Asthma is a major public health problem with a huge social and economic burden affecting 300 million people worldwide. Viral respiratory infections are the major cause of acute asthma exacerbations and may contribute to asthma inception in high risk young children with susceptible genetic background. Acute exacerbations are associated with decreased lung growth or accelerated loss of lung function and, as such, add substantially to both the cost and morbidity associated with asthma. While the importance of preventing viral infection is well established, preventive strategies have not been well explored. Good personal hygiene, hand-washing and avoidance of cigarette smoke are likely to reduce respiratory viral infections. Eating a healthy balanced diet, active probiotic supplements and bacterial-derived products, such as OM-85, may reduce recurrent infections in susceptible children. There are no practical anti-viral therapies currently available that are suitable for widespread use.Hand hygiene is the best measure to prevent the common cold. A healthy balanced diet, active probiotic supplements and immunostimulant OM-85 may reduce recurrent infections in asthmatic children. In other words, do viruses cause or simply unmask asthma?Asthma is a major public health problem with a huge social and economic burden affecting 300 million people worldwide . Viral rRespiratory viruses first infect nasal epithelial cells which triggers an antiviral response. This response is driven by type I (α/β) and III (λ) interferons (IFN) that are induced following recognition of viral ribonucleic acid (RNA) by pattern recognition receptors (PRRs). Toll-like receptors (TLRs) are cell surface and endosomal PRRs, whilst the RNA helicase receptors (RIG-I and MDA-5) and NOD-like receptors (NOD2), detect viral RNA in the cytoplasm. Signalling via the PRRs activates transcription factors , which lead to the production and secretion of type I and III IFN. The IFNs then bind to cell surface receptors to activate a separate pathway leading to the production of interferon stimulated genes (ISGs) which encode antiviral proteins that combat infection, as well as PRRs and transcriptional factors which further amplify IFN production. The respiratory syncytial virus (RSV), human meta-pneumovirus (hMPV) and human rhinovirus (HRV) are all single stranded RNA viruses but engage differently with cell signalling pathways. In airway epithelial cells RSV and hMPV RNA are primarily detected by RIG-I in the cytoplasm ,15. RSV A successful antiviral response would see the infection limited to the upper airway, as is the case clinically with the majority of viral infections in healthy individuals. Should such a response be deficient, then predominantly upper-airway viral infections, such as HRV, may spread to the lower airways, causing lower respiratory symptoms and an exacerbation of asthma in predisposed individuals. in vitro  infection of primary airway epithelial cells from asthmatic and healthy adults with HRV have demonstrated that asthmatic cells produce less IFN-β [While definitive data are yet to be produced, experimental HRV infections in adult volunteers initially suggested that asthmatics were more likely to develop lower respiratory infections (LRI) than healthy adults, i.e. less likely to be able to limit viral replication to the upper airways ,20. Subsss IFN-β  and IFN-ss IFN-β  making tss IFN-β ,15 and Iss IFN-β . It is lss IFN-β . Such deEach year, at the end of summer, parents of asthmatic children are concerned about acute asthma exacerbations following a common cold, asking how to minimize the risk during the winter viral season. It is a valid concern as up to 70% of asthmatic children have an intermittent or wheeze which is mostly symptomatic after viral infections ,26. AsthMuch research interest has focused on the potential role respiratory viral infections play in the inception of asthma. It is well established that hospitalization for RSV bronchiolitis is a risk factor for asthma during childhood ,38. EpidThe crucial period, with respect to asthma initiation, appears to be the first two to three years of life during which the growth and remodelling of lung and airways proceeds at maximum rates. Pulmonary inflammation resulting from atopy and sLRI occurring during this vulnerable time is hypothesized to perturb underlying tissue differentiation programs, resulting in deleterious long term effects on respiratory functions. As a result, there is widespread belief amongst the paediatric respiratory community that intervention measures that can lower the frequency and/or intensity of sLRI in early life amongst the high risk atopic subgroup of children are likely to be successful at preventing asthma. If successful, these strategies would have major implications for reducing the high impact of this chronic disease on the community ,41,42.Recent studies using culture-independent techniques have challenged the long-held dogma that lungs are sterile and have demonstrated that a microbiota community exists in the lung -45. The Children are important vectors for HRV transmission to family members particularly siblings ,48. HRV There are three ways of common cold transmission in children. First, inhalation of small particles aerosolized by coughing; second, large particle droplets from saliva expelled while sneezing; and third, self-inoculation of one’s own conjunctivae or nasal mucosa after touching a person or object contaminated with the cold viruses. The first two methods are inefficient , while tMeticulous hand hygiene is the best measure to prevent the common cold; frequent hand washing and avoid touching one's nose and eyes -53. The A recent Cochrane review which included data from 67 randomised controlled trials and observational studies, investigated the effectiveness of physical interventions to reduce the spread of respiratory viruses. The authors concluded that respiratory virus spread can be reduced by hygiene measures (such as handwashing), especially around younger children and can reduce transmission from children to other family members . ControvAsthmatic children should avoid close contact with people who have colds especially during the first three days of their illness. There is little evidence to support the effectiveness of face masks to reduce the risk of viral respiratory infections and consequently, the use of mask is generally not recommended for prevention of common cold ,61.Immune function and anti-viral defenses have a number of components, both specific and non-specific. Asthmatic children can improve their immune function by following some simple advice including a healthy life style with regular exercise, a balanced diet, adequate sleep and avoiding environmental tobacco smoke, stress and unnecessary antibiotics.Exercise has anti-inflammatory effects and in the long term can protect the development of chronic diseases and obesity . RegularMalnutrition is the most common cause of immune deficiency worldwide and a balanced diet is fundamental for a healthy immune system. Vitamin D deficiency has been associated with increased risk of infections, early-life wheeze and reduced asthma control ,70. VitaSleep and the circadian system exert a regulatory influence on immune functions. Sleep deprivation can affect immune function in several ways including reduced natural killer cell activity, suppressed interleukin-2 production and increased levels of circulating proinflammatory cytokines ,84. TherAir pollutants  and environmental tobacco smoke (ETS) have long been correlated with multiple adverse effects on the immune system and susceptibility to viral respiratory tract infections in children -90. StudThere is strong evidence that some pharmacological preparations can help prevent viral infection by specific effects on immune system. These results have been promising with a hope that using these strategies can attenuate the role of viruses in asthma inception.Ancient physicians of the Middle East prescribed yogurt for curing disorders of the stomach, intestines and for stimulation of appetite. It is written in the old Persian Testament that “Abraham owed his longevity to the consumption of sour milk” . The popUp to 100 trillion bacteria from different species colonize the human gut . This miReduced bacterial diversity in the infant’s gastrointestinal tract has been associated with an increased risk of allergic sensitization and allergic rhinitis but not asthma or atopic dermatitis . In the There are a growing number of clinical trials using probiotics for the prevention and management of respiratory infections. While the precise mechanisms are largely unknown, speculations include: probiotics compete against pathogens; increase the barrier function in respiratory epithelium; immunostimulatory effects by enhancing cellular immunity with increased activity of natural killer cells and macrophages in airways . ProbiotOne study demonstrated that that daily probiotic supplementation for six winter months in children three to five years of age reduced the incidence of fever, coughing and rhinorrhea by 32-43% with no notable adverse events . ProbiotAlthough there are several clinical trials that showed the preventive effect of probiotic, prebiotic  or synbiSeveral immunostimulants, including herbal extracts, bacterial extracts, synthetic compounds, have been promoted as increasing the immune defences of the respiratory tract. A recent Cochrane review included data from 35 placebo-controlled trials including 4060 participants below the age of 18 years in which various types of “immunostimulants” were used to reduce acute respiratory tract infections, involving either upper or lower airways. The authors concluded that immunostimulants reduced the incidence of acute respiratory infections by 40% on average in susceptible children, but that trial quality was generally poor and a “high level of statistical heterogeneity was evident”. A subgroup analysis focusing on bacterial immunostimulants, including OM85, produced similar results with lower statistical heterogeneity . Haemophilus influenzae, Diplococcus pneumoniae, Klebsiella pneumoniae and ozaenae, Staphylococcus aureus, Streptococcus pyogenes and viridans ,  Neisseria catarrhalis  and has been used in several countries around the world for as long as 20 years [ p  = 0.001) [OM-85 BV (Broncho-Vaxom) is an immunostimulant extracted from eight common bacterial pathogens of the upper respiratory tract: 20 years . Recent 20 years . Of part= 0.001) .Again, direct evidence of the mechanisms involved are lacking from human studies. However, recent data from rodents shows that baseline regulatory T lymphocyte activity in the airways can be boosted by microbe-derived stimulation of the gut . BacteriAs discussed above, evidence exists for an impaired innate immune response to respiratory viral infections in asthmatics . Entry oVitamin D deficiency is a common worldwide problem ,150. BesMacrolides possess anti-inflammatory and immunomodulatory properties extending beyond their antibacterial activity . Indeed,As the majority of respiratory viral infections in young children are caused by HRV or RSV, we will briefly discuss anti-viral strategies to prevent HRV or RSV infections in asthmatic children. Because there are more than 100 serotypes of HRV, antiviral drugs are considered to be more effective than vaccination. Antiviral agents have been designed to inhibit rhinovirus attachment, entry to the cell, viral uncoating, and RNA and protein synthesis . Table 1HRV has the icosahedrally shaped capsid formed by 60 identical copies of viral capsid structural proteins VP1-4. The capsid protects the single-stranded, positive sense RNA genome. While HRV-A and -B most often induce a self-limited upper respiratory infection, the recently discovered HRV-C was associated with sLRIs in infants, bronchiolitis, and asthma exacerbations in children ,171.HRV deposits on nasal or conjunctival mucosa and is transported to the posterior nasopharynx by mucociliary action of epithelial cells . The so-Endothelial cells express histamine receptors and increased adhesion molecule expression, such as ICAM-1, was demonstrated by histamine infusion. Second-generation H1-antihistamines decrease expression of ICAM-1 on cultured bronchial epithelial cells . Zinc ma in vitro  potency against HRV [The monoclonal antibody to the cellular ICAM-1 was not effective. CFY196 (Coldsol) is a nasal spray multivalent Fab fusion proteins against ICAM-1 with a better avidity and inst HRV . Tremacainst HRV . Pleconainst HRV . in vitro . Ruprintrivir nasal spray (2% solution) prophylaxis reduced the proportion of subjects with positive viral culture by 26% and reduce viral titers, but did not decrease the frequency of colds [Despite extensive research, no agent has been approved for prevention and/or therapy of rhinovirus-induced diseases so far. Ruprintrivir selectively inhibits HRV 3C protease and shows potent, broad-spectrum anti-HRV activity of colds . HRV RNAof colds , morpholof colds , and smaof colds . The novMaternally-derived RSV neutralizing antibodies help to protect infants against RSV hospitalization . PalivizVaccination against HRV and RSV have been in development for quite some time, but there are no safe and effective vaccines at present ,191. HigRespiratory viral infections are major contributors to the global burden imposed by asthma. In early life, they contribute to the inception of asthma and are responsible for most of the acute exacerbations for asthma in childhood. While the debate is not completely settled, children at high risk of developing asthma and those with established asthma may be at increased risk of acquiring respiratory viral infections and may be less able to contain these to the upper airway. Several simple general strategies can be used to help prevent respiratory viral infections in asthmatic children Table , with goWhile research continues on specific anti-viral therapies, including vaccination, there are no currently available practical therapies that are suitable for widespread use. The role of preventative strategies in primary prevention of asthma in high risk children is of considerable academic interest and a number of studies are currently in the pipeline. The results are awaited with interest.IFN: Interferon; PRRs: Pattern recognition receptors; TLRs: Toll-like receptors; HRV: Human rhinovirus; RSV: Respiratory syncytial virus; hMPV: Human meta-pneumovirus; LRI: Lower respiratory infections; sLRI: Severe lower respiratory infections; ETS: Environmental tobacco smoke; ICAM-1: Intercellular adhesion molecule-1.The authors declare they have no competing interests.HA and PDS conceived and designed the review. All authors reviewed the articles, abstracted data, and participated in the data synthesis. HA, PDS, YSC drafted the current manuscript, with critical review by PDS and CMJ. All authors contributed, read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2431/12/147/prepub


Nutritional and endocrine conditions in utero are known to have pronounced and long-term effects on progeny, but the ways in which the progeny has sustained physiological effects on the dam have received little attention to date.Mammalian females pay high energetic costs for reproduction, the greatest of which is imposed by lactation. The synthesis of milk requires, in part, the mobilization of bodily reserves to nourish developing young. Numerous hypotheses have been advanced to predict how mothers will differentially invest in sons and daughters, however few studies have addressed sex-biased milk synthesis. Here we leverage the dairy cow model to investigate such phenomena. Using 2.39 million lactation records from 1.49 million dairy cows, we demonstrate that the sex of the fetus influences the capacity of the mammary gland to synthesize milk during lactation. Cows favor daughters, producing significantly more milk for daughters than for sons across lactation. Using a sub-sample of this dataset  we further demonstrate that the effects of fetal sex interact dynamically across parities, whereby the sex of the fetus being gestated can enhance or diminish the production of milk during an established lactation. Moreover the sex of the fetus gestated on the first parity has persistent consequences for milk synthesis on the subsequent parity. Specifically, gestation of a daughter on the first parity increases milk production by ∼445 kg over the first two lactations. Our results identify a dramatic and sustained programming of mammary function by offspring  Since the 1970s, biologists have directed substantial research effort to understanding adaptive sex-biased allocation of maternal resources in animals and plants. Biologists have proposed numerous hypotheses for sex-biases, including local resource competition Equus caballusCervus elaphus hispanicus, Myodes glareolusMacaca mulattaHomo sapiensMacropus eugenii,Cervus elaphus hispanicus) and rhesus macaques (Macaca mulatta). Landete-Castillejos and colleagues showed that hinds favored sons by producing more milk with higher protein content for them Although sex-ratio at birth has been the primary outcome investigated, post-natal maternal physiological transfer and behavioral care afford females substantial flexibility in sex-biased resource allocation Myodes glareolus) to demonstrate that all-female litters received significantly greater milk yield than did all-male litters, regardless of litter size or maternal condition Mother’s milk, however, is particularly difficult to evaluate when investigating adaptive allocation of maternal resources. Milk synthesis is unlikely to be at the maternal optimum because of parent-offspring conflict Bos taurus. Although intensive artificial selection has shaped cattle during recent centuries, domesticated cattle are derived from large-bodied, sexually-dimorphic aurochs (Bos primigenius) We investigated the magnitude and direction of sex-biased milk synthesis in the Holstein breed of http://www.drms.org). Whole-lactation milk yield and composition data were derived from monthly yield and composition data collected on commercial dairy farms across the United States. Standardized lactation curves, characterized over 5 decades of research, were then used to predict production between the monthly data points. Production is adjusted for breed, region, season and parity during the calculation of whole-lactation milk and component production, which was standardized to a 305-day lactation. These records are used daily by most of the 50,000 dairy farmers in the U.S. to make management decisions. Detailed discussions of the program and data analysis have been published elsewhere To investigate sex-biased milk synthesis, we acquired all lactation records from 1995 to 1999 in the database managed by Dairy Records Management Systems  were excluded to enable repeated measures analysis of lactations with adequate representation in the database. If at least 1 of the first 5 test days, typically conducted monthly, were flagged for bST administration, then the lactation was considered bST-positive . The final database consisted of 2.39 million lactation records, representing 1.49 million individual Holstein cows, however due to missing data in certain fields, some analyses included fewer lactations and final analysis sample sizes are reported for each analysis. Mixed models were used to evaluate the fixed effects of calf sex, parity, bST, and interactions and the random effect of year according to the following model:ijkl is a dependent variable, µ is the overall mean, Si is the fixed effect of calf sex (i = 1 to 2), Pj is the fixed effect of parity (j = 1 to 5), Bk is the fixed effect of bST (k = 1 to 2), Yl is the random effect of year (l = 1 to 5), SPij is the interaction of calf sex and parity, SBik is the interaction of calf sex and bST, SPBijk is the interaction of calf sex, parity, and bST, and eijkl is the residual error. Repeated lactations within cow were fit to a heterogeneous autoregressive  and means were separated using the PDIFF option of SAS, with significance declared at P <0.05.To exclude potentially confounding effects of dystocia and bST treatment on results and to evaluate carryover effects of calf sex on multiple lactations, a more conservative data set was generated. All bST-positive lactations were deleted, and only those beginning with a calving difficulty score of 1 or 2  were retained. Finally, the data were narrowed to only those cows with both lactations 1 and 2 represented, leaving 113,750 cows. Data for 305-day milk yield in lactations 1 and 2 were modeled with the fixed effects of calf sexP<0.001). Similar, though marginally smaller, effects were observed in parities 2–5 , but sex-biased milk synthesis was not observed in parities 2–5 . Nevertheless, sex-biased milk synthesis remained when analysis was restricted to a subset of the dataset  that excluded cases of bST and dystocia, and included information on individual cows across the first and second parity. On first parity, cows producing daughters had significantly greater 305-day milk yield, with an advantage of 1.6% relative to cows producing sons . The daughter advantage was also observed in parity 2, although the magnitude of the difference was reduced . These results indicate that the milk production advantage associated with birth of a daughter is not attributable to prevention of dystocia.Male calves are typically larger than females, and pose a greater risk of dystocia 1son2, son1daughter2, daughter1son2, and daughter1daughter2. Cows that had first produced a son and were gestating a son for their second pregnancy synthesized significantly less milk over 305 days than did all other groups . Gestation of a daughter on the second pregnancy could partially “rescue” milk synthesis on the first lactation if a son had been produced previously , but remained significantly less than cows that had produced a daughter on their first pregnancy (P<0.001). Fetal sex on the second pregnancy didn’t have any effect for cows that produced a daughter on pregnancy 1 .Milk production on first lactation was associated with the sex of the fetus on the second pregnancy because the two overlapped temporally . Across P<0.001), particularly if they gestated a son on the second pregnancy as well . Production of a daughter on the second parity partially increased milk production on second lactation . Cows that produced a daughter on their first parity produced significantly more milk on their second lactation (P<0.001), regardless of the sex of the calf on the second parity .Fetal sex on the first parity had persistent effects on milk production during the second lactation . Cows thin utero are known to have pronounced and long-term effects on progeny Holstein dairy cows demonstrate a significant biological effect of sex-biased milk production in favor of daughters. In dairying, calves are removed on the day of birth and standardized mechanical procedures are used for milking, therefore post-natal sex-bias does not explain the results presented here. Instead milk production varied as a function of fetal sex, indicating that functional development of the mammary gland is influenced pre-natally. Importantly, lower milk yield for sons was not compensated by higher protein and fat production; total production of milk energy was greater in cows that gestated daughters. Among rhesus monkeys, mothers rearing daughters produce more milk, but of significantly lower milk energy density- the aggregated calories derived from fat, protein, and sugar- than do mothers of sons Sex-differentiated programming of the mammary gland is further substantiated by the greater effect of bST administration in cows gestating sons than cows gestating daughters. Postnatal administration of recombinant bovine somatotropin (bST) in multiparous cows overwhelmed the prenatal effects of offspring sex, but had a greater effect in cows gestating sons. Somatotropin, or growth hormone (GH), is produced in the anterior pituitary, stimulated by GH-releasing hormone. Most notably, GH influences metabolism in hepatic and adipose tissues, shunting more maternal bodily reserves to milk synthesis These biological findings may have economic impact for the modern dairy industry. With the widespread availability of sexed-selected semen for use in artificial breeding programs, dairy managers have the option of achieving approximately 90% female pregnancies rather than a natural rate near 47% nd trimester had a lower probability of sustaining breastfeeding to three months post-partum The precise mechanistic pathways through which fetal sex influences mammary gland development remain unknown. Fetal-origin hormones may translocate via maternal circulation to bind directly to receptors in the dam’s mammary gland influencing functional development and subsequent milk synthesis. Among ungulates, ruminants may be especially valuable for understanding mammary gland development during pregnancy as a function of fetal sex because of their cotyledonary placenta. Klisch and Mess posited that for ruminants, an evolutionary “arms race” between the mother and fetus Daughter-biased milk synthesis may reflect adaptive maternal allocation in response to fetal sex, adaptive fetal manipulation of maternal physiology, or may be a by-product of the maternal-fetal interface. Importantly, uniformly biased milk production in favor of daughters across maternal conditions does not support the Trivers-Willard hypothesis The question remains though, under natural conditions how do bull calves grow faster during the post-natal period if their dams are producing less milk, and therefore lower total protein and fat production? One explanation may be that females bias nursing behavior such that milk production is up-regulated for sons, a tactic we could not evaluate in conventional dairying as calves are removed after birth. Landete-Castillejos and colleagues revealed that among captive Iberian red deer, dams rearing sons had greater total milk production and total protein production


Punctatus include developmental stages such as blastula, gastrula, and hatchlings, and represent the most complete developmental sequence of animals available from the earliest Cambrian. Despite the extremely well-preserved specimens, the evolutionary position of Punctatus has relied only on their conical remains and they have been tentatively assigned to cnidarians. We present a new interpretation of the Punctatus body plan based on the developmental reconstruction aided by recent advances in developmental biology.Microfossils of the genus Punctatus developed from a rather large egg, gastrulated in a mode of invagination from a coeloblastura, and then formed a mouth directly from the blastopore. Spiny benthic hatchlings were distinguishable from swimming or crawling ciliate larvae found in cnidarians and sponges. A mouth appeared at the perihatching embryonic stage and was renewed periodically during growth, and old mouths transformed into the body wall, thus elongating the body. Growing animals retained a small blind gut in a large body cavity without partitioning by septa and did not form tentacles, pedal discs or holdfasts externally. A growth center at the oral pole was sufficient for body patterning throughout life, and the body patterning did not show any bias from radial symmetry.Punctatus body plan has basic differences from that of cnidarians, especially concerning a spacious body cavity separating ectoderm from endoderm. The lack of many basic cnidarian characters in the body patterning of Punctatus leads us to consider its own taxonomic group, potentially outside of Cnidaria.Contrary to proposed cnidarian affinity, the  Punctatus emeiensis , have provided exceptional fossils comprising reliable developmental series for some species Punctatus interpreted conical forms as test or thecal remains, although they were suggested to be flexible Punctatus have also been assigned to conulariids because their transverse ribbings and longitudinal sulci between flat sides (faces) were regarded as homologous, though the pyramidal forms were six-sided Previous reconstructions of Punctatus in previous studies relied only on the gross morphology of conical remains in spite of their rich developmental data, though recently ephyra-like fossils have been proposed to be a life stage of this animal Punctatus, Hexaconularia had a preserved bivalve-like structure at the apical end. This structure was recently reinterpreted as an embryonic shell and it was suggested to be an implicated difference from conulariids Classification of Punctatus fossils have provided detailed information of this animal both externally and internally. We have reinvestigated the developmental pattern of Punctatus by observing more than 10,000 fossils. Our findings show; (1) early development to gastrulation of Punctatus was similar to that of some cnidarians with a coeloblastula that started gastrulation as invagination or unipolar ingression Punctatus.Well-preserved developmental series of Punctatus was reconstructed starting from the hatchling stage, as this stage displays characters linking the embryonic and post-hatching stages , suggesting that they were diagenetic structures produced by bacterial activities Punctatus raises the possibility that the animal was secondarily derived from a cnidarian ancestor with symbionts, or it was small enough to simplify its gastric cavity. It is also possible to interpret Punctatus as a naked animal allied with conulariids. However, among SSFs, a partitioned gastric cavity has been found in a cnidarian species smaller than PunctatusPunctatus, but being tetra-radail with a seemingly distinct mode of development Punctatus may be questioned as taphonomic vias. Several well-grown individuals have preserved and intact oral ruffle soft tissues Punctatus was an animal without tentacles.The very simple gut of Punctatus hatchling was immotile and benthic and thus did not require directional movement, making them free from selection pressures of body organization guided by antero-posterior polarity, unlike pelagic or epibenthic crawling cnidarian planulae, sponge larvae, and bilaterians. For Punctatus, a morphogenetic center at the future oral pole was sufficient to build a conical body if it could repeatedly produce oral ruffles  Collapsing cleavage stage (Sn34-47), possibly same stage as specimen in Embryonic fossils assignable to early developmental stages of (TIF)Click here for additional data file.Figure S2Micro-CT sections showing small gut. (A) Young juvenile with a short blind gut and empty blastocoel (Sn24-88). (B) Irregular trabeculae connecting the column and short gut (Sn80-20). Trabeculae are thought to be metamorphic structures that appeared during fossilization. atr, artifactual trabecula; bc, blastocoel; col, collar; fr, fringe; g, gut; mo, mouth.(TIF)Click here for additional data file.Movie S1Micro-CT 3D-reconstruction of cleaving embryo. Note the wide blastocoel and the size gradient of blastomeres, suggesting an animal-vegetal axis.(MOV)Click here for additional data file.Movie S2Punctatus emeiensis with four annular fringes. Note the very small gut suspended from the mouth and the nearly empty and spacious body cavity.Micro-CT 3D-reconstruction of young (MOV)Click here for additional data file.


Oophaga histrionica and O. lehmanni) at five localities: two (one per species) alongside noisy streams, two away from streams, and one interspecific hybrid. We recorded the calls of 47 males and characterized the microgeographic variation in their spectral and temporal features, measuring ambient noise level, body size, and body temperature as covariates. As predicted, frogs living in noisy habitats uttered high frequency calls and, in one species, were much smaller in size. These results support a previously unconsidered role of noise on streams as a selective force promoting an increase in call frequency and pleiotropic effects in body size. Regarding hybrid frogs, their calls overlapped in the signal space with the calls of one of the parental lineages. Our data support acoustic adaptation following two evolutionary routes but do not support the presumed role of hybridization in promoting phenotypic diversity.According to the acoustic adaptation hypothesis, communication signals are evolutionary shaped in a way that minimizes its degradation and maximizes its contrast against the background noise. To compare the importance for call divergence of acoustic adaptation and hybridization, an evolutionary force allegedly promoting phenotypic variation, we compared the mate recognition signal of two species of poison frogs ( AnolisSpecies exhibiting intraspecific geographic or microgeographic variation in habitat use offer excellent opportunities to understand the very initial steps of evolutionary divergence in auditory signals Cross-breeding between taxa (hybridization) also can favor the evolution of communication signal diversity because hybrids often exhibit distinctive phenotypes The role of natural selection on the divergence of auditory communication signals has been compared to the role of other evolutionary forces such as sexual selection and genetic drift Among vertebrates, poison frogs (Dendrobatidae) represent an increasingly important model in which to study the evolutionary biology of complex signals and its implications in speciation. They use multimodal  and multicomponent (e.g. color and pattern) communication signals Oophaga histrionica and O. lehmanni inhabit tropical wet forests where males usually establish territories, call, and mate near forest gaps and forest edges O. histrionica and O. lehmanni living alongside noisy streams should exhibit higher call frequencies than individuals living away from streams, where the broadband low-frequency noise is significantly lower in intensity. On the other hand, cross-breeding experiments and microsatellite analyses have shown that at least one population currently assigned to O. histrionica resulted from hybridization between O. histrionica and O. lehmanniO. histrionica could exhibit either intermediate or novel call traits relative to the parental lineages, perhaps distinctive enough to increase the probability of incipient speciation. Our aim here was to estimate the effect of stream noise and hybridization on microgeographic divergence of an acoustic mating signal. To reach this goal, we 1) characterized geographic variation on spectral and temporal traits of the advertisement calls of O. histrionica and O. lehmanni, and then used multivariate analyses to 2) compare each species' calls between individuals occurring at streams and away from them, and 3) compare the calls of a hybrid population against the call of the parental lineages.The frogs Procedures for recordings, capture and handling of live animals in the field were approved by the Colombian Ministry of Environment under the research permit 004 of July 27–2007. The Unidad Administrativa Especial de Parques Nacionales Naturales de Colombia and the Corporación Autónoma Regional del Valle del Cauca CVC gave us permission and logistic support for working in all study localities.Oophaga histrionica and O. lehmanni (Dendrobatidae) inhabit tropical wet forests, on the pacific slope of the western Colombian Andes. Whereas O. histrionica is widely distributed between sea level and 1000 m elevation, O. lehmanni is scattered within less than 400 km2, between 550–1010 m elevation O. histrionica and two populations to O. lehmanni, all of them in the Departamento del Valle del Cauca. We publish here the approximate position rather than the precise coordinates of the study sites because both species are heavily trafficked for the international pet market and O. lehmanni  where the acoustic environment is highly influenced by high-intensity and low-frequency noise produced by rocky streams. Another population of each species occurs away from fast flowing streams , in less noisy habitats (O. histrionica (hereafter Oh-Hybrid) has arisen from natural cross-breeding between the latter two populations of O. histrionica and O. lehmanniThe poison frogs habitats . The thiOh-Stream, males called from the ground and from crevices in rocky areas along the streams. Once a calling male was located, we recorded its advertisement calls by positioning a unidirectional microphone (Sennheiser K6/ME66), connected to a digital recorder (Marantz PMD660), 30–150 cm from a calling male. Immediately after recording we measured male body temperature to the nearest 0.1°C with an infrared thermometer (Oakton model 35629) and captured him to measure body size  to the nearest 0.1 mm with a digital caliper. At the end of each recording session, we measured ambient noise level  at the calling position to the nearest dB (re 20 µPa) with a sound level meter Roline RO-1350.Most individuals (40/47) were recorded in the field during ten field trips between April 2009 and September 2012; seven were recorded under lab conditions due to significant security concerns in the study area, which has been historically occupied by guerrillas. Calling males were generally found during daytime hours in elevated positions on the forest floor upon fallen trunks, leaves, and abundant leaflitter; in O. histrionica and O. lehmanni, we repeated these measurements in three pulses located at the initial, middle and final portions of a single call and then used the corresponding average values for further analyses. From power spectra , we measured peak frequency. For each call parameter, the average of measurements taken on three calls per male was used as the smallest unit of statistical analysis.Digital recordings were analyzed with Raven 1.4 sound analysis software Oh-Stream and Ol-Stream, we ran an ANOVA. Because part of the call divergence could be merely explained by co-variation in body size, we repeated the ANOVA after removing concomitant variation in male body size Since sound pressure levels were measured in dB, which represent a logarithmic scale, the calculation of the average values and further statistical analyses were conducted after converting dB values to a linear scale . Differences in ambient noise level among populations were tested with an Analysis of Variance ANOVA using significant level of α<0.05. Since the other measured acoustic traits are usually intercorrelated, we reduced redundancy by conducting a principal component analysis (PCA) with Varimax-rotation. Because body temperature may affect calling performance and thereby acoustic trait values, we removed its effect by conducting linear regression on PCAs, and then used the regression residuals as new temperature-independent acoustic traits in subsequent statistical analyses. To estimate the degree of acoustic divergence among the five studied populations we ran a canonical discriminant analysis. To test whether abiotic noise originated from streams was correlated with high call frequency in Oh-Stream and Ol-Stream) and frogs living away from streams   from three populations with low frequency calls  (Oh-Stream and Oh-Away) from three populations with shorter calls . Considering both functions, the calls of hybrid frogs, Oh-Hybrid, overlapped almost completely with the calls of Ol-Away. Since populations differed in average body size . As a result (Oh-Stream) moved towards the signal spaces of Ol-Away and Oh-Hybrid and overlapped completely within them (Ol-Stream) remained statistically distinguishable from any other population, because of its high frequency calls. There was no intrapopulation relationship between ambient noise level and any of the call characteristics .The first discriminant function , mostly -Hybrid) . The seca result , one of hin them . The othOur results demonstrate uncoupled divergence in call traits among the studied populations. Concerning the spectral call traits, populations were grouped independently of their phylogenetic relationship, but according to similarities on their habitat acoustic environment. In contrast, concerning temporal call traits, populations were grouped according to their phylogenetic affinity rather than their acoustic environment. Hybridization did not appear to promote call diversity in our study system.Oh-Stream and Ol-Stream) produced calls with higher frequency than males living away from streams. The pattern suggests parallel adaptation in both species. Since stream noise contains most of its energy at low frequencies . Their calls remained distinguishable in frequency from the calls of any other population even after statistically removing the negative effect of body size , ay calls . StudiesO. histrionica and O. lehmanni on a microgeographic scale. The evolutionary route, however, apparently differed between the species: it implied pleiotropical differences in body size in O. histrionica but not in O. lehmanni. Stream noise appears to be a selective force strong enough to promote microgeographic divergence in calls despite the counteracting effect of genetic flow at the microgeographic scale Summing up, our data support the hypothesis that stream noise has favored the evolution of high frequency calls in the poison frogs Table S1Oophaga histrionica , O. lehmanni , and a hybrid population between both species (Oh-Hybrid). Values are Mean ± standard deviation.Statistical summary of advertisement call traits, made for the studied populations of poison frogs: (DOC)Click here for additional data file.


Alcohol consumption alters factors that modify gene expression without changing the DNA code  in many organ systems, including the immune system. Alcohol enhances the risk for developing several serious medical conditions related to immune system dysfunction, including acute respiratory distress syndrome (ARDS), liver cancer, and alcoholic liver disease (ALD). Binge and chronic drinking also render patients more susceptible to many infectious pathogens and advance the progression of HIV infection by weakening both innate and adaptive immunity. Epigenetic mechanisms play a pivotal role in these processes. For example, alcohol-induced epigenetic variations alter the developmental pathways of several types of immune cells  and through these and other mechanisms promote exaggerated inflammatory responses. In addition, epigenetic mechanisms may underlie alcohol’s ability to interfere with the barrier functions of the gut and respiratory systems, which also contribute to the heightened risk of infections. Better understanding of alcohol’s effects on these epigenetic processes may help researchers identify new targets for the development of novel medications to prevent or ameliorate alcohol’s detrimental effects on the immune system. Extensive clinical and experimental data suggest that alcohol consumption has dose-dependent modulatory effects on the immune system that influence the two arms of the immune response . In many other organ systems, such as the brain and liver, alcohol consumption has been shown to alter factors that can modify gene expression without changing the DNA code  and which play critical roles in mediating alcohol’s effects. However, very few studies have focused on the effects of alcohol-mediated epigenetic alterations on immunity. Because chronic alcohol consumption is correlated with an exacerbated state of chronic inflammation (which is part of the innate immune response), researchers can apply knowledge of how epigenetic factors are dysregulated in inflammatory and autoimmune disorders to identify potential epigenetic targets that can be used to develop therapies for treating alcohol-abusing patients. This review summarizes how inflammatory mediators and both innate and adaptive immune responses are modulated by moderate, binge, and chronic alcohol consumption. The discussion further identifies and highlights exciting potential avenues to explore epigenetic regulation of these immune responses.All cells within an organism carry identical genetic information in the form of DNA, yet a multitude of individual cell types arises during the course of development. These individualized cellular morphologies, characteristics, and functions result from the unique gene expression profiles of the different cell types. Regulation of gene expression profiles is critical not only during development, but also for cellular proliferation, differentiation, environmental adaptation, stress, and immune responses throughout the individual’s lifetime and is largely dependent on epigenetic mechanisms. An epigenetic trait is a stably heritable observable characteristic  that results from changes in a chromosome without alterations in the DNA sequence . EpigeneIt is well documented that alcohol exposure prior to an injury or infection dampens the immune system, resulting in a range of adverse outcomes, such as delaying infection clearance, extending hospital stays, and increasing morbidity and mortality compared with nonintoxicated patients  (Specific enzymes are responsible for adding or removing acetyl or methyl moieties from histone tails. Histone acetyl transferases (HATs) and histone deacetylases (HDACs) add and remove acetyl groups, respectively. Similarly, methylation is tightly regulated by enzymes that add methyl groups to  or remove methyl groups from  specific lysine residues . So far,e (NAD+) .Several approaches may potentially be used to prevent or correct the epigenetic effects of alcohol consumption, such as alcohol-mediated immune defects. For example, inhibition of HDACs by molecular HDAC inhibitors (HDACis), alteration of DNA methylation on cytosine residues, or miRNA modulation all represent branches of possible therapeutic targets for restoring immune defects caused by alcohol exposure. These approaches will be discussed later in this review.Ethanol exposure enhances the activity of a HAT called p300 in the liver of rats fed a chronic ethanol diet, which leads to heightened histone acetylation .Elevated ROS levels resulting from ethanol metabolism increase histone H3 acetylation in liver cells  .+ (NADH) to NAD+, and this reduced redox state suppresses the activity of the redox-sensitive HDAC, SIRT1, thus augmenting histone acetylation in rats , which serves as a methyl-group donor for both DNA and histone methylation .Chronic ethanol exposure decreases the levels of the antioxidant glutathione, which serves as the predominant scavenger of ROS in the liver ; this glChronic ethanol exposure in rats leads to inhibition of a set of reactions called the ubiquitin–proteasome pathway, which helps eliminate molecules that are defective or no longer needed from the cell. This inhibition of the ubiquitin–proteasome pathway likely alters protein turnover of transcription factors and histone-modifying enzymes and is associated with epigenetic alteration at a specific lysine residue (K9) of histone H3 (i.e. increased H3K9-ac and reduced H3K9-me2) as well as DNA hypomethylation .Acetylation of H3K9 also is associated with increased ADH1 expression in cultured rat hepatocytes treated with 100 mM ethanol for 24 hours, suggesting that ethanol (and its metabolites) may amplify ethanol metabolism .Beverage alcohol  is predominantly metabolized by the enzymes alcohol dehydrogenase (ADH), cytochrome p450 (CYP 450), and aldehyde dehydrogenase (ALDH) in the liver . This prThrough the various mechanisms discussed above, alcohol consumption can lead to multifactorial, dose-dependent, and tissue-specific epigenetic effects. For example, cultured primary rat hepatocytes demonstrated a dose- and time-dependent histone-acetylation response to ethanol exposure. Thus, cells treated with 5–100 mM ethanol for 24 hours exhibited a maximal, eightfold increase in H3K9-ac levels at 24 hours following treatment with the highest ethanol concentration . FurtherEthanol metabolites, including acetaldehyde and acetate, also could cause H3K9-specific acetylation in rat hepatocytes. Interestingly, the signaling pathways activated by acetate and ethanol seemed to modulate H3K9-ac via different mechanisms. Thus, certain molecules  prevented acetylation by ethanol but had no effect on the acetate-dependent formation of H3K9-ac . In addiRat models of acute/binge and chronic alcohol exposure have been utilized to examine the relationship between epigenetic gene regulation and alcohol exposure in vivo. In one of those models, a single dose of ethanol diluted in sterile water resulting in a concentration of 6 grams ethanol per kilogram bodyweight (g/kg) was injected directly into the stomachs of 8-week-old male Sprague-Dawley rats. This high-dose binge-alcohol exposure model was used to compare H3K9 modification status across 11 different tissues at 1, 3, and 12 hours following ethanol exposure . The invOther investigators evaluated changes in gene expression levels after chronic ethanol treatment using in vivo models. One of these models is the Tsukamoto-French rat model of alcoholic liver disease , in whicIn summary, both acute/binge and chronic alcohol exposure can result in tissue- and cell-specific patterns of epigenetic responses. Future studies to determine the precise role of alcohol-mediated chromatin modifications hopefully will identify new epigenetic targets and pathophysiological mechanisms for regulating gene expression in diseases associated with alcohol consumption. The factors contributing to altered epigenetic modifications arising from acute versus chronic alcohol exposure may differ, because chronic alcohol exposure has been strongly correlated with nutrient deficiencies and a shift in the redox state. This implies that potential therapeutic interventions targeting epigenetic modifiers may need to differ depending on the degree of alcohol consumption. Furthermore, understanding the role of nutrients in regulating epigenetic modifications will provide insight into potential dietary supplementation in chronic alcohol-abusing patients.A recent report from the Centers for Disease Control and Prevention (CDC) stated that alcohol abuse in the form of binge drinking (defined by the CDC as four or more drinks for women and five or more drinks for men in a short period of time) is the third-leading preventable cause of death in the United States, resulting in more than 80,000 deaths each year and enormous economic costs  (CDC 2012). A significant, positive correlation exists between the duration and amount of alcohol consumed and the risk for developing several serious medical conditions, including acute respiratory distress syndrome (ARDS) ; liver cThe mammalian immune system is an elaborate network of molecules and cells that identify, combat, and eliminate harmful agents; it can be divided into two branches: innate and adaptive immunity. The innate immunity is present from birth, whereas the adaptive immunity develops over the organism’s life course with the continuous exposure to pathogens and other potentially harmful compounds.Following pathogen or toxin exposure, the ancient innate immune response is responsible for immediate recognition, rapid attack, and destruction of foreign intruders and involves inflammatory reactions. Innate immune cells carry special molecules called Toll-like receptors (TLRs) on their surface that recognize and bind highly conserved structures on bacterial, fungal, or viral surfaces, including peptidoglycan, flagellin, zymosan, and lipopolysaccharide  . The innClassically activated (M1) macrophages, whose activation results in a proinflammatory response.Alternatively activated (M2) macrophages, whose activation results in an anti-inflammatory response.An important subset of innate immune cells are macrophages; they eliminate pathogens by a process called phagocytosisAfter challenge to the immune system occurs , macrophages are generated by the maturation of precursor cells called monocytes. During this process, the macrophages can become either M1 or M2 macrophages; this is called macrophage polarization. The ratio of M1 to M2 macrophages changes depending on the presence of a variety of factors; this variability is known as macrophage plasticity and allows the organism to modulate the immune response. Accordingly, controlling macrophage plasticity is critical to first battle pathogens and then resolve the resulting inflammation to prevent tissue damage. Alcohol exposure skews macrophage polarization towards M1  in the liver , resultiDendritic cells (DCs) are an additional component of the innate immune response. They have an important role in linking the innate and adaptive branches of the immune system. To this end, the DCs exhibit proteins called major histocompatibility complex (MHCs) on their surface. With the MHC proteins, DCs present antigens to other cells that are part of the adaptive immune system—that is, B and T lymphocytes . DCs mature following stimulation by whole bacteria or LPS or after exposure to various signaling molecules, such as interleukin 1β (IL-1β), granulocyte macrophage colony–stimulating factor (GM-CSF), and tumor necrosis factor-α (TNFα) . The matAlcohol consumption has a variety of effects on innate immune cells. For example, alcohol decreases the phagocytic activity of monocytes, macrophages, Kupffer cells, microglia, and DCs and diminishes their capacity to present antigens and produce the molecules necessary for microbe killing. In addition, alcohol alters expression of other proteins  on their cell surface that are required for cell–cell interactions among immune cells  ultimately gain the ability to recognize and kill pathogens . Conversely, CD4+ T-cells give rise to several T helper (Th) cell subsets, including Th1, Th2, and Th17 cells, that will produce mutually exclusive groups of cytokines which help mount specific immune responses by stimulating other immune cells  are key players of the adaptive immune response. Like DCs, APCs present antigen to B and T-cells that have not yet been activated , contributing to their maturation and differentiation. Naïve T-cells are classified based on expression of specific proteins on their surface called cluster of differentiation (CD) proteins. Two of those proteins important in distinguishing different T-cell populations are CD4 and CD8. T-cells carrying the CD8 protein , the developing cells fall into one of two developmental paths: the myeloid lineage, which includes granulocytes and monocytes (which then further differentiate into macrophages or DCs), and the lymphoid lineage, which includes B- and T-lymphocytes. This myeloid versus lymphoid lineage commitment corresponds with global and reduced DNA methylation, respectively . During H3K20me3 . In the lar fate .Alcohol alters macrophage polarization in the liver—that is, it alters the normal ratio of M1 to M2 macrophages. Chronic alcohol exposure sensitizes Kupffer cells to LPS stimulation, leading to prolonged and predominant M1 polarization and the exacerbated release of pro-inflammatory cytokines . This shAnother potential strategy for shifting Kupffer cell polarization is the use of therapeutic reagents that target epigenetic modifiers because epigenetic processes play central roles in the regulation of immune-system functions. For example, one critical mechanism to restore the internal balance  of the immune system in response to infection involves miRNA-dependent post-transcriptional regulation. Researchers found that expression of one specific miRNA called miR-155 was dramatically increased when macro -phages derived from the bone marrow were stimulated by LPS. This enhanced miRNA expression served to fine-tune the expression of pro-inflammatory mediators and promote M2 polarization . SimilarBesides ethanol-induced production of miR-155, histone modifications also can regulate macrophage polarization. As mentioned earlier, macrophages and other innate immune cells carry TLRs on their surface that can interact with LPS and other molecules, leading to the activation of the TLRs. Studies have demonstrated that when TLR4 was stimulated by LPS, histone acetylation and H3K4 tri-methylation (both of which are associated with active gene transcription) occurred in DNA regions encoding several pro-inflammatory cytokines . MacrophIL-12 gene and thereby regulating DC activation  and inhibition of deacetylation accordingly could promote IL-4 production , thereby counteracting the decreased HDAC activity induced by chronic ethanol exposure .Although drugs that modulate epigenetic targets have not yet been used specifically to treat alcohol-induced inflammation, research of other inflammatory and autoimmune diseases suggest that epigenetic modulation plays a critical role in regulating the inflammatory cytokine network . AccordiIt is important to note that the development of selective HDACis may be complicated by the fact that most HDACs are components of multi-protein complexes, which often include other HDACs . TherefoAnother important component of the innate immune system are the epithelial cells that line the outer surfaces of exposed tissues, such as the skin, respiratory, gastrointestinal (GI), and urogenital tracts. These cells provide a physical barrier that impedes pathogen invasion by forming strong intercellular associations . AnotherChronic alcohol consumption increases microbial colonization and LPS accumulation in the small intestine by decreasing gastric acid secretion in the stomach and delaying GI motility . The intThe multifactorial contributions of chronic alcohol consumption to the development of ALD largely have been deciphered using rodent models. For example, investigators demonstrated a direct translocation of LPS across the gut mucosa in rats continuously administered alcohol directly into the stomach for 9 weeks . Other sAlthough alcohol itself does not alter intestinal permeability, one of the products of alcohol metabolism  increases barrier permeability in a dose-dependent manner  by disruStreptococcus pneumoniae, to invade the tissues and become pathogenic  . This lethogenic . In factthogenic . Furtherthogenic  and incrthogenic  in a dosthogenic . Alcoholthogenic .S. pneumoniae (+ T-cells, which increased the morbidity and mortality associated with influenza virus infection (Klebsiella pneumoniae (When an infection occurs, neutrophils and monocytes are recruited to the lungs . Upon aceumoniae . Rodent eumoniae . Alcoholeumoniae . Furthernfection , and deceumoniae  in murinK. pneumoniae infection was protective in mouse models (Several strategies targeting epigenetic regulatory mechanisms may be effective in the treatment of alcohol-induced lung infections. For example, therapies that restore neutrophil recruitment to infected lungs through regulation of cytokine production would be beneficial. In support of this notion, it was demonstrated that pretreatment with G-CSF prior to alcohol exposure and e models . Targetie models . Severale models , 2010. AThe relationship between alcohol exposure and altered immune responses is complex. Chronic alcohol abuse is correlated with increased susceptibility to infection and causes tissue damage from an overactive innate immune response, excessive oxidative stress, and exacerbated or prolonged inflammation. Alcohol exposure has tissue- and immune cell-type–specific effects, such as influencing cell recruitment to infected or inflamed tissue, altering cytokine and chemokine production and secretion, skewing differentiation towards a particular cell fate or preventing cell replication, impairing antigen presentation, interfering with phagocytosis and granulopoiesis, or inducing apoptosis. Although the specific role of epigenetic modulation in this alcohol-induced immune dysregulation has not yet been determined, research in related fields strongly suggests that experimental and clinical studies are warranted.


Background: Because human exposure to persistent organic pollutants (POPs) occurs mainly through ingestion of contaminated food, regulatory bodies issue dietary consumption advisories to describe safe intake levels for food items of concern, particularly fish.Objectives: Our study goal was to estimate the effectiveness of fish consumption advisories in reducing exposure of infants and children to POPs.Methods: We used the time-variant mechanistic model CoZMoMAN to estimate and compare prenatal, postnatal, and childhood exposure to polychlorinated biphenyl congener PCB-153 under different scenarios of maternal guideline adherence for both hypothetical constant and realistic time-variant chemical emissions. The scenarios differed in terms of length of compliance (1 vs. 5 years), extent of fish substitution , and replacement diet (uncontaminated produce vs. beef). We also estimated potential exposure reductions for a range of theoretical chemicals to explore how guideline effectiveness varies with a chemical’s partitioning and degradation properties.Results: When assuming realistic time periods of advisory compliance, our findings suggest that temporarily eliminating or reducing maternal fish consumption is largely ineffective in reducing pre- and postnatal exposure to substances with long elimination half-lives in humans, especially during periods of decreasing environmental emissions. Substituting fish with beef may actually result in higher exposure to certain groups of environmental contaminants. On the other hand, advisories may be highly effective in reducing exposure to substances with elimination half-lives in humans shorter than the length of compliance.Conclusions: Our model estimates suggest that fish consumption advisories are unlikely to be effective in reducing prenatal, postnatal, and childhood exposures to compounds with long elimination half-lives in humans.http://dx.doi.org/10.1289/ehp.1206380Citation: Binnington MJ, Quinn CL, McLachlan MS, Wania F. 2014. Evaluating the effectiveness of fish consumption advisories: modeling prenatal, postnatal, and childhood exposures to persistent organic pollutants. Environ Health Perspect 122:178–186;  We performed simulations using the time-variant contaminant fate model CoZMoMAN (CoZMoMAN  to estimCoZMoMAN  with theCoZMoMAN  and is pCoZMoMAN . For PCBCoZMoMAN .–6/hr) for PCB-153, corresponding to a 15.1-year half-life)  . We usedDefining scenarios of fish consumption guideline compliance. As discussed, fish consumption advisories often target populations designated as sensitive to the deleterious neurological effects of PCB exposure, particularly those within major developmental stages: prenatal, postnatal, and childhood growth. Because specific advisories vary quantitatively in recommended daily fish intake based on the target community, we adopted the framework outlined in the Guide to Eating Ontario Sport Fish based on its simplicity; generally, children and women of childbearing age are advised to regularly consume half or none of the suggested serving sizes for the general population, which vary among fish of different species, size, and lake  because fruits and vegetables are not a significant vector for human PCB exposure , or a prCalculations for PCB-153 assuming hypothetical constant or historical time-variant emissions. We focused primarily on PCB-153 because previous CoZMoMAN estimates of this congener exhibited the greatest consistency with measured time trends in the Western Baltic drainage basin  of contaminants for which adjustments of fish intake elicit the greatest effect on human exposure. We performed simulations for multiple combinations of octanol–air (Koa) and octanol–water (Kow) partition coefficients that define the chemical partitioning space. According to Kow < 11 and log Kow > 7. Therefore, we focused on this area of the chemical partitioning space in our calculations; log Kow was varied from 2 to 12 and log Koa from 7 to 12 by half log-unit increments. From an analysis of dietary bioaccumulation potential by Kow of 6 to 9 and a log Koa of 9 to 12  to be most affected by advisory compliance because fish intake was estimated to result in > 50% of the exposure to chemicals possessing these properties. For our chemical partitioning space analyses, we assumed that compounds were emitted at a constant rate directly to the atmosphere, and we then calculated exposure reductions once human concentrations reached steady state. Initial simulations assumed that chemicals were perfectly persistent, exhibiting negligible breakdown in environmental compartments and organisms (including humans), which allowed us to evaluate the influence of chemical partitioning properties and compliance scenarios in the absence of any modifying effects of metabolism. In other words, we simulated constant chemical emissions until no difference in contaminant exposure was estimated between human generations (steady-state assumption), and contaminants were also not subject to any degradation processes in environmental and biotic compartments (perfect persistence assumption).post hoc analyses to compare fish advisory effectiveness assuming an organic chemical biotransformation half-life approaching that of the elimination half-life for methylmercury (40–50 days)  , becauseFish advisory impact assuming steady-state emissions of PCBs. We also performed estimates for a best-case scenario of lifetime guideline adherence because dietary POP exposure during childhood has been shown to contribute significantly to body burdens among children . When fiFish advisory impact assuming time-variant emissions of PCBs. To analyze the influence of time-variant emissions on PCB-153 exposures following maternal fish replacement, we focused on long-term (5-year) compliance scenarios because the 1-year compliance scenarios had limited impact when assuming steady-state emissions. Reductions in postnatal PCB-153 exposure are not displayed in The impact of fish consumption advisory compliance scenarios assuming steady-state emissions of hypothetical chemicals. When contaminants were assumed to be perfectly persistent, model projections of prenatal exposure assuming 5-year advisory compliance and constant chemical emissions indicated both increasing and decreasing exposures, depending on chemical properties and the specific fish substitution scenario , PCB-126, and PCB-169. This region of chemicals also roughly corresponds to those with properties previously identified to possess elevated human bioaccumulation potential , whose lipid-normalized PCB-153 blood lipid concentrations ranged over 7.1-fold (84–600 ng/g lipid). The greatest prenatal exposure reduction for PCB-153 we calculated was for children born in 1965 to mothers who completely replaced fish with produce for 5 years, assuming time-variant emissions. For these individuals we estimated a prenatal PCB-153 exposure decline of 59%, from 43 to 18 ng/g lipid, or merely a 2.4-fold decrease. Finally, our lifetime fish advisory compliance results suggest that periods of adherence that include childhood and childbearing age , only when long-term (5-year) advisory compliance was assumed. The lack of substantial impact for short-term compliance (1-year) suggests that current advisories based predominantly on long-lived POPs are likely to be ineffective in reducing human exposure to many chemical groups for which they are designed . As an example, previous epidemiological work by ring age  would liring age .A further consideration for the effectiveness of POP-based fish advisories is that any minor chemical exposure benefit of short-term compliance is likely accompanied by a reduced intake of beneficial nutrients from fish, because many consumers following guidelines do in fact reduce overall fish intake rather than replace higher-contaminated fish with lesser-contaminated fish . ReductiEvidence that fish consumption advisory effectiveness rises and declines as contaminant emissions increase and decrease is of particular interest when considering the policy context in which food consumption guidelines for POPs are typically developed. Environmental contaminants identified as food-chain-bioaccumulation and biota-toxicity risks undergo rigorous study to understand any exposure concerns. Thus, issuing of advisories will often lag behind regulations to reduce emissions of the contaminant to the environment. Although it would be most advantageous to define consumption recommendations for chemicals undergoing increasing or peak emissions, the issuing of advisories well before undertaking emission reduction is unlikely.Our model estimates suggest that the success of advisory adherence will be greatest for chemicals with a high metabolic transformation rate , because exposure reductions would be elevated if efficient contaminant removal occurred during the period of reduced chemical intake. This high impact of chemical half-life on guideline effectiveness is consistent with expectations, because a mother’s reduction in intake of chemicals with a 1-year transformation half-life for > 5 years should have a substantial effect on resultant exposures in her child. However, POP-based dietary advisories are often based on compounds characterized by high persistence, such as PCBs and PCDDs . An impoThe greater importance of advisories targeting methylmercury compared with those for POPs has also been previously addressed in the risk–benefit analysis literature e.g., . In partOWK < 4)  . Undemanobenzene . Given tFinally, several limitations of our analysis based on inherent model assumptions merit mention. As discussed, our simulations rely on lipid partitioning to estimate food chain bioaccumulation, and we therefore cannot comment on the effectiveness of methylmercury-based fish advisory compliance, beyond predictions based solely on the elimination half-life of this chemical. Also, we did not address potential effects of predicted POP exposure reductions on health outcomes. Other limitations include the assumption that the entirety of contaminant load is derived from local food sources, that contaminated dietary proportions are unchanged throughout an individual’s lifetime, and that interindividual and intergenerational differences in reproductive behaviors or metabolic degradation rates are not considered. Collectively these assumptions likely limit the generalizability of absolute exposure reduction results to specific populations and contexts beyond the Baltic watershed, but our principal relative findings are expected to remain applicable to any temperate fish-eating community.Kow < 11 and log Koa > 7, a region including several highly bioaccumulative groups of compounds in humans . Notably, the rate of chemical transformation in humans was a much greater influence on guideline effectiveness than the assumed degree of advisory compliance, because effectiveness increased as human transformation half-life decreased. Evaluation of fish consumption guidelines remains necessary for their improvement, and, as our study demonstrates, effective modeling practice may help inform this pursuit. Ultimately, for fish advisories targeting members of the general population sensitive to POP neurodevelopmental and reproductive deficits , we recommend a focus on guidelines for chemicals with human elimination half-lives that are ≤ 1 year. We also propose that advisories include specific recommendations for healthy alternatives to fish, focusing on replacement food items that will reduce the likelihood that contaminant exposures will persist or even increase. Finally, a complete ban on fish consumption may be preferable to targeted, life stage–based fish consumption advisories when fish are heavily contaminated with substances that have long human elimination half-lives.We conducted a series of simulations to investigate the efficacy of maternal fish consumption advisory adherence in reducing exposures of offspring to PCB-153, as well as a range of hypothetical organic contaminants. Our results suggest that the main variables determining the impact on PCB-153 exposure are the degree of advisory compliance (to what extent fish is removed from the diet and the length of time for which the advisory is followed) and the time from peak contaminant emissions. Notably, PCB-153 exposure reductions were limited for the majority of dietary scenarios when maternal dietary adjustments lasted for either 1 year or 5 years before birth. This suggests that, for compounds with long half-lives, temporary fish advisory-related decreases in daily contaminant intake will not necessarily translate to appreciable decreases in maternal POP body burdens, a highly relevant parameter in determining prenatal dose. For the range of hypothetical chemicals assessed, the estimated effect of following guidelines was greatest and relatively uniform for chemicals within the region bounded by 2 < log (426 KB) PDFClick here for additional data file.


Developmental stuttering is a disorder of speech fluency with an unknown pathogenesis. The similarity of its phenotype and natural history with other childhood neuropsychiatric disorders of frontostriatal pathology suggests that stuttering may have a closely related pathogenesis. We investigated in this study the potential involvement of frontostriatal circuits in developmental stuttering. We collected functional magnetic resonance imaging data from 46 persons with stuttering and 52 fluent controls during performance of the Simon Spatial Incompatibility Task. We examined differences between the two groups of blood-oxygen-level-dependent activation associated with two neural processes, the resolution of cognitive conflict and the context-dependent adaptation to changes in conflict. Stuttering speakers and controls did not differ on behavioral performance on the task. In the presence of conflict-laden stimuli, however, stuttering speakers activated more strongly the cingulate cortex, left anterior prefrontal cortex, right medial frontal cortex, left supplementary motor area, right caudate nucleus, and left parietal cortex. The magnitude of activation in the anterior cingulate cortex correlated inversely in stuttering speakers with symptom severity. Stuttering speakers also showed blunted activation during context-dependent adaptation in the left dorsolateral prefrontal cortex, a brain region that mediates cross-temporal contingencies. Frontostriatal hyper-responsivity to conflict resembles prior findings in other disorders of frontostriatal pathology, and therefore likely represents a general mechanism supporting functional compensation for an underlying inefficiency of neural processing in these circuits. The reduced activation of dorsolateral prefrontal cortex likely represents the inadequate readiness of stuttering speakers to execute a sequence of motor responses. Developmental stuttering is a disorder of speech fluency that disrupts the rhythm and timing of speech, producing frequent repetitions, and prolongations and blocks of syllables and words. Stuttering symptoms follow a distinctive developmental trajectory, with speech dysfluency typically beginning in early childhood, then gradually attenuating with the development of language skills, and remitting by adolescence in approximately 75% of persons who stutter Prior fMRI studies of stuttering have focused nearly exclusively on assessing brain activation during tasks that involve speech processing or production in attempts to identify the neural circuits that produce stuttering. Studies using language-related tasks in persons who stutter, however, have been unable to distinguish the abnormalities in brain activation that contribute to generating speech dysfluencies in stuttering speakers from the activations that derive from the attempts to control those dysfluencies. Recent stuttering research has revealed impairments in capacities that can broadly be described as self-regulation, including regulation of attention, emotion, and motor activity We therefore aimed to study self-regulatory control systems in the brains in stuttering speakers across the lifespan, from early childhood through adulthood, independent of the speech-based abnormalities that define stuttering behaviors. Self-regulatory functions are commonly studied using tasks in which participants must respond to a task-relevant feature of a stimulus for which processing is less automatic rather than a task-irrelevant feature of that same stimulus for which processing is more automatic. These differing features of the same stimulus produce cognitive interference We hypothesized that we would detect evidence in clinically identified stutterers for functional compromise of self-regulatory control systems based within frontostriatal circuits of the brain. Similar to activation in various other childhood-onset behavioral disorders We recruited 46 participants with developmental stuttering through advertisements posted online and at local clinics, hospitals, and stuttering support groups. All participants in the stuttering group had been diagnosed by a licensed speech-language pathologist before enrollment in the study. Fifty-two fluent controls to match for age and sex were recruited randomly from a telemarketing list of 10,000 names in the local community, excluding those with lifetime Axis I disorders, or any language disorders.We administered the Kiddie-Schedule for Affective Disorders and Schizophrenia, Present and Lifetime Version for participants under age 18 Additional exclusionary criteria for all participants included a history of premature birth, prior head trauma with loss of consciousness, past seizures, mental retardation, pervasive developmental disorders, or chronic medical illness. Study procedures were approved by the Institutional Review Board of the New York State Psychiatric Institute. Written informed consent was obtained from all adult participants and the parents of child participants. Children also provided written or verbal (at or younger than 6 years) assent for their participation.http://www.pstnet.com). The reaction time (RT) and accuracy of each button-press were recorded.We used the Simon Spatial Incompatibility Task to investigate alterations of self-regulatory functions in stuttering Images were collected on a 3-Telsa GE Signa EXCITE scanner . Head positioning was standardized using the canthomeatal line. A sagittal T1-weighted localizing scan was obtained to determine anterior-posterior commissure line, to which the axial functional images were aligned. Following the localizing scan, the functional images were acquired using a T2*-weighted single-shot echo-planar imaging sequence with repetition time 2200 msec, echo time 30 msec, image matrix 64×64, field of view 24×24 cm, flip angle = 90°, 34 slices, slice thickness 3.5 mm without gap, 140 volumes collected per run. Each run lasted 5 minutes 8 seconds, totaling 15 minutes 24 seconds for three runs.http://www.fil.ion.ucl.ac.uk/spm). Preprocessing procedures consisted of spatial realignment, slice timing correction, normalization to the standard template in Montreal Neurological Institute space, and smoothing with a 8 mm full-width-at-half-maximum Gaussian kernel. A high pass filter (1/128 Hz cutoff) was applied subsequently to remove low-frequency noise from the fMRI time series.Image processing was performed using Statistical Parametric Mapping software  and current trial. Each event of interest was modeled by its time series convolved with the canonical hemodynamic response function. Incorrect trials, omitted trials, and RT outliers were modeled as regressors of no interest and excluded from further analyses. Linear contrasts of event regressors were constructed to estimate neural activity corresponding to conflict resolution and context-dependent adaptation.Resolving conflict in this task requires engagement of frontostriatal circuits to enact a controlled response to direction while withholding the automatic response tendency to position of the arrow stimulus. We compared brain activation between “cI” trials  and “cC” trials  so as to identify neural systems involved in resolving conflict.A well-replicated observation in this task is that RTs shorten during trains of repeated stimuli, so that RTs to “iI” trials  are shorter than RTs to “cI” trials , even though the current stimulus in both trials is incongruent. Improved responses to current conflict following a previous encounter of a conflict is thought to stem from the self-regulatory function integrating the stimulus context from the recent past with the current stimulus . We used the iI vs cI contrast to assess the functional integrity of brain regions in mediating these cross-temporal contingencies, a crucial function in the execution of motor and linguistic sequences.Contrast maps were further entered into a group-level analysis, in which diagnosis (stutterers or controls) was considered as a random effect. Two-sample t-tests were applied to assess group differences in conflict resolution and context-dependent adaptation while covarying for age, sex, IQ scores, and RT interference (the difference in mean RTs for incongruent and congruent stimuli). Furthermore, we correlated parameter estimates of task-related activation with total score (standardized by z-transform) of ACES/OASES subscaleIII (Communication in Daily Situations), the most representative index of speech difficulties in various social settings. Results from voxel-based analyses were thresholded at a p-value<0.025 for individual voxels and a spatial extent of at least 30 voxels  (A−>B)), measures the predictability of current values of B from previous values of A. In fMRI time series analyses, the mathematical definition of GCI = 1-var(eAB)/var(eB) produces positive results when the variance of error in prediction of current B values using previous values of A and B (var(eAB)) is reduced compared to that using B previous values alone (var(eB)) in an autoregressive model We also performed an analysis of effective connectivity using Granger causality. The Granger Causality Index of A on B, denoted by , but no interactions with diagnosis , indicating that stuttering speakers and controls both required more time to respond to incongruent than to congruent stimuli. Furthermore, response latency on the current trial was modulated by congruency of the previous trial, as represented by a significant interaction of current trial and preceding trial congruency . Post hoc analyses indicated that this interaction derived from responses to iI stimuli that were faster than to cI stimuli , or from context-dependent adaptation. The absence of significant three-way interactions  indicated that both groups similarly speeded their RTs to incongruent-incongruent stimulus trials. Both groups maintained a high level of accuracy throughout the task (>70% across three runs); groups did not differ in accuracy during incongruent  or congruent trials .Behavioral performance was analyzed using a repeated measures analysis of variance with factors of congruency of preceding and current trial and diagnosis . We deteNeural activity indexing conflict resolution was examined using the cI-cC contrast. Greater activation was detected in stuttering speakers in the anterior cingulate cortex (ACC), left anterior prefrontal cortex, right medial frontal cortex (MFC), left supplementary motor area (SMA), right caudate nucleus, and left parietal cortex , Fig. 3ANeural activity associated with context-dependent adaptation was assessed by comparing BOLD signal between iI and cI trials, revealing decreased activation of the dorsolateral prefrontal cortex (DLPFC) in stuttering speakers compared with controls , Fig. 3BThe magnitude of activation in stuttering speakers during conflict resolution correlated inversely with score of ACES/OASES subscaleIII in the anterior cingulate cortex , left in(DLPFC->ACC)) and vice versa (GCI(ACC->DLPFC)). Bidirectional GCIs remained significant after Bonferroni adjustment for multiple comparisons. The strength of causal influences, in contrast, did not differ between the two groups in any causality metric  would be expected to generate compensatory activation of the ACC to support correct behavioral responding to conflict in the current trial.Brain regions that subserve self-regulatory processes in the Simon task, including the ACC, adjacent motor areas, and DLPFC, also support the production of normal speech Our study has several limitations. First, the range of ages of the participants was wide, potentially increasing the variance of brain activations. We covaried for age in all our analyses, however, and we additionally detected no age-dependent effects on our findings, suggesting that the findings obtain for all ages in the sample, consistent with findings from prior behavioral studies that impaired capacities are present throughout development Conventional theories tend to conceptualize stuttering as a discrete symptom confined mostly to the domain of language. The limitations of this singular focus on studying disturbances in language systems are clear when considering the robust abnormalities in neural responses of persons who stutter in our non-language task, especially the strong correlations of activation with stuttering severity. Our findings suggest that the pathogenesis of stuttering involves more than speech dysfluency. In particular, altered capacities for self-regulation may be important in determining the severity of symptoms. In addition, our assessment of the neural support for context-dependent adaptation was intended to probe processes that may also support the dynamic regulation of speech fluency across time. Reduced DLPFC activation during cross-temporal adaptation may therefore also contribute to the difficulty that stuttering speakers have in binding speech segments into fluent sequences across time. Inefficient DLPFC functioning presumably produces a compensatory heightened activation of the ACC and related frontal regions to support better behavioral regulation in persons who stutter.Figure S1(A) Stronger activation in frontostriatal regions during conflict resolution of stuttering speakers who were not taking psychoactive medications relative to fluent speakers . (B) Blunted activation in the dorsolateral prefrontal cortex during context-dependent adaptation of stuttering speakers who were not taking psychoactive medications relative to fluent speakers .(TIF)Click here for additional data file.Figure S2(A) After controlling for ethnicity, activation remains stronger in frontostriatal regions of stuttering speakers during conflict resolution relative to fluent speakers . (B) Activation remains blunted in the dorsolateral prefrontal cortex of stuttering speakers during context-dependent adaptation after controlling for ethnicity .(TIF)Click here for additional data file.