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.     

Neutralizing antibody titers against dengue virus correlate with protection from symptomatic infection in a longitudinal cohort

The four dengue virus serotypes (DENV1–4) are mosquito-borne flaviviruses that infect ∼390 million people annually; up to 100 million infections are symptomatic, and 500,000 cases progress to severe disease. Exposure to a heterologous DENV serotype, the specific infecting DENV strains, and the interval of time between infections, as well as age, ethnicity, genetic polymorphisms, and comorbidities of the host, are all risk factors for severe dengue. In contrast, neutralizing antibodies (NAbs) are thought to provide long-lived protection against symptomatic infection and severe dengue. The objective of dengue vaccines is to provide balanced protection against all DENV serotypes simultaneously. However, the association between homotypic and heterotypic NAb titers and protection against symptomatic infection remains poorly understood. Here, we demonstrate that the titer of preinfection cross-reactive NAbs correlates with reduced likelihood of symptomatic secondary infection in a longitudinal pediatric dengue cohort in Nicaragua. The protective effect of NAb titers on infection outcome remained significant when controlled for age, number of years between infections, and epidemic force, as well as with relaxed or more stringent criteria for defining inapparent DENV infections. Further, individuals with higher NAb titers immediately after primary infection had delayed symptomatic infections compared with those with lower titers. However, overall NAb titers increased modestly in magnitude and remained serotype cross-reactive in the years between infections, possibly due to reexposure. These findings establish that anti-DENV NAb titers correlate with reduced probability of symptomatic DENV infection and provide insights into longitudinal characteristics of antibody-mediated immunity to DENV in an endemic setting.Dengue virus (DENV) is a mosquito-borne flavivirus that infects up to 390 million individuals each year (1). Although most infections are inapparent, ∼25% of infections cause acute febrile illness, which progresses to severe disease in half a million individuals annually (2). DENV consists of four evolutionarily distinct, antigenically related DENV serotypes, DENV1–4, and neutralizing antibodies (NAbs) against the four serotypes are considered a critical component of the protective immune response (3, 4). Primary (1°) DENV infection induces a NAb response that is described as increasingly type-specific over time, providing long-term protection against the 1° infecting serotype, but only transient protection against other DENV serotypes (5, 6). Cross-serotype protection against symptomatic infection is observed for up to 2 years after 1° infection, after which point individuals are at increased risk of symptomatic infection and severe dengue upon subsequent heterologous infection (7–10). Over time, cross-serotype–reactive antibodies are thought to decay to subneutralizing levels, binding, but not neutralizing, DENV and contributing to enhanced replication during heterologous infection by facilitating virus entry into target cells expressing Fc receptors (11). However, after subsequent infection with a different serotype, the NAb response becomes broadly neutralizing and is thought to reduce incidence of severe disease (12).There has been limited success in establishing the relationship between the level of preinfection NAb titers to DENV and risk of disease upon subsequent DENV infection in endemic settings. In recent vaccine trials, symptomatic disease was observed in individuals with relatively high NAb titers, raising concerns that the current immunologic assays do not measure the NAbs critical for protection (13). In studies of infants, who receive IgG antibodies by transplacental transfer from DENV-immune mothers, infants with higher NAb titers at birth generally, although not always, experienced symptomatic disease later than those with lower titers (14–16). Recent studies in children and adults have made important advances in demonstrating an association between the quantity of cross-reactive preinfection NAb titers and reduced risk of symptomatic secondary (2°) infection, defined as two or more infections, but have not been conclusive: the association did not hold for all DENV serotypes (15, 17); exposure could not be proven for DENV-negative individuals (18); or the magnitude of preinfection NAb titers was not directly studied (12, 19). Thus, there is an urgent need to definitively establish whether NAb titers correlate with protection in endemic settings. Here, we estimated the relationship between preinfection NAb titers and probability of symptomatic infection and characterized determinants of long-term protection in children with multiple DENV infections in a pediatric dengue cohort study in Nicaragua.     

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.     

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).     

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.     

Identification of simple arylfluorosulfates as potent agents against resistant bacteria

Sulfur fluoride exchange (SuFEx), a next generation of click chemistry, opens an avenue for drug discovery. We report here the discovery and structure–activity relationship studies of a series of arylfluorosulfates, synthesized via SuFEx, as antibacterial agents. Arylfluorosulfates 3, 81, and 101 showed potency to overcome multidrug resistance and were not susceptible to the generation of resistance. They exhibited rapid bactericidal potency and selectively killed gram-positive bacterial strains. These compounds also exhibited the ability to disrupt established bacterial biofilm and kill persisters derived from biofilm. Furthermore, arylfluorosulfate 3 had a synergistic effect with streptomycin and gentamicin. In addition, their anti-MRSA potency was evaluated and determined by the Caenorhabditis elegans model.

Antibiotic resistance is a tremendous threat to global health. Some of the most concerning multidrug-resistant pathogen strains include methicillin- and vancomycin-resistant Staphylococcus aureus (MRSA and VRSA, respectively), vancomycin-resistant Enterococcus faecium (VRE), Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp (1–3). Antibiotic resistance is one of the biggest public health challenges and is a leading cause of death with at least 2.8 million infected cases and more than 35,000 deaths every year in the United States (4). Therefore, the development of novel antibacterial drugs with the ability to overcome drug-resistance is urgently needed. Furthermore, our general understanding of the role of our microbiomes (i.e., skin, oral, gut, etc.) in antibiotic resistance and responses is ever increasing (5–7). The next generation of antibacterial agents will require limited drug promiscuity to eliminate resistance and decrease unwanted off-target effects on our symbiotic commensal organisms and immunity (8, 9).Sulfur fluoride exchange (SuFEx) (10), a new generation of click chemistry, has found diverse applications to chemical synthesis (11–16), materials science (17–22), chemical biology (23–28), and drug discovery (29, 30). In our previous studies, we demonstrated that SuFEx modification is a highly reliable approach for the late-stage functionalization of drugs and drug-like molecules to generate new compounds with improved properties (31, 32). Later on, Ravindar et al. reported the synthesis of arylfluorosulfate analogs and screened them for antimicrobial activity (33).Here we report further screening studies on arylfluorosulfate derivatives (Ar-O-SO₂-F) in our laboratory and have found several simple molecules which are potent against methicillin- and vancomycin-resistant strains (Fig. 1 and SI Appendix, Figs. S1 and S2). Through structure and activity relationship (SAR) studies, we determined that the -OSO₂F moiety is essential for these compounds’ antibacterial activities. Not only are they capable of inhibiting bacterial biofilm formation, but they are also able to disrupt established bacterial biofilm and induce the killing of persister cells. Significantly, these arylfluorosulfates are effective against MRSA infection in a Caenorhabditis elegans-based infection model. Our findings reported here thus may serve as the foundation toward the development of arylfluorosulfate-based antibacterial agents.Open in a separate windowFig. 1.(A) Arylfluorosulfates were derived from phenols or phenol’s precursors. (B) The structures of antibacterial arylfluorosulfates 3, 81, and 101 and their MIC values against MRSA.     

Noncontact rotation,levitation, and acceleration of flowing liquid metal wires

This paper reports the noncontact manipulation of free-falling cylindrical streams of liquid metals into unique shapes, such as levitated loops and squares. Such cylindrical streams form in aqueous media by electrochemically lowering the interfacial tension. The electrochemical reactions require an electrical current that flows through the streams, making them susceptible to the Lorentz force. Consequently, varying the position and shape of a magnetic field relative to the stream controls these forces. Moreover, the movement of the metal stream relative to the magnetic field induces significant forces arising from Lenz’s law that cause the manipulated streams to levitate in unique shapes. The ability to control streams of liquid metals in a noncontact manner will enable strategies for shaping electronically conductive fluids for advanced manufacturing and dynamic electronic structures.

Noncontact methods of manufacturing and manipulation can minimize disrupting objects of interest. Objects can be manipulated in a noncontact manner by magnetic methods (levitation and tweezers) (1, 2), acoustic manipulation (3, 4), optical tweezers (5), and other techniques (6, 7). However, to date, free-flowing liquid streams have been particularly difficult to manipulate in a noncontact manner. Realizing highly controlled changes in directionality or complex shaping of liquids, especially without disrupting the cross-sectional shape of the stream, is a challenge. Here, we explore the noncontact manipulation of free-flowing streams of liquid metals (LMs). Gallium-based LMs (Galinstan, the eutectic alloy of gallium indium and tin used in this work) have recently received significant attention due to their promises of soft and stretchable metallic conductors, low melting points, and simultaneous fluidity and metallic properties at room temperature as well as low toxicity (8–15).LM alloys are seemingly unlikely candidates to form stable fluid streams due to their enormous surface tension and water-like viscosity, which favor the formation of droplets (Fig. 1A). However, electrochemical oxidation of the surface of the LM in basic solution lowers the effective tension of the LM to extremely low values (16, 17). This electrochemical manipulation of interfacial tension enables various fascinating phenomena, such as reversible deformation (18), patterning (19), heartbeat effects (20), “superfluid-like” penetration through porous media (21), and other electrochemical effects (22–29). Most importantly, the presence of oxide species on the LM also enables long, stable wire-like streams of metal to form as it exits a nozzle into the solution (17, 30) (Fig. 1B). Because of their cylindrical cross-section and metallic conductivity, we call these fluidic streams liquid metal wires (LMWs), which form narrow diameters (∼100 to 200 µm). Although normally LM is not responsive to magnetic fields, the current passing through the wire to drive the electrochemical reactions makes it susceptible to magnetic forces via the Lorentz force (Fig. 1C). The Lorentz force arises by applying a magnetic field normal to the direction of electrical current. The Lorentz force is normal to both the current and magnetic field, as described by the so-called "left-hand rule."Open in a separate windowFig. 1.Shaping free-flowing liquid metal wires by the Lorentz force and Lenz''s law: (A) drops form at 0 V and (B) a liquid metal wire at 1.5 V. (C) Current-carrying LMW rotated by the Lorentz force within a magnetic field in which N and S refer to the north and south poles of the magnet. (D) Schematic illustration of the experimental setup; a blue piece of paper covered one wall of the vessel to facilitate imaging. (E) Photographs showing the LMW path resulting from different positions of the magnet with the N pole outward. The dotted lines indicate the location and the shape of the magnet. (F) False-colored images of LM (white) showing four sequences of frames with a force diagram and motion analysis. The yellow dotted line denotes the periphery of the magnet.In this work, we control the displacement of free-falling LMWs at room temperature using the Lorentz force. Because LM is soft, it provides almost no resistance to manipulation via the Lorentz force and therefore, accelerates radially. The displacement of the LMWs relative to the magnet also induces a secondary force according to Lenz’s law (i.e., a drag force that opposes the motion at the periphery of the magnet). Thus, the combination effects of the Lorentz force and Lenz’s law drive the metal into shapes that mirror the circumference of the magnet while levitating the metal. As shown here, the behavior depends on the location of the magnet relative to the LMW. We demonstrate and characterize the unique ability to manipulate LM streams in a noncontact manner using only a relatively low applied voltage and a common magnet.     

Mutually opposing forces during locomotion can eliminate the tradeoff between maneuverability and stability

A surprising feature of animal locomotion is that organisms typically produce substantial forces in directions other than what is necessary to move the animal through its environment, such as perpendicular to, or counter to, the direction of travel. The effect of these forces has been difficult to observe because they are often mutually opposing and therefore cancel out. Indeed, it is likely that these forces do not contribute directly to movement but may serve an equally important role: to simplify and enhance the control of locomotion. To test this hypothesis, we examined a well-suited model system, the glass knifefish Eigenmannia virescens, which produces mutually opposing forces during a hovering behavior that is analogous to a hummingbird feeding from a moving flower. Our results and analyses, which include kinematic data from the fish, a mathematical model of its swimming dynamics, and experiments with a biomimetic robot, demonstrate that the production and differential control of mutually opposing forces is a strategy that generates passive stabilization while simultaneously enhancing maneuverability. Mutually opposing forces during locomotion are widespread across animal taxa, and these results indicate that such forces can eliminate the tradeoff between stability and maneuverability, thereby simplifying neural control.Animals routinely produce muscle commands that result in “antagonistic” (mutually opposing) forces during locomotion that either cancel out at each instant of time or average to zero over each gait cycle (1–5). This may seem surprising because these antagonistic forces do not contribute to the cycle-averaged movement of the center of mass of the animal. Such antagonistic forces are not only present during forward locomotion but in hovering for animals such as hummingbirds, hawkmoths, and electric fish; these animals produce large antagonistic forces and exhibit extraordinary maneuverability during station keeping (6–9). In this study, we demonstrate that active generation and differential control of such antagonistic forces can eliminate the tradeoff between stability and maneuverability during locomotion.Stability is generally defined as the resistance to, and recovery from, disturbances to an intended trajectory (10). Although maneuverability can be defined in several ways (11, 12), it is perhaps most generally recognized as the relative amplitude of the control signal required to change movement direction (13). That is, if a small change in the control amplitude effects a rapid change in direction, the system would be considered highly maneuverable. The potential for a tradeoff between the resistance to changes in direction and the ability to change direction appears self-evident (5, 10, 13, 14), and this tradeoff is indeed considered a fundamental challenge for the engineering design of airborne, submarine, and terrestrial vehicles (14–17). Many swimming, flying, and running animals, however, appear to use locomotor strategies that are extremely stable and yet facilitate the control of extraordinary maneuvers (4, 10, 18, 19).To investigate the relationship between antagonistic forces and locomotor control, we studied the glass knifefish, Eigenmannia virescens, that hovers and rapidly changes direction while producing opposing forces using a single elongated fin (Fig. 1A). Glass knifefish, like other knifefish, generate thrust force primarily through undulatory motions of an elongated anal fin (20–22). The ribbon fin consists of 217 ± 27 downward-pointing rays (table 4 in ref. 23; all statistics are quoted as mean ± SD unless otherwise noted), with each ray independently controlled by a set of muscles. These rays are oscillated in a plane transverse to the body axis and can be coordinated to produce a wave that travels longitudinally along the fin. In this study, we integrate biological experiments (Fig. 2), computational modeling, and experiments with a biomimetic robot (Fig. 1B and Fig. S1) to understand how the fish achieves both stability and maneuverability during rapid adjustments of its fore–aft position. Eigenmannia and other similar species of knifefish often partition their ribbon fin into two inward-counterpropagating waves (22) (Movie S1). The fin kinematics can be idealized as a pair of inward-traveling waves with parameters including oscillation frequency (f), wavelength (λ), and angular amplitude (θ) (Fig. 1C). We term the point where these two waves meet the “nodal point.” Although much is understood about the kinematics and mechanics of unidirectional traveling waves in a fluid (20, 24–28), far less is known (22, 29) about counterpropagating waves, particularly in relation to control.Open in a separate windowFig. 1.Three testbeds considered in this paper include the glass knifefish, a biomimetic robot, and a model of the swimming dynamics. (A) The glass knifefish E. virescens. Experiments with a biomimetic robot match force measurements predicted by a computational model of ribbon fin propulsion. (B) A biomimetic robot that has a ventral ribbon fin to emulate the fin of knifefish. The biomimetic robotic fin consists of 32 independently controlled rays, allowing for a wide range of fin kinematics, such as counterpropagating waves. (C) Fin is modeled as a pair of inward-traveling waves. Directions of head and tail waves and kinematics of the ribbon fin are shown in this schematic: angular deflection (θ), wavelength (λ), lengths of the two waves (L_head and L_tail), length of whole fin (L_fin), temporal frequency (f), and nodal point (red circle).Open in a separate windowFig. 2.Experimental apparatus. (A) The steady-state flow (0–12 cm/s) direction is shown. The fish keeps itself stationary relative to the PVC tube, and the kinematics of the ribbon fin are recorded from below through an angled mirror. (B) One annotated frame recorded from the experiment is shown. Both ends of the fin and nodal point are shown in red. All peaks and troughs of head and tail waves are shown with green and orange dots, respectively.     

From the Cover: Earliest economic exploitation of chicken outside East Asia: Evidence from the Hellenistic Southern Levant

Chicken (Gallus gallus domesticus) is today one of the most widespread domesticated species and is a main source of protein in the human diet. However, for thousands of years exploitation of chickens was confined to symbolic and social domains such as cockfighting. The question of when and where chickens were first used for economic purposes remains unresolved. The results of our faunal analysis demonstrate that the Hellenistic (fourth–second centuries B.C.E.) site of Maresha, Israel, is the earliest site known today where economic exploitation of chickens was widely practiced. We base our claim on the exceptionally high frequency of chicken bones at that site, the majority of which belong to adult individuals, and on the observed 2:1 ratio of female to male bones. These results are supported further by an extensive survey of faunal remains from 234 sites in the Southern Levant, spanning more than three millennia, which shows a sharp increase in the frequency of chicken during the Hellenistic period. We further argue that the earliest secure evidence for economic exploitation of chickens in Europe dates to the first century B.C.E. and therefore is predated by the finds in the Southern Levant by at least a century. We suggest that the gradual acclimatization of chickens in the Southern Levant and its gradual integration into the local economy, the latter fully accomplished in the Hellenistic period, was a crucial step in the adoption of this species in European husbandry some 100 y later.In the modern world, the chicken (Gallus gallus domesticus) is one of the most widespread livestock species and is a major source of animal protein in the human diet. The ancestor of the domestic chicken is the red jungle fowl (Gallus gallus), originating in Southeast Asia, with possible genetic contributions from closely related species through hybridization (1–5). Intensive hybridization between the modern chicken and its wild ancestor caused a loss of the wild progenitor genes (6, 7). Consequently, recent studies usually have focused either on the genetics of the chicken progenitor (8–12) or on zooarchaeological evidence for the domestication of chickens (13–15).The dispersal trajectory of chickens to West Asia, to the Mediterranean, and to Europe following its initial domestication in Southeast Asia remains largely unknown. Moreover, there are only very partial data, and thus there is great uncertainty regarding the place and time of the earliest economic exploitation of chickens: When and where did chickens move from being an exotic species, used only sporadically for symbolic and ritual purposes, to an important livestock species in the Mediterranean and European economies (16, 17)? Our study of chicken remains from the Southern Levant (Israel, the Palestinian Authority, and Jordan) and particularly from the Hellenistic site of Maresha in Southern Israel sheds new light on these issues.We define three main phases in the cultural history of chicken use, based on archaeological, historical, and iconographic evidence (Fig. 1). The early phase (Fig. 1, phase A) may have already begun around the sixth millennium B.C.E. when the chicken was initially domesticated during several independent domestication events in Southeast Asia and China (1, 2, 4, 11, 12). On the Indian subcontinent, which also constitutes a part of the natural dispersal range of the jungle fowl, chicken remains were recorded at a few second millennium B.C.E. sites, and it is commonly assumed that domestication occurred there independently (1, 14, 15, 18, 19). The second phase took place in the third–second millennia B.C.E. and includes the dispersal of the chicken out of its natural distribution range to West Asia (Fig. 1, phase B). The earliest chicken remains in the Near East were retrieved in Iran, Anatolia, and Syria and dated to the third millennium B.C.E. or slightly earlier (20). In Egypt, the oldest known chicken remains are possibly even earlier (16). At this early phase, chicken remains in archaeological sites are very sparse and often are not associated with domestic contexts. Historical and iconographic records demonstrate an acquaintance with the chicken from the mid-second millennium B.C.E. in Egypt, Mesopotamia, and the Levant (21). All these sources relate to chickens (almost exclusively cocks) as an exotic bird, used inter alia for cockfighting and displayed as exotica in royal zoos. The third phase includes its introduction to Europe (Fig. 1, phase C1) and the intensification of its use mainly on this continent (Fig. 1, phase C2).Open in a separate windowFig. 1.The dispersal of chickens in the Old World: the area marked “A” is the geographical range of the jungle fowl in South Asia and its initial domestication, which already may have begun around the sixth millennium B.C.E. in Southeast Asia and possibly in China; The area marked “B” maps the dispersal of chickens to West Asia during the third and second millennia B.C.E. C1 represents the first wave of chicken dispersal into Europe: introduction to Europe during the eighth century B.C.E. (chicken remains have low representation in sites). C2 represents the second wave of chicken dispersal into Europe and other regions from the first century B.C.E. (chicken remains have higher representation in sites). The location of Maresha is marked in the enlarged map (Inset).Archaeologically, chicken remains are first observed in Europe only in late ninth and eighth century B.C.E. contexts. The introduction of chickens to this region usually is attributed to the Phoenicians who brought chickens from their homeland to their colonies in the West (17, 22). This hypothesis is based on the fact that the earliest chicken remains in Europe were retrieved from Phoenician sites, mostly (although not only) in Iberia (23–25). The oldest reliable dated remains of chickens from central Europe (in the Czech Republic) are from the eighth century B.C.E. (26). The continued presence of chickens has been confirmed in Iberia (27, 28), as well as in southern France and Greece (24, 29), during the second half of the first millennium B.C.E. (Fig. 1, phase C1). However, a survey of the zooarchaeological literature of Europe demonstrates that before the first century B.C.E. the proportion of chicken remains in archaeological sites was extremely low and hardly ever exceeded 3% of the total faunal remains (25, 30, 31).The historical evidence also marks the eighth century B.C.E. (or even slightly later) as the arrival date of chickens in Europe. The arrival of chickens in Greece likely postdates Homer (around the eighth century B.C.E.), because the Greek poet does not mention this bird, but chickens are mentioned by Theognis of Megara in the sixth century (32). From the seventh century B.C.E., cocks are depicted on Greek coins and vases (28). In the fifth century B.C.E., the Greek playwright Aristophanes refers to the chicken as the “Persian bird” or “Median bird” (33), possibly indicating that in this period chickens were imported to Greece from Persia (14, 34). By the third century cocks became portrayed more frequently in Egypt (14, 22, 35 and references therein), but in Ptolemaic papyri chickens are hardly mentioned compared with other domesticated species (36). The symbolic role of cocks is well demonstrated by the Roman writer Cicero in his De Divinatione (37), where he mentions that cocks accompanied the Roman armies in 249 B.C.E. and that their behavior was observed carefully before battle as a sign of defeat or victory. Finally, fighting cocks are mentioned by Roman writers such as Varro (38) and Columella (39) (see also refs. 14 and 17).Returning to faunal data, from the first century B.C.E., more sites with chicken remains are known in Europe, and the proportions of chickens at these sites are higher (Fig. 1, phase C2). This increase is apparent in Roman sites in Italy (40) and later in Southern Britain (13) and Sweden (41, 42). Significant proportions of chicken remains are observed in some first century B.C.E. locations in the Near East, such as in Sagalassos in Anatolia (43, 44) and Petra in Jordan (45, 46), and at Berenike (47) and Mons Claudianus (48) in Egypt. Indeed, the relative number of chicken remains in Berenike during Roman times is almost threefold that of the Ptolemaic period (49).Unlike chicken bones, chicken egg shells often are overlooked during excavation (50). The first archaeological evidence for chicken eggs in the Mediterranean is from the first century B.C.E. This evidence includes some examples from Mons Claudianus and a high percentage of medullary bones from Berenike, indicative of females during laying time (47).Although the faunal evidence points to the first century B.C.E. as a turning point in patterns of chicken exploitation in the Mediterranean, the historical and iconographic records imply a slightly earlier date for its economic utilization. For example, a Roman law in the Lex Faunia (161 B.C.E.) banned the consumption of more than a single chicken per meal. Other remarkable testimonies for the integration of the chicken into European livestock in the first century B.C.E. are provided by the Greek historian Diodorus Siculus, who described the sophisticated technique of artificial incubation of chicken eggs in Ptolemaic Egypt (51), and by the Roman historian Varro, who offered advice on how to treat hens during laying time (38). Subsequently, in the first century C.E. the Roman writer Columella and the Roman culinary Apicius mention chicken eggs among the ingredients in culinary recipes (39, 52).We propose that the intensification in chicken exploitation in Europe during phase C2, as reflected by the archaeological and historical records, is related to our new data regarding chicken husbandry in the Southern Levant. The main new data we present here are from the site of Maresha, a national park situated in the Judean foothills in Southern Israel (Fig. 1 and Fig. S1) and dated to the Hellenistic period (fourth–second centuries B.C.E.). Located on an important trading route, Maresha flourished as a leading city of the region of Idumea, and its population comprised a complex ethnic mosaic (53). The town was in ruins by the late second century B.C.E. and was never resettled. In Hellenistic Maresha we note that, in addition to the symbolic cock painted in the so-called “Sidonian” tomb there (54), unisex chicken figurines are more common than any other animal figurines except for riders on horses (55, 56).Open in a separate windowFig. S1.(A) Plan of Maresha in the Hellenistic period with analyzed subterranean complexes 1, 169, 89, 147, and 57 indicated. Image courtesy of ref. 76. (B) Examples of subterranean complexes at Maresha. (Upper) Olive press, subterranean complex no. 44 in Maresha. (Lower) Columbarium, subterranean complex no. 30 in Maresha. Image courtesy of Boaz Zissu.The unprecedented amount of chicken remains revealed at Maresha, far outside the original distribution of the domestic fowl, coupled with the clear chronology of the findings and the excellent preservation of the chicken bones, render Hellenistic Maresha a key site for understanding the new role of the chicken in the Mediterranean during this period. The study of the faunal evidence at Maresha is followed by a comparative chronological and regional study, based on the frequency of chicken remains as presented in 234 faunal reports from the Southern Levant, spanning all periods until early modern times. This study provides diachronic data on the process of introduction and subsequent widespread adoption of the chicken in Levantine economies. We offer suggestions based on these data regarding the time and mode of expansion of chickens from Southwest Asia to Europe and throughout the Mediterranean.     

Diploid-dominant life cycles characterize the early evolution of Fungi

Most of the described species in kingdom Fungi are contained in two phyla, the Ascomycota and the Basidiomycota (subkingdom Dikarya). As a result, our understanding of the biology of the kingdom is heavily influenced by traits observed in Dikarya, such as aerial spore dispersal and life cycles dominated by mitosis of haploid nuclei. We now appreciate that Fungi comprises numerous phylum-level lineages in addition to those of Dikarya, but the phylogeny and genetic characteristics of most of these lineages are poorly understood due to limited genome sampling. Here, we addressed major evolutionary trends in the non-Dikarya fungi by phylogenomic analysis of 69 newly generated draft genome sequences of the zoosporic (flagellated) lineages of true fungi. Our phylogeny indicated five lineages of zoosporic fungi and placed Blastocladiomycota, which has an alternation of haploid and diploid generations, as branching closer to the Dikarya than to the Chytridiomyceta. Our estimates of heterozygosity based on genome sequence data indicate that the zoosporic lineages plus the Zoopagomycota are frequently characterized by diploid-dominant life cycles. We mapped additional traits, such as ancestral cell-cycle regulators, cell-membrane– and cell-wall–associated genes, and the use of the amino acid selenocysteine on the phylogeny and found that these ancestral traits that are shared with Metazoa have been subject to extensive parallel loss across zoosporic lineages. Together, our results indicate a gradual transition in the genetics and cell biology of fungi from their ancestor and caution against assuming that traits measured in Dikarya are typical of other fungal lineages.

Fungi and Metazoa evolved from a common protist-like ancestor, yet the two kingdoms have diverged in ways that make their kinship as Opisthokonts barely recognizable. Fungi grow on or within their food and feed by external digestion (osmotrophy), while animals mostly eat things smaller than themselves via ingestion. This difference is the basis for massive changes in morphology, including loss of motility during feeding and polarized cell growth in fungi (1, 2). The two kingdoms are also considered intrinsically different in life cycles, because fungi are characterized as being haplontic (haploid-dominant life cycle) while animals are diplontic (diploid-dominant). However, this textbook difference is inaccurate in two ways. First, the subkingdom Dikarya, with the majority of fungal species diversity, comprises lineages that spend some or most of their life cycles in a dikaryotic phase wherein two haploid nuclei undergo conjugate division, a cell type genetically analogous to a diploid (3). Further, life cycles have not been carefully investigated in most early-diverging fungal lineages (EDF), which include many phyla outside of Dikarya (e.g., non-Dikarya fungi). EDF have retained ancestral traits also retained in Metazoa, such as flagellation, actin structures used for crawling, presence of cholesterol in cell membranes, vitamin dependencies, and cell-cycle genes (4–8). However, life-cycle transitions between the Opisthokont ancestor and the extant Fungi are shrouded due to a lack of information on the genetic characteristics of EDF and the undersampling of their genomic diversity (9–11). The goal of this paper is to provide a robust and comprehensive phylogeny of the Fungi, emphasizing zoosporic taxa, to reassess the evolution of life-cycle and cellular characters during early fungal diversification using genomic data.Although fungi are often considered to have haploid-dominant life cycles, there are many variations observed (Fig. 1). In a haplontic life cycle, mitosis is restricted to the haploid phase, and meiosis ensues immediately following sex and nuclear fusion (Fig. 1A). In contrast, in the diplontic life cycle that generally characterizes Metazoa, mitosis is restricted to diploid cells (Fig. 1B). The alternation between haploid and diploid mitotic cycles, which generally characterizes plants, is documented, albeit rarely, in fungi, such as baker’s yeast and the water mold Allomyces (Fig. 1C). Despite this general avoidance of diploid mitosis in fungi, many Dikarya show a distinctive dikaryotic life cycle wherein, following mating, haploid nuclei of the two partners remain paired and undergo synchronous mitoses (Fig. 1D). This life cycle is analogous to diploidy with respect to genetic dominance (12) and would provide some of the proposed advantages of diploidy, such as buffering against somatic mutation (13). Overall, although we appreciate that fungal life cycles have great potential to vary, we have a poor understanding of life cycles of the EDF which represent the majority of the phylogenetic diversity of Fungi.Open in a separate windowFig. 1.Illustrated life cycles observed in fungi. (A) In haplontic life cycles mitosis is limited to the haploid phase, with plasmogamy of gametes followed by meiosis. (B) In diplontic life cycles, mitosis only occurs in the diploid phase with haploid cells only functioning as gametes. (C) Life cycles may alternative between haploid and diploid mitotic phases and may show morphological differences between ploidies as in Allomyces. (D) The dikaryotic life cycle is an alternative to alternation of haploid and diploid generations which lacks diploid mitosis and instead has a phase with two nuclear genotypes undergoing synchronous division.We consider EDF to comprise 11 phyla, including 8 zoosporic phyla that reproduce with swimming spores and form a contentious paraphyletic grade along the backbone of the fungal tree (9, 10, 14–16). The deeply diverging phyla, Rozellomycota/Cryptomycota and Aphelidiomycota, are endoparasites that have the ability to phagocytize, which enables them to ingest host cytoplasm, a trait presumably retained from the most recent common ancestor (MRCA) of Opisthokonta (17, 18). The remaining free-living zoosporic phyla have microscopic vegetative thalli that may be unicellular or mycelial (SI Appendix, Fig. S1), and the greatest species diversity is found in the Chytridiomycota, which has an estimated 14 orders (9). Chytridiomycota is united with the phyla Monoblepharidomycota + Neocallimastigomycota in subkingdom Chytridiomyceta, though the branching order of the three phyla is uncertain (14, 15, 19).Blastocladiomycota is an enigmatic group with a life cycle alternating between morphologically distinctive haploid and diploid thalli (Fig. 1C) (20, 21). Members include the water mold, Allomyces, that has been used as a model system for genetics and physiology (22) and a genus of obligate fatal parasites, Coelomomyces, that has a haploid phase in copepods and a diploid phase in mosquitoes (23). The precise phylogenetic placement of the Blastocladiomycota has been controversial (10, 15, 19, 24), with nearly equal support for the Blastocladiomycota diverging before the Chytridiomyceta or after the Chytridiomyceta. Several traits of Blastocladiomycota ally them with the terrestrial fungi (here defined as the nonzoosporic phyla Mucoromycota and Zoopagomycota and subkingdom Dikarya): closed mitosis, the presence of a Spitzenkörper, beta 1,3 glucans in the cell wall, and true mycelial growth in some members (22, 25). The detection of mating types in Coelomomyces (26), which have only been otherwise documented in terrestrial fungi, may be indicative that Blastocladiomycota is more closely related to Dikarya than Chytridiomyceta.Mating and sexuality are poorly described in zoosporic fungi beyond the well-characterized water mold model Allomyces. According to mycological textbooks, life cycles of Chytridiomycota are characterized as being haplontic with zygotic meiosis (27–29), but the majority of assumptions of meiotic stages are unconfirmed by cytology. Moreover, the requisite genetic studies using molecular markers to confirm ploidy cycling have not been accomplished for these presumably sexual species. Importantly, the best-studied chytrid fungus, Batrachochytrium dendrobatidis, has a life cycle that appears to be dominated by asexually reproducing diploid, or aneuploid, thalli (30). More recently, additional studies have indicated that non-Dikarya phyla have heterozygosity indicative of diploidy or higher ploidy (31–34), suggesting that the assumption of haplontic life cycles for the Chytridiomyceta and other EDF may be false.Current sequencing technologies now create the potential for leveraging genomic sequencing to broadly sample fungal genomes for estimating ploidy and other cellular traits in a robust phylogenomic framework. Here, we sampled 69 zoosporic fungal genomes using both culture and single-cell approaches. Our genome analyses provide a strongly supported phylogeny for understanding taxonomy and the evolution of ploidy and other traits which had previously been held to be distinctive between Fungi and Metazoa. These data bolster the growing picture that many traits including motility, feeding modes, and life cycles changed gradually during the early diversification of fungi. The high levels of heterozygosity estimated from genomes analyzed in this study reveal that somatic diploidy is much more common in Fungi than previously appreciated.     

Prediction errors disrupt hippocampal representations and update episodic memories

The brain supports adaptive behavior by generating predictions, learning from errors, and updating memories to incorporate new information. Prediction error, or surprise, triggers learning when reality contradicts expectations. Prior studies have shown that the hippocampus signals prediction errors, but the hypothesized link to memory updating has not been demonstrated. In a human functional MRI study, we elicited mnemonic prediction errors by interrupting familiar narrative videos immediately before the expected endings. We found that prediction errors reversed the relationship between univariate hippocampal activation and memory: greater hippocampal activation predicted memory preservation after expected endings, but memory updating after surprising endings. In contrast to previous studies, we show that univariate activation was insufficient for understanding hippocampal prediction error signals. We explain this surprising finding by tracking both the evolution of hippocampal activation patterns and the connectivity between the hippocampus and neuromodulatory regions. We found that hippocampal activation patterns stabilized as each narrative episode unfolded, suggesting sustained episodic representations. Prediction errors disrupted these sustained representations and the degree of disruption predicted memory updating. The relationship between hippocampal activation and subsequent memory depended on concurrent basal forebrain activation, supporting the idea that cholinergic modulation regulates attention and memory. We conclude that prediction errors create conditions that favor memory updating, prompting the hippocampus to abandon ongoing predictions and make memories malleable.

In daily life, we continuously draw on past experiences to predict the future. Expectation and surprise shape learning across many situations, such as when we discover misinformation in the news, receive feedback on an examination, or make decisions based on past outcomes. When our predictions are incorrect, we must update our mnemonic models of the world to support adaptive behavior. Prediction error is a measure of the discrepancy between expectation and reality; this surprise signal is both evident in brain activity and related to learning (1–6). The brain dynamically reconstructs memories during recall, recreating and revising past experiences based on current information (7). The intuitive idea that surprise governs learning has long shaped our understanding of memory, reward learning, perception, action, and social behavior (2, 8–14). Yet, the neural mechanisms that allow prediction error to update memories remain unknown.Past research has implicated the hippocampus in each of the mnemonic functions required for learning from prediction errors: retrieving memories to make predictions, identifying discrepancies between past and present, and encoding new information (2, 15–20). Functional MRI (fMRI) studies have shown that hippocampal activation increases after predictions are violated; this surprise response has been termed “mismatch detection” (18, 19, 21–23) or “mnemonic prediction error” (20). These past studies have shown that the hippocampus detects mnemonic prediction errors. Several theoretical frameworks have hypothesized that this hippocampal prediction error signal could update memories (17, 20, 24–27), but this crucial link for understanding how we learn from error has not yet been demonstrated.What mechanisms could link hippocampal prediction errors to memory updating? A leading hypothesis is that prediction errors shift the focus of attention and adjust cognitive processing (20, 28–32). After episodes that align with expectations, we should continue generating predictions and shift attention internally, sustaining and reinforcing existing memories. However, after mnemonic prediction errors, we should reset our expectations and shift attention externally, preparing to encode new information and update memories. Consistent with this idea, mnemonic prediction errors have been shown to enhance the hippocampal input pathway that supports encoding, but suppress the output pathway that supports retrieval (20). We propose that surprising events may also change intrinsic hippocampal processing, changing the effect of hippocampal activation on memory outcomes.Neuromodulation may be a critical factor that regulates hippocampal processing and enables memory updating. Currently, there is mixed evidence supporting two hypotheses: acetylcholine or dopamine could act upon the hippocampus to regulate processing after surprising events (24–27, 29, 31, 33, 34). Several models have proposed that acetylcholine from the medial septum (within the basal forebrain) regulates the balance between input and output pathways in the hippocampus (27–29, 35–38), thus allowing stored memories to be compared with perceptual input (31, 38, 39). After prediction errors, acetylcholine release could change hippocampal processing and enhance encoding or memory updating (26, 29, 33, 37, 39). On the other hand, dopamine released from the ventral tegmental area (VTA), if transmitted to the hippocampus, could also modulate hippocampal plasticity after prediction errors. Past studies have shown that the hippocampus and VTA are coactivated after surprising events (40, 41). Other work has shown that coactivation of the hippocampus and VTA predicts memory encoding and integration (42–45). Overall, basal forebrain and VTA neuromodulation are both candidate mechanisms for regulating hippocampal processing and memory updating.In the present study, we used an fMRI task with human participants to examine trial-wise hippocampal responses to prediction errors during narrative videos. During the “encoding phase,” participants viewed 70 full-length videos that featured narrative episodes with salient endings (e.g., a baseball batter hitting a home run) (Fig. 1A). During the “reactivation phase” the following day, participants watched the videos again (Fig. 1B). We elicited mnemonic prediction errors by interrupting half of the videos immediately before the expected narrative ending (e.g., the video ends while the baseball batter is midswing). These surprising interruptions were comparable to the prediction errors employed in prior studies of memory updating (1). Half of the videos were presented in full-length form (Full, as previously seen during the encoding phase) and half were presented in interrupted form (Interrupted, eliciting prediction error).Open in a separate windowFig. 1.Overview of experimental paradigm. (A) During the encoding phase, all videos were presented in full-length form. Here we show example frames depicting a stimulus video. (B) During the reactivation phase, participants viewed the 70 videos again, but half (35 videos) were interrupted to elicit mnemonic prediction error. Participants were cued with the video name, watched the video (Full or Interrupted), and then viewed a fixation screen. The “baseball” video was interrupted when the batter was midswing. fMRI analyses focused on the postvideo fixation periods (red highlighted boxes). Thus, visual and auditory stimulation were matched across Full and Interrupted conditions, allowing us to compare postvideo neural activation while controlling for perceptual input. (C) During the test phase, participants answered structured interview questions about all 70 videos, and were instructed to answer based on their memory of the Full video originally shown during the Encoding phase. Here we show example text illustrating the memory test format and scoring of correct details (our measure of memory preservation) and false memories (our measure of memory updating, because false memories indicate that the memory has been modified). The void response (“I don’t remember”) is not counted as a false memory. (D) Overview of the experiment. All participants completed encoding, reactivation, and test phases of the study. The Delayed group (fMRI participants) completed the test phase 24 h after reactivation, because prior studies have shown that memory updating becomes evident only after a delay (e.g., to permit protein synthesis). The Immediate group completed the test phase immediately after reactivation and was not scanned. The purpose of the Immediate group was to test the behavioral prediction that memory updating required a delay.During the “test phase,” participants completed a memory test in the form of a structured interview (Fig. 1C). On each trial, participants were cued with the name of the video and recalled the narrative. The experimenter then probed for further details with predetermined questions (e.g., “Can you describe the baseball batter’s ethnicity, age range, or clothing?”). Our critical measure of memory updating was “false memories,” because the presence of a false memory indicates that the original memory was changed in some way. Although it can be adaptive to update real-world memories by incorporating relevant new information, we expected that our laboratory paradigm would induce false memories because participants would integrate interfering details across similar episodes (1, 7). Because we were interested in false memories as a measure of memory updating, we instructed participants not to guess and permitted them to skip details they could not recall.Prior research in human and animals has shown that some memory-updating effects only emerge after delays that allow protein synthesis to occur during consolidation and reconsolidation (1, 46–48). Therefore, to test our primary question about the neural correlates of memory updating, fMRI participants completed the encoding, reactivation, and test phases over 3 d, with 24-h between each session (Delayed group, n = 24). In addition, we tested the behavioral prediction that memory updating would require a delay (i.e., because transforming a memory trace requires protein synthesis) by recruiting a separate group of participants who completed the test phase immediately after the reactivation phase on day 2 (Immediate group, n = 24) (Fig. 1D). Delayed group participants completed the reactivation phase while undergoing an fMRI scan, whereas Immediate group participants (n = 24) were not scanned. Our primary fMRI analyses examined the fixation period immediately following the offset of Full and Interrupted videos (postvideo period) (Fig. 1 B, Right) during the reactivation phase in the Delayed group. Importantly, this design compares neural responses to surprising and expected video endings while controlling for visual and auditory input.Our approach allowed us to test several questions set up by the prior literature. First, we used naturalistic video stimuli to examine the effect of mnemonic prediction error on hippocampal activation and episodic memories. Second, to investigate hippocampal processing, we used multivariate analyses to track how episodic representations were sustained or disrupted over time. Third, to test hypotheses about neuromodulatory mechanisms, we related hippocampal activation and memory updating to activation in the basal forebrain and VTA.     

The embryonic node behaves as an instructive stem cell niche for axial elongation

In warm-blooded vertebrate embryos (mammals and birds), the axial tissues of the body form from a growth zone at the tail end, Hensen’s node, which generates neural, mesodermal, and endodermal structures along the midline. While most cells only pass through this region, the node has been suggested to contain a small population of resident stem cells. However, it is unknown whether the rest of the node constitutes an instructive niche that specifies this self-renewal behavior. Here, we use heterotopic transplantation of groups and single cells and show that cells not destined to enter the node can become resident and self-renew. Long-term resident cells are restricted to the posterior part of the node and single-cell RNA-sequencing reveals that the majority of these resident cells preferentially express G2/M phase cell-cycle–related genes. These results provide strong evidence that the node functions as a niche to maintain self-renewal of axial progenitors.

In higher vertebrate embryos the body axis forms in head-to-tail direction from a growth zone at the tail end, which is present from gastrula stages through to the end of axis elongation, several days later. Hensen’s node is part of this growth zone. Rather than defining a distinct cell population arising very early in development, the node represents a dynamic region at the tip of the primitive streak, which appears as a morphological “node” from HH4 (1) in chick. The initial cells that make up this region are derived from two distinct cell populations, which meet at the tip of the elongating primitive streak (HH3 to 3+ in chick) (2–4). These are then joined by cells from the epiblast lateral to the anterior streak and node (5) (at stages HH3+ to HH4) and from the primitive streak immediately caudal to the node during regression (from stage HH5) (6, 7). Although ingression of cells from adjacent epiblast along most of the length of the streak continues later into development (6), this ceases at the level of the node by HH4+ (5, 8, 9). After stage 5, the node begins to regress caudally (7), while cells exit the node to lay down the midline of the developing head–tail axis, contributing to axial (notochord) and paraxial (medial somite) mesoderm, definitive endoderm, and neural midline (floorplate) tissues (Fig. 1 A–C) (5, 10–12).Open in a separate windowFig. 1.The node confers resident behavior. (A–C) Node replacement using a GFP donor showing normal node axial fates. (D and E) Epiblast lateral to the HH3+/4 node ingresses into it and gives rise to the axis and to regressing node as resident cells. (F and G) Anterior epiblast not normally fated to enter the node behaves as lateral epiblast when forced to do so. (H and I) Anterior epiblast normally gives rise to head structures. (J and K) Lateral epiblast no longer gives rise to node-derived axial structures when prevented from entering the node. (L) Quantifying tissue contribution of lateral (D, green) versus anterior (F, blue) epiblast grafts to the host. E, endoderm; F, floorplate; MS, medial-somite; N, notochord; RC, resident cell. Transverse dashed lines show levels of accompanying sections. The field of view of the wholemount images (C, E, G, I, K) is approximately 2 mm x 5 mm.Therefore, most cells pass transiently though the node, temporarily gaining a node-like gene-expression signature, which they lose upon leaving the node (5). However, transplantation of cell groups and fate-mapping experiments in chick (10, 13–15) and mouse (16–20) during early development have suggested that the node may also contain a few resident self-renewing cells that persist within the node during axial elongation, while other cells leave (Fig. 1C, “RC”). In particular, labeling of single cells in the node has provided a few examples of cells that contribute to midline structures and appear to self-renew because one or more cells remain at the site of labeling after some progeny have left (10, 17, 21, 22). At a cell-population level, grafts of groups of cells transplanted repeatedly between older and younger tailbud regions can contribute to midline structures over two or more hosts, while again some cells remain in the tailbud (14, 19). These findings have led to the idea that some cells in the node (most likely a very small subset) may have the ability to self-renew, perhaps indefinitely, thus displaying stem cell behavior.Are the self-renewing cells a special population that arose in earlier development, or might the node act as an environment (niche) (23–25) that captures a subset of the cells that enter it and instructs them to become resident and acquire self-renewal behavior and act as stem cells (26–28)? To demonstrate self-renewal and to test whether the node is an instructive stem cell niche, it is critical to test whether an individual cell can acquire this behavior when introduced to the node environment; this has not yet been attempted. Here we address this question using transplantation of groups of cells and of single cells in vivo and single-cell RNA sequencing (scRNA-seq). We find that the tip of the primitive streak is able to impart notochord and somite identity to most or all cells that enter it, while capturing a small subset to become resident and acquire self-renewal behavior. Cells from epiblast that would never have entered the node region during normal development are able to read these cues. We also define the developmental stage at which epiblast cells lose competence to respond to node signals. Long-term resident cells are preferentially located in the posterior part of the node, and display enriched expression of G2/M cell cycle markers.     

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).     

A family of starch-active polysaccharide monooxygenases

The recently discovered fungal and bacterial polysaccharide monooxygenases (PMOs) are capable of oxidatively cleaving chitin, cellulose, and hemicelluloses that contain β(1→4) linkages between glucose or substituted glucose units. They are also known collectively as lytic PMOs, or LPMOs, and individually as AA9 (formerly GH61), AA10 (formerly CBM33), and AA11 enzymes. PMOs share several conserved features, including a monocopper center coordinated by a bidentate N-terminal histidine residue and another histidine ligand. A bioinformatic analysis using these conserved features suggested several potential new PMO families in the fungus Neurospora crassa that are likely to be active on novel substrates. Herein, we report on NCU08746 that contains a C-terminal starch-binding domain and an N-terminal domain of previously unknown function. Biochemical studies showed that NCU08746 requires copper, oxygen, and a source of electrons to oxidize the C1 position of glycosidic bonds in starch substrates, but not in cellulose or chitin. Starch contains α(1→4) and α(1→6) linkages and exhibits higher order structures compared with chitin and cellulose. Cellobiose dehydrogenase, the biological redox partner of cellulose-active PMOs, can serve as the electron donor for NCU08746. NCU08746 contains one copper atom per protein molecule, which is likely coordinated by two histidine ligands as shown by X-ray absorption spectroscopy and sequence analysis. Results indicate that NCU08746 and homologs are starch-active PMOs, supporting the existence of a PMO superfamily with a much broader range of substrates. Starch-active PMOs provide an expanded perspective on studies of starch metabolism and may have potential in the food and starch-based biofuel industries.Polysaccharide monooxygenases (PMOs) are enzymes secreted by a variety of fungal and bacterial species (1–5). They have recently been found to oxidatively degrade chitin (6–8) and cellulose (8–14). PMOs have been shown to oxidize either the C1 or C4 atom of the β(1→4) glycosidic bond on the surface of chitin (6, 7) or cellulose (10–12, 14), resulting in the cleavage of this bond and the creation of new chain ends that can be subsequently processed by hydrolytic chitinases and cellulases. Several fungal PMOs were shown to significantly enhance the degradation of cellulose by hydrolytic cellulases (9), indicating that these enzymes can be used in the conversion of plant biomass into biofuels and other renewable chemicals.There are three families of PMOs characterized thus far: fungal PMOs that oxidize cellulose (9–12) (also known as GH61 and AA9); bacterial PMOs that are active either on chitin (6, 8) or cellulose (8, 13) (also known as CBM33 and AA10); and fungal PMOs that oxidize chitin (AA11) (7). Sequence homology between these three families is very low. Nevertheless, the available structures of PMOs from all three families reveal a conserved fold, including an antiparallel β-sandwich core and a highly conserved monocopper active site on a flat protein surface (Fig. 1A) (2, 6, 7, 9, 10, 15–17). Two histidine residues in a motif termed the histidine brace coordinate the copper center. The N-terminal histidine ligand binds in a bidentate mode, and its imidazole ring is methylated at the N_ε position in fungal PMOs (Fig. 1A).Open in a separate windowFig. 1.(A) Representative overall and active site structures of fungal PMOs (PDB ID code 2YET) (10). (B) Structure of cellulose (18, 19). Chitin also contains β(1→4) linkages and has similar crystalline higher order structure to cellulose. (C) Model structure of amylopectin (23–25). Hydrogen bonds are shown with green dashed lines.Considering the conserved structural features, it is not surprising that the currently known PMOs act on substrates with similar structures. Cellulose and chitin contain long linear chains of β(1→4) linked glucose units and N-acetylglucosamine units, respectively (Fig. 1B). The polymer chains form extensive hydrogen bonding networks, which result in insoluble and very stable crystalline structures (18–21). PMOs are thought to bind to the substrate with their flat active site surface, which orients the copper center for selective oxidation at the C1 or C4 position (6, 16, 22). Some bacterial chitin-binding proteins are cellulose-active PMOs (8, 13, 14), further suggesting that the set of PMO substrates is restricted to β(1→4) linked polymers of glucose and glucose derivatives.Here, we report on the identification of new families of PMOs that contain several key features of previously characterized PMOs, but act on substrates different from cellulose or chitin. A member of one of these novel families of PMOs, NCU08746, was shown to oxidatively cleave amylose, amylopectin, and starch. We designate the NCU08746 family as starch-active PMOs. Both amylose and amylopectin contain linear chains of α(1→4) linked glucose, whereas the latter also contains α(1→6) glycosidic linkages at branch points in the otherwise α(1→4) linked polymer. Unlike cellulose and chitin, amylose and amylopectin do not form microcrystals; instead, they exist in disordered, single helical, and double helical forms (23–27) (see Fig. 1C for example). Starch exists partially in nanocrystalline form, but lacks the flat molecular surfaces as those found in chitin and cellulose. The discovery of starch-active PMOs shows that this oxidative mechanism of glycosidic bond cleavage is more widespread than initially expected.