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.     

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.     

Inside-out regulation of E-cadherin conformation and adhesion

Cadherin cell–cell adhesion proteins play key roles in tissue morphogenesis and wound healing. Cadherin ectodomains bind in two conformations, X-dimers and strand-swap dimers, with different adhesive properties. However, the mechanisms by which cells regulate ectodomain conformation are unknown. Cadherin intracellular regions associate with several actin-binding proteins including vinculin, which are believed to tune cell–cell adhesion by remodeling the actin cytoskeleton. Here, we show at the single-molecule level, that vinculin association with the cadherin cytoplasmic region allosterically converts weak X-dimers into strong strand-swap dimers and that this process is mediated by myosin II–dependent changes in cytoskeletal tension. We also show that in epithelial cells, ∼70% of apical cadherins exist as strand-swap dimers while the remaining form X-dimers, providing two cadherin pools with different adhesive properties. Our results demonstrate the inside-out regulation of cadherin conformation and establish a mechanistic role for vinculin in this process.

E-cadherins (Ecads) are essential, calcium-dependent cell–cell adhesion proteins that play key roles in the formation of epithelial tissue and in the maintenance of tissue integrity. Ecad adhesion is highly plastic and carefully regulated to orchestrate complex movement of epithelial cells, and dysregulation of adhesion is a hallmark of numerous cancers (1). However, little is known about how cells dynamically regulate the biophysical properties of individual Ecads.The extracellular region of Ecads from opposing cells bind in two distinct trans orientations: strand-swap dimers and X-dimers (Fig. 1 A and B). Strand-swap dimers are the stronger cadherin adhesive conformation and are formed by the exchange of conserved tryptophan (Trp) residues between the outermost domains of opposing Ecads (2–4). In contrast, X-dimers, which are formed by extensive surface interactions between opposing Ecads, are a weaker adhesive structure and serve as an intermediate during the formation and rupture of strand-swap dimers (5–7). Using cell-free, single-molecule experiments we previously showed that X-dimers and strand-swap dimers can be distinguished based on their distinctly different response to mechanical force. When a strand-swap dimer is pulled, its lifetime decreases with increasing force, resulting in the formation of a slip bond (8, 9) (Fig. 1B). In contrast, an X-dimer responds to pulling force by forming a catch bond, where bond lifetime initially increases up to a threshold force and then subsequently decreases (8, 10) (Fig. 1B). It has also been shown that wild-type Ecad ectodomains in solution can interconvert between X-dimer and strand-swap dimer conformations (9, 11). However, the biophysical mechanisms by which Ecad conformations (and adhesion) are regulated on the cell surface are unknown.Open in a separate windowFig. 1.Overview of experiment. (A) The extracellular region of Ecad from opposing cells mediates adhesion. The cytoplasmic region of Ecad associates either directly or indirectly with p120 catenin, β-catenin, α-catenin, vinculin, and F-actin. (B) Strand-swap dimers form slip bonds (blue) and X-dimers form catch bonds (red). Ecads interconvert between these two dimer conformations. Structures were generated from the crystal structure of mouse Ecad (PDB ID code 3Q2V); the X-dimer was formed by alignment to an X-dimer crystal structure (PDB ID code 3LNH). (C) Graphics showing the cell lines used in experiments and Western blot analysis of corresponding cell lysates.The cytoplasmic region of Ecad associates with the catenin family of proteins, namely, p120-catenin, β-catenin, and α-catenin. The Ecad–catenin complex, in turn, links to filamentous actin (F-actin) either by the direct binding of α-catenin and F-actin or by the indirect association of α-catenin and F-actin via vinculin (12) (Fig. 1A). Adhesive forces transmitted across intercellular junctions by Ecad induce conformational changes in α-catenin (13, 14), strengthen F-actin binding (15), and recruit vinculin to the sites of force application (16, 17). However, vinculin and α-catenin do not merely serve as passive cytoskeletal linkers; they also dynamically modulate cytoskeletal rearrangement and recruit myosin to cell–cell junctions (13, 18–20). Studies show that α-catenin and vinculin play important roles in strengthening and stabilizing Ecad adhesion: bead-twisting experiments show force-induced stiffening of Ecad-based junctions and cell doublet stretching experiments demonstrate reinforcement of cell–cell adhesion in vinculin- and α-catenin–dependent manners (18, 19, 21).Currently, actin anchorage and cytoskeletal remodeling are assumed to be the exclusive mechanisms by which α-catenin and vinculin strengthen Ecad adhesion (22–24). Here, we directly map the allosteric effects of cytoplasmic proteins on Ecad ectodomain conformation and demonstrate, at the single-molecule level, that vinculin association with the Ecad cytoplasmic region switches X-dimers to strand-swap dimers. We show that cytoskeletal tension, due to vinculin-mediated recruitment of myosin II, regulates Ecad ectodomain structure and adhesion. Finally, we demonstrate that only ∼50% of Ecads are linked to the underlying cytoskeleton and that while about 70% of Ecads form strand-swap dimers the remaining form X-dimers, which provides cells with two Ecad pools with different adhesive properties.     

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.     

Coevolution can reverse predator–prey cycles

A hallmark of Lotka–Volterra models, and other ecological models of predator–prey interactions, is that in predator–prey cycles, peaks in prey abundance precede peaks in predator abundance. Such models typically assume that species life history traits are fixed over ecologically relevant time scales. However, the coevolution of predator and prey traits has been shown to alter the community dynamics of natural systems, leading to novel dynamics including antiphase and cryptic cycles. Here, using an eco-coevolutionary model, we show that predator–prey coevolution can also drive population cycles where the opposite of canonical Lotka–Volterra oscillations occurs: predator peaks precede prey peaks. These reversed cycles arise when selection favors extreme phenotypes, predator offense is costly, and prey defense is effective against low-offense predators. We present multiple datasets from phage–cholera, mink–muskrat, and gyrfalcon–rock ptarmigan systems that exhibit reversed-peak ordering. Our results suggest that such cycles are a potential signature of predator–prey coevolution and reveal unique ways in which predator–prey coevolution can shape, and possibly reverse, community dynamics.Population cycles, e.g., predator–prey cycles, and their ecological drivers have been of interest for the last 90 y (1–4). Classical models of predator–prey systems, developed first by Lotka (5) and Volterra (6), share a common prediction: Prey oscillations precede predator oscillations by up to a quarter of the cycle period (7). When plotted in the predator–prey phase plane, these cycles have a counterclockwise orientation (4). These cycles are driven by density-dependent interactions between the populations. When predators are scarce, prey increase in abundance. As their food source increases, predators increase in abundance. When the predators reach sufficiently high densities, the prey population is driven down to low numbers. With a scarcity of food, the predator population crashes and the cycle repeats.While many cycles, like the classic lynx–hare cycles (Fig. 1A) (3), exhibit the above characteristics, predator–prey cycles with different characteristics have also been observed. For example, antiphase cycles where predator oscillations lag behind prey oscillations by half of the cycle period (Fig. 1B) (8) and cryptic cycles where the predator population oscillates while the prey population remains effectively constant (Fig. 1C) (9) have been observed in experimental systems. This diversity of cycle types motivates the question, “Why do cycle characteristics differ across systems?”Open in a separate windowFig. 1.Examples of different kinds of predator–prey cycles. (A) Counterclockwise lynx–hare cycles (3). (B) Antiphase rotifer–algal cycles (8). (C) Cryptic phage-bacteria cycles (9). In all time series, red and blue correspond to predator and prey, respectively. See SI Text, section C for data sources.In Lotka–Volterra and other ecological models, predator and prey life history traits are assumed to be fixed. However, empirical studies across taxa have shown that prey (9–16) and predators (17–20) can evolve over ecological time scales. That is, changes in allele frequencies (and associated phenotypes) can occur at the same rate as changes in population densities or spatial distributions and alter the ecological processes driving the changes in population densities or distributions; this phenomenon has been termed “eco-evolutionary dynamics” (21, 22). Furthermore, predator–prey coevolution is important for driving community composition and dynamics (16, 19, 20, 23–26). This body of work suggests that the interaction between ecological and evolution processes has the potential to alter the ecological dynamics of communities.Experimental (8, 9, 13, 14) and theoretical studies (13, 27, 28) have shown that prey or predator evolution alone can alter the characteristics of predator–prey cycles and drive antiphase (Fig. 1B) and cryptic (Fig. 1C) cycles. Additional theoretical work has shown that predator–prey coevolution can also drive antiphase and cryptic cycles (29). Thus, evolution in one or both species is one mechanism through which antiphase or cryptic predator–prey cycles can arise. However, it is unclear if coevolution can drive additional kinds of cycles with characteristics different from those in Fig. 1.The main contribution of this study is to show that predator–prey coevolution can drive unique cycles where peaks in predator abundance precede peaks in prey abundance, the opposite of what is predicted by classical ecological models. We refer to these reversed cycles as “clockwise cycles.” The theoretical and empirical finding of clockwise cycles represents an example of how evolution over ecological time scales can alter community-level dynamics.     

Diffusion of a disordered protein on its folded ligand

Intrinsically disordered proteins often form dynamic complexes with their ligands. Yet, the speed and amplitude of these motions are hidden in classical binding kinetics. Here, we directly measure the dynamics in an exceptionally mobile, high-affinity complex. We show that the disordered tail of the cell adhesion protein E-cadherin dynamically samples a large surface area of the protooncogene β-catenin. Single-molecule experiments and molecular simulations resolve these motions with high resolution in space and time. Contacts break and form within hundreds of microseconds without a dissociation of the complex. The energy landscape of this complex is rugged with many small barriers (3 to 4 k_BT) and reconciles specificity, high affinity, and extreme disorder. A few persistent contacts provide specificity, whereas unspecific interactions boost affinity.

Specific molecular interactions orchestrate a multitude of simultaneous cellular processes. The discovery of intrinsically disordered proteins (IDPs) (1, 2) has substantially aided our understanding of such interactions. More than two decades of research revealed a plethora of functions and mechanisms (2–6) that complemented the prevalent structure-based view on protein interactions. Even the idea that IDPs always ought to fold upon binding has largely been dismantled by recent discoveries of high-affine–disordered complexes (7, 8). Classical shape complementary is indeed superfluous in the complex between prothymosin-α and histone H1, in which charge complementary is the main driving force for binding (7). However, complexes between IDPs and folded proteins can also be highly dynamic [e.g., Sic1 and Cdc4 (9), the Na⁺/H⁺ exchanger tail and ERK2 (10), nucleoporin tails, and nuclear transport receptors (11)]. Yet timescales of motions and their spatial amplitudes are often elusive, such that it is unclear how precisely the surfaces of folded proteins alter the dynamics of bound IDPs. Answering this question is a key step in understanding how specificity, affinity, and flexibility can be simultaneously realized in such complexes.To address this question, we focused on the dynamics of the cell adhesion complex between E-cadherin (E-cad) and β-catenin (β-cat), which is involved in growth pathologies and cancer (12). E-cad is a transmembrane protein that mediates cell–cell adhesions by linking actin filaments of adjacent epithelial cells (Fig. 1A). Previous NMR results showed that the cytoplasmic tail of E-cad is intrinsically disordered (13). E-cad binds β-cat, which establishes a connection to the actin-associated protein α-catenin (14–16). β-cat, on the other hand, is a multifunctional repeat protein (17–20) that mediates cadherin-based cell adhesions (21) and governs cell fate decisions during embryogenesis (22). It contains three domains: an N-terminal domain (130 amino acids [aa]), a central repeat domain (550 aa), and a C-terminal domain (100 aa). Whereas the N- and C-terminal domains of β-cat are in large parts unstructured (17), with little effect on the affinity of the E-cad/β-cat complex (23), the 12 repeats of the central domain arrange in a superhelix (24). The X-ray structure showed that the E-cad wraps around this central domain of β-cat (24) (Fig. 1B). However, not only is half of the electron density of E-cad missing, the X-ray unit cell also comprises two structures with different resolved parts of E-cad (Fig. 1B). In fact, only 45% of all resolved E-cad residues are found in both structures (Fig. 1C). Although this ambiguity together with the large portion of missing residues (25) suggests that E-cad is highly dynamic in the complex with β-cat, the timescales and amplitudes of these dynamics are unknown.Open in a separate windowFig. 1.Complex between the cytoplasmic tail of E-cad and β-cat. (A) Schematics of cell–cell junctions mediated by E-cad and β-cat. (B) The two X-ray structures of the complex between the tail of E-cad (red) and the central repeat domain of β-cat (white) resolve different parts of E-cad (Protein Data Bank: 1i7x), indicating the flexibility of E-cad in the complex. (Bottom) Cartoon representation of the resolved E-cad parts. (C) Scheme showing the resolved parts of E-cad (red).Here, we integrated single-molecule Förster resonance energy transfer (smFRET) experiments with molecular simulations to directly measure the dynamics of E-cad on β-cat with high spatial and temporal resolution. In our bottom-up strategy, we first probed intramolecular interactions within E-cad using smFRET to parameterize a coarse-grained (CG) model. In a second step, we monitored E-cad on β-cat, integrated this information into the CG model, and obtained a dynamic picture of the complex. We found that all segments of E-cad diffuse on the surface of β-cat at submillisecond timescales and obtained a residue-resolved understanding of these motions: A small number of persistent interactions provide specificity, whereas many weak multivalent contacts boost affinity, which confirms the idea that regulatory enzymes access their recognition motifs on E-cad and β-cat without requiring the complex to dissociate (24).     

Tuning a riboswitch response through structural extension of a pseudoknot

Structural and dynamic features of RNA folding landscapes represent critical aspects of RNA function in the cell and are particularly central to riboswitch-mediated control of gene expression. Here, using single-molecule fluorescence energy transfer imaging, we explore the folding dynamics of the preQ₁ class II riboswitch, an upstream mRNA element that regulates downstream encoded modification enzymes of queuosine biosynthesis. For reasons that are not presently understood, the classical pseudoknot fold of this system harbors an extra stem–loop structure within its 3′-terminal region immediately upstream of the Shine–Dalgarno sequence that contributes to formation of the ligand-bound state. By imaging ligand-dependent preQ₁ riboswitch folding from multiple structural perspectives, we reveal that the extra stem–loop strongly influences pseudoknot dynamics in a manner that decreases its propensity to spontaneously fold and increases its responsiveness to ligand binding. We conclude that the extra stem–loop sensitizes this RNA to broaden the dynamic range of the ON/OFF regulatory switch.A variety of small metabolites have been found to regulate gene expression in bacteria, fungi, and plants via direct interactions with distinct mRNA folds (1–4). In this form of regulation, the target mRNA typically undergoes a structural change in response to metabolite binding (5–9). These mRNA elements have thus been termed “riboswitches” and generally include both a metabolite-sensitive aptamer subdomain and an expression platform. For riboswitches that regulate the process of translation, the expression platform minimally consists of a ribosomal recognition site [Shine–Dalgarno (SD)]. In the simplest form, the SD sequence overlaps with the metabolite-sensitive aptamer domain at its downstream end. Representative examples include the S-adenosylmethionine class II (SAM-II) (10) and the S-adenosylhomocysteine (SAH) riboswitches (11, 12), as well as prequeuosine class I (preQ₁-I) and II (preQ₁-II) riboswitches (13, 14). The secondary structures of these four short RNA families contain a pseudoknot fold that is central to their gene regulation capacity. Although the SAM-II and preQ₁-I riboswitches fold into classical pseudoknots (15, 16), the conformations of the SAH (17) and preQ₁-II counterparts are more complex and include a structural extension that contributes to the pseudoknot architecture (14). Importantly, the impact and evolutionary significance of these “extra” stem–loop elements on the function of the SAH and preQ₁-II riboswitches remain unclear.PreQ₁ riboswitches interact with the bacterial metabolite 7-aminomethyl-7-deazaguanine (preQ₁), a precursor molecule in the biosynthetic pathway of queuosine, a modified base encountered at the wobble position of some transfer RNAs (14). The general biological significance of studying the preQ₁-II system stems from the fact that this gene-regulatory element is found almost exclusively in the Streptococcaceae bacterial family. Moreover, the preQ₁ metabolite is not generated in humans and has to be acquired from the environment (14). Correspondingly, the preQ₁-II riboswitch represents a putative target for antibiotic intervention. Although preQ₁ class I (preQ₁-I) riboswitches have been extensively investigated (18–28), preQ₁ class II (preQ₁-II) riboswitches have been largely overlooked despite the fact that a different mode of ligand binding has been postulated (14).The consensus sequence and the secondary structure model for the preQ₁-II motif (COG4708 RNA) (Fig. 1A) comprise ∼80–100 nt (14). The minimal Streptococcus pneumoniae R6 aptamer domain sequence binds preQ₁ with submicromolar affinity and consists of an RNA segment forming two stem–loops, P2 and P4, and a pseudoknot P3 (Fig. 1B). In-line probing studies suggest that the putative SD box (AGGAGA; Fig. 1) is sequestered by pseudoknot formation, which results in translational-dependent gene regulation of the downstream gene (14).Open in a separate windowFig. 1.PreQ₁ class II riboswitch. (A) Chemical structure of 7-aminomethyl-7-deazaguanosine (preQ₁); consensus sequence and secondary structure model for the COG4708 RNA motif (adapted from reference 14). Nucleoside presence and identity as indicated. (B) S. pneumoniae R6 preQ₁-II RNA aptamer investigated in this study. (C) Schematics of an H-type pseudoknot with generally used nomenclature for comparison.Here, we investigated folding and ligand recognition of the S. pneumoniae R6 preQ₁-II riboswitch, using complementary chemical, biochemical, and biophysical methods including selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE), mutational analysis experiments, 2-aminopurine fluorescence, and single-molecule fluorescence resonance energy transfer (smFRET) imaging. In so doing, we explored the structural and functional impact of the additional stem–loop element in the context of its otherwise “classical” H-type pseudoknot fold (29–32) (Fig. 1C). Our results reveal that the unique 3′-stem–loop element in the preQ₁-II riboswitch contributes to the process of SD sequestration, and thus the regulation of gene expression, by modulating both its intrinsic dynamics and its responsiveness to ligand binding.     

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.     

Evidence that dimethyl sulfide facilitates a tritrophic mutualism between marine primary producers and top predators

Tritrophic mutualistic interactions have been best studied in plant–insect systems. During these interactions, plants release volatiles in response to herbivore damage, which, in turn, facilitates predation on primary consumers or benefits the primary producer by providing nutrients. Here we explore a similar interaction in the Southern Ocean food web, where soluble iron limits primary productivity. Dimethyl sulfide has been studied in the context of global climate regulation and is an established foraging cue for marine top predators. We present evidence that procellariiform seabird species that use dimethyl sulfide as a foraging cue selectively forage on phytoplankton grazers. Their contribution of beneficial iron recycled to marine phytoplankton via excretion suggests a chemically mediated link between marine top predators and oceanic primary production.Many plant species interact with carnivores to gain protection from herbivory. Such mutualistic tritrophic interactions have been studied extensively in plant–insect systems, and are frequently mediated by plant volatiles released in response to insect feeding (1). One example that has received detailed study is the interaction between the phytophagous two-spotted spider mite Tetranychus urticae, the lima bean plant Phaseolus lunatus, and the predatory mite Phytoseiulus persimilis (2, 3). In this model system, grazing by the herbivorous spider mite has been demonstrated to elicit a cascade of biochemical reactions within the afflicted plants, stimulating the release of a suite of volatile terpenoids such as (E)-4,8-dimethyl-l,3,7-nonatriene, (E)-β-ocimene, and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (3). These volatiles attract olfactory-searching P. persimilis that prey upon herbivorous spider mites.The possibility of tritrophic mutualisms involving plant volatiles has received considerable attention in terrestrial communities (2–5); however, similar interactions have rarely been suggested for marine systems (6). Dimethyl sulfide (DMS) and its precursor dimethylsulfoniopropionate (DMSP) are well-established infochemicals in the marine environment, and as such are good candidate molecules for mediating tritrophic interactions between phytoplankton and carnivores (7–10). DMS arises as a catabolic breakdown product of DMSP, and has been studied extensively for its putative role as a global climate regulator (11). DMSP is produced by marine algae, where it has been proposed to function as an osmolyte (12) and a cryoprotectant (13). When algal cells lyse, due to biotic or abiotic stress, one of the fates of DMSP is catabolism by the enzyme DMSP lyase to DMS and acrylic acid (14–16). This process may also occur during autocatalytic cell death (17). It has been proposed that acrylic acid is the biologically salient product of this reaction due to its antimicrobial properties (18).DMS production has also been shown to increase during zooplankton grazing (14). It has been previously proposed that this phytoplankton-derived odorant is an important infochemical for marine apex predators including whale sharks (19), harbor seals (20), penguins (21–23), and procellariiform (tube-nosed) seabirds (24). Procellariiform seabirds have been the best-studied in this regard, and many species have been shown to detect and respond to biogenic concentrations of DMS in foraging contexts (24, 25). Members of this order share highly pelagic lifestyles and are central-place foragers associated with land only during incubation and chick rearing (26). Procellariiformes routinely range thousands of kilometers to forage (27) and have large olfactory bulbs compared with other avian clades (28), and some species have been shown to track their prey using their sense of smell (29). Some procellariiform species are attracted to DMS, whereas others are not (24, 30) (Fig. 1); however, the relationship between DMS behavioral sensitivity and the consumption of herbivorous crustacea has not previously been shown.Open in a separate windowFig. 1.Phylogenetic relationships between the species included in the meta-analysis, mapped with DMS responsiveness. DMS responsiveness is thought to be ancestral in this lineage (30). Certain species in the outgroup, sphenisciformes (penguins), have also been shown to be responsive to DMS (21–23).The Southern Ocean is the largest marine ecosystem in the world, with the polar front forming a distinct northern boundary to this ecoregion (31). Our rationale for using this system is twofold: (i) A majority of the world’s procellariiform species breed or forage in the Southern Ocean (32), and (ii) food web relationships are relatively simple by comparison with other marine systems. Phaeocystis antarctica and several siliceous diatom species are the dominant DMS-producing phytoplankton species in this ecosystem, and Antarctic krill (Euphasia superba) and other small crustaceans (copepods, decapods, amphipods, etc.) are their major consumers.Here we take advantage of a 50-y dietary database of Southern Ocean seabirds (33) to explore whether DMS mediates a mutualistic tritrophic interaction in the Southern Ocean pelagic ecosystem. If this is the case, then we predict that (i) carnivorous species, such as seabirds, that are attracted to this infochemical should specialize on primary consumers, such as crustaceans, and (ii) primary producers should gain some benefit from this interaction.     

Early guenon from the late Miocene Baynunah Formation,Abu Dhabi,with implications for cercopithecoid biogeography and evolution

A newly discovered fossil monkey (AUH 1321) from the Baynunah Formation, Emirate of Abu Dhabi, United Arab Emirates, is important in a number of distinct ways. At ∼6.5–8.0 Ma, it represents the earliest known member of the primate subfamily Cercopithecinae found outside of Africa, and it may also be the earliest cercopithecine in the fossil record. In addition, the fossil appears to represent the earliest member of the cercopithecine tribe Cercopithecini (guenons) to be found anywhere, adding between 2 and 3.5 million y (∼50–70%) to the previous first-appearance datum of the crown guenon clade. It is the only guenon—fossil or extant—known outside the continent of Africa, and it is only the second fossil monkey specimen so far found in the whole of Arabia. This discovery suggests that identifiable crown guenons extend back into the Miocene epoch, thereby refuting hypotheses that they are a recent radiation first appearing in the Pliocene or Pleistocene. Finally, the new monkey is a member of a unique fauna that had dispersed from Africa and southern Asia into Arabia by this time, suggesting that the Arabian Peninsula was a potential filter for cross-continental faunal exchange. Thus, the presence of early cercopithecines on the Arabian Peninsula during the late Miocene reinforces the probability of a cercopithecoid dispersal route out of Africa through southwest Asia before Messinian dispersal routes over the Mediterranean Basin or Straits of Gibraltar.Cercopithecine monkeys (Order Primates, Superfamily Cercopithecoidea, Family Cercopithecidae, Subfamily Cercopithecinae), also known as cheek-pouch monkeys, are the most speciose and widely distributed group of living Old World primates. Recent molecular estimates date the divergence of Cercopithecinae from Colobinae (leaf-eating monkeys) to between 17.6 Ma (range 21.5–13.9 Ma) and 14.5 Ma (range 16.2–12.8 Ma) and the origin of crown Cercopithecinae to around 11.5 Ma (range 13.9–9.2 Ma) (1, 2). However, the earliest known fossil cercopithecines only appear much later, around 7.4 Ma in the Turkana Basin of East Africa (3, 4).Cercopithecine monkeys are divided into two tribes: Cercopithecini, including African guenons (Allenopithecus, Miopithecus, Chlorocebus, Erythrocebus, Allochrocebus, Cercopithecus), and Papionini, which includes African and Eurasian macaques (Macaca) as well as African papionins (Papio, Lophocebus, Rungwecebus, Theropithecus, Mandrillus, Cercocebus). Of the living cercopithecines, only two genera are known outside of the African continent, both of them papionins: Papio (found on the Arabian Peninsula) and Macaca (found throughout Southern and Southeast Asia, and introduced in Gibraltar). The earliest fossil cercopithecines known outside of Africa are attributed to the genus Macaca and appear to be latest Miocene or early Pliocene in age (∼6.0–5.0 Ma) (Fig. 1) (5–9). Until now, no guenons, extant or extinct, have ever been known outside of the African continent.Open in a separate windowFig. 1.Hypothesized cercopithecoid dispersal routes out of Africa in relation to the known late Miocene fossil record. The oldest cercopithecine, Parapapio lothagamensis (light blue circles), is known from ∼7.4–6.1 Ma in the Turkana Basin and Tugen Hills, Kenya (3, 4, 41). An unnamed fossil papionin (purple circle) is known from the late Miocene of Ongoliba, Congo (5, 57). Macaca spp. (dark blue circles) are located throughout North Africa at sites ranging in age from ∼6.5–5.5 Ma (5, 8, 58, 59), and Macaca spp. first appear in Europe ∼6.0–5.3 Ma and in China in the early Pliocene (5–9). The oldest colobine outside of Africa, Mesopithecus (green circles), is known from a number of late Miocene sites securely dated between ∼8.5 and 5.3 Ma in Greece, Macedonia, Italy, Ukraine, Iran, Afghanistan, possibly Pakistan, and China (46–48). Three dispersal routes for cercopithecoids can be hypothesized: route 1 imagines a dispersal event over the Straits of Gibraltar or Mediterranean Basin into Europe and Asia; route 2 postulates a dispersal event through the Arabian Sinai Peninsula; and route 3 suggests a migration over the Arabian Straits of Bab el Mandeb. The discovery of AUH 1321 and AUH 35 in Abu Dhabi at >6.5–8 Ma (red circle), contemporaneous with the first appearance of Mesopithecus sp. in Eurasia and ∼1–2 million y earlier than the appearance of Macaca spp. in Eurasia, suggests scenarios 2 and 3 were possible before scenario 1. None of these scenarios is mutually exclusive and may have occurred in combination or succession.Three possible routes can be reasonably hypothesized for cercopithecine (and cercopithecoid) dispersal out of Africa and into Europe and Asia during the late Miocene: (i) over the Mediterranean Basin or Straits of Gibraltar to the north/northwest, (ii) across the Arabian Sinai Peninsula to the northeast, or (iii) across the Arabian Straits of Bab el Mandeb to the east (Fig. 1). Fossil Macaca specimens from the terminal Miocene of Spain and Italy have been suggested to provide evidence for the use of a route across the Mediterranean Basin or the Straits of Gibraltar via an ephemeral land bridge either immediately before—or perhaps associated with—the drop in Mediterranean sea levels during the Messinian (∼6.0–5.3 Ma) (8–19). Paleontological evidence for an Arabian route has been lacking, but paleogeographic and paleoenvironmental work on circum-Arabia suggests that the region did not present a persistent ecological barrier to some amount of intercontinental exchange during the late Miocene (20). In fact, an established land connection through Sinai was probably present during this time period, and oceanic spreading is not estimated to have begun in the southern Red Sea until around 5 Ma, with progressive development of open marine conditions throughout the Pliocene. Thus, before 6.5 Ma, a southern route in the region of the Straits of Bab el Mandeb was also possible (Fig. 1) (21).Although Arabia is a large area of the earth, fossil monkeys have so far been represented by only a single specimen, an isolated male lower canine (AUH 35), discovered in 1989 by A.H. and Peter Whybrow in the late Miocene Baynunah Formation, Abu Dhabi, United Arab Emirates (22–25). The specimen came from Jebel Dhanna, site JDH-3 (JD-3 in refs. 24 and 26) (Fig. 2), a locality now lost to industrial development. Because male cercopithecid lower canines are not metrically identifiable beyond the Family level of classification (23), AUH 35 was described as a cercopithecid with indeterminate affinities. Here we report the discovery of a second monkey specimen from the Baynunah Formation in Abu Dhabi (AUH 1321), found almost 20 y after the first. AUH 1321 clearly represents a cercopithecine and, because it is dated to between 6.5 and 8.0 Ma, it is the oldest cercopithecine yet known outside of Africa and possibly the oldest cercopithecine in the fossil record. Thus, the discovery of AUH 1321 provides the earliest paleontological evidence of cercopithecine dispersal out of continental Africa and possibly hints at an Arabian cercopithecoid dispersal route into Eurasia during the Late Miocene (Fig. 1). Furthermore, we believe AUH 1321 can be attributed to the Cercopithecini (guenons) and, therefore, it represents the only record of this tribe, living or fossil, yet known outside of Africa.Open in a separate windowFig. 2.Map illustrating the location of the two fossil sites in the Baynunah Formation that have produced fossil monkeys. Top Right Inset shows the location of the SHU 2–2 excavation (kite aerial photography by Nathan Craig).     

Mechanical force effect on the two-state equilibrium of the hyaluronan-binding domain of CD44 in cell rolling

CD44 is the receptor for hyaluronan (HA) and mediates cell rolling under fluid shear stress. The HA-binding domain (HABD) of CD44 interconverts between a low-affinity, ordered (O) state and a high-affinity, partially disordered (PD) state, by the conformational change of the C-terminal region, which is connected to the plasma membrane. To examine the role of tensile force on CD44-mediated rolling, we used a cell-free rolling system, in which recombinant HABDs were attached to beads through a C-terminal or N-terminal tag. We found that the rolling behavior was stabilized only at high shear stress, when the HABD was attached through the C-terminal tag. In contrast, no difference was observed for the beads coated with HABD mutants that constitutively adopt either the O state or the PD state. Steered molecular dynamics simulations suggested that the force from the C terminus disrupts the interaction between the C-terminal region and the core of the domain, thus providing structural insights into how the mechanical force triggers the allosteric O-to-PD transition. Based on these results, we propose that the force applied from the C terminus enhances the HABD–HA interactions by inducing the conformational change to the high-affinity PD transition more rapidly, thereby enabling CD44 to mediate lymphocyte trafficking and hematopoietic progenitor cell homing under high-shear conditions.Leukocyte extravasation from blood to sites of infection and inflammation or to specific organs is achieved by a sequential adhesion cascade: (i) rolling, (ii) chemokine-induced activation, (iii) firm adhesion, and (iv) transcellular migration. Rolling is mediated by specialized cell surface adhesion molecules, such as selectins, CD44, and specific types of integrins (1, 2).Under conditions of hydrodynamic flow, receptor–ligand bonds are subjected to tensile mechanical force, which disrupts the receptor–ligand bond (Fig. 1A). In general, the lifetime of the receptor–ligand bond exponentially decreases with an increase of the mechanical force (3). However, there is growing evidence demonstrating that the lifetimes of some receptor–ligand bonds increase when moderate levels of force are applied (4–9). However, the underlying mechanism of this phenomenon is still elusive and in some cases controversial. For example, integrin and bacterial adhesin FimH-mediated adhesion have been explained by an “allosteric model,” in which mechanical force induces allosteric changes of the receptor, resulting in the stabilization of the high-affinity state (10, 11). Although selectin-mediated adhesion has been explained by the allosteric model (12), a different “sliding-rebinding model” was also reported (13). This model proposes that force tilts the binding interface to make it parallel to the direction of force, allowing the selectin ligand to slide on the selectin and to form new contacts. The sliding-rebinding model has also been used to explain the force-induced activation of von Willebrand factor-mediated adhesion and actin depolymerization (6, 8).Fig. 1.The effect of the tensile force on the two-state conformations of CD44 HABD. (A) Illustration of the tensile force applied between the receptor on the cells and the immobilized ligand under the fluid shear force. (B) The crystal structure of CD44 HABD ...CD44 is a transmembrane receptor for hyaluronan (HA) (14). CD44–HA interactions are involved in various physiological and pathological processes mediated over a wide range of hydrodynamic forces, including T-lymphocyte trafficking on the endothelium (15, 16), hematopoietic progenitor cell homing into bone marrow niches (17), and the progression of atherosclerosis (18). The HA-binding domain (HABD) of CD44 adopts two distinctive conformations representing the low- and high-affinity states for HA (19–21). HABD is composed of a conserved Link module and the N- and C-terminal extension segment (22). In the ordered (O) state, the C-terminal segment is well folded (Fig. 1B) (19), whereas it becomes disordered in the partially disordered (PD) state upon ligand binding (Fig. 1C) (20). In addition, solution NMR analyses demonstrated that HABD exists in an equilibrium between the O and PD states in both the HA-unbound and HA-bound states, with a transition rate of ∼500 ms, and that HA binding induces an equilibrium shift toward the PD state (21) (Fig. 1D). The Y161A mutant, which constitutively adopts the PD state, exhibits a higher affinity than wild-type HABD, indicating that the O and PD states represent the low- and high-affinity states for HA, respectively (21) (Fig. 1E). Cells expressing the Y161A mutant exhibited firm adhesion and impaired rolling on an HA substrate, suggesting that the two-state conformations are essential for the CD44-mediated rolling under flow conditions (21).Despite the importance of the mechanical force in rolling, the means by which it affects the CD44-mediated rolling remain poorly characterized. Recently, it was reported that the rolling of CD44-expressing cells is enhanced at the higher shear stress (23), raising the possibility that CD44 possesses some mechanochemical specializations to resist higher tensile force. Considering the fact that the C terminus of CD44 HABD is connected to the plasma membrane, the force applied from the C terminus of HABD would induce the allosteric transition from the O to the PD state, thereby providing the resistance to the applied force. On the other hand, our previous NMR studies demonstrated that more than 90% of HABD adopts the PD state in the HA-bound state (21), indicating that the free energy of the PD state can be lowered upon HA binding, regardless of the presence or absence of the tensile force. Therefore, it is worthwhile to investigate whether the CD44–HA interaction is strengthened by the tensile force.To assess the effect of the tensile force on the CD44-mediated rolling, we established a cell-free rolling system using cell-sized beads, which are coated with recombinant HABDs. The effect of the tensile force can be investigated by comparing the rolling activity of the beads coated with the ligand-binding domain via the N-terminal or the C-terminal tag (Fig. 1G) (10). We compared the rolling behavior of the beads with N- or C-terminally attached HABD and found that the rolling behavior was stabilized only at higher shear stress, when HABD was attached to the beads via the C-terminal tag. Steered molecular dynamics (SMD) simulations suggested that the force from the C terminus induces the dissociation of the “mechanosensitive latch” in the C-terminal region, which triggers the conversion from the O to the PD state. Based on these results, we propose that the tensile force from the C terminus stabilizes the CD44–HA bond by inducing a rapid transition from the O to the PD state, thereby sustaining the CD44-mediated cell rolling under higher shear stress conditions.     

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.     

Studies on enmetazobactam clarify mechanisms of widely used β-lactamase inhibitors

β-Lactams are the most important class of antibacterials, but their use is increasingly compromised by resistance, most importantly via serine β-lactamase (SBL)-catalyzed hydrolysis. The scope of β-lactam antibacterial activity can be substantially extended by coadministration with a penicillin-derived SBL inhibitor (SBLi), i.e., the penam sulfones tazobactam and sulbactam, which are mechanism-based inhibitors working by acylation of the nucleophilic serine. The new SBLi enmetazobactam, an N-methylated tazobactam derivative, has recently completed clinical trials. Biophysical studies on the mechanism of SBL inhibition by enmetazobactam reveal that it inhibits representatives of all SBL classes without undergoing substantial scaffold fragmentation, a finding that contrasts with previous reports on SBL inhibition by tazobactam and sulbactam. We therefore reinvestigated the mechanisms of tazobactam and sulbactam using mass spectrometry under denaturing and nondenaturing conditions, X-ray crystallography, and NMR spectroscopy. The results imply that the reported extensive fragmentation of penam sulfone–derived acyl–enzyme complexes does not substantially contribute to SBL inhibition. In addition to observation of previously identified inhibitor-induced SBL modifications, the results reveal that prolonged reaction of penam sulfones with SBLs can induce dehydration of the nucleophilic serine to give a dehydroalanine residue that undergoes reaction to give a previously unobserved lysinoalanine cross-link. The results clarify the mechanisms of action of widely clinically used SBLi, reveal limitations on the interpretation of mass spectrometry studies concerning mechanisms of SBLi, and will inform the development of new SBLi working by reaction to form hydrolytically stable acyl–enzyme complexes.

β-Lactamases are a major mechanism of resistance to the clinically vital β-lactam antibiotics, with >2,000 different β-lactamases reported (1). β-Lactamases are grouped into classes A, C, and D, which employ a nucleophilic serine in catalysis (serine β-lactamases, SBLs), and class B, which employ metal ions in catalysis (2). Presently, SBLs are the most important β-lactamases from a clinical perspective. SBL inhibitors (SBLi) have been developed for use in combination with a β-lactam antibiotic, with tazobactam (3), sulbactam (4), and clavulanic acid (5) being the most widely used SBLi. These SBLi all contain a β-lactam ring which reacts with SBLs to produce an acyl–enzyme complex (AEC) intermediate, as is also the case for efficient SBL substrates (Fig. 1A). With efficient substrates the β-lactam–derived AEC is readily hydrolyzed. With SBLi the reaction bifurcates at the AEC stage; in addition to hydrolysis, reaction of the AEC via opening of the β-lactam fused five-membered ring occurs to give one or more relatively hydrolytically stable species (Figs. 1B and ​and2).2). The nature of these species is central to SBLi inhibition and has been studied by crystallography (6–11) and ultraviolet-visible (UV/Vis) (10, 12) and Raman (6, 7, 9, 12–15) spectroscopy, as well as different types of mass spectrometry (MS) (10, 16–22).Open in a separate windowFig. 1.Sulfone derivatives of penicillins are potent clinically used mechanism-based inhibitors of SBLs. (A) Outline mechanism for penicillin hydrolysis as catalyzed by SBLs; reaction proceeds via an AEC, which is efficiently hydrolyzed. (B) Sulfone derivatives of penicillins are SBLi that react to give one or more hydrolytically stable complex(es), the nature of which was the focus of our work.Open in a separate windowFig. 2.Pathways for reactions of penam sulfones with SBLs. Following initial acyl–enzyme 2 formation the main transient inactivation pathway occurs via thiazolidine ring opening to give species 3-5 which are relatively stable to hydrolysis. Fragmentation of 3-5 can occur in rare cases and is promoted by acid to give 6-8 or heat to give 11. In rare cases fragmentation of 2-5 can result in irreversible inactivation of the SBL to give 9 and 10. Efficient hydrolysis of the β-lactam occurs to give a β-amino acid product 12, which in solution fragments to give 13-16. Our results imply biologically relevant inhibition involves 3-5, or equivalent mass species.The structures of tazobactam and sulbactam are closely related to those of the penicillins; they differ by lack of a C-6 side chain, functionalization of the pro-S methyl group (in case of tazobactam), and by oxidation of the thiazolidine to a sulfone. These differences result in a loss of useful antibacterial activity but a gain of potent SBL inhibition. Although the presence of sulfur in drugs is common [e.g., sulfonamide antibiotics (23)] and there is growing interest in covalently acting drugs (24, 25), sulfones are rare in drugs and, as far as we are aware, sulbactam and tazobactam are the only clinically approved sulfone-containing drugs working by covalent reaction with their targets (26–28).Since the clinical introduction of the pioneering SBLi, β-lactamases have evolved and SBLi use is increasingly compromised by extended spectrum β-lactamases (ESBLs) and inhibitor-resistant SBLs (29). Efforts have been made to develop new SBLi, including those with and without a β-lactam. The latter include diazabicyclooctanes (30) and cyclic boronates (31, 32). However, β-lactam–containing SBLi remain of most clinical importance. Among SBLi in clinical development, enmetazobactam (formerly AAI-101; Fig. 1) is of particular interest because it is a “simple” N-methylated derivative of the triazole ring of tazobactam (33). In combination with cefepime, enmetazobactam is reported to manifest substantially better antimicrobial properties against class A ESBL-producing strains than the commonly used piperacillin/tazobactam combination (20, 33, 34).We report studies on the mechanism of SBL inhibition by enmetazobactam using denaturing and nondenaturing (native) MS methods, NMR spectroscopy, and crystallography. The results led us to reevaluate the mechanisms of SBL inhibition by the clinically important sulfone-containing SBLi, i.e., tazobactam and sulbactam, and reveal limitations on the interpretation of MS studies concerning SBL inhibition.     

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.     

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.