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.     

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.     

Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS.Although proteins adopt structures determined by their amino acid sequences, they are not static objects and fluctuate among ensembles of conformations (1). Transitions between these states can occur on a variety of length scales (Å to nm) and time scales (ps to s) and have been linked to functionally relevant phenomena such as allosteric signaling, enzyme catalysis, and protein–protein interactions (2–4). Indeed, protein conformational fluctuations and dynamics, often associated with static and dynamic inhomogeneity, are thought to play a crucial role in biomolecular functions (5–11). It is difficult to characterize such spatially and temporally inhomogeneous dynamics in bulk solution by an ensemble-averaged measurement, especially in proteins that undergo multiple-conformation transformations. In such cases, single-molecule spectroscopy is a powerful approach to analyze protein conformational states and dynamics under physiological conditions, and can provide a molecular-level perspective on how a protein’s structural dynamics link to its functional mechanisms (12–21).A case in point is the nitric oxide synthase (NOS) enzymes (22–24), whose nitric oxide (NO) biosynthesis involves electron transfer reactions that are associated with relatively large-scale movement (tens of Å) of the enzyme domains (Fig. 1A). During catalysis, NADPH-derived electrons first transfer into an FAD domain and an FMN domain in NOS that together comprise the NOS reductase domain (NOSr), and then transfer from the FMN domain to a heme group that is bound in a separate attached “oxygenase” domain, which then enables NO synthesis to begin (22, 25–27). The electron transfers into and out of the FMN domain are the key steps for catalysis, and they appear to rely on the FMN domain cycling between electron-accepting and electron-donating conformational states (28, 29) (Fig. 1B). In this model, the FMN domain is suggested to be highly dynamic and flexible due to a connecting hinge that allows it to alternate between its electron-accepting (FAD→FMN) or closed conformation and electron-donating (FMN→heme) or open conformation (Fig. 1 A and B) (28, 30–36). In the electron-accepting closed conformation, the FMN domain interacts with the NADPH/FAD domain (FNR domain) to receive electrons, whereas in the electron donating open conformation the FMN domain has moved away to expose the bound FMN cofactor so that it may transfer electrons to a protein acceptor like the NOS oxygenase domain, or to a generic protein acceptor like cytochrome c. In this way, the reductase domain structure cycles between closed and open conformations to deliver electrons, according to a conformational equilibrium that determines the movements and thus the electron flux capacity of the FMN domain (25, 28, 32, 34, 35, 37). A similar conformational switching mechanism is thought to enable electron transfer through the FMN domain in the related flavoproteins NADPH-cytochrome P450 reductase and methionine synthase reductase (38–42).Open in a separate windowFig. 1.(A) The nNOSr ribbon structure (from PDB: 1TLL) showing bound FAD (yellow) in FNR domain (green), FMN (orange) in FMN domain (yellow), connecting hinge (blue), and the Cy3–Cy5 label positions (pink) and distance (42 Å, dashed line). (B) Cartoon of an equilibrium between the FMN-closed and FMN-open states, with Cy dye label positions indicated. (C) Cytochrome c reductase activity of nNOSr proteins in their CaM-bound and CaM-free states. Color scheme of bar graphs: Black, WT nNOSr unlabeled; Red, Cys-lite (CL) nNOSr unlabeled; Blue, E827C/Q1268C CL nNOSr unlabeled; and Dark cyan, E827C/Q1268C CL nNOSr labeled.NOS enzymes also contain a calmodulin (CaM) binding domain that is located just before the N terminus of the FMN domain (Fig. 1B), and this provides an important layer of regulation (25, 27). CaM binding to NOS enzymes increases electron transfer from NADPH through the reductase domain and also triggers electron transfer from the FMN domain to the NOS heme as is required for NO synthesis (31, 32). The ability of CaM, or similar signaling proteins, to regulate electron transfer reactions in enzymes is unusual, and the mechanism is a topic of interest and intensive study. It has long been known that CaM binding alters NOSr structure such that, on average, it populates a more open conformation (43, 44). Recent equilibrium studies have detected a buildup of between two to four discreet conformational populations in NOS enzymes and in related flavoproteins, and in some cases, have also estimated the distances between the bound FAD and FMN cofactors in the different species (26, 36, 37, 39, 40), and furthermore, have confirmed that CaM shifts the NOS population distribution toward more open conformations (34, 36, 45). Although valuable, such ensemble-averaged results about conformational states cannot explain how electrons transfer through these enzymes, or how CaM increases the electron flux in NOS, because answering these questions requires a coordinate understanding of the dynamics of the conformational fluctuations. Indeed, computer modeling has indicated that a shift toward more open conformations as is induced by CaM binding to nNOS should, on its own, actually diminish electron flux through nNOS and through certain related flavoproteins (38). Despite its importance, measuring enzyme conformational fluctuation dynamics is highly challenging, and as far as we know, there have been no direct measures on the NOS enzymes or on related flavoproteins, nor studies on how CaM binding might influence the conformational fluctuation dynamics in NOS.To address this gap, we used single-molecule fluorescence energy resonance transfer (FRET) spectroscopy to characterize individual molecules of nNOSr that had been labeled at two specific positions with Cyanine 3 (Cy3) donor and Cyanine 5 (Cy5) acceptor dye molecules, regarding their conformational states distribution and the associated conformational fluctuation dynamics, which in turn enabled us to determine how CaM binding impacts both parameters. This work provides a unique perspective and a novel study of the NOS enzymes and within the broader flavoprotein family, which includes the mammalian enzymes methionine synthase reductase (MSR) and cytochrome P450 reductase (CPR), and reveals how CaM’s control of the conformational behaviors may regulate the electron transfer reactions of NOS catalysis.     

Dynamical malaria models reveal how immunity buffers effect of climate variability

Assessing the influence of climate on the incidence of Plasmodium falciparum malaria worldwide and how it might impact local malaria dynamics is complex and extrapolation to other settings or future times is controversial. This is especially true in the light of the particularities of the short- and long-term immune responses to infection. In sites of epidemic malaria transmission, it is widely accepted that climate plays an important role in driving malaria outbreaks. However, little is known about the role of climate in endemic settings where clinical immunity develops early in life. To disentangle these differences among high- and low-transmission settings we applied a dynamical model to two unique adjacent cohorts of mesoendemic seasonal and holoendemic perennial malaria transmission in Senegal followed for two decades, recording daily P. falciparum cases. As both cohorts are subject to similar meteorological conditions, we were able to analyze the relevance of different immunological mechanisms compared with climatic forcing in malaria transmission. Transmission was first modeled by using similarly unique datasets of entomological inoculation rate. A stochastic nonlinear human–mosquito model that includes rainfall and temperature covariates, drug treatment periods, and population variability is capable of simulating the complete dynamics of reported malaria cases for both villages. We found that under moderate transmission intensity climate is crucial; however, under high endemicity the development of clinical immunity buffers any effect of climate. Our models open the possibility of forecasting malaria from climate in endemic regions but only after accounting for the interaction between climate and immunity.Climate plays a key role in driving the seasonal outbreaks of malaria in areas of low or unstable malaria transmission (1–4). Recent studies have shown the possibility of forecasting malaria outbreaks on the basis of climate information and disease features in these low-transmission settings (3, 5). For instance, in highland malaria the role of warming temperatures is vividly debated (4, 6–8) and in desert-epidemic fringes early studies reported predictions of a widespread increase in malaria transmission (9–12). Recent malaria models also predict a global net increase of the population at risk (13); however, others suggest a shift in spatial distribution rather than a large net increase in total malaria incidence worldwide (14, 15). In epidemic fringes, variation in the incidence of disease is largely determined by the seasonal variation of the mosquito population’s occurrence and density, which are essentially modulated by local rainfall [e.g., if water limited (3, 16)] or temperature [e.g., if altitude limited (2, 4, 8)]. This is not the case in holoendemic transmission settings, where incidence of disease is determined not only by external forces, but also by the development of clinical and antiparasite immunity. Under intense transmission, clinical immunity develops during childhood after many infections (17, 18), whereby the individual can tolerate nonnegligible parasite densities without showing symptoms. Subsequently, antiparasite immunity, which enables control of parasite density, develops much more slowly (19), leading to a state of premunition, whereby individuals harbor chronic, potentially subpatent infections (20). Continued exposure to the parasite is seemingly required to maintain such premunition (21). Complete protection from further infections is rarely, if ever, achieved. In such high-transmission regions, the relationship between local climate and disease is difficult to disentangle.In this study, two unique long-term cohort datasets from villages separated by 5 km but with markedly different malaria transmission intensity (Fig. 1, Upper) enable us to showcase the relative roles of internal and external factors in malaria epidemiology, assess the potential degree of predictability emanating from climatic variability, and generate estimates of key parameters in determining malaria population dynamics. To this end, we use a recently developed inference methodology for nonlinear stochastic dynamical systems, successfully applied to epidemic dynamics (3, 16) but never applied to endemic settings. A general coupled mosquito–human compartment model that includes possible key mechanisms common to both villages serves our aim of disentangling differences related to immunity, infectivity, superinfection, and asymptomatic infections as well as to measure the relevance of local climate for each village.Open in a separate windowFig. 1.(Upper) P. falciparum malaria incidence for Dielmo (red) and Ndiop (green). Vertical dotted black lines separate the four different drug regimes (from left to right: Quinine, Chloroquine, Fansidar, and ACT). Incidence units are episodes per person per month. (Lower) Average annual cycles computed as the average month by month for the whole time series of P. falciparum monthly incidence for Dielmo (red) and Ndiop (green), local rainfall (blue), and temperature (orange). Shaded regions correspond to the SD.     

Evolution of stickleback in 50 years on earthquake-uplifted islands

How rapidly can animal populations in the wild evolve when faced with sudden environmental shifts? Uplift during the 1964 Great Alaska Earthquake abruptly created freshwater ponds on multiple islands in Prince William Sound and the Gulf of Alaska. In the short time since the earthquake, the phenotypes of resident freshwater threespine stickleback fish on at least three of these islands have changed dramatically from their oceanic ancestors. To test the hypothesis that these freshwater populations were derived from oceanic ancestors only 50 y ago, we generated over 130,000 single-nucleotide polymorphism genotypes from more than 1,000 individuals using restriction site-associated DNA sequencing (RAD-seq). Population genomic analyses of these data support the hypothesis of recent and repeated, independent colonization of freshwater habitats by oceanic ancestors. We find evidence of recurrent gene flow between oceanic and freshwater ecotypes where they co-occur. Our data implicate natural selection in phenotypic diversification and support the hypothesis that the metapopulation organization of this species helps maintain a large pool of genetic variation that can be redeployed rapidly when oceanic stickleback colonize freshwater environments. We find that the freshwater populations, despite population genetic analyses clearly supporting their young age, have diverged phenotypically from oceanic ancestors to nearly the same extent as populations that were likely founded thousands of years ago. Our results support the intriguing hypothesis that most stickleback evolution in fresh water occurs within the first few decades after invasion of a novel environment.On March 27, 1964, the largest earthquake ever recorded in North America struck the south coast of Alaska (1, 2). This catastrophic event uplifted islands in Prince William Sound and the Gulf of Alaska in just a few minutes, creating ponds from formerly marine habitat and setting the stage for the diversification of threespine stickleback fish (Gasterosteus aculeatus) in these new freshwater sites. This seismic disturbance provides an excellent opportunity to address long-standing evolutionary questions regarding how often dramatic phenotypic shifts can happen over contemporary timescales (3–7).Despite examples of rapid divergence in wild populations, evolutionary rates may often be constrained by a suite of factors (8). For example, evolution in new habitats may be limited by waiting times for new beneficial mutations (9–11). Even when adaptation occurs from standing genetic variation, evolution via selection of numerous independent loci of small effect may be time consuming (12–16). We know, however, that evolution can occur rapidly, particularly under artificial selection or in human-altered landscapes (17–21). In addition, empirical studies in the wild—particularly in response to significant environmental changes—have demonstrated that strong selection and rapid evolution over decades may be more common than once thought (22–24).A rapid evolutionary response is predicted when the intensity of directional selection is strong (11, 25), a scenario likely to occur immediately after a habitat shift or environmental disturbance (26, 27). However, because of previous technological limitations, few studies of rapid differentiation in the wild have included genetic data to fully disentangle evolution from induced phenotypic plasticity. The small numbers of markers previously available for most population genetic studies have not provided the necessary precision with which to analyze very recently diverged populations (but see refs. 28 and 29). As a consequence, the frequency of contemporary evolution in the wild is still poorly defined, and its genetic and genomic basis remains unclear (30).Advances in sequencing technology now allow the precise inference from genomic data of colonization history and evolutionary patterns that have occurred over just a few generations (31, 32). The threespine stickleback system is ideal for testing hypotheses about contemporary evolution. Postglacial adaptive radiations over the last 12,000–20,000 y in newly available freshwater habitats have spawned divergent phenotypes that demonstrate parallel phenotypic evolution (33, 34), with underlying parallel genetic (35–39) and genomic (40–43) bases. An open question, however, is whether this parallel divergence in stickleback actually requires thousands of years, or whether it can occur in nature over decadal timescales, as is implied by studies of a small number of recently formed artificial and wild stickleback populations (44–50). Also unknown is how often the countless populations of stickleback in geographically close ponds represent invasion followed by local dispersal or independent founding from the sea.To address these questions, we identified populations from three islands (Middleton, Montague, and Danger) in Prince William Sound and the Gulf of Alaska that could have been founded only after the 1964 earthquake (Fig. 1 and SI Appendix, Table S1). Middleton Island was uplifted 3.4 m, creating a new terrace with ponds from a previously submarine platform (1). Similarly, Danger and Montague Islands experienced uplift and creation of new ponds (51). Stickleback now can be found in many of the habitats produced by the earthquake (52). We first analyzed a subset of populations from Middleton Island to describe the pattern of multivariate phenotypic divergence. We then produced and analyzed restriction site-associated DNA sequencing (RAD-seq) data (53, 54) from 25,000 RAD loci in 1,057 individuals collected from a total of 20 populations from all three islands and one mainland population. Deep sequencing yielded a set of 130,000 single-nucleotide polymorphisms (SNPs) and a total of 146 million genotypes. This large genomic dataset allowed us to ask whether phenotypic and genetic divergence in stickleback, thought to require thousands of years, can occur in a fraction of that time. Unlike previous studies that have made inroads into this question (47–50), the high level of biological replication of individually genotyped samples, within and across populations, in the present study avails a battery of population genomic analyses such as analysis of molecular variance (AMOVA), principal component analysis (PCA), and STRUCTURE. These approaches are most appropriate for defining (and assigning individuals to) genetic groupings across recently formed populations potentially still experiencing gene flow, such as those that are the focus of our study. We use this robust dataset to test the parallel origin of several populations against a precisely dated geological event—the Great Alaskan Earthquake of 1964—to ask whether replicated colonization of a large number of newly formed freshwater habitats by oceanic stickleback ancestors occurred independently on different islands and even amid close geographic locales within individual islands.Open in a separate windowFig. 1.Sampling locations. (A) Prince William Sound and the Gulf of Alaska, with Danger (B), Montague (C), and Middleton (D) Islands boxed. (Inset) Alaska with box representing sampling area. Sites are coded by whether they are freshwater or oceanic habitat and by the dominant ecotype found in the population. Dark gray shading within each island cartoon delineates the approximate pre-1964 shoreline.     

Cortical oscillations arise from contextual interactions that regulate sparse coding

Precise spike times carry information and are important for synaptic plasticity. Synchronizing oscillations such as gamma bursts could coordinate spike times, thus regulating information transmission in the cortex. Oscillations are driven by inhibitory neurons and are modulated by sensory stimuli and behavioral states. How their power and frequency are regulated is an open question. Using a model cortical circuit, we propose a regulatory mechanism that depends on the activity balance of monosynaptic and disynaptic pathways to inhibitory neurons: Monosynaptic input causes more powerful oscillations whereas disynaptic input increases the frequency of oscillations. The balance of stimulation to the two pathways modulates the overall distribution of spikes, with stronger disynaptic stimulation (e.g., preferred stimuli inside visual receptive fields) producing high firing rates and weak oscillations; in contrast, stronger monosynaptic stimulation (e.g., suppressive contextual stimulation from outside visual receptive fields) generates low firing rates and strong oscillatory regulation of spike timing, as observed in alert cortex processing complex natural stimuli. By accounting for otherwise paradoxical experimental findings, our results demonstrate how the frequency and power of oscillations, and hence spike times, can be modulated by both sensory input and behavioral context, with powerful oscillations signifying a cortical state under inhibitory control in which spikes are sparse and spike timing is precise.Individual neurons can precisely time their spikes when driven by temporally fluctuating synaptic inputs (1). Narrowband oscillations mediated by inhibitory neurons are thought to be a key source of coordinated fluctuating discharges from input neurons, and they vary in power and frequency during wakeful behavior and sleep. Oscillations in the gamma range (30–80 Hz), thought to be mediated by fast-spiking inhibitory neurons expressing the calcium-binding protein parvalbumin (2, 3), are modulated by the sensory environment (4–6), attention (7), and volition (8), as well as by specific memory tasks, causing changes in sensory responses (2) and information transfer (3) in the cortex. The modulation is observed both in the oscillation power, which we define as the peak of a distinct “bump” in the power spectrum of the local field potential (LFP), as well as the oscillation frequency, which is the frequency at this peak in the power spectrum (5, 6). In current models of oscillations in neuronal networks, oscillations are regulated by stimulation of inhibitory neurons such that increasing stimulation mainly increases their frequency (9–11) or power (12). In the visual cortex, both the contrast and size of visual stimuli increase the stimulation to local inhibitory neurons (13, 14), but the former increases the frequency of gamma-range oscillations (6), and the latter decreases it (5). The power of gamma oscillations increases in the somatosensory, medial temporal (15), motor (8), olfactory (16), and primary visual cortex (5) with increased stimulation to local inhibitory neurons. However, the peak power of oscillations decreases with increased stimulation of inhibitory neurons with attention (17) in some cortical areas (7). In a third scenario, whereas the broadband power in the LFP signal increases with increasing visual contrast (6, 18), peak narrowband power shows no significant trend in response to increasing contrast (8), which is thought to increase the stimulation to the local inhibitory neurons (13).We show that these diverse experimental observations can be explained by the following hypothesis: The balance of two distinct pathways that activate local inhibitory neurons mediates bidirectional regulation of oscillations (Fig. 1A). We classify these pathways as monosynaptic (MS), those that make direct excitatory synaptic connections to the inhibitory neurons, and disynaptic (DS), those that act through the local excitatory neurons.Fig. 1.Relative strength of MS and DS stimulation to inhibitory neurons determines the power and frequency of oscillations in spiking activity. (A) Schematic of local network and the monosynaptic (solid black) and disynaptic (dotted black) pathways for stimulating ...     

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.     

Contingency and entrenchment in protein evolution under purifying selection

The phenotypic effect of an allele at one genetic site may depend on alleles at other sites, a phenomenon known as epistasis. Epistasis can profoundly influence the process of evolution in populations and shape the patterns of protein divergence across species. Whereas epistasis between adaptive substitutions has been studied extensively, relatively little is known about epistasis under purifying selection. Here we use computational models of thermodynamic stability in a ligand-binding protein to explore the structure of epistasis in simulations of protein sequence evolution. Even though the predicted effects on stability of random mutations are almost completely additive, the mutations that fix under purifying selection are enriched for epistasis. In particular, the mutations that fix are contingent on previous substitutions: Although nearly neutral at their time of fixation, these mutations would be deleterious in the absence of preceding substitutions. Conversely, substitutions under purifying selection are subsequently entrenched by epistasis with later substitutions: They become increasingly deleterious to revert over time. Our results imply that, even under purifying selection, protein sequence evolution is often contingent on history and so it cannot be predicted by the phenotypic effects of mutations assayed in the ancestral background.Whether a heritable mutation is advantageous or deleterious to an organism often depends on the evolutionary history of the population. A mutation that is beneficial at the time of its introduction may confer its beneficial effect only in the presence of other potentiating or permissive mutations (1–9). Thus, the fate of a mutation arising in a population may be contingent on previous mutations (10–13). Conversely, once a mutation has fixed in a population, the mutation becomes part of the genetic background onto which subsequent modifications are introduced. Because the beneficial effects of the subsequent modifications may depend on the focal mutation, as time passes reversion of the focal mutation may become increasingly deleterious, leading to a type of evolutionary conservatism, or entrenchment (14–18).In the context of protein evolution, the effects of contingency and entrenchment are most easily studied by considering a sequence of single amino acid changes (19) that extends both forward and backward in time from some focal substitution. To assess the roles of contingency and entrenchment we can study the degree to which each focal substitution was facilitated by previous substitutions, and the degree to which the focal substitution influences the subsequent course of evolution (Fig. 1A).Open in a separate windowFig. 1.(A) A schematic model indicating how a focal substitution may be contingent on prior substitutions and may constrain future substitutions along an evolutionary trajectory, owing to epistasis. (B) A model of protein evolution under weak mutation and purifying selection for thermodynamic stability. Starting from the wild-type sequence of argT we propose 10 random 1-aa point mutations. For each of the proposed mutants we compute its predicted stability (ΔG) using FoldX, and its associated fitness. The fitness function is assumed to be either Gaussian or semi-Gaussian, with a maximum at the wild-type stability. One of the proposed mutants fixes in the population, based on its relative fixation probability under the Moran model with effective population size N_e. This process is iterated for 30 consecutive substitutions to produce an evolutionary trajectory. We simulate 100 replicate trajectories, each initiated at the wild-type argT sequence.Dependencies within a sequence of substitutions are closely connected to the concept of epistasis—that is, the idea that the phenotypic effect of a mutation at a particular genetic site may depend on the genetic background in which it arises (20–24). In the absence of epistasis, a mutation has the same effect regardless of its context and therefore regardless of any prior history or subsequent evolution. By contrast, in the presence of epistasis, each substitution may be contingent on the entire prior history of the protein, and it may constrain all subsequent evolution.The potential for epistasis to play an important role in evolution, including protein evolution, has not been overlooked by researchers (1, 8, 25–34), nor have the concepts of contingency (3, 4, 9, 12, 35–38) and, more recently, entrenchment (18, 39, 40). However, most studies have addressed the role of epistasis in the context of adaptive evolution (1–9, 27, 30, 31, 36, 38), whereas the consequences of epistasis under purifying selection have received less attention (18, 41–44). Indeed, although some more sophisticated models have been proposed (e.g., refs. 45–50), all commonly used phylogenetic models of long-term protein evolution assume that epistasis is absent so that sites evolve independently (51–56).Here we explore the relationships between epistasis, contingency, and entrenchment under long-term purifying selection on protein stability. Our analysis combines computational models for protein structures with population-genetic models for evolutionary dynamics. We use a force-field-based model, FoldX (57), to characterize the effects of point mutations on a protein’s stability and fitness. This approach allows us to simulate evolutionary trajectories of protein sequences under purifying selection, by the sequential fixation of nearly neutral mutations. We can then dissect the epistatic relationships between these substitutions by systematically inserting or reverting particular substitutions at various time points along the evolutionary trajectory.Our analysis considers epistasis both at the level of protein stability and at the level of fitness. Whereas empirical studies in diverse proteins have demonstrated that the stability effects of point mutations are typically additive across sites (58, 59), in this study we are specifically interested in epistasis for stability among the mutations that fix during evolution. Even if most random mutations are virtually additive in their effects on stability, the mutations that fix under purifying selection are highly nonrandom, and so there is reason to suspect that epistasis for stability may be enriched among such mutations. Moreover, because the mapping from stability to fitness is itself nonlinear (18, 26, 60, 61) and because selection is sensitive to selection coefficients as small as the inverse of the population size (62), even slight variation in the stability effects of mutations across different genetic backgrounds may be sufficient to influence the course of evolution.Using the computational approach summarized above, we will demonstrate that the nearly neutral mutations that fix under purifying selection are, indeed, often epistatic with each other for both stability and fitness. In particular, we find that each mutation that fixes is typically permitted to fix by the presence of preceding substitutions—that is, most substitutions would be too deleterious to fix were it not for epistasis with preceding substitutions. Conversely, we also find that mutations that fix typically become entrenched over time by epistasis—so that a substitution that was nearly neutral when it fixed becomes increasingly deleterious to revert as subsequent substitutions accumulate (18, 39). These results imply an important role for epistasis in shaping the course of sequence evolution in a protein under selection to maintain thermodynamic stability.     

Aggregation propensities of superoxide dismutase G93 hotspot mutants mirror ALS clinical phenotypes

Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misassembly and aggregation in cells affected by the motor neuron disease ALS. However, the mechanistic relationship between superoxide dismutase 1 (SOD1) mutations and human disease is controversial, with many hypotheses postulated for the propensity of specific SOD mutants to cause ALS. Here, we experimentally identify distinguishing attributes of ALS mutant SOD proteins that correlate with clinical severity by applying solution biophysical techniques to six ALS mutants at human SOD hotspot glycine 93. A small-angle X-ray scattering (SAXS) assay and other structural methods assessed aggregation propensity by defining the size and shape of fibrillar SOD aggregates after mild biochemical perturbations. Inductively coupled plasma MS quantified metal ion binding stoichiometry, and pulsed dipolar ESR spectroscopy evaluated the Cu²⁺ binding site and defined cross-dimer copper–copper distance distributions. Importantly, we find that copper deficiency in these mutants promotes aggregation in a manner strikingly consistent with their clinical severities. G93 mutants seem to properly incorporate metal ions under physiological conditions when assisted by the copper chaperone but release copper under destabilizing conditions more readily than the WT enzyme. Altered intradimer flexibility in ALS mutants may cause differential metal retention and promote distinct aggregation trends observed for mutant proteins in vitro and in ALS patients. Combined biophysical and structural results test and link copper retention to the framework destabilization hypothesis as a unifying general mechanism for both SOD aggregation and ALS disease progression, with implications for disease severity and therapeutic intervention strategies.ALS is a lethal degenerative disease of the human motor system (1). Opportunities for improved understanding and clinical intervention arose from the discovery that up to 23.5% of familial ALS cases and 7% of spontaneous cases are caused by mutations in the superoxide dismutase 1 (SOD1) gene encoding human Cu, Zn SOD (2–4). SOD is a highly conserved (5), dimeric, antioxidant metalloenzyme that detoxifies superoxide radicals (6, 7), but overexpression of SOD1 ALS mutants is sufficient to cause disease in mice (8). Misfolded and/or aggregated SOD species are deposited within mouse neuronal and glial inclusions (9, 10), even before symptoms appear (11, 12). Although human familial ALS has a symptomatic phenotype indistinguishable from sporadic cases (13), individual SOD1 mutations can result in highly variable disease progression and penetrance (14, 15).Many nongeneral mechanisms, including loss of activity or gain of function, were postulated to explain the roles of SOD mutants in ALS (3, 16–19). Recently, however, an initial hypothesis proposing that SOD manifests disease symptoms by framework destabilization (protein instability caused by structural defects) and consequent protein misassembly and aggregation has gained renewed support (2, 10, 14, 20–23). Ironically, WT SOD is an unusually stable protein (7, 24–26), and precisely how SOD mutations cause disease remains unclear. For instance, human SOD free cysteine residues C6 and C111 have been implicated in protein aggregation by promoting cross-linking (27, 28) and/or stability changes associated with oxidative modifications (29–33). Mutation of the chemically reactive thiols significantly decreases the irreversible denaturation rate for human and bovine SOD (24, 34). However, ALS mutants in a C6A/C111S SOD (AS-SOD) background (35, 36) maintain the native C57–C146 disulfide bond but can still undergo aggregation, and mutations of the free cysteines can cause ALS (37, 38). These results imply that free cysteines are not strictly required but rather, may alter aggregation kinetics (20). SOD also contains two metal ion cofactors in each subunit: a catalytic copper ion (6) and a structurally stabilizing zinc ion (34, 39, 40) (Fig. 1A). In higher eukaryotes, a copper chaperone for SOD (CCS) plays an important role in catalyzing both the copper incorporation and native disulfide bond formation (41). Structural analyses of apo WT SOD point to greater flexibility or increased solvent accessibility of C6 otherwise buried in the stable dimer interface (42, 43), and molecular dynamics simulations also suggest a critical role for metal ions in protein structure, because SOD’s β-sheet propensity decreases in the absence of metals (44). As a result, apo SOD readily forms protein aggregates (45, 46), but the molecular structures of SOD aggregates are likely polymorphic and represent a controversial topic (23, 47–51). The intertwined effects of the aggregation-enhancing free cysteines, dimer-stabilizing metal ions, and CCS maturation of SOD complicate the study of the ALS-causing SOD mutations themselves, and therefore, a clear cause-and-effect relationship remains obscure and requires deconvolution.Open in a separate windowFig. 1.Comparison of crystallographic and solution structures of WT and G93A SOD. (A) Overall architecture of the WT SOD dimer is displayed in 90° rotated views. G93 (small red spheres) resides on a surface-exposed interstrand loop between the fifth and sixth sequential β-strands of SOD and is expected to be innocuous in facilitating protein stability; however, this site harbors the most substitutions observed to result in ALS. G93 is also distant from both (Upper) the dimer interface and (Lower Left) the SOD active site (gold and silver spheres), which are generally implicated as the major determinants for SOD stability. Small blue spheres denote free cysteines. (Lower Right) The close-up view of the mutation site (boxed region in Lower Left tilted forward) shows high similarity between WT (purple) and G93A (red) SOD crystal structures [Protein Data Bank ID codes 1PU0 (WT) and 2ZKY (G93A)]. Hydrogen bonds characteristic of a β-bulge motif are indicated, whereby G93 (or A93) represents position 1. The main chain carbonyl group of β-barrel cork residue L38 is adjacent to the G93 site. (B) SAXS-derived electron pair P(r) distributions from WT (purple) and G93A (red) SOD samples in solution are compared with the theoretical curve for 1PU0. P(r) plots are normalized to peak height. Ab initio models of WT SOD derived from P(r) data are depicted in purple, with crystal structure docked into mesh envelope. Contributions to major and minor peaks from subunit and dimer dimensions are indicated.To better understand the structural effects of ALS mutations on SOD architecture, we coupled the wealth of crystallographic knowledge on SOD structure (7, 52, 53) with small-angle X-ray scattering (SAXS) experiments to characterize misassembly aggregates of ALS mutant SODs in solution. Over 20 y ago, we solved the first atomic structure of the human WT SOD protein (Fig. 1A) (20, 34) and proposed the framework destabilization hypothesis to explain how diverse mutations located throughout the 153-residue β-barrel enzyme might produce a similar disease phenotype (2), albeit with distinctions in the progression trajectory. Since that time, a staggering number of ALS mutations has been documented in patients [178 (mostly missense) (54)], with a similar phenotype in dogs (55, 56). Solution-based techniques are increasingly being applied to connect structure to biological outcome, for instance, through examination of intermolecular interactions within stress-activated pathways, for instance (57, 58). SAXS, which can probe structures for a wide size range of species, also provides higher resolution insights (59), for instance, over visible light-scattering techniques, readily distinguishing unfolded from folded proteins (60).Here, we monitor the initial events of protein aggregation in a subset of ALS mutants localized to a mutational hotspot site at glycine 93. Specifically, we wished to test a possible structural basis for how G93 mutations (to A, C, D, R, S, or V) modulate age of onset and clinical severity in ALS patients (14, 15). The G93 substitution occurs in a β-bulge region (61) between sequential β-strands of the protein (Fig. 1A) on a protruding loop roughly ∼20 Å from T54, the nearest residue of the opposing subunit, and the metal-containing active site (Fig. S1). A priori, mutation of this outer loop position would not be expected to interfere with active site chemistry or buried molecular interfaces. However, we discovered correlations of aggregation nucleation kinetics of SOD proteins with ALS mutations at this site, the stabilizing effects of metal ion retention, and available data for clinical phenotypes in patients with the same mutation. Furthermore, by measuring and exploiting the dimer geometry to observe intrinsic SOD conformers, we show that G93 mutant proteins natively reveal increased intradimer conformational flexibility in the absence of aggregation, which may reflect an increased tendency for ALS mutants to become metal-deficient and misfolding-prone and further explain the correlation to disease severity. Collective results on G93 mutants, thus, support and extend the framework destabilization hypothesis.     

Single-molecule analysis reveals widespread structural variation in multiple myeloma

Multiple myeloma (MM), a malignancy of plasma cells, is characterized by widespread genomic heterogeneity and, consequently, differences in disease progression and drug response. Although recent large-scale sequencing studies have greatly improved our understanding of MM genomes, our knowledge about genomic structural variation in MM is attenuated due to the limitations of commonly used sequencing approaches. In this study, we present the application of optical mapping, a single-molecule, whole-genome analysis system, to discover new structural variants in a primary MM genome. Through our analysis, we have identified and characterized widespread structural variation in this tumor genome. Additionally, we describe our efforts toward comprehensive characterization of genome structure and variation by integrating our findings from optical mapping with those from DNA sequencing-based genomic analysis. Finally, by studying this MM genome at two time points during tumor progression, we have demonstrated an increase in mutational burden with tumor progression at all length scales of variation.Multiple myeloma (MM) is the malignancy of B lymphocytes that terminally differentiate into long-lived, antibody-producing plasma cells. Like other cancers, it is characterized by many genomic aberrations, including single nucleotide variants (SNVs) (1, 2), translocations (most notably involving the Ig heavy chain locus on chr14), and copy number changes, including aneuploidy (3). Recent large-scale sequencing studies have described widespread inter- and intra-tumor genomic heterogeneity (1, 2), clonal evolution (4, 5) and clonal tides (4) in MM. However, most of this work focuses on point mutations and large-scale copy number changes. Although the role of structural variation in normal human genome polymorphism (6, 7) and diseases (8) is widely appreciated, a comprehensive analysis of structural variation in MM is yet to be reported.The therapeutic landscape for MM over the past decade has been transformed with the introduction of proteasome inhibitors (bortezomib, carfilzomib) and thalidomide analogs (9, 10). Consequently, patient survival rates have vastly improved (11). However, MM remains an incurable cancer, and almost all patients with symptomatic MM die of their disease because acquired drug resistance limits the efficacy of current therapies and shortens overall survival (12). Therefore, understanding the impact of contemporary treatments on MM genomic selection may provide fundamental insights for preventing and/or circumventing drug resistance through judicious use of existing therapies and/or rational design of novel agents.To address these issues, we have used optical mapping (7, 13–19) and DNA sequencing to comprehensively characterize structural variation in a primary MM genome at two stages of tumor progression and drug response. The two stages represent a sensitive relapse (MM-S; patient responded to subsequent treatments) and a subsequent refractory relapse (MM-R; no response to any treatments) (SI Materials and Methods and Fig. 1). Optical mapping is a single-molecule system that constructs large datasets comprising ordered restriction maps (Rmaps; 1 Rmap is a restriction map of a single DNA molecule) from individual genomic DNA molecules (Fig. S1). These datasets are submitted to a computational pipeline powered by cluster computing for genome assembly (15) and discovery of structural variants (7, 14, 16, 19). The final assembly presents a relatively unbiased, long-range view of the genome, free of amplification and cloning artifacts, which supports the identification of structural variants and large-scale copy number changes. Previously, optical mapping has been used to uncover structural variation in normal (7), disease risk (17), and cancerous (18) human genomes. Here, we connect long-range structural variation findings from optical mapping with results from whole genome DNA sequencing data analysis (Fig. 1). Such analysis has enabled us to comprehensively identify somatic variation in these tumor samples across all length scales, including structural, copy number, and single nucleotide variation. Additionally, by analyzing these tumor samples at two time points during tumor progression, we have highlighted an increase in mutational burden with tumor progression.Open in a separate windowFig. 1.Overview of cancer genome analysis pipeline comprising optical mapping and DNA sequencing data. Red text indicates that the method identifies somatic variation directly by comparing the tumor to the normal sample. Colored outlines highlight different variation types analyzed by integrating data from both approaches; for example, deletions from optical mapping (blue outline) were analyzed along with deletions from BreakDancer, Pindel, and CNVnator.     

Shape control and compartmentalization in active colloidal cells

Small autonomous machines like biological cells or soft robots can convert energy input into control of function and form. It is desired that this behavior emerges spontaneously and can be easily switched over time. For this purpose we introduce an active matter system that is loosely inspired by biology and which we term an active colloidal cell. The active colloidal cell consists of a boundary and a fluid interior, both of which are built from identical rotating spinners whose activity creates convective flows. Similarly to biological cell motility, which is driven by cytoskeletal components spread throughout the entire volume of the cell, active colloidal cells are characterized by highly distributed energy conversion. We demonstrate that we can control the shape of the active colloidal cell and drive compartmentalization by varying the details of the boundary (hard vs. flexible) and the character of the spinners (passive vs. active). We report buckling of the boundary controlled by the pattern of boundary activity, as well as formation of core–shell and inverted Janus phase-separated configurations within the active cell interior. As the cell size is increased, the inverted Janus configuration spontaneously breaks its mirror symmetry. The result is a bubble–crescent configuration, which alternates between two degenerate states over time and exhibits collective migration of the fluid along the boundary. Our results are obtained using microscopic, non–momentum-conserving Langevin dynamics simulations and verified via a phase-field continuum model coupled to a Navier–Stokes equation.Active matter describes particulate systems with the characteristic that each “particle” (agent) converts energy into motion (1, 2). Active matter covers a range of length scales that include molecular motors in the cytoskeleton (3–5), swimming bacteria (6–8), driven colloids (9, 10), flocks of birds and fish (11–14), and people and vehicles in motion (15). Over the last decade, studies of active matter have demonstrated behavior not seen in equilibrium systems, including giant number fluctuations (16, 17), emergent attraction and superdiffusion (18–20), clustering (21, 22), swarming (23–27), and self-assembled motifs (28, 29). These systems provide interesting theoretical and engineering challenges as well as opportunities to explore and target novel behaviors that proceed outside of thermodynamic equilibrium.Of particular interest are systems found in nature or inspired by natural phenomena. Biological systems usually operate in confined regions of space––think of intracellular space, interfaces and membranes, and the crowding of cells near surfaces. The role of hydrodynamics in confinement has been studied for biological swimmers, such as bacteria and sperm, showing accumulation at the walls (30–32) and upstream swimming along surfaces (33) or in a spiral vortex (34–36). Attraction to walls has also been reported in the absence of hydrodynamics for disks (37, 38), spheres (39), and dumbbell swimmers (40). But, whereas these examples study the behavior under the influence of hard boundaries, biological swimmers typically interact with soft boundaries, such as membranes and biofilms. Another design variable is the possibility that the boundary itself is active, as in the surface of a bacterium covered with flagellae or, as demonstrated recently, active nematic vesicles (41).In this work, we propose and investigate an active matter system under flexible, active confinement. We call this system an active colloidal cell. Our realization of an active colloidal cell consists of independent particles, called spinners (42), that translate and rotate in two dimensions and are constrained within a finite area by a flexible boundary that is also built from spinners. Each spinner has a gear-like geometry, which consists of a large central disk and four smaller satellite disks (Fig. 1A). Similar gear-shaped rigid aggregates of self-propelled particles have been formed experimentally (43). Spinners are freely mobile in the cell interior. On the cellular boundary, spinners are connected to one another by a flexible chain of beads attached by finitely extensible springs. Both the interior and the boundary spinners can be subject to a clockwise or counterclockwise driving torque, which makes them active.Open in a separate windowFig. 1.Schematic of the confined spinner models. (A) The active colloidal cell is made up of spinners driven counterclockwise (blue) or clockwise (yellow). Boundary spinners are connected by a flexible bead–spring chain (gray). We compare the behavior of a continuum model (B) to a microscopic model (C). The compartmentalization of interior spinners is visualized by coloring the Voronoi tessellation in the microscopic model.Rotationally driven particles can synchronize and self-organize (44, 45) in the absence (42) and in the presence (46–48) of hydrodynamic interactions. Crystallization has recently been observed in rotating magnetic Janus colloids (49) and fast-moving bacteria (50). Spinners in the interior of the cell resemble molecular motors that push themselves forward on their neighbors and, thus, sustain convective dynamics. The effect of the boundary spinners is similar to that found in the cilia of living tissues, which stir nearby fluid. Our results demonstrate that a natural consequence of the activity present in the colloidal cell is control over both its external shape and internal structure. We report compartmentalization into regions of clockwise and counterclockwise spinners––a behavior which is affected by, and can be controlled via, properties of the enclosing boundary configuration as previously suggested (51). Transitions in the internal structure of the colloidal cell occur as its radius increases, and as the composition of the interior spinners and the patterning of the boundary are varied.A previous study of spinners in bulk (42) showed phase separation into clockwise- and counterclockwise domains. Cates and collaborators (6, 52, 53) have suggested that phase separation is a generic consequence of local energy input in an otherwise equilibrium system. Here and in the study of bulk spinners we demonstrate phase separation due to local rotational, rather than translational, energy input. We obtain our results using a particulate, microscopic model (Fig. 1C) as well as a continuum model (Fig. 1B). This allows us to conclude that the phenomena we observe are robust with respect to details of the model.In this study we use two models to study the behavior of an active colloidal cell, illustrated in Fig. 1. The microscopic model describes spinners as individual particles and simulates their motion using Langevin dynamics. It resolves the behavior of individual spinners but does not include hydrodynamic effects. In contrast, the continuum model describes the spinner system as a viscous binary fluid, which is governed by an incompressible Navier–Stokes equation coupled to a Cahn–Hilliard equation. Both models are described in detail in Materials and Methods below. Note that the microscopic model was introduced in earlier work using Brownian dynamics (42) and is extended here to include boundaries.     

Asymmetrical effects of unilateral right or left amygdala damage on auditory cortical processing of vocal emotions

We tested whether human amygdala lesions impair vocal processing in intact cortical networks. In two functional MRI experiments, patients with unilateral amygdala resection either listened to voices and nonvocal sounds or heard binaural vocalizations with attention directed toward or away from emotional information on one side. In experiment 1, all patients showed reduced activation to voices in the ipsilesional auditory cortex. In experiment 2, emotional voices evoked increased activity in both the auditory cortex and the intact amygdala for right-damaged patients, whereas no such effects were found for left-damaged amygdala patients. Furthermore, the left inferior frontal cortex was functionally connected with the intact amygdala in right-damaged patients, but only with homologous right frontal areas and not with the amygdala in left-damaged patients. Thus, unilateral amygdala damage leads to globally reduced ipsilesional cortical voice processing, but only left amygdala lesions are sufficient to suppress the enhanced auditory cortical processing of vocal emotions.Socially relevant and emotionally charged stimuli evoke increased activation in sensory cortices, both during the visual processing of emotional pictures or facial expressions (1, 2) and during the auditory processing of vocally expressed emotions (3–5). Such increases are assumed to be remotely driven by the amygdala, which is critically involved in decoding the emotional value of stimuli (6–8). Moreover, these effects seem to be predominantly (although not exclusively) mediated by ipsilateral anatomical (9) and functional connections between amygdala and sensory areas (10, 11).In line with this view, recent studies conducted in patients with amygdala lesions reported impairments in the recognition of facial expressions (12), emotional words (13), or vocal emotions (14, 15). Furthermore, studies in both human patients (16, 17) and monkeys (18) showed significant changes in visual cortical activations to facial expressions following lesions of the amygdala. These changes in cortical processing are assumed to be remotely driven by the impaired emotional processing in the amygdala (10, 16). Distant effects of amygdala damage have also been observed for visual stimuli in cats (19) and for auditory stimuli in rats (8). However, other results have challenged this view, with some studies reporting no impairment in recognition (20–25) or changes in cortical processing for emotional stimuli in patients with amygdala lesions (26). Notably, Edmiston et al. (26) observed normal visual increases in response to emotional scenes for patients with unilateral amygdala resection, arguing against a direct role for the amygdala in modulating activity in sensory cortical areas. However, in that study (26), such increases could be related to attentional effects driven by greater interest or complexity of emotional scenes (27, 28).Thus, evidence for impaired cortical responses to emotional stimuli after unilateral amygdala damage in humans remains inconsistent. In addition, unlike in rodents (8), to date, no study has investigated how the cortical processing of emotionally salient auditory stimuli might be affected by amygdala lesions in humans. Here, we tested for the first time, to our knowledge, whether unilateral amygdala damage in patients with left or right medial temporal lobe (MTL) lesions would modify auditory responses in intact cortical areas to voices and vocally expressed emotions. Previous studies consistently found differential activity in several subregions of auditory cortex in response to vocal emotions (29, 30), as well as in the amygdala (4, 5, 29, 31, 32), especially for angry voices (3–5). These auditory effects predominate in the superior temporal gyrus (STG) and superior temporal sulcus (STS), attributed to the processing of emotional valence in the amygdala (10) and presumably mediated by direct anatomical connections between the latter and auditory cortex (9, 33). Previous studies also consistently reported a response to emotional voices in the inferior frontal cortex, which may support higher level categorization processes (34) and thus constitutes an important component of the distributed network involved in detecting and decoding vocal emotions (29, 35).We therefore hypothesized that cortical processing of human vocalizations in general, and of vocal emotions in particular, might be impaired in patients with lesions to the amygdala. This impairment is thought to result from a reduced emotional decoding of affective vocal cues in the amygdala, which is generally sensitive to emotional cues in voices (4, 5, 29, 31, 36) and usually is assumed to enhance cortical processing remotely (10, 16, 19). We also hypothesized that left and right amygdala lesions might have different effects. Whereas a right MTL lesion may strongly impair the processing of facial expressions due to well-known hemispheric asymmetries in face processing (12, 17, 37), the left amygdala seems to be more strongly involved in the decoding of emotional cues expressed in speech (13) or speech-like material (3, 4, 29). In two experiments, we tested brain responses to human vocalizations in general (experiment 1) and to emotional vocalizations embedded in pseudolanguage (experiment 2) (Fig. 1) while 10 patients with unilateral left amygdala lesions and 10 patients with unilateral right amygdala lesions (SI Results, Fig. S1A, and Table S1) underwent functional MRI (fMRI) scanning. In experiment 2, emotional voices were presented in either the attended or unattended ear during a dichotic listening task (38). We expected, first, that vocalizations, as socially salient stimuli, would generally produce weaker cortical processing in interconnected regions due to unilateral amygdala damage (experiment 1) (39). Second, in keeping with predominant left amygdala activity in healthy individuals during the processing of vocal emotions, we expected more severe impairment in cortical processing of emotional cues in patients with left amygdala lesions compared with right amygdala lesions (experiment 2) (29, 40).Open in a separate windowFig. 1.Experiment 2 included three emotion conditions, with angry voices presented in the left or right ear or neither. On an trials, an angry voice was heard on the task-relevant side, whereas on na trials, an angry voice was heard on the task-irrelevant side. On nn trials, neutral voices were presented to each ear. (A) Examples show all three conditions when attention was focused on the right ear. The same trials were also performed when attention was focused on the left ear (not shown here). (B) RTs and error rates for gender decisions on the attended voice revealed a main effect for the factor condition, indicating increased RTs and error rates during the an condition, as indicated by the asterisks.     

From the Cover: Presynaptic mitochondrial morphology in monkey prefrontal cortex correlates with working memory and is improved with estrogen treatment

Humans and nonhuman primates are vulnerable to age- and menopause-related decline in working memory, a cognitive function reliant on the energy-demanding recurrent excitation of neurons within Brodmann’s Area 46 of the dorsolateral prefrontal cortex (dlPFC). Here, we tested the hypothesis that the number and morphology (straight, curved, or donut-shaped) of mitochondria in dlPFC presynaptic boutons are altered with aging and menopause in rhesus monkeys (Macaca mulatta) and that these metrics correlate with delayed response (DR) accuracy, a well-characterized measure of dlPFC-dependent working memory. Although presynaptic bouton density or size was not significantly different across groups distinguished by age or menses status, DR accuracy correlated positively with the number of total and straight mitochondria per dlPFC bouton. In contrast, DR accuracy correlated inversely with the frequency of boutons containing donut-shaped mitochondria, which exhibited smaller active zone areas and fewer docked synaptic vesicles than those with straight or curved mitochondria. We then examined the effects of estrogen administration to test whether a treatment known to improve working memory influences mitochondrial morphology. Aged ovariectomized monkeys treated with vehicle displayed significant working memory impairment and a concomitant 44% increase in presynaptic donut-shaped mitochondria, both of which were reversed with cyclic estradiol treatment. Together, our data suggest that hormone replacement therapy may benefit cognitive aging, in part by promoting mitochondrial and synaptic health in the dlPFC.Working memory is a type of executive function that involves the storage, organization, and update of information which together guide decision making and goal-directed behavior (1, 2). This complex function is highly vulnerable to age- and menopause-related decline in humans and nonhuman primates and can be assessed in rhesus monkeys using the well-characterized delayed response (DR) test of visuospatial working memory (3–6). Rhesus monkeys are exceptionally valuable models of human aging, menopause, and related cognitive decline, because their brain anatomy, neuronal gene expression, reproductive physiology, and patterns of endocrine senescence closely resemble those of humans (4, 7–10). Importantly, they fail to develop the histopathological features of Alzheimer’s disease (11–13). Thus, we can investigate the neurobiological parameters that are coupled to age- and menopause-related cognitive dysfunction in the absence of confounding factors inherent to pathology.Performance on DR is mediated in part by layer III neurons of the dorsolateral prefrontal cortex (dlPFC) Brodmann’s Area 46, which exhibit persistent spatially tuned firing during the delay period of the DR when the spatial position is held in working memory (1, 14). A recent electrophysiological study showed that firing of these “delay cells” in Brodmann’s Area 46 is markedly decreased in aged monkeys (15, 16). This loss of firing can be accounted for partly by structural changes that occur with aging and surgical menopause (ovariectomy), both of which are associated with a dramatic loss of the plastic, thin dendritic spines on dlPFC neurons (17, 18). Intriguingly, cyclic estradiol treatment improves cognitive function in aged ovariectomized monkeys while concurrently restoring these thin spines (18). There is evidence for postsynaptic structural and molecular features that promote working memory (15, 18–20), but the contribution of presynaptic morphology has received less attention.Because the recurrent firing necessary for working memory is highly energy demanding, the prefrontal cortex (PFC) contains more mitochondria than other cortical regions (15, 21). Mitochondria are very dynamic organelles that perform various metabolic functions, including energy production and facilitation of synaptic transmission (22, 23). In neurons, mitochondria are trafficked throughout the dendritic and axonal extents and are more stationary at synaptic sites, where energy demand is especially high (24, 25). Although postsynaptic dendritic spines lack mitochondria, many presynaptic boutons contain them and promote neurotransmission by accelerating recovery from synaptic depression following activity (26). In cell cultures across many tissue types, mitochondrial stressors such as rotenone (an inhibitor of mitochondrial complex I) or hypoxia can induce the formation of donut-shaped mitochondria, which generate increased levels of reactive oxygen species (ROS) (27, 28).Here, we tested the hypothesis that mitochondrial number and morphology (Fig. 1) in dlPFC presynaptic boutons of female rhesus monkeys are altered with aging and menopause and correlate with DR accuracy. In addition, we used a cyclic estradiol manipulation known to improve working memory to test whether mitochondrial morphology is regulated in relation to the treatment.Open in a separate windowFig. 1.3D reconstructions of serial electron micrographs displaying straight (A), curved (B), and donut-shaped (C) mitochondria within monkey dlPFC axonal boutons. (Scale bar: 1 μm.)     

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.