Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles

A central challenge to the development of protein-based therapeutics is the inefficiency of delivery of protein cargo across the mammalian cell membrane, including escape from endosomes. Here we report that combining bioreducible lipid nanoparticles with negatively supercharged Cre recombinase or anionic Cas9:single-guide (sg)RNA complexes drives the electrostatic assembly of nanoparticles that mediate potent protein delivery and genome editing. These bioreducible lipids efficiently deliver protein cargo into cells, facilitate the escape of protein from endosomes in response to the reductive intracellular environment, and direct protein to its intracellular target sites. The delivery of supercharged Cre protein and Cas9:sgRNA complexed with bioreducible lipids into cultured human cells enables gene recombination and genome editing with efficiencies greater than 70%. In addition, we demonstrate that these lipids are effective for functional protein delivery into mouse brain for gene recombination in vivo. Therefore, the integration of this bioreducible lipid platform with protein engineering has the potential to advance the therapeutic relevance of protein-based genome editing.Therapeutic proteins are an expanding class of biologics that can be used for specific and transient manipulation of cell function (1). Recently, the programmable nuclease Cas9 and other genome-editing proteins have been shown to mediate editing of disease-associated alleles in the human genome, facilitating new treatments for many genetic diseases (2–5). The transient nature of therapeutic protein delivery makes it an attractive method for delivery of genome-editing proteins (4). A challenge to efficient delivery of genome-editing proteins is their proteolytic instability and poor membrane permeability (6). Developing delivery vehicles to transport active protein to their intracellular target site is thus essential to advance protein-based genome editing. The last few years have witnessed tremendous progress in designing nanocarriers for intracellular protein delivery (7, 8). However, the lack of an effective, general approach to load protein into a stable nanocomplex and the inefficient release of protein from endocytosed nanoparticles pose challenges for protein delivery (6). There remains a great demand for the development of novel platforms that efficiently assemble protein into nanoparticles for intracellular delivery while maintaining biological activity of the protein.Recently, we developed lipid-like nanoparticles that can be synthesized in a combinatorial manner as highly effective protein and gene delivery vehicles (9–13). We found that electrostatic self-assembly between lipid and protein is essential to form a stable nanocomplex for protein delivery (11). Further, we demonstrated that the integration of a bioreducible disulfide bond into the hydrophobic tail of the lipid enhances efficiency of small interfering RNA (siRNA) delivery (14), due to the improved endosomal escape and cargo release following lipid degradation in the reductive intracellular environment. Meanwhile, we engineered supercharged proteins shown to enhance protein delivery by fusing superpositively charged GFP to a protein of interest (15–17) and using cationic-lipid mediated delivery of supernegatively charged proteins (4). We hypothesized that combining cationic bioreducible lipids and supernegatively charged proteins would drive electrostatic self-assembly of a supramolecular nanocomplex to deliver the genome-editing protein (Fig. 1). In addition, we hypothesized that the bioreduction of these lipid/protein nanocomplexes inside cells in response to the reductive intracellular environment (e.g., high concentration of glutathione) could facilitate endosomal escape of the protein cargo, enabling protein to enter the nucleus for effective genome editing.Open in a separate windowFig. 1.Design of bioreducible lipid-like materials and negatively supercharged protein for effective protein delivery and genome editing.In this study, we synthesized 12 bioreducible lipids by a Michael addition of primary or secondary amines and an acrylate that features a disulfide bond and a 14-carbon hydrophobic tail (Fig. 2). Our combinatorial synthesis allows facile generation of lipids with chemically diverse head groups, enabling study of the structure–activity relationship of the head groups. We fused several negatively supercharged GFP variants to Cre recombinase (4) with the aim of enhancing the electrostatic interaction between protein and cationic lipid. Our work also demonstrates that the anionic ribonuceloprotein complex formed between Cas9 and single-guide RNA (sgRNA) (2) is able to form a nanocomplex with our bioreducible lipids for efficient genome editing in human cells. We find that the bioreducible lipids can efficiently deliver active negatively charged proteins complexes with a higher efficiency than commercially available lipids. Our bioreducible lipids enable Cre- and Cas9:sgRNA-mediated gene recombination and gene knockout with efficiencies higher than 70% in cultured human cells. Finally, we demonstrate that these lipid nanoparticles can deliver genome-editing protein into the mouse brain for effective DNA recombination in vivo.Open in a separate windowFig. 2.Synthesis of bioreducible lipid-like materials. (A) Synthesis route and lipid nomenclature. (B) Chemical structures of amines used as head groups for lipid synthesis.     

Self-assembly of amphiphilic Janus dendrimers into uniform onion-like dendrimersomes with predictable size and number of bilayers

A constitutional isomeric library synthesized by a modular approach has been used to discover six amphiphilic Janus dendrimer primary structures, which self-assemble into uniform onion-like vesicles with predictable dimensions and number of internal bilayers. These vesicles, denoted onion-like dendrimersomes, are assembled by simple injection of a solution of Janus dendrimer in a water-miscible solvent into water or buffer. These dendrimersomes provide mimics of double-bilayer and multibilayer biological membranes with dimensions and number of bilayers predicted by the Janus compound concentration in water. The simple injection method of preparation is accessible without any special equipment, generating uniform vesicles, and thus provides a promising tool for fundamental studies as well as technological applications in nanomedicine and other fields.Most living organisms contain single-bilayer membranes composed of lipids, glycolipids, cholesterol, transmembrane proteins, and glycoproteins (1). Gram-negative bacteria (2, 3) and the cell nucleus (4), however, exhibit a strikingly special envelope that consists of a concentric double-bilayer membrane. More complex membranes are also encountered in cells and their various organelles, such as multivesicular structures of eukaryotic cells (5) and endosomes (6), and multibilayer structures of endoplasmic reticulum (7, 8), myelin (9, 10), and multilamellar bodies (11, 12). This diversity of biological membranes inspired corresponding biological mimics. Liposomes (Fig. 1) self-assembled from phospholipids are the first mimics of single-bilayer biological membranes (13–16), but they are polydisperse, unstable, and permeable (14). Stealth liposomes coassembled from phospholipids, cholesterol, and phospholipids conjugated with poly(ethylene glycol) exhibit improved stability, permeability, and mechanical properties (17–20). Polymersomes (21–24) assembled from amphiphilic block copolymers exhibit better mechanical properties and permeability, but are not always biocompatible and are polydisperse. Dendrimersomes (25–28) self-assembled from amphiphilic Janus dendrimers and minidendrimers (26–28) have also been elaborated to mimic single-bilayer biological membranes. Amphiphilic Janus dendrimers take advantage of multivalency both in their hydrophobic and hydrophilic parts (23, 29–32). Dendrimersomes are assembled by simple injection (33) of a solution of an amphiphilic Janus dendrimer (26) in a water-soluble solvent into water or buffer and produce uniform (34), impermeable, and stable vesicles with excellent mechanical properties. In addition, their size and properties can be predicted by their primary structure (27). Amphiphilic Janus glycodendrimers self-assemble into glycodendrimersomes that mimic the glycan ligands of biological membranes (35). They have been demonstrated to be bioactive toward biomedically relevant bacterial, plant, and human lectins, and could have numerous applications in nanomedicine (20).Open in a separate windowFig. 1.Strategies for the preparation of single-bilayer vesicles and multibilayer onion-like vesicles.More complex and functional cell mimics such as multivesicular vesicles (36, 37) and multibilayer onion-like vesicles (38–40) have also been discovered. Multivesicular vesicles compartmentalize a larger vesicle (37) whereas multibilayer onion-like vesicles consist of concentric alternating bilayers (40). Currently multibilayer vesicles are obtained by very complex and time-consuming methods that do not control their size (39) and size distribution (40) in a precise way. Here we report the discovery of “single–single” (28) amphiphilic Janus dendrimer primary structures that self-assemble into uniform multibilayer onion-like dendrimersomes (Fig. 1) with predictable size and number of bilayers by simple injection of their solution into water or buffer.     

CO and CN? syntheses by [FeFe]-hydrogenase maturase HydG are catalytically differentiated events

The synthesis and assembly of the active site [FeFe] unit of [FeFe]-hydrogenases require at least three maturases. The radical S-adenosyl-l-methionine HydG, the best characterized of these proteins, is responsible for the synthesis of the hydrogenase CO and CN⁻ ligands from tyrosine-derived dehydroglycine (DHG). We speculated that CN⁻ and the CO precursor ⁻:CO₂H may be generated through an elimination reaction. We tested this hypothesis with both wild type and HydG variants defective in second iron-sulfur cluster coordination by measuring the in vitro production of CO, CN⁻, and ⁻:CO₂H-derived formate. We indeed observed formate production under these conditions. We conclude that HydG is a multifunctional enzyme that produces DHG, CN⁻, and CO at three well-differentiated catalytic sites. We also speculate that homocysteine, cysteine, or a related ligand could be involved in Fe(CO)_x(CN)_y transfer to the HydF carrier/scaffold.Many microorganisms can either oxidize molecular hydrogen or reduce protons according to the reaction H₂ = 2H⁺ + 2 e⁻. The enzymes that catalyze this reaction fall into two phylogenetically unrelated groups, the [NiFe]- and [FeFe]-hydrogenases (1, 2). Initial crystallographic studies of the [FeFe]-hydrogenases from Clostridium pasteuranium (3) and Desulfovibrio desulfuricans (4) showed that the active site is composed of a conventional [4Fe-4S] cubane connected by a cysteine thiolate to a binuclear FeFe unit, in which each iron ion is terminally coordinated by one CN⁻ ligand and one CO ligand and by a third CO molecule that bridges the two metals (5). Unexpectedly, we also found that a small molecule first postulated (6), and now indirectly confirmed (7), to be dithiomethylamine (DTMA) bridges the two Fe ions (Fig. 1).Open in a separate windowFig. 1.[FeFe]-hydrogenase H-cluster from D. desulfuricans (6). Only the cysteine residue bridging the [4Fe-4S] and [FeFe] subsites is depicted as Cys. The cysteine ligands of the [4Fe-4S] cluster are shown as straight lines.The [4Fe-4S] cubane bridged to the binuclear [FeFe] unit has been collectively called the H-cluster (1). Work from several laboratories has shown that the maturation of the [FeFe] center requires at least three protein maturases: HydF that has GTPase activity and appears to be both a [FeFe] center scaffold and carrier (8, 9), HydG that synthesizes CO and CN⁻ from tyrosine (10–13), and HydE that, by elimination, should be involved in the synthesis of the DTMA bridge (14, 15). Both HydE and HydG are members of the large radical S-adenosyl-l-methionine (SAM) protein family (16, 17). With the recent reports of HydG crystal structures from Carboxydothermus hydrogenoformans (Ch) by us (18) and from Thermoanaerobacter italicus (Ti) by Dinis et al. (19), X-ray models are now available for the three maturases (20, 21); however, unambiguous structure-function relationships have been proposed only in the case of HydG. Indeed, site-directed mutational studies have shown that CO and CN⁻ syntheses are affected by either the deletion of the maturase C-terminal region, where a second iron-sulfur cluster binds (22), or Cys-to-Ser mutations in its corresponding CxxCx₂₂C binding motif (10, 13). In addition, it has been shown that HydG synthesizes Fe(CO)_x(CN)_y precursors (x = 1 or 2; y = 1) of the [FeFe] catalytic unit (23). The two HydG crystal structures are very similar at the SAM and [4Fe-4S] cluster-containing (β/α)₈ TIM-like barrel, common to several radical SAM proteins (16) (Fig. 2). Conversely, there are significant differences in the composition of the extra C-terminal second (s) iron-sulfur cluster. In our crystals, ChHydG lacks this center (18), whereas TiHydG coordinates a [4Fe-4S]_s cluster in one of the two molecules of the asymmetric unit and a second center with a fifth iron in the other molecule (19). This fifth iron has been described as being bound by His265, a putative alanine molecule and a sulfide bridge to a [4Fe-4S] unit coordinated by the CxxCx₂₂C motif. Two water molecules complete the octahedral Fe coordination (19). Here the different second cluster structures are collectively called [FeS]_s.Open in a separate windowFig. 2.Structures of (A) Ch HydG depicting tunnel I, the SAM cofactor, and [4Fe-4S] cluster (Top Right), the tyrosine active site cavity (Top Center), tunnel II, and the Cl⁻ binding cavity (Bottom Center) (18) and (B) Ti HydG with its additional second iron-sulfur cluster (19).     

Biasing moral decisions by exploiting the dynamics of eye gaze

Eye gaze is a window onto cognitive processing in tasks such as spatial memory, linguistic processing, and decision making. We present evidence that information derived from eye gaze can be used to change the course of individuals’ decisions, even when they are reasoning about high-level, moral issues. Previous studies have shown that when an experimenter actively controls what an individual sees the experimenter can affect simple decisions with alternatives of almost equal valence. Here we show that if an experimenter passively knows when individuals move their eyes the experimenter can change complex moral decisions. This causal effect is achieved by simply adjusting the timing of the decisions. We monitored participants’ eye movements during a two-alternative forced-choice task with moral questions. One option was randomly predetermined as a target. At the moment participants had fixated the target option for a set amount of time we terminated their deliberation and prompted them to choose between the two alternatives. Although participants were unaware of this gaze-contingent manipulation, their choices were systematically biased toward the target option. We conclude that even abstract moral cognition is partly constituted by interactions with the immediate environment and is likely supported by gaze-dependent decision processes. By tracking the interplay between individuals, their sensorimotor systems, and the environment, we can influence the outcome of a decision without directly manipulating the content of the information available to them.Moral cognition arises from the interplay between emotion and reason (1–5), between cultural and personal values (6), and in the competition between different cognitive representations (7–9). Many studies have explored these tensions, finding that moral decisions can be influenced by priming, highlighting, or framing one factor over another (4–6, 9). Despite this, almost no attention has been devoted to how moral deliberation is played out in the very moment of choice or what effect this might have on the decision process itself. In the current experiments we focused on the temporal dynamics of moral cognition. We hypothesized that tracking the gaze of participants while they decided between two options would provide sufficient knowledge that could be exploited to influence the outcome of the moral deliberation.Our hypothesis is derived from an understanding of human cognition that emphasizes dynamic interaction between cognition and environment through sensorimotor activation, a position supported by converging lines of evidence (10–31). Gaze patterns in humans reflect the course of reasoning during spatial indexing tasks both in adults (10, 11) and in infants (12). Evidence from neural stimulation shows that saccadic programming and perceptual decisions develop together in the monkey brain (15, 16). In decision tasks, before asserting their preference for faces or similarly valued snack foods people look more toward the alternative they are going to choose (17, 19). For example, the attentional drift-diffusion model (aDDM) proposes a computational mechanism underlying choice whereby gaze direction biases the decision process (19, 31). Similarly, studies measuring hand and eye movements show that attitudes and preferences are dynamically constructed over the course of a trial (20, 29, 30).In this paper we extend the study of gaze and decision making from simple preferences to complex moral choices. Together, past research suggests that moment by moment the alternative that participants look at while making a decision will be the alternative that they are considering at that point in time. However, rather than using priming or stimuli presentation to control what participants saw or thought during their moral deliberation we controlled only when the decision was made and predicted that we could systematically influence their choices (Fig. 1).Open in a separate windowFig. 1.Hypotheses. We hypothesized that participants’ eye gaze reveals their decision process owing to general coupling between sensorimotor decision processes. By using a gaze-contingent probe and selecting when a decision is prompted the resulting choice can be biased toward a randomly predetermined option.We used an experimental paradigm where participants sat in front of a computer while we monitored their gaze using a remote eye-tracking system. Through headphones they heard statements such as “Murder is sometimes justifiable.” Subsequently two response alternatives were presented simultaneously on-screen, in this case “sometimes justifiable” and “never justifiable.” We instructed the participants to choose the alternative that they considered to be morally right (Fig. 2 and Materials and Methods). We told the participants that they would view the alternatives a short but random amount of time, after which we would remove the alternatives from view and prompt them to indicate their choice. During their deliberation, participants looked freely between the two alternatives before making their choice, a design allowing us to demonstrate that gaze reflects decision-making processes even for moral choices (experiment 1). To show that knowledge of these dynamics can be exploited to influence the decision itself, without the knowledge of participants, we timed the decision prompt to be directly dependent on their gaze distribution according to fixed rules (experiments 2 and 3).Open in a separate windowFig. 2.Trial design. Participants first hear a moral statement being read out loud while viewing a central fixation point. When the statement has been read completely two alternatives appear on the screen randomly assigned to the left or right position. During this portion of the trial the participants’ gaze is monitored by a remote eye tracker. Participants view the alternatives until their choice is prompted, either by fulfilling the experiment-specific conditions or 3,000-ms passes. Participants indicate which alternative they choose by clicking the right or left mouse button, respectively. Finally, a 7-point continuous confidence scale is presented. There is a 1,000-ms pause between participants’ last response and the start of the next trial. Experiment 1: While participants view both alternatives their gaze is being tracked; once one alternative has accumulated at least 750 ms of gaze and the other at least 250 ms the decision prompt is activated and the trial is interrupted. Whatever alternative has the most accumulated gaze time at the time of interruption is designated the target. Experiment 2: The target is randomly determined before trial onset. Participants’ gaze is measured while they view alternatives and once the target alternative has been viewed for at least 750 ms and the other alternative has been viewed for at least 250 ms the decision prompt is activated and the trial is interrupted.     

From the Cover: Mode of action uncovered for the specific reduction of methane emissions from ruminants by the small molecule 3-nitrooxypropanol

Ruminants, such as cows, sheep, and goats, predominantly ferment in their rumen plant material to acetate, propionate, butyrate, CO₂, and methane. Whereas the short fatty acids are absorbed and metabolized by the animals, the greenhouse gas methane escapes via eructation and breathing of the animals into the atmosphere. Along with the methane, up to 12% of the gross energy content of the feedstock is lost. Therefore, our recent report has raised interest in 3-nitrooxypropanol (3-NOP), which when added to the feed of ruminants in milligram amounts persistently reduces enteric methane emissions from livestock without apparent negative side effects [Hristov AN, et al. (2015) Proc Natl Acad Sci USA 112(34):10663–10668]. We now show with the aid of in silico, in vitro, and in vivo experiments that 3-NOP specifically targets methyl-coenzyme M reductase (MCR). The nickel enzyme, which is only active when its Ni ion is in the +1 oxidation state, catalyzes the methane-forming step in the rumen fermentation. Molecular docking suggested that 3-NOP preferably binds into the active site of MCR in a pose that places its reducible nitrate group in electron transfer distance to Ni(I). With purified MCR, we found that 3-NOP indeed inactivates MCR at micromolar concentrations by oxidation of its active site Ni(I). Concomitantly, the nitrate ester is reduced to nitrite, which also inactivates MCR at micromolar concentrations by oxidation of Ni(I). Using pure cultures, 3-NOP is demonstrated to inhibit growth of methanogenic archaea at concentrations that do not affect the growth of nonmethanogenic bacteria in the rumen.Since the agricultural and industrial revolution 200 y ago, the methane concentration in the atmosphere has increased from less than 0.6 to 1.8 ppm. The present concentration is only 0.45% of that of CO₂, but because methane has a 28- to 34-fold higher global warming potential than CO₂ on a 100-y horizon, it contributes significantly to global warming (1). On the other hand, the lifetime of atmospheric methane is relatively short relative to CO₂. Accordingly, the climate response to reductions of methane emissions will be relatively rapid. Thus, measures targeting methane emissions are considered paramount to mitigate climate change (2).One of the main anthropogenic sources of atmospheric methane are ruminants (cattle, sheep, goats), the number of which has grown in parallel with the world population. Presently, there are about 1.5 billion cattle, 1.1 billion sheep, and 0.9 billion goats raised by humans (3). Ruminants emit about 100 million tons of methane per year, which corresponds to ∼20% of global methane emissions (4).In the rumen (Fig. 1), plant material is fermented by anaerobic bacteria, protozoa, fungi, and methanogenic archaea in a trophic chain, predominantly yielding acetate, propionate, butyrate, CO₂, and methane with H₂ as intermediate (5, 6). Whereas organic acids are absorbed and metabolized by the animals, methane escapes the rumen into the atmosphere via eructation and breathing of the animals. The generation of methane by methanogenic archaea in the intestine of domestic ruminants lessens feed efficacy, as up to 12% of the gross energy ingested by the animal is lost this way (7).Fig. 1.Methane formation in the rumen of a dairy cow and its inhibition by 3-nitrooxypropanol (3-NOP). The H₂ concentration in the rumen fluid is near 1 µM (≙140 Pa = 0.14% in the gas phase).Methane (CH₄) formation is the main H₂ sink in the rumen. It is formed by methanogenic archaea at the bottom of the trophic chain mainly from carbon dioxide (CO₂) and hydrogen (H₂) (Fig. 1). However, the methane eructated by ruminants contains only minute amounts of H₂; the concentration of dissolved H₂ in the rumen is near 1 µM (8), equivalent to a H₂ partial pressure of near 140 Pa. Because at 1 µM, H₂ formation from most substrates in the rumen is exergonic (9), the low H₂ concentration indicates that H₂ is consumed in the rumen by the methanogens more rapidly than it is formed by other microorganisms (10). The H₂ concentration increases substantially only when methane formation from H₂ and CO₂ is specifically inhibited by more than 50% (10, 11). Already a small increase in the H₂ concentration (8) leads to both down-regulation of H₂-generating pathways (12) and up-regulation of H₂-neutral and H₂-consuming pathways such as propionate formation, resulting in additional energy supply to the host animal (13–15). Thus, the H₂ concentration stays constant, although its consumption by methanogens is partially inhibited in the rumen.The amount of methane formation per unit of ingested feedstuff can differ significantly between individual animals as it is a heritable trait (16). Understanding these differences has been the scientific motivation to pursue the development of selective inhibitors of methanogenesis that are nontoxic to animals (17, 18). Only recently, a compound has been described that apparently can both substantially decrease CH₄ and increase propionate productions in the rumen without compromising animal performance and health (19). It is the small molecule 3-nitrooxypropanol (3-NOP) (chemical structure shown in Fig. 1) that has been found to persistently decrease enteric methane emissions from sheep (20), dairy cows (21), and beef cattle (22) without apparent negative side effects (19). 3-NOP, given to high-producing dairy cows at 60 mg/kg feed dry matter (Fig. 1), not only decreased methane emissions by 30% but also increased body weight gain significantly without negatively affecting feed intake nor milk production and composition (19).Methane formation in methanogenic archaea is catalyzed by methyl-coenzyme M reductase (MCR), involving methyl-coenzyme M and coenzyme B as substrates (Fig. 2A). MCR is a nickel enzyme in which the nickel is bound in a tetrapyrrole derivative named cofactor F₄₃₀ (23, 24). This nickel-containing cofactor has to be in the Ni(I) oxidation state for the enzyme to be active. Because the redox potential E^o′ of the F₄₃₀(Ni²⁺)/ F₄₃₀(Ni¹⁺) couple is −600 mV, the enzyme is very susceptible to inactivation by oxidants (23, 24). MCR has been well characterized by high-resolution X-ray structures (25–27) and EPR spectroscopy (28) with either substrates or products bound.Fig. 2.Binding of 3-NOP to methyl-coenzyme M reductase (MCR) as suggested by molecular docking. The crystal structure of inactive isoenzyme I from M. marburgensis was used in the docking experiments (25). (A) MCR-catalyzed reaction. CH₃-S-CoM, methyl-coenzyme ...The molecular shape of 3-NOP (Fig. 1) is similar to that of methyl-coenzyme M (Fig. 2A). This fact and the moderate oxidation potential of 3-NOP suggested that inhibition of methanogenesis in ruminants is achieved by targeting the active site of MCR, for which we now provide experimental evidence. We start by describing how the development of 3-NOP was facilitated by molecular modeling.     

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii

Ethanol is the most widely used renewable transportation biofuel in the United States, with the production of 13.3 billion gallons in 2012 [John UM (2013) Contribution of the Ethanol Industry to the Economy of the United States]. Despite considerable effort to produce fuels from lignocellulosic biomass, chemical pretreatment and the addition of saccharolytic enzymes before microbial bioconversion remain economic barriers to industrial deployment [Lynd LR, et al. (2008) Nat Biotechnol 26(2):169–172]. We began with the thermophilic, anaerobic, cellulolytic bacterium Caldicellulosiruptor bescii, which efficiently uses unpretreated biomass, and engineered it to produce ethanol. Here we report the direct conversion of switchgrass, a nonfood, renewable feedstock, to ethanol without conventional pretreatment of the biomass. This process was accomplished by deletion of lactate dehydrogenase and heterologous expression of a Clostridium thermocellum bifunctional acetaldehyde/alcohol dehydrogenase. Whereas wild-type C. bescii lacks the ability to make ethanol, 70% of the fermentation products in the engineered strain were ethanol [12.8 mM ethanol directly from 2% (wt/vol) switchgrass, a real-world substrate] with decreased production of acetate by 38% compared with wild-type. Direct conversion of biomass to ethanol represents a new paradigm for consolidated bioprocessing, offering the potential for carbon neutral, cost-effective, sustainable fuel production.Increasing demand for fuels, geopolitical instability, the limitation of global petroleum reserves, and the impact on climate change induced by greenhouse gases have increased the need for renewable and sustainable biofuels (1–5). First-generation biofuels produced from food crops, such as corn, are limited by cost and competition with food supply (6, 7). Switchgrass is a perennial grass native to North America, and its high productivity on marginal farmlands and low agricultural input requirements make it an attractive feedstock for the production of biofuels and biochemicals (8). A yield of 36.7 Mg⋅ha⁻¹ was achieved in field trials in Oklahoma (9), and switchgrass has the potential to produce 500% or more energy than is used for its cultivation (10). The use of abundant lignocellulosic plant biomass as feedstock is environmentally desirable and economically essential for enabling a viable biofuels industry (11). Current strategies for bioethanol production from lignocellulosic feedstocks require three major operational steps: physicochemical pretreatment, enzymatic saccharification, and fermentation (Fig. 1) (6, 12). Pretreatment and enzymatic hydrolysis represent substantial cost and it is estimated that the use of cellulolytic microbes for consolidated bioprocessing and eliminating pretreatment would reduce bioprocessing costs by 40% (2). Considerable effort has been made to develop single microbes capable of both saccharification and fermentation to avoid the substantial expense of using saccharolytic enzyme mixtures (13). Heterologous expression of saccharolytic enzymes has been demonstrated in a number of organisms, including Saccharomyces cerevisiae, Zymomonas mobilis, Escherichia coli, and Bacillus subtilis to ferment various model cellulosic and hemicellulosic substrates (13–15). Although these approaches have resulted in progress in cellulose utilization, the overall enzyme activity is still very low compared with that of naturally cellulolytic organisms and the rates of hydrolysis are not sufficient for an industrial process (13).Open in a separate windowFig. 1.Comparison of bioethanol production strategies and a predicted fermentative pathway in C. bescii. Depiction of “single step bioprocessing” accomplished by engineered C. bescii. CBP, consolidated bioprocessing.High-temperature fermentations facilitate biomass deconstruction and may reduce contamination and volatilize toxic products, such as alcohols. Clostridium thermocellum and Thermoanaerobacterium saccharolyticum have been used in mixed culture fermentations successfully for laboratory-scale demonstration of first-generation consolidated bioprocessing (13, 16) (Fig. 1). C. thermocellum is one promising candidate for consolidated bioprocessing because it is naturally cellulolytic, able to hydrolyze cellulose at 2.5 g⋅L⁻¹⋅h⁻¹, and produces ethanol as one fermentation product, but it has not yet been engineered to produce ethanol at high yield and lacks the ability to ferment hemicellulosic sugars (13, 17). Caldicellulosiruptor bescii, on the other hand, is the most thermophilic cellulolytic bacterium so far described, growing optimally at ∼80 °C with the ability to use a wide range of substrates, such as cellulose, hemicellulose, and lignocellulosic plant biomass without harsh and expensive chemical pretreatment (17, 18), efficiently fermenting both C₅ and C₆ sugars derived from plant biomass (17, 18). C. bescii uses the Embden–Meyerhof–Parnas pathway for conversion of glucose to pyruvate, and the predominant end-products are acetate, lactate, and hydrogen (Fig. 2) (18). A mutant strain of C. bescii (JWCB018) was recently isolated in which the lactate dehydrogenase gene (ldh) was disrupted spontaneously via insertion of a native transposon (19, 20). A complete deletion of ldh was also engineered (21), and this strain no longer produced lactate, instead diverting metabolic flux to additional acetate and H₂, demonstrating the utility of the newly developed tools to provide a platform for further strain engineering. The recent development of genetic methods for the manipulation of this organism (19, 21, 22) opens the door for metabolic engineering for the direct conversion of unpretreated plant biomass to liquid fuels, such as ethanol, via “single step bioprocessing” (Fig. 1).Open in a separate windowFig. 2.Overview of C. bescii fermentative pathways for bioconversion of hexose sugars. Pathway 1 (blue) results in 2 mol of acetic acid and 4 mol of H₂ per mole of glucose. Pathway 2 (green) produces 2 mol of lactic acid per mole of glucose. Pathway 3 (red) is a new pathway resulting from heterologous expression of the C. thermocellum adhE gene to synthesize 2 mol of ethanol per mole of glucose.     

De novo production of the plant-derived alkaloid strictosidine in yeast

The monoterpene indole alkaloids are a large group of plant-derived specialized metabolites, many of which have valuable pharmaceutical or biological activity. There are ∼3,000 monoterpene indole alkaloids produced by thousands of plant species in numerous families. The diverse chemical structures found in this metabolite class originate from strictosidine, which is the last common biosynthetic intermediate for all monoterpene indole alkaloid enzymatic pathways. Reconstitution of biosynthetic pathways in a heterologous host is a promising strategy for rapid and inexpensive production of complex molecules that are found in plants. Here, we demonstrate how strictosidine can be produced de novo in a Saccharomyces cerevisiae host from 14 known monoterpene indole alkaloid pathway genes, along with an additional seven genes and three gene deletions that enhance secondary metabolism. This system provides an important resource for developing the production of more complex plant-derived alkaloids, engineering of nonnatural derivatives, identification of bottlenecks in monoterpene indole alkaloid biosynthesis, and discovery of new pathway genes in a convenient yeast host.Monoterpene indole alkaloids (MIAs) are a diverse family of complex nitrogen-containing plant-derived metabolites (1, 2). This metabolite class is found in thousands of plant species from the Apocynaceae, Loganiaceae, Rubiaceae, Icacinaceae, Nyssaceae, and Alangiaceae plant families (2, 3). Many MIAs and MIA derivatives have medicinal properties; for example, vinblastine, vincristine, and vinflunine are approved anticancer therapeutics (4, 5). These structurally complex compounds can be difficult to chemically synthesize (6, 7). Consequently, industrial production relies on extraction from the plant, but these compounds are often produced in small quantities as complex mixtures, making isolation challenging, laborious, and expensive (8–10). Reconstitution of plant pathways in microbial hosts is proving to be a promising approach to access plant-derived compounds as evidenced by the successful production of terpenes, flavonoids, and benzylisoquinoline alkaloids in microorganisms (11–19). Microbial hosts can also be used to construct hybrid biosynthetic pathways to generate modified natural products with potentially enhanced bioactivities (8, 20, 21). Across numerous plant species, strictosidine is believed to be the core scaffold from which all 3,000 known MIAs are derived (1, 2). Strictosidine undergoes a variety of redox reactions and rearrangements to form the thousands of compounds that comprise the MIA natural product family (Fig. 1) (1, 2). Due to the importance of strictosidine, the last common biosynthetic intermediate for all known MIAs, we chose to focus on heterologous production of this complex molecule (1). Therefore, strictosidine reconstitution represents the necessary first step for heterologous production of high-value MIAs.Open in a separate windowFig. 1.Strictosidine, the central intermediate in monoterpene indole alkaloid (MIA) biosynthesis, undergoes a series of reactions to produce over 3,000 known MIAs such as vincristine, quinine, and strychnine.     

Cortical thickness of the dorsolateral prefrontal cortex predicts strategic choices in economic games

Human prosociality has been traditionally explained in the social sciences in terms of internalized social norms. Recent neuroscientific studies extended this traditional view of human prosociality by providing evidence that prosocial choices in economic games require cognitive control of the impulsive pursuit of self-interest. However, this view is challenged by an intuitive prosociality view emphasizing the spontaneous and heuristic basis of prosocial choices in economic games. We assessed the brain structure of 411 players of an ultimatum game (UG) and a dictator game (DG) and measured the strategic reasoning ability of 386. According to the reflective norm-enforcement view of prosociality, only those capable of strategically controlling their selfish impulses give a fair share in the UG, but cognitive control capability should not affect behavior in the DG. Conversely, we support the intuitive prosociality view by showing for the first time, to our knowledge, that strategic reasoning and cortical thickness of the dorsolateral prefrontal cortex were not related to giving in the UG but were negatively related to giving in the DG. This implies that the uncontrolled choice in the DG is prosocial rather than selfish, and those who have a thicker dorsolateral prefrontal cortex and are capable of strategic reasoning (goal-directed use of the theory of mind) control this intuitive drive for prosociality as a means to maximize reward when there are no future implications of choices.Humans are a cooperative species, and the question of why humans are so cooperative has been a subject of considerable interest in social and biological sciences (1–4). The traditional answer in the social sciences highlights critical roles of social norms and cultural values internalized as personal values and social preferences (5, 6). Recent neuroscientific studies of brain structure and activity extended this traditional view of human prosociality by showing that players of economic games act prosocially when they cognitively control selfish impulses (7–13). Experimental evidence shows that prosocial choices in economic games positively relate to local gray matter volume and thickness and the activation of brain areas that control selfish impulsive drives, such as the dorsolateral prefrontal cortex (DLPFC) and temporoparietal junction (TPJ) (7–9). Furthermore, impairment of cognitive control by disruption of DLPFC function prevents rejection of unfair offers in the ultimatum game (UG), which some authors considered prosocial and fairness-seeking behavior (10–13). Recently, this reflective view of human prosociality has been challenged by an alternative view emphasizing the intuitive nature of prosocial behavior, subsumed under intuitive prosociality (14) or heuristic cooperation (15–17). Support for the intuitive and automatic nature of prosocial behavior is provided by findings that prosocial choices are promoted under time pressure (15, 16, 18), under cognitive load (19–21), or after priming by successful experiences of intuitive decision making (15, 22). Also, participants who expressed more positive emotional words and less inhibitory words during and after an economic game cooperated more (23). Additionally, increased activity in the lateral prefrontal cortex was negatively related to fairness-seeking behavior in an economic game (24). According to the heuristic prosociality model (14–17), humans are predisposed to cooperate in social exchange situations. People fail to behave in a prosocial manner in social exchanges when this predisposition is overridden by strategic reasoning to secure their self-interest. By comparing participants’ behaviors in two economic games with brain structural differences and strategic reasoning abilities, we provide evidence that strategic reasoning controls, and thus reduces rather than promotes, game players’ prosocial behavior.The contrast between two simple, two-person economic games—namely, the dictator game (DG) and the UG—is often used to support the reflective prosocial model by demonstrating how strategic reasoning affects game players’ decisions. In both games, one player freely decides how much of a fixed reward to take and how much to leave for the other player. The difference between the two games is that the other player in the UG (termed “responder”) has the option to reject the decision made by the first player (termed “proposer”), causing both to earn nothing. This option is not provided to the second player in the DG, who plays the role of a “recipient”. The recipient simply receives whatever the first player (“dictator”) gives. The level of giving by the proposer in the UG is usually higher than that by the dictator in the DG (25). This is attributed to the proposer’s strategic reasoning, which requires inference of the recipient’s internal state and prediction of the resulting response (e.g., anger on the basis of unfair giving and subsequent rejection) (8, 9, 13). Given that neuroimaging and neuroendocrinological studies showed that negative emotions are associated with rejection of unfair offers (24, 26, 27), UG proposers may anticipate negative responses to unfair offers. UG proposers anticipate norm-enforcing responses (rejection of the offer) to norm-violating behavior (taking most of the reward) and strategically adjust giving behavior to secure acceptance by the responder. Thus, those capable of using strategic reasoning are expected to make fair offers in the UG compared with those who struggle to control their selfish drive for immediate reward.In contrast, in the DG, which requires no strategic reasoning to earn as much as possible, strategic control over selfish impulses is expected not to influence the player’s choices. Spitzer et al. (9) confirmed this by showing a positive correlation between the difference in giving in the UG and the DG (i.e., a measure of strategic reasoning) and activity of the right DLPFC and the lateral orbitofrontal cortex. Given earlier findings implicating the DLPFC in cognitive control of impulsive behavior (28–33), this is taken to support the reflective model of prosociality, in which prosocial behavior requires cognitive control of the impulsive drive toward selfish behavior. Steinbeis et al. (8) provided further support via a comparison of young children’s choices in the two economic games. Children took a large share in the DG while providing fairer amounts to responders in the UG. The children’s more generous giving in the UG may be based upon strategic reasoning regarding the possible consequences of not giving enough in the UG—that is, receiving no reward due to rejection by the other child—which plays no role in the DG. Thus, the difference in giving between the UG and the DG is considered to reflect the use of strategic reasoning in the UG. The strategic choices of more giving in the UG than in the DG is related to children’s age, cortical thickness, and activity of their left DLPFC. As children age and their DLPFC develops further, they become able to control their selfish drive and adjust their behavior to the anticipated negative consequences.This interpretation of UG–DG difference in prosocial giving as a reflection of strategic reasoning (8, 9) assumes that the default choice in the DG is impulsive and selfish. Younger children and those with a thinner DLPFC are presumably less capable of strategically adjusting their decisions to deal with anticipated responses and would impulsively pursue their own benefits in both the UG and the DG. In contrast, older children and those with a thicker DLPFC are more likely to have enhanced cognitive control, which can be used to strategically adjust their choices, especially in the UG but not in the DG. Therefore, a UG–DG positive reward transfer difference is produced by strategists’ control over the selfish impulses in the UG, whereas those who fail to control such impulses in the UG claim a considerable share in both games (Fig. 1A). In contrast, the alternative, intuitive prosociality model assumes that the uncontrolled choice is prosocial in both the UG and the DG, rather than selfish. Strategists control this impulse toward prosociality in the DG where immediate pursuit of self-interest causes no strategic problem (Fig. 1B). Nonstrategists do not control this impulse and provide a fair share in both games. Thus, a difference due to strategic reasoning is predicted to exist in the DG but not in the UG. The reflective and intuitive prosociality models thus make distinct predictions regarding the relationship between DLPFC thickness and behavior in the UG and DG. The reflective model predicts a positive relationship between DLPFC thickness and giving in the UG, whereas the intuitive model predicts a negative relationship between DLPFC thickness and giving in the DG.Open in a separate windowFig. 1.Schematic representations of how strategic considerations generate the difference between strategists (ST) and nonstrategists (Non-ST) in the UG and DG. A shows the prediction that strategic considerations should improve fair behavior in the UG. B shows the prediction that strategic considerations should depress fair behavior in the DG.These two alternative accounts of differences in giving in the UG and DG (8, 9) provide a way to test the intuitive selfishness assumption against the intuitive prosociality assumption. We first successfully replicated earlier findings that strategic behavior is more pronounced among those who had a thicker DLPFC than those who had a thinner DLPFC (8) in a study of 411 adult, nonstudent participants who played both the UG and DG and from whom brain structural images were obtained. Then, we found for the first time, to our knowledge, that local gray matter thickness of the DLPFC negatively correlated with giving in the DG but was not correlated with giving in the UG (Fig. 2 C and D). We further measured the strategic reasoning of 386 of these participants using a newly developed test of strategic reasoning, measured 411 participants’ Machiavellianism (34, 35) score, and found that those exhibiting better strategic reasoning behaved more selfishly in the DG than those with poor strategic reasoning, but no relationship was found between task performance and fairness in the UG. These striking findings provide strong evidence supporting the intuitive prosociality prediction depicted in Fig. 1B but not the reflective prosociality prediction shown in Fig. 1A.Open in a separate windowFig. 2.Brain areas in the Destrieu Atlas (A), the relationship of cortical thickness of the DLPFC (middle frontal gyrus) and strategic choice (UG–DG) (B), the relationship of its right cortical thickness and giving in the UG and DG (C), and the relationship of its left cortical thickness and giving in the UG and DG (D). The horizontal axis represents the residual cortical thickness adjusted for participants’ age, sex, and ICV. The vertical axis represents the mean strategic choice of the players, giving in the UG or the DG within 0.1-mm intervals of residual cortical thickness. Each interval spans 0.1 mm on the horizontal axis segment. The size of each circle shows the number of players who fell within the interval. Error bars are SEs. n = 411. Correlations are after adjusting for age, sex, and ICV.     

Bacterial lipids activate,synergize, and inhibit a developmental switch in choanoflagellates

In choanoflagellates, the closest living relatives of animals, multicellular rosette development is regulated by environmental bacteria. The simplicity of this evolutionarily relevant interaction provides an opportunity to identify the molecules and regulatory logic underpinning bacterial regulation of development. We find that the rosette-inducing bacterium Algoriphagus machipongonensis produces three structurally divergent classes of bioactive lipids that, together, activate, enhance, and inhibit rosette development in the choanoflagellate Salpingoeca rosetta. One class of molecules, the lysophosphatidylethanolamines (LPEs), elicits no response on its own but synergizes with activating sulfonolipid rosette-inducing factors (RIFs) to recapitulate the full bioactivity of live Algoriphagus. LPEs, although ubiquitous in bacteria and eukaryotes, have not previously been implicated in the regulation of a host–microbe interaction. This study reveals that multiple bacterially produced lipids converge to activate, enhance, and inhibit multicellular development in a choanoflagellate.The foundational event in animal origins—the transition to multicellularity (1–3)—occurred in oceans filled with diverse bacteria (4–7). There is a growing appreciation that specific bacteria direct diverse animal developmental processes, including light organ development in the Hawaiian bobtail squid and immune system development and maturation in organisms as diverse as cnidaria and mammals (8–20). However, the multicellularity of animals and the complex communities of bacteria with which they often interact hinder the complete characterization of many host–microbe dialogues.Choanoflagellates, a group of microbial eukaryotes that are the closest living relatives of animals (21–24), promise to help illuminate the mechanisms by which bacteria influence animal development. As did cells in the first animals, choanoflagellates use a distinctive collar of actin-filled microvilli surrounding a flow-generating apical flagellum to capture bacteria as prey (25–27). Indeed, choanoflagellate-like cells likely formed the basis for the evolution of animal epithelial cells that today provide a selective barrier for mediating interactions with bacteria (27–29).In many choanoflagellates, including Salpingoeca rosetta, a developmental program can be initiated such that single cells develop into multicellular rosettes. Importantly, rosette development does not occur through cell aggregation. Instead, as in the development of an animal from a zygote, rosettes develop from a single founding cell that undergoes serial rounds of oriented cell division, with the sister cells remaining stably adherent (Fig. 1). The orientation of the nascently divided cells around a central focus, the production of extracellular matrix, and the activity of a C-type lectin called Rosetteless, ultimately result in the formation of spherical, multicellular rosettes (30–32). Rosettes resemble morula-stage embryos, and the transition to multicellularity in S. rosetta evokes ancestral events that spawned the first animals (26, 27, 33).Open in a separate windowFig. 1.Stages of rosette development in S. rosetta. During rosette development, a single founding cell undergoes serial rounds of cell division, resulting in a structurally integrated rosette. Importantly, rosette development does not involve cell aggregation. Shown are a single cell (A), a pair of cells (B), a 4-cell rosette (C), an 8-cell rosette (D) and a 16-cell rosette (E).The initiation of rosette development was recently found to be induced by a coisolated environmental bacterium, Algoriphagus machipongonensis (phylum Bacteroidetes) (34, 35). The ecological relevance of the interaction between A. machipongonensis (hereafter, Algoriphagus) and S. rosetta is evidenced by the coexistence of these organisms in nature (35) and the predator–prey relationship between choanoflagellates and bacteria (25, 36). Indeed, rosettes likely have a fitness advantage over single cells in some environments, as multicellular choanoflagellates are predicted to produce increased flux of water past each cell (37), and prey capture studies reveal that rosettes collect more bacterial prey/cell/unit time than do single cells (38). However, in other environments, rosette development would likely reduce fitness as rosettes have reduced motility relative to single cells. Therefore, we hypothesize that choanoflagellates use bacterially produced molecules to identify environments in which rosette development might provide a fitness advantage.The simplicity of the interaction between S. rosetta and Algoriphagus, in which both members can be cultured together or independently, offers a biochemically tractable model for investigating the molecular bases of bacteria–eukaryote interactions. Using rosette development as a bioassay, the first rosette-inducing molecule, Rosette Inducing Factor-1 (RIF-1), was isolated from Algoriphagus. The observation that RIF-1 fails to fully recapitulate the bioactivity of the live bacterium (Fig. 2 A and C) raised the possibility that additional molecules might be required (35). To gain a more complete understanding of the molecules and regulatory logic by which bacteria regulate rosette development, we set out to identify the minimal suite of molecules produced by Algoriphagus that are necessary and sufficient to regulate rosette development in S. rosetta.Open in a separate windowFig. 2.Maximal rosette development requires lipid cofactor interactions. (A) When treated with media that lack necessary bacterial signals (Media Control), S. rosetta does not produce rosettes. In contrast, when treated with live Algoriphagus, Algoriphagus-conditioned media, OMVs from Algoriphagus, or bulk lipids extracted from Algoriphagus, rosettes develop at maximal (∼90% cells in rosettes) or near-maximal levels. (B) A heat map depicts the rosette-inducing activity of Algoriphagus lipid fractions used to treat SrEpac, either in isolation or in combination, at a final lipid concentration of 2 μg/mL Sulfonolipid-enriched fraction 11 was the only fraction sufficient to induce rosette development when tested alone (30% of cells in rosettes). Tests of each of the lipid fractions in combination revealed previously unidentified inhibitory and enhancing activity. Fractions 4 and 5 decreased rosette development (to 12% and 8%, respectively) in fraction 11-treated cells, whereas fraction 7 increased rosette development to 65%. (C) The RIF mix (solid square) and purified RIF-2 (solid circle) induced rosette development at micromolar concentrations. (Inset) RIF-1 (open circle) is active at femtomolar to nanomolar concentrations, but induces 10-fold lower levels of rosette development than RIF-2. The long gray box in the main graph indicates the range of concentrations at which RIF-1 is active and the range of its rosette-inducing activity. Rosette development was quantified 24 h after induction. Minor ticks on x axis are log-spaced.     

Entropic factors provide unusual reactivity and selectivity in epoxide-opening reactions promoted by water

Despite the myriad of selective enzymatic reactions that occur in water, chemists have rarely capitalized on the unique properties of this medium to govern selectivity in reactions. Here we report detailed mechanistic investigations of a water-promoted reaction that displays high selectivity for what is generally a disfavored product. A combination of structural and kinetic data indicates not only that synergy between substrate and water suppresses undesired pathways but also that water promotes the desired pathway by stabilizing charge in the transition state, facilitating proton transfer, doubly activating the substrate for reaction, and perhaps most remarkably, reorganizing the substrate into a reactive conformation that leads to the observed product. This approach serves as an outline for a general strategy of exploiting solvent-solute interactions to achieve unusual reactivity in chemical reactions. These findings may also have implications in the biosynthesis of the ladder polyether natural products, such as the brevetoxins and ciguatoxins.Given its simple structure and low molecular weight, water is a remarkably complex substance. Several landmark investigations (1) have revealed the ability of water to self-assemble to form sophisticated dynamic hydrogen bond networks, which accounts for its unusual properties, such as its high boiling point and high surface tension (2). Despite the unique properties that it offers and that nature has exploited, water is generally eschewed in synthetic chemistry largely because of chemical incompatibility with many commonly used reagents and the low aqueous solubility of many organic molecules coupled with the attendant assumption that homogeneity is required for reactivity. Although several important examples of remarkable reactivity have been documented for reactions carried out in water (3–5), on the surface of water (6, 7), or in micelles suspended in water (8, 9), utilization of the “biological solvent” in organic reactions remains uncommon.We became acutely aware of the remarkable properties of water when we discovered that neutral aqueous solutions of epoxy alcohol 1a underwent a spontaneous and selective “endo” cyclization reaction to form 6,6-fused bicyclic product 2a (Fig. 1) (10–12). Exceptional reactivity and selectivity were observed only when two criteria were satisfied: The substrate contained a six-membered tetrahydropyran ring, and the solvent used was water at pH 7.0. These surprising results were counter to a set of general empirical rules put forth by Baldwin for cyclization reactions, which state that the smaller ring product resulting from what is commonly called an exo cyclization is favored for similar reactions (13). The tetrahydropyranol ring, therefore, appeared to “template” the endo cyclization pathway to form the larger ring product with water playing a critical yet heretofore unknown role.Open in a separate windowFig. 1.Endo selective cyclization of epoxy alcohols templated by a tetrahydropyran ring(s) and promoted by water.Moreover, 2a is a substructure found in a large family of natural products commonly referred to as the ladder polyethers (e.g., brevetoxin B, S) (14–16). These constituents of harmful algal blooms (a.k.a., red tide) have attracted significant attention (17) because of the remarkable structural regularities that temper their apparent complexity. Several biogenetic pathways for the construction of rings of these natural products have been proposed and generally involve a cascade of cyclizations by way of epoxide-opening reactions (Fig. 2) (18–23). However, this hypothesis requires that all of the epoxide ring-opening events proceed with atypical endo regioselectivity.Open in a separate windowFig. 2.Biosynthetic proposal for the formation of the ladder polyether natural product brevetoxin B, a potent neurotoxin.Our discovery that reactions of 1a in water preferentially lead to the endo product 2a rather than exo product 3a provided a possible means to overcome this obstacle. Further support was provided when we demonstrated that cascade reactions akin to those proposed for the biosynthesis of the natural products was possible with the selective endo cyclization of di- and triepoxide analogs, such as 4 and 6, respectively (Fig. 1). Once again selective reactions were only observed when reactions were carried out in neutral water.These striking results and their possible relevance to the biogenesis of the ladder polyether natural products prompted us to investigate the mechanism of the cyclization reaction. Here we report the culmination of these efforts. They reveal an intimate connection between water and the tetrahydropyran ring (template) of 1a, provide support for the feasibility of the biosynthetic proposal for the ladder polyether natural products, and demonstrate the importance of solvent–substrate interactions for promoting selectivity. We believe that this last concept may have broad implications and a different means for chemists to attain unusual selectivity for a variety of chemical reactions.     

Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel

Flux-dependent inactivation that arises from functional coupling between the inner gate and the selectivity filter is widespread in ion channels. The structural basis of this coupling has only been well characterized in KcsA. Here we present NMR data demonstrating structural and dynamic coupling between the selectivity filter and intracellular constriction point in the bacterial nonselective cation channel, NaK. This transmembrane allosteric communication must be structurally different from KcsA because the NaK selectivity filter does not collapse under low-cation conditions. Comparison of NMR spectra of the nonselective NaK and potassium-selective NaK2K indicates that the number of ion binding sites in the selectivity filter shifts the equilibrium distribution of structural states throughout the channel. This finding was unexpected given the nearly identical crystal structure of NaK and NaK2K outside the immediate vicinity of the selectivity filter. Our results highlight the tight structural and dynamic coupling between the selectivity filter and the channel scaffold, which has significant implications for channel function. NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating.Ion conduction through the pore domain of cation channels is regulated by two gates: an inner gate at the bundle crossing of the pore-lining transmembrane helices and an outer gate located at the selectivity filter (Fig. 1 B and C). These two gates are functionally coupled as demonstrated by C-type inactivation, in which channel opening triggers loss of conduction at the selectivity filter (1–4). A structural model for C-type inactivation has been developed for KcsA, with selectivity filter collapse occurring upon channel opening (4–10). In the reverse pathway, inactivation of the selectivity filter has been linked to changes at the inner gate (5–14). However, flux-dependent inactivation occurs in Na⁺ and Ca²⁺ channels as well and would likely require a structurally different mechanism to explain coupling between the selectivity filter and inner gate (7, 13–18).Open in a separate windowFig. 1.Crystal structures of the nonselective cation channel NaK and the potassium-selective NaK2K mutant show structural changes restricted to the area of the selectivity filter. Alignment of the WT NaK (gray; PDB 3E8H) and NaK2K (light blue; PDB 3OUF) selectivity filters shows a KcsA-like four-ion-binding-site selectivity filter is created by the NaK2K mutations (D66Y and N68D) (A), but no structural changes occur outside the vicinity of the selectivity filter (B). (C) Full-length NaK (green; PDB 2AHZ) represents a closed conformation. Alignment of this structure with NaK (gray) highlights the changes in the M2 hinge (arrow), hydrophobic cluster (residues F24, F28, and F94 shown as sticks), and constriction point (arrow; residue Q103 shown as sticks) upon channel opening. Two (A) or three monomers (B and C) from the tetramer are shown for clarity.This study provides experimental evidence of structural and dynamic coupling between the inner gate and selectivity filter in the NaK channel, a nonselective cation channel from Bacillus cereus (19). These results were entirely unexpected given the available high-resolution crystal structures (20, 21). The NaK channel has the same basic pore architecture as K⁺ channels (Fig. 1 B and C) and has become a second model system for investigating ion selectivity and gating due to its distinct selectivity filter sequence (₆₃TVGDGN₆₈) and structure (19–23). Most strikingly, there are only two ion binding sites in the selectivity filter of the nonselective NaK channel (Fig. 1A) (21, 24). However, mutation of two residues in the selectivity filter sequence converts the NaK selectivity filter to the canonical KcsA sequence (₆₃TVGYGD₆₈; Fig. 1 A and B), leading to K⁺ selectivity and a KcsA-like selectivity filter structure with four ion binding sites (21, 23). This K⁺-selective mutant of NaK is called NaK2K. Outside of the immediate vicinity of the two mutations in the selectivity filter, high-resolution crystal structures of NaK and NaK2K are essentially identical (Fig. 1B) with an all-atom rmsd of only 0.24 Å.NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating because there is no evidence for any collapse or structural change in the selectivity filter. The NaK selectivity filter structure is identical in Na⁺ or K⁺ (22) and even in low-ion conditions (25), consistent with its nonselective behavior. Even the selective NaK2K filter appears structurally stable in all available crystal structures (25). Here we use NMR spectroscopy to study bicelle-solubilized NaK. Surprisingly, we find significant differences in the NMR spectra of NaK and NaK2K that extend throughout the protein and are not localized to the selectivity filter region. This, combined with NMR dynamics studies of NaK, suggests a dynamic pathway for transmembrane coupling between the inner gate and selectivity filter of NaK.     

Insect nicotinic receptor interactions in vivo with neonicotinoid,organophosphorus, and methylcarbamate insecticides and a synergist

The nicotinic acetylcholine (ACh) receptor (nAChR) is the principal insecticide target. Nearly half of the insecticides by number and world market value are neonicotinoids acting as nAChR agonists or organophosphorus (OP) and methylcarbamate (MC) acetylcholinesterase (AChE) inhibitors. There was no previous evidence for in vivo interactions of the nAChR agonists and AChE inhibitors. The nitromethyleneimidazole (NMI) analog of imidacloprid, a highly potent neonicotinoid, was used here as a radioligand, uniquely allowing for direct measurements of house fly (Musca domestica) head nAChR in vivo interactions with various nicotinic agents. Nine neonicotinoids inhibited house fly brain nAChR [³H]NMI binding in vivo, corresponding to their in vitro potency and the poisoning signs or toxicity they produced in intrathoracically treated house flies. Interestingly, nine topically applied OP or MC insecticides or analogs also gave similar results relative to in vivo nAChR binding inhibition and toxicity, but now also correlating with in vivo brain AChE inhibition, indicating that ACh is the ultimate OP- or MC-induced nAChR active agent. These findings on [³H]NMI binding in house fly brain membranes validate the nAChR in vivo target for the neonicotinoids, OPs and MCs. As an exception, the remarkably potent OP neonicotinoid synergist, O-propyl O-(2-propynyl) phenylphosphonate, inhibited nAChR in vivo without the corresponding AChE inhibition, possibly via a reactive ketene metabolite reacting with a critical nucleophile in the cytochrome P450 active site and the nAChR NMI binding site.The nicotinic nervous system has two principal sites of insecticide action, the nicotinic receptor (nAChR) activated by acetylcholine (ACh) and neonicotinoid agonists (1–6), and acetylcholinesterase (AChE) inhibited by organophosphorus (OP) and methylcarbamate (MC) compounds to generate and maintain localized toxic ACh levels (Fig. 1) (7). The nAChR and AChE targets have been identified in insects by multiple techniques but not by direct assays of the ACh binding site in the brain of poisoned insects. Here we use the outstanding insecticidal potency of the nitromethyleneimidazole (NMI) analog of imidacloprid (IMI) (8) as a radioligand (9), designated [³H]NMI, to directly measure the house fly (Musca domestica) nAChR not only in vitro but also in vivo, allowing us to validate by a previously undescribed method the neonicotinoid direct and OP/MC indirect nAChR targets (Fig. 2). This approach also helped solve the intriguing mechanism by which an O-(2-propynyl) phosphorus compound strongly synergizes neonicotinoid insecticidal activity (10) by dual inhibition of cytochrome P450 (CYP) (11–13) and the nAChR agonist site (described herein). Insecticide disruption at the insect nAChR can now be readily studied in vitro and in vivo with a single radioligand allowing better understanding of the action of several principal insecticide chemotypes (Fig. 3).Open in a separate windowFig. 1.The insect nicotinic receptor is the direct or indirect target for neonicotinoids, organophosphorus compounds and methylcarbamates, which make up about 45% of the insecticides by number and world market value (2, 7).Open in a separate windowFig. 2.In this study, Musca nicotinic receptor in vivo interactions with major insecticide chemotypes are revealed by a [³H]NMI radioligand reporter assay. *Position of tritium label.Open in a separate windowFig. 3.Two neonicotinoid nicotinic agonists and two anticholinesterase insecticides.     

Cleavage and polyadenylation specificity factor 30: An RNA-binding zinc-finger protein with an unexpected 2Fe–2S cluster

Cleavage and polyadenylation specificity factor 30 (CPSF30) is a key protein involved in pre-mRNA processing. CPSF30 contains five Cys₃His domains (annotated as “zinc-finger” domains). Using inductively coupled plasma mass spectrometry, X-ray absorption spectroscopy, and UV-visible spectroscopy, we report that CPSF30 is isolated with iron, in addition to zinc. Iron is present in CPSF30 as a 2Fe–2S cluster and uses one of the Cys₃His domains; 2Fe–2S clusters with a Cys₃His ligand set are rare and notably have also been identified in MitoNEET, a protein that was also annotated as a zinc finger. These findings support a role for iron in some zinc-finger proteins. Using electrophoretic mobility shift assays and fluorescence anisotropy, we report that CPSF30 selectively recognizes the AU-rich hexamer (AAUAAA) sequence present in pre-mRNA, providing the first molecular-based evidence to our knowledge for CPSF30/RNA binding. Removal of zinc, or both zinc and iron, abrogates binding, whereas removal of just iron significantly lessens binding. From these data we propose a model for RNA recognition that involves a metal-dependent cooperative binding mechanism.Zinc-finger proteins (ZFs) are a large class of proteins that use zinc as structural cofactors (1–4). ZFs perform a variety of functions ranging from the modulation of gene expression through specific interactions with DNA or RNA to the control of signaling pathways via protein–protein interactions. The general feature that defines a ZF protein is the presence of one or more domains that contain a combination of four cysteine and/or histidine residues that serve as ligands for zinc. When zinc binds to these ligands, the domain adopts the structure necessary for function (1–4).ZFs are typically identified by the presence of cysteine and histidine residues in regular repeats and are categorized into classes based upon the number of cysteine and histidine residues, and the spacing between the residues (1, 2). At least 14 distinct classes of ZFs have been identified to date. ZFs are highly abundant, with more than 3% of the proteins in the human genome annotated as ZFs, based upon their sequences (1, 5–9). In some cases, there are considerable in vitro and in vivo data that support the annotation of proteins as ZFs, whereas in other cases the only evidence that a protein is a ZF comes from its amino acid sequence. The best-studied class of ZFs comprises the “classical” ZFs. This class is composed of ZFs that contain a Cys₂His₂ domain (CysX_2–5CysX_12–13HisX_3–5His). Classical ZFs adopt an alpha-helical/antiparallel beta-sheet structure when zinc is coordinated and bind DNA in a sequence-specific manner (2, 4). The remaining classes of ZFs are collectively called “nonclassical” ZFs (1). One class of nonclassical ZFs is the Cys₃His class (CysX_7–9CysX_4–6CysX₃His). The first protein of this class to be identified was tristetraprolin, which contains two Cys₃His domains and regulates cytokine mRNAs via a specific ZF domain/RNA binding interaction (1). With the publication of genome sequences this domain has been found in a myriad of proteins. The National Center for Biotechnology Information (NCBI) conserved domain architecture tool identifies 404 distinct proteins (both hypothetical and experimentally validated) that contain this domain, and humans contain at least 60 (Fig. 1). As a class, these proteins are predicted to be involved in RNA regulation; however, the function(s) of most of these proteins have not yet been established (1, 2, 10, 11).Open in a separate windowFig. 1.Survey of the CCCH domain containing proteins in H. sapiens.One important Cys₃His ZF protein is cleavage and polyadenylation specificity factor 30 (CPSF30) (2, 12). CPSF30 contains five Cys₃His domains. CPSF30 is part of a complex of proteins, collectively called CPSF, that are involved in the polyadenylation step of pre-mRNA processing (16). The other members of CPSF are CPSF160, CPSF73, CPSF100, Fip1, and Wdr33 (16). Polyadenylation is a 3′ end maturation step that all eukaryotic mRNAs (except histones) undergo (Fig. 2) (12). It involves endonucleolytic cleavage of the pre-mRNA followed by addition of a polyadenosine tail. Polyadenylation occurs at a specific region of the pre-mRNA called the polyadenylation cleavage site (PAS). The PAS consists of an upstream element with the conserved sequence AAUAAA (also called the AU-hexamer), a stretch of bases where cleavage occurs, after which a conserved GU-rich or U-rich sequence is present (usually between 40–60 nt after the cleavage site) (12, 13). CPSF73 is the endonuclease that cleaves the RNA; the roles of the other CPSF proteins are less clear (12, 13). Initially, CPSF160 was identified as the protein within the CPSF complex that recognizes the AU-hexamer (14–16); however, two recent studies using cell-based methods found that CPSF160 does not play this role (17, 18). Instead, CPSF30 and Wdr33 were identified as the proteins involved in AU-hexamer recognition (17, 18). These findings are intriguing in light of evidence that the H1N1 human influenza virus protein NS1A targets CPSF30 to obstruct cellular mRNA processing (19–21), suggesting that the link between NS1A and cellular mRNA processing is RNA recognition by CPSF30.Open in a separate windowFig. 2.Cartoon of pre-mRNA processing, with possible roles of CPSF30 highlighted in blue.Given these cell-based results that CPSF30 is involved in recognition of the AU-hexamer of pre-mRNA along with our emerging understanding that CCCH-type ZFs are a general ZF motif involved in AU-rich RNA sequence recognition, we sought to determine whether CPFS30 directly recognizes the pre-mRNA AU-hexamer sequence via its CCCH domains by isolating CPSF30 and examining its RNA binding properties at the molecular level. CPSF30 contains five CCCH domains, and our hypothesis was that CPSF30 would bind five zinc ions at these CCCH domains and selectively recognize the AU-rich hexamer of pre-mRNA. To our surprise, CPSF30 was a reddish-colored protein upon isolation and purification, which suggested the presence of an iron cofactor. Here, we report that CPSF30 contains a 2Fe–2S site, with a CCCH ligand set, in addition to zinc. We also report that CPSF30 selectively recognizes the polyadenylation hexamer (AAUAAA) of pre-mRNA in a cooperative and metal-dependent manner. These findings are discussed in the context of CCCH “zinc” domains, iron, and recognition of AU-rich RNA sequences.     

High-energy chemistry of formamide: A unified mechanism of nucleobase formation

The coincidence of the Late Heavy Bombardment (LHB) period and the emergence of terrestrial life about 4 billion years ago suggest that extraterrestrial impacts could contribute to the synthesis of the building blocks of the first life-giving molecules. We simulated the high-energy synthesis of nucleobases from formamide during the impact of an extraterrestrial body. A high-power laser has been used to induce the dielectric breakdown of the plasma produced by the impact. The results demonstrate that the initial dissociation of the formamide molecule could produce a large amount of highly reactive CN and NH radicals, which could further react with formamide to produce adenine, guanine, cytosine, and uracil. Based on GC-MS, high-resolution FTIR spectroscopic results, as well as theoretical calculations, we present a comprehensive mechanistic model, which accounts for all steps taking place in the studied impact chemistry. Our findings thus demonstrate that extraterrestrial impacts, which were one order of magnitude more abundant during the LHB period than before and after, could not only destroy the existing ancient life forms, but could also contribute to the creation of biogenic molecules.As the Sun formed from its molecular cloud, it was accompanied by a disk of material that consisted of gas and small dust particles. Over several tens of millions of years, these dust particles accumulated and formed the planets we see today. This process occurred in several stages in the terrestrial planet zone, eventually culminating in massive, potentially moon-forming impacts on the proto-Earth (1). Then, following the solidification of the Moon ∼4.5 Ga, the initially heavy impactor flux declined (2) and increased again during the Late Heavy Bombardment (LHB) some ∼4−3.85 Ga (2). Our best models for the origin of the LHB link the LHB to a dynamic instability in the outer solar system (the so-called Nice model) (3, 4), when Jupiter’s orbit changed as a result of close encounters with ice giants and small cometary bodies. These changes resulted in the release of impactors from their previously stable asteroidal and cometary reservoirs. The synthesis of observation and theoretical constraints indicates that the impactor flux on Earth was ∼10 times higher at the LHB than in the period immediately preceding the LHB and that this flux slowly decayed afterward (5–7). At the peak, the LHB most likely involved an impact frequency of 10⁹ tons of material per year (ref. 5 and Fig. 1A). The typical impact speeds are estimated to have increased from ∼9 to ∼21 km/s once the LHB began. The ratio of the gravitational cross-sections of Earth and the Moon is found to be ∼17:1. Thus, for every lunar basin, such as Orientale or Imbrium, ∼17 basins should have formed on Earth (8).Fig. 1.Impactor flux on the Moon surface according Koeberl et al. (5) is shown in A. It is estimated that Earth was exposed to an impactor flux that was at least 1 order of magnitude higher than that of the Moon. The conversion formula for such an estimate is ...Such huge impact activity also had extensive implications for the evolution of early Earth (selected milestones in early Earth’s history are shown in Fig. 1B) (9): The atmosphere was partly eroded and transformed (10, 11), and the hydrosphere was enriched by water (12, 13). Crucially, these impact-related processes most likely also contributed to the transformation of biomolecules and their precursors on Earth’s surface, which would have relevant consequences on the origin of life (14, 15).Although the impact energies were most likely not large enough to produce ocean evaporation or globally sterilizing events (16), they could have served as local energy sources for biomolecule synthesis (17–19) Therefore, the high-impact activity may not have been harmful for the formation of biomolecules and the first living structures. Conversely, it may have been the source of energy required to initiate chemical reactions, such as the synthesis of biomolecules (20).One of the current landmarks of prebiotic chemistry is the proposal that formamide could be the parent compound of the components of the first informational polymers (21). Saladino et al. extensively studied the formamide-based chemistry that can lead to the synthesis of nucleobases and nucleotides and their metabolic products (22–24). By choosing the appropriate catalyst, purine, adenine, guanine, and cytosine (catalyzed by limestone, kaolin, silica, alumina, or zeolite), thymine (irradiated by sunlight and catalyzed by TiO₂), and hypoxanthine and uracil (in the presence of montmorillonites) were obtained (23–27).Our recent studies reported the formation of the canonical nucleobases, as well as purine and glycine, during the dielectric breakdown induced by the high-power laser Asterix in the presence of catalytic materials (meteorites, TiO₂, clay) (17, 18). Formamide-based synthesis in the high-density energy event (impact plasma) can solve the long-standing enigma of the simultaneous formation of all four nucleobases. The main objective of the present study is to demonstrate a unified mechanism of the formation of the nucleobases through the reaction of formamide and its dissociation products in a high-energy impact event relevant to LHB.The plasma formed by the impact of an extraterrestrial body was simulated using the high-power chemical iodine Prague Asterix Laser System (PALS). During the dielectric breakdown in gas (laser-induced dielectric breakdown or LIDB) generated by a laser pulse of energy of 150 J (time interval of ∼350 ps, wavelength of 1.315 μm, and output density of 10¹⁴ to 10¹⁶ W/cm²), all expected outcomes for a high-energy density event occurred: shock rises in temperature to 4,500 K (28), the formation of a shock wave, and the generation of secondary hard radiation (UV-VUV, XUV, and X-ray). LIDB generated by the PALS laser is shown in Fig. 2A, and the laser hall is shown in Fig. 2B.Fig. 2.(A) Experimental set-up used for the formamide irradiation. (B) Laser hall with the sample placed at the end of the beam line.The dielectric of gaseous nitrogen represents an atmosphere in which hot, dense plasma is formed, and the liquid sample represents a lagoon containing formamide (29). The unstable radicals produced in the formamide dissociation have been identified and quantified using time-resolved discharge emission spectroscopy (30, 31) and the models of plasma chemistry described in our previous work (17). The stable products were analyzed using high-resolution IR spectroscopy. The impact event was simulated using LIDB in formamide liquid. Finally, the canonical nucleobases were detected using GC-MS, and quantum chemical calculations were used to propose a plausible mechanism for their formation (see Materials and Methods for experimental setup and quantum chemical calculations).