Outer membrane permeability: Antimicrobials and diverse nutrients bypass porins in Pseudomonas aeruginosa

Gram-negative bacterial pathogens have an outer membrane that restricts entry of molecules into the cell. Water-filled protein channels in the outer membrane, so-called porins, facilitate nutrient uptake and are thought to enable antibiotic entry. Here, we determined the role of porins in a major pathogen, Pseudomonas aeruginosa, by constructing a strain lacking all 40 identifiable porins and 15 strains carrying only a single unique type of porin and characterizing these strains with NMR metabolomics and antimicrobial susceptibility assays. In contrast to common assumptions, all porins were dispensable for Pseudomonas growth in rich medium and consumption of diverse hydrophilic nutrients. However, preferred nutrients with two or more carboxylate groups such as succinate and citrate permeated poorly in the absence of porins. Porins provided efficient translocation pathways for these nutrients with broad and overlapping substrate selectivity while efficiently excluding all tested antibiotics except carbapenems, which partially entered through OprD. Porin-independent permeation of antibiotics through the outer-membrane lipid bilayer was hampered by carboxylate groups, consistent with our nutrient data. Together, these results challenge common assumptions about the role of porins by demonstrating porin-independent permeation of the outer-membrane lipid bilayer as a major pathway for nutrient and drug entry into the bacterial cell.

Antimicrobial resistance is a major worldwide threat to human health. The World Health Organization has classified Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii as the most concerning pathogens (“critical priority”) (1). All three pathogens are Gram-negative bacteria with the characteristic inner and outer membranes. The outer membrane is a stringent permeability barrier that restricts the entry of most molecules and therefore presents a major challenge for the development of urgently needed novel antibiotics (2–5).The outer membrane consists of an asymmetric lipid bilayer with lipopolysaccharide (LPS) in the outer leaflet and phospholipids in the inner leaflet and various outer-membrane proteins that are embedded in, or attached to, the lipid bilayer. LPS contains negatively charged phosphate and carboxylate groups that are cross-linked by divalent Mg²⁺ and Ca²⁺ cations, resulting in stable clusters of LPS molecules that reduce the permeation of small molecules by 10- to 100-fold compared to phospholipid bilayers (6). Some outer membrane proteins form water-filled channels (so-called porins) that facilitate translocation of molecules through the outer membrane (4, 5). Enterobacteriaceae have general “unspecific” porins that permit the entry of molecules with a size of up to 600 Da. By contrast, P. aeruginosa and A. baumannii have a large set of “specific” porins that permit the entry of only few molecules with sizes below 200 Da. In addition, all three pathogens have porins with mainly structural roles in stabilizing the link between outer membrane and the underlying peptidoglycan layer (OmpA and OprF). It has been proposed that a small fraction of these structural porin molecules form large unspecific pores that permit entry of larger molecules at low rates (7), but this model remains controversial.Antimicrobials and nutrients can penetrate the outer membrane by two different pathways, through the lipid bilayer or through porins. Hydrophobic molecules might predominantly use the lipid pathway, while hydrophilic molecules might prefer porins. However, the quantitative relevance of each pathway for outer-membrane permeability remains unknown (3, 8, 9). Even slow permeation pathways that mediate concentration-equilibration times in the order of minutes (instead of seconds) can yield relevant intracellular drug concentrations in bacteria with generation times of more than 20 min, unless drug-efflux pumps and/or hydrolases diminish drug levels (2).Translocation pathways and their selectivity for specific physicochemical properties of molecules are crucial for the rational improvement of drug entry into Gram-negative bacteria. The important contribution of large cation-selective porins such as OmpF and OmpC for outer-membrane translocation into Enterobacteriaceae enabled the establishment of rules for medicinal chemistry to improve whole-cell activities of antimicrobials against these bacteria (10–12). These porins have been extensively studied, and in particular OmpF has a major impact on susceptibility to various β-lactam antibiotics (13). However, an Escherichia coli ΔompC ΔompF double mutant retains substantial susceptibility to diverse other antibiotics (9), suggesting alternative translocation pathways.For P. aeruginosa, physicochemical parameters favoring translocation have been more difficult to identify (10, 14, 15). Both P. aeruginosa and A. baumannii have lower outer-membrane permeability than Enterobacteriaceae for hydrophilic molecules because they lack unspecific porins (16), making antimicrobial development particularly difficult for these critical pathogens. Specific porins might facilitate antibiotic entry into P. aeruginosa (17), but clear evidence for standard assay conditions is only available for penetration of carbapenems through OprD (18). Functional studies of individual porins in P. aeruginosa are hampered by the large diversity of specific porins that are thought to each enable uptake of a few nutrients (19). Phenotypes of inactivating one particular porin might be masked by the numerous remaining other porins. To circumvent these issues, individual porins have been purified and reconstituted in artificial membranes, or expressed in E. coli, to determine their substrate specificity. However, the results might not reflect porin functions in their native context because their channel properties differ depending on the lipid environment (20, 21).In this study, we overcame these difficulties using extensive mutagenesis. In contrast to previous assumptions, we show that wild-type P. aeruginosa PA14 and a PA14 Δ40 mutant that lacks all identifiable 40 porin genes have indistinguishable susceptibility to diverse antibiotics. Moreover, the Δ40 strain grew normally on rich media, and nutrient consumption assays revealed substantial porin-independent uptake of diverse hydrophilic nutrients. Bringing back individual porins accelerated uptake of some neutral/zwitterionic molecules and was essential for efficient consumption of negatively charged carboxylate-containing compounds. Instead of narrow substrate specificity, porins actually had broad overlapping substrate selectivity. These results demonstrate an unexpected but efficient porin-independent translocation pathway through the outer-membrane lipid bilayer for diverse hydrophilic compounds and all antipseudomonal antibiotics. A detailed understanding of this pathway will facilitate the development of novel antibiotics.     

ATP disrupts lipid-binding equilibrium to drive retrograde transport critical for bacterial outer membrane asymmetry

The hallmark of the gram-negative bacterial envelope is the presence of the outer membrane (OM). The OM is asymmetric, comprising lipopolysaccharides (LPS) in the outer leaflet and phospholipids (PLs) in the inner leaflet; this critical feature confers permeability barrier function against external insults, including antibiotics. To maintain OM lipid asymmetry, the OmpC-Mla system is believed to remove aberrantly localized PLs from the OM and transport them to the inner membrane (IM). Key to the system in driving lipid trafficking is the MlaFEDB ATP-binding cassette transporter complex in the IM, but mechanistic details, including transport directionality, remain enigmatic. Here, we develop a sensitive point-to-point in vitro lipid transfer assay that allows direct tracking of [¹⁴C]-labeled PLs between the periplasmic chaperone MlaC and MlaFEDB reconstituted into nanodiscs. We reveal that MlaC spontaneously transfers PLs to the IM transporter in an MlaD-dependent manner that can be further enhanced by coupled ATP hydrolysis. In addition, we show that MlaD is important for modulating productive coupling between ATP hydrolysis and such retrograde PL transfer. We further demonstrate that spontaneous PL transfer also occurs from MlaFEDB to MlaC, but such anterograde movement is instead abolished by ATP hydrolysis. Our work uncovers a model where PLs reversibly partition between two lipid-binding sites in MlaC and MlaFEDB, and ATP binding and/or hydrolysis shift this equilibrium to ultimately drive retrograde PL transport by the OmpC-Mla system. These mechanistic insights will inform future efforts toward discovering new antibiotics against gram-negative pathogens.

The cell envelope of gram-negative bacteria is composed of two lipid bilayers. The inner membrane (IM) is a phospholipid (PL) bilayer while the outer membrane (OM) contains both PLs and lipopolysaccharides (LPS). The OM is asymmetric with LPS residing in the outer leaflet and PLs in the inner leaflet; such lipid asymmetry allows the membrane to function as an effective barrier against the entry of toxic substances, including antibiotics (1, 2). Extensive efforts have contributed toward an improved understanding of the molecular mechanisms involved in OM assembly and homeostasis. We now know that the Lol, Bam, and Lpt pathways are responsible for the unidirectional transport and/or assembly of OM lipoproteins, β-barrel proteins, and LPS, respectively (3–5). However, mechanisms for PL transport (6), which occur in both directions for assembly and homeostasis of the OM (7–9), remain largely elusive.The first pathway implicated in bacterial PL transport is the OmpC-Mla system (10, 11). Cells lacking this pathway accumulate PLs in the outer leaflet of the OM, indicating a role in the maintenance of OM lipid asymmetry. The OmpC-Mla system is believed to mediate retrograde PL transport where the periplasmic MlaC chaperone shuttles PLs from the OmpC-MlaA complex in the OM to the MlaFEDB ATP-binding cassette (ABC) transporter in the IM. MlaA is an OM lipoprotein that interacts and works with trimeric osmoporin OmpC (11, 12) but forms a separate hydrophilic channel in the OM for transbilayer PL translocation (12, 13). While MlaA also interacts with OmpF, only removing OmpC, but not OmpF, results in OM lipid asymmetry defects in cells (11); therefore, OmpC is functionally important in the Mla system, but its exact role is unclear. MlaC is found to bind PLs with high affinity in a deep hydrophobic pocket (14–16), presumably enabling extraction of PLs from the OM via OmpC-MlaA. In the IM ABC transporter, MlaE and MlaF constitute the permease and nucleotide binding domains, respectively. MlaD is a substrate-binding protein containing a single transmembrane helix, and its periplasmic MCE domain (17) forms a hexamer with a hydrophobic pore and copurifies with PLs (14, 18). Recent structural studies of the MlaFEDB complex revealed that the MlaE permease domains complement the MlaD hydrophobic pore to form a contiguous cavity for binding and transporting PLs (19–23). MlaB is a cytoplasmic protein containing the STAS domain (24) and has been shown to be essential for the proper assembly and activity of the IM complex (18, 25). How ATP hydrolysis may drive PL movement within the MlaFEDB complex is not known.Apart from the lack of mechanistic details, there is still substantial controversy with regards to the directionality of lipid transport mediated by the OmpC-Mla system. Genetic studies in Escherichia coli and Acinetobacter baumannii are more consistent with the retrograde (OM-to-IM) model (10, 26). In particular, E. coli mla or ompC mutants accumulate outer leaflet PLs in the OM, a phenotype that can be corrected by overexpression of PldA, the OM phospholipase (10, 11). In addition, A. baumannii strains lacking lipooligosaccharides have enhanced growth and restored OM barrier function when the Mla system and PldA are removed, presumably because these cells need enough PLs to maintain the outer leaflet of the OM (26, 27). Nevertheless, these experiments in cells lack evidence that demonstrate the direct transport of PLs. While there was a report suggesting the system in A. baumannii may be involved in anterograde (IM-to-OM) PL transport (28), it has recently been shown that the mla mutant strains used had secondary mutations in them, thus rendering the claim invalid (29). A key experiment that sheds light on transport is when the overexpression of MlaFEDB together with MlaC partially rescued defects in retrograde transport of PLs, observed in E. coli strains lacking the Tol-Pal complex (30). Despite this, however, in vitro reconstitution demonstrated ATP-independent (anterograde) transfer of PLs from the MlaFEDB complex (in liposomes) to MlaC (31). These conflicting data throw into question the biological significance of ATP hydrolysis and argue for the need to provide additional in vitro evidence to truly define the directionality of lipid transport mediated by the OmpC-Mla system.In this study, we utilize [¹⁴C]-labeled lipids to directly track the point-to-point transfer of PLs between MlaC and nanodisc-reconstituted MlaFEDB complex. We show that PLs bound to MlaC can be spontaneously transferred to MlaFEDB in an MlaD-dependent manner. The presence of ATP further enhances such retrograde transfer; we demonstrate that ATP hydrolysis is coupled to PL transport, and this process too is modulated by MlaD. Interestingly, spontaneous PL transfer can also happen from MlaFEDB to MlaC, but ATP hydrolysis prevents this “anterograde” transfer, ultimately driving PL transport in the retrograde direction. Our work establishes that the OmpC-Mla system powers the retrograde transport of PLs via ATP hydrolysis to maintain OM lipid asymmetry.     

Phase separation in the outer membrane of Escherichia coli

Gram-negative bacteria are surrounded by a protective outer membrane (OM) with phospholipids in its inner leaflet and lipopolysaccharides (LPS) in its outer leaflet. The OM is also populated with many β-barrel outer-membrane proteins (OMPs), some of which have been shown to cluster into supramolecular assemblies. However, it remains unknown how abundant OMPs are organized across the entire bacterial surface and how this relates to the lipids in the membrane. Here, we reveal how the OM is organized from molecular to cellular length scales, using atomic force microscopy to visualize the OM of live bacteria, including engineered Escherichia coli strains and complemented by specific labeling of abundant OMPs. We find that a predominant OMP in the E. coli OM, the porin OmpF, forms a near-static network across the surface, which is interspersed with barren patches of LPS that grow and merge with other patches during cell elongation. Embedded within the porin network is OmpA, which forms noncovalent interactions to the underlying cell wall. When the OM is destabilized by mislocalization of phospholipids to the outer leaflet, a new phase appears, correlating with bacterial sensitivity to harsh environments. We conclude that the OM is a mosaic of phase-separated LPS-rich and OMP-rich regions, the maintenance of which is essential to the integrity of the membrane and hence to the lifestyle of a gram-negative bacterium.

Diderm bacteria, such as Escherichia coli, are surrounded by an outer membrane (OM) that protects cells against the immune systems of plants and animals, contributes to the mechanical stability of the cell, and excludes many classes of antibiotics, thereby contributing to antimicrobial resistance (1, 2). The OM is comprised of an asymmetric bilayer of phospholipids in the inner leaflet, lipopolysaccharides (LPS) in the outer leaflet, and many outer-membrane proteins (OMPs). OMPs are hugely diverse β-barrel proteins that can be present at hundreds to hundreds of thousands of copies per cell (3). They have been shown to be relatively static (4), probably due to promiscuous protein–protein interactions and binding of LPS that exists in a slow-moving, liquid-crystalline state (5, 6). Using fluorescent labels, some OMPs have been shown to cluster into supramolecular islands of ∼0.3- to 0.5-μm sizes (4, 7–9). However, it remains unknown how abundant OMPs are organized across the entire bacterial surface and how this relates to the lipids in the membrane.To address this fundamental question, we have imaged the entire surface of live and metabolically active bacteria at nanometer resolution using atomic force microscopy (AFM). Applying such large-scale, high-resolution imaging on engineered E. coli strains and complementing it by specific labeling of abundant OMPs, we identify large-scale and near-static protein-rich networks interspersed with nanoscale domains that are enriched in LPS. Key components of the protein-rich networks are abundant trimeric porins such as OmpF, in addition to (the monomeric) OmpA, which forms noncovalent interactions to the underlying cell wall (10). By contrast, no significant protein content is detected in the LPS-rich domains, which are also found to grow and merge with other patches during cell elongation. When the LPS–phospholipid asymmetry of the OM is perturbed by mislocalization of phospholipids to the outer leaflet (11), we find deformation of the membrane rather than expansion of LPS patches, indicating the appearance of a new, phospholipid-enriched phase at the bacterial surface.     

High-throughput suppressor screen demonstrates that RcsF monitors outer membrane integrity and not Bam complex function

The regulator of capsule synthesis (Rcs) is a complex signaling cascade that monitors gram-negative cell envelope integrity. The outer membrane (OM) lipoprotein RcsF is the sensory component, but how RcsF functions remains elusive. RcsF interacts with the β-barrel assembly machinery (Bam) complex, which assembles RcsF in complex with OM proteins (OMPs), resulting in RcsF’s partial cell surface exposure. Elucidating whether RcsF/Bam or RcsF/OMP interactions are important for its sensing function is challenging because the Bam complex is essential, and partial loss-of-function mutations broadly compromise the OM biogenesis. Our recent discovery that, in the absence of nonessential component BamE, RcsF inhibits function of the central component BamA provided a genetic tool to select mutations that specifically prevent RcsF/BamA interactions. We employed a high-throughput suppressor screen to isolate a collection of such rcsF and bamA mutants and characterized their impact on RcsF/OMP assembly and Rcs signaling. Using these mutants and BamA inhibitors MRL-494 and darobactin, we provide multiple lines of evidence against the model in which RcsF senses Bam complex function. We show that Rcs activation in bam mutants results from secondary OM and lipopolysaccharide defects and that RcsF/OMP assembly is required for this activation, supporting an active role of RcsF/OMP complexes in sensing OM stress.

The bacterial cell envelope is an essential structure, acting as a first line of defense against environmental assault. The gram-negative cell envelope is complex, consisting of an inner (IM) and outer (OM) membrane that encloses the cell wall in an aqueous periplasmic space (1). The OM is asymmetric, with phospholipids and lipopolysaccharides (LPS) in the inner and outer leaflets, respectively. The cation cross-bridged LPS molecules confer extreme resistance to detergents and many antibiotics (2).The regulator of capsule synthesis (Rcs) signaling cascade is one of several envelope stress responses that monitor envelope integrity and biogenesis (Fig. 1) (3). Rcs involves at least six proteins spanning all cellular compartments from the cell surface to the cytoplasm. At the core of this pathway is the RcsCDB Histidine-Aspartate phosphorelay complex consisting of the IM hybrid histidine protein kinase RcsC, the IM phosphotransferase protein RcsD, and the cytoplasmic DNA-binding response regulator RcsB (4–6). The activity of this Rcs phosphorelay is regulated by interactions with two upstream components, IgaA and RcsF. IgaA is a polytopic IM protein with a large periplasmic domain, and it inhibits the phosphorelay through RcsD (7, 8). The OM lipoprotein RcsF is a sensory component of the Rcs cascade, which activates downstream signaling in response to stress by releasing IgaA inhibition (8–12). However, sensing by RcsF and signal transduction to IgaA are poorly understood at a molecular level, in part because many distinct genetic and chemical stimuli can induce Rcs, including defects in lipoprotein biogenesis (13–15), cell wall biogenesis (12, 16–19), and the defects of LPS at the cell surface (as a result of Polymyxin B [PMB] treatment, for example) (19–22).Open in a separate windowFig. 1.Proposed mechanistic models for the Rcs stress response. Rcs components (orange) are shown in the context of the envelope structure and biogenesis pathways. The sensory lipoprotein RcsF and the negative regulator IgaA are central to the regulation of RcsCDB phosphorelay. RcsF is exported to the OM by the Lol pathway; the Bam complex assembles RcsF with partner OMPs, leading to a partially surface-exposed topology. Red arrows represent proposed signaling events in response to stress (red stars) that are not yet fully understood. (A) Proposed model for the OM/LPS sensing by RcsF. Cell surface localization of RcsF NTD enables RcsF to monitor the integrity of the outer leaflet. Upon LPS stress (e.g., PMB treatment), the signal is transduced to the periplasmic CTD through the conformational change in the RcsF/OMP complex stimulating downstream signaling. (B) Proposed model for the Bam complex sensing function of RcsF. Envelope stress by an unknown mechanism inhibits the Bam complex function; as a result, RcsF/BamA interaction is prevented, and RcsF is accumulated in the periplasmic-facing orientation stimulating downstream signaling.At the OM, RcsF forms a complex with β-barrel OM proteins (OMPs) such as OmpA, OmpC, and OmpF, adopting a transmembrane orientation in which RcsF is partially surface exposed (12, 23). The β-barrel assembly machinery (Bam complex) that assembles all OMPs also assembles RcsF/OMP complexes, and RcsF interacts with its central and essential component, BamA (12, 23).Defective lipoprotein biogenesis results in the retention of RcsF at the IM, promoting physical association with IgaA and the constitutive activation of signaling (12, 13). Two hypotheses have been proposed to explain how RcsF signals from the OM (Fig. 1 A and B): the first suggests that the surface-exposed N-terminal domain (NTD) of RcsF in an RcsF/OMP complex monitors the integrity of LPS at the outer leaflet, transmitting the signal to the periplasmic carboxyl-terminal domain (CTD) to induce downstream signaling (19, 23) (Fig. 1A); the second argues that the RcsF/OMP complex plays no active role in signal transduction, with stress signals altering the RcsF/BamA interaction to retain RcsF in a periplasm-facing orientation, allowing downstream signaling (12) (Fig. 1B). This altering of the RcsF/BamA interaction is thought to allow RcsF to monitor Bam complex activity (12). Testing these hypotheses has proven to be challenging, as the Bam complex is essential, and there was no clear path to identifying point mutations that specifically disrupt RcsF/OMP or RcsF/BamA interactions without compromising OMP biogenesis and OM integrity.The Bam complex consists of five components, A through E (24): BamA is a β-barrel with five periplasmic Potra domains that scaffold four regulatory lipoproteins, BamB through E. An essential lipoprotein, BamD, recruits OMP substrates to the Bam complex and activates BamA for OMP folding and insertion into the OM (25–29). Coordination of BamA and BamD activities is essential for the OMP assembly and is mediated by their direct interaction at the Potra 5 interface, for which the salt bridge between BamA E373 and BamD R197 is critically important (29, 30). Previously, we reported that the loss of the nonessential Bam component BamE results in a significant decrease in RcsF/OMP assembly (SI Appendix, Fig. S1) (19). The ΔbamE strain and an assembly-defective rcsF^A55Y mutant strain (SI Appendix, Table S1) are both significantly deficient in the detection of PMB-induced LPS stress, providing the first evidence to support an active role for RcsF/OMP in signaling under conditions of LPS stress (19). In the absence of BamE, BamA binds RcsF but is unable to engage with BamD to complete RcsF/OMP assembly (31) (SI Appendix, Fig. S1). As a result, RcsF accumulates on BamA, preventing it from functioning in OMP assembly (31, 32). This RcsF-dependent “jamming” of BamA is the reason for the synthetic lethal interaction of ΔbamE and various bam mutants, including a bamB null (SI Appendix, Table S1) (31, 32).We exploited the lethal interaction between BamA and RcsF in the bamE bamB double mutant to select for mutations that disrupt this RcsF/BamA interaction. Our characterization of the effects of the rcsF and bamA suppressor mutations identified on Rcs signaling and RcsF/OMP assembly demonstrates that assembly of the RcsF/OMP complex is required for Rcs signaling and argues against the model that proposes that RcsF monitors BamA activity. Moreover, our data suggest that the recently published RcsF/BamA structure corresponds to the "jammed" RcsF-BamA complex and not an assembly intermediate, as suggested (33).     

Microscopic origins of the crystallographically preferred growth in evaporation-induced colloidal crystals

Unlike crystalline atomic and ionic solids, texture development due to crystallographically preferred growth in colloidal crystals is less studied. Here we investigate the underlying mechanisms of the texture evolution in an evaporation-induced colloidal assembly process through experiments, modeling, and theoretical analysis. In this widely used approach to obtain large-area colloidal crystals, the colloidal particles are driven to the meniscus via the evaporation of a solvent or matrix precursor solution where they close-pack to form a face-centered cubic colloidal assembly. Via two-dimensional large-area crystallographic mapping, we show that the initial crystal orientation is dominated by the interaction of particles with the meniscus, resulting in the expected coalignment of the close-packed direction with the local meniscus geometry. By combining with crystal structure analysis at a single-particle level, we further reveal that, at the later stage of self-assembly, however, the colloidal crystal undergoes a gradual rotation facilitated by geometrically necessary dislocations (GNDs) and achieves a large-area uniform crystallographic orientation with the close-packed direction perpendicular to the meniscus and parallel to the growth direction. Classical slip analysis, finite element-based mechanical simulation, computational colloidal assembly modeling, and continuum theory unequivocally show that these GNDs result from the tensile stress field along the meniscus direction due to the constrained shrinkage of the colloidal crystal during drying. The generation of GNDs with specific slip systems within individual grains leads to crystallographic rotation to accommodate the mechanical stress. The mechanistic understanding reported here can be utilized to control crystallographic features of colloidal assemblies, and may provide further insights into crystallographically preferred growth in synthetic, biological, and geological crystals.

As an analogy to atomic crystals, colloidal crystals are highly ordered structures formed by colloidal particles with sizes ranging from 100 nm to several micrometers (1–6). In addition to engineering applications such as photonics, sensing, and catalysis (4, 5, 7, 8), colloidal crystals have also been used as model systems to study some fundamental processes in statistical mechanics and mechanical behavior of crystalline solids (9–14). Depending on the nature of interparticle interactions, many equilibrium and nonequilibrium colloidal self-assembly processes have been explored and developed (1, 4). Among them, the evaporation-induced colloidal self-assembly presents a number of advantages, such as large-size fabrication, versatility, and cost and time efficiency (3–5, 15–18). In a typical synthesis where a substrate is immersed vertically or at an angle into a colloidal suspension, the colloidal particles are driven to the meniscus by the evaporation-induced fluid flow and subsequently self-assemble to form a colloidal crystal with the face-centered cubic (fcc) lattice structure and the close-packed {111} plane parallel to the substrate (2, 3, 19–23) (see Fig. 1A for a schematic diagram of the synthetic setup).Open in a separate windowFig. 1.Evaporation-induced coassembly of colloidal crystals. (A) Schematic diagram of the evaporation-induced colloidal coassembly process. “G”, “M”, and “N” refer to “growth,” “meniscus,” and “normal” directions, respectively. The reaction solution contains silica matrix precursor (tetraethyl orthosilicate, TEOS) in addition to colloids. (B) Schematic diagram of the crystallographic system and orientations used in this work. (C and D) Optical image (Top Left) and scanning electron micrograph (SEM) (Bottom Left) of a typical large-area colloidal crystal film before (C) and after (D) calcination. (Right) SEM images of select areas (yellow rectangles) at different magnifications. Corresponding fast-Fourier transform (see Inset in Middle in C) shows the single-crystalline nature of the assembled structure. (E) The 3D reconstruction of the colloidal crystal (left) based on FIB tomography data and (right) after particle detection. (F) Top-view SEM image of the colloidal crystal with crystallographic orientations indicated.While previous research has focused on utilizing the assembled colloidal structures for different applications (4, 5, 7, 8), considerably less effort is directed to understand the self-assembly mechanism itself in this process (17, 24). In particular, despite using the term “colloidal crystals” to highlight the microstructures’ long-range order, an analogy to atomic crystals, little is known regarding the crystallographic evolution of colloidal crystals in relation to the self-assembly process (3, 22, 25). The underlying mechanisms for the puzzling—yet commonly observed—phenomenon of the preferred growth along the close-packed <110> direction in evaporation-induced colloidal crystals are currently not understood (3, 25–29). The <110> growth direction has been observed in a number of processes with a variety of particle chemistries, evaporation rates, and matrix materials (3, 25–28, 30), hinting at a universal underlying mechanism. This behavior is particularly intriguing as the colloidal particles are expected to close-pack parallel to the meniscus, which should lead to the growth along the <112> direction and perpendicular to the <110> direction (16, 26, 31)^*.Preferred growth along specific crystallographic orientations, also known as texture development, is commonly observed in crystalline atomic solids in synthetic systems, biominerals, and geological crystals. While current knowledge recognizes mechanisms such as the oriented nucleation that defines the future crystallographic orientation of the growing crystals and competitive growth in atomic crystals (32–34), the underlying principles for texture development in colloidal crystals remain elusive. Previous hypotheses based on orientation-dependent growth speed and solvent flow resistance are inadequate to provide a universal explanation for different evaporation-induced colloidal self-assembly processes (3, 25–29). A better understanding of the crystallographically preferred growth in colloidal self-assembly processes may shed new light on the crystal growth in atomic, ionic, and molecular systems (35–37). Moreover, mechanistic understanding of the self-assembly processes will allow more precise control of the lattice types, crystallography, and defects to improve the performance and functionality of colloidal assembly structures (38–40).     

An image-computable model of how endogenous and exogenous attention differentially alter visual perception

Attention alters perception across the visual field. Typically, endogenous (voluntary) and exogenous (involuntary) attention similarly improve performance in many visual tasks, but they have differential effects in some tasks. Extant models of visual attention assume that the effects of these two types of attention are identical and consequently do not explain differences between them. Here, we develop a model of spatial resolution and attention that distinguishes between endogenous and exogenous attention. We focus on texture-based segmentation as a model system because it has revealed a clear dissociation between both attention types. For a texture for which performance peaks at parafoveal locations, endogenous attention improves performance across eccentricity, whereas exogenous attention improves performance where the resolution is low (peripheral locations) but impairs it where the resolution is high (foveal locations) for the scale of the texture. Our model emulates sensory encoding to segment figures from their background and predict behavioral performance. To explain attentional effects, endogenous and exogenous attention require separate operating regimes across visual detail (spatial frequency). Our model reproduces behavioral performance across several experiments and simultaneously resolves three unexplained phenomena: 1) the parafoveal advantage in segmentation, 2) the uniform improvements across eccentricity by endogenous attention, and 3) the peripheral improvements and foveal impairments by exogenous attention. Overall, we unveil a computational dissociation between each attention type and provide a generalizable framework for predicting their effects on perception across the visual field.

Endogenous and exogenous spatial attention prioritize subsets of visual information and facilitate their processing without concurrent eye movements (1–3). Selection by endogenous attention is goal-driven and adapts to task demands, whereas exogenous attention transiently and automatically orients to salient stimuli (1–3). In most visual tasks, both types of attention typically improve visual perception similarly [e.g., acuity (4–6), visual search (7, 8), perceived contrast (9–11)]. Consequently, models of visual attention do not distinguish between endogenous and exogenous attention (e.g., refs. 12–19). However, stark differences also exist. Each attention type differentially modulates neural responses (20, 21) and fundamental properties of visual processing, including temporal resolution (22, 23), texture sensitivity (24), sensory tuning (25), contrast sensitivity (26), and spatial resolution (27–34).The effects of endogenous and exogenous attention are dissociable during texture segmentation, a visual task constrained by spatial resolution [reviews (1–3)]. Whereas endogenous attention optimizes spatial resolution to improve the detection of an attended texture (32–34), exogenous attention reflexively enhances resolution even when detrimental to perception (27–31, 34). Extant models of attention do not explain these well-established effects.Two main hypotheses have been proposed to explain how attention alters spatial resolution. Psychophysical studies ascribe attentional effects to modulations of spatial frequency (SF) sensitivity (30, 33). Neurophysiological (13, 35, 36) and neuroimaging (37, 38) studies bolster the idea that attention modifies spatial profiles of neural receptive fields (RFs) (2). Both hypotheses provide qualitative predictions of attentional effects but do not specify their underlying neural computations.Differences between endogenous and exogenous attention are well established in segmentation tasks and thus provide an ideal model system to uncover their separate roles in altering perception. Texture-based segmentation is a fundamental process of midlevel vision that isolates regions of local structure to extract figures from their background (39–41). Successful segmentation hinges on the overlap between the visual system’s spatial resolution and the levels of detail (i.e., SF) encompassed by the texture (39, 41, 42). Consequently, the ability to distinguish between adjacent textures varies as resolution declines toward the periphery (43–46). Each attention type differentially alters texture segmentation, demonstrating that their effects shape spatial resolution [reviews (1–3)].Current models of texture segmentation do not explain performance across eccentricity and the distinct modulations by attention. Conventional models treat segmentation as a feedforward process that encodes the elementary features of an image (e.g., SF and orientation), transforms them to reflect the local structure (e.g., regions of similarly oriented bars), and then pools across space to emphasize texture-defined contours (39, 41, 47). Few of these models account for variations in resolution across eccentricity (46, 48, 49) or endogenous (but not exogenous) attentional modulations (18, 50). All others postulate that segmentation is a “preattentive” (42) operation whose underlying neural processing is impervious to attention (39, 41, 46–49).Here, we develop a computational model in which feedforward processing and attentional gain contribute to segmentation performance. We augment a conventional model of texture processing (39, 41, 47). Our model varies with eccentricity and includes contextual modulation within local regions in the stimulus via normalization (51), a canonical neural computation (52). The defining characteristic of normalization is that an individual neuron is (divisively) suppressed by the summed activity of neighboring neurons responsive to different aspects of a stimulus. We model attention as multiplicative gains [attentional gain factors (15)] that vary with eccentricity and SF. Attention shifts sensitivity toward fine or coarse spatial scales depending on the range of SFs enhanced.Our model is image-computable, which allowed us to reproduce behavior directly from grayscale images used in psychophysical experiments (6, 26, 27, 29–33). The model explains three signatures of texture segmentation hitherto unexplained within a single computational framework (Fig. 1): 1) the central performance drop (CPD) (27–34, 43–46) (Fig. 1A), that is, the parafoveal advantage of segmentation over the fovea; 2) the improvements in the periphery and impairments at foveal locations induced by exogenous attention (27–32, 34) (Fig. 1B); and 3) the equivalent improvements across eccentricity by endogenous attention (32–34) (Fig. 1C).Open in a separate windowFig. 1.Signatures of texture segmentation. (A) CPD. Shaded region depicts the magnitude of the CPD. Identical axis labels are omitted in B and C. (B) Exogenous attention modulation. Exogenous attention improves segmentation performance in the periphery and impairs it near the fovea. (C) Endogenous attention modulation. Endogenous attention improves segmentation performance across eccentricity.Whereas our analyses focused on texture segmentation, our model is general and can be applied to other visual phenomena. We show that the model predicts the effects of attention on contrast sensitivity and acuity, i.e., in tasks in which both endogenous and exogenous attention have similar or differential effects on performance. To preview our results, model comparisons revealed that normalization is necessary to elicit the CPD and that separate profiles of gain enhancement across SF (26) generate the effects of exogenous and endogenous attention on texture segmentation. A preferential high-SF enhancement reproduces the impairments by exogenous attention due to a shift in visual sensitivity toward details too fine to distinguish the target at foveal locations. The transition from impairments to improvements in the periphery results from exogenous attentional gain gradually shifting to lower SFs that are more amenable for target detection. Improvements by endogenous attention result from a uniform enhancement of SFs that encompass the target, optimizing visual sensitivity for the attended stimulus across eccentricity.     

Electrochemical borylation of carboxylic acids

A simple electrochemically mediated method for the conversion of alkyl carboxylic acids to their borylated congeners is presented. This protocol features an undivided cell setup with inexpensive carbon-based electrodes and exhibits a broad substrate scope and scalability in both flow and batch reactors. The use of this method in challenging contexts is exemplified with a modular formal synthesis of jawsamycin, a natural product harboring five cyclopropane rings.

Boronic acids are among the most malleable functional groups in organic chemistry as they can be converted into almost any other functionality (1–3). Aside from these versatile interconversions, their use in the pharmaceutical industry is gaining traction, resulting in approved drugs such as Velcade, Ninlaro, and Vabomere (4). It has been shown that boronic acids can be rapidly installed from simple alkyl halides (5–19) or alkyl carboxylic acids through the intermediacy of redox-active esters (RAEs) (Fig. 1A) (20–24). Our laboratory has shown that both Ni (20) and Cu (21) can facilitate this reaction. Conversely, Aggarwal and coworkers (22) and Li and coworkers (23) demonstrated photochemical variations of the same transformation. While these state-of-the-art approaches provide complementary access to alkyl boronic acids, each one poses certain challenges. For example, the requirement of excess boron source and pyrophoric MeLi under Ni catalysis is not ideal. Although more cost-effective and operationally simple, Cu-catalyzed borylation conditions can be challenging on scale due to the heterogeneity resulting from the large excess of LiOH•H₂O required. In addition to its limited scope, Li and coworkers’ protocol requires 4 equivalence of B₂pin₂ and an expensive Ir photocatalyst. The simplicity of Aggarwal and coworkers’ approach is appealing in this regard and represents an important precedent for the current study.Open in a separate windowFig. 1.(A) Prior approaches to access alkyl boronic esters from activated acids. (B) Inspiration for initiating SET events electrochemically to achieve borylation. (C) Summary of this work.At the heart of each method described above, the underlying mechanism relies on a single electron transfer (SET) event to promote decarboxylation and form an alkyl radical species. In parallel, the related borylation of aryl halides via a highly reactive aryl radical can also be promoted by SET. While numerous methods have demonstrated that light can trigger this mechanism (Fig. 1B) (16, 25–31), simple electrochemical cathodic reduction can elicit the same outcome (32–35). It was postulated that similar electrochemically driven reactivity could be translated to alkyl RAEs. The development of such a transformation would be highly enabling, as synthetic organic electrochemistry allows the direct addition or removal of electrons to a reaction, representing an incredibly efficient way to forge new bonds (36–40). This disclosure reports a mild, scalable, and operationally simple electrochemical decarboxylative borylation (Fig. 1C) not reliant on transition metals or stoichiometric reductants. In addition to mechanistic studies of this interesting transformation, applications to a variety of alkyl RAEs, comparison to known decarboxylative borylation methods, and a formal synthesis of the polycyclopropane natural product jawsamycin [(–)-FR-900848] are presented.     

A multidomain connector links the outer membrane and cell wall in phylogenetically deep-branching bacteria

Deinococcus radiodurans is a phylogenetically deep-branching extremophilic bacterium that is remarkably tolerant to numerous environmental stresses, including large doses of ultraviolet (UV) radiation and extreme temperatures. It can even survive in outer space for several years. This endurance of D. radiodurans has been partly ascribed to its atypical cell envelope comprising an inner membrane, a large periplasmic space with a thick peptidoglycan (PG) layer, and an outer membrane (OM) covered by a surface layer (S-layer). Despite intense research, molecular principles governing envelope organization and OM stabilization are unclear in D. radiodurans and related bacteria. Here, we report a electron cryomicroscopy (cryo-EM) structure of the abundant D. radiodurans OM protein SlpA, showing how its C-terminal segment forms homotrimers of 30-stranded β-barrels in the OM, whereas its N-terminal segment forms long, homotrimeric coiled coils linking the OM to the PG layer via S-layer homology (SLH) domains. Furthermore, using protein structure prediction and sequence-based bioinformatic analysis, we show that SlpA-like putative OM–PG connector proteins are widespread in phylogenetically deep-branching Gram-negative bacteria. Finally, combining our atomic structures with fluorescence and electron microscopy of cell envelopes of wild-type and mutant bacterial strains, we report a model for the cell surface of D. radiodurans. Our results will have important implications for understanding the cell surface organization and hyperstability of D. radiodurans and related bacteria and the evolutionary transition between Gram-negative and Gram-positive bacteria.

Deinococcus radiodurans is an evolutionarily deep-branching bacterium with several distinctive characteristics (1). It is remarkably tolerant to large doses of ionizing and ultraviolet (UV) radiation, extreme temperatures, osmotic pressure, oxidative stress, and desiccation, primarily owing to its extensive DNA repair system (2), complex cell envelope (3), and antioxidation systems, such as the one involving the carotenoid deinoxanthin (4, 5). In fact, D. radiodurans can even survive for years in outer space (6). Due to its ability to survive under extreme environmental conditions and its deep position in the bacterial tree of life, D. radiodurans has been of tremendous interest for several synthetic biology and evolutionary studies (2).The cell envelope of D. radiodurans is atypical. While it stains Gram positive, its architecture resembles that of Gram-negative bacteria, containing an inner membrane (IM) covered by a peptidoglycan (PG) layer in a large periplasmic space (7–9) and an outer membrane (OM). However, this OM lacks lipopolysaccharide and common phospholipids typical of Gram-negative bacterial OMs, and instead has a lipid composition similar to the IM (10). The D. radiodurans OM is also covered by a regularly spaced, hexagonal surface layer or S-layer (11, 12). Previous studies have suggested that the S-layer is made of a protein called hexagonally packed intermediate-layer (HPI) surface protein (3, 8, 11, 13–17), while newer studies have suggested that a heterocomplex with gating properties, termed the S-layer deinoxanthin-binding complex (SDBC), forms a large part of the D. radiodurans cell envelope, including the S-layer (18, 19). A previously identified abundant protein called SlpA (UniProtKB {"type":"entrez-protein","attrs":{"text":"Q9RRB6","term_id":"81550587"}}Q9RRB6) is suggested to be the main component of this complex. Recently, an 11-Å resolution structure of this complex was reported using electron cryomicroscopy (cryo-EM), showing how it exhibits a triangular base partly embedded in the OM and a stalk departing orthogonally from the base, presumably away from the membrane (18). Deletion of slpA leads to substantial disruption of the D. radiodurans cell envelope, suggesting its important role in the maintenance of cell envelope integrity (20). Finally, it has been shown using biochemical experiments that the N-terminus of D. radiodurans SlpA binds to the PG-containing cell wall, demonstrating that at least the N-terminal segment of the molecule resides in the periplasmic space (21).In addition to the experimental observations introduced above, from an evolutionary perspective, an ortholog of D. radiodurans SlpA (UniProtKB {"type":"entrez-protein","attrs":{"text":"Q5SH37","term_id":"62287492"}}Q5SH37) has also been characterized from the closely related thermophilic model bacterium Thermus thermophilus (22, 23). In line with data from D. radiodurans, deletion or truncation of slpA from T. thermophilus leads to remarkable disruption of the cell envelope (20, 24), underpinning its importance in cell surface organization. At the sequence level, SlpA contains a signal peptide, an SLH domain, a long, predicted α-helical region, and a C-terminal β-strand–rich domain, which is thought to fold into an OM β-barrel (18, 19) (Fig. 1A). Due to the presence of the N-terminal SLH domain, which commonly attaches S-layer proteins (SLPs) (23, 25–28) of Gram-positive bacteria to PG-linked pyruvylated secondary cell wall polymers (SCWPs), it has been suggested that SlpA constitutes the S-layer. Conversely, in T. thermophilus, SlpA has been shown to interact with PG through its SLH domain, suggesting a role for it as a periplasmic spacer (29). The role of SlpA in organizing the cell envelope of D. radiodurans and related deep-branching bacteria such as T. thermophilus is, therefore, still enigmatic.Open in a separate windowFig. 1.Cryo-EM reconstruction of D. radiodurans SlpA. (A) The SlpA protein contains a tripartite structure, including an N-terminal SLH domain, which is connected to a C-terminal β-barrel by a long coiled-coil segment. (B) Two-dimensional class averages of the trimeric SlpA specimen used for cryo-EM structure determination. Characteristic top and side views are shown. (C) Density map of the SlpA trimer (contour level on the Lower Left) shown from the Top. The resolution of the OMBB portion of the map is 2.9 Å, and resolution decreases toward the N-terminus, with a global resolution of 3.3 Å. Two subunits are shown as blue ribbons inside white envelope outlines and one as gray density (model hidden). Distance measurement includes the micelle density. (D) An orthogonal view of C, with the SlpA trimer shown from the side. The extended coiled coil degrades in resolution toward the N-terminus (see also SI Appendix, Fig. S1), presumably due to flexibility of the long stalk. (Scale bars in B, 100 Å; in C and D, 25 Å.)In this study, we report the cryo-EM structure of the SlpA protein complex from D. radiodurans. Our structure shows that SlpA exhibits a tripartite organization, with its C-terminal part forming a homotrimeric 30-stranded OM β-barrel (OMBB), its central part forming a trimeric coiled coil that can traverse the large periplasmic space, and the extreme N-terminal part forming an SLH domain trimer that can interact with the PG layer. Our structure- and sequence-based bioinformatic analyses further show the presence of SlpA-like proteins in several phyla of phylogenetically deep-branching Gram-negative bacteria. Finally, combining our atomic structures and bioinformatic results with microscopy of wild-type and mutant cells, we report a model for the cell envelope of D. radiodurans, showing how this Gram-negative (diderm) bacterial SlpA protein shares several characteristics commonly found in Gram-positive (monoderm) SLPs, with connotations on prokaryotic evolution.     

Electrophilic aromatic substitution reactions of compounds with Craig-Möbius aromaticity

Electrophilic aromatic substitution (EAS) reactions are widely regarded as characteristic reactions of aromatic species, but no comparable reaction has been reported for molecules with Craig-Möbius aromaticity. Here, we demonstrate successful EAS reactions of Craig-Möbius aromatics, osmapentalenes, and fused osmapentalenes. The highly reactive nature of osmapentalene makes it susceptible to electrophilic attack by halogens, thus osmapentalene, osmafuran-fused osmapentalene, and osmabenzene-fused osmapentalene can undergo typical EAS reactions. In addition, the selective formation of a series of halogen substituted metalla-aromatics via EAS reactions has revealed an unprecedented approach to otherwise elusive compounds such as the unsaturated cyclic chlorirenium ions. Density functional theory calculations were conducted to study the electronic effect on the regioselectivity of the EAS reactions.

Aromaticity, a core concept in chemistry, was initially introduced to account for the bonding, stability, reactivity, and other properties of many unsaturated organic compounds. There have been many elaborations and extensions of the concept of aromaticity (1, 2). The concepts of Hückel aromaticity and Möbius aromaticity are widely accepted (Fig. 1A). A π-aromatic molecule of the Hückel type is planar and has 4n + 2 conjugated π-electrons (n = 0 or an integer), whereas a Möbius aromatic molecule has one twist of the π-system, similar to that in a Möbius strip, and 4n π-electrons (3, 4). Since the discovery of naphthalene in 1821, aromatic chemistry has developed into a rich field and with a variety of subdisciplines over the course of its 200-y history, and the concept of aromaticity has been extended to other nontraditional structures with “cyclic delocalization of mobile electrons” (5). For example, benzene-like metallacycles—predicted by Hoffmann et al. as metallabenzenes—in which a metal replaces a C–H group in the benzene ring (6), have garnered extensive research interest from both experimentalists and theoreticians (7–12). As paradigms of the metalla-aromatic family, most complexes involving metallabenzene exhibit thermodynamic stability, kinetic persistence, and chemical reactivity associated with the classical aromaticity concept (13–15). Typically, like benzene, metallabenzene can undergo characteristic reactions of aromatics such as electrophilic aromatic substitution (EAS) reactions (16–18) (Fig. 1B, I) and nucleophilic aromatic substitution reactions (19–21).Open in a separate windowFig. 1.Schematic representations of aromaticity classification (A) and EAS reactions (B) of benzene, metallabenzene, and polycyclic metallacycles with Craig-Möbius aromaticity.The incorporation of transition metals has also led to an increase in the variety of the aromatic families (22–25). We have reported that stable and highly unusual bicyclic systems, metallapentalenes (osmapentalenes), benefit from Craig-Möbius aromaticity (26–30). In contrast to other reported Möbius aromatic compounds with twisted topologies, which are known as Heilbronner-Möbius aromatics (31–34), the involvement of transition metal d orbitals in π-conjugation switches the Hückel anti-aromaticity of pentalene into the planar Craig-Möbius aromaticity of metallapentalene (35–38) (Fig. 1A, III). Both the twisted topology and the planar Craig-Möbius aromaticity are well established and have been accepted as reasonable extensions of aromaticity (39–43). There has been no experimental evidence, however, as to whether these Möbius aromatic molecules can undergo classical aromatic substitution reactions, such as EAS reactions, instead of addition reactions. Given the key role of EAS in aromatic chemistry to obtain various derivatives, we sought to extend the understanding of the reactivity paradigm in the metalla-aromatic family.Our recent synthetic efforts associated with the metallapentalene system prompted us to investigate whether typical EAS reactions could proceed in these Craig-Möbius aromatics. If so, how could substitution be achieved in the same way that it is with traditional Hückel aromatics such as benzenes? In this paper, we present EAS reactions, mainly the halogenation of osmapentalene, osmafuran-fused osmapentalene, and osmabenzene-fused osmapentalene, which follow the classic EAS mechanistic scheme (Fig. 1B). With the aid of density functional theory (DFT) calculations, we characterized the effects on EAS reactivity and regioselectivity.     

Chlorination of arenes via the degradation of toxic chlorophenols

Aryl chlorides are among the most versatile synthetic precursors, and yet inexpensive and benign chlorination techniques to produce them are underdeveloped. We propose a process to generate aryl chlorides by chloro-group transfer from chlorophenol pollutants to arenes during their mineralization, catalyzed by Cu(NO₃)₂/NaNO₃ under aerobic conditions. A wide range of arene substrates have been chlorinated using this process. Mechanistic studies show that the Cu catalyst acts in cooperation with NO_x species generated from the decomposition of NaNO₃ to regulate the formation of chlorine radicals that mediate the chlorination of arenes together with the mineralization of chlorophenol. The selective formation of aryl chlorides with the concomitant degradation of toxic chlorophenol pollutants represents a new approach in environmental pollutant detoxication. A reduction in the use of traditional chlorination reagents provides another (indirect) benefit of this procedure.

Chlorophenols are widely encountered moieties present in herbicides, drugs, and pesticides (1). Owing to the high dissociation energies of carbon‒chloride bonds, chlorophenols biodegrade very slowly, and their prolonged exposure leads to severe ecological and environmental problems (Fig. 1A) (2–4). Several well-established technologies have been developed for the treating of chlorophenols, including catalytic oxidation (5–11), biodegradation (12–15), solvent extraction (16, 17), and adsorption (18–20) Among these methods, adsorption is the most versatile and widely used method due to its high removal efficiency and simple operation, but the resulting products are of no value, and consequently, these processes are not viable.Open in a separate windowFig. 1.Background and reaction design. (A) Examples of chlorophenol pollutants. (B) Examples of aryl chlorides. (C) The chlorination process reported herein was based on chloro-group transfer from chlorophenol pollutants.With the extensive application of substitution reactions (21, 22), transfunctionalizations (23, 24), and cross-coupling reactions (25, 26), aryl chlorides are regarded as one of the most important building blocks widely used in the manufacture of polymers, pharmaceuticals, and other types of chemicals and materials (Fig. 1B) (27–31). Chlorination of arenes is usually carried out with toxic and corrosive reagents (32–34). Less toxic and more selective chlorination reagents tend to be expensive [e.g., chloroamides (35, 36)] and are not atom economic (37–39). Consequently, from the perspective of sustainability, the ability to transfer a chloro group from unwanted chlorophenols to other substrates would be advantageous.Catalytic isofunctional reactions, including transfer hydrogenation and alkene metathesis, have been widely exploited in organic synthesis. We hypothesized that chlorination of arenes also could be achieved by chloro-group transfer, and since stockpiles of chlorophenols tend to be destroyed by mineralization and chlorophenol pollutants may be concentrated by adsorption (18–20), they could be valorized as chlorination reagents via transfer of the chloro group to arene substrates during their mineralization, thereby adding value to the destruction process (Fig. 1C). Although chlorophenol pollutants are not benign, their application as chlorination reagents, with their concomitant destruction to harmless compounds, may be considered as not only meeting the criteria of green chemistry but also potentially surpassing it. Herein, we describe a robust strategy to realize chloro-group transfer from chlorophenol pollutants to arenes and afford a wide range of value-added aryl chlorides.     

Emerging predictable features of replicated biological invasion fronts

Biological dispersal shapes species’ distribution and affects their coexistence. The spread of organisms governs the dynamics of invasive species, the spread of pathogens, and the shifts in species ranges due to climate or environmental change. Despite its relevance for fundamental ecological processes, however, replicated experimentation on biological dispersal is lacking, and current assessments point at inherent limitations to predictability, even in the simplest ecological settings. In contrast, we show, by replicated experimentation on the spread of the ciliate Tetrahymena sp. in linear landscapes, that information on local unconstrained movement and reproduction allows us to predict reliably the existence and speed of traveling waves of invasion at the macroscopic scale. Furthermore, a theoretical approach introducing demographic stochasticity in the Fisher–Kolmogorov framework of reaction–diffusion processes captures the observed fluctuations in range expansions. Therefore, predictability of the key features of biological dispersal overcomes the inherent biological stochasticity. Our results establish a causal link from the short-term individual level to the long-term, broad-scale population patterns and may be generalized, possibly providing a general predictive framework for biological invasions in natural environments.What is the source of variance in the spread rates of biological invasions? The search for processes that affect biological dispersal and sources of variability observed in ecological range expansions is fundamental to the study of invasive species dynamics (1–10), shifts in species ranges due to climate or environmental change (11–13), and, in general, the spatial distribution of species (3, 14–16). Dispersal is the key agent that brings favorable genotypes or highly competitive species into new ranges much faster than any other ecological or evolutionary process (1, 17). Understanding the potential and realized dispersal is thus key to ecology in general (18). When organisms’ spread occurs on the timescale of multiple generations, it is the byproduct of processes that take place at finer spatial and temporal scales that are the local movement and reproduction of individuals (5, 10). The main difficulty in causally understanding dispersal is thus to upscale processes that happen at the short-term individual level to long-term and broad-scale population patterns (5, 18–20). Furthermore, the large fluctuations observed in range expansions have been claimed to reflect an intrinsic lack of predictability of the phenomenon (21). Whether the variability observed in nature or in experimental ensembles might be accounted for by systematic differences between landscapes or by demographic stochasticity affecting basic vital rates of the organisms involved is an open research question (10, 18, 21, 22).Modeling of biological dispersal established the theoretical framework of reaction–diffusion processes (1–3, 23–25), which now finds common application in dispersal ecology (5, 14, 22, 26–30) and in other fields (17, 23, 25, 31–36). Reaction–diffusion models have also been applied to model human colonization processes (31), such as the Neolithic transition in Europe (25, 37, 38). The classical prediction of reaction–diffusion models (1, 2, 24, 25) is the propagation of an invading wavefront traveling undeformed at a constant speed (Fig. 1E). Such models have been widely adopted by ecologists to describe the spread of organisms in a variety of comparative studies (5, 10, 26) and to control the dynamics of invasive species (3, 4, 6). The extensive use of these models and the good fit to observational data favored their common endorsement as a paradigm for biological dispersal (6). However, current assessments (21) point at inherent limitations to the predictability of the phenomenon, due to its intrinsic stochasticity. Therefore, single realizations of a dispersal event (as those addressed in comparative studies) might deviate significantly from the mean of the process, making replicated experimentation necessary to allow hypothesis testing, identification of causal relationships, and to potentially falsify the models’ assumptions (39).Open in a separate windowFig. 1.Schematic representation of the experiment. (A) Linear landscape. (B) Individuals of the ciliate Tetrahymena sp. move and reproduce within the landscape. (C) Examples of reconstructed trajectories of individuals (Movie S1). (D) Individuals are introduced at one end of a linear landscape and are observed to reproduce and disperse within the landscape (not to scale). (E) Illustrative representation of density profiles along the landscape at subsequent times. A wavefront is argued to propagate undeformed at a constant speed v according to the Fisher–Kolmogorov equation.Here, we provide replicated and controlled experimental support to the theory of reaction–diffusion processes for modeling biological dispersal (23–25) in a generalized context that reproduces the observed fluctuations. Firstly, we experimentally substantiate the Fisher–Kolmogorov prediction (1, 2) on the existence and the mean speed of traveling wavefronts by measuring the individual components of the process. Secondly, we manipulate the inclusion of demographic stochasticity in the model to reproduce the observed variability in range expansions. We move from the Fisher–Kolmogorov equation (Materials and Methods) to describe the spread of organisms in a linear landscape (1, 2, 24, 25). The equation couples a logistic term describing the reproduction of individuals with growth rate r and carrying capacity K and a diffusion term accounting for local movement, epitomized by the diffusion coefficient D . These species’ traits define the characteristic scales of the dispersal process. In this framework, a population initially located at one end of a linear landscape is predicted to form a wavefront of colonization invading empty space at a constant speed (1, 2, 24, 25), which we measured in our dispersal experiment (Fig. 1D and SI Text).     

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

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

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

INAUGURAL ARTICLE by a Recently Elected Academy Member:Chirality-matched catalyst-controlled macrocyclization reactions

Macrocycles, formally defined as compounds that contain a ring with 12 or more atoms, continue to attract great interest due to their important applications in physical, pharmacological, and environmental sciences. In syntheses of macrocyclic compounds, promoting intramolecular over intermolecular reactions in the ring-closing step is often a key challenge. Furthermore, syntheses of macrocycles with stereogenic elements confer an additional challenge, while access to such macrocycles are of great interest. Herein, we report the remarkable effect peptide-based catalysts can have in promoting efficient macrocyclization reactions. We show that the chirality of the catalyst is essential for promoting favorable, matched transition-state relationships that favor macrocyclization of substrates with preexisting stereogenic elements; curiously, the chirality of the catalyst is essential for successful reactions, even though no new static (i.e., not “dynamic”) stereogenic elements are created. Control experiments involving either achiral variants of the catalyst or the enantiomeric form of the catalyst fail to deliver the macrocycles in significant quantity in head-to-head comparisons. The generality of the phenomenon, demonstrated here with a number of substrates, stimulates analogies to enzymatic catalysts that produce naturally occurring macrocycles, presumably through related, catalyst-defined peripheral interactions with their acyclic substrates.

Macrocyclic compounds are known to perform a myriad of functions in the physical and biological sciences. From cyclodextrins that mediate analyte separations (1) to porphyrin cofactors that sit in enzyme active sites (2, 3) and to potent biologically active, macrocyclic natural products (4) and synthetic variants (5–7), these structures underpin a wide variety of molecular functions (Fig. 1A). In drug development, such compounds are highly coveted, as their conformationally restricted structures can lead to higher affinity for the desired target and often confer additional metabolic stability (8–13). Accordingly, there exists an entire synthetic chemistry enterprise focused on efficient formation and functionalization of macrocycles (14–18).Open in a separate windowFig. 1.(A) Examples of macrocyclic compounds with important applications. HCV, hepatitis C virus. (B) Use of chiral ligands in metal-catalyzed or mediated stereoselective macrocyclization reactions. (C) Remote desymmetrization using guanidinylated ligands via Ullmann coupling. (D) This work: use of copper/peptidyl complexes for macrocyclization and the exploration of matched and mismatched effect.In syntheses of macrocyclic compounds, the ring-closing step is often considered the most challenging step, as competing di- and oligomerization pathways must be overcome to favor the intramolecular reaction (14). High-dilution conditions are commonly employed to favor macrocyclization of linear precursors (19). Substrate preorganization can also play a key role in overcoming otherwise high entropic barriers associated with multiple conformational states that are not suited for ring formation. Such preorganization is most often achieved in synthetic chemistry through substrate design (14, 20–22). Catalyst or reagent controls that impose conformational benefits that favor ring formation are less well known. Yet, critical precedents include templating through metal-substrate complexation (23, 24), catalysis by foldamers (25) or enzymes (26–29), or, in rare instances, by small molecules (discussed below). Characterization of biosynthetic macrocyclization also points to related mechanistic issues and attributes for efficient macrocyclizations (30–34). Coupling macrocyclization reactions to the creation of stereogenic elements is also rare (35). Metal-mediated reactions have been applied toward stereoselective macrocyclizations wherein chiral ligands transmit stereochemical information to the products (Fig. 1B). For example, atroposelective ring closure via Heck coupling has been applied in the asymmetric total synthesis of isoplagiochin D by Speicher and coworkers (36–40). Similarly, atroposelective syntheses of (+)-galeon and other diarylether heptanoid natural products were achieved via Ullman coupling using N-methyl proline by Salih and Beaudry (41). Finally, Reddy and Corey reported the enantioselective syntheses of cyclic terpenes by In-catalyzed allylation utilizing a chiral prolinol-based ligand (42). While these examples collectively illustrate the utility of chiral ligands in stereoselective macrocyclizations, such examples remain limited.We envisioned a different role for chiral catalysts when addressing intrinsically disfavored macrocyclization reactions. When unfavorable macrocyclization reactions are confronted, we hypothesized that a catalyst–substrate interaction might provide transient conformational restriction that could promote macrocyclization. To address this question, we chose to explore whether or not a chiral catalyst-controlled macrocyclization might be possible with peptidyl copper complexes. In the context of the medicinally ubiquitous diarylmethane scaffold, we had previously demonstrated the capacity for remote asymmetric induction in a series of bimolecular desymmetrizations using bifunctional, tetramethylguanidinylated peptide ligands. For example, we showed that peptidyl copper complexes were able to differentiate between the two aryl bromides during C–C, C–O, and C–N cross-coupling reactions (Fig. 1C) (43–45). Moreover, in these intermolecular desymmetrizations, a correlation between enantioselectivity and conversion was observed, revealing the catalyst’s ability to perform not only enantiotopic group discrimination but also kinetic resolution on the monocoupled product as the reaction proceeds (44). This latter observation stimulated our speculation that if an internal nucleophile were present to undergo intramolecular cross-coupling to form a macrocycle, stereochemically sensitive interactions (so-called matched and mismatched effects) (46) could be observed (Fig. 1D). Ideally, we anticipated that transition state–stabilizing interactions might even prove decisive in matched cases, and the absence of catalyst–substrate stabilizing interactions might account for the absence of macrocyclization for these otherwise intrinsically unfavorable reactions. Herein, we disclose the explicit observation of these effects in chiral catalyst-controlled macrocyclization reactions.     

Volatile-consuming reactions fracture rocks and self-accelerate fluid flow in the lithosphere

Hydration and carbonation reactions within the Earth cause an increase in solid volume by up to several tens of vol%, which can induce stress and rock fracture. Observations of naturally hydrated and carbonated peridotite suggest that permeability and fluid flow are enhanced by reaction-induced fracturing. However, permeability enhancement during solid-volume–increasing reactions has not been achieved in the laboratory, and the mechanisms of reaction-accelerated fluid flow remain largely unknown. Here, we present experimental evidence of significant permeability enhancement by volume-increasing reactions under confining pressure. The hydromechanical behavior of hydration of sintered periclase [MgO + H₂O → Mg(OH)₂] depends mainly on the initial pore-fluid connectivity. Permeability increased by three orders of magnitude for low-connectivity samples, whereas it decreased by two orders of magnitude for high-connectivity samples. Permeability enhancement was caused by hierarchical fracturing of the reacting materials, whereas a decrease was associated with homogeneous pore clogging by the reaction products. These behaviors suggest that the fluid flow rate, relative to reaction rate, is the main control on hydromechanical evolution during volume-increasing reactions. We suggest that an extremely high reaction rate and low pore-fluid connectivity lead to local stress perturbations and are essential for reaction-induced fracturing and accelerated fluid flow during hydration/carbonation.

Hydration and carbonation reactions in the crust and mantle transport H₂O and CO₂ from Earth’s surface to the interior and control volatile budgets within the Earth (1–6). These reactions are characterized by solid-volume increase, by up to several tens of vol%, which induces stress that may lead to fracturing (7–10). The driving force of such stress generation is the thermodynamic free energy released when metastable anhydrous/noncarbonate minerals react with fluids (7). The stress generated by the reaction has the potential to cause rock fracture and fragmentation (7, 11–13), thereby increasing the reactive surface area and fluid flow and further accelerating the reactions (7, 8, 14). Such chemical breaking of rocks, or reaction-induced fracturing, appears to be important in driving hydration and carbonation reactions to completion (8, 15, 16) in an otherwise self-limiting process where reaction products can clog pores and suppress fluid flow, thereby hindering the reaction (15, 17).Observations of naturally serpentinized and fractured ultramafic rocks indicate a volume increase of 20 to 60% during hydration reactions (13, 18–20), providing evidence of an accelerated supply of fluids during hydration (Fig. 1 A and B). Natural carbonation of ultramafic rocks is also associated with extensive fracture networks, and reaction-induced fracturing is considered a key process in mineral carbonation (Fig. 1C) (7, 8, 21). Numerical simulations indicate a positive feedback between volume-increasing reaction, fracturing, and fluid flow (10, 22–32). Laboratory experiments partially reproduce fracturing during peridotite carbonation, serpentinization, and periclase hydration (29, 33–36); however, hydrothermal flow-through experiments of peridotite serpentinization and carbonation show a decrease in permeability and deceleration of fluid flow and reaction rate (37–42). Observations of the natural carbonation of serpentinized peridotite indicate the decrease in permeability and reduced fluid flow and reaction rate are a consequence of pore clogging related to carbonation (43). Until now, no experimental studies have shown a clear increase in permeability during expansive fluid–rock reactions under confining pressure. As such, despite their geological and environmental importance, the evolution of expansive fluid–rock reactions remains difficult to predict, owing to the complex hydraulic–chemical–mechanical feedbacks underlying these reactions (15, 16, 44). The processes controlling the self-acceleration or deceleration of these reactions remain largely unknown.Open in a separate windowFig. 1.Reaction-induced fractures related to natural hydration/carbonation. (A) Polygonal block of serpentinite cut by planar lizardite veins, extracted from a serpentinite body, San Andreas Lake, California. (B) Photomicrograph of mesh structure in partly serpentinized peridotite, Redwood City serpentinite, California [crossed-polarized light (61)]. (C) Quartz veins in silica–carbonate rocks (i.e., listvenite, a carbonated ultramafic rock) that occur along the boundaries of serpentinite bodies, San Jose, California. ol, olivine; serp, serpentine (lizardite ± antigorite mixture); br, brucite.Here, we use the hydration of periclase to brucite [MgO + H₂O → Mg(OH)₂] as an analog for solid-volume–increasing reactions in the Earth. This reaction produces an extreme solid-volume increase of 119%, with a high reaction rate at 100 to 600 °C (45). Previous experimental studies on periclase hydration have revealed that extensive fracturing occurs under certain conditions (29, 33, 35), yet the links between fracturing experiments (periclase hydration), nonfracturing experiments (peridotite hydration/carbonation), and natural observations are unknown. On the basis of in situ observations of fluid flow during the reactions, we clearly show that fluid flow and associated permeability are strongly enhanced by solid-volume–increasing reactions under confining pressure (i.e., at simulated depth). Based on the experimental results and nondimensional parameterization, we propose that the ratio of the initial fluid flow rate to the reaction rate has a primary control on the self-acceleration and deceleration of fluid flow and reactions during hydration and carbonation within the Earth.     

Conjugated polymers based on metalla-aromatic building blocks

Conjugated polymers usually require strategies to expand the range of wavelengths absorbed and increase solubility. Developing effective strategies to enhance both properties remains challenging. Herein, we report syntheses of conjugated polymers based on a family of metalla-aromatic building blocks via a polymerization method involving consecutive carbyne shuttling processes. The involvement of metal d orbitals in aromatic systems efficiently reduces band gaps and enriches the electron transition pathways of the chromogenic repeat unit. These enable metalla-aromatic conjugated polymers to exhibit broad and strong ultraviolet–visible (UV–Vis) absorption bands. Bulky ligands on the metal suppress π–π stacking of polymer chains and thus increase solubility. These conjugated polymers show robust stability toward light, heat, water, and air. Kinetic studies using NMR experiments and UV–Vis spectroscopy, coupled with the isolation of well-defined model oligomers, revealed the polymerization mechanism.

Conjugated polymers are macromolecules usually featuring a backbone chain with alternating double and single bonds (1–3). These characteristics allow the overlapping p-orbitals to form a system with highly delocalized π-electrons, thereby giving rise to intriguing chemical and physical properties (4–6). They have exhibited many applications in organic light-emitting diodes, organic thin film transistors, organic photovoltaic cells, chemical sensors, bioimaging and therapies, photocatalysis, and other technologies (7–10). To facilitate the use of solar energy, tremendous efforts have been devoted in recent decades to developing previously unidentified conjugated polymers exhibiting broad and strong absorption bands (11–13). The common strategies for increasing absorption involve extending π-conjugation by incorporating conjugated cyclic moieties, especially fused rings; modulating the strength of intramolecular charge transfer between donor and acceptor units (D–A effect); increasing the coplanarity of π conjugation through weak intramolecular interactions (e.g., hydrogen bonds); and introducing heteroatoms or heavy atoms into the repeat units of conjugated polymers (11–16). Additionally, appropriate solubility is a prerequisite for processing and using polymers and is usually achieved with the aid of long alkyl or alkoxy side chains (12, 17).Aromatic rings are among the most important building blocks for conjugated polymers. In addition to aromatic hydrocarbons, a variety of aromatic heterocycles composed of main-group elements have been used as fundamental components. These heteroatom-containing conjugated polymers show unique optical and electronic properties (4–10). However, while metalla-aromatic systems bearing a transition metal have been known since 1979 due to the pioneering work by Thorn and Hoffmann (18), none of them have been used as building blocks for conjugated polymers. The HOMO–LUMO gaps (E_g) of metalla-aromatics are generally narrower (Fig. 1) than those of their organic counterparts (19–22). We reasoned that this feature should broaden the absorption window if polymers stemming from metalla-aromatics are achievable.Open in a separate windowFig. 1.Comparison of traditional organic skeletons with metalla-aromatic building blocks (the computed energies are in eV). (A) HOMO–LUMO gaps of classic aromatic skeletons. (B) Carbolong frameworks as potential building blocks for novel conjugated polymers with broad absorption bands and improved solubility.In recent years, we have reported a series of readily accessible metal-bridged bicyclic/polycyclic aromatics, namely carbolong complexes, which are stable in air and moisture (23–25). The addition of osmium carbynes (in carbolong complexes) and alkynes gave rise to an intriguing family of d_π–p_π conjugated systems, which function as excellent electron transport layer materials in organic solar cells (26, 27). These observations raised the following question: Can this efficient addition reaction be used to access metalla-aromatic conjugated polymers? It is noteworthy that incorporation of metalla-aromatic units into conjugated polymers is hitherto unknown. In this contribution, we disclose a polymerization reaction involving M≡C analogs of C≡C bonds, which involves a unique carbyne shuttling strategy (Fig. 2A). This led to examples of metalla-aromatic conjugated polymers (polycarbolongs) featuring metal carbyne units in the main chain. On the other hand, the development of polymerization reactions plays a crucial role in involving certain building blocks in conjugated polymers (28–32). These efficient, specific, and feasible polymerizations could open an avenue for the synthesis of conjugated polymers.Open in a separate windowFig. 2.Design of polymers and synthesis of monomers. (A) Schematic illustration of the polymerization strategy. (B) Preparation of carbolong monomers. Insert: X-ray molecular structure for the cations of complex 3. Ellipsoids are shown at the 50% probability level; phenyl groups in PPh₃ are omitted for clarity.     

A 5,000-year vegetation and fire history for tierra firme forests in the Medio Putumayo-Algodón watersheds,northeastern Peru

This paper addresses an important debate in Amazonian studies; namely, the scale, intensity, and nature of human modification of the forests in prehistory. Phytolith and charcoal analysis of terrestrial soils underneath mature tierra firme (nonflooded, nonriverine) forests in the remote Medio Putumayo-Algodón watersheds, northeastern Peru, provide a vegetation and fire history spanning at least the past 5,000 y. A tree inventory carried out in the region enables calibration of ancient phytolith records with standing vegetation and estimates of palm species densities on the landscape through time. Phytolith records show no evidence for forest clearing or agriculture with major annual seed and root crops. Frequencies of important economic palms such as Oenocarpus, Euterpe, Bactris, and Astrocaryum spp., some of which contain hyperdominant species in the modern flora, do not increase through prehistoric time. This indicates pre-Columbian occupations, if documented in the region with future research, did not significantly increase the abundance of those species through management or cultivation. Phytoliths from other arboreal and woody species similarly reflect a stable forest structure and diversity throughout the records. Charcoal ¹⁴C dates evidence local forest burning between ca. 2,800 and 1,400 y ago. Our data support previous research indicating that considerable areas of some Amazonian tierra firme forests were not significantly impacted by human activities during the prehistoric era. Rather, it appears that over the last 5,000 y, indigenous populations in this region coexisted with, and helped maintain, large expanses of relatively unmodified forest, as they continue to do today.

More than 50 y ago, prominent scholars argued that due to severe environmental constraints (e.g., poor natural resources), prehistoric cultures in the Amazon Basin were mainly small and mobile with little cultural complexity, and exerted low environmental impacts (1, 2). Contentious debates ensued and have been ongoing ever since. Empirical data accumulated during the past 10 to 20 y have made it clear that during the late Holocene beginning about 3,000 y ago dense, permanent settlements with considerable cultural complexity had developed along major watercourses and some of their tributaries, in seasonal savannas/areas of poor drainage, and in seasonally dry forest. These populations exerted significant, sometimes profound, regional-scale impacts on landscapes, including with raised agricultural fields, fish weirs, mound settlements, roads, geometric earthworks called geoglyphs, and the presence of highly modified anthropic soils, called terra pretas or “Amazonian Dark Earths” (Fig. 1) (e.g., refs. 3–15).Open in a separate windowFig. 1.Location of study region (MP-A) and other Amazonian sites discussed in the text. River names are in blue. The black numbers represent major pre-Columbian archaeological sites with extensive human alterations (1, Marajó Island; 2, Santarém; 3, Upper Xingu; 4, Central Amazon Project; 5, Bolivian sites) (3, 5–10, 14, 15). ADE, terra preta locations (e.g., refs. 19 and 20); triangles are geoglyph sites (6, 8). The white circles are terrestrial soil locations previously studied by Piperno and McMichael (29, 31–33, 54) (Ac, Acre; Am, Amacayacu; Ay, Lake Ayauchi; B, Barcelos; GP, lakes Gentry-Parker; Iq, Iquitos to Nauta; LA, Los Amigos; PVM, Porto Velho to Manaus; T, Tefe).An important, current debate that frames this paper centers not on whether some regions of the pre-Columbian Amazon supported large and complex human societies, but rather on the spatial scales, degrees, and types of cultural impacts across this continental-size landscape. Some investigators drawing largely on available archaeological data and studies of modern floristic composition of selected forests, argue that heavily modified “domesticated” landscapes were widespread across Amazonia at the end of prehistory, and these impacts significantly structure the vegetation today, even promoting higher diversity than before (e.g., refs. 14–21). It is believed that widespread forms of agroforestry with planted, orchard-like formations or other forest management strategies involving the care and possible enrichment of several dozens of economically important native species have resulted in long-term legacies left on forest composition (e.g., refs. 14–22). Some (20) propose that human influences played strong roles in the enrichment of “hyperdominant” trees, which are disproportionately common elements in the modern flora (sensu ref. 23). Some even argue that prehistoric fires and forest clearance were so spatially extensive that post-Columbian reforestation upon the tragic consequences of European contact was a principal contributor to decreasing atmospheric CO₂ levels and the onset of the “Little Ice Age” (24, 25).However, modern floristic studies are often located in the vicinity of known archaeological sites and/or near watercourses (26). Many edible trees in these studies are early successional and would not be expected to remain as significant forest elements for hundreds of years after abandonment. Historic-period impacts well-known in some regions to have been profound have been paid little attention and may be mistaken for prehistoric legacies (26–28). Moreover, existing phytolith and charcoal data from terrestrial soils underneath standing tierra firme forest in some areas of the central and western Amazon with no known archaeological occupations nearby exhibit little to no evidence for long-term human occupation, anthropic soils, agriculture, forest clearing or other significant vegetation change, or recurrent/extensive fires during the past several thousand years (Fig. 1) (29–33). Even such analyses of terrestrial soils of lake watersheds in western Amazonia known to have been occupied and farmed in prehistory revealed no spatially extensive deforestation of the watersheds, as significant human impacts most often occurred in areas closest to the lakes (Fig. 1) (34). Furthermore, vast areas have yet to be studied by archaeologists and paleoecologists, particularly the tierra firme forests that account for 95% of the land area of Amazonia.To further inform these issues, we report here a vegetation and fire history spanning 5,000 y derived from phytolith and charcoal studies of terrestrial soils underneath mature tierra firme forest in northeastern Peru. Phytoliths, the silica bodies produced by many Neotropical plants, are well preserved in terrestrial soils unlike pollen, and are deposited locally. They can be used to identify different tropical vegetational formations, such as old-growth forest, early successional vegetation typical of human disturbances including forest clearings, a number of annual seed and root crops, and trees thought to have been cultivated or managed in prehistory (e.g., refs. 29–33 and 35).     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Click-to-lead design of a picomolar ABA receptor antagonist with potent activity in vivo

Abscisic acid (ABA) is a key plant hormone that mediates both plant biotic and abiotic stress responses and many other developmental processes. ABA receptor antagonists are useful for dissecting and manipulating ABA’s physiological roles in vivo. We set out to design antagonists that block receptor–PP2C interactions by modifying the agonist opabactin (OP), a synthetically accessible, high-affinity scaffold. Click chemistry was used to create an ∼4,000-member library of C4-diversified opabactin derivatives that were screened for receptor antagonism in vitro. This revealed a peptidotriazole motif shared among hits, which we optimized to yield antabactin (ANT), a pan-receptor antagonist. An X-ray crystal structure of an ANT–PYL10 complex (1.86 Å) reveals that ANT’s peptidotriazole headgroup is positioned to sterically block receptor–PP2C interactions in the 4′ tunnel and stabilizes a noncanonical closed-gate receptor conformer that partially opens to accommodate ANT binding. To facilitate binding-affinity studies using fluorescence polarization, we synthesized TAMRA–ANT. Equilibrium dissociation constants for TAMRA–ANT binding to Arabidopsis receptors range from ∼400 to 1,700 pM. ANT displays improved activity in vivo and disrupts ABA-mediated processes in multiple species. ANT is able to accelerate seed germination in Arabidopsis, tomato, and barley, suggesting that it could be useful as a germination stimulant in species where endogenous ABA signaling limits seed germination. Thus, click-based diversification of a synthetic agonist scaffold allowed us to rapidly develop a high-affinity probe of ABA–receptor function for dissecting and manipulating ABA signaling.

The phytohormone abscisic acid (ABA) controls numerous physiological processes in plants ranging from seed development, germination, and dormancy to responses for countering biotic and abiotic stresses (1). ABA binds to the PYR/PYL/RCAR (Pyrabactin Resistance 1/PYR1-like/Regulatory Component of ABA Receptor) soluble receptor proteins (2, 3) and triggers a conformational change in a flexible “gate” loop flanking the ligand-binding pocket such that the ABA–receptor complex can then bind to and inhibit clade A type II C protein phosphatases (PP2Cs), which normally dephosphorylate and inactivate SNF1-related protein kinase 2 (SnRK2). This, in turn, leads to SnRK2 activation, phosphorylation of downstream targets, and multiple cellular outputs (4, 5).Chemical modulators of ABA perception have been sought as both research tools for dissecting ABA’s role in plant physiology and for their potential agricultural utility (6, 7). Dozens of ABA receptor agonists, which reduce transpiration and water use by inducing guard cell closure, have been developed and are being explored as chemical tools for mitigating the effects of drought on crop yields (7–23), most of them either being analogs of ABA or sulfonamides similar to quinabactin (24). ABA receptor antagonists could conceivably be useful in cases where water is not limiting, for example, to increase transpiration and gas exchange under elevated CO₂ in glasshouse agriculture, as germination stimulators, and for studying the ABA dependence of physiological processes, among other applications (25–31). Thus, both ABA receptor agonists and antagonists have potential uses as research tools and for plant biotechnology.In principle, there are at least two mechanisms for blocking ABA receptor activation: by preventing gate closure, which is necessary for PP2C binding, or by sterically disrupting the activated, closed-gate receptor conformer from binding to PP2Cs. Prior efforts to design antagonists have focused on the latter strategy and include multiple ABA-derived ligands such as AS6 (25), PanMe (26), 3′-alkyl ABA (30–32), 3′-(phenyl alkynyl) ABA (33), or ligands derived from tetralone ABA (34) with varying degrees of conformational restriction (27, 28, 35). With the exception of PanMe, these antagonists have linkers attached to the 3′ carbon of ABA or 11′ carbon of tetralone ABA, which is positioned to disrupt receptor–PP2C interactions by protruding through the 3′ tunnel. PanMe was created by modifying ABA’s C4′ (Fig. 1) with a toluylpropynyl ether substituent designed to occupy the 4′ tunnel, a site of close receptor–PP2C contact (26). Structural studies showed that this 4′ moiety adopts two conformations, one that resides in the 4′ tunnel and another that occupies the adjacent 3′ tunnel (26). Collectively, these elegant studies have demonstrated that antagonists of receptor–PP2C interactions can be designed by modifying agonists at sites situated proximal to the 3′ or 4′ tunnels. Despite these advances, current antagonists have limitations. For example, PanMe, which has low nanomolar affinity for the subfamily II receptor PYL5, is limited by relatively low activity on subfamily I and III ABA receptors, and as we show here, the ABA antagonist AA1 (36) (Fig. 1) lacks detectable antagonist activity in vitro and is, therefore, unlikely to be a true ABA receptor antagonist. Together, these data suggest that higher-affinity pan-antagonists and/or molecules with increased bioavailability will be necessary to more efficiently block endogenous ABA signaling. We set out to address these limitations by modifying the scaffold of the synthetic ABA agonist opabactin (OP), which has an approximately sevenfold increase in both affinity and bioactivity relative to ABA (21). We describe an OP derivative called antabactin (ANT) and show that it is a high-affinity binder and antagonist of ABA receptors that disrupts ABA-mediated signaling in vivo.Open in a separate windowFig. 1.Structures of ABA, PanMe, and AA1.     

From the Cover: 1.36 million years of Mediterranean forest refugium dynamics in response to glacial–interglacial cycle strength

The sediment record from Lake Ohrid (Southwestern Balkans) represents the longest continuous lake archive in Europe, extending back to 1.36 Ma. We reconstruct the vegetation history based on pollen analysis of the DEEP core to reveal changes in vegetation cover and forest diversity during glacial–interglacial (G–IG) cycles and early basin development. The earliest lake phase saw a significantly different composition rich in relict tree taxa and few herbs. Subsequent establishment of a permanent steppic herb association around 1.2 Ma implies a threshold response to changes in moisture availability and temperature and gradual adjustment of the basin morphology. A change in the character of G–IG cycles during the Early–Middle Pleistocene Transition is reflected in the record by reorganization of the vegetation from obliquity- to eccentricity-paced cycles. Based on a quantitative analysis of tree taxa richness, the first large-scale decline in tree diversity occurred around 0.94 Ma. Subsequent variations in tree richness were largely driven by the amplitude and duration of G–IG cycles. Significant tree richness declines occurred in periods with abundant dry herb associations, pointing to aridity affecting tree population survival. Assessment of long-term legacy effects between global climate and regional vegetation change reveals a significant influence of cool interglacial conditions on subsequent glacial vegetation composition and diversity. This effect is contrary to observations at high latitudes, where glacial intensity is known to control subsequent interglacial vegetation, and the evidence demonstrates that the Lake Ohrid catchment functioned as a refugium for both thermophilous and temperate tree species.

Identification and protection of past forest refugia, supporting a relict population, has gained interest in light of projected forest responses to anthropogenic climate change (1–4). Understanding the past and present composition of Mediterranean forest refugia is central to the study of long-term survival of tree taxa and the systematic relation between forest dynamics and climate (5). The Quaternary vegetation history of Europe, studied for over a century, is characterized by successive loss of tree species (6–8). Species loss was originally explained by the repeated migration across east–west oriented mountain chains during glacial–interglacial (G–IG) cycles (9). Later views gave more importance to the survival of tree populations during warm and arid stages in southern refugia (10, 11). Tree survival likely depends on persistence of suitable climate and tolerable levels of climate variability, as well as niche differentiation and population size at the refugium (12), although the precise relation between regional extinctions, climate variability, and local edaphic factors is not well known (13). Mediterranean mountain regions are considered to serve as forest refugia over multiple glacial cycles and frequently coincide with present-day biodiversity hotspots (14). Across the Mediterranean, increases in aridity and fire occurrence have impacted past vegetation communities (15–18). Comprehensive review of available Quaternary Mediterranean records indicates that Early (2.58 to 0.77 Ma) and Middle Pleistocene (0.77 to 0.129 Ma) tree diversity was higher compared to the present (13, 19–21). Particularly drought intolerant, thermophilic taxa were more abundant and diverse (8) but with strong spatial and temporal variations in tree diversity across the region. Long-term relationships between refugia function and environmental change over multiple G–IG cycles are hard to quantify due to the rarity of long, uninterrupted records.The Early–Middle Pleistocene Transition (EMPT), between 1.4 and 0.4 Ma (22), is of particular importance for understanding the relation between past climate change, vegetation dynamics, and biodiversity in the Mediterranean region. The EMPT is characterized by a gradual transition of G–IG cycle duration from obliquity (41 thousand years; kyr) to eccentricity (100 kyr) scale with increasing amplitude of each G–IG cycle (e.g., refs. 23, 24). The EMPT was accompanied by long-term cooling of the deep and surface ocean and was likely caused by atmospheric CO₂ decline and ice-sheet feedbacks (25–30). In Europe, the EMPT is associated with pronounced vegetation changes and local extinction and isolation of small tree populations (31).Here, we document vegetation history of the last 1.36 Ma in the Lake Ohrid (LO) catchment, located at the Albanian/North Macedonian border at 693 m above sea level (m asl, Fig. 1), the longest continuous sedimentary lake record in Europe (32, 33). The chronology of the DEEP core (International Continental Scientific Drilling Program site 5045-1; 41°02’57’’ N, 20°42’54’’ E, Fig. 1) is based on tuning of biogeochemical proxy data to orbital parameters with independent tephrostratigraphic and paleomagnetic age control (32, 33). The Balkan Peninsula has long been considered an important glacial forest refugium for presently widespread taxa such as Abies, Picea, Carpinus, Corylus, Fagus Ostrya, Quercus, Tilia, and Ulmus (7, 34–36). More than 60% of the Balkans is currently located >1,000 m asl (36), providing steep latitudinal and elevational gradients to support refugia under both cold and warm conditions. Today, the LO catchment is dominated by (semi) deciduous oak (Quercus) and hornbeam (Carpinus/Ostrya) forests. Above 1,250 m elevation, mixed mesophyllous forest with montane elements occurs (Fagus and at higher elevations Abies), which above 1,800 m elevation develops into subalpine grassland with Juniperus shrubs (see ref. 37 for site details). Isolated populations of Pinus peuce and Pinus nigra currently grow in the area (37–40).Open in a separate windowFig. 1.(A) Location of LO and TP on the Balkan Peninsula. (B) Local setting around LO, bathymetry (81), and DEEP coring site (adapted from ref. 32).Previous analysis of pollen composition of the last 500 kyr at the DEEP site revealed that the LO has been an important refugium. Arboreal pollen (AP) is deposited continuously and changes in abundance on multimillennial timescales in association with G–IG cycles, whereas millennial-scale variability is tightly coupled to Mediterranean sea-surface temperature variations (37, 41–45). Subsequent studies confirm the refugial character of the site recording Early Pleistocene (1.365 to 1.165 Ma) high relict tree diversity and abundance—and significant hydrological changes, including an increase in lake size and depth (38). Here, we present a continuous palynological record from LO with millennial resolution (∼2 kyr) back to 1.36 Ma to assess the systematic relationships between tree pollen abundance, forest diversity, and G–IG climate variability.Our objective is as follows: 1) infer the impact of past climate variability on local vegetation across the EMPT, 2) estimate tree species diversity in the catchment, and 3) examine how the amplitude and duration of preceding G–IG intervals affected the vegetation development and plant species diversity in this refugial area.