Convergent evolution toward an improved growth rate and a reduced resistance range in Prochlorococcus strains resistant to phage

Prochlorococcus is an abundant marine cyanobacterium that grows rapidly in the environment and contributes significantly to global primary production. This cyanobacterium coexists with many cyanophages in the oceans, likely aided by resistance to numerous co-occurring phages. Spontaneous resistance occurs frequently in Prochlorococcus and is often accompanied by a pleiotropic fitness cost manifested as either a reduced growth rate or enhanced infection by other phages. Here, we assessed the fate of a number of phage-resistant Prochlorococcus strains, focusing on those with a high fitness cost. We found that phage-resistant strains continued evolving toward an improved growth rate and a narrower resistance range, resulting in lineages with phenotypes intermediate between those of ancestral susceptible wild-type and initial resistant substrains. Changes in growth rate and resistance range often occurred in independent events, leading to a decoupling of the selection pressures acting on these phenotypes. These changes were largely the result of additional, compensatory mutations in noncore genes located in genomic islands, although genetic reversions were also observed. Additionally, a mutator strain was identified. The similarity of the evolutionary pathway followed by multiple independent resistant cultures and clones suggests they undergo a predictable evolutionary pathway. This process serves to increase both genetic diversity and infection permutations in Prochlorococcus populations, further augmenting the complexity of the interaction network between Prochlorococcus and its phages in nature. Last, our findings provide an explanation for the apparent paradox of a multitude of resistant Prochlorococcus cells in nature that are growing close to their maximal intrinsic growth rates.Large bacterial populations are present in the oceans, playing important roles in primary production and the biogeochemical cycling of matter. These bacterial communities are highly diverse (1–4) yet form stable and reproducible bacterial assemblages under similar environmental conditions (5–7).These bacteria are present together with high abundances of viruses (phages) that have the potential to infect and kill them (8–11). Although studied only rarely in marine organisms (12–16), this coexistence is likely to be the result of millions of years of coevolution between these antagonistic interacting partners, as has been well documented for other systems (17–20). From the perspective of the bacteria, survival entails the selection of cells that are resistant to infection, preventing viral production and enabling the continuation of the cell lineage. Resistance mechanisms include passively acquired spontaneous mutations in cell surface molecules that prevent phage entry into the cell and other mechanisms that actively terminate phage infection intracellularly, such as restriction–modification systems and acquired resistance by CRISPR-Cas systems (21, 22). Mutations in the phage can also occur that circumvent these host defenses and enable the phage to infect the recently emerged resistant bacterium (23).Acquisition of resistance by bacteria is often associated with a fitness cost. This cost is frequently, but not always, manifested as a reduction in growth rate (24–27). Recently, an additional type of cost of resistance was identified, that of enhanced infection whereby resistance to one phage leads to greater susceptibility to other phages (14, 15, 28).Over the years, a number of models have been developed to explain coexistence in terms of the above coevolutionary processes and their costs (16, 29–32). In the arms race model, repeated cycles of host mutation and virus countermutation occur, leading to increasing breadths of host resistance and viral infectivity. However, experimental evidence generally indicates that such directional arms race dynamics do not continue indefinitely (25, 33, 34). Therefore, models of negative density-dependent fluctuations due to selective trade-offs, such as kill-the-winner, are often invoked (20, 33, 35, 36). In these models, fluctuations are generally considered to occur between rapidly growing competition specialists that are susceptible to infection and more slowly growing resistant strains that are considered defense specialists. Such negative density-dependent fluctuations are also likely to occur between strains that have differences in viral susceptibility ranges, such as those that would result from enhanced infection (30).The above coevolutionary processes are considered to be among the major mechanisms that have led to and maintain diversity within bacterial communities (32, 35, 37–39). These processes also influence genetic microdiversity within populations of closely related bacteria. This is especially the case for cell surface-related genes that are often localized to genomic islands (14, 40, 41), regions of high gene content, and gene sequence variability among members of a population. As such, populations in nature display an enormous degree of microdiversity in phage susceptibility regions, potentially leading to an assortment of subpopulations with different ranges of susceptibility to coexisting phages (4, 14, 30, 40).Prochlorococcus is a unicellular cyanobacterium that is the numerically dominant photosynthetic organism in vast oligotrophic expanses of the open oceans, where it contributes significantly to primary production (42, 43). Prochlorococcus consists of a number of distinct ecotypes (44–46) that form stable and reproducible population structures (7). These populations coexist in the oceans with tailed double-stranded DNA phage populations that infect them (47–49).Previously, we found that resistance to phage infection occurs frequently in two high-light–adapted Prochlorococcus ecotypes through spontaneous mutations in cell surface-related genes (14). These genes are primarily localized to genomic island 4 (ISL4) that displays a high degree of genetic diversity in environmental populations (14, 40). Although about a third of Prochlorococcus-resistant strains had no detectable associated cost, the others came with a cost manifested as either a slower growth rate or enhanced infection by other phages (14). In nature, Prochlorococcus seems to be growing close to its intrinsic maximal growth rate (50–52). This raises the question as to the fate of emergent resistant Prochlorococcus lineages in the environment, especially when resistance is accompanied with a high growth rate fitness cost.To begin addressing this question, we investigated the phenotype of Prochlorococcus strains with time after the acquisition of resistance. We found that resistant strains evolved toward an improved growth rate and a reduced resistance range. Whole-genome sequencing and PCR screening of many of these strains revealed that these phenotypic changes were largely due to additional, compensatory mutations, leading to increased genetic diversity. These findings suggest that the oceans are populated with rapidly growing Prochlorococcus cells with varying degrees of resistance and provide an explanation for how a multitude of presumably resistant Prochlorococcus cells are growing close to their maximal known growth rate in nature.     

Rate,spectrum, and evolutionary dynamics of spontaneous epimutations

Stochastic changes in cytosine methylation are a source of heritable epigenetic and phenotypic diversity in plants. Using the model plant Arabidopsis thaliana, we derive robust estimates of the rate at which methylation is spontaneously gained (forward epimutation) or lost (backward epimutation) at individual cytosines and construct a comprehensive picture of the epimutation landscape in this species. We demonstrate that the dynamic interplay between forward and backward epimutations is modulated by genomic context and show that subtle contextual differences have profoundly shaped patterns of methylation diversity in A. thaliana natural populations over evolutionary timescales. Theoretical arguments indicate that the epimutation rates reported here are high enough to rapidly uncouple genetic from epigenetic variation, but low enough for new epialleles to sustain long-term selection responses. Our results provide new insights into methylome evolution and its population-level consequences.Plant genomes make extensive use of cytosine methylation to control the expression of transposable elements (TEs) and genes (1). Despite its tight regulation, methylation losses or gains at individual cytosines or clusters of cytosines can emerge spontaneously, in an event termed “epimutation” (2, 3). Many examples of segregating epimutations have been documented in experimental and wild populations of plants and in some cases contribute to heritable variation in phenotypes independently of DNA sequence variation (4, 5). These observations have led to much speculation about the role of DNA methylation in plant evolution (6–8), and its potential in breeding programs (9). In the model plant Arabidopsis thaliana, spontaneous methylation changes at CG dinucleotides accumulate in a rapid but nonlinear fashion over generations (2, 3, 10), thus pointing to high forward–backward epimutation rates (11). Precise estimates of these rates are necessary to be able to quantify the long-term dynamics of epigenetic variation under laboratory or natural conditions, and to understand the molecular mechanisms that drive methylome evolution (12–14). Here we combine theoretical modeling with high-resolution methylome analysis of multiple independent A. thaliana mutation accumulation (MA) lines (15), including measurements of methylation changes in continuous generations, to obtain robust estimates of forward and backward epimutation rates.     

Mechanisms of NDV-3 vaccine efficacy in MRSA skin versus invasive infection

Increasing rates of life-threatening infections and decreasing susceptibility to antibiotics urge development of an effective vaccine targeting Staphylococcus aureus. This study evaluated the efficacy and immunologic mechanisms of a vaccine containing a recombinant glycoprotein antigen (NDV-3) in mouse skin and skin structure infection (SSSI) due to methicillin-resistant S. aureus (MRSA). Compared with adjuvant alone, NDV-3 reduced abscess progression, severity, and MRSA density in skin, as well as hematogenous dissemination to kidney. NDV-3 induced increases in CD3+ T-cell and neutrophil infiltration and IL-17A, IL-22, and host defense peptide expression in local settings of SSSI abscesses. Vaccine induction of IL-22 was necessary for protective mitigation of cutaneous infection. By comparison, protection against hematogenous dissemination required the induction of IL-17A and IL-22 by NDV-3. These findings demonstrate that NDV-3 protective efficacy against MRSA in SSSI involves a robust and complementary response integrating innate and adaptive immune mechanisms. These results support further evaluation of the NDV-3 vaccine to address disease due to S. aureus in humans.The bacterium Staphylococcus aureus is the leading cause of skin and skin structure infections (SSSIs), including cellulitis, furunculosis, and folliculitis (1–4), and a common etiologic agent of impetigo (5), erysipelas (6), and superinfection in atopic dermatitis (7). This bacterium is a significant cause of surgical or traumatic wound infections (8, 9), as well as decuibitus and diabetic skin lesions (10). Moreover, SSSI is an important risk factor for systemic infection. The skin is a key portal of entry for hematogenous dissemination, particularly in association with i.v. catheters. S. aureus is now the second most common bloodstream isolate in healthcare settings (11), and SSSI is a frequent source of invasive infections such as pneumonia or endocarditis (12, 13). Despite a recent modest decline in rates of methicillin-resistant S. aureus (MRSA) infection in some cohorts (13), infections due to S. aureus remain a significant problem (14, 15). Even with appropriate therapy, up to one-third of patients diagnosed with S. aureus bacteremia succumb—accounting for more attributable annual deaths than HIV, tuberculosis, and viral hepatitis combined (16).The empiric use of antibiotics in healthcare-associated and community-acquired settings has increased S. aureus exposure to these agents, accelerating selection of resistant strains. As a result, resistance to even the most recently developed agents is emerging at an alarming pace (17, 18). The impact of this trend is of special concern in light of high rates of mortality associated with invasive MRSA infection (e.g., 15–40% in bacteremia or endocarditis), even with the most recently developed antistaphylococcal therapeutics (19, 20). Moreover, patients who experience SSSI due to MRSA exhibit high 1-y recurrence rates, often prompting surgical debridement (21) and protracted antibiotic treatment.Infections due to MRSA are a special concern in immune-vulnerable populations, including hemodialysis (22), neutropenic (23, 24), transplantation (25), and otherwise immunosuppressed patients (26, 27), and in patients with inherited immune dysfunctions (28–31) or cystic fibrosis (32). Patients having deficient interleukin 17 (IL-17) or IL-22 responses (e.g., signal transduction mediators STAT3, DOCK8, or CARD9 deficiencies) exhibit chronic or “cold” abscesses, despite high densities of pathogens such as S. aureus (33, 34). For example, patients with Chronic Granulomatous Disease (CGD; deficient Th1 and oxidative burst response) have increased risk of disseminated S. aureus infection. In contrast, patients with Job’s Syndrome (deficient Th17 response) typically have increased risk to SSSI and lung infections, but less so for systemic S. aureus bacteremia (35, 36). This pattern contrasts that observed in neutropenic or CGD patients (37). These themes suggest efficacious host defenses against MRSA skin and invasive infections involve complementary but distinct molecular and cellular immune responses.From these perspectives, vaccines or immunotherapeutics that prevent or lessen severity of MRSA infections, or that enhance antibiotic efficacy, would be significant advances in patient care and public health. However, to date, there are no licensed prophylactic or therapeutic vaccine immunotherapies for S. aureus or MRSA infection. Unfortunately, efforts to develop vaccines targeting S. aureus capsular polysaccharide type 5 or 8 conjugates, or the iron-regulated surface determinant B protein, have not been successful thus far (38, 39). Likewise, passive immunization using monoclonal antibodies targeting the S. aureus adhesin clumping factor A (ClfA, tefibazumab) (40) or lipoteichoic acid (pagibaximab) (41) have not shown efficacy against invasive infections in human clinical studies to date. Moreover, the striking recurrence rates of SSSI due to MRSA imply that natural exposure does not induce optimal preventive immunity or durable anamnestic response to infection or reinfection. Thus, significant challenges exist in the development of an efficacious vaccine targeting diseases caused by S. aureus (42) that are perhaps not optimally addressed by conventional approaches.The NDV-3 vaccine reflects a new strategy to induce durable immunity targeting S. aureus. Its immunogen is engineered from the agglutinin-like sequence 3 (Als3) adhesin/invasin of Candida albicans, which we discovered to be a structural homolog of S. aureus adhesins (43). NDV-3 is believed to cross-protect against S. aureus and C. albicans due to sequence (T-cell) and conformational (B-cell) epitopes paralleled in both organisms (44). Our prior data have shown that NDV-3 is efficacious in murine models of hematogenous and mucosal candidiasis (45), as well as S. aureus bacteremia (46–48). Recently completed phase I clinical trials demonstrate the safety, tolerability, and immunogenicity of NDV-3 in humans (49).     

Distinct dopamine neurons mediate reward signals for short- and long-term memories

Drosophila melanogaster can acquire a stable appetitive olfactory memory when the presentation of a sugar reward and an odor are paired. However, the neuronal mechanisms by which a single training induces long-term memory are poorly understood. Here we show that two distinct subsets of dopamine neurons in the fly brain signal reward for short-term (STM) and long-term memories (LTM). One subset induces memory that decays within several hours, whereas the other induces memory that gradually develops after training. They convey reward signals to spatially segregated synaptic domains of the mushroom body (MB), a potential site for convergence. Furthermore, we identified a single type of dopamine neuron that conveys the reward signal to restricted subdomains of the mushroom body lobes and induces long-term memory. Constant appetitive memory retention after a single training session thus comprises two memory components triggered by distinct dopamine neurons.Memory of a momentous event persists for a long time. Whereas some forms of long-term memory (LTM) require repetitive training (1–3), a highly relevant stimulus such as food or poison is sufficient to induce LTM in a single training session (4–7). Recent studies have revealed aspects of the molecular and cellular mechanisms of LTM formation induced by repetitive training (8–11), but how a single training induces a stable LTM is poorly understood (12).Appetitive olfactory learning in fruit flies is suited to address the question, as a presentation of a sugar reward paired with odor induces robust short-term memory (STM) and LTM (6, 7). Odor is represented by a sparse ensemble of the 2,000 intrinsic neurons, the Kenyon cells (13). A current working model suggests that concomitant reward signals from sugar ingestion cause associative plasticity in Kenyon cells that might underlie memory formation (14–20). A single activation session of a specific cluster of dopamine neurons (PAM neurons) by sugar ingestion can induce appetitive memory that is stable over 24 h (19), underscoring the importance of sugar reward to the fly.The mushroom body (MB) is composed of the three different cell types, α/β, α′/β′, and γ, which have distinct roles in different phases of appetitive memories (11, 21–25). Similar to midbrain dopamine neurons in mammals (26, 27), the structure and function of PAM cluster neurons are heterogeneous, and distinct dopamine neurons intersect unique segments of the MB lobes (19, 28–34). Further circuit dissection is thus crucial to identify candidate synapses that undergo associative modulation.By activating distinct subsets of PAM neurons for reward signaling, we found that short- and long-term memories are independently formed by two complementary subsets of PAM cluster dopamine neurons. Conditioning flies with nutritious and nonnutritious sugars revealed that the two subsets could represent different reinforcing properties: sweet taste and nutritional value of sugar. Constant appetitive memory retention after a single training session thus comprises two memory components triggered by distinct reward signals.     

Recombinant transfer in the basic genome of Escherichia coli

An approximation to the ∼4-Mbp basic genome shared by 32 strains of Escherichia coli representing six evolutionary groups has been derived and analyzed computationally. A multiple alignment of the 32 complete genome sequences was filtered to remove mobile elements and identify the most reliable ∼90% of the aligned length of each of the resulting 496 basic-genome pairs. Patterns of single base-pair mutations (SNPs) in aligned pairs distinguish clonally inherited regions from regions where either genome has acquired DNA fragments from diverged genomes by homologous recombination since their last common ancestor. Such recombinant transfer is pervasive across the basic genome, mostly between genomes in the same evolutionary group, and generates many unique mosaic patterns. The six least-diverged genome pairs have one or two recombinant transfers of length ∼40–115 kbp (and few if any other transfers), each containing one or more gene clusters known to confer strong selective advantage in some environments. Moderately diverged genome pairs (0.4–1% SNPs) show mosaic patterns of interspersed clonal and recombinant regions of varying lengths throughout the basic genome, whereas more highly diverged pairs within an evolutionary group or pairs between evolutionary groups having >1.3% SNPs have few clonal matches longer than a few kilobase pairs. Many recombinant transfers appear to incorporate fragments of the entering DNA produced by restriction systems of the recipient cell. A simple computational model can closely fit the data. Most recombinant transfers seem likely to be due to generalized transduction by coevolving populations of phages, which could efficiently distribute variability throughout bacterial genomes.The increasing availability of complete genome sequences of many different bacterial and archaeal species, as well as metagenomic sequencing of mixed populations from natural environments, has stimulated theoretical and computational approaches to understand mechanisms of speciation and how prokaryotic species should be defined (1–8). Much genome analysis and comparison has been at the level of gene content, identifying core genomes (the set of genes found in most or all genomes in a group) and the continually expanding pan-genome. Population genomics of Escherichia coli has been particularly well studied because of its long history in laboratory research and because many pathogenic strains have been isolated and completely sequenced (9–14). Proposed models of how related groups or species form and evolve include isolation by ecological niche (7–9, 11, 15), decreased homologous recombination as divergence between isolated populations increases (2–4, 8, 14, 16), and coevolving phage and bacterial populations (6).E. coli genomes are highly variable, containing an array of phage-related mobile elements integrated at many different sites (17), random insertions of multiple transposable elements (18), and idiosyncratic genome rearrangements that include inversions, translocations, duplications, and deletions. Although E. coli grows by binary cell division, genetic exchange by homologous recombination has come to be recognized as a significant factor in adaptation and genome evolution (9, 10, 19). Of particular interest has been the relative contribution to genome variability of random mutations (single base-pair differences referred to as SNPs) and replacement of genome regions by homologous recombination with fragments imported from other genomes (here referred to as recombinant transfers or transferred regions). Estimates of the rate, extent, and average lengths of recombinant transfers in the core genome vary widely, as do methods for detecting transferred regions and assessing their impact on phylogenetic relationships (12–14, 20, 21).In a previous comparison of complete genome sequences of the K-12 reference strain MG1655 and the reconstructed genome of the B strain of Delbrück and Luria referred to here as B-DL, we observed that SNPs are not randomly distributed among 3,620 perfectly matched pairs of coding sequences but rather have two distinct regimes: sharply decreasing numbers of genes having 0, 1, 2, or 3 SNPs, and an abrupt transition to a much broader exponential distribution in which decreasing numbers of genes contain increasing numbers of SNPs from 4 to 102 SNPs per gene (22). Genes in the two regimes of the distribution are interspersed in clusters of variable lengths throughout what we referred to as the basic genome, namely, the ∼4 Mbp shared by the two genomes after eliminating mobile elements. We speculated that genes having 0 to 3 SNPs may primarily have been inherited clonally from the last common ancestor, whereas genes comprising the exponential tail may primarily have been acquired by horizontal transfer from diverged members of the population.The current study was undertaken to extend these observations to a diverse set of 32 completely sequenced E. coli genomes and to analyze how SNP distributions in the basic genome change as a function of evolutionary divergence between the 496 pairs of strains in this set. We have taken a simpler approach than those of Touchon et al. (13), Didelot et al. (14), and McNally et al. (21), who previously analyzed multiple alignments of complete genomes of E. coli strains. The appreciably larger basic genome derived here is not restricted to protein-coding sequences and retains positional information.     

Novel serologic biomarkers provide accurate estimates of recent Plasmodium falciparum exposure for individuals and communities

Tools to reliably measure Plasmodium falciparum (Pf) exposure in individuals and communities are needed to guide and evaluate malaria control interventions. Serologic assays can potentially produce precise exposure estimates at low cost; however, current approaches based on responses to a few characterized antigens are not designed to estimate exposure in individuals. Pf-specific antibody responses differ by antigen, suggesting that selection of antigens with defined kinetic profiles will improve estimates of Pf exposure. To identify novel serologic biomarkers of malaria exposure, we evaluated responses to 856 Pf antigens by protein microarray in 186 Ugandan children, for whom detailed Pf exposure data were available. Using data-adaptive statistical methods, we identified combinations of antibody responses that maximized information on an individual’s recent exposure. Responses to three novel Pf antigens accurately classified whether an individual had been infected within the last 30, 90, or 365 d (cross-validated area under the curve = 0.86–0.93), whereas responses to six antigens accurately estimated an individual’s malaria incidence in the prior year. Cross-validated incidence predictions for individuals in different communities provided accurate stratification of exposure between populations and suggest that precise estimates of community exposure can be obtained from sampling a small subset of that community. In addition, serologic incidence predictions from cross-sectional samples characterized heterogeneity within a community similarly to 1 y of continuous passive surveillance. Development of simple ELISA-based assays derived from the successful selection strategy outlined here offers the potential to generate rich epidemiologic surveillance data that will be widely accessible to malaria control programs.Many countries have extensive programs to reduce the burden of Plasmodium falciparum (Pf), the parasite responsible for most malaria morbidity and mortality (1). Effectively using limited resources for malaria control or elimination and evaluating interventions require accurate measurements of the risk of being infected with Pf (2–15). To reflect the rate at which individuals are infected with Pf in a useful way, metrics used to estimate exposure in a community need to account for dynamic changes over space and time, especially in response to control interventions (16–18).A variety of metrics can be used to estimate Pf exposure, but tools that are more precise and low cost are needed for population surveillance. Existing metrics have varying intrinsic levels of precision and accuracy and are subject to a variety of extrinsic factors, such as cost, time, and availability of trained personnel (19). For example, entomological measurements provide information on mosquito to human transmission for a community but are expensive, require specially trained staff, and lack standardized procedures, all of which reduce precision and/or make interpretation difficult (19–22). Parasite prevalence can be measured by detecting parasites in the blood of individuals from a cross-sectional sample of a community and is, therefore, relatively simple and inexpensive to perform, but results may be imprecise, especially in areas of low transmission (19, 23), and biased by a number of factors, including immunity and access to antimalarial treatment (5, 6, 19, 23–25). The burden of symptomatic disease in a community can be estimated from routine health systems data; however, such data are frequently unreliable (5, 26–28) and generally underestimate the prevalence of Pf infection in areas of intense transmission. Precise and quantitative information about exposure at an individual level can be reliably obtained from cohort studies by measuring the incidence of asymptomatic and/or symptomatic Pf infection (i.e., by measuring the molecular force of infection) (29–35). Unfortunately, the expense of cohort studies limits their use to research settings. The end result is that most malaria-endemic regions lack reliable, timely data on Pf exposure, limiting the capabilities of malaria control programs to guide and evaluate interventions.Serologic assays offer the potential to provide incidence estimates for symptomatic and asymptomatic Pf infection, which are currently obtained from cohort studies, at the cost of cross-sectional studies (36–38). Although Pf infections are transient, a record of infection remains detectable in an individual’s antibody profile. Thus, appropriately chosen antibody measurements integrated with age can provide information about an individual’s exposure history. Antibodies can be measured by simple ELISAs and obtained from dried blood spots, which are easy to collect, transport, and store (39–41). Serologic responses to Pf antigens have been explored as potential epidemiological tools (42–45), and estimated rates of seroconversion to well-characterized Pf antigens accurately reflect stable rates of exposure in a community, whereas distinct changes in these rates are obtained from successful interventions (22, 39, 41, 46–53). However, current serologic assays are not designed to detect short-term or gradual changes in Pf exposure or measure exposure to infection at an individual level. The ability to calibrate antibody responses to estimates of exposure in individuals could allow for more flexible sampling of a population (e.g., not requiring age stratification), improve accuracy of exposure estimates from small sample sizes, and better characterize heterogeneity in exposure within a community.Different Pf antigens elicit antibody responses with different magnitudes and kinetics, providing a large and diverse set of potential biomarkers for exposure (38, 54–58). We hypothesized that new and more highly informative serologic biomarkers better able to characterize an individual’s recent exposure history could be identified by analyzing antibody responses to a large number of candidate Pf antigens in participants with well-characterized exposure histories. To test this hypothesis, we probed plasma from participants in two cohort studies in Uganda against a protein microarray containing 856 Pf antigens. The primary aim of this analysis was to identify responses to select antigens that were most informative of recent exposure using robust, data-adaptive statistical methods. Each participant’s responses to these selected antigens were used as predictors for two primary outcomes of their recent exposure to Pf: (i) days since last Pf infection and (ii) the incidence of symptomatic malaria in the last year. These individual-level estimates were then aggregated across a population to assess community-level malaria exposure. The selection strategy presented here identified accurate biomarkers of exposure for children living in areas of moderate to high Pf exposure and illustrates the utility of this flexible and broadly applicable approach.     

Hyperpolarization-activated,cyclic nucleotide-gated cation channels in Aplysia: Contribution to classical conditioning

Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels are critical regulators of neuronal excitability, but less is known about their possible roles in synaptic plasticity and memory circuits. Here, we characterized the HCN gene organization, channel properties, distribution, and involvement in associative and nonassociative forms of learning in Aplysia californica. Aplysia has only one HCN gene, which codes for a channel that has many similarities to the mammalian HCN channel. The cloned acHCN gene was expressed in Xenopus oocytes, which displayed a hyperpolarization-induced inward current that was enhanced by cGMP as well as cAMP. Similarly to its homologs in other animals, acHCN is permeable to K⁺ and Na⁺ ions, and is selectively blocked by Cs⁺ and ZD7288. We found that acHCN is predominantly expressed in inter- and motor neurons, including LFS siphon motor neurons, and therefore tested whether HCN channels are involved in simple forms of learning of the siphon-withdrawal reflex in a semiintact preparation. ZD7288 (100 μM) significantly reduced an associative form of learning (classical conditioning) but had no effect on two nonassociative forms of learning (intermediate-term sensitization and unpaired training) or baseline responses. The HCN current is enhanced by nitric oxide (NO), which may explain the postsynaptic role of NO during conditioning. HCN current in turn enhances the NMDA-like current in the motor neurons, suggesting that HCN channels contribute to conditioning through this pathway.Hyperpolarization-activated, cyclic nucleotide-gated (HCN), cation nonselective ion channels generate hyperpolarization-activated inward currents (I_h) and thus tend to stabilize membrane potential (1–3). In addition, binding of cyclic nucleotides (cAMP and cGMP) to the C-terminal cyclic nucleotide binding domain (CNBD) enhances I_h and thus couples membrane excitability with intracellular signaling pathways (2, 4). HCN channels are widely important for numerous systemic functions such as hormonal regulation, heart contractility, epilepsy, pain, central pattern generation, sensory perception (4–15), and learning and memory (16–24).However, in previous studies it has been difficult to relate the cellular effects of HCN channels directly to their behavioral effects, because of the immense complexity of the mammalian brain. We have therefore investigated the role of HCN channels in Aplysia, which has a numerically simpler nervous system (25). We first identified and characterized an HCN gene in Aplysia, and showed that it codes for a channel that has many similarities to the mammalian HCN channel. We found that the Aplysia HCN channel is predominantly expressed in motor neurons including LFS neurons in the siphon withdrawal reflex circuit (26, 27). We therefore investigated simple forms of learning of that reflex in a semiintact preparation (28–30) and found that HCN current is involved in classical conditioning and enhances the NMDA-like current in the motor neurons. These results provide a direct connection between HCN channels and behavioral learning and suggest a postsynaptic mechanism of that effect. HCN current in turn is enhanced by nitric oxide (NO), a transmitter of facilitatory interneurons, and thus may contribute to the postsynaptic role of NO during conditioning.     

Activation of Big Grain1 significantly improves grain size by regulating auxin transport in rice

Grain size is one of the key factors determining grain yield. However, it remains largely unknown how grain size is regulated by developmental signals. Here, we report the identification and characterization of a dominant mutant big grain1 (Bg1-D) that shows an extra-large grain phenotype from our rice T-DNA insertion population. Overexpression of BG1 leads to significantly increased grain size, and the severe lines exhibit obviously perturbed gravitropism. In addition, the mutant has increased sensitivities to both auxin and N-1-naphthylphthalamic acid, an auxin transport inhibitor, whereas knockdown of BG1 results in decreased sensitivities and smaller grains. Moreover, BG1 is specifically induced by auxin treatment, preferentially expresses in the vascular tissue of culms and young panicles, and encodes a novel membrane-localized protein, strongly suggesting its role in regulating auxin transport. Consistent with this finding, the mutant has increased auxin basipetal transport and altered auxin distribution, whereas the knockdown plants have decreased auxin transport. Manipulation of BG1 in both rice and Arabidopsis can enhance plant biomass, seed weight, and yield. Taking these data together, we identify a novel positive regulator of auxin response and transport in a crop plant and demonstrate its role in regulating grain size, thus illuminating a new strategy to improve plant productivity.Because it is one of the most important staple food crops cultivated worldwide, improvement of grain yield is a major focus of rice-breeding programs (1). Grain size is one of the determining factors of grain yield (2, 3). A number of quantitative trait loci (QTLs) controlling rice grain size have been identified in recent years (4–11). However, functional mechanisms of these genes remain largely unknown. Because QTLs usually have important functions in determining grain size, many of them have been widely selected in breeding processes or existed in modern elite varieties, and a certain QTL could be only applicable in certain varieties (12). Thus, exploration of new grain size-associated genes and elucidation of their functional mechanisms have great significance for further improvement of rice yield (12).Seed size, as well as other organ size, is controlled by various plant hormones, such as auxin, brassinosteroid, and cytokinin (10, 13, 14). A number of studies have demonstrated that auxin plays a vital role in organ size determination by affecting cell division, cell expansion, and differentiation (15–17). Auxin exists predominantly as indole-3-acetic acid (IAA) in plants, and genetic studies of its biosynthetic genes in Arabidopsis have demonstrated that IAA regulates many aspects of plant growth and development, including stem elongation, lateral branching, vascular development, and tropic growth responses (18, 19). Combined with biochemical studies, the tryptophan (Trp)-dependent IAA biosynthesis pathway has been clearly established involving the YUCCA family flavin monooxgenases (20). Importantly, the two-step pathway is highly conserved throughout the plant kingdom (21). Until very recently, the Trp-independent auxin biosynthetic pathway was elucidated as contributing to early embryogenesis in Arabidopsis (22). Primary auxin signaling is a rapid process initiated from the hormone perception by receptor TIR1, an F-box protein, followed by degradation of the negative regulator AUX/IAA proteins, and further release the downstream auxin response factors (ARFs) (23–26). However, how the ARFs work in plants remains elusive. Auxin transport, generally referring to the cell-to-cell transportation of the hormone directed basipetally from shoots to roots in vascular tissues, plays a critical role in auxin response (18). The transport involves a number of membrane-associated proteins, such as PINs (protein inhibitor of nNOS), AUX1 (AUXIN TRANSPORTER PROTEIN 1), and ABCBs (ATP-BINDING CASSETTE, SUB-FAMILY B PROTEINS) as efflux or influx carriers (27–30). Disruption of auxin transport induced by either gene mutations or chemical inhibitor treatment will lead to diverse development defects, such as decreased lateral organ initiation and defective tropic growth responses (27, 31–34).In this study, we identify a rice mutant, named big grain1-D (Bg1-D) because it is a dominant mutant having extralarge grain size. BG1 encodes a novel plasma membrane-associated protein, and is specifically induced by auxin treatment. We show that BG1 is a new positive regulator of auxin response involved in auxin transport, and demonstrate that manipulation of BG1 expression can greatly improve grain size and plant productivity.