of primarily biofilm structural taxa has been maintained throughout African hominid evolution, and these microbial groups are also shared with howler monkeys, suggesting that they have been important oral members since before the catarrhine–platyrrhine split ca. 40 Mya. However, community structure and individual microbial phylogenies do not closely reflect host relationships, and the dental biofilms of Homo and chimpanzees are distinguished by major taxonomic and functional differences. Reconstructing oral metagenomes from up to 100 thousand years ago, we show that the microbial profiles of both Neanderthals and modern humans are highly similar, sharing functional adaptations in nutrient metabolism. These include an apparent Homo-specific acquisition of salivary amylase-binding capability by oral streptococci, suggesting microbial coadaptation with host diet. We additionally find evidence of shared genetic diversity in the oral bacteria of Neanderthal and Upper Paleolithic modern humans that is not observed in later modern human populations. Differences in the oral microbiomes of African hominids provide insights into human evolution, the ancestral state of the human microbiome, and a temporal framework for understanding microbial health and disease.

The oral cavity is colonized by one of the most diverse sets of microbial communities of the human body, currently estimated at over 600 prevalent taxa (1). Dental diseases, such as caries and periodontitis, remain health burdens in all human populations despite hygiene interventions (2, 3), and oral microbes are often implicated in extraoral inflammatory diseases (4, 5). To date, most oral microbiome research has focused on clinical samples obtained from industrialized populations that have daily oral hygiene routines and access to antibiotics (1, 6), but far less is known about the global diversity of the oral microbiome, especially from diverse past and present nonindustrialized societies (7). The oral cavity contains at least six distinct habitats, but dental biofilms, including both supra- and subgingival dental plaque, are among the most diverse and clinically important (1, 6, 8). During life, these dental biofilms naturally and repeatedly calcify, forming dental calculus (tooth tartar) (9), a robust, long-term record of the oral microbiome (10). Archaeological dental calculus has been shown to preserve authentic oral bacterial metagenomes in a wide range of historic and prehistoric populations and up to 50 thousand years ago (ka) (10–13). As such, dental calculus presents an opportunity to directly investigate the evolution of the hominid microbiome and to reconstruct ancestral states of the modern human oral microbiome. In addition, because research has shown that evolutionary traits, diet, and cultural behaviors shape modern human microbiome structure and function at other body sites, such as the gut and skin microbiomes (14–18), investigating ancient oral metagenomes has the potential to reveal valuable information about major events in modern human evolution and prehistory, such as predicted dietary changes during the speciation of Homo (19–21) and the direct interaction of Neanderthals and modern humans during the Late Pleistocene (22).To better understand the evolutionary ecology of the African hominid microbiome, we generated and analyzed 109 dental calculus metagenomes from present-day modern humans (n = 8), gorillas (Gorilla, n = 29), chimpanzees (Pan, n = 20), Neanderthals (n = 13), and two groups of archaeological modern humans associated with major lifestyle transitions (preagricultural, n = 20; preantibiotic, n = 14), as well as New World howler monkeys (n = 5) for comparison (SI Appendix, Fig. S1). To account for potential sampling biases, we analyzed multiple subspecies and populations of each African great ape genus, which were obtained from C20^th or C21^st-collected museum collections, and for modern humans we sampled multiple populations from both Africa and Europe. To this, we added previously published microbiome data from chimpanzees (n = 1) (13), Neanderthals (n = 4) (13), and present-day modern humans (n = 10) (23), for a total dataset of 124 individuals (Fig. 1A, SI Appendix, Table S1, and Dataset S1). We also generated eight new radiocarbon dates for archaeological individuals, for a total of 44 directly or indirectly dated ancient individuals in this study (Dataset S1).Open in a separate windowFig. 1.Sample locations and oral microbiome authentication of ancient dental calculus. (A) Sample locations. (B) PCoA comparing euclidean distances of microbial genera of well-preserved ancient and present-day dental calculus to environmental proxy controls (degraded archaeological bone) and present-day dental plaque and feces. Ancient dental calculus is distinct from gut and archaeological bone but overlaps with present-day dental plaque. (C) Representative DNA damage patterns for Neanderthals and ancient and present-day modern humans for four oral-specific bacterial species. The Neanderthal and upper Paleolithic modern human individuals show expected damage patterns consistent with authentic aDNA, whereas the present-day individual does not. See also SI Appendix, Fig. S4.Here, we investigate the structure, function, and core microbial members of the human oral microbiome within an evolutionary framework, seeking to determine whether a core microbiome can be defined for each African hominid group, whether the core is phylogenetically coherent, and whether some members of the core are specific to certain host groups. We test whether the oral microbiome of hominids reflects host phylogeny, finding that African hominid oral microbiota are distinguished by major taxonomic and functional differences that only weakly reflect host relationships and are likely influenced by other physiological, dietary, or behavioral factors. We compare the microbial profiles of Neanderthals and modern humans and, contrary to expectations (12, 13), find a high consistency of oral microbiome structure within Homo, regardless of geography, time period, or diet/lifestyle. We detect the persistence of shared genetic diversity in core taxa between Neanderthals and Upper Paleolithic humans prior to 14 ka, supporting a growing body of evidence for earlier admixture and interaction in Ice Age Europe (24, 25). Finally, we explore possible implications of our findings on Homo-associated encephalization (19, 26) and the role of dietary starch in human evolution (20, 21) by investigating the evolutionary history of amylase-binding capability by oral streptococci. We find that amylase binding is an apparent Homo-specific trait, suggestive of microbial coadaptation to starch-rich diets early in human evolution.     

From the Cover: Aquatic biodiversity enhances multiple nutritional benefits to humans

Humanity depends on biodiversity for health, well-being, and a stable environment. As biodiversity change accelerates, we are still discovering the full range of consequences for human health and well-being. Here, we test the hypothesis—derived from biodiversity–ecosystem functioning theory—that species richness and ecological functional diversity allow seafood diets to fulfill multiple nutritional requirements, a condition necessary for human health. We analyzed a newly synthesized dataset of 7,245 observations of nutrient and contaminant concentrations in 801 aquatic animal taxa and found that species with different ecological traits have distinct and complementary micronutrient profiles but little difference in protein content. The same complementarity mechanisms that generate positive biodiversity effects on ecosystem functioning in terrestrial ecosystems also operate in seafood assemblages, allowing more diverse diets to yield increased nutritional benefits independent of total biomass consumed. Notably, nutritional metrics that capture multiple micronutrients and fatty acids essential for human well-being depend more strongly on biodiversity than common ecological measures of function such as productivity, typically reported for grasslands and forests. Furthermore, we found that increasing species richness did not increase the amount of protein in seafood diets and also increased concentrations of toxic metal contaminants in the diet. Seafood-derived micronutrients and fatty acids are important for human health and are a pillar of global food and nutrition security. By drawing upon biodiversity–ecosystem functioning theory, we demonstrate that ecological concepts of biodiversity can deepen our understanding of nature’s benefits to people and unite sustainability goals for biodiversity and human well-being.

Species losses and range shifts because of climate change, harvesting, and other human activities are altering aquatic biodiversity locally and globally (1–5). In aquatic ecosystems, not only are some species severely depleted because of overfishing or habitat loss (3, 6–8), the ecosystem-level dimensions of biodiversity such as the total number of species and their functional diversity have also changed (9). Beyond the loss of particular species, changes in ecosystem-level dimensions of biodiversity threaten numerous ecosystem services to humans, which include the cultural, economic, or health benefits people derive from nature (10–13). In many regions, such as tropical coastal systems, the cumulative impacts of human activities are severe and associated with strong declines in taxonomic and ecological functional diversity (6) and coincide with regions with a high dependence of people upon wild-caught seafood for food and nutrition (14). In temperate regions, where some coastal communities depend on local wild seafood harvests to meet their nutritional needs (15, 16), species richness may be increasing as species recover from exploitation and warmer oceans allow species to expand their ranges into new territory (1, 2, 17).There is growing concern that biodiversity change leads to changes in human health and well-being (10, 13, 18). Specific and quantitative links between aquatic biodiversity and human health that distinguish contributions of species diversity from those of biomass, as predicted by biodiversity–ecosystem functioning theory, have not been established. At a time of unprecedented global change and increasing reliance on seafood to meet nutritional demands (19), there is an urgent need to understand how changing aquatic ecosystem structure may alter the provisioning of seafood-derived human nutrition.Seafood, consisting of wild-caught marine and freshwater finfish and invertebrates, provides an important source of protein and calories to humans. Additionally, unlike staple foods such as rice or other grains, seafood can address multiple dimensions of food and nutritional security simultaneously by providing essential micronutrients, such as vitamins, minerals, and polyunsaturated essential fatty acids critical to human health (19–22). Given the multiple attributes of seafood that are valuable to human health, it is possible that the diversity of an aquatic assemblage, distinct from the inclusion of any particularly nutritious species, could support human well-being consistent with a large body of evidence for biodiversity’s major contributions to ecological functions (11, 23–26). Dietary diversity is a basic tenet of a nutritious diet (27) and it is widely appreciated that diets composed of more food groups and more species are more nutritious (28–31). Ecological measures of dietary diversity (diet diversity, species richness, functional diversity, and Simpson’s index of evenness) have been associated with the nutritional value of diets in a range of contexts (27, 29, 32–38). These studies rely on relationships between species included in the diet (or other food intake measures) and nutritional adequacy of reported diets. However, a simple correlation between dietary diversity and a measure of dietary benefits provides only partial support for a claim that biodiversity benefits human well-being, consistent with the same ecological processes by which biodiversity supports numerous ecosystem functions and services (23, 26). We build upon this foundation of empirical relationships between diet diversity and diet quality by placing this question in the quantitative ecological theoretical framework that relates biodiversity to function (24, 25), thereby laying the groundwork for additional development of links between biodiversity science and our understanding of human well-being.Ecological theory predicts that biodiversity can be ecologically and economically important, apart from the importance of total biomass or the presence of particular species (23, 39). According to theory and over 500 explicit experimental tests (23, 40, 41), diversity in ecological communities and agricultural systems enhances ecosystem functioning by two mechanisms: 1) more diverse assemblages may outperform less diverse assemblages of the same density or biomass of individuals because more diverse assemblages will include more of the possible species and are therefore more likely to include high-performing species, assuming random processes of including species from the species pool (a selection effect), or 2) more diverse assemblages of a given density (or biomass) contain species with complementary functional traits, allowing them to function more efficiently (a complementarity effect) (25, 39). For aquatic animals, increased diversity enhances productivity of fish biomass (42) and also enhances temporal stability of biomass production and total yields (43, 44), providing economic and nutritional benefits to humans related to increased stability of harvests and production of biomass for consumption (43). However, when considering aquatic species from the perspective of human nutrition, functions other than biomass production become relevant because total seafood biomass consumption is not predictive of micronutrient benefits from seafood (45, 46).Here, we test a hypothesis central to ecological theory in the 21st century: whether biodiversity per se (species richness and ecological functional diversity), distinct from the identities and abundance of species, enhances human well-being (Fig. 1). We chose a measure of human well-being distinct from provision of protein, calories, or total yields—the micronutrient and essential fatty acid benefits of seafood. For increasing biodiversity per se (as opposed to increasing total seafood consumption) to enhance nutritional benefits as predicted by biodiversity–ecosystem functioning theory (25, 47), the amounts of various nutrients within edible tissues must differ among species, and furthermore, nutrient concentrations must trade off among species, such that species that have relatively high concentrations of some nutrients also have relatively low concentrations of others (25). Specifically, a “biodiversity effect” (sensu ref. 25) on nutritional benefits requires that concentrations of multiple nutrients are negatively correlated with each other, or uncorrelated, when compared among species, creating a complementary distribution of nutrients across species. In contrast, if nutrient concentrations in edible tissue are positively correlated for multiple nutrients across species such that, for example, a species containing high amounts of iron also has a high essential fatty acid concentration, thereby containing multiple nutrients in high concentrations simultaneously, seafood species or ecological functional diversity in the diet would not be important. In the case of positive correlations among nutrient concentrations, the ecosystem service of nutritional benefits would be enhanced by consuming more fish biomass or by selecting a few highly nutritious species, without considering species richness or ecological functional diversity.Open in a separate windowFig. 1.Aquatic biodiversity increases human well-being because edible species have distinct and complementary multinutrient profiles (A) and differ in mean micro- and macronutrient content (shown here relative to 10 and 25% thresholds of recommended dietary allowance, RDA, guidelines) for representative finfish (Abramis brama, Mullus surmuletus), mollusc (Mytilus galloprovincialis), and crustacean species (Nephrops norvegicus). Biodiversity–ecosystem functioning theory predicts that nutritional benefits, including the number of nutrient RDA targets met per 100 g portion (NT; i, iii) and minimum portion size (P_min; ii, iv) (B and E), are enhanced with increasing seafood species richness. Orange dots in B and E correspond to potential diets of high and low biodiversity levels. Seafood consumers with limited access to seafood each day may not reach RDA targets if diets are low in diversity (D–F versus A–C; gray shading indicates proportion of population that meets nutrient requirements). DHA: docosahexaenoic acid, EPA: eicosapentaenoic acid.We aimed to bridge two distinct theoretical frameworks—the biodiversity–ecosystem functioning theory and human nutrition science—by quantitatively testing for effects of aquatic species richness and ecological functional diversity (48, 49) in seafood diets on nutritional benefits via complementarity or selection effects. We used the public health measure of recommended dietary allowance (RDA) index to quantify nutritional benefits. RDAs are nutrient-based reference values that indicate the average daily dietary intake level that is sufficient to meet the nutrient requirement of nearly all (97 to 98%) healthy individuals in a particular life stage and gender group (50). Here, we used the RDA for females aged 19 to 50 y (SI Appendix, Tables S1 and S2; see SI Appendix, Table S1 for definitions of key terms). We measured nutritional value in terms of concentrations relative to RDAs, and we refer to these recommended amounts (or portions thereof) as “RDA targets” (SI Appendix, Tables S1 and S2 and Metrics). We quantified nutritional value in two ways: 1) the minimum amount of seafood tissue (in grams) required to meet given RDA targets (either for a single nutrient or the five micronutrients and fatty acids simultaneously; referred to as “minimum portion size required,” P_min [SI Appendix, Table S1, Eq. A1, and Metrics]) and 2) the number of nutrients that meet an RDA target in a single 100 g seafood portion (NT, SI Appendix, Table S1, Eq. A2). By considering nutritional value per unit biomass in both metrics, we avoided confounding diversity of seafood consumed with the total amount consumed (Metrics). We first tested two hypotheses: 1) seafood species richness increases NT because of complementarity in nutrient concentrations among species, and 2) seafood species richness increases the nutritional value of a 100 g edible portion of seafood, thereby lowering the minimum portion size, P_min, and improving the efficiency with which seafood consumers reach nutritional targets (Fig. 1). Following biodiversity–ecosystem functioning theory, we predicted that increased species richness is correlated with ecological functional diversity (51) in potential seafood diets and that ecological functional diversity is related to diversity in the concentration of essential elements and fatty acids that have nutritional value to human consumers, such that species and ecological functional diversity yields increased nutritional benefits. We also tested the hypothesis that seafood diversity increases total intake of heavy metal contaminants because some aquatic animals are known to bioaccumulate toxic metals in their tissues. For this reason, variation in bioaccumulation among species could lead to a biodiversity effect on contaminant intake that is detrimental to human health.In a global analysis of over 5,040 observations of nutrient concentrations in 547 aquatic species (SI Appendix, Fig. S1), we considered the provision of nutritional benefits to human consumers. To assess whether the relationships between biodiversity and human nutrition benefits depend on the geographic extent (global or local) over which seafood are harvested or accessed (11), we tested whether seafood species richness is associated with higher nutritional value at local scales (versus global scale) in traditional Indigenous seafood diets in North America (SI Appendix, Methods 1.4). Seafood is critical for Indigenous groups, who on average consume seafood at a rate that is 15 times higher than the global average per capita consumption rate (16). To test our hypotheses at the geographic scale of local consumer communities, we complemented our global analysis with additional analyses of 25 to 57 species in 14 geographically constrained groups of species consumed together as part of traditional Indigenous diets (SI Appendix, Methods 1.4).     

Transfer-printed,tandem microscale light-emitting diodes for full-color displays

Inorganic semiconductor-based microscale light-emitting diodes (micro-LEDs) have been widely considered the key solution to next-generation, ubiquitous lighting and display systems, with their efficiency, brightness, contrast, stability, and dynamic response superior to liquid crystal or organic-based counterparts. However, the reduction of micro-LED sizes leads to the deteriorated device performance and increased difficulties in manufacturing. Here, we report a tandem device scheme based on stacked red, green, and blue (RGB) micro-LEDs, for the realization of full-color lighting and displays. Thin-film micro-LEDs (size ∼100 μm, thickness ∼5 μm) based on III–V compound semiconductors are vertically assembled via epitaxial liftoff and transfer printing. A thin-film dielectric-based optical filter serves as a wavelength-selective interface for performance enhancement. Furthermore, we prototype arrays of tandem RGB micro-LEDs and demonstrate display capabilities. These materials and device strategies provide a viable path to advanced lighting and display systems.

Microscale light-emitting diodes (micro-LEDs) based on inorganic semiconductors have been widely regarded as one of the most promising solutions to the next generation of emissive display technologies for versatile applications, from televisions, smartphones and wearable watches, to advanced virtual, augmented, and mixed realities (1–4). Constructed from single-crystalline–compound semiconductors like gallium arsenide (GaAs), gallium phosphide (GaP), and gallium nitride (GaN), these micro-LEDs present significant benefits over conventional liquid crystal displays (LCDs) (5), organic LEDs (OLEDs) (6–9), and more recent quantum dot (10, 11) and perovskite-based LEDs (12), in terms of their efficiencies, brightness, contrast, dynamic response, and long-time stability. High-resolution displays rely on arrays of polychromatic (red, green, and blue [RGB]) emissive elements with dimensions of less than 100 μm or even 10 μm, which are massively and heterogeneously assembled onto silicon, glass, or plastic substrates (13–17). This scaling down of device sizes enhances the display resolution and reduces the raw material cost; however, it is also accompanied by significant challenges. First, reducing LED size causes dramatic efficiency decreases for all types of LEDs (18–21), particularly GaAs- or GaP-based red LEDs that are more susceptible to sidewall defects (SI Appendix, Fig. S1). Second, device shrinkage also results in deteriorated uniformity, which influences the reliability and performance of display systems. Third, fabricating and transferring smaller devices demand higher accuracies for alignment and positioning (22), imposing greatly increased manufacturing expenses (SI Appendix, Fig. S2).Alternatively, device arrays with a single-pixel, spectrally tunable LED (23) or vertically stacked tandem LEDs (24) relieve the above constraints associated with conventional laterally arranged RGB micro-LEDs. However, color-tunable LEDs based on voltage-dependent spectral change can neither achieve full-range tunability nor obtain independent color/intensity controllability (25, 26). On the other hand, the current state-of-the-art wafer-bonding–based assembly methods only realize double-layer stacking with the capability of dual-color tuning (green/blue or red/blue) (22, 27, 28). Although there are some reports on full-color stacked inorganic and organic RGB LEDs (24, 29–31), these demonstrations are only limited to a few of chip-scale, large or thick LEDs for lighting purposes.In this paper, we report a tandem thin-film RGB micro-LED architecture with full-range color tunability to overcome the limitations of previously explored technologies. Based on the epitaxial liftoff and transfer printing method, arrays of thin-film, high-performance, inorganic RGB micro-LEDs made of different III–V compounds are assembled in a vertical stack. Embedded into the stacked structure, a thin-film dielectric filter serves as a wavelength selective interlayer to improve the LED light output. Independently addressable active arrays show full-color electroluminescent patterns, demonstrating the display capability of the tandem micro-LEDs.     

Robust folding of a de novo designed ideal protein even with most of the core mutated to valine

Protein design provides a stringent test for our understanding of protein folding. We previously described principles for designing ideal protein structures stabilized by consistent local and nonlocal interactions, based on a set of rules relating local backbone structures to tertiary packing motifs. The principles have made possible the design of protein structures having various topologies with high thermal stability. Whereas nonlocal interactions such as tight hydrophobic core packing have traditionally been considered to be crucial for protein folding and stability, the rules proposed by our previous studies suggest the importance of local backbone structures to protein folding. In this study, we investigated the robustness of folding of de novo designed proteins to the reduction of the hydrophobic core, by extensive mutation of large hydrophobic residues (Leu, Ile) to smaller ones (Val) for one of the designs. Surprisingly, even after 10 Leu and Ile residues were mutated to Val, this mutant with the core mostly filled with Val was found to not be in a molten globule state and fold into the same backbone structure as the original design, with high stability. These results indicate the importance of local backbone structures to the folding ability and high thermal stability of designed proteins and suggest a method for engineering thermally stabilized natural proteins.

The de novo design of protein structures, starting from pioneering work (1, 2), has been achieved in tandem with our understanding of how amino acid sequences determine folded structures (3–16). A breakthrough in protein design methodology was a finding of principles for encoding funnel-shaped energy landscapes into amino acid sequences (7, 10, 17, 18). Based on studies of protein folding, it had been suggested that naturally occurring proteins have evolved to have funnel-shaped energy landscapes toward their folded structures (19–23). However, complicated structures of naturally occurring proteins with nonideal features for folding—for example, kinked α-helices, bulged β-strands, long or strained loops, and buried polar groups—make it difficult to understand how the funnels are encoded in amino acid sequences. By focusing on protein structures without such nonideal features, we proposed principles for designing ideal protein structures stabilized by completely consistent local and nonlocal interactions (24), based on a set of rules relating local backbone structures to preferred tertiary motifs (7, 10). These design rules describe the relation of the lengths or torsion patterns of two secondary structure elements and the connecting loop to favorable packing geometries (SI Appendix, Fig. S1A). The design principles enable to encode strongly funneled energy landscapes into amino acid sequences, by the stabilization of folded structures (positive design) and by the destabilization of nonnative conformations (negative design) due to the restriction of folding conformational space by the rules (SI Appendix, Fig. S1C). In the design procedure, backbone structures for a target topology are generated based on a blueprint (SI Appendix, Fig. S1B), in which either the lengths or backbone torsion patterns of the secondary structures and loops are determined using the rules so that the tertiary motifs present in the target topology are favored, and then amino acid sequences stabilizing the generated backbone structures are designed. The designed amino acid sequences stabilize their folded structures both with nonlocal interactions such as hydrophobic core packing and with local interactions favoring the secondary structures and loops specified in the blueprint, which destabilize a myriad of nonnative topologies through local backbone strain captured by the rules, thereby resulting in funnel-shaped energy landscapes (SI Appendix, Fig. S1C). The principles have enabled the de novo design of ideal protein structures for various topologies with atomic-level accuracy (Fig. 1) (6, 7, 10, 13).Open in a separate windowFig. 1.In silico energy landscapes and far-UV circular dichroism (CD) spectra for 10 de novo designed ideal proteins. (A–E) Five designs by Koga et al. in 2012 (7). (F–I) Four designs by Lin et al. in 2015 (10). (J) Top7 by Kuhlman et al. in 2003 (6). (Top) Design models. (Middle) Energy landscapes obtained from Rosetta ab initio structure prediction simulations (41). Red points represent the lowest energy structures obtained in independent Monte Carlo structure prediction trajectories starting from an extended chain for each sequence; the y axis is the Rosetta all-atom energy; the x axis is the Cα root-mean-square deviation (RMSD) to the design model. Green points represent the lowest energy structures obtained in trajectories starting from the design model. (Bottom) The far-UV CD spectra during thermal denaturation with the melting temperature T_m, which is obtained by fitting to the denaturation curves shown in SI Appendix, Fig. S2.Interestingly, the de novo designs exhibit prominent characteristics in terms of thermal stability when compared with naturally occurring proteins. The circular dichroism (CD) measurements up to 170 °C conducted in this study revealed the melting temperature (T_m), which was above 100 °C for most of the designs (Fig. 1) (6, 7, 10). Therefore, the designs have great potential for use as scaffolds to engineer proteins with specific functions of interest. Indeed, miniprotein structures (∼40 residues) designed de novo according to the rules were applied as scaffolds for creating protein binders specific for influenza hemagglutinin and botulinum neurotoxin, displaying high thermal stability (>70 °C) despite the small size (25).The rules in the principles described above emphasize the importance of local backbone structures not the details of amino acid side chains to protein folding, which is also supported by studies using simple calculations with the hydrophobic-polar lattice model or the snake-cube model (26, 27). On the other hand, it is known that hydrophobic interactions are the dominant driving force for folding (28, 29) and the cores of naturally occurring proteins are tightly packed with hydrophobic amino acid residues (30, 31) like a jigsaw puzzle. Indeed, in our design principles, protein cores were designed to be tightly packed and as “fat” as possible with larger hydrophobic residues so that energy landscapes were sculpted to be deeply funneled into a target topology by lowering its energy (SI Appendix, Fig. S1C).Which factor, the local backbone structures encoded by the rules or the tight core packing with fat hydrophobic residues, contributes more to the generation of funnels in the designs? Here, we studied the contribution of hydrophobic core packing to folding ability and thermal stability by investigating the robustness of folding against the reduction of packing, using the design with the highest thermal stability among our nine de novo designs (Fig. 1, except Top7), Rsmn2x2_5_6 (10). We started to study single-residue mutants from Leu or Ile to Val that prune one carbon atom from the aliphatic side chain, which lose the tight packing like a jigsaw puzzle and decrease the hydrophobicity, and then, we combined the mutations. Consequently, we found that a mutant with 10 residue substitutions of Leu or Ile with Val still has the folding ability and high thermal stability despite its reduced and loosened hydrophobic core packing. This result suggests the importance of the local backbone structures for the folding ability and stability of the de novo designs.     

Cryo-EM structure of Mycobacterium smegmatis DyP-loaded encapsulin

Encapsulins containing dye-decolorizing peroxidase (DyP)-type peroxidases are ubiquitous among prokaryotes, protecting cells against oxidative stress. However, little is known about how they interact and function. Here, we have isolated a native cargo-packaging encapsulin from Mycobacterium smegmatis and determined its complete high-resolution structure by cryogenic electron microscopy (cryo-EM). This encapsulin comprises an icosahedral shell and a dodecameric DyP cargo. The dodecameric DyP consists of two hexamers with a twofold axis of symmetry and stretches across the interior of the encapsulin. Our results reveal that the encapsulin shell plays a role in stabilizing the dodecameric DyP. Furthermore, we have proposed a potential mechanism for removing the hydrogen peroxide based on the structural features. Our study also suggests that the DyP is the primary cargo protein of mycobacterial encapsulins and is a potential target for antituberculosis drug discovery.

Compartmentalization is used by cells to overcome many difficult metabolic and physiological challenges (1). Eukaryotes employ membrane-bound organelles such as the mitochondrion (2); however, most prokaryotes rely on alternative proteinaceous compartments to achieve spatial control (3), one of which is the encapsulin nanocompartment.Encapsulins are newly identified nanocompartments but have already been applied in various scientific fields due to the unique structures (4, 5). It has been reported that more than 900 putative encapsulin systems in bacteria and archaea exist and are distributed across 15 bacterial and two archaeal phyla (6, 7), suggesting they are functionally diverse. Encapsulins are made of one type of shell protein, as opposed to several as is observed in many bacterial microcompartments (8, 9). The key feature of encapsulin systems is that cargo proteins can be specifically encapsulated and targeted to the encapsulin capsid interior, using a selective C-terminal sequence referred to as targeting peptides (TPs) (10). The functions of the nanocompartment are associated with the functions of its protein cargo. Many functionally diverse cargo proteins are associated with encapsulins, including dye-decolorizing peroxidases (DyPs) (11), ferritin-like proteins (FLP) (12), hydroxylamine oxidoreductase (HAO) (13), and cysteine desulfurases (14). Moreover, it has been shown that some encapsulin systems may possess multiple cargo proteins, which are made up of one core cargo protein and up to three secondary cargo proteins according to the TPs (6). Notably, a large proportion of native cargo proteins are DyP-type peroxidases, conferring the resistance of the cell to oxidative stress (6, 7, 11, 15–18). However, to date, the structural information on the cargo-encapsulated encapsulins is not yet available (SI Appendix, Table S1), and thus, little is known about the structural arrangement and mechanistic features of the cargo proteins loaded in the encapsulins.Actinobacteria harbors the largest number of encapsulin or encapsulin-like systems (6). DyP-containing encapsulins have already been reported from mycobacteria, including Mycobacterium smegmatis (15) and Mycobacterium tuberculosis (19). These have been considered as potential biomarkers to detect active tuberculosis (TB) (20). In the present study, we have isolated and characterized a DyP-loaded encapsulin system from M. smegmatis, which is commonly used as a model organism in studying the biology of the M. tuberculosis (21). We have determined its complete high-resolution structure by cryogenic electron microscopy (cryo-EM). Our results have revealed the interactions between the CFP-29 (a 29 kDa culture filtrate protein) shell and DyP cargo and a potential antioxidation mechanism. Our study also lays the foundation for the discovery of new diagnosis protocols and treatments of TB.     

Deep time diversity and the early radiations of birds

Reconstructing the history of biodiversity has been hindered by often-separate analyses of stem and crown groups of the clades in question that are not easily understood within the same unified evolutionary framework. Here, we investigate the evolutionary history of birds by analyzing three supertrees that combine published phylogenies of both stem and crown birds. Our analyses reveal three distinct large-scale increases in the diversification rate across bird evolutionary history. The first increase, which began between 160 and 170 Ma and reached its peak between 130 and 135 Ma, corresponds to an accelerated morphological evolutionary rate associated with the locomotory systems among early stem birds. This radiation resulted in morphospace occupation that is larger and different from their close dinosaurian relatives, demonstrating the occurrence of a radiation among early stem birds. The second increase, which started ∼90 Ma and reached its peak between 65 and 55 Ma, is associated with rapid evolution of the cranial skeleton among early crown birds, driven differently from the first radiation. The third increase, which occurred after ∼40 to 45 Ma, has yet to be supported by quantitative morphological data but gains some support from the fossil record. Our analyses indicate that the bird biodiversity evolution was influenced mainly by long-term climatic changes and also by major paleobiological events such as the Cretaceous–Paleogene (K–Pg) extinction.

The evolution of global biodiversity is a focal area of study in both evolutionary biology and paleontology, but its examination has been approached in different ways. Neontological studies reconstruct the history of biodiversity mainly by analyzing the tempo and mode of diversification based on molecular timetrees composed of extant species (1). By contrast, paleontologists normally measure past biodiversity by investigating morphological evolution and taxic diversity from the fossil record (2). This dichotomy in both methodology and data sources is best exemplified by recent studies on the evolution of bird biodiversity (the vernacular term “birds” is equivalent to the phylogenetic taxon “Avialae” in the present paper; see Methods and SI Appendix, Supplemental Text A). For example, the time-calibrated phylogeny of extant birds and related diversification rate analyses have revealed a rapid diversification of crown birds (equivalent to the phylogenetic taxon “Aves”) near the Cretaceous–Paleogene (K–Pg) boundary followed by a period of low-level net diversification rates starting about 50 Ma (3–5). Paleontological studies have revealed high morphological evolutionary rates both prior to and after the origin of Avialae (6–14), and an increased taxonomic diversity in the Early Cretaceous based on the known Mesozoic fossil record (15) (SI Appendix, Supplemental Text B). However, these results are not directly comparable and are difficult to be evaluated within the same evolutionary framework given that they are based on different evaluation parameters of bird diversity.     

Integrative analysis reveals unique structural and functional features of the Smc5/6 complex

Structural maintenance of chromosomes (SMC) complexes are critical chromatin modulators. In eukaryotes, the cohesin and condensin SMC complexes organize chromatin, while the Smc5/6 complex directly regulates DNA replication and repair. The molecular basis for the distinct functions of Smc5/6 is poorly understood. Here, we report an integrative structural study of the budding yeast Smc5/6 holo-complex using electron microscopy, cross-linking mass spectrometry, and computational modeling. We show that the Smc5/6 complex possesses several unique features, while sharing some architectural characteristics with other SMC complexes. In contrast to arm-folded structures of cohesin and condensin, Smc5 and Smc6 arm regions do not fold back on themselves. Instead, these long filamentous regions interact with subunits uniquely acquired by the Smc5/6 complex, namely the Nse2 SUMO ligase and the Nse5/Nse6 subcomplex, with the latter also serving as a linchpin connecting distal parts of the complex. Our 3.0-Å resolution cryoelectron microscopy structure of the Nse5/Nse6 core further reveals a clasped-hand topology and a dimeric interface important for cell growth. Finally, we provide evidence that Nse5/Nse6 uses its SUMO-binding motifs to contribute to Nse2-mediated sumoylation. Collectively, our integrative study identifies distinct structural features of the Smc5/6 complex and functional cooperation among its coevolved unique subunits.

Structural maintenance of chromosomes (SMC) complexes regulate genome organization and maintenance in both prokaryotic and eukaryotic cells. Each complex contains a pair of SMC subunits and a set of non-SMC subunits (1). Studies of several SMC proteins reveal that they form tripartite filamentous structures. An SMC subunit folds back on itself at its middle “hinge” region, enabling its N- and C-terminal ATPase domains to associate forming a “head” region, and its two long coiled-coil regions located in between the hinge and the head to pair in an antiparallel manner, forming an “arm” region (SI Appendix, Fig. S1A) (1). The two SMC subunits of each complex form its backbone and can associate with each other at hinge, head, and arm regions (1).Much of the molecular understanding of SMC complexes has come from studies of those acting as DNA organization and separation factors, such as prokaryotic Smc-ScpAB and MukBEF complexes and eukaryotic cohesin and condensin. These complexes can entrap and loop DNA, resulting in DNA tethering and folding (2–4). One emerging feature of these complexes is that their long arm regions bend sharply at so-called “elbow” sites (SI Appendix, Fig. S1A). Elbow bending causes the hinge to contact the head-proximal coiled-coil or head-bound non-SMC proteins, a conformation thought to facilitate DNA loop extrusion (5–8). In these SMCs, the head and hinge regions that associate with other proteins and DNA are conserved, while the arm regions are not and act mainly as connecting elements (9).Differing from cohesin (containing Smc1/3) and condensin (containing Smc2/4), the third eukaryotic SMC complex, containing Smc5 and Smc6, does not appear to affect chromatid intertwining or mitotic chromosome structures (10–12). Rather, the Smc5/6 complex directly regulates DNA replication and recombinational repair (13–15). These unique functions correlate with the acquisition of a special set of six subunits, namely non-SMC elements (Nse)1 to 6. Three Nse subunits, Nse2, Nse5, and Nse6, are not found in any other SMC complexes in either prokaryotes or eukaryotes. Nse2 (also known as Mms21) is a SUMO ligase that promotes the sumoylation of more than a dozen genome maintenance factors (16–19). Nse5 and Nse6 are thought to act distinctly from Nse2 by forming a subcomplex that recruits the Smc5/6 complex to DNA damage sites (13–15, 20–22). The acquisition of the Nse2, Nse5, and Nse6 subunits is one of the most unique features that sets the Smc5/6 complex apart from other SMC complexes.Our understanding of how the Smc5/6 complex gained unique functions among the SMC family of complexes is hindered by the limited structural information of its holo-complex. Studies of subunits and their fragments or subcomplexes have provided insights into potential intersubunit interactions (23–25). However, these data may not reflect structures and interactions within the entire complex. Thus, it is imperative to determine, in the context of the holo-complex, whether Smc5 and Smc6 adopt distinct conformations relative to other SMCs, how they associate with Nse subunits, and what the functional relationships are among the complex-specific Nse2, -5, and -6 subunits. Here, we provide an integrative structural analysis of the Smc5/6 holo-complex isolated from budding yeast that addresses these challenges. We use negative-staining electron microscopy (EM), cross-linking mass spectrometry (CL-MS), single-particle cryo-EM, structural modeling, and functional analyses to identify several unique features of the Smc5/6 complex that distinguish it from the other SMC complexes. We also provide evidence that the coevolved Nse2, -5, and -6 subunits are connected at both structural and functional levels.     

From the Cover: Crop wild relatives of the United States require urgent conservation action

The contributions of crop wild relatives (CWR) to food security depend on their conservation and accessibility for use. The United States contains a diverse native flora of CWR, including those of important cereal, fruit, nut, oil, pulse, root and tuber, and vegetable crops, which may be threatened in their natural habitats and underrepresented in plant conservation repositories. To determine conservation priorities for these plants, we developed a national inventory, compiled occurrence information, modeled potential distributions, and conducted threat assessments and conservation gap analyses for 600 native taxa. We found that 7.1% of the taxa may be critically endangered in their natural habitats, 50% may be endangered, and 28% may be vulnerable. We categorized 58.8% of the taxa as of urgent priority for further action, 37% as high priority, and 4.2% as medium priority. Major ex situ conservation gaps were identified for 93.3% of the wild relatives (categorized as urgent or high priority), with 83 taxa absent from conservation repositories, while 93.1% of the plants were equivalently prioritized for further habitat protection. Various taxonomic richness hotspots across the US represent focal regions for further conservation action. Related needs include facilitating greater access to and characterization of these cultural-genetic-natural resources and raising public awareness of their existence, value, and plight.

Wild plants related to domesticated crops provide important genetic resources for plant breeding (1, 2). Owing to their close evolutionary relationships with cultivated species, traits from crop wild relatives (CWR) can be introgressed into domesticates with relative ease (3, 4). These plants are central to research on domestication, evolution, and anthropology (5–8) and may themselves be attractive candidates for de novo domestication (9). Furthermore, many of these species are collected for direct dietary and other cultural uses (10, 11). As populations of some of these taxa are adapted to extreme climates, adverse soil types, and significant pests and diseases, they have been identified as key contributors in breeding for sustainability and climate adaptation (12). As characterization and breeding technologies advance, their use in crop improvement will also become more efficient (1, 13).Knowledge gaps regarding CWR, including information on their taxonomy, relatedness to pertinent crops, geographic distribution, ecological interactions, agriculturally relevant traits, and degree of representation in conservation systems, constrain their potential use in crop improvement (1). These gaps likewise affect conservation efforts, which are essential to protect vulnerable populations from habitat destruction, climate change, pollution, invasive species, and overharvesting in their natural habitats (in situ), and to ensure that these cultural-genetic-natural resources are safeguarded over the long term and available for research and education in ex situ plant conservation repositories (i.e., gene banks and botanical gardens) (14–16). Previous analyses indicate that many CWR are poorly conserved both in situ and ex situ, highlighting the urgency of addressing fundamental information gaps to support efforts related to their conservation and accessibility for use (16–18).Here we develop a national inventory of CWR of the United States, wherein taxa are classified based on current knowledge of their relation to agricultural crops and their significance as wild food sources (SI Appendix, Table S1). We use occurrence information combined with climatic and topographic data to model the potential distributions of 600 prioritized native wild taxa, including wild relatives of apples (Malus Mill.), barley (Hordeum L.) beans (Phaseolus L.), blueberries and cranberries (Vaccinium L.), chile peppers (Capsicum L.), cotton (Gossypium L.), currants (Ribes L.), grapes (Vitis L.), hops (Humulus L.), onions (Allium L.), pecans (Carya Nutt.), plums (Prunus L.), potatoes (Solanum L.), pumpkins and zucchini (Cucurbita L.), raspberries and blackberries (Rubus L.), strawberries (Fragaria L.), sunflowers (Helianthus L.), sweetpotatoes (Ipomoea L.), and other crops (SI Appendix, Table S2).We then use ecogeographic tools to conduct preliminary threat assessments and conservation gap analyses for the CWR. These are based on an approximation of the distribution of species’ genetic diversity, using the extent of geographic and ecological variation in their predicted native ranges as a proxy, which has been shown to be an effective surrogate (19, 20), facilitating conservation planning despite pervasive gaps in population-level genetic data (20–22). The ecogeographic variation evident in the locations where ex situ conservation samples have been collected and evident in species’ ranges distributed within protected natural areas is measured against the variation found within species’ overall predicted native ranges. Geographic and ecological gaps in current conservation are then identified, providing baseline information for conservation planning, prioritization, and action.     

Structural basis of Chikungunya virus inhibition by monoclonal antibodies

Chikungunya virus (CHIKV) is an emerging viral pathogen that causes both acute and chronic debilitating arthritis. Here, we describe the functional and structural basis as to how two anti-CHIKV monoclonal antibodies, CHK-124 and CHK-263, potently inhibit CHIKV infection in vitro and in vivo. Our in vitro studies show that CHK-124 and CHK-263 block CHIKV at multiple stages of viral infection. CHK-124 aggregates virus particles and blocks attachment. Also, due to antibody-induced virus aggregation, fusion with endosomes and egress are inhibited. CHK-263 neutralizes CHIKV infection mainly by blocking virus attachment and fusion. To determine the structural basis of neutralization, we generated cryogenic electron microscopy reconstructions of Fab:CHIKV complexes at 4- to 5-Å resolution. CHK-124 binds to the E2 domain B and overlaps with the Mxra8 receptor-binding site. CHK-263 blocks fusion by binding an epitope that spans across E1 and E2 and locks the heterodimer together, likely preventing structural rearrangements required for fusion. These results provide structural insight as to how neutralizing antibody engagement of CHIKV inhibits different stages of the viral life cycle, which could inform vaccine and therapeutic design.

Chikungunya virus (CHIKV), a single-stranded positive-sense RNA envelope virus, is an emerging alphavirus transmitted to humans by Aedes species mosquitoes (1, 2). CHIKV consists of three related genotypes: Asian, East/Central/South African (ECSA), and West African (3). According to the Centers for Disease Control and Prevention, there have been millions of cases reported in approximately 100 countries. CHIKV infection causes an acute febrile illness accompanied by musculoskeletal disease (4, 5). A subset of cases (∼30%) showed that chronic arthritis can develop and persist for months to years (6, 7).The 12-kb positive-sense RNA genome is packaged within an icosahedral nuclear capsid core composed of 240 copies of capsid proteins, which is surrounded by a host-derived lipid bilayer. The surface of the mature CHIKV particle (diameter ∼700 Å) has 80 trimeric envelope E1-E2 heterodimer protein spikes anchored on the lipid bilayer membrane (SI Appendix, Fig. S1A) arranged in T = 4 icosahedral symmetry. E1 and E2 protein ectodomains each consist of three domains: E1-DI; E1-DII and E1-DIII; and E2-DA, E2-DB, and E2-DC (SI Appendix, Fig. S1B). The fusion loop on the distal end of E1-DII mediates endosomal membrane fusion. The groove formed by E2-DA and E2-DB shields the fusion loop of E1 protein from premature membrane fusion at neutral pH (8). Multiple attachment factors have been implicated in CHIKV entry of cells (9), and E2-DB reportedly contains receptor-binding sites (10, 11). Mxra8, a recently identified alphavirus receptor (12), recognizes an epitope spanning both the E1 and E2 proteins (13, 14).The virus infection cycle starts with the E1-E2 proteins binding to the cell-surface receptors (12). The virion is then internalized into the endosome (15, 16). The acidic condition of the endosome causes E1-E2 heterodimers to undergo conformational changes, exposure of the E1 fusion loop for insertion into the endosomal membrane, and subsequent reorganization of the E1 protein into E1 trimers to allow endosomal membrane fusion (17, 18). After fusion, the capsid and RNA genome are released into the cytoplasm (19) to allow translation and replication of the viral genome. The newly synthesized virus buds at the plasma membrane (20).Currently, there exist no licensed CHIKV vaccine or therapeutics. Neutralizing antibodies have been shown to confer both prophylactic and therapeutic protection in animal models (21–28). Here we show the potencies of two CHIKV antibodies, CHK-124 and CHK-263, in vivo and demonstrate that they inhibit multiple steps in the virus infection cycle in vitro. We also determined the cryogenic electron microscopy (cryo-EM) structures of their Fab fragments complexed with CHIKV to 4- to 5-Å resolution. For CHK-124, the predominant neutralization mechanisms are aggregation of virus particles and inhibition of receptor binding. For CHK-263, the mechanism is the inhibition of fusion by locking E1 and E2 proteins together. Altogether, our study provides a structural understanding as to how potent antibodies block CHIKV infection.     

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.     

From the Cover: Invasiveness is linked to greater commercial success in the global pet trade

The pet trade has become a multibillion-dollar global business, with tens of millions of animals traded annually. Pets are sometimes released by their owners or escape, and can become introduced outside of their native range, threatening biodiversity, agriculture, and health. So far, a comprehensive analysis of invasive species traded as pets is lacking. Here, using a unique dataset of 7,522 traded vertebrate species, we show that invasive species are strongly overrepresented in trade across mammals, birds, reptiles, amphibians, and fish. However, it is unclear whether this occurs because, over time, pet species had more opportunities to become invasive, or because invasive species have a greater commercial success. To test this, we focused on the emergent pet trade in ants, which is too recent to be responsible for any invasions so far. Nevertheless, invasive ants were similarly overrepresented, demonstrating that the pet trade specifically favors invasive species. We show that ant species with the greatest commercial success tend to have larger spatial distributions and more generalist habitat requirements, both of which are also associated with invasiveness. Our findings call for an increased risk awareness regarding the international trade of wildlife species as pets.

The extraordinary movement of our own species through migration, colonization, and travel has driven the geographic expansion of countless other species since prehistoric times (1). Humans have deliberately introduced a diverse range of species, in particular domesticated crops and animals that have contributed to our success (1). Today, however, the trade in live organisms for nonutilitarian reasons has rocketed (2–4). In the last decade alone, billions of plants and animals comprising thousands of species were traded annually, fueling a multibillion-dollar global business (2, 3, 5, 6). In particular, the demand for nontraditional (also known as “exotic”) ornamentals and pets, i.e., organisms without a long history of domestication, has grown (2). These species are sometimes released into the wild or escape and may survive and reproduce (2, 7–9). Species with populations that have established outside of their native range are referred to as invasive species hereafter (see Table 1 for terminology). Some invasive species can have severe impacts on global biodiversity (10–13) and impose tremendous costs on society by damaging physical infrastructure, agriculture, forestry, and human health (14, 15). However, even though it is undisputed that the trade in pets and ornamentals contributes to the global movement of invasive animals (16–19), it is still unclear whether this trade specifically favors invasive species.Table 1.Glossary

Vagus nerve stimulation activates two distinct neuroimmune circuits converging in the spleen to protect mice from kidney injury

Acute kidney injury is highly prevalent and associated with high morbidity and mortality, and there are no approved drugs for its prevention and treatment. Vagus nerve stimulation (VNS) alleviates inflammatory diseases including kidney disease; however, neural circuits involved in VNS-induced tissue protection remain poorly understood. The vagus nerve, a heterogeneous group of neural fibers, innervates numerous organs. VNS broadly stimulates these fibers without specificity. We used optogenetics to selectively stimulate vagus efferent or afferent fibers. Anterograde efferent fiber stimulation or anterograde (centripetal) sensory afferent fiber stimulation both conferred kidney protection from ischemia–reperfusion injury. We identified the C1 neurons–sympathetic nervous system–splenic nerve–spleen–kidney axis as the downstream pathway of vagus afferent fiber stimulation. Our study provides a map of the neural circuits important for kidney protection induced by VNS, which is critical for the safe and effective clinical application of VNS for protection from acute kidney injury.

Acute kidney injury (AKI) is characterized by a rapid loss of kidney function that is indicated by increased serum creatinine and/or decreased urine output. This debilitating condition affects ∼10 to 15% of patients admitted to hospitals, and its incidence in intensive care units can exceed 50% (1). AKI is associated with high morbidity and mortality as well as major complications including fluid overload, electrolyte disturbances, uremic complications, and drug toxicity (2). Recent epidemiological and experimental observations have also demonstrated that AKI can lead to chronic kidney disease and kidney failure (3, 4), conditions associated with a lower health–related quality of life and with an increased risk of mortality and cardiovascular morbidity (5, 6). Despite these severe consequences, there are no Food and Drug Administration–approved drugs for the prevention and treatment of AKI. Novel therapies with innovative approaches are desperately needed to address this growing concern.Mechanisms of neuroimmune regulation have been attracting significant attention for their potential to benefit patients with inflammatory disease (7, 8). Activation of the cholinergic anti-inflammatory pathway (CAP) by vagus nerve stimulation (VNS) is one of the most promising strategies to harness neuroimmune interactions and attenuate inflammation associated with various diseases including kidney disease (9). The vagus nerve (10th cranial nerve) is a bilateral nerve bundle composed of axons of efferent (motor) and afferent (sensory) neurons; the former provides input to thoracic/abdominal organs, and the latter transmits sensory information from these organs to the central nervous system (CNS). The canonical CAP is elicited by the activation of the parasympathetic efferent vagus nerve and requires the spleen; the parasympathetic signal is thought to activate splenic ganglionic noradrenergic neurons (10) via synapses located within the celiac/suprarenal/superior mesenteric ganglia (11–15). Norepinephrine is released from splenic nerve terminals and binds to β₂ adrenergic receptors expressed on a population of choline acetyltransferase (ChaT)–positive CD4⁺ CD44^high CD62L^low memory T cells. This leads to the release of acetylcholine from these cells (16) that binds to α7 nicotinic acetylcholine receptors (α7nAChRs) expressed on macrophages, resulting in the suppressed production of proinflammatory cytokines (e.g., tumor necrosis factor-α) by macrophages and suppressed inflammation (10, 17). Although activation of this canonical CAP (efferent vagus nerve–splenic nerve–spleen axis) is effective in reducing the severity of many inflammatory disease models, including endotoxemia (10, 16, 17) and colitis (18), other pathways elicited by VNS have also been shown to have an anti-inflammatory effect. For example, the efferent vagus nerve synapses on cholinergic myenteric neurons that are in close contact with muscularis macrophages expressing α7nAChRs in the intestine, and VNS activates these macrophages to ameliorate surgery-induced intestinal inflammation (19). Interestingly, electrical stimulation of the central end of the transected vagus nerve (“central VNS”) also produces an anti-inflammatory effect in experimental arthritis (20) and endotoxemia (21). These findings suggest that VNS can engage multiple neural circuits in a context-dependent manner to attenuate inflammation.VNS was also shown to be effective in kidney transplantation. VNS in brain-dead donor rats reduced inflammation in the donors and immune cell infiltration in the transplanted kidneys in recipients, leading to improved long-term transplant kidney function and recipient survival (22, 23). We previously demonstrated that electrical stimulation of the cervical vagus nerve in mice 24 h before kidney ischemia–reperfusion injury (IRI) markedly attenuated AKI and that the kidney protection was through α7nAChR⁺ splenocytes (24). However, the precise neural circuit(s) involved in the spleen-dependent kidney protection by VNS remain unknown to date. When the vagus nerve is electrically stimulated, action potentials are transmitted in two directions (anterograde and retrograde) in both efferent and afferent fibers (SI Appendix, Fig. S1A): 1) Anterograde efferent fiber stimulation provides input to thoracic/abdominal organs and elicits the canonical CAP. 2) In retrograde efferent fiber stimulation, action potentials propagate back to the cell bodies in the medulla oblongata, which could alter the function of vagus efferent neurons. 3) Anterograde activation of vagal sensory afferents stimulates first-order neurons located in the nucleus tractus solitarius (NTS) and subsequently countless brain regions (25, 26). 4) Retrograde afferent fiber stimulation causes the release of a variety of neuropeptides (e.g., substance P, calcitonin gene-related peptide [CGRP]) at nerve terminals in thoracic/abdominal organs and those peptides have proinflammatory or anti-inflammatory effects on immune cells (27). The neuropeptide release at nerve terminals of afferent sensory neurons may contribute to the development of inflammatory diseases especially in the skin (e.g., psoriasis) (28). On the other hand, release of CGRP from vagal and somatic sensory afferents has direct anti-inflammatory effects on immune cells in the lung and skin (29–31). Thus, retrograde vagal afferent fiber stimulation could contribute to anti-inflammation and organ protection by VNS, a concept that has not previously been explored, due in part to prior methodological constraints. For example, transecting or applying a local anesthetic to the vagus nerve to block nerve conduction prior to electrical stimulation enables a distinction between distal and central VNS effects, but this strategy is still not selective because VNS elicits a combination of anterograde and retrograde stimulation in efferent and afferent fibers in this setting (SI Appendix, Fig. S1B).As a first step to identify the neural circuit(s) involved in the kidney protection by VNS, we utilized optogenetics for selective stimulation of vagus efferent or afferent fibers and demonstrated that anterograde stimulation of either efferent or afferent fibers is sufficient to protect the kidneys from IRI. We also showed that the protection is mediated by splenocytes in both cases. Since the protective effect of anterograde efferent fiber stimulation is consistent with the CAP activation, we further sought to identify the downstream pathway of anterograde afferent fiber stimulation. Vagal sensory afferents innervate the NTS, which projects in turn to many other regions in the CNS (32). We explored three downstream pathways to the periphery: 1) vagal efferents (a vagovagal reflex), 2) sympathetic efferents (a vagosympathetic reflex), and 3) hypothalamo–pituitary–adrenocortical (HPA) axis, all of which can have immunomodulatory effects upon activation (9, 33, 34), and our findings strongly suggest that a vagosympathetic reflex plays an important role in the kidney protection by vagal afferent fiber stimulation. We further identified the splenic nerve as a critical branch of the sympathetic nerve in mediating kidney protection, which is consistent with the importance of splenocytes in this context.Next, we sought to identify a central node in the CNS that mediates the vagosympathetic reflex to confer kidney protection. C1 neurons, which reside in the rostral ventrolateral medulla (RVLM), are glutamatergic, catecholaminergic, and peptidergic neurons, and receive direct input from neurons of the NTS (35). The C1 neurons are activated by a variety of physical stressors and circulating inflammatory molecules and serve as a central regulator of autonomic function (36, 37). They innervate sympathetic preganglionic neurons in the intermediolateral cell column of the spinal cord as well as neurons in the dorsal motor nucleus of the vagus (where most of the neurons of the efferent vagus nerve originate) and neurons in the paraventricular nucleus of the hypothalamus (the primary driver of the HPA axis) (36). We previously demonstrated that selective stimulation of C1 neurons protected the kidneys from AKI (38). Here we demonstrate that C1 neurons are an integrative center for the neural pathway in vagus afferent fiber stimulation and kidney protection. Our study defines a neural circuit involved in kidney protection by VNS, which is critical for the safe and effective clinical application of VNS for protection from AKI.     

From the Cover: Ancient noeggerathialean reveals the seed plant sister group diversified alongside the primary seed plant radiation

Noeggerathiales are enigmatic plants that existed during Carboniferous and Permian times, ∼323 to 252 Mya. Although their morphology, diversity, and distribution are well known, their systematic affinity remained enigmatic because their anatomy was unknown. Here, we report from a 298-My-old volcanic ash deposit, an in situ, complete, anatomically preserved noeggerathialean. The plant resolves the group’s affinity and places it in a key evolutionary position within the seed plant sister group. Paratingia wuhaia sp. nov. is a small tree producing gymnospermous wood with a crown of pinnate, compound megaphyllous leaves and fertile shoots each with Ω-shaped vascular bundles. The heterosporous (containing both microspores and megaspores), bisporangiate fertile shoots appear cylindrical and cone-like, but their bilateral vasculature demonstrates that they are complex, three-dimensional sporophylls, representing leaf homologs that are unique to Noeggerathiales. The combination of heterospory and gymnospermous wood confirms that Paratingia, and thus the Noeggerathiales, are progymnosperms. Progymnosperms constitute the seed plant stem group, and Paratingia extends their range 60 My, to the end of the Permian. Cladistic analysis resolves the position of the Noeggerathiales as the most derived members of a heterosporous progymnosperm clade that are the seed plant sister group, altering our understanding of the relationships within the seed plant stem lineage and the transition from pteridophytic spore-based reproduction to the seed. Permian Noeggerathiales show that the heterosporous progymnosperm sister group to seed plants diversified alongside the primary radiation of seed plants for ∼110 My, independently evolving sophisticated cone-like fertile organs from modified leaves.

The origin of the seed in the Late Devonian, ∼365 Mya, represents a key innovation in land plant evolution. Seeds provided a fundamentally new reproductive strategy that overcame the limitations of free-sporing, pteridophytic reproduction and enabled colonization of drier habitats (1‒3). Progymnosperms, the evolutionary stem group leading to seed plants, display a mosaic of evolutionary characters combining free-sporing reproduction with production of secondary xylem (wood) through a bifacial vascular cambium characteristic of seed plants (2‒4). Although not representing a monophyletic evolutionary group, progymnosperms are important for our present understanding of the origin of the seed and represent intermediates between pteridophytes and seed plants (1, 2, 4). Progymnosperms include the basally divergent homosporous Aneurophytales, as well as the more derived heterosporous Archaeopteridales, Protopityales (1, 4), and the enigmatic Carboniferous cone Cecropsis (5). The transition to seed plants requires multiple character state transitions from known progymnosperm sister groups comprising either Archaeopteris (6, 7) or Archaeopteris + Cecropsis (e.g., ref. 8). For each of these cases, viable intermediates are absent from the fossil record.In contrast, Noeggerathiales (9) have at times been proposed as progymnosperms (9–12), but this has been controversial. Comprising ∼20 genera and 50 species, Noeggerathiales are known from the late Carboniferous–Permian (323 to 251 Ma) tropical floras in North America, Europe, and East Asia (10), where they are identified as a related group based on shared features of heterospory, adaxial sporangial attachment to “sporophylls,” longitudinal sporangial dehiscence, plagiotropic pinnule attachment to the rachis, and once-pinnate compound megaphylls (9–12). However, their systematic position remained uncertain because details of their stem anatomy were unknown. In the absence of this anatomical information, they have been postulated as close relatives of the progymnosperms, leptosporangiate ferns, sphenopsids, the extant fern Tmesipteris, or as a distinct class of their own (see ref. 9 for summary). Recently, leaves of Plagiozamites oblongifolius that were interpreted as noeggerathialean (9) have also been interpreted as cycads (13). Wang et al. (9) considered the Noeggerathiales to be putative progymnosperms, but as their specimens lacked wood, a more confident assignment was not possible. This uncertainty meant that even when considered as progymnosperms, their relationship to other progymnosperms and seed plants has remained speculative.Here, we report a species that we name Paratingia wuhaia J. Wang et al. sp. nov. and ascribe to the Noeggerathiales. The fossils were collected from a single 66-cm-thick volcanic ash bed in the Chinese “vegetational Pompeii” from the Taiyuan Formation at Wuda open coalmine, Inner Mongolia (14, 15) (SI Appendix, Geological Information, Materials). The ash preserved in situ the morphology and anatomy of plants in exquisite detail (Fig. 1 A–N) and has been dated to 298.34 ± 0.09 Ma during the Asselian stage of the early Permian (16).Open in a separate windowFig. 1.P. wuhaia J. Wang et al. sp. nov. from the early Permian Taiyuan Formation of Wuda Coalfield, Inner Mongolia. (A) Holotype with an entire crown consisting of pseudostrobili and leaves. (B) Once-pinnate compound leaf with both large and small pinnules visible. Reprinted with permission from ref. 15. (C) Cross-section of a crown illustrating pseudostrobili around the stem. (D) Cross-section of pseudostrobilus with microsporangia around the axis with bilateral, inversed Ω-shaped vascular bundle. (E) Cross-section of a leaf rachis showing the same form of vascular bundle as that of pseudostrobili axes. (F–H) Partial cross, radial, and tangential sections of the stem showing the secondary xylem (wood). (I) Tangential section of pseudostrobilus showing sporangial arrangement with single line of megasporangia along with the axis (middle-right pseudostrobilus of the fragmental crown illustrated SI Appendix, Fig. S8; specimen PB22132; magnified in SI Appendix, Fig. S21C.) (J) Radial section of pseudostrobilus showing adaxial sporangia and axis lacking nodes (upmost-left pseudostrobilus of the fragmental crown in SI Appendix, Fig. S8; magnified in SI Appendix, Fig. S21A). (K) Tangential section showing adaxial sporangia and a single line of megasporangia along with the axis (SI Appendix, Fig. S7; specimen PB22131, second up-right pseudostrobilus). (L) Tangential section through same specimen as K showing megasporangial arrangement (magnified in SI Appendix, Figs. S21B and S23A). (M) Detail of the middle part of L showing the megasporangia and microsporangia. (N) Single spore macerated from the holotype. (Scale bars: A, 10 cm; B, 3 cm; C–E, 1 cm; F, 100 μm; G and H, 200 μm; I–L, 5 mm; M, 2 mm; N, 10 μm.)Isolated noeggerathialean fertile shoots (SI Appendix, Figs. S3–S8) and megaphyllous leaves (SI Appendix, Figs. S9‒S14) are common in the assemblage. The species P. wuhaia is based on >200 specimens, including >10 specimens that were preserved with intact crowns containing both leaves and fertile shoots in organic attachment to the stems, demonstrating that they belong to a single species (Fig. 1A and SI Appendix, Figs S2‒S4).     

Optical vector field rotation and switching with near-unity transmission by fully developed chiral photonic crystals

State-of-the-art nanostructured chiral photonic crystals (CPCs), metamaterials, and metasurfaces have shown giant optical rotatory power but are generally passive and beset with large optical losses and with inadequate performance due to limited size/interaction length and narrow operation bandwidth. In this work, we demonstrate by detailed theoretical modeling and experiments that a fully developed CPC, one for which the number of unit cells N is high enough that it acquires the full potentials of an ideal (N → ∞) crystal, will overcome the aforementioned limitations, leading to a new generation of versatile high-performance polarization manipulation optics. Such high-N CPCs are realized by field-assisted self-assembly of cholesteric liquid crystals to unprecedented thicknesses not possible with any other means. Characterization studies show that high-N CPCs exhibit broad transmission maxima accompanied by giant rotatory power, thereby enabling large (>π) polarization rotation with near-unity transmission over a large operation bandwidth. Polarization rotation is demonstrated to be independent of input polarization orientation and applies equally well on continuous-wave or ultrafast (picosecond to femtosecond) pulsed lasers of simple or complex (radial, azimuthal) vector fields. Liquid crystal–based CPCs also allow very wide tuning of the operation spectral range and dynamic polarization switching and control possibilities by virtue of several stimuli-induced index or birefringence changing mechanisms.

Optical vector field (more commonly called polarization) rotators and switches are essential components of all modern optical and photonic systems for communications, ellipsometry, metrology, biological/chemical detection, and quantum processing/computing (1–10). There are, however, some inherent limitations. Wave plates made with birefringent crystals, for example, require strict alignment of the optic axis with respect to the polarization orientation of incident light and generally do not work with laser vector beams of complex polarization fields; Faraday rotators that do not have this requirement are generally too cumbersome and bulky due to their weak optical rotatory powers. One promising approach to circumvent these limitations is to employ chiral optical materials such as chiral photonic crystals and metasurfaces. Nevertheless, structural chirality, such as chiral metamaterials, metasurfaces, and photonic crystals that are capable of very large optical rotatory power (up to ∼100,000°/mm), are inevitably accompanied by large absorption losses (11–15). In metamaterials/surfaces, the intrinsic noncircular absorption and nanofabrication difficulty also add to the limitation of their practical scalability in the interaction length, resulting in small (<π) net polarization rotation angle, very small aperture, and narrow operating spectral bandwidth (11–13). Similar issues confront most chiral photonic crystals (CPCs) due to the limitations of molecular self-assembly or nanofabrication/processing technique and high transmission loss associated with operation near the Bragg reflection band (14, 15).Here, we show by theory and experimental corroborations that a fully developed liquid crystal–based CPC, one for which the number of unit cells N approaches that (N → ∞) of an ideal crystal, can circumvent all the aforementioned limitations and possess several advantageous characteristics impossible with conventional low-N thin counterparts. Such high-period–number chiral photonic crystals (HN-CPCs) are achieved by fabricating cholesteric liquid crystals (CLCs) to thicknesses several hundred times that of conventional ones using a refined field-assisted self-assembly (FASA) technique (16, 17; see SI Appendix, Note 1, for more details). Optical properties of CLCs as CPCs arise from complex “collective” responses from many unit cells. While thicker crystals obviously give rise to larger effects, the resulting properties as the crystal thickness or period number N evolves from low values to a very high value do not lend themselves to such simple linear extrapolation; as a function of N, pleasant surprises and new insights and possibilities abound. Our studies show that for N > 500, these CLCs exhibit simultaneously broad transmission maxima and large polarization rotation power in the off-Bragg-resonance spectral regime. Polarization rotation is independent of input polarization orientation and acts equally well on simple or complex vector fields (18–22) of continuous-wave (CW) or ultrafast pulsed laser beams. Liquid crystal–based CPCs also allow dynamic polarization switching and control by virtue of field–induced index/birefringence changing mechanisms at modest or ultrafast (picosecond to femtosecond) speeds (23–34).