Intestinal permeability,gut-bacterial dysbiosis,and behavioral markers of alcohol-dependence severity

Alcohol dependence has traditionally been considered a brain disorder. Alteration in the composition of the gut microbiota has recently been shown to be present in psychiatric disorders, which suggests the possibility of gut-to-brain interactions in the development of alcohol dependence. The aim of the present study was to explore whether changes in gut permeability are linked to gut-microbiota composition and activity in alcohol-dependent subjects. We also investigated whether gut dysfunction is associated with the psychological symptoms of alcohol dependence. Finally, we tested the reversibility of the biological and behavioral parameters after a short-term detoxification program. We found that some, but not all, alcohol-dependent subjects developed gut leakiness, which was associated with higher scores of depression, anxiety, and alcohol craving after 3 wk of abstinence, which may be important psychological factors of relapse. Moreover, subjects with increased gut permeability also had altered composition and activity of the gut microbiota. These results suggest the existence of a gut–brain axis in alcohol dependence, which implicates the gut microbiota as an actor in the gut barrier and in behavioral disorders. Thus, the gut microbiota seems to be a previously unidentified target in the management of alcohol dependence.Alcohol consumption is the world’s third largest risk factor for disease and disability and accounts for 5.9% of all deaths worldwide (1). Although alcohol exerts large deleterious effects on health, studies to date on the pathophysiology of alcohol dependence have mainly focused on the influence of alcohol consumption on neuronal functions in the brain (2). A limited number of studies have, however, suggested that gut functions might also be altered by chronic alcohol consumption (3, 4). Accordingly, we and others have shown that actively drinking alcohol-dependent (AD) subjects exhibited increased intestinal permeability (IP) and increased plasma levels of gut-derived bacterial products such as lipopolysaccharides and peptidoglycans (5–8). These bacterial products activate specific inflammatory pathways that partially recover after a 3-wk period of alcohol abstinence (5, 6). These recent observations indirectly suggest the possibility that the composition of gut microbiota could be altered in AD subjects and related to behavioral symptoms.The human gut microbiota consists of a complex community exceeding 100 trillion microorganisms (9) whose collective genome—the microbiome—encodes 100 times more genes than the human genome (10). It is now widely accepted that the gut microbiota should be considered an “exteriorized” organ placed within the body, which provides important physiological functions and is indispensable for human life (10–12). However, the microbial composition or activity of the gut can be modified by diet, antibiotic use, host genetics, and other environmental factors (13). Data suggest that an imbalance of the intestinal microbiota, known as dysbiosis, may contribute to a variety of somatic diseases such as obesity (14), type 2 diabetes (15), inflammatory bowel diseases (16, 17), and allergy (18).Recent studies suggest that the gut bacteria also influence brain functions and behavior and may therefore play a role in the development of psychiatric disorders (19). Indeed, in experimental studies, researchers observed that germfree mice displayed reduced anxiety-like behavior compared with mice with normal gut microbiota, demonstrating evidence of gut-to-brain interactions (20, 21). Further studies brought forward evidence that the pathways underlying the gut–brain axis are multiple and highly complex, involving brain biochemistry, the vagus nerve, proinflammatory cytokines, and tryptophan metabolism (22). Furthermore, inflammation and tryptophan/kynurenine pathways have been related to the development of depression-like behavior (23–26). In addition, gut bacteria produce neurotransmitters (serotonin, GABA, dopamine, acetylcholine), and bacterial fermentation of dietary fiber induces the release of short-chain fatty acids, which are metabolites with potential neuroactive properties (22). Recent evidence also suggests that Bacteroides fragilis may prevent autism spectrum disorder in a mouse model (27) and that administration of probiotic Bifidobacterium infantis may have antidepressant properties in rats through changes in the tryptophan/kynurenine pathway (26). Although several animal studies support a relation between the gut microbiota and behavior, major questions remain regarding this relation in human health.Depression and anxiety frequently develop in actively drinking AD subjects and play an important role in the negative reinforcement of drinking tendency (28). These factors are strongly related to the urge to drink, hereafter referred to as alcohol craving (29, 30), an important predictor of relapse after detoxification (31). The possibility that these psychological symptoms of addiction are related to a dysbiosis has so far never been investigated. The aim of the present study was to determine whether gut permeability could be associated to the severity of psychological symptoms (depression, anxiety, and craving) developed by human AD subjects. Then, we assessed the composition and activity of the gut microbiota and tested whether they are related to gut permeability. Finally, we analyzed whether alterations in gut permeability, microbiota composition, and metabolome are reversible after 3 wk of alcohol withdrawal, which is known to induce partial recovery of psychiatric symptoms (32).     

Composition of the gut microbiota modulates the severity of malaria

Plasmodium infections result in clinical presentations that range from asymptomatic to severe malaria, resulting in ∼1 million deaths annually. Despite this toll on humanity, the factors that determine disease severity remain poorly understood. Here, we show that the gut microbiota of mice influences the pathogenesis of malaria. Genetically similar mice from different commercial vendors, which exhibited differences in their gut bacterial community, had significant differences in parasite burden and mortality after infection with multiple Plasmodium species. Germfree mice that received cecal content transplants from “resistant” or “susceptible” mice had low and high parasite burdens, respectively, demonstrating the gut microbiota shaped the severity of malaria. Among differences in the gut flora were increased abundances of Lactobacillus and Bifidobacterium in resistant mice. Susceptible mice treated with antibiotics followed by yogurt made from these bacterial genera displayed a decreased parasite burden. Consistent with differences in parasite burden, resistant mice exhibited an elevated humoral immune response compared with susceptible mice. Collectively, these results identify the composition of the gut microbiota as a previously unidentified risk factor for severe malaria and modulation of the gut microbiota (e.g., probiotics) as a potential treatment to decrease parasite burden.Infection by Plasmodium species remain a global health burden causing over 200 million cases of malaria and around 1 million deaths annually, with the vast majority of fatalities being children under the age of 5 y living in sub-Saharan Africa (1). Many Plasmodium infections are either asymptomatic or cause only mild malaria. However, some infections progress to severe malaria that most often manifests as impaired consciousness (cerebral malaria), respiratory distress, and severe anemia (2). The best correlate of disease severity following Plasmodium falciparum infection in humans is parasite density (3, 4).The gut microbiota has an impact on multiple facets of host physiology (5), including shaping susceptibility to numerous diseases (6–14). The effects of the gut microbiota on the host are strongly influenced by the collective composition of the bacterial populations (15), and commensal florae are known to affect local pathogen burdens and host immunity (16–18). In addition to influencing local gut immunity, the gut microbiome affects host immunity to extragastrointestinal tract viral infections (19).Recent studies also support that the gut microbiome modulates Plasmodium infections in humans. Anti–α-gal Abs, induced by the gut pathobiont Escherichia coli O86:B7, cross-react with sporozoites from human and rodent Plasmodium species that impair transmission of the parasite between the vector and vertebrate host; however, this cross-reactive immunity did not affect blood stage parasite burden (20). Additionally, the stool bacteria composition of Malian children correlated prospectively with risk of P. falciparum infection, but not progression to febrile malaria (21). Importantly, it remains unclear whether the gut microbiome also contributes to the development of severe malaria. Using the murine model of malaria, these data demonstrate that the gut microbiome affects blood stage parasite burden and the subsequent severity of malaria.     

Distinct antimicrobial peptide expression determines host species-specific bacterial associations

Animals are colonized by coevolved bacterial communities, which contribute to the host’s health. This commensal microbiota is often highly specific to its host-species, inferring strong selective pressures on the associated microbes. Several factors, including diet, mucus composition, and the immune system have been proposed as putative determinants of host-associated bacterial communities. Here we report that species-specific antimicrobial peptides account for different bacterial communities associated with closely related species of the cnidarian Hydra. Gene family extensions for potent antimicrobial peptides, the arminins, were detected in four Hydra species, with each species possessing a unique composition and expression profile of arminins. For functional analysis, we inoculated arminin-deficient and control polyps with bacterial consortia characteristic for different Hydra species and compared their selective preferences by 454 pyrosequencing of the bacterial microbiota. In contrast to control polyps, arminin-deficient polyps displayed decreased potential to select for bacterial communities resembling their native microbiota. This finding indicates that species-specific antimicrobial peptides shape species-specific bacterial associations.Epithelial surfaces of most animals are colonized by complex bacterial communities (1–4). This commensal microbiota has been shown to be beneficial for a broad range of host-physiological functions, including facilitation of nutrient supply (5–7), immune system maturation (8–10), gut development (11), and colonization resistance against pathogens (12). This finding is supported by observations of severe fitness disadvantages in germ-free animals (13) and evidence that dysregulation of host–bacterial homeostasis is involved in the occurrence of disorders, such as inflammatory bowel disease (14, 15). However, the processes that determine community membership in the microbiota are not fully understood, which has encouraged discussions as to what extent the microbiota is controlled by the host through top-down mechanisms, relative to microbiota-intrinsic or environmental-mediated factors (16, 17).In 2007, Fraune and Bosch uncovered that two species of the cnidarian Hydra are colonized by remarkably different bacterial communities, despite being cultured under identical laboratory conditions for decades (1). These laboratory cultures were colonized by microbial communities similar to that of the same Hydra species freshly isolated from the wild, indicating strong host-mediated selective forces on the associated microbiota (1).Compelling evidence for host-control over commensal bacteria also comes from reciprocal microbiota transplantations of zebrafish and mice into germ-free recipients (18). In that study, the authors demonstrated that the recipient host shapes the community structure of the transferred, foreign microbiota to resemble their native bacterial community (18). However, the study did not elucidate the factors responsible for host-mediated community control. Several host-factors are suggested to have influence on microbiota composition, ranging from oxygen conditions in the gut, nutrient intake, temperature, mucus barriers, and immunity (reviewed in ref. 17). All of these factors are likely to differ drastically between mouse and zebrafish.Several studies have shown an active cross-talk between the host’s immune system and its associated microbiota. Commensal microbes are able to drive fundamental aspects of innate and adaptive immunity, such as T-cell maturation (9, 19), production of IgA, mucus secretion (20), and induction of innate immunity-effector molecules, such as antimicrobial peptides (AMPs) (21). Similarly, the host’s immune system appears to regulate the abundance and composition of the microbiota (22–26). Studies in mice have shown that the expression level of AMPs of the α-defensin family greatly affects community composition (25). In the cnidarian Hydra, sequentially expressed AMPs of the periculin family mediate the establishment of the bacterial microbiota during embryogenesis (27).In Hydra, AMPs of the arminin peptide family are among the most highly expressed genes. Multiple arminins have been identified in Hydra magnipapillata, all of them being short, secreted peptides (28). The propeptide consists of a highly conserved, negatively charged N-terminal region and a rather nonconserved, highly cationic C-terminal part, which was predicted to be cleaved to generate the bacteriocidal fragment (28). Consistent with that prediction, the synthetically produced C-terminal fragment of Arminin 1a showed strong antibacterial activity against Escherichia coli, Bacillus megaterium, and several methicillin-resistant Staphylococcus aureus strains in concentrations equal or lower than 0.4 µM (28).In the present study, we addressed the question whether species-specific AMPs shape species-specific bacterial communities. In particular, we investigated the effect of arminin deficiency in the cnidarian host Hydra. Arminin-deficient and control polyps were inoculated with native as well as foreign bacterial communities characteristic for the closely related species Hydra oligactis and Hydra viridissima. Whereas control polyps selected for bacterial communities resembling their native microbiota, this host-driven selection was significantly less pronounced in arminin-deficient polyps. These data provide strong evidence for a role of species-specific AMPs in selecting suitable bacterial partners, leading to host-species specific bacterial associations.     

Patterned progression of bacterial populations in the premature infant gut

In the weeks after birth, the gut acquires a nascent microbiome, and starts its transition to bacterial population equilibrium. This early-in-life microbial population quite likely influences later-in-life host biology. However, we know little about the governance of community development: does the gut serve as a passive incubator where the first organisms randomly encountered gain entry and predominate, or is there an orderly progression of members joining the community of bacteria? We used fine interval enumeration of microbes in stools from multiple subjects to answer this question. We demonstrate via 16S rRNA gene pyrosequencing of 922 specimens from 58 subjects that the gut microbiota of premature infants residing in a tightly controlled microbial environment progresses through a choreographed succession of bacterial classes from Bacilli to Gammaproteobacteria to Clostridia, interrupted by abrupt population changes. As infants approach 33–36 wk postconceptional age (corresponding to the third to the twelfth weeks of life depending on gestational age at birth), the gut is well colonized by anaerobes. Antibiotics, vaginal vs. Caesarian birth, diet, and age of the infants when sampled influence the pace, but not the sequence, of progression. Our results suggest that in infants in a microbiologically constrained ecosphere of a neonatal intensive care unit, gut bacterial communities have an overall nonrandom assembly that is punctuated by microbial population abruptions. The possibility that the pace of this assembly depends more on host biology (chiefly gestational age at birth) than identifiable exogenous factors warrants further consideration.The vertebrate digestive system hosts a profound transition from a state of complete or near-sterility in utero to dense bacterial colonization within weeks of birth. This event has lasting effects on the host (1), influencing health and development (2–4), infection resistance (5, 6), predisposition to inflammatory (7) and metabolic disorders (8), and immune function (9), but remarkably little is known about this process. Gut colonization has been partly characterized in term infants (10–12) who reside in open venues, and who will, therefore, experience many exposures (e.g., contact with older children, adults, and pets, varying diets, oral antibiotics) that could drive microbial population assembly (1, 11–13).A delineation of the dynamics of the natural de novo assembly of this microbial community would form a basis for better understanding how the gut acquires its founding microbiome, and how the bacteria in the gut start their transition to population equilibrium (1, 14, 15). In view of the importance of bacterial gut colonization, we sought to determine if the initial assembly of host intestinal microbial populations follows discernible patterns, and if interventions such as antibiotics or nutrition alter this progression. A discernibly patterned progression would suggest that host biology influences bacterial community assembly more than do random encounters of individuals with microbes, whereas stochastic assembly would suggest that random encounters sculpt population structure. In this latter scenario, the gut serves as a passive culture chamber. Fine interval enumeration of gut contents from multiple subjects in as controlled an environment as possible is needed to answer this question.Here, we demonstrate that the gut microbiota of premature infants residing in a tightly controlled environment of a neonatal intensive care unit (NICU) progresses through a choreographed succession of bacterial classes from Bacilli to Gammaproteobacteria to Clostridia interrupted by abrupt population changes. The rate of assembly is slowest for the most premature of these infants.     

Polymers in the gut compress the colonic mucus hydrogel

Colonic mucus is a key biological hydrogel that protects the gut from infection and physical damage and mediates host–microbe interactions and drug delivery. However, little is known about how its structure is influenced by materials it comes into contact with regularly. For example, the gut abounds in polymers such as dietary fibers or administered therapeutics, yet whether such polymers interact with the mucus hydrogel, and if so, how, remains unclear. Although several biological processes have been identified as potential regulators of mucus structure, the polymeric composition of the gut environment has been ignored. Here, we demonstrate that gut polymers do in fact regulate mucus hydrogel structure, and that polymer–mucus interactions can be described using a thermodynamic model based on Flory–Huggins solution theory. We found that both dietary and therapeutic polymers dramatically compressed murine colonic mucus ex vivo and in vivo. This behavior depended strongly on both polymer concentration and molecular weight, in agreement with the predictions of our thermodynamic model. Moreover, exposure to polymer-rich luminal fluid from germ-free mice strongly compressed the mucus hydrogel, whereas exposure to luminal fluid from specific-pathogen-free mice—whose microbiota degrade gut polymers—did not; this suggests that gut microbes modulate mucus structure by degrading polymers. These findings highlight the role of mucus as a responsive biomaterial, and reveal a mechanism of mucus restructuring that must be integrated into the design and interpretation of studies involving therapeutic polymers, dietary fibers, and fiber-degrading gut microbes.Biological hydrogels (including mucus, blood clots, and the extracellular matrix) provide critical functions, yet little is known about how their structure is influenced by materials they come into contact with regularly. For example, the environments of many hydrogels abound in polymers, such as dietary fibers (1, 2) or administered therapeutics (3–5) in the gut and soluble glycoproteins in tissues. Whether such polymers interact with these hydrogels, and if so, how, remains unclear. An important example is the case of colonic mucus, which protects the gut from infection and physical damage (6–8), mediates drug delivery (9), and mediates host–microbe interactions (10) in a structure-dependent manner; for example, a “tighter” mesh could impede the infiltration of microorganisms from the intestinal lumen (6, 11–13). Mucus restructuring is typically attributed solely to changes in secretion (14–16), or to the activity of specific enzymes (8, 17), detergents (18), or dextran sulfate sodium-induced inflammation (19). However, the physicochemical properties of the gut environment itself—particularly its polymeric composition—have not been considered as a potential regulator of mucus structure. We therefore sought to characterize the structure of the colonic mucus hydrogel in the absence and in the presence of polymers.     

Rhythmicity of the intestinal microbiota is regulated by gender and the host circadian clock

In mammals, multiple physiological, metabolic, and behavioral processes are subject to circadian rhythms, adapting to changing light in the environment. Here we analyzed circadian rhythms in the fecal microbiota of mice using deep sequencing, and found that the absolute amount of fecal bacteria and the abundance of Bacteroidetes exhibited circadian rhythmicity, which was more pronounced in female mice. Disruption of the host circadian clock by deletion of Bmal1, a gene encoding a core molecular clock component, abolished rhythmicity in the fecal microbiota composition in both genders. Bmal1 deletion also induced alterations in bacterial abundances in feces, with differential effects based on sex. Thus, although host behavior, such as time of feeding, is of recognized importance, here we show that sex interacts with the host circadian clock, and they collectively shape the circadian rhythmicity and composition of the fecal microbiota in mice.The composition of intestinal microbiota is influenced by host genetics (1), aging (2), antibiotic exposure (3), lifestyle (4), diet (5), pet ownership (6), and concomitant disease (7, 8). The impact of diet in shaping the composition of the microbiota has been well established in both humans and mice (9, 10). The type of food consumed and also the feeding behavior of the host influence the microbiota. For example, a 24-h fast increases the abundance of Bacteroidetes and reduces that of Firmicutes in mouse cecum, without altering the communal microbial diversity (11). Bacteroidetes are also dominant in the microbiota of the fasted Burmese python, whereas ingestion of a meal shifts the intestinal composition toward Firmicutes (12).The rotation of the earth results in the oscillation of light during the 24-h cycle. Organisms adapted to this cycle by developing a circadian rhythm, an endogenous and entrainable mechanism that times daily events such as feeding, temperature, sleep-wakefulness, hormone secretion, and metabolic homeostasis (13, 14). In mammals, this rhythm is controlled by a master clock that resides in the suprachiasmatic nucleus of the hypothalamus. It responds to the changing light cycle and signals this information to peripheral clocks in most tissues (15). The core mammalian clock is comprised of activators BMAL1 and CLOCK as well as repressors PERIOD (PER) and CRYPTOCHROME (CRY), forming an interlocked regulatory loop (14).Circadian rhythms also exist in fungi and cyanobacteria (16). For example, a pacemaker in cyanobacteria transduces the oscillating daylight signal to regulate gene expression and to time cell division (17, 18). Hence, the synchronization of endogenous circadian rhythms with the environment is crucial for the survival of the bacteria as well as metazoa.Recent studies show that the intestinal microbiota undergo diurnal oscillation under the control of host feeding time, and that ablation of the host molecular clock Per genes causes dysbiosis (19, 20). Here, we report that microbial composition and its oscillation are influenced by the host clock, including the Bmal1-dependent forward limb of the signaling pathway. We also find that rhythmicity is conditioned by the sex of the host, being more pronounced in females than in males.     

Temporal and spatial variation of the human microbiota during pregnancy

Despite the critical role of the human microbiota in health, our understanding of microbiota compositional dynamics during and after pregnancy is incomplete. We conducted a case-control study of 49 pregnant women, 15 of whom delivered preterm. From 40 of these women, we analyzed bacterial taxonomic composition of 3,767 specimens collected prospectively and weekly during gestation and monthly after delivery from the vagina, distal gut, saliva, and tooth/gum. Linear mixed-effects modeling, medoid-based clustering, and Markov chain modeling were used to analyze community temporal trends, community structure, and vaginal community state transitions. Microbiota community taxonomic composition and diversity remained remarkably stable at all four body sites during pregnancy (P > 0.05 for trends over time). Prevalence of a Lactobacillus-poor vaginal community state type (CST 4) was inversely correlated with gestational age at delivery (P = 0.0039). Risk for preterm birth was more pronounced for subjects with CST 4 accompanied by elevated Gardnerella or Ureaplasma abundances. This finding was validated with a set of 246 vaginal specimens from nine women (four of whom delivered preterm). Most women experienced a postdelivery disturbance in the vaginal community characterized by a decrease in Lactobacillus species and an increase in diverse anaerobes such as Peptoniphilus, Prevotella, and Anaerococcus species. This disturbance was unrelated to gestational age at delivery and persisted for up to 1 y. These findings have important implications for predicting premature labor, a major global health problem, and for understanding the potential impact of a persistent, altered postpartum microbiota on maternal health, including outcomes of pregnancies following short interpregnancy intervals.The human body harbors diverse, complex, and abundant microbiota whose composition is determined largely by body site but also by host genetics, environmental exposures, and time (1, 2). The microbiota plays critical roles in health and in disease, including nutrient acquisition, immune programming, and protection from pathogens (3). Normal pregnancy represents a unique, transient, and dynamic state of altered anatomy, physiology, and immune function. Preterm birth, i.e., before 37 wk of gestation, occurs in 11% of pregnancies and is the leading cause of neonatal death (4). In both term and preterm pregnancies, the interplay between the microbiota and the host remains poorly understood.Approximately 25% of preterm births are associated with occult microbial invasion of the amniotic cavity (5). Evidence suggests that the most common source of invading microbes is the host microbiota. In studies of amniotic fluid from women with preterm labor and either intact or ruptured membranes, 16S ribosomal RNA (rRNA) sequences of known vaginal, gut, and oral indigenous bacterial species have been recovered in 15–50% of cases, and their relative abundances have correlated directly with markers of inflammation and inversely with time to delivery (6–9). Preterm birth also is associated with bacterial vaginosis, a community-wide alteration of the vaginal microbiota (10, 11) that increases the risk of preterm birth approximately twofold (12, 13).Several studies have examined the vaginal microbiota during pregnancy using cultivation-independent techniques (14–19). Collectively, these studies found the vaginal communities of pregnant women to be dominated by Lactobacillus species and characterized by lower richness and diversity than in nonpregnant women but with higher stability. Of the two studies that evaluated pregnancy outcomes, one found preterm birth to be linked with higher intracommunity (alpha) diversity in the vagina (16), but the other found no significant association between preterm birth and any specific community type or microbial taxon (17).Other (nonvaginal) body sites have been even less well studied in the setting of pregnancy. The subgingival crevice has been investigated only with cultivation (20, 21) or with taxon-specific molecular approaches (22). Two studies of the fecal microbiota reported differences in bacterial community structure between the first and third trimesters (23, 24); in each study, however, samples were collected at only two time points. These limited findings support the need for longitudinal investigations of the microbiota at multiple body sites during pregnancy.As part of a larger ongoing study, we examined a total of 49 women who were divided into two groups, each of which included controls (term deliveries) and cases (preterm deliveries). We characterized the temporal dynamics of microbiota composition based on prospective weekly sampling during pregnancy from four body sites: vagina, distal gut (stool), saliva, and tooth/gum, as well as after delivery. Our data reveal microbiota compositional stability during pregnancy at all body sites, a diverse vaginal community state early during pregnancy in women who subsequently delivered prematurely, and a dramatic shift in vaginal microbiota composition at the time of delivery that in some cases persisted for the maximum duration of postpartum sampling (1 y).     

Intramolecular allosteric communication in dopamine D2 receptor revealed by evolutionary amino acid covariation

The structural basis of allosteric signaling in G protein-coupled receptors (GPCRs) is important in guiding design of therapeutics and understanding phenotypic consequences of genetic variation. The Evolutionary Trace (ET) algorithm previously proved effective in redesigning receptors to mimic the ligand specificities of functionally distinct homologs. We now expand ET to consider mutual information, with validation in GPCR structure and dopamine D2 receptor (D2R) function. The new algorithm, called ET-MIp, identifies evolutionarily relevant patterns of amino acid covariations. The improved predictions of structural proximity and D2R mutagenesis demonstrate that ET-MIp predicts functional interactions between residue pairs, particularly potency and efficacy of activation by dopamine. Remarkably, although most of the residue pairs chosen for mutagenesis are neither in the binding pocket nor in contact with each other, many exhibited functional interactions, implying at-a-distance coupling. The functional interaction between the coupled pairs correlated best with the evolutionary coupling potential derived from dopamine receptor sequences rather than with broader sets of GPCR sequences. These data suggest that the allosteric communication responsible for dopamine responses is resolved by ET-MIp and best discerned within a short evolutionary distance. Most double mutants restored dopamine response to wild-type levels, also suggesting that tight regulation of the response to dopamine drove the coevolution and intramolecular communications between coupled residues. Our approach provides a general tool to identify evolutionary covariation patterns in small sets of close sequence homologs and to translate them into functional linkages between residues.Identifying residues that coevolved to maintain or acquire fitness properties is critical for understanding protein structure, function, and evolution (1). Previous studies have shown that covarying residue pairs, those that exhibit correlated amino acid changes in large multiple sequence alignments, tend to form structural contacts (2–7), enhancing predictions of protein 3D structures (8–11). Covariation can also involve distal residues, but the function of these at-a-distance couplings is elusive and has been attributed to background noise, alternative protein conformations, or subunit interactions of protein homooligomers (5, 7, 12). Alternately, distal covarying residue pairs could indicate allosteric couplings (6, 13–18).The possibility of capturing intramolecular allosteric communication by amino acid covariation analysis of protein family sequences has not been extensively explored. Nonproximal thermodynamic coupling between correlated residue pairs was noted in 274 PDZ domains (14), but the relationship to allostery is still debated (19, 20). It may be that distinctive allosteric mechanisms, even among close homologs, limit the extraction of allosteric couplings from sequences (13). Our previous identification of residues important for allosteric signaling within G protein-coupled receptors (GPCRs) using Evolutionary Trace (ET) (21–24) and strong conservation of some of the residues implicated led us to ask whether ET could also uncover couplings among protein sequence positions not in direct contact.ET estimates the relative functional sensitivity of a protein to variations at each residue position using phylogenetic distances to account for the functional divergence among sequence homologs (25, 26). Similar ideas can be applied to pairs of sequence positions to recompute ET as the average importance of the couplings between a residue and its direct structural neighbors (27). To measure the evolutionary coupling information between residue pairs, we present a new algorithm, ET-MIp, that integrates the mutual information metric (MIp) (5) to the ET framework. We used dopamine D2 receptor (D2R), a target of drugs for neurological and psychiatric diseases (28), to test whether ET-MIp could elucidate the allosteric functional communications from amino acid covariation patterns and resolve the evolutionary distance at which the allosteric pathways of D2R homologs are sufficiently conserved to detect residue−residue coupling signatures. D2R is expressed in the central nervous system and responds to dopamine, the major catecholamine neurotransmitter. Canonical D2R signaling is effected by G_i/o class G proteins, which regulate ion channels (29, 30), MAPK kinases (31), phospholipase C (32), and inhibition of adenylyl cyclase (33). D1 class receptors (D1R and D5R) have lower affinities for dopamine (34–36) and activate adenylyl cyclase through G_s class G proteins. To characterize allosteric communication between covarying pairs of residues ranked as important by ET (ET residue pairs), we examined functional coupling for ligand binding affinities and downstream G_i activation induced by agonist-stimulated D2R.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

A priori calculations of the free energy of formation from solution of polymorphic self-assembled monolayers

Modern quantum chemical electronic structure methods typically applied to localized chemical bonding are developed to predict atomic structures and free energies for meso-tetraalkylporphyrin self-assembled monolayer (SAM) polymorph formation from organic solution on highly ordered pyrolytic graphite surfaces. Large polymorph-dependent dispersion-induced substrate−molecule interactions (e.g., −100 kcal mol⁻¹ to −150 kcal mol⁻¹ for tetratrisdecylporphyrin) are found to drive SAM formation, opposed nearly completely by large polymorph-dependent dispersion-induced solvent interactions (70–110 kcal mol⁻¹) and entropy effects (25–40 kcal mol⁻¹ at 298 K) favoring dissolution. Dielectric continuum models of the solvent are used, facilitating consideration of many possible SAM polymorphs, along with quantum mechanical/molecular mechanical and dispersion-corrected density functional theory calculations. These predict and interpret newly measured and existing high-resolution scanning tunnelling microscopy images of SAM structure, rationalizing polymorph formation conditions. A wide range of molecular condensed matter properties at room temperature now appear suitable for prediction and analysis using electronic structure calculations.A priori calculations of the free energies of chemical reactions using density functional theory (DFT) and/or ab initio methods are now well established for gas-phase processes (1, 2), gas surface reactions (3), and, using continuum self-consistent reaction field (SCRF) methods, for condensed phase processes also (4). Over the last few years, a major advance in computational methods has occurred, however, allowing for rapid and accurate evaluation of intermolecular dispersion interactions. This makes feasible similar SCRF calculations for physisorption, macromolecule structuring, and other self-assembly processes in condensed phases. We present the first application of this type, to our knowledge, considering the free energy of formation of various polymorphs of tetraalkylporphyrin self-assembled monolayers (SAMs) at solid/liquid interfaces on highly ordered pyrolytic graphite (HOPG) surfaces. Calculated structures and free energies are used to interpret new and existing high-resolution scanning tunneling microscopy (STM) images, focusing on the critical roles played by the dispersion interaction in driving SAM formation and by entropy and dispersion-based desolvation effects that oppose it.Calculating a priori free energies for SAM formation from solution is a significant challenge. Accurate representations of the substrate−molecule energies, the intermolecular energies, the solvent interaction energies, and the effects of solvent structure are required, a set of tasks that, with a few exceptions (see e.g., refs. 5 and 6), has remained prohibitive a priori. Alternatively, model calculations have been useful for identifying key qualitative features (7–11), whereas some full molecular dynamics simulations of the free energy using empirical force fields have been revealing for SAMs (12–15) and also for macromolecules (16), and DFT molecular dynamics approaches are appearing (17). A priori computational methods have significant advantages in that they do not require parameterization, they treat interactions both generally and accurately, and, through use of implicit solvation models, they can avoid extensive liquid structure simulations. However, applications to self-assembly processes have historically been prevented by difficulties in treating discrete and continuum van der Waals dispersive forces both quantitatively and economically. Modern methods (18–25) can now describe discrete interactions well, and self-assembly processes such as molecular crystal formation (22) and host−guest chemistry (26) have now been successfully modeled. Dispersive interactions with a solvent continuum (18, 27) are also now included in many codes, including Gaussian (28) and, very recently, VASP (29). Like discrete dispersion interactions, the best method for including continuum solvent dispersion is a topic of considerable current interest (30–32). In both cases, many options are available, and consideration of SAM formation offers avenues for finding the most robust approaches. Herein we consider three possible approaches obtained by combining commonly used options for different aspects of the calculations, as described in Methods. In overview, our computational approach is to (i) systematically search through conformational options to establish sets of possibly feasible SAM structures; (ii) optimize these SAM structures typically in the absence of solvent; (iii) determine SAM solvation energy contributions at these optimized structures for comparison with bare-surface and free-molecule results; and (iv) determine zero-point energy, entropy, and enthalpy contributions to the free energy of SAM formation, including evaluating both the molecular and phonon vibration frequencies in situ in the SAM as well as the vibration frequencies of the gas-phase monomer molecules.To be useful, a new computational strategy must address relevant experimental challenges, and indeed the measurement of free energies for SAM formation is in itself a significant challenge facing modern nanotechnology (33). A critical experiment, the first measurement of the temperature dependence of composition of SAM components, has recently been reported (34), opening this field to future quantitative analysis. Indeed, in total, only a few studies of SAM temperature dependence have ever been performed, as recently reviewed (12, 34, 35). An important conclusion drawn is that the nature of observed SAM composition can be controlled by either kinetic and/or thermodynamic effects (9, 12, 14, 15, 33–44). SAMs in thermodynamic equilibrium can have free energies determined directly from dynamics observed in the STM (17). More generally, although methods for measuring enthalpies of SAM formation are becoming available (45), it was recently concluded that “The Achilles’ heel of [the current] approach is the semi-theoretical evaluation of the dewetting enthalpy which at this point necessarily relies on plausible assumptions” (46). A priori calculations can not only directly calculate the required quantities but are also relevant to this field of endeavor as they can directly examine postulates concerning kinetic versus thermodynamic control. They can also reveal the factors that control observed free energies, providing insight into SAM synthetic strategies and functional properties.Herein our techniques for a priori SAM simulation are applied to understand the factors controlling 2D polymorphism. This is attractive to study as it involves only one type of adsorbate molecule, simplifying analysis. We consider room temperature meso-tetraalkylporphyrin (M-C_mP) SAMs on HOPG as they show controllable polymorphism and synthetic diversity, considering (see Fig. 1) C₁₃P and Co−C₁₃P, presenting their syntheses and STM measurements, C₁₂P, reprocessing observed images (47, 48) using recently (49) developed internal calibration techniques to provide improved quantitative analysis, Zn−C₁₂P (47), C₁₁P (50), and Cu−C₁₁P (50, 51), as well as, briefly, C₁₉P (49).Open in a separate windowFig. 1.The meso-tetraalkylporphyrins considered.Different 2D polymorphs of Cu−C₁₁P have been produced by varying the porphyrin concentration in the supernatant solution, with higher-coverage polymorphs forming at higher solution concentrations (51, 52). Increasing the porphyrin concentration once a SAM has formed brings about dynamic changes to the SAM only around defects, however, indicating that the SAMs are kinetically trapped, and this trapping takes only seconds to manifest after first application of the solution to the substrate. Similar results have recently been observed for porphyrin SAMs on Au(111) (34). This kinetic trapping makes thermodynamic quantities difficult to measure and sets a key challenge for future experimental work. As what happens in those first few seconds qualitatively reflects thermodynamics (high-coverage polymorphs develop at high porphyrin concentrations), we introduce the simplistic hypothesis that indeed thermodynamics completely controls this aspect.Experimentally testing this hypothesis requires quantitative information (7, 8, 33), as has been measured, e.g., for codeposition of single SAMs containing mixed porphyrins (40) to reveal that molecules inside SAM phases are not in equilibrium with those in solution. However, only limited data are available concerning relative polymorph propensities. Here, we test the hypothesis by comparing observed structures and crudely estimated formation free energies to calculated ones. Although the uncertainties in the experimentally derived free energies are large, they are obtained using new and existing data of the type commonly available from STM measurements. Our method therefore has widespread application, providing starting points for quantitative analyses of the factors driving SAM formation.The computational studies also focus on the factors that control free energies of SAM formation and polymorphism. For meso-tetraalkylporphyrin SAMs, chain length variation can be exploited to obtain understanding of the entropy/enthalpy balance (39), and we consider three different chain lengths, C₁₁ to C₁₃. Solvent dependence can also provide direct means of modulating free energies of polymorphism (43), but herein we do not dwell on this effect, considering only solvents that are likely to have similar critical dielectric and density parameters, ignoring the differences between them.     

Microbial biogeography of wine grapes is conditioned by cultivar,vintage, and climate

Wine grapes present a unique biogeography model, wherein microbial biodiversity patterns across viticultural zones not only answer questions of dispersal and community maintenance, they are also an inherent component of the quality, consumer acceptance, and economic appreciation of a culturally important food product. On their journey from the vineyard to the wine bottle, grapes are transformed to wine through microbial activity, with indisputable consequences for wine quality parameters. Wine grapes harbor a wide range of microbes originating from the surrounding environment, many of which are recognized for their role in grapevine health and wine quality. However, determinants of regional wine characteristics have not been identified, but are frequently assumed to stem from viticultural or geological factors alone. This study used a high-throughput, short-amplicon sequencing approach to demonstrate that regional, site-specific, and grape-variety factors shape the fungal and bacterial consortia inhabiting wine-grape surfaces. Furthermore, these microbial assemblages are correlated to specific climatic features, suggesting a link between vineyard environmental conditions and microbial inhabitation patterns. Taken together, these factors shape the unique microbial inputs to regional wine fermentations, posing the existence of nonrandom “microbial terroir” as a determining factor in regional variation among wine grapes.Microbial biogeography, the study of microbial biodiversity over time and space (1), uncovers the role that geospatial dispersion patterns play in human health (2), environmental inhabitation (3), indoor environments (4), and agriculture (5), revealing important links between environmental conditions, microbial communities, and macroscopic phenomena. Vitis vinifera (wine grape) represents an economically and culturally important agricultural crop for which microbial activity plays critical roles in grape (6) and wine production and quality formation (7, 8). Indeed, regional variation in grape- and wine-quality characteristics is a critical feature of perceived product identity (terroir), with significant consequences for consumer preference and economic appreciation (9). However, scant evidence exists for nonrandom microbial distribution patterns in grapes and wines (6, 10) and the factors driving microbial assemblages on grape surfaces are unknown. Given that many of the same environmental conditions that govern regional variations in grapevine growth and development (11) also alter microbial communities across space and time (1), it follows that biogeographical assemblages of grape-surface microbiota may exist, potentially influencing grapevine health and wine quality.The V. vinifera phyllosphere is colonized by bacteria and fungi that substantially modulate grapevine health, development, and grape and wine qualities (6). Many microbes inhabiting the grape surface cannot survive the low-pH, ethanolic, anaerobic conditions of wine fermentations, but their metabolic activity on the grape surface can have long-ranging consequences, such as the metabolic changes wrought by phytopathogenic fungi (12, 13). However, some grape-surface microbes can grow and survive in wine fermentations (14), and several are implicated in wine spoilage downstream (7). This is particularly true of grapes damaged by disease or pest pressure, which have been shown to contain elevated populations of bacteria such as Acetobacteraceae and yeasts such as Zygosaccharomyces (15, 16). The impact of grape microbiota is often beneficial, and the participation of indigenous microbiota in wine fermentations is often considered to enhance the sensory complexity of wines (17). Bioprospecting the microbiota of wine fermentations has led to the discovery of several species with positive enological properties, which are now commercially available as coinocula with Saccharomyces yeasts in winemaking (17). Consequently, a growing number of yeasts and bacteria are being recognized as active participants in wine fermentations with important contributions to wine sensory qualities (17).Nevertheless, whether grape-surface microbiota are nonrandomly distributed in the environment is disputed (6), and little work has been done to test the impact of natural factors, such as climate, growing region, and cultivar on grape microbiota. Geographical delineations among Saccharomyces cerevisiae populations and cultivable yeast communities in New Zealand vineyards have been documented previously, providing evidence for regional dispersion of vineyard yeasts (18). However, it is unknown whether nonrandom geographical dispersion patterns exist among the complete grape-surface microbiota and how these communities are formed, explaining relationships between environmental growing conditions and the microbial consortium living on and interacting with wine grapes. Historically, microbial surveillance efforts have been limited by the throughput and methodological biases of culture-based techniques and low resolution of early molecular ecology techniques (19, 20). However, recent advances in massively parallel, short-amplicon sequencing technologies have launched a breakthrough in microbial ecology studies of wine- and food-fermentation systems (14, 21–27), putting hitherto untenable ecological questions within reach.     

Structural interactions of a voltage sensor toxin with lipid membranes

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.Protein toxins from venomous organisms have been invaluable tools for studying the ion channel proteins they target. For example, in the case of voltage-activated potassium (Kv) channels, pore-blocking scorpion toxins were used to identify the pore-forming region of the channel (1, 2), and gating modifier tarantula toxins that bind to S1–S4 voltage-sensing domains have helped to identify structural motifs that move at the protein–lipid interface (3–5). In many instances, these toxin–channel interactions are highly specific, allowing them to be used in target validation and drug development (6–8).Tarantula toxins are a particularly interesting class of protein toxins that have been found to target all three families of voltage-activated cation channels (3, 9–12), stretch-activated cation channels (13–15), as well as ligand-gated ion channels as diverse as acid-sensing ion channels (ASIC) (16–21) and transient receptor potential (TRP) channels (22, 23). The tarantula toxins targeting these ion channels belong to the inhibitor cystine knot (ICK) family of venom toxins that are stabilized by three disulfide bonds at the core of the molecule (16, 17, 24–31). Although conventional tarantula toxins vary in length from 30 to 40 aa and contain one ICK motif, the recently discovered double-knot toxin (DkTx) that specifically targets TRPV1 channels contains two separable lobes, each containing its own ICK motif (22, 23).One unifying feature of all tarantula toxins studied thus far is that they act on ion channels by modifying the gating properties of the channel. The best studied of these are the tarantula toxins targeting voltage-activated cation channels, where the toxins bind to the S3b–S4 voltage sensor paddle motif (5, 32–36), a helix-turn-helix motif within S1–S4 voltage-sensing domains that moves in response to changes in membrane voltage (37–41). Toxins binding to S3b–S4 motifs can influence voltage sensor activation, opening and closing of the pore, or the process of inactivation (4, 5, 36, 42–46). The tarantula toxin PcTx1 can promote opening of ASIC channels at neutral pH (16, 18), and DkTx opens TRPV1 in the absence of other stimuli (22, 23), suggesting that these toxin stabilize open states of their target channels.For many of these tarantula toxins, the lipid membrane plays a key role in the mechanism of inhibition. Strong membrane partitioning has been demonstrated for a range of toxins targeting S1–S4 domains in voltage-activated channels (27, 44, 47–50), and for GsMTx4 (14, 50), a tarantula toxin that inhibits opening of stretch-activated cation channels in astrocytes, as well as the cloned stretch-activated Piezo1 channel (13, 15). In experiments on stretch-activated channels, both the d- and l-enantiomers of GsMTx4 are active (14, 50), implying that the toxin may not bind directly to the channel. In addition, both forms of the toxin alter the conductance and lifetimes of gramicidin channels (14), suggesting that the toxin inhibits stretch-activated channels by perturbing the interface between the membrane and the channel. In the case of Kv channels, the S1–S4 domains are embedded in the lipid bilayer and interact intimately with lipids (48, 51, 52) and modification in the lipid composition can dramatically alter gating of the channel (48, 53–56). In one study on the gating of the Kv2.1/Kv1.2 paddle chimera (53), the tarantula toxin VSTx1 was proposed to inhibit Kv channels by modifying the forces acting between the channel and the membrane. Although these studies implicate a key role for the membrane in the activity of Kv and stretch-activated channels, and for the action of tarantula toxins, the influence of the toxin on membrane structure and dynamics have not been directly examined. The goal of the present study was to localize a tarantula toxin in membranes using structural approaches and to investigate the influence of the toxin on the structure of the lipid bilayer.     

Gut bacteria mediate aggregation in the German cockroach

Aggregation of the German cockroach, Blattella germanica, is regulated by fecal aggregation agents (pheromones), including volatile carboxylic acids (VCAs). We demonstrate that the gut microbial community contributes to production of these semiochemicals. Chemical analysis of the fecal extract of B. germanica revealed 40 VCAs. Feces from axenic cockroaches (no microorganisms in the alimentary tract) lacked 12 major fecal VCAs, and 24 of the remaining compounds were represented at extremely low amounts. Olfactory and aggregation bioassays demonstrated that nymphs strongly preferred the extract of control feces over the fecal extract of axenic cockroaches. Additionally, nymphs preferred a synthetic blend of 6 fecal VCAs over a solvent control or a previously identified VCA blend. To test whether gut bacteria contribute to the production of fecal aggregation agents, fecal aerobic bacteria were cultured, isolated, and identified. Inoculation of axenic cockroaches with individual bacterial taxa significantly rescued the aggregation response to the fecal extract, and inoculation with a mix of six bacterial isolates was more effective than with single isolates. The results indicate that the commensal gut microbiota contributes to production of VCAs that act as fecal aggregation agents and that cockroaches discriminate among the complex odors that emanate from a diverse microbial community. Our results highlight the pivotal role of gut bacteria in mediating insect–insect communication. Moreover, because the gut microbial community reflects the local environment, local plasticity in fecal aggregation pheromones enables colony-specific odors and fidelity to persistent aggregation sites.Diverse microbial communities inhabit the alimentary tract and other tissues of many insect species. Their effects on the host vary, ranging from facultative provision of essential nutrients to stimulation of the immune system and exclusion of pathogenic microbes (1–6). Insect-symbiotic associations, some obligatory, are common, where hosts are nutritionally and immunologically dependent on their symbiotic microbes: Buchnera in aphids (7), nitrogen-fixing bacteria in termites (8), Blattabacterium in cockroaches (e.g., ref. 9), lactic acid bacteria in honey bees (10) and Wolbachia, which affects sex determination (11), immune function (e.g., ref. 12) and nutrition (13) in many insect species. The alimentary tract, and especially the hindgut of many (possibly all) insects, is persistently colonized by opportunistic, facultative, and commensal microbiota largely structured by exogenous (diet and local environment) and endogenous (gut environment) factors. The commensal gut microbiota can modulate various aspects of insect biology, including behavior (e.g., refs. 14–16), host–parasite and host–pathogen interactions (e.g., refs. 2 and 4), and various life history traits (1, 17).The German cockroach, Blattella germanica is a major pest of the built environment, where it can acquire and transmit pathogens, contaminate food, and produce allergenic proteins that cause human morbidity (18, 19). The German cockroach lives in aggregations (20), and contact with conspecifics accelerates nymphal development (21) and reproductive maturation in both sexes (22, 23). Younger nymphs benefit from coprophagy in aggregations (24), and gregarious behavior may also facilitate mate location, predator avoidance, thermoregulation, and prevention of water loss. Fidelity to the resting/aggregation site may also facilitate group foraging in the rapidly changing human environment. Aggregation behavior is mediated by at least two types of chemical cues: endogenous compounds produced by the insect and compounds contained in feces. Cuticular hydrocarbons facilitate aggregations (25), and salivary compounds contribute to dissolution of aggregations (26); both are examples of endogenous signals. Feces-associated compounds function as powerful attractants and arrestants in all life stages of the German cockroach (27, 28).Identification of the fecal aggregation pheromones of cockroaches has been fraught with controversy. Candidate pheromones are thought to be endogenously produced by rectal pads (29), with arrestment agents, including blattellastanoside A and B (30) and volatile carboxylic acids (VCAs) (31, 32), and attractants, including ammonia, alkyl amines, amino alcohols, alcohols (33), and VCAs (31, 32). However, the chemical profiles of aggregation-inducing agents vary greatly among reports. The structures of blattellastanosides may be an artifact of chemical isolation and fractionation (34). Some compounds are inconsistently detected in feces, and behavioral responses to them range from attraction to neutral to avoidance (32, 35). More than 150 compounds, including 57 carboxylic acids, have been identified from feces of the German cockroach (31). Because methylation decreased the aggregation response (31), a mix of VCAs was considered the likely aggregation stimuli (32).Symbiotic and commensal bacteria modulate the production of sex pheromones in grass grub beetles (36) and Drosophila (15) and the aggregation pheromone in locusts (37). We hypothesized that the fecal VCAs that mediate aggregation in the German cockroach originate from the bacterial community in the feces, and, because gut-associated bacteria are acquired from the environment, we posited that both the VCA profiles and the behavioral responses to them depend on environmental conditions. Our behavioral assays and chemical analysis revealed that the feces of axenically reared cockroaches (no microorganisms in the alimentary tract) contained many fewer VCAs and failed to elicit aggregation behavior. Inoculation with fecal aerobic bacteria rescued the aggregation activity of fecal extracts of axenic cockroaches. A synthetic blend of VCAs was an effective aggregation stimulus for German cockroaches. We propose that gut bacteria impact the production of fecal VCAs as aggregation agents and that cockroaches use fecal VCAs from commensal microbes as aggregation cues that reflect their colony odor.     

Minimal model for collective kinetochore–microtubule dynamics

Chromosome segregation during cell division depends on interactions of kinetochores with dynamic microtubules (MTs). In many eukaryotes, each kinetochore binds multiple MTs, but the collective behavior of these coupled MTs is not well understood. We present a minimal model for collective kinetochore–MT dynamics, based on in vitro measurements of individual MTs and their dependence on force and kinetochore phosphorylation by Aurora B kinase. For a system of multiple MTs connected to the same kinetochore, the force–velocity relation has a bistable regime with two possible steady-state velocities: rapid shortening or slow growth. Bistability, combined with the difference between the growing and shrinking speeds, leads to center-of-mass and breathing oscillations in bioriented sister kinetochore pairs. Kinetochore phosphorylation shifts the bistable region to higher tensions, so that only the rapidly shortening state is stable at low tension. Thus, phosphorylation leads to error correction for kinetochores that are not under tension. We challenged the model with new experiments, using chemically induced dimerization to enhance Aurora B activity at metaphase kinetochores. The model suggests that the experimentally observed disordering of the metaphase plate occurs because phosphorylation increases kinetochore speeds by biasing MTs to shrink. Our minimal model qualitatively captures certain characteristic features of kinetochore dynamics, illustrates how biochemical signals such as phosphorylation may regulate the dynamics, and provides a theoretical framework for understanding other factors that control the dynamics in vivo.Microtubule (MT) dynamics are critical for cell division. Plus ends of spindle MTs interact with kinetochores, protein complexes that assemble at the centromere of each chromosome, and these dynamic MTs exert forces to move chromosomes. Individual MTs are “dynamically unstable,” spontaneously switching between a polymerizing state and a depolymerizing state (1) with growth, shortening, and switching rates that are regulated by the forces exerted at the MT tips (2–6). For many eukaryotes, however, multiple MTs are connected to each kinetochore, giving rise to collective MT behavior that is not well understood and can be entirely different from the behavior of individual MTs. Here, we develop a model of collective MT dynamics based on the measured force-dependent dynamics of individual MTs.Accurate chromosome segregation depends on correctly biorienting the kinetochore pairs by attaching sister kinetochores to opposite spindle poles. Properly attached kinetochores undergo center-of-mass (CM) and breathing oscillations that are regulated by collective MT dynamics (7–12). Incorrect attachments—such as syntelic attachment of both kinetochores to the same pole—must be corrected (13–17). Tension may cue this process because bioriented kinetochore pairs are under tension while syntelically attached kinetochores are not (7, 9, 15, 17, 18). Error correction is also mediated by Aurora B kinase phosphorylating MT-binding kinetochore proteins (13–17, 19–21). A consistent theory of metaphase kinetochore–MT dynamics should capture CM and breathing oscillations for correctly attached pairs and elucidate the contributions of tension and phosphorylation to syntelic error correction.Several models suggest that chromosome oscillations result from competition between poleward MT-based pulling and antipoleward “polar ejection” forces (22–24). Another model proposes that oscillations occur via a general mechanobiochemical feedback (25). Models of force-dependent MTs interacting with the same object also exhibit cooperative behavior (5, 26–29). However, these models do not explain error correction dynamics. Thus, the underlying physical mechanisms coordinating metaphase chromosome motions are unclear.We address these issues by developing a minimal model for collective MT dynamics based on in vitro measurements of single MTs interacting dynamically with kinetochore proteins (4, 6, 20, 21). In the model, MT polymerization and rescue are promoted by tension and inhibited by compression, whereas depolymerization and catastrophe are enhanced by compression and reduced by tension. With just these features, we find a robust and versatile mechanism by which force-dependent MTs coupled to the same kinetochore may drive metaphase chromosome motions. The force–velocity relation for a MT bundle is fundamentally different from that of a single dynamically unstable MT, exhibiting bistable behavior. Bistability gives rise to kinetochore oscillations and is shifted by phosphorylation to produce error correction. The model qualitatively predicts kinetochore motions in our experiments in which Aurora B is hyperactivated in bioriented kinetochore pairs. Thus, we find that many characteristics of metaphase kinetochore dynamics emerge simply from the force coupling of many MTs to the same kinetochore, and chemical signals such as phosphorylation can regulate this physical mechanism.