Metabolic dependencies drive species co-occurrence in diverse microbial communities

Microbial communities populate most environments on earth and play a critical role in ecology and human health. Their composition is thought to be largely shaped by interspecies competition for the available resources, but cooperative interactions, such as metabolite exchanges, have also been implicated in community assembly. The prevalence of metabolic interactions in microbial communities, however, has remained largely unknown. Here, we systematically survey, by using a genome-scale metabolic modeling approach, the extent of resource competition and metabolic exchanges in over 800 communities. We find that, despite marked resource competition at the level of whole assemblies, microbial communities harbor metabolically interdependent groups that recur across diverse habitats. By enumerating flux-balanced metabolic exchanges in these co-occurring subcommunities we also predict the likely exchanged metabolites, such as amino acids and sugars, that can promote group survival under nutritionally challenging conditions. Our results highlight metabolic dependencies as a major driver of species co-occurrence and hint at cooperative groups as recurring modules of microbial community architecture.Microbial communities are ubiquitous in nature and exert a large influence on our environment and health (1–5). These communities exhibit a great compositional variety, ranging from hot-spring assemblies with low species diversity (6) to the human gut microbiota harboring hundreds of species (7, 8). Competition for metabolic resources can affect community composition through competitive exclusion or by facilitating niche differentiation (9–11). Cooperative and syntrophic interactions, such as beneficial metabolic exchanges, are also likely to play an important role because they can substantially alter the nutritional quality of the habitat (8, 9, 11–15). For example, cross-feeding of metabolic by-products such as ethanol and acetate is central to the diversity of cellulose-degrading communities (16). However, such metabolic exchanges are difficult to discover in natural communities, because the metabolites in the environment cannot be easily attributed to a particular donor species or to the abiotic sources. Moreover, species can often use and secrete a large number of metabolites (17, 18), further hampering the elucidation of metabolic exchanges. Here, we tackle these challenges by introducing a modeling approach applicable to large microbial communities. Currently available methods for simulating metabolic exchanges (8, 19–22) are not directly relevant to communities occurring in nature. Whereas some of these methods use only topological information, ignoring mass balance and growth constraints, the others require prior knowledge of metabolic objective functions of the member species (i.e., evolutionarily selected beneficial characteristics such as high growth rate or optimal ATP production)—information that is often not available. In contrast, our modeling approach, termed “species metabolic interaction analysis,” or SMETANA, can be readily applied with as little information as species identity and their genome sequences. Starting with a community metabolic model assembled from the member-species-level models, SMETANA maps all possible interspecies metabolic exchanges. The methodology thus provides an unbiased estimate of the metabolic interaction potential of a community as well as identifies likely exchanged metabolites. We used this approach to interrogate over 800 microbial communities and co-occurring subcommunities therein. To capture interacting species modules beyond pairs, we also considered subcommunities with simultaneous co-occurrence of up to four species. Our results highlight metabolic dependencies as a key biotic force shaping the composition of diverse microbial communities in nature.     

Self-affirmation alters the brain’s response to health messages and subsequent behavior change

Health communications can be an effective way to increase positive health behaviors and decrease negative health behaviors; however, those at highest risk are often most defensive and least open to such messages. For example, increasing physical activity among sedentary individuals affects a wide range of important mental and physical health outcomes, but has proven a challenging task. Affirming core values (i.e., self-affirmation) before message exposure is a psychological technique that can increase the effectiveness of a wide range of interventions in health and other domains; however, the neural mechanisms of affirmation’s effects have not been studied. We used functional magnetic resonance imaging (fMRI) to examine neural processes associated with affirmation effects during exposure to potentially threatening health messages. We focused on an a priori defined region of interest (ROI) in ventromedial prefrontal cortex (VMPFC), a brain region selected for its association with self-related processing and positive valuation. Consistent with our hypotheses, those in the self-affirmation condition produced more activity in VMPFC during exposure to health messages and went on to increase their objectively measured activity levels more. These findings suggest that affirmation of core values may exert its effects by allowing at-risk individuals to see the self-relevance and value in otherwise-threatening messages.Promoting physical activity and decreasing sedentary behavior are major strategies to manage and prevent chronic diseases (1–16). In particular, sedentary behavior increases risk, independent of other types of activity, and exchanging sedentary for even light activity has physiological and psychological benefits (17–23). However, sedentary lifestyle is still prevalent despite worldwide efforts to increase activity; according to the World Health Organization, “60% to 85% of people in the world—from both developed and developing countries—lead sedentary lifestyles” (24). Thus, effective, theory-driven behavior change interventions are critical (25, 26).One major difficulty in decreasing sedentary and other health risk behaviors through health communication tools is that self-relevant health messages can be perceived to be threatening to self-worth and are often met with resistance. This phenomenon speaks to a classic and problematic paradox: those at highest risk are likely to be defensive, reducing openness to altering risk behaviors (27).     

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.     

From the Cover: Why the proportion of transmission during early-stage HIV infection does not predict the long-term impact of treatment on HIV incidence

Antiretroviral therapy (ART) reduces the infectiousness of HIV-infected persons, but only after testing, linkage to care, and successful viral suppression. Thus, a large proportion of HIV transmission during a period of high infectiousness in the first few months after infection (“early transmission”) is perceived as a threat to the impact of HIV “treatment-as-prevention” strategies. We created a mathematical model of a heterosexual HIV epidemic to investigate how the proportion of early transmission affects the impact of ART on reducing HIV incidence. The model includes stages of HIV infection, flexible sexual mixing, and changes in risk behavior over the epidemic. The model was calibrated to HIV prevalence data from South Africa using a Bayesian framework. Immediately after ART was introduced, more early transmission was associated with a smaller reduction in HIV incidence rate—consistent with the concern that a large amount of early transmission reduces the impact of treatment on incidence. However, the proportion of early transmission was not strongly related to the long-term reduction in incidence. This was because more early transmission resulted in a shorter generation time, in which case lower values for the basic reproductive number (R₀) are consistent with observed epidemic growth, and R₀ was negatively correlated with long-term intervention impact. The fraction of early transmission depends on biological factors, behavioral patterns, and epidemic stage and alone does not predict long-term intervention impacts. However, early transmission may be an important determinant in the outcome of short-term trials and evaluation of programs.Recent studies have confirmed that effective antiretroviral therapy (ART) reduces the transmission of HIV among stable heterosexual couples (1–3). This finding has generated interest in understanding the population-level impact of HIV treatment on reducing the rate of new HIV infections in generalized epidemic settings (4). Research, including mathematical modeling (5–10), implementation research (11), and major randomized controlled trials (12–14), are focused on how ART provision might be expanded strategically to maximize its public health benefits (15, 16).One concern is that if a large fraction of HIV transmission occurs shortly after a person becomes infected, before the person can be diagnosed and initiated on ART, this will limit the potential impact of HIV treatment on reducing HIV incidence (9, 17, 18). Data suggest that persons are more infectious during a short period of “early infection” after becoming infected with HIV (19–22), although there is debate about the extent, duration, and determinants of elevated infectiousness (18, 23). The amount of transmission that occurs also will depend on patterns of sexual behavior and sexual networks (17, 24–27). There have been estimates for the contribution of early infection to transmission from mathematical models (7, 17, 21, 24–26) and phylogenetic analyses (28–31), but these vary widely, from 5% to above 50% (23).In this study, we use a mathematical model to quantify how the proportion of transmission that comes from persons who have been infected recently affects the impact of treatment scale-up on HIV incidence. The model is calibrated to longitudinal HIV prevalence data from South Africa using a Bayesian framework. Thus, the model accounts for not only the early epidemic growth rate highlighted in previous research (5, 9, 18), but also the heterogeneity and sexual behavior change to explain the peak and decline in HIV incidence observed in sub-Saharan African HIV epidemics (32, 33).The model calibration allows uncertainty about factors that determine the amount of early transmission, including the relative infectiousness during early infection, heterogeneity in propensity for sexual risk behavior, assortativity in sexual partner selection, reduction in risk propensity over the life course, and population-wide reductions in risk behavior in response to the epidemic (32, 33). This results in multiple combinations of parameter values that are consistent with the observed epidemic and variation in the amount of early transmission. We simulated the impact of a treatment intervention and report how the proportion of early transmission correlates with the reduction in HIV incidence from the intervention over the short- and long-term.     

Predictive Value of Brain Natriuretic Peptides in Patients on Peritoneal Dialysis: Results from the ADEMEX Trial

Background and objectives: Natriuretic peptides have been suggested to be of value in risk stratification in dialysis patients. Data in patients on peritoneal dialysis remain limited.Design, setting, participants, & measurements: Patients of the ADEMEX trial (ADEquacy of peritoneal dialysis in MEXico) were randomized to a control group [standard 4 × 2L continuous ambulatory peritoneal dialysis (CAPD); n = 484] and an intervention group (CAPD with a target creatinine clearance ≥60L/wk/1.73 m²; n = 481). Natriuretic peptides were measured at baseline and correlated with other parameters as well as evaluated for effects on patient outcomes.Results: Control group and intervention group were comparable at baseline with respect to all measured parameters. Baseline values of natriuretic peptides were elevated and correlated significantly with levels of residual renal function but not with body size or diabetes. Baseline values of N-terminal fragment of B-type natriuretic peptide (NT-proBNP) but not proANP(1–30), proANP(31–67), or proANP(1–98) were independently highly predictive of overall survival and cardiovascular mortality. Volume removal was also significantly correlated with patient survival.Conclusions. NT-proBNP have a significant predictive value for survival of CAPD patients and may be of value in guiding risk stratification and potentially targeted therapeutic interventions.Plasma levels of cardiac natriuretic peptides are elevated in patients with chronic kidney disease, owing to impairment of renal function, hypertension, hypervolemia, and/or concomitant heart disease (1–7). Atrial natriuretic peptide (ANP) and particularly brain natriuretic peptide (BNP) levels are linked independently to left ventricular mass (3–5,8–16) and function (3,6–17) and predict total and cardiovascular mortality (1,3,8,10,12,18) as well as cardiac events (12,19). ANP and BNP decrease significantly during hemodialysis treatment but increase again during the interdialytic interval (1,2,4,6,7,14,17,20–23). Levels in patients on peritoneal dialysis (PD) have been found to be lower than in patients on hemodialysis (11,24–26), but the correlations with left ventricular function and structure are maintained in both types of dialysis modalities (11,15,27,28).The high mortality of patients on peritoneal dialysis and the failure of dialytic interventions to alter this mortality (29,30) necessitate renewed attention into novel methods of stratification and identification of patients at highest risk to be targeted for specific interventions. Cardiac natriuretic peptides are increasingly considered to fulfill this role in nonrenal patients. Evaluations of cardiac natriuretic peptides in patients on PD have been limited by small numbers (3,9,11,12,15,24–26) and only one study examined correlations between natriuretic peptide levels and outcomes (12). The PD population enrolled in the ADEMEX trial offered us the opportunity to evaluate cardiac natriuretic peptides and their value in predicting outcomes in the largest clinical trial ever performed on PD (29,30). It is hoped that such an evaluation would identify patients at risk even in the absence of overt clinical disease and hence facilitate or encourage interventions with salutary outcomes.     

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.     

α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation

Physiologically, α-synuclein chaperones soluble NSF attachment protein receptor (SNARE) complex assembly and may also perform other functions; pathologically, in contrast, α-synuclein misfolds into neurotoxic aggregates that mediate neurodegeneration and propagate between neurons. In neurons, α-synuclein exists in an equilibrium between cytosolic and membrane-bound states. Cytosolic α-synuclein appears to be natively unfolded, whereas membrane-bound α-synuclein adopts an α-helical conformation. Although the majority of studies showed that cytosolic α-synuclein is monomeric, it is unknown whether membrane-bound α-synuclein is also monomeric, and whether chaperoning of SNARE complex assembly by α-synuclein involves its cytosolic or membrane-bound state. Here, we show using chemical cross-linking and fluorescence resonance energy transfer (FRET) that α-synuclein multimerizes into large homomeric complexes upon membrane binding. The FRET experiments indicated that the multimers of membrane-bound α-synuclein exhibit defined intermolecular contacts, suggesting an ordered array. Moreover, we demonstrate that α-synuclein promotes SNARE complex assembly at the presynaptic plasma membrane in its multimeric membrane-bound state, but not in its monomeric cytosolic state. Our data delineate a folding pathway for α-synuclein that ranges from a monomeric, natively unfolded form in cytosol to a physiologically functional, multimeric form upon membrane binding, and show that only the latter but not the former acts as a SNARE complex chaperone at the presynaptic terminal, and may protect against neurodegeneration.α-Synuclein is an abundant presynaptic protein that physiologically acts to promote soluble NSF attachment protein receptor (SNARE) complex assembly in vitro and in vivo (1–3). Point mutations in α-synuclein (A30P, E46K, H50Q, G51D, and A53T) as well as α-synuclein gene duplications and triplications produce early-onset Parkinson''s disease (PD) (4–10). Moreover, α-synuclein is a major component of intracellular protein aggregates called Lewy bodies, which are pathological hallmarks of neurodegenerative disorders such as PD, Lewy body dementia, and multiple system atrophy (11–14). Strikingly, neurotoxic α-synuclein aggregates propagate between neurons during neurodegeneration, suggesting that such α-synuclein aggregates are not only intrinsically neurotoxic but also nucleate additional fibrillization (15–18).α-Synuclein is highly concentrated in presynaptic terminals where α-synuclein exists in an equilibrium between a soluble and a membrane-bound state, and is associated with synaptic vesicles (19–22). The labile association of α-synuclein with membranes (23, 24) suggests that binding of α-synuclein to synaptic vesicles, and its dissociation from these vesicles, may regulate its physiological function. Membrane-bound α-synuclein assumes an α-helical conformation (25–32), whereas cytosolic α-synuclein is natively unfolded and monomeric (refs. 25, 26, 31, and 32; however, see refs. 33 and 34 and Discussion for a divergent view). Membrane binding by α-synuclein is likely physiologically important because in in vitro experiments, α-synuclein remodels membranes (35, 36), influences lipid packing (37, 38), and induces vesicle clustering (39). Moreover, membranes were found to be important for the neuropathological effects of α-synuclein (40–44).However, the relation of membrane binding to the in vivo function of α-synuclein remains unexplored, and it is unknown whether α-synuclein binds to membranes as a monomer or oligomer. Thus, in the present study we have investigated the nature of the membrane-bound state of α-synuclein and its relation to its physiological function in SNARE complex assembly. We found that soluble monomeric α-synuclein assembles into higher-order multimers upon membrane binding and that membrane binding of α-synuclein is required for its physiological activity in promoting SNARE complex assembly at the synapse.     

Southern East Asian origin and coexpansion of Mycobacterium tuberculosis Beijing family with Han Chinese

The Beijing family is the most successful genotype of Mycobacterium tuberculosis and responsible for more than a quarter of the global tuberculosis epidemic. As the predominant genotype in East Asia, the Beijing family has been emerging in various areas of the world and is often associated with disease outbreaks and antibiotic resistance. Revealing the origin and historical dissemination of this strain family is important for understanding its current global success. Here we characterized the global diversity of this family based on whole-genome sequences of 358 Beijing strains. We show that the Beijing strains endemic in East Asia are genetically diverse, whereas the globally emerging strains mostly belong to a more homogenous subtype known as “modern” Beijing. Phylogeographic and coalescent analyses indicate that the Beijing family most likely emerged around 30,000 y ago in southern East Asia, and accompanied the early colonization by modern humans in this area. By combining the genomic data and genotyping result of 1,793 strains from across China, we found the “modern” Beijing sublineage experienced massive expansions in northern China during the Neolithic era and subsequently spread to other regions following the migration of Han Chinese. Our results support a parallel evolution of the Beijing family and modern humans in East Asia. The dominance of the “modern” Beijing sublineage in East Asia and its recent global emergence are most likely driven by its hypervirulence, which might reflect adaption to increased human population densities linked to the agricultural transition in northern China.Tuberculosis has plagued human beings since ancient times and remains a leading cause of global morbidity and mortality. The causative agent of human tuberculosis is the Mycobacterium tuberculosis complex (MTBC), a group of organisms that harbor little genetic diversity compared with other bacteria (1). MTBC most likely originated in Africa, although its age is being debated (2–4). The human-adapted MTBC is highly clonal and is classified into seven main phylogenetic groups, designated lineage 1 through lineage 7 (2). These seven lineages show strong biogeographic associations that have been proposed to result from codiversification with different human populations (2, 5). Lineage 2 that dominates in East Asia is one of the most successful MTBC variants; more than a quarter of the global tuberculosis epidemic is caused by this lineage (6, 7). Lineage 2 contains strains that mostly belong to the so-called Beijing family (8, 9). This strain family has attracted great attentions due to its global emergence in recent decades (6, 7, 10–12), its tendency to cause disease outbreak (13–17), and its association with antibiotic resistance (12, 18). Experimental and clinical evidences suggest a hypervirulent phenotype of Beijing strains (12, 19), and a higher mutation rate compared with other strains (20).According to genotyping data from previous molecular-epidemiology studies, most Beijing strains from widespread geographic areas showed a remarkable degree of genetic similarity (6, 21), suggesting this strain family might have emerged from recent expansions. It was hypothesized that vaccination with Bacille Calmette Guerin (bacillus Calmette–Guérin) that has been widely implemented in East Asian countries might be the force driving the dominance of this strain family in this area (21). Moreover, the global emergence of the Beijing family may have been due to its hypervirulence and association with drug resistance (7, 18). However, there were discrepant results regarding the relative protective effect of bacillus Calmette–Guérin vaccination against Beijing strains from animal infection experiments (19), and many epidemiological studies failed to find any association between bacillus Calmette–Guérin vaccination and Beijing strains (22–25). The link between drug resistance and the Beijing family has primarily been observed in regions where this family has emerged recently (e.g., Cuba, South Africa, countries of the former Soviet Union) but not in East Asian, where the Beijing family has been endemic for a long time (18, 26). Furthermore, more recent studies indicate that the expansion of the Beijing family may have started long before the introduction of vaccination and antibiotic treatment (2, 3, 27).With the increased availability of genotyping data, the Beijing strains were proved more heterogeneous than initially estimated, and several Beijing sublineages have been identified (28–31). However, a full understanding of the genetic diversity of Beijing family is constrained by the low amount of nucleotide variation (8, 32). Whole-genome sequencing provides an ideal tool to study the genetic diversity of MTBC, and new insights into the origin and evolution of MTBC have been gained (2, 4, 20, 33–35). The genomic diversity of Beijing family was initially studied in a most recent study, in which a general East Asian origin and recent expansions of this strain family were suggested (36). However, the details about the origin and primary dissemination of Beijing family remain unclear. Answering of these questions is important to better understand the virulence of this lineage and its global success. Here, we combined whole-genome sequencing of key strains with detailed single nucleotide polymorphism (SNP) typing of a large collection of clinical MTBC strains isolated from across China. Our results strongly support a southern East Asian origin of the MTBC Beijing family and suggest a parallel evolution of this family with modern humans in East Asia during the last 30,000 y.     

Evolutionary limits to cooperation in microbial communities

Microbes produce many compounds that are costly to a focal cell but promote the survival and reproduction of neighboring cells. This observation has led to the suggestion that microbial strains and species will commonly cooperate by exchanging compounds. Here, we examine this idea with an ecoevolutionary model where microbes make multiple secretions, which can be exchanged among genotypes. We show that cooperation between genotypes only evolves under specific demographic regimes characterized by intermediate genetic mixing. The key constraint on cooperative exchanges is a loss of autonomy: strains become reliant on complementary genotypes that may not be reliably encountered. Moreover, the form of cooperation that we observe arises through mutual exploitation that is related to cheating and “Black Queen” evolution for a single secretion. A major corollary is that the evolution of cooperative exchanges reduces community productivity relative to an autonomous strain that makes everything it needs. This prediction finds support in recent work from synthetic communities. Overall, our work suggests that natural selection will often limit cooperative exchanges in microbial communities and that, when exchanges do occur, they can be an inefficient solution to group living.

‘Benefit-of-the-species’ arguments … provide for the reader an escape from inner conflict, exacting nothing emotionally beyond what most of us learn to accept in childhood, that most forms of life exploit and prey on one another.

Hamilton, 1975 (1)

Microbes typically live in dense communities containing many strains and species. These genetically diverse societies are widespread and central to how microbes affect us, including examples such as the gut microbiome, polymicrobial infections, and communities vital to bioremediation and nutrient cycling (2, 3). In these collectives, ecological interactions are thought to be both common and strong given that cell density is typically high and that microbes possess many phenotypes that influence the reproduction and survival of surrounding cells (4, 5). Such social traits include many secretions, such as extracellular enzymes and scavenging molecules (4–6), and other beneficial “leaky” traits, such as detoxification agents (7) or amino acids (8, 9).A central explanation for cooperative phenotypes in microbes is that they function to help cells of the same genotype (10, 11), which is backed up by a growing body of theory and experiments (12–18). However, it is also clear that, in nature, microbes commonly interact with many different genotypes (both different strains and species) in complex ecological networks (19–21). Do these different microbial genotypes cooperate with one another? Understanding this question is central to building models of microbial communities and how they will respond to both environmental and anthropogenic perturbations (22).Studies involving genetically engineered (8, 9, 23, 24) and/or artificially selected communities (23, 25, 26) emphasize how easily cooperation between genotypes can be achieved in the laboratory. Additionally, there are a growing number of suggestions that cooperation should commonly evolve between microbial strains and species (27–30). This view contrasts with empirical surveys of natural bacterial communities, which suggest that competitive interactions predominate over cooperative interactions (31). However, it has also been suggested that cooperation between different genotypes may explain the unculturability of many species in the laboratory when in monoculture (32–34). If correct, studies with culturable species could underestimate cooperativity in microbial communities.The potential for cooperation between different microbial genotypes then remains unclear. Indeed, we even lack clear predictions of what to expect. There is a need for general theory on cooperation between microbial genotypes. One microbial interaction that has been explored theoretically is syntrophy, where one species produces a toxic waste product that another species consumes (35–38). Syntrophy is likely to be ecologically important and under some conditions (36), can benefit both species. However, syntrophic species need not pay energetic costs to interact: one species is producing waste, and the other species is feeding. Such byproduct cooperation can, therefore, readily evolve but is fundamentally different to the exchange of costly secretions (39, 40). Other models have analyzed when costly cooperation between species is expected in microbes and other organisms (35, 39, 41). However, these models assume that there is no opportunity for one partner to express the beneficial trait of the other. Although this constraint will sometimes occur, there is considerable functional overlap in the cooperative traits of microbial species (7). In addition, the potential for horizontal gene transfer in microbes means that there is a broad scope for a focal strain to pick up the phenotypes of co-occurring strains and species (42–44).Here, we examine the potential for microbial cooperation between different strains and species. We base our work on the well-established models of within-genotype microbial cooperation for a single public good (12, 18, 45–47) so that the relationship to previous work is clear (SI Materials and Methods). We add one key feature to these models: we allow cells to invest in multiple distinct cooperative secretions, such that there is the potential for different genotypes to exchange secretions with one another. Our analysis shows that the degree of genetic mixing defines the potential for cooperation both within and between genotypes. Low mixing favors genotypes that produce all secretions, whereas high mixing favors genotypes that do not produce any at all. Only for intermediate levels of genetic mixing do we find between-genotype cooperation, where strains produce a subset of secretions and rely on other genotypes for the complementary traits. Moreover, the form of cooperation that emerges is inefficient and results in a loss of productivity relative to one genotype making all secretions. Natural selection limits both the occurrence and effectiveness of cooperation within microbial communities.     

Kinesin processivity is gated by phosphate release

Kinesin-1 is a dimeric motor protein, central to intracellular transport, that steps hand-over-hand toward the microtubule (MT) plus-end, hydrolyzing one ATP molecule per step. Its remarkable processivity is critical for ferrying cargo within the cell: over 100 successive steps are taken, on average, before dissociation from the MT. Despite considerable work, it is not understood which features coordinate, or “gate,” the mechanochemical cycles of the two motor heads. Here, we show that kinesin dissociation occurs subsequent to, or concomitant with, phosphate (P_i) release following ATP hydrolysis. In optical trapping experiments, we found that increasing the steady-state population of the posthydrolysis ADP·P_i state (by adding free P_i) nearly doubled the kinesin run length, whereas reducing either the ATP binding rate or hydrolysis rate had no effect. The data suggest that, during processive movement, tethered-head binding occurs subsequent to hydrolysis, rather than immediately after ATP binding, as commonly suggested. The structural change driving motility, thought to be neck linker docking, is therefore completed only upon hydrolysis, and not ATP binding. Our results offer additional insights into gating mechanisms and suggest revisions to prevailing models of the kinesin reaction cycle.Since its discovery nearly 30 years ago (1), kinesin-1—the founding member of the kinesin protein superfamily—has emerged as an important model system for studying biological motors (2, 3). During “hand-over-hand” stepping, kinesin dimers alternate between a two–heads-bound (2-HB) state, with both heads attached to the microtubule (MT), and a one–head-bound (1-HB) state, where a single head, termed the tethered head, remains free of the MT (4, 5). The catalytic cycles of the two heads are maintained out of phase by a series of gating mechanisms, thereby enabling the dimer to complete, on average, over 100 steps before dissociating from the MT (6–8). A key structural element for this coordination is the neck linker (NL), a ∼14-aa segment that connects each catalytic head to a common stalk (9). In the 1-HB state, nucleotide binding is thought to induce a structural reconfiguration of the NL, immobilizing it against the MT-bound catalytic domain (2, 3, 10–17). This transition, called “NL docking,” is believed to promote unidirectional motility by biasing the position of the tethered head toward the next MT binding site (2, 3, 10–17). The completion of an 8.2-nm step (18) entails the binding of this tethered head to the MT, ATP hydrolysis, and detachment of the trailing head, thereby returning the motor to the ATP-waiting state (2, 3, 10–17). Prevailing models of the kinesin mechanochemical cycle (2, 3, 10, 14, 15, 17), which invoke NL docking upon ATP binding, explain the highly directional nature of kinesin motility and offer a compelling outline of the sequence of events following ATP binding. Nevertheless, these abstractions do not speak directly to the branching transitions that determine whether kinesin dissociates from the MT (off-pathway) or continues its processive reaction cycle (on-pathway). The distance moved by an individual motor before dissociating—the run length—is limited by unbinding from the MT. The propensity for a dimer to unbind involves a competition among multiple, force-dependent transitions in the two heads, which are not readily characterized by traditional structural or bulk biochemical approaches. Here, we implemented high-resolution single-molecule optical trapping techniques to determine transitions in the kinesin cycle that govern processivity.     

Quantitative analysis of vesicle recycling at the calyx of Held synapse

Vesicle recycling is pivotal for maintaining reliable synaptic signaling, but its basic properties remain poorly understood. Here, we developed an approach to quantitatively analyze the kinetics of vesicle recycling with exquisite signal and temporal resolution at the calyx of Held synapse. The combination of this electrophysiological approach with electron microscopy revealed that ∼80% of vesicles (∼270,000 out of ∼330,000) in the nerve terminal are involved in recycling. Under sustained stimulation, recycled vesicles start to be reused in tens of seconds when ∼47% of the preserved vesicles in the recycling pool (RP) are depleted. The heterogeneity of vesicle recycling as well as two kinetic components of RP depletion revealed the existence of a replenishable pool of vesicles before the priming stage and led to a realistic kinetic model that assesses the size of the subpools of the RP. Thus, our study quantified the kinetics of vesicle recycling and kinetically dissected the whole vesicle pool in the calyceal terminal into the readily releasable pool (∼0.6%), the readily priming pool (∼46%), the premature pool (∼33%), and the resting pool (∼20%).Synaptic vesicle recycling ensures synaptic transmission during sustained neuronal activity (1–3). Despite its crucial role, the cycle is poorly understood. In contrast to vesicle exocytosis and endocytosis, which can be directly assayed by presynaptic capacitance measurements and postsynaptic current recordings, vesicle recycling is usually investigated by fluorescence imaging and electron microscopy (EM) with limited signal or temporal resolution (4–7). Likely owing to technical difficulties, the basic properties of vesicle recycling, such as the size of the recycling pool (RP) (3, 6, 8–11), the kinetics of vesicle recycling (6, 8–12), and how the RP supports synaptic transmission (1, 13–15) remain to be elucidated. Classically, presynaptic vesicles can be functionally divided into three populations: the readily releasable pool (RRP), the reserve pool, and the resting pool (3, 16, 17). The RRP is defined as being composed of docked and immediately releasable vesicles (17), which are usually depleted by high-frequency stimulation, prolonged presynaptic depolarization, or the application of hypertonic solution (18–21). The reserve pool functions as a reservoir and serves to maintain vesicle refilling into the RRP (2, 3). These two pools together are commonly referred to as the RP. The resting pool serves as a depot of vesicles for backup use (16, 22). However, it has been debated for a decade whether nerve terminals use the majority (∼100%, from electrophysiology) or only a small fraction (5–40%, from fluorescence imaging and EM) of vesicles in recycling, and whether the RP size undergoes dynamic changes during varied neuronal activity (6, 7, 23–28).The use of vesicles in recycling is a critical determinant of synaptic transmission (1, 13–15). However, it has never been rigorously determined how fast recently recaptured vesicles are organized to recycle and whether vesicles in the RP are homogeneously ready for use (25). Two forms of vesicle retrieval, “kiss-and-run” and full collapse, have been reported for many years. It is still ambiguous whether the rapidly recaptured vesicles in the kiss-and-run mode can be rapidly reused (29–31).Here, we addressed the above issues by developing a new approach to quantify the basic properties of vesicle recycling with unparalleled precision. Different from previous studies in cultured cell systems, the present work combined electrophysiological measurements and EM observations at the calyx of Held synapse in acute brain slices, quantitatively analyzed synaptic vesicle recycling, and kinetically dissected the recycling vesicle pool. We propose a realistic kinetic model and provide new insights into the mechanism that ensures rate-limited but sustainable synaptic transmission.     

From the Cover: PNAS Plus: Adverse childhood experiences and physiological wear-and-tear in midlife: Findings from the 1958 British birth cohort

Allostatic load (AL) is a measure of overall physiological wear-and-tear over the life course, which could partially be the consequence of early life exposures. AL could allow a better understanding of the potential biological pathways playing a role in the construction of the social gradient in adult health. To explore the biological embedding hypothesis, we examined whether adverse childhood experiences (ACEs) are associated with elevated AL in midlife. We used imputed data on 3,782 women and 3,753 men of the National Child Development Study in Britain followed up seven times. ACEs were measured using prospective data collected at ages 7, 11, and 16. AL was operationalized using data from the biomedical survey collected at age 44 on 14 parameters representing four biological systems. We examined the role of adult health behaviors, body mass index (BMI), and socioeconomic status as potential mediators using a path analysis. ACEs were associated with higher AL for both men and women after adjustment for early life factors and childhood pathologies. The path analysis showed that the association between ACEs and AL was largely explained by early adult factors at age 23 and 33. For men, the total mediated effect was 59% (for two or more ACEs) via health behaviors, education level, and wealth. For women, the mediated effect represented 76% (for two or more ACEs) via smoking, BMI, education level, and wealth. Our results indicate that early psychosocial stress has an indirect lasting impact on physiological wear-and-tear via health behaviors, BMI, and socioeconomic factors in adulthood.Health disparities are observed for a wide range of health indicators from risk factors, incidence of chronic diseases, and mortality across the world (1). According to Hertzman, the socioeconomic gradient in health “is capable of replicating itself on new disease processes as they emerge in society” (2). Recently, epidemiological studies have shown that classic determinants are not sufficient for explaining the social gradient in health (3). This may point toward the existence of other mechanisms influencing health, like a potential biological pathway. The notion of allostatic load (AL) may be useful to explore how experiences over the life course may “get under the skin” and become biologically embedded (2, 4, 5).In the last two decades, epidemiological research has used the concept of AL to explain how chronic stress can lead to physiological dysregulation and disease (6–12). AL is a measure of overall physiological wear-and-tear over the life course, which could be the consequence of early life exposures (13, 14). According to AL theory, cumulative and repeated activation of compensatory physiological mechanism in response to chronic stress can lead to a multisystem predisease state represented by a dysregulation of neuroendocrine, metabolic, inflammatory, or cardiovascular parameters (15, 16). Empirical evidence shows that AL has strong correlations to subclinical conditions, morbidity, and mortality (12, 17), and may be a useful measure of overall health, rather than considering each biomarker separately (6, 18).Several studies have suggested that exposure to chronic stress during sensitive periods of development may alter the balance and responsiveness of physiological systems and have long-term effects on health (2, 13, 14, 19–21). Early life exposure to adverse childhood experiences (ACEs), like trauma, abuse, or maltreatment has been linked to alterations in brain structure and neurobiological stress–response systems, which have consequences for health and emotional well-being (22, 23). Exposure to ACE could influence health through a broad range of behavioral and socioeconomic mechanisms. For instance, the ACE study explored the relationship between ACE and health behaviors, linking childhood trauma to long-term effects on health via health risk behaviors such as alcohol consumption, smoking, and sexual behaviors, among others. Felitti et al. (24) also suggested that ACE could be a common pathway to social, emotional, and cognitive impairments that may lead to increased risky behaviors. It has been established that the adoption of health behaviors may also be explained by wide and complex psychological processes such as self-regulation, self-efficacy, and self-management mechanisms (25, 26). Furthermore, socioeconomic and material conditions in childhood appear to be linked to later brain development and cognition (27). Lately, epidemiological studies have shown that ACEs were associated with mortality and health even after adjusting for socioeconomic and behavioral factors, suggesting that a direct biological effect occurring from early life is plausible (28, 29). It has been suggested that psychosocial factors could protect and buffer early adverse circumstances, such as parental warmth and psychological resources, reducing physiological responses and mitigating disease processes (30, 31). However, only a few studies have analyzed the influence of ACE on health over the life course by examining these different pathways, and fewer have used an AL index.The main hypothesis tested in this study is that chronic stress resulting from ACE may be biologically embedded (2, 19) and lead to a cumulative multisystem dysregulation via three broad and intertwined pathways across the life course: (i) an indirect health behaviors pathway, (ii) an indirect socioeconomic/materialist and/or psychosocial pathway, (iii) through a direct biological pathway via alterations of physiological stress systems (e.g., hypothalamic-pituitary-adrenal axis) that could influence health in the long term. The aim of this paper is to explore whether ACEs are associated with elevated AL in midlife. We will examine whether cumulative socioeconomic conditions and/or health behaviors mediate such a relationship, and if the relationships still persists after adjusting for these mediating factors.     

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating (reviewed in refs. 1–3). Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology.In Drosophila melanogaster, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (reviewed in refs. 2 and 3). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (4, 5), and the seminal protein sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating (6–10). However, although a receptor for SP has been identified (11), along with elements of the neural circuit in which it is required (12–14), SP’s mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses?We addressed this question by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics (15–27). In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine (17, 18, 28). Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB) (19, 20). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates (17). Noradrenaline, the vertebrate structural and functional equivalent to OA (29, 30), is important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders (31–38). In this paper we investigate the role of neurons that release OA and tyramine in ovulin’s action. For simplicity, we refer to these neurons as “OA neurons” to reflect the well-established role of OA in ovulation behavior (16–20, 22).We investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology (39–41). We found that ovulin increases ovulation and egg laying through OA neuronal signaling. We also found that ovulin relaxes oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects we detected an ovulin-dependent increase in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin’s stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology.     

Local generation of multineuronal spike sequences in the hippocampal CA1 region

Sequential activity of multineuronal spiking can be observed during theta and high-frequency ripple oscillations in the hippocampal CA1 region and is linked to experience, but the mechanisms underlying such sequences are unknown. We compared multineuronal spiking during theta oscillations, spontaneous ripples, and focal optically induced high-frequency oscillations (“synthetic” ripples) in freely moving mice. Firing rates and rate modulations of individual neurons, and multineuronal sequences of pyramidal cell and interneuron spiking, were correlated during theta oscillations, spontaneous ripples, and synthetic ripples. Interneuron spiking was crucial for sequence consistency. These results suggest that participation of single neurons and their sequential order in population events are not strictly determined by extrinsic inputs but also influenced by local-circuit properties, including synapses between local neurons and single-neuron biophysics.A hypothesized hallmark of cognition is self-organized sequential activation of neuronal assemblies (1). Self-organized neuronal sequences have been observed in several cortical structures (2–5) and neuronal models (6–7). In the hippocampus, sequential activity of place cells (8) may be induced by external landmarks perceived by the animal during spatial navigation (9) and conveyed to CA1 by the upstream CA3 region or layer 3 of the entorhinal cortex (10). Internally generated sequences have been also described in CA1 during theta oscillations in memory tasks (4, 11), raising the possibility that a given neuronal substrate is responsible for generating sequences at multiple time scales. The extensive recurrent excitatory collateral system of the CA3 region has been postulated to be critical in this process (4, 7, 12, 13).The sequential activity of place cells is “replayed” during sharp waves (SPW) in a temporally compressed form compared with rate modulation of place cells (14–20) and may arise from the CA3 recurrent excitatory networks during immobility and slow wave sleep. The SPW-related convergent depolarization of CA1 neurons gives rise to a local, fast oscillatory event in the CA1 region (“ripple,” 140–180 Hz; refs. 8 and 21). Selective elimination of ripples during or after learning impairs memory performance (22–24), suggesting that SPW ripple-related replay assists memory consolidation (12, 13). Although the local origin of the ripple oscillations is well demonstrated (25, 26), it has been tacitly assumed that the ripple-associated, sequentially ordered firing of CA1 neurons is synaptically driven by the upstream CA3 cell assemblies (12, 15), largely because excitatory recurrent collaterals in the CA1 region are sparse (27). However, sequential activity may also emerge by local mechanisms, patterned by the different biophysical properties of CA1 pyramidal cells and their interactions with local interneurons, which discharge at different times during a ripple (28–30). A putative function of the rich variety of interneurons is temporal organization of principal cell spiking (29–32). We tested the “local-circuit” hypothesis by comparing the probability of participation and sequential firing of CA1 neurons during theta oscillations, natural spontaneous ripple events, and “synthetic” ripples induced by local optogenetic activation of pyramidal neurons.     

N terminus of ASPP2 binds to Ras and enhances Ras/Raf/MEK/ERK activation to promote oncogene-induced senescence

The ASPP2 (also known as 53BP2L) tumor suppressor is a proapoptotic member of a family of p53 binding proteins that functions in part by enhancing p53-dependent apoptosis via its C-terminal p53-binding domain. Mounting evidence also suggests that ASPP2 harbors important nonapoptotic p53-independent functions. Structural studies identify a small G protein Ras-association domain in the ASPP2 N terminus. Because Ras-induced senescence is a barrier to tumor formation in normal cells, we investigated whether ASPP2 could bind Ras and stimulate the protein kinase Raf/MEK/ERK signaling cascade. We now show that ASPP2 binds to Ras–GTP at the plasma membrane and stimulates Ras-induced signaling and pERK1/2 levels via promoting Ras–GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation. These functions require the ASPP2 N terminus because BBP (also known as 53BP2S), an alternatively spliced ASPP2 isoform lacking the N terminus, was defective in binding Ras–GTP and stimulating Raf/MEK/ERK signaling. Decreased ASPP2 levels attenuated H-RasV12–induced senescence in normal human fibroblasts and neonatal human epidermal keratinocytes. Together, our results reveal a mechanism for ASPP2 tumor suppressor function via direct interaction with Ras–GTP to stimulate Ras-induced senescence in nontransformed human cells.ASPP2, also known as 53BP2L, is a tumor suppressor whose expression is altered in human cancers (1). Importantly, targeting of the ASPP2 allele in two different mouse models reveals that ASPP2 heterozygous mice are prone to spontaneous and γ-irradiation–induced tumors, which rigorously demonstrates the role of ASPP2 as a tumor suppressor (2, 3). ASPP2 binds p53 via the C-terminal ankyrin-repeat and SH3 domain (4–6), is damage-inducible, and can enhance damage-induced apoptosis in part through a p53-mediated pathway (1, 2, 7–10). However, it remains unclear what biologic pathways and mechanisms mediate ASPP2 tumor suppressor function (1). Indeed, accumulating evidence demonstrates that ASPP2 also mediates nonapoptotic p53-independent pathways (1, 3, 11–15).The induction of cellular senescence forms an important barrier to tumorigenesis in vivo (16–21). It is well known that oncogenic Ras signaling induces senescence in normal nontransformed cells to prevent tumor initiation and maintain complex growth arrest pathways (16, 18, 21–24). The level of oncogenic Ras activation influences its capacity to activate senescence; high levels of oncogenic H-RasV12 signaling leads to low grade tumors with senescence markers, which progress to invasive cancers upon senescence inactivation (25). Thus, tight control of Ras signaling is critical to ensure the proper biologic outcome in the correct cellular context (26–28).The ASPP2 C terminus is important for promoting p53-dependent apoptosis (7). The ASPP2 N terminus may also suppress cell growth (1, 7, 29–33). Alternative splicing can generate the ASPP2 N-terminal truncated protein BBP (also known as 53BP2S) that is less potent in suppressing cell growth (7, 34, 35). Although the ASPP2 C terminus mediates nuclear localization, full-length ASPP2 also localizes to the cytoplasm and plasma membrane to mediate extranuclear functions (7, 11, 12, 36). Structural studies of the ASPP2 N terminus reveal a β–Grasp ubiquitin-like fold as well as a potential Ras-binding (RB)/Ras-association (RA) domain (32). Moreover, ASPP2 can promote H-RasV12–induced senescence (13, 15). However, the molecular mechanism(s) of how ASPP2 directly promotes Ras signaling are complex and remain to be completely elucidated.Here, we explore the molecular mechanisms of how Ras-signaling is enhanced by ASPP2. We demonstrate that ASPP2: (i) binds Ras-GTP and stimulates Ras-induced ERK signaling via its N-terminal domain at the plasma membrane; (ii) enhances Ras-GTP loading and B-Raf/C-Raf dimerization and forms a ASPP2/Raf complex; (iii) stimulates Ras-induced C-Raf phosphorylation and activation; and (iv) potentiates H-RasV12–induced senescence in both primary human fibroblasts and neonatal human epidermal keratinocytes. These data provide mechanistic insight into ASPP2 function(s) and opens important avenues for investigation into its role as a tumor suppressor in human cancer.     

Application of desorption electrospray ionization mass spectrometry imaging in breast cancer margin analysis

Distinguishing tumor from normal glandular breast tissue is an important step in breast-conserving surgery. Because this distinction can be challenging in the operative setting, up to 40% of patients require an additional operation when traditional approaches are used. Here, we present a proof-of-concept study to determine the feasibility of using desorption electrospray ionization mass spectrometry imaging (DESI-MSI) for identifying and differentiating tumor from normal breast tissue. We show that tumor margins can be identified using the spatial distributions and varying intensities of different lipids. Several fatty acids, including oleic acid, were more abundant in the cancerous tissue than in normal tissues. The cancer margins delineated by the molecular images from DESI-MSI were consistent with those margins obtained from histological staining. Our findings prove the feasibility of classifying cancerous and normal breast tissues using ambient ionization MSI. The results suggest that an MS-based method could be developed for the rapid intraoperative detection of residual cancer tissue during breast-conserving surgery.Breast cancer is the most commonly diagnosed carcinoma in women in the United States and Western countries. Breast conservation surgery (BCS) has become the preferred treatment option for many women with early-stage breast cancer (1). BCS entails resection of the tumor, with a clean margin of normal tissue around it. Surgery is usually followed by radiation therapy. Results from seven large randomized prospective studies, with the largest two having over 20 y of follow-up, have shown equal survival when comparing BCS coupled with whole-breast radiation and mastectomy (2, 3).Normally, breast surgeons aim to remove a patient’s tumor, along with a rim of normal tissue that is free of cancer. Preoperative mammography, ultrasonography, or MRI may be used by the surgeon to guide adequate resection (4–6). Despite numerous improvements in imaging and surgical technique, the need for reexcision to achieve complete tumor resection in the United States typically ranges from 20–40% (7–15), and has been reported as being as high as 60% (16). The importance of reexcision is underscored by numerous studies, which have shown that incomplete resection of tumor and positive margins are associated with increased locoregional recurrence compared with negative margins (12, 17–20). Furthermore, the landmark meta-analysis performed by the Early Breast Cancer Trialists’ Collaborative Group (18, 21) directly linked local recurrence to survival, placing great emphasis on the surgeon’s role in minimizing local recurrence by obtaining adequate margins.Breast tumor reexcisions are accompanied by a number of undesirable problems: The completion of therapy is delayed, infection rates are increased, cost is increased, there can be a negative psychological impact on the patient, and there can be diminished aesthetic outcomes (22–24). The development of an intraoperative technique that allows the fast and accurate identification of residual tumor at surgical resection margins could decrease the reexcision rate, and therefore improve the care delivered to patients with cancer who are receiving BCS.To this end, multiple intraoperative methods have been explored, with various benefits as well as limitations. These methods include touch frozen section analysis (25), touch preparation cytology (26), specimen radiography (27, 28), rf spectroscopy (29, 30), Raman spectroscopy (31), radioguided occult lesion localization (32), near-IR fluorescence (33, 34), and high-frequency ultrasound (35–37). The intraoperative application of MRI, which has been successfully applied in brain surgery (38–42), is limited in its application in BCS. These limitations include MRI interpretation in the presence of acute surgical changes; lack of real-time imaging, requiring the interruption of surgery; and accurate localization of tumor based on images requiring development of fiducials (43–46).Mass spectrometry imaging (MSI) has been applied to investigate the molecular distribution of proteins, lipids, and metabolites without the use of labels (47, 48). In particular, the newly developed ambient ionization technique of desorption electrospray ionization (DESI) allows direct tissue analysis with little to no sample preparation (49, 50). Therefore, with the advantage of easy use, DESI-MSI has great potential in the application of intraoperative tumor assessment. The development of DESI-MSI enables the correlation of lipid distribution in two or three dimensions with tissue morphology (47, 51) and the distinction of cancerous from noncancerous tissues based on lipidomic information (52–54). Distinctive lipid profiles associated with different human cancers have been investigated by DESI-MSI (55–58). Moreover, the grades and subtypes of human brain tumors have been discriminated using this technique. Additionally, tumor margins have been delineated using DESI-MSI, and the results have been correlated with histopathological examination (59, 60).It has been reported that breast cancer demonstrates metabolic profiles that are distinct from those metabolic profiles found in normal breast tissue. This finding suggests a potential for using metabolite information for breast cancer diagnosis and tumor margin identification (61, 62). Here, we demonstrate an MS-based methodology for using lipidomic information to distinguish cancerous from noncancerous tissue and to delineate tumor boundaries.