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+{"text": "Environmental Science & Technology, raising concerns about the fate of this and other pharmaceuticals that end up in water supplies. Acetaminophen is one of the most widely used over-the-counter painkillers in the world\u2014in the United States alone, some 37,000 metric tons are produced each year, says coauthor Mary Bedner, a research chemist at the National Institute of Standards and Technology. \u201cSome of this is reaching the environment,\u201d she says, \u201cbut no one really knows what happens to it or what effect it might ultimately have on ecosystems or people.\u201dAcetaminophen is turned into at least two toxic compounds by chlorination treatment, researchers report in the 15 January 2006 issue of Environmental Science & Technology a USGS team reported detecting it in nearly a quarter of the water bodies it sampled. \u201cIt gets there through wastewater  and in some cases through poor disposal practices,\u201d says Nick Voulvoulis, a senior lecturer in natural sciences at Imperial College London. Only 22% of Britons and just 1.4% of Americans return unwanted medicines to pharmacies, says Voulvoulis. More than 35% of U.S. nonreturners flush unused drugs down the toilet, while most British drugs end up in landfills, from which they can leach into water bodies.Reports of acetaminophen in European rivers have appeared since the 1990s, and in the 15 March 2002 issue of N-acetyl-p-benzoquinone imine . Together, these compounds represented the fate of nearly 27% of the original drug concentration.Concerned that acetaminophen\u2019s structure renders it likely to react with chlorine, Bedner and colleague William MacCrehan used reversed-phase liquid chromatography to follow its interaction with the chlorinating agent hypochlorite. Under simulated treatment conditions in samples of distilled water and wastewater, 11 new compounds were formed from acetaminophen within an hour, the time the reactants would likely be in contact at any plant. Among them were 1,4-benzoquinone (a mutagen) and \u201cFortunately, these are unstable compounds, especially in the presence of sulfite, which is sometimes used to dechlorinate treated water, so they are unlikely to persist long in the environment,\u201d Bedner says. \u201cHowever, they could accumulate where treated wastewater is returned to rivers, and the effects of resupply over long periods are unknown.\u201d The results also raise the question of what other drug-derived toxicants are out there, she says.\u201cThis work shows we need to know much more about the fate of the drugs that contaminate our water supplies,\u201d says Dami\u00e0 Barcel\u00f3, a professor of environmental chemistry at Barcelona\u2019s Centre for Research and Development. \u201cWe also have to look for what they turn into. Searching only for the original compounds themselves will not reveal all the dangers these contaminants may pose.\u201d"}
+{"text": "As drought and growing populations cause water supplies to dwindle in areas around the world, reclaimed wastewater offers a possible solution. Indeed, some communities in California already use reclaimed wastewater to irrigate crops, water golf courses, and augment freshwater aquifers to block saltwater intrusion. Critics are concerned about the potential health hazards of the pharmaceuticals, hormones, and other contaminants that even treated wastewater has been shown to contain. But recent research reveals that the process of reverse osmosis may remove some of these contaminants.Journal of Chromatography A, Joel Pedersen, an environmental chemist at the University of Wisconsin\u2013Madison, and his colleagues used gas chromatography\u2013mass spectrometry to look for 19 compounds in effluent samples collected from reclaimed wastewater plants in California. They found detectable concentrations for 13, including food preservatives, painkillers, oral contraceptive hormones, and prescription medications. However, at the 228th American Chemical Society meeting held in Philadelphia in August 2004, Pedersen further reported that gas chromatography confirmed all 13 compounds to have been eliminated at two pilot plants testing reverse osmosis for contaminant removal.As described in the 12 March 2004 issue of the Nonetheless, Pedersen cautions that it\u2019s too early to recommend that all reclaimed wastewater facilities employ reverse osmosis. \u201cThis is a case where the analytical chemistry is ahead of the toxicology,\u201d he says.\u201cLittle is known about the toxicity of trace concentrations of these compounds,\u201d agrees Shane Snyder, project manager of research and development at the Southern Nevada Water Authority (SNWA) in Las Vegas. Snyder has monitored the flow of treated wastewater effluent into nearby Lake Mead since 1997. He says fish in Las Vegas Bay are the healthiest in all of Lake Mead because they thrive on nutrients in the effluent. Snyder and colleagues at the U.S. Fish and Wildlife Service are writing a paper on this topic.Often used to remove salts, reverse osmosis requires electricity to pump water through semipermeable membranes. \u201cA lot of work is involved to perform reverse osmosis correctly,\u201d says Pedersen. \u201cLarge-scale reverse osmosis may not be economically feasible in some areas.\u201d Salts, contaminants, and biofilms can clog the pores of membranes, raising maintenance costs.Still other costs can make the process prohibitively expensive for inland cities in particular. Reverse osmosis generates brine. While coastal California wastewater facilities dump brine into the ocean, inland facilities must heat the brine to evaporate the water, then dispose of the dry salt in a landfill. \u201cThe cost of brine disposal is often more expensive than the cost of reverse osmosis itself,\u201d says Snyder. About 30% of treated water ends up as brine during reverse osmosis. That water loss \u201cis not acceptable when you live in the desert,\u201d Snyder says. By comparison, standard treatment results in less than 1% water loss, according Snyder.N-nitrosodimethylamine, a disinfectant by-product of wastewater treatment, breaches reverse osmosis membranes. However, dangerous compounds may be removed with less expensive treatments than reverse osmosis. For example, advanced oxidation methods can destroy N-nitrosodimethylamine.Moreover, \u201creverse osmosis membranes are not infallible,\u201d says Snyder. For instance, the carcinogen But it\u2019s too soon to count reverse osmosis out just yet. Newer models require less pressure to pump water through. \u201cMore efficient membranes will lower the energy costs of reverse osmosis,\u201d Snyder predicts, \u201cand likely make the process more cost-effective.\u201d"}
+{"text": "HIV avoids elimination by cytotoxic T-lymphocytes (CTLs) through the evolution of escape mutations. Although there is mounting evidence that these escape pathways are broadly consistent among individuals with similar human leukocyte antigen (HLA) class I alleles, previous population-based studies have been limited by the inability to simultaneously account for HIV codon covariation, linkage disequilibrium among HLA alleles, and the confounding effects of HIV phylogeny when attempting to identify HLA-associated viral evolution. We have developed a statistical model of evolution, called a phylogenetic dependency network, that accounts for these three sources of confounding and identifies the primary sources of selection pressure acting on each HIV codon. Using synthetic data, we demonstrate the utility of this approach for identifying sites of HLA-mediated selection pressure and codon evolution as well as the deleterious effects of failing to account for all three sources of confounding. We then apply our approach to a large, clinically-derived dataset of Gag p17 and p24 sequences from a multicenter cohort of 1144 HIV-infected individuals from British Columbia, Canada (predominantly HIV-1 clade B) and Durban, South Africa (predominantly HIV-1 clade C). The resulting phylogenetic dependency network is dense, containing 149 associations between HLA alleles and HIV codons and 1386 associations among HIV codons. These associations include the complete reconstruction of several recently defined escape and compensatory mutation pathways and agree with emerging data on patterns of epitope targeting. The phylogenetic dependency network adds to the growing body of literature suggesting that sites of escape, order of escape, and compensatory mutations are largely consistent even across different clades, although we also identify several differences between clades. As recent case studies have demonstrated, understanding both the complexity and the consistency of immune escape has important implications for CTL-based vaccine design. Phylogenetic dependency networks represent a major step toward systematically expanding our understanding of CTL escape to diverse populations and whole viral genes. One of the enduring challenges facing HIV vaccine design is the remarkable rate of viral mutation and adaptation that limits the ability of the immune system to mount a lasting effective response. This rapid rate of mutation leads to extensive within- and between-host viral diversity that makes creation of a broadly reactive vaccine difficult. A first step in overcoming this challenge is to identify consistent patterns in viral adaptation. Recently, several studies have analyzed large groups of HIV-infected individuals and looked for correlations between HIV polymorphisms and the HLA class I alleles that restrict the cellular immune response. Here, we point out a limitation of previous approaches: correlations among HLA alleles and HIV codons lead to statistical confounding if not taken into consideration. In response, we develop two statistical models of evolution that explicitly represent stochastic selection pressure from multiple sources. After validating these models on synthetic data, we analyze the patterns of immune escape in a multicenter cohort of over 1000 individuals. Our results identify a dense network of interactions between HLA alleles and HIV codons, as well as among HIV codons, reflecting both a complexity and a promising consistency in the way that HIV adapts to the human immune response. These chains of interactions are almost certainly the norm in codon coevolution, as observed covariation is often driven by the constraints of three-dimensional physical interaction The comparative method for discrete traits has received much attention in the study of protein evolution. Here, the comparative method is used to identify coevolving codons within a protein or between proteins in the hopes of identifying structural or functional codon interactions and their resulting constraints on protein evolution  will further confound the detection of HLA-mediated escape mutations. Because the HLA class I loci are located in close proximity on chromosome 6, the alleles tend to be in tight LD, meaning inheritance of (e.g.) a specific HLA-B allele is strongly correlated with inheritance of a specific HLA-C allele phylogenetic dependency network (PDN), that accounts for phylogenetic relationships among the data by conditioning those relationships on a model of evolution. In addition, we describe the Noisy Add distribution, a parsimonious distribution  that is suitable for modeling HIV escape and codon covariation. This Noisy Add distribution is a generalization of the distribution described by Carlson et al. In summary, when detecting HIV escape mutations, there are at least two sources of confounding in addition to the phylogenetic structure of the HIV sequences: (1) covariation among HIV codons, and (2) HLA linkage disequilibrium. There is therefore a potential need for a statistical model that can accurately account for both sources of confounding in a phylogenetic context. In what follows, we describe a variation of the dependency network clades) and determine the power available for different cohort sizes. Finally, we undertake an HIV/HLA cross sectional analysis with the largest cohort to date, combining Gag p17/p24 sequences and individual HLA data from the HOMER cohort from British Columbia, Canada (predominantly HIV-1 clade B) We demonstrate the utility of phylogenetic dependency networks for modeling HLA-mediated escape in HIV. We first examine synthetic data generated from the Brumme et al. study target attributes, denoted Y, correspond to the presence or absence of amino acids at all codons in an HIV protein. For a given Y in Y, the predictor attributes, denoted X, correspond to the presence or absence of amino acids at all codons other than that for Y and the presence or absence of all HLA alleles. Note that all attributes are binary. We have found that this choice yields more statistical power in practice.Here, we describe phylogenetic dependency networks generally and use the domain of HLA-mediated HIV codon evolution to illustrate the concepts. A dependency network represents the probabilistic dependencies among a set of predictor and target attributes. In our domain, structure of a dependency network, is a directed graph linking nodes, where each node corresponds to one of the attributes in the domain.  An arc from X to Y in the graph is a statement that the probability distribution for Y depends on X. Thus, in our domain, a dependency network graphically depicts which HLA and codon attributes predict each codon. The second component is a collection of conditional or local probability distributions, one for every target attribute of interest. The local probability distribution for target attribute Y is P(Y|X\u02c6), where X\u02c6\u2286X are the parents of Y in the graph. Therefore, in our domain, a dependency network contains a probability distribution for each codon attribute conditioned on various HLA and codon attributes. When constructing a dependency network, each local probability distribution is learned independently. This approach is computationally efficient, although it can lead to a decrease in statistical efficiency  has two components. The first component, sometimes referred to as the ency see .X\u02c6, but also on where that individual's HIV sequence sits in the phylogeny (P\u03a8(Y|X\u02c6), one for each Y in Y, where P\u03a8 refers to a distribution corrected for phylogeny.A phylogenetic dependency network (PDN) for our HIV application is a dependency network in which each local probability distribution is corrected for the phylogenetic structure of the HIV sequences. That is, the probability that a codon in an individual is a given amino acid depends on not only the attributes hylogeny . SpecifiX\u02c6, the set of parents for Y. Specifically, we use significance tests\u2014False Discovery Rate (FDR) thresholds based on likelihood-ratio tests (LRTs)\u2014to determine X\u02c6. To avoid the inappropriate use of an LRT, we exclude attributes as possible predictors when the corresponding predictor-target pair has a 2\u00d72 contingency table that includes at least one bin where both the observed and expected value is at most three. This parameter was chosen based on performance with independent data (not shown).In this paper, we use a model-selection approach to identify univariate model, are described in the section \u201cModel Details\u201d and evaluated in A simple approach for identifying a set of attributes that predict a given codon  is to test for pairwise correlations between a target codon and each predictor attribute. The details of a statistical model that follows this approach, hereafter referred to as the X and target amino acid Y, we compare the likelihood of a null model that reflects the assertion that Y is under no selection pressure to an alternative model that reflects the assertion that Y is under selection pressure induced by a single predictor attribute X. The null model assumes the target codon Y can be described completely by a model of independent evolution along a phylogenetic tree .  Because each target attribute is binary, a natural null model is the two-state version of the continuous time Markov process, commonly used in phylogenetics To determine whether there is a significant pairwise correlation between predictor attribute tic tree . The leaX , and do not explicitly name X for the unseen individuals represented in the interior of the phylogenetic tree. Because X may not share the same evolutionary history as Y, we assume X influences Y only at the leaves of the tree. In particular, we assume that, among the variables corresponding to attribute X, only iX influences iY for each i. This assumption was evaluated more fully by Carlson et al. X and Y share the same evolutionary history. To model selection pressure at the leaves, we extend the null model by adding a hidden attribute H  that represents what Y would have been had there been no selection pressure. The probability distribution for iY then depends on iH and iX. When the values of iH and iY are different, we say that a transition conditioned on iX has taken place. The precise rules governing the transitions conditioned on iX are given by the univariate leaf distribution P\u03a8(Y|X)\u200a=\u200aP. We assume that this leaf distribution is not a function of i\u2014that is, this distribution is the same for each individual i\u200a=\u200a1,\u2026, N. Also note that the subscript \u03a8 is a reminder that iY depends not only on iX, but also on the phylogeny through variable iH.The alternative model adds a component of selection pressure derived from the predictor attribute X . We use Escape means an individual may transition to iY\u200a=\u200a0 only when iX\u200a=\u200a1. Reversion means an individual may transition to iY\u200a=\u200a1 only when iX\u200a=\u200a0. Attraction means an individual may transition to iY\u200a=\u200a1 only when iX\u200a=\u200a1. Repulsion means an individual may transition to iY\u200a=\u200a0 only when iX\u200a=\u200a0. Given a univariate leaf distribution, a single parameter s specifies the probability that the transition occurs given the appropriate state of iX. Note that attraction/repulsion correspond to a positive correlation between iX and iY, whereas escape/reversion correspond to a negative correlation.In the univariate case, we define four possible leaf distributions. susceptible threonine at position 242 of the HIV Gag protein H\u200a=\u200a1, Y\u200a=\u200a0) with a non-zero probability only when the individual has the B*57 allele (X\u200a=\u200a1), which corresponds to the escape distribution just described. In addition, escape from threonine bears a fitness cost that leads to reversion in B*57-negative individuals H\u200a=\u200a0, Y\u200a=\u200a1) with non-zero probability only when the individual lacks B*57 (X\u200a=\u200a0), corresponding to the reversion distribution. The codon for threonine usually escapes to the resistant amino acid asparagine H\u200a=\u200a0, Y\u200a=\u200a1) only when the individual has the B*57 allele (X\u200a=\u200a1), which corresponds to the attraction distribution. Finally, the amino acid can transition from asparagine to not asparagine  only when the individual lacks the B*57 allele (X\u200a=\u200a0), which corresponds to the repulsion distribution. Although there is a natural pairing between escape/reversion and attraction/repulsion, in that the former indicates a negative correlation and the latter a positive correlation, the processes are each distinct and may provide information as to the underlying mechanism , which depends on multiple predictor attributes X\u02c6. As in the univariate case, this leaf distribution is the same for each individual i\u200a=\u200a1,\u2026, N.The univariate model works well when there are no correlations among predictor attributes or among target attributes X\u02c6 is iteratively augmented with the most significantly associated attribute at each iteration. For each added attribute, we record only the most significant leaf distribution . The significance of a predictor X with respect to target attribute Y is computed using false discovery rates based on an LRT in which both the null and alternative models are conditioned on all significant predictors that were identified in previous iterations of forward selection. For practical purposes, we terminate forward selection when the most significant association has a p-value greater than or equal to some threshold to be described.The set of significant predictor attributes can be identified by a number of methods including forward, backward, and forward/backward selection. In this work, we concentrate on forward selection, wherein P\u03a8(Y|X\u02c6). In this paper, we consider two distributions: Decision Tree and Noisy Add.There any many possibilities for the form of the multivariate leaf distribution P\u03a8(Y|X\u02c6) is to list the probability distribution for Y given every possible instance of the attributes H and X\u02c6. Unfortunately, the length of this list grows exponentially with the number of predictor attributes. An alternative is to use a Decision Tree, which is a compact representation of such a list. The use of the Decision Tree as a multivariate leaf distribution was recently employed by Matthews et al. A straightforward way to represent the multivariate leaf distribution tip to refer to the bottom points on the Decision Tree. Each path in the tree from root to tip defines a particular instance of a subset of the attributes X\u02c6, which in turn defines a conditioning event for the distribution of the target attribute. For example, in X\u02c6\u200a=\u200a, with each branch labeled 0 or 1. The path that follows the value 0 for the attribute B57, the value 0 for the attribute C06, and the value 1 for the attribute M28 corresponds to the instance \u2014that is, the individual has M28 but not B57 or C06. At the tip of this path sits the corresponding conditional probability distribution P\u03a8. In general, each tip k in the Decision Tree is associated with the conditional distribution P\u03a8(iY|X\u02c6\u200a=\u200apathk), where pathk is the conditioning event corresponding to the kth path. The collection of these conditional distributions over all tips constitutes the multivariate leaf distribution.A graphical depiction of the Decision Tree leaf distribution is shown in splitting) a tip node k on some attribute not already in the path to the tip. When splitting tip node k on an attribute X, the node is replaced with two branches and two corresponding tip nodes. The left and right branches correspond to adding X\u200a=\u200a1 and X\u200a=\u200a0, respectively, to the conditioning event associated with the original tip node. The split is made if the resulting local distribution is a significantly better estimate than that prior to the split, as measured by an LRT. The LRT is computed using the univariate model applied to those individuals whose attribute values match those described by pathk. To make the process more efficient in our HIV application, we consider splitting the tip node only under the path X\u200a=\u200a0 for all X in X\u02c6. That is, we repeatedly apply the univariate model to all individuals for whom X\u200a=\u200a0 for all the previously identified significant predictor attributes. We iterate this process until no significant predictors are found, using a threshold of p<0.05.A Decision Tree leaf distribution can be constructed in many ways. As mentioned, we use forward greedy search. First, we initialize the tree to a single root node, which is simply the univariate leaf distribution for the most significant attribute. We then grow the tree iteratively. At each iteration, we consider extending  selection pressure leads to iY\u200a=\u200a1 (attraction or reversion); or (3) selection pressure leads to iY\u200a=\u200a0 (escape or repulsion). We can represent these three possible events by a hidden attribute I that takes on the values 0, 1, and \u22121, respectively. Given a set of M predictor attributes, we can associate a hidden variable jth attribute in the ith individual. Then, assuming that selection pressure across the predictor attributes contributes independently and equally to the outcome of iY, we can determine the outcome of iY by summing the values of the i is 0, then it is as if no selection occurred. If \u03a3i<0, then negative selection (escape/repulsion) has occurred, and the target variable iY will be zero. If \u03a3i>0, then positive selection (attraction/reversion) has occurred, and the target variable will be one. Of course, we don't know the actual values of jI for each predictor variable, so we must sum over the possibilities, resulting in a probability distribution over \u03a3i. The strength or frequency of selection pressure contributed by each predictor attribute j is captured by the parameter js. Like the corresponding parameter s in the univariate model, js is the probability that the predictor attribute exerts selection pressure : (1) the selection pressure is absent, either because the state of the predictor attribute excludes selection pressure or, with probability 1\u2212jX\u2208X\u02c6 as a predictor of target Y is quantified using an LRT against the null model consisting of X\u02c6\u2212jX. The most significant predictor attribute is added to the Noisy Add model on each iteration, stopping when the most significant predictor fails to achieve p<0.005. The contribution of a given predictor attribute q-values p-value. The FDR is defined to be the expected proportion of false positives among results called significant at a given threshold t. The q-value of t is the minimum FDR observed for all t\u2032\u2265tS(t) is the number of associations called significant at t and F(t) is the number of true nulls  at t. To estimate the numerator, we order the p-values of the association tests in increasing order p1,\u2026, mp and use the approximation E[S(ip)]\u2248S(ip)\u200a=\u200ai. To compute E[F(t)], Storey and Tibshirani point out that uniformity of p-values allows the approximationa priori that the vast majority of the many hypotheses tested will be null , and so conservatively set We identify significance using p-value. In order to achieve a valid p-value, all model assumptions must be reasonably met. In particular, all sources of confounding must be accounted for. In principle, our multivariate models can account for these sources, provided the input phylogeny is reasonable and all other sources of confounding are provided as predictor attributes in X\u02c6. To confirm this argument for the Noisy Add leaf distribution, we constructed QQ plots using the mixed clade dataset and a synthetic dataset as described in the following section  shifts the distribution as expected. Likewise, the distribution on real data follows the pattern observed on synthetic data that includes planted associations.The asymptotic conservative guarantee of (1) requires a conservative estimate of (2), which requires a valid   section . On nullF(t)] can be estimated from null data by (e.g.) permuting the predictor attributes Alternatively, E[q-values using the method of Storey and Tibshirani for Noisy Add for the model that corrects for phylogeny, LD, and covariation, and the permutation test for all other models, as these models fail to account for key sources of confounding, rendering their p-values stochastically liberal. For these other models, we found that across all datasets tested, the permutation test was more conservative than using equation (2), which consistently yielded overly liberal q-values (data not shown).In what follows, we compute HLA-codon associations at that p-value are expected to be null, but that 20% of all associations (including codon-codon associations) are expected to be null. Thus, we find it useful to compute FDR separately for each class of association. In what follows, we compute q-values for HLA-codon and codon-codon associations separately. The final combined association lists are then ordered by the q-values, with ties broken by p-values.Finally, FDR represents the expected proportion of tests called significant that are null. When different classes of predictor attributes are considered, this may lead to confusion. For example, an FDR of 20% for an association between an HLA allele and codon does not imply that 20% of HOMER cohort from British Columbia, Canada, consisting of 567 predominantly clade B gag sequences Durban cohort, consisting of 522 predominantly clade C p17/p24 gag sequences from Durban, South Africa These methods were applied to population-based HIV sequences from chronically infected, antiretroviral na\u00efve and HLA-typed individuals from two cohorts: the q-values for each predictor-target pair. We then planted predictor-target pairs for each significant (q\u22640.2) predictor-target pair identified from the real data. Specifically, we generated a synthetic target amino acid for each consensus amino acid in the sequence, such that (1) if the amino acid had no significant (q\u22640.2) associations, then the amino acid was generated according to the parameters of the independent evolution model (the null model from the univariate case), and (2) if the amino acid had M>0 associations, then the amino acid was generated according to the given multivariate model with the predictor parameters s1,\u2026, Ms, taken from the real data. When an observation was missing in the real data, the corresponding observation in the synthetic data was also made to be missing. We treated amino acid insertions/deletions and mixtures as missing data.Synthetic datasets were designed to mimic the real datasets as closely as possible. We first fit a specified model to the real data to identify parameters and q\u22640.2 compared to 0.65% of all predictor-target pairs in the real data for the combined HOMER-Durban cohort.Our goal was to generate data that is as realistic as possible, both in the values of the parameters used and the number of predictors deemed correlated with the target. Because our recall rate is less than 100% (see section on synthetic results), planting only those associations that are found in the real data would result in a smaller proportion of synthetic predictor-target pairs called significant than real predictor-target pairs called significant. We therefore planted two associations for every observed significant association in the real data and reduced the number of independently evolving codons accordingly. For the Noisy Add model, this procedure planted 72 HLA-codon and 612 codon-codon associations in the HOMER cohort and 114 HLA-codon and 952 codon-codon associations in the combined HOMER-Durban cohort. In hindsight, doubling the number of planted associations was an overcompensation, as experiments on this synthetic data yielded a 75% recall rate. Nonetheless, the doubling produced a reasonable result, as Noisy Add declared 0.56% of all synthetic predictor-target pairs significant at HLA-codon associations refer to the most significant associations between an HLA allele and any observed amino acid at the codon under any of the four leaf distributions. Likewise, codon-codon associations refer to the most significant association between the codons over all the associations computed for the complete repertoire of observed amino acids and possible leaf distributions at those codons. This approach was taken exclusively in the synthetic studies, though the results were similar when we looked at exact associations .As mentioned, we binarized all data. For example, if three amino acids were observed at a given sequence position, we created three binary attributes corresponding to the presence and absence of each amino acid. When reporting results, however, we assumed that the most relevant information was at the codon level. Thus, unless stated otherwise, recall (TP/(FN+TP)) and the y-axis is precision (TP/(TP+FP)), where TP is the number of true positives, FP is the number of false positives, and FN is the number of false negatives. To construct PR curves, we computed precision and recall for every observed q-value for each method. We used as a gold standard the synthetic data as described in the previous section. Accuracy of q-values, called calibration, is plotted as (1\u2212Precision) versus q-value. A perfectly calibrated result is a line with slope one. To compare two PR curves, we computed p-values using the absolute value of the difference between the areas under the two curves as the statistic. The null distribution assumes the two curves will on average provide the same ranking over the predictor target pairs and is constructed using a permutation test in which two pseudo-curves are generated by randomly swapping the ranks between the two methods for each predictor-target pair. That is, if methods r1 and r2, respectively, for a predictor target pair PT, then with probability 0.5, r2 and r1 for PT. Resulting ties in ranks were broken at random. Ranks were used rather than q-values so that the scores of two uncalibrated methods could be compared directly. 10,000 permutation tests were run to compute each p-value.We report power results as Precision-Recall (PR) curves, where the x-axis is susceptible form. If the association is attraction or repulsion, then T242 is the resistant form. The PDN will often find complementary associations. For example, T242 is the susceptible form with respect to B*57, and N242 is the resistant form. We will sometimes refer to such associations as T242N. For simplicity, we will usually report only the smaller q-value of the two associations. If only the susceptible association is significant (q\u22640.2), we will sometimes write T242X. Likewise, if only the resistant is significant, we will sometimes write X242N.When we refer to associations involving codons, we will sometimes find the following notation useful. T242 will refer to an amino acid (in this case threonine) at a specific codon (242). If the association is escape or reversion, then T242 is the http://www.hiv.lanl.gov/content/immunology/tables/optimal_ctl_summary.html, accessed on December 21, 2007. To allow the inclusion of processing mutations Optimally defined epitopes In this section, we provide details regarding the univariate and Noisy Add models, in addition to a brief discussion on computational requirements for the models.Y that denotes the presence (Y\u200a=\u200a1) or absence (Y\u200a=\u200a0) of a particular amino acid at a particular codon. We use variable iY, i\u200a=\u200a1,\u2026, N to denote the attribute Y for the ith individual in the study. (We use corresponding notation for predictor attributes and variables.) It is quite common to assume that the variables Y1,\u2026, NY are independent and identically distributed (IID). In our application, however, the variables are related through a phylogenetic tree. We can model these relationships using a probabilistic phylogenetic model as shown in Y1,\u2026, NY correspond to the variables with the same name.  Unlabeled nodes in the interior of the tree correspond to events of divergence. We use \u03a8 to denote the structure (branchings and branch lengths) of the tree.First, let us consider the null model. Consider target attribute B in this phylogenetic tree is a conditional probability distribution P(B|A), where A is the parent node of B. As in the probabilistic model of Felsenstein \u03b8\u200a=\u200a, where \u03c0 is the stationary distribution of Y\u200a=\u200a1 and \u03bb is the rate of mutation. The conditional probability table of the CTMP from parent node A to child node B along a branch of length d is given byb\u03c0\u200a=\u200a\u03c0 when b\u200a=\u200a1, and b\u03c0\u200a=\u200a1\u2212\u03c0 when b\u200a=\u200a0. This evolution model is reversible, making the choice of root in the tree arbitrary Associated with each variable (or node) Y1,\u2026, NY, there are several criteria that can be used to identify good values for the parameters \u03c0 and \u03bb and the structure \u03a8 of this model . For this and all models discussed in this paper, we choose parameters and structure using the maximum likelihood criterion, as is done in (e.g.) \u03b8. To learn the structure \u03a8, we apply PhyML to the (gag p17\u2013p24) nucleotide sequences using the general time reversible GTR model with all other parameters estimated from the data Given a set of observations for  P\u03a8(Y|\u03b8), as it represents a phylogenetically corrected distribution for Y. Note that this model includes the situation where the observations of Y1,\u2026, NY are IID as a special case We denote this null model X. To construct this model, shown in iY to iH, which represents what iY would have been had there been no influence from iX. Then, we assume that, for each individual i, the probability distribution for iY depends on iX and iH. Further, we assume that these conditional distributions P are the same for each individual i, and collectively denote them by \u03a8P(Y|X). In general, this univariate leaf distribution can have four parameters corresponding to the four states of the conditional variables iH and iX. In our experience, however, use of such a distribution leads to loss of power. Consequently, we consider four separate distributions \u200a=\u200as>0; P\u200a=\u200a0; P\u200a=\u200a1. That is, iH and iY can be in different states only when iH\u200a=\u200a1 and iX\u200a=\u200a1.ReversionP\u200a=\u200as>0; P\u200a=\u200a0; P\u200a=\u200a1. That is, iH and iY can be in different states only when iH\u200a=\u200a0 and iX\u200a=\u200a0.AttractionP\u200a=\u200as>0; P\u200a=\u200a0; P\u200a=\u200a1. That is, iH and iY can be in different states only when iH\u200a=\u200a0 and iX\u200a=\u200a1.RepulsionP\u200a=\u200as>0; P\u200a=\u200a0; P\u200a=\u200a1. That is, iH and iY can be in different states only when iH\u200a=\u200a1 and iX\u200a=\u200a0.\u03b8\u200a=\u200a and the structure \u03a8 can be optimized simultaneously. In practice, however, we find that using the structure \u03a8 learned in the absence of information about X works well, and is computationally more efficient. In addition, it may seem counter-intuitive that the HLA alleles of the individuals corresponding to the interior nodes of the phylogeny are not being taken into account. A path from one node to the next in the phylogeny, however, presumably reflects a series of infections over many individuals, some who will have the allele and some who will not. Thus, there will be some net evolution, which we account for by optimizing the parameters \u03c0 and \u03bb for each codon individually. Finally, we note that this model can be thought of as a (discrete) mixed-effects model, wherein the predictor variables iX correspond to the fixed effects and the hidden variables iH correspond to the random effects This model is reversible in the sense that the choice of root node among non-leaf nodes does not affect the likelihood of the data. We also note that, in principle, all parameters generative or directed acyclic graphical (DAG) model. In general, a generative model consists of a structure, a directed acyclic graph, in which nodes correspond to variables and missing arcs specify conditional independencies among the variables, and a set of conditional probability distributions, one distribution for each node. The conditional probability distribution for a given node is the distribution of the node given its parents. The conditional independences specified by the structure of the graph allow the joint distribution of the data to be written as the product over the nodes of their conditional distributions. The independences represented by the model facilitate computationally efficient inference, parameter estimation, and structure learning Both the null and alternative models are instances of what is known as a In the following section, we consider the multiple-predictor case and again use graphical models to represent phylogenetically corrected distributions. As we shall see, the computational efficiencies afforded by graphical models will play an even more important role.i. In the text that follows, we describe this and the generalized process for an arbitrary individual i. In the corresponding figures, we drop the subscript i to simplify the notation.) If iX\u200a=\u200a1 , a coin weighted with probability s for heads is flipped. If the coin lands heads, then the intermediate variable iI gets the value 1 . Otherwise, iI gets the value 0, corresponding to no selection pressure. The value of iI is then copied to the value of another variable \u03a3i.  Finally, the target variable iY is assigned a value based on the deterministic function shown in To understand the Noisy Add leaf distribution, let us recast the univariate distribution as the generative process shown in js to denote the weight for predictor variable s to represent the collection of parameters . The variable \u03a3i is now a sum of the intermediate variables iY is a deterministic function of \u03a3i and iH as given in The generalization of this process to multiple predictor variables i\u200a=\u200a1,\u2026, N, we obtain the conditional distribution \u03b8\u200a=\u200a are the parameters of the model. Maximum likelihood values for these parameters can be inferred efficiently. The summation iY for any instance of the variables X\u02c6i and iH in time that is quadratic in M. Furthermore, given any instance of the predictor variables X\u02c6i, iH, and iY, the probability distributions for M. Consequently, we can use the EM algorithm to estimate the parameters s efficiently. To estimate the full set of Noisy Add parameters \u03b8, we embed this estimation procedure within an outer loop as follows.Applying this generative process to individuals Compute ls e.g., .Iterate to convergence in likelihood:For\u2003j\u200a=\u200a1,\u2026, MComputes to maximize the likelihoodGiven these probabilities, choose \u03c0 and \u03bb to maximize the likelihood using a standard M-step for CTMP , as we run one test for each X\u2212Y pair and likelihood calculations on a tree are O(N log2N). The number of EM iterations required to converge is roughly independent of the size of the data. In the case of the multivariate models, there is an additional penalty due to the forward selection procedure that requires a complete pass through all predictors to identify the most significant predictor for each iteration. Likelihood maximization is slower for Noisy Add, due to the increased number of iterations required for EM to converge for large numbers of significant predictor attributes, and inference being quadratic in the number of significant predictors.In this section, we briefly outline the theoretical and practical computational requirements of the models. The running times are primarily a function of the number of target attributes |In practice, all of the models were run on a 320 node Windows HPC cluster and completed in 1\u201324 hours, with the shortest times corresponding to the univariate model run on the smaller HOMER cohort and the longest times corresponding to the Noisy Add model run on the combined HOMER-Durban cohort.and covariation among the amino acids will lead to a significant drop in power and inflation in estimates of significance.In this section, we use synthetic data to demonstrate the power and calibration of the proposed models and to demonstrate that failure to account for the phylogenetic tree, linkage disequilibrium (LD) among HLA alleles, p<0.0001)  the Decision Tree model fit to real data (<0.0001) . Thus, tP), linkage disequilibrium among HLA alleles (L), and covariation among HIV codons (C). Previous approaches to finding HLA-associated polymorphisms have accounted for LD but not phylogeny As we have discussed, there are at least three major sources of statistical confounding for HIV-HLA association tests: phylogeny . This model is similar to the one used by Moore et al. HLA LD only (\u03bb set to infinity). This model is similar to a second model in Moore et al., who suggested adding other codons as covariates to their logistic regression model HLA LD and covariation only  increase in power over accounting only for linkage disequilibrium and codon covariation , despite the relative homogeneity of the data. Furthermore, accounting only for phylogeny , indicating that there is shared information at both the HLA-codon and codon-codon levels and that power can be increased by merging datasets from disparate cohorts as long as all three sources of confounding are accounted for.As in the previous experiments, we fit the full Noisy Add model to this combined dataset and then generated synthetic data from the resulting model. We then attempted to learn back the associations using the full dataset and then, for comparison, by stratifying the data and running the Noisy Add model separately for each clade. As indicated by the PR and calibration curves , the NoiWe then applied the remaining five models to this mixed-clade dataset, observing the founder effects demonstrated by Bhattacharya et al. ciations . Inspectp\u200a=\u200a0.34 for HLA-codon associations and p\u200a=\u200a0.09 for all associations). Nevertheless, it is interesting to note that, in the HLA-codon case, after the founder effects are incorporated into the model, the non-stratified version of Given the prominent structure of the multiclade data, a natural solution is to stratify the data by clade, running the phylogeny-na\u00efve model (p<0.0001 in both cases) and did not suffer from founder effects. Finally, it is striking that for codon coevolution, it is better to account only for protein-wide codon covariation than to use a sophisticated phylogenetic-correction algorithm that is limited to pairwise associations, especially if the data can be stratified by gross tree topology , although accounting for both phylogeny and codon covariation is clearly a more powerful approach.In contrast, accounting for phylogeny with the full model  in the absence of CTL-mediated selection pressure. Consistent with these interpretations, Matthews et al. Previous studies have differed widely in their results, in part because they employed different methods, and in part because they used different sample sizes 473 , which aN. Here, power is defined to be the ability to detect associations at 80% precision regardless of the model's reported q-value. In the range tested, the Noisy Add model has an approximately linear increase in the power to detect associations (HLA-codon and codon-codon associations combined) as the sample size increases. In contrast, increasing sample size for the other models has a limited effect. In particular, failure to account for codon covariation leads to a flat power curve for all models that do not account for codon covariation. The model that accounts for codon covariation and HLA allele LD but not phylogeny  N\u200a=\u200a572, those associations will presumably be stronger and hence the measured power should be greater. To demonstrate this supposition, we ran Noisy Add on a random subsample of the mixed clade cohort, and then generated new synthetic datasets based on these associations. The dashed blue lines  in N\u200a=\u200a572 compared to planted associations originally found at N\u200a=\u200a1144. Thus, if associations of the strength detected by Brumme et al. N\u2248550) are desired, then N\u200a=\u200a1150 will provide sufficient power to recover 90% of the associations. If, however, more subtle effects are sought, then larger cohorts are necessary. It is our opinion that only post hoc analyses of larger cohorts will determine the minimum relevant effect size. Furthermore, it should be noted that, whereas power can be increased by combining data from multiple cohorts, if only associations for a single cohort are of interest, then greater power will clearly be achieved from a single-clade cohort of size N than from a multi-clade cohort of size N. Nevertheless, the power increase from combining cohorts of different clades will prove useful in situations where single-clade cohorts cannot be expanded in practice.Statistical power is a function of sample size and effect size. In the results just described, the planted associations came from those detected at 20% q\u22640.2, representing 100 distinct HLA-codon pairs and 716 distinct codon-codon pairs. To explore these dense networks we developed a dependency-network viewer, PhyloDv, designed for intra-protein networks. PhyloDv draws the protein as a circle, with the N-terminus at the \u201c3 o'clock\u201d position and the protein extending counter-clockwise around the circle. Codon-codon associations are drawn as headless arcs (or edges) within the circle, whereas HLA-codon associations are drawn as external edges. Edge color reflects the strength of the association. q-value. The program, which includes interactive detailed views of each codon to explore the specific associations, is available upon request. The individual associations are available as Having established the Noisy Add model's ability to reliably recover associations in mixed clade datasets, we now turn to an analysis of the actual associations that were recovered on the mixed clade B/C data. The Noisy Add model found 149 HLA-codon associations and 1386 codon-codon associations at p<10\u221231), and a disproportionate number of p17 codons (24%) are associated with HLA alleles than are p24 codons . Not surprisingly, a disproportionate number of covarying codons were within 10 positions of each other . The dense PDN reveals broad patterns of codon covariation and HLA-mediated substitutions. For example, pairs of codons are more likely to covary within a subprotein (N\u200a=\u200a528) than between subproteins  predict substitutions at other codons. Furthermore, on average, each HLA-associated codon predicts substitutions at 7.0 other codons on average (range 0\u201325). These two observations highlight the complexity of HLA-mediated escape. Also of note is that, of the 181 codons that covary with at least one other codon, 60 (33%) have an association with at least one HLA allele, suggesting that HLA-mediated selection pressure will confound codon coevolution when unaccounted for.Among the 68 HLA-codon associations with escape/reversion leaf distributions, 33 represent escape/reversion from a residue that is consensus in both clades B and C, 7 represent escape/reversion from clade B consensus only, and 11 represent escape from consensus C only, where we define clade B consensus based on the HOMER cohort and clade C consensus based on the Durban cohort. Interestingly, of the 11 clade C susceptible associations, 5 had predicted resistant forms matching clade B consensus , and 2 of the 3 clade B susceptible associations had a predicted resistant form matching clade C consensus . In all, there were 21 HLA-codon associations for which the predicted resistant form was clade B or C consensus . These ain vitro fitness As noted in the synthetic results, Noisy Add can distinguish with 85% accuracy the difference between reversion and escape leaf distributions (though it cannot discern whether both are present). On the current dataset, 5 HLA-codon associations were identified as primarily reversion: B*14 K302, B*15 K26, B*57 T242, B*58 G55, and B*81 L184. These associations most likely have a corresponding escape association that we are not detecting, but these associations are nonetheless notable in that there is a strong statistical pull towards the \u201csusceptible\u201d form in the absence of the associated allele, which may suggest that fitness costs are associated with the resistant form In some cases, CTL escape requires a set of secondary substitutions that may stabilize protein structure, compensate for lost protein function, or facilitate further escape q\u200a=\u200a0.01), with the result being evenly distributed between K (q\u200a=\u200a0.08) and G (q\u200a=\u200a0.11). Kelleher et al. later reported that the R264K but not R264G mutation was typically preceded by L268M. Accordingly, in the PDN, we find that L268M predicts R264K (q<0.001) but not R264G, and that L268M is itself predicted by B*27 (q\u200a=\u200a0.001). Kelleher et al. also reported that the R264G was associated with E260D, and Schneidewind et al. The first HIV escape pathway that was described in detail is escape from the B*27-restricted KK10 epitope in p24 Gag 263\u2013272 q<0.001). In addition, we note that R264K is strongly associated with substitution I267V (q<0.001) within the KK10 epitope and with L215M (q\u200a=\u200a0.01). Residue 264 is within 3 angstroms of both codons 215 and 173 in folded p24 Recently, Schneidewind et al. demonstrated that S173A compensates for the loss of replicative capacity incurred by R264K q\u200a=\u200a0.0001), a substitution which also strongly predicts the D260E substitution of the R264G pathway (q<0.001). In addition, A146P is associated with maintaining wild type L268 (not the R264K pathway) whereas A163X predicts I267V (R264K pathway). Both A146P Finally, although it is not known if there are any determinants that predict whether KK10 escape occurs via the R264K or R264G pathway q<0.001). The T242N substitution predicts (q<0.001) further escape at G248A (position 9 of the TW10 epitope) and a single compensatory mutation at codon E210D (q\u200a=\u200a0.01) in the CypA binding loop, whereas the G248A substitution predicts compensatory substitutions V218A (q\u200a=\u200a0.02) and M228V (q\u200a=\u200a0.08), and G248T predicts H219Q (q\u200a=\u200a0.07) and M228I (q<0.001), of the CypA binding loop. Although 228 is  in direct contact with 248 (3 angstroms) The B*57 and B*5801 alleles have been strongly associated with effective HIV control Previous studies have reported alternative escape pathways in the A*02-restricted SLYNTVATL epitope (Gag positions 77\u201385) at epitope positions 3, 6, and 8 (though the Y79F escape at epitope position 3 is clade C consensus) \u03c6X174. No studies have systematically explored this phenomenon for immune escape in HIV Gag or other viruses, but the case studies of B*27 KK10 and B*57 TW10 make it clear that compensation happens in response to at least some CTL escape mutations. Poon and Chao further reported that compensatory mutations tended to cluster in linear and/or three-dimensional space, though many exceptions were noted. Indeed, the KK10 and TW10 case studies reveal two patterns of compensation: distal compensation in the case of TW10, where compensatory mutations are distal in three-dimensional space but alter functional dependencies, and proximal compensation in the case of KK10, where codon pairs in a compensatory pathway are in close proximity and are likely required to maintain structural fidelity. Although the PDN predicted both known pathways, only the latter form of compensation can be easily and independently verified computationally by computing distances between covarying codon pairs.In the first study of its kind, Poon and Chao null codon pairs, which we defined to be all codon pairs for which no significant direct associations were found even though both codons exhibited enough variability to pass our minimum count filter.To determine the proportion of covarying codon pairs that are in direct contact, we computed codon-codon distances against the p17 trimer crystal structure q\u22640.2) linearly distal codon pairs (>10 codons apart) that could be mapped to at least one structure, 37  were within 5 \u00c5 of each other and 113  were within 10 \u00c5. Even among linearly proximal codon pairs (2\u201310 codons apart), covarying pairs were more likely  than non-covarying pairs  to be within 5 \u00c5 in the three-dimensional structure (p\u200a=\u200a0.0001).Among the 424 significant (p<0.0001). The direct codon-codon associations were also significantly closer than those of p\u200a=\u200a0.003; 5.3%<5 \u00c5, p\u200a=\u200a0.04), which doesn't account for phylogeny, and p<10\u221212; 3.2%<5 \u00c5, p<10\u22125), which computes pairwise, phylogenetically-corrected associations. Fisher's exact test (median 22.1 \u00c5) was indistinguishable from the null codon pairs .To further validate the ability of the model to distinguish direct associations within a chain of interactions, we computed pairwise distances among all linearly distal one-hop associations, excluding instances where a direct association was also inferred. The median distance between direct association codon pairs (15.9 \u00c5) was significantly smaller than the median distance between one-hop codon pairs  are strongly targeted by the T-cell response. However, a shift in immunodominance patterns occurs over the course of infection, as the T-cell response broadens to target additional epitopes and the presence of the T242N TW10 escape mutation, suggesting that escape in IW9 often occurs in the context of escape in TW10 . Similarly, A163G KF11 escape is predicted by escape substitutions T242N (TW10) and I147M (IW9), and lack of escape at 310 (QW9), reflecting previous reports of the targeting order of Gag B*57-restricted epitopes The epitopes targeted by CTL are not a simple function of the individual's HLA repertoire. Rather, specific patterns of epitope targeting are often observed. For example, epitope targeting by CTL often follows patterns of immunodominance CTL-mediated codon covariation. Another may be the overall strength of the CTL response and/or the strength of the CTL response to epitopes targeted by a given allele. In the most extreme example, epitopes are either targeted or not depending on the strength of the immune system. Individuals who are targeting the epitopes will tend to select for escape mutations in all the epitopes, whereas individuals who have weakened immune systems may not target any of the epitopes (or will target with less strength). In this scenario, escape from epitope A implies that the immune system is active, thus increasing the likelihood of escape from epitope B, and vice versa. In a less extreme example, suppose that some individuals with a given allele mount a response to epitopes restricted by that allele, whereas other individuals do not. This situation will lead to codon-codon dependencies among associations in epitopes restricted by the allele. In addition, several studies have noted the accumulation of multiple or alternative escape substitutions within the same epitope It is important to note that the order in which direct escape associations arise cannot be inferred from the PDN. Rather the presence of arcs between epitopes suggests that targeting of epitopes restricted by the same allele is somehow correlated. Immunodominance is one biological mechanism that may induce such p\u200a=\u200a0.0002). 213 of 554  linearly distal codons pairs occurred within different optimally-defined epitopes restricted by the same HLA allele (compared to 32.3% of null codon pairs). If we also include predicted epitopes, defined here as the region \u00b15 codons from a direct HLA-codon association, then 304  linearly distal codon pairs are in known or predicted epitopes restricted by the same HLA allele (compared to 36.6% of null codon pairs). We thus conclude that a majority of codon covariation in Gag p17/p24 is attributable to CTL-mediated selection pressures, though the specific mechanism of CTL-mediated covariation cannot be identified from this study.To determine how much of the observed codon covariation may be CTL-mediated, we looked at covariation in the PDN with regard to known, optimally defined epitopes. Among linearly proximal codon pairs, both co-evolving codons were within the same HLA-restricted optimal epitope 138 of 162 times . Thus, we categorized every direct and one-hop association based on whether or not it was within three codons of an optimally-defined epitope, using a strict matching criterion that required that an optimal epitope exactly matched the consensus sequence among either clade B or clade C HIV sequences that had the predicted susceptible amino acid and the codon in question.The observation that a majority of codon-codon associations occur within or proximal to epitopes restricted by the same HLA allele suggests that CTL escape is driving much of the observed HIV codon variation. Indeed, Brumme et al. q-value rank of the association. To prevent double counting, only the most significant association per HLA-epitope pair was considered (see clean one-hop). Only four such associations were found with q\u22640.4 (p<0.0001 compared to direct associations with a permutation test), indicating that most of the one-hop epitopes were epitopes that additionally had direct associations. It is therefore not surprising that ered see . The plol model  .p<0.0001). The models that roughly account for clade differences, either through codon covariation  HLA escape polymorphisms from indirect or, more specifically, one-hop (H\u2192B) escape polymorphisms in situations where the true interaction is the chain H\u2192A\u2192B. Although the direct\u2013indirect distinction can arise under several mechanisms, the explicit statistical interpretation is as follows: a direct HLA-polymorphism H\u2192A association means the HLA allele H is a strong predictor of the polymorphism A, whereas an indirect HLA-polymorphism association H\u2192A\u2192B means the polymorphism B is better predicted by the polymorphism A than by the HLA allele H. Although B is in a sense HLA-associated, the distinction of direct versus indirect associations may have important biological implications. For example, many of the indirect associations identified by the dependency network for the B*57-restricted TW10 and B*27-restricted KK10 epitopes are consistent with known compensatory mutations associated with escape in these epitopes This study also represents a significant step forward by providing a statistical approach that can help differentiate direct  dramatically outperformed the phylogenetic pairwise approach. Although many phylogeny-aware comparative methods have been developed for codon-covariation directed acyclic graphical (DAG) model rather than a dependency network. In a DAG model, arcs from predictor to target attributes form a directed acyclic graph and local distributions take the same form as in a PDN.  When learning the distributions in the DAG model, Poon et al. took phylogeny into account, although in a way different from our approach. In particular, when learning the distribution of an attribute given its parents in the DAG model, they imputed for each individual the value of the attribute corresponding to the ancestor of that individual in the phylogeny. These imputed values were then treated as observed data and fed to a standard DAG model structure learning algorithm.The first approach to identifying chains of interactions in a phylogenetic context was recently provided by Poon et al. The PDN provides an alternative approach that leverages the strengths of dependency networks. The most apparent difference is that dependency networks allow cycles, resulting in a network that is easier for the non-expert to interpret than is the DAG model A given B and the local distribution for B given A are closely related, yet are learned independently.) This independent learning leads to a decrease in statistical efficiency. In practice, however, this decrease is typically minimal One drawback of a dependency network relative to a DAG model is that the local distributions among the target attributes overlap and yet are learned independently.  species, a phylogeny is a reasonable model of the population structure. Even among individuals of the same species, a phylogeny may be a good representation of the structure, insofar as the structure reflects a hierarchical clustering of the individuals that may be due to a number of genetic and environmental factors http://www.codeplex.com/MSCompBio.We have introduced phylogenetic dependency networks for modeling multiple sources of selection pressure on evolutionary traits, and have applied this approach to the characterization of patterns of immune escape in HIV. In so doing, we have identified broad patterns of covariation and CTL adaptation, the verification of which should broadly inform vaccine design. Although the specific distributions described here may not be suited for all applications, dependency networks that are conditioned on appropriate models of population structure are widely applicable, and represent a powerful tool for efficiently combining multiple effects in population-based analyses. The programs used in this paper are available at Table S1q<0.2.List of associations with (0.29 MB XLS)Click here for additional data file.Table S2q<0.2.Distance in Angstroms between codons with (0.08 MB XLS)Click here for additional data file."}
+{"text": "We show that in the absence of Lgl1, production of mature white blood cell lineages and long-term survival of mice are not affected. Additionally, loss of Lgl1 does not alter leukaemia driven by constitutive Notch, c-Myc or Jak2 signalling. These results suggest that the role of Lgl1 in the haematopoietic lineage might be restricted to specific co-operating mutations and a limited number of cellular contexts.In epithelial and stem cells, lethal giant larvae (Lgl) is a potent tumour suppressor, a regulator of Notch signalling, and a mediator of cell fate via asymmetric cell division. Recent evidence suggests that the function of Lgl is conserved in mammalian haematopoietic stem cells and implies a contribution to haematological malignancies. To date, direct measurement of the effect of Lgl expression on malignancies of the haematopoietic lineage has not been tested. In Lgl1 Drosophila, the Scribble complex protein, Lethal giant larvae (Lgl) is a potent tumour suppressor. Lgl maintains epithelial cell polarity Drosophila develop epithelial tumours \u2212/\u2212 mice also display neural tube hyperplasia associated with ectopic Notch signalling In Lgl1 has recently been described to affect cell cycle in mouse HSC, and gene signatures associated with Lgl1 deficiency in mouse HSC identify a group of cytogenetically normal AML with poor prognosis + or PTPRCA-Ly5.1/ly5.2 mice were purchased from WEHI, . Lgl1+/\u2212The animal experiments in this study were performed as approved by the animal experimentation ethics committee of the Peter MacCallum Cancer Centre. PTPRCA-Ly5.16 of foetal liver single cell suspensions were injected intravenous (I.V). To prevent infection, reconstituted mice were maintained on neomycin sulfate\u2013supplemented drinking water for 6 weeks post irradiation. 6\u201310 weeks post reconstitution peripheral blood was isolated from mice and successful reconstitution determined by percentage of Ly5.2+ cells.Timed matings were established between heterozygote polarity deficient animals. The following procedure was conducted on the day of harvest. Embryos were harvested from these matings at embryonic day 14.5 and genotyped by PCR on single cell suspensions from foetal livers. 8\u201314 week female PTPRCA-Ly5.1+ recipients received two doses of 5.5 Gy administered greater than three hours apart. 1\u00d710Automated cell counts were performed on blood collected from the retro-orbital plexus collected in eppendorf tubes containing a small volume of 20 mM EDTA. Samples were diluted 1\u223612 with PBS and run on an Advia 2120 analyser (Siemens). For donor reconstitution and lymphocyte population analysis, peripheral blood was lysed to remove red blood cells then stained with antibodies against Ly5.2, B220, CD19, CD4 and CD8 . Samples were analysed on an LSR II flow cytometer (BD).6 un-fractionated bone marrow cells. Donor and recipient cells were identified on the basis of Ly5.1/5.2 expression. For secondary transplants, Ly5.1/2 heterozygote mice were used as recipients to identify primary foetal liver donor cells and primary recipients from secondary recipients.Whole bone marrow was isolated from femur and tibia of donor mice and transplanted as described in+/+ and Lgl1\u2212/\u2212 single cell suspensions were prepared from whole foetal livers isolated from 14.5 embryos. Suspensions were cultured in IL-3, IL-6, and stem cell factor conditioned media with 20% FCS for 3 days. Phoenix-E cells were transfected by calcium phosphate with MigR1 plasmids containing either GFP only or GFP with NotchICN\u0394Ram\u0394P as described in 6 GFP+ve foetal liver cells by intravenous injection into the tail vein. Cohorts of reconstituted mice were the result of 2 independent foetal liver isolations and independent transfections.For generation of Notch driven leukemia, Lgl19/L or greater. For Notch driven leukemias, peripheral lymphoid organs were analysed by flow cytometry for Ly5.2, GFP, CD3, CD4 and CD8 expression. For E\u00b5Myc driven leukemia, peripheral lymphoid organs were assayed for Ly5.2 and B220. For TEL-JAK2 leukemia, peripheral lymphoid organs were analysed by flow cytometry for Ly5.2, CD3, CD4 and CD8 expression.Mice reconstituted with either NotchICN\u0394Ram\u0394P transduced or E\u00b5Myc/TEL-JAK2 foetal livers were monitored daily for signs of leukemia onset or other signs of ill health. Mice were killed when deemed moribund by an experienced animal technician blinded to the genotype. Typically, mice presented with several of the following features: hunched posture, laboured breathing, weight loss, enlarged lymph nodes and/or spleen, peripheral white blood cell cellularity of 13\u00d710(\u2212\u0394\u0394Ct).RNA was prepared and real time quantitative PCR performed as described previouslyt test, and p values <0.05 were considered significant. For survival analysis, Kaplan-Meier survival curves were analysed using Log-rank (Mantel-Cox) test. P<0.05 were considered significant.Statistical differences between the means of two data groups was determined by using two-tailed unpaired Student's \u2212/\u2212 embryos into lethally irradiated recipients. Following transplantation, and as we previously observed \u2212/\u2212 mice  were present at normal frequencies , E\u00b5-Myc- (Log rank Mantel-Cox test P\u200a=\u200a0.4607) or TEL-JAK2 induced leukaemia (Log rank Mantel-Cox test P\u200a=\u200a0.9071 \u2013 +/+ or Lgl\u2212/\u2212 control chimeric mice. In adult mice, Lgl1 is expressed globally whereas the homologue Lgl2 is restricted to heart, kidney, liver, lung, skin, intestine and stomach. To eliminate the possibility that increased expression of Lgl2 compensated for loss of Lgl1 in the haematopoietic system, we isolated peripheral immune cells from primary chimeric animals and B cell lymphomas from Myc+ primary foetal liver chimeras and assayed for expression of Lgl1 and Lgl2 (We next investigated whether the tumour suppressor function of Lgl1 was capable of modifying disease progression in well-characterized mouse leukaemia models. As Lgl expression has been suggested to regulate Notch signalling Lgl1 has recently been demonstrated to regulate HSC cell cycle and depletion of Lgl1 in HSC to confer a reconstitution advantage when in competition with wild type HSC Interestingly, we observed no development of leukaemia in primary chimeric mice consistent with previous studies using conditional Lgl1 deletion"}
+{"text": "Breastfeeding has a major impact on CMV epidemiology. Postnatal CMV reactivation's incidence during lactation is nearby the maternal seroprevalence. Although perinatal CMV infection has practically no consequences in term newborn, it may cause, in some cases, a severe symptomatic disease in preterm newborns.The aims of the present study are to evaluate the rate and clinical expression of CMV infection breast milk transmitted in preterm infants and to check the safety of the freezing treated breast milk.st to 9th postpartum week. Both fresh breast milk and 72, 96, 120 hours frozen samples have been examined, checking the presence of CMV; urine samples have been tested too.The study included fifty-seven preterm infants and their CMV seropositive mothers. Fresh breast milk samples have been collected from 170.2% of tested mothers showed reactivation of the infection, and CMV-positive breast milk during the six weeks postpartum has been found. However, only one infant was infected by CMV, developing hepatic affection concomitantly with a multi-system involvement, as shown CMV DNA detection in urine, saliva, blood, gastric aspirate, and stools.Freezing breast milk at -20\u00b0C and pasteurization may respectively reduce or eliminate the viral load. Human breast milk is considered as an ideal food for newborns, both for term and preterm infants, because of its nutritional value, unique qualities and properties. However it can also be a vehicle for viral and bacterial infections, as the breastfeeding is known to have a major impact on the epidemiology of postnatal cytomegalovirus (CMV) infection . BecauseThe aim of the study is to evaluate the rate and clinical expression of CMV infection transmitted by breast milk in preterm infants, born before 32 gestational weeks and/or weighting \u22642000 g at birth, checking the safety of the freezing treated breast milk.st January 2004 to 31st December 2007. Fifty seven preterm infants and their mothers, who were found CMV seropositive, were included in the prospective study. The study have been included preterm infants aged of < 32 complete weeks, or weighting < 2000 g at birth. Since the second life day. each newborn has been fed by naso-gastric tube (NGT).The study has been carried out by Neonatology Intensive Care Unit (NICU) of the Teaching Hospital, in Perugia  from 1Peripheral blood sample has been taken from mothers after delivery, testing CMV antibodies, to determine maternal serology. CMV immunoglobulin IgG and IgM antibodies have been determined, using enzyme immunoassay kits .Fresh breast milk samples have been collected from the 1th to 9th postpartum week. Fresh breast milk and 72, 96, 120 hours frozen samples have been tested, checking the presence of CMV. Milk samples have been filed and separated into cellular and aqueous fractions by centrifugation for obtaining. viral isolation. CMV culture has been undertaken on aqueous fraction, using the rapid shell vial tissue culture on MRC-5 cells . After two days incubation at 37\u00b0C, the cultures have been tested checking the presence of immediate-early IE1 and IE2 (MW 68-72 kDa) CMV antigens by direct immunofluorescence, using FITC-labelled monoclonal antibodies . Milk samples DNA have been extracted by easyMAG  as molecular tests Nested PCR has been used as screening test, and quantitative real time PCR (RT-PCR) has been used to quantify the viral DNA load . All samples have been assayed one by one. A negative control (distilled water) and a positive control (a DNA construct from CMV genome) have been included in each nested PCR. The lowest limit of detection was 10 copies/5 \u03bcl and 105 genomes/ml, for nested PCR and RT-PCR, respectively. Milk has been collected together with urine samples, to determine the viruria through quantitative and qualitative methods. Each infant has been fed by thawed maternal 72 hours frozen breast milk. Clinical status and laboratory tests have been documented during the study time. By informed consent, the parents of participating infants agreed them to be included in this study.Previous viral infected mothers have been identified by IgG seropositivity towards CMV, in absence of IgM. In 40 out of 57 seropositive mothers (70.2%) the virus reactivated and was excreted in the milk during the six weeks postpartum lactation period. Maternal CMV reactivation was shown by viral shedding in breast milk Figure  concomit4 to 106 copies/mL, were shown from the 4th to the 6th week after delivery. In this period the average DNA load was 61958,8 \u00b1 15818,3 copies/mL. Thereafter, DNA copy number decreased progressively. The 72 h freezing process was capable of decreasing significantly the infectivity, as shown by viral culture, and the viral load  in the values of CMV kinetics, comparing positive samples to total samples examined in the different weeks. The majority of the colostrum samples were CMV DNA positive  and most milk samples became CMV DNA positive two weeks after delivery . During the period of the study 109 samples were tested and the highest values of CMV DNA copies, ranging between 10The average gestational age of the newborns was 29 weeks (range 23-34 weeks). The average birth weight was 1158 \u00b1 436.3 g. Among the infants who have been fed with CMV DNA-positive breast milk, only one became infected with CMV. Considering all the checked women, the rate of postnatal infection in exposed newborns was 2.5%: the rate of perinatal infection was 1.7%. The postnatal rate infection (2.5%) has been considered, because the infant became CMV positive 8 weeks after birth and the mother's serological test did not change, showing CMV IgM negative and IgG positive. This results indicate that maternal CMV reactivation was not systemic but occurred only in the breast.rd, on 6th, on 12th, on 24th, on 36th month and on 5th of correct gestation age). To date (after at least 3 years from the enrolment), none of them presents health problems correlated with CMV infection, including the symptomatic infants.All the infants were followed up to detect long-term sequele as described for congenital and perinatal cytomegalovirus infection: each infant underwent serial monitoring  followed by administration of valganciclovir (25 mg/Kg/die for six months). -8. 6-8].The mechanism of CMV reactivation in human milk, and the role of milk cells and cell-free virus in vertical transmission are still unknown . MaternaIn a previous study, all term infants shed CMV into urine over a long period, and all of them had normal clinical courses without sequelae, but two preterm infants developed pneumonia . WithoutIn conclusion, our study confirms that CMV transmission through breast milk can cause a severe clinical course in preterm infants but freezing could highly decrease the transmission rate. Because breastfeeding is healthy and wide spread, and the number of preterm infants is increasing in the most developed countries, a new procedure for gentle virus inactivation of seropositive breast milk is being assessed in our laboratory, to prevent CMV transmission to extremely preterm infants in the future ,20.CMV: CitoMegaloVirus; LBW: Low Birth Weight; NGT: NasoGastric Tube; NICU: Neonatology Intensive Care Unit; PCR: Polymerase Chain Reaction; RT-PCR: Real Time PCR;The authors declare that they have no competing interests.MC carried out alignment and drafted the manuscript and performed the statistical analysis. PB conceived of the study, and participated in its design and coordination. AS participated in the design of the study. EC carried out CMV culture.RC carried out the immunoassays and PCR. MJR participated in the design of the study. AF participated in the sequence alignment.All authors read and approved the final manuscript"}
+{"text": "Background. There is no FDA-approved medication for cocaine dependence or consensus on the statistical approach for analyzing data from cocaine dependence pharmacotherapy trials. The goal of this paper is to illustrate the importance of understanding medication's pharmacodynamics when specifying the statistical model to test its efficacy. Method. Data from a double-blind placebo controlled trial of reserpine for cocaine dependence are analyzed. Since the antihypertensive properties of reserpine are well established, blood pressure data are utilized to evaluate the ability of two statistical models, one that does not take the pharmacodynamics of reserpine into account and one that does, to detect reserpine's antihypertensive effect. Results. The statistical model specified without regard to reserpine's pharmacodynamics failed to find a significant medication effect for either systolic (P = 0.49) or diastolic (P = 0.59) blood pressure. Contrariwise, the model based on the pharmacodynamics of reserpine found a significant effect for both systolic (P = 0.002) and diastolic (P = 0.004) blood pressure. Conclusions. If the pharmacodynamics of a study medication are not considered when specifying statistical models, then erroneous conclusions may be reached. This trial is registered with NCT00033033. According to the Office of National Drug Control Policy, there are over 3 million long-term cocaine users in the USA . BecauseThe trend over the past 15 years has been the use of increasingly sophisticated statistical analytic approaches, about which the study investigators, who have posed the hypotheses to be tested, may have only a superficial understanding. With this increasing gap between the statistical knowledge of the investigators and the analytic approaches being used, there is a danger that investigators will choose to \u201cleave the statistics to the statisticians.\u201d The problem with this approach was summarized in an article entitled \u201cMedical statistics-no time for complacency\u201d in which Aalen  outlinedWe encountered the reality of this possibility in analyzing the results of our double-blind placebo controlled trial of reserpine for cocaine dependence . ReserpiVital signs data were collected from 119 participants randomized into a double-blind placebo controlled trial of reserpine for cocaine dependence . Three sEligible participants were at least 18 years of age and in good physical health as determined by a medical history, physical exam, electrocardiogram, and standard laboratory tests. Participants were required to have at least one positive urine toxicology screen for the cocaine metabolite benzoylecgonine (BE)  during the two-week screening period, to meet DSM-IV criteria for cocaine dependence as assessed by the structured clinical interview for DSM-IV (SCID) [Vital Signs Measurement. During the two-week baseline period and the twelve-week active trial, participants were scheduled to attend three research visits per week. Sitting blood pressure was assessed three times per week during baseline and the first two weeks of the active phase and then weekly thereafter.Study candidates signing informed consent entered the screening and baseline phase which entailed the completion of six clinic visits within two consecutive weeks. Stratified randomization, balancing for gender and self-report of cocaine use (<18 or \u226518 days of use in the last 30 days), was used to assign eligible participants to reserpine or placebo within each study site. All participants were scheduled to receive one tablet per day  during dose escalation (week 1) and dose taper (week 13). During weeks 2 through 12, all participants were scheduled to receive two tablets per day . During the twelve week active trial participants were scheduled to attend three research visits per week. Study participants received $10 in retail scrip or vouchers per research visit; at the end-of-study evaluation (first visit of week twelve), participants received an additional $25 in retail scrip or vouchers because of the larger assessment burden associated with the visit. A master's level clinician provided each study participant with an hour of individual cognitive behavioral therapy on a weekly basis.There are many elements to consider when defining the analytic approach and statistical model for analyzing data from a cocaine pharmacotherapy trial. Understanding of some of these elements, for example, the occurrence of missing data or the distributions associated with common outcome measures, can be gained through familiarity with the data sets collected from multiple cocaine dependence pharmacotherapy trials. An element that is more specific to a given trial is the study medication being tested, the pharmacodynamics of which need to be considered in order to define an optimal statistical analysis. The investigator needs to consider and to clearly communicate to the statistician what is known about the effective dose of the medication and the speed with which the medication will exert its effect on the outcome variable. In terms of \u201cspeed\u201d one could consider medications like methylphenidate, valium, and nicotine patch, all of which begin working almost immediately, at one end of the continuum and drugs like antidepressants, such as SSRIs, tricyclic antidepressants, and MAO inhibitors, which take up to six weeks to exert an effect, at the other end of the continuum.Inclusion of Titration Data. Understanding the effective dose of the medication will also help determine whether or not the data collected during the medication titration phase should be included in the analysis. If a medication does not exert an effect until a certain dose is reached, then including the data collected prior to that point could weaken the ability of the statistical test to detect either a Medication main effect or a Medication-by-Time interaction effect. Conversely, if a medication exerts an effect at relatively low doses and with relatively quick speed, excluding the titration phase data could result in weakening a Medication main effect and very possibly eliminating the chance for a significant Medication-by-Time interaction effect. In cocaine dependence trials, we often cannot specify, with certainty, the effective dose of the medication being studied. Given this uncertainty, if the particular medication being studied has a relatively rapid speed of action, then it would likely be important to include all postrandomization study data, along with data collected during titration.Defining the Statistical Effect of Interest and Model Terms. The speed with which a medication exerts its effect on a given outcome should be considered when defining the statistical effect of interest. Currently, a common method for testing the efficacy of a medication in a clinical trial is to compare the impact of medication on the participants' respective rates of improvement (or deterioration) during treatment. In a regression, this translates into determining efficacy solely on the basis of the Medication-by-Time interaction effect. However, it is important to note that even when a medication has a substantial effect, that effect is unlikely to continue increasing indefinitely over time. At some point, the effect would very probably reach a maximum and level off. If this plateau occurs too early in the participants' treatment, then tests based solely on a linear Medication-by-Time interaction would fail to detect the medication effect, whereas an evaluation of the Medication main effect would reveal the efficacy of the medication. When investigators are unable to reasonably rule out the possibility of a medication reaching its peak effect early in the treatment phase, the Medication main effect and the Medication-by-Time interaction effect can be evaluated jointly to determine efficacy. A significant Medication-by-Time effect would indicate medication efficacy, and in the reasonably certain absence of a Medication-by-Time effect, a significant Medication effect would also indicate efficacy. Of course, basing efficacy on two hypotheses instead of one can reduce statistical power in that one should adjust the alpha level to correct for multiple comparisons; Bonferroni would suggest alpha levels of 0.025 for each hypothesis rather than usual alpha of 0.05 [ of 0.05 .If the Medication main effect is of interest, then one needs to consider how to include baseline data in the model. For outcome measures with significant intrasubject variability, including a summary of baseline values as a covariate can significantly reduce the standard error for a Medication main effect, thus increasing the statistical power of the test. It is important to note that this reduction in standard error occurs even in cases where the two treatment groups did not differ during baseline. Including baseline as a covariate also helps to mitigate possible imperfections in randomization.Reserpine destabilizes the membranes of the vesicles which store catecholamines in neurons. As a result, the storage vesicles rupture causing the neurotransmitters  to spill out into the cytoplasm and to be metabolized by the enzyme monoamine oxidase (MAO). This is an immediate and irreversible effect of reserpine even at the 0.25\u2009mg dose . No new The protocol-specified statistical analysis plan for the reserpine trial called for the use of generalized estimating equations (GEE) , 12 to cThere were no significant baseline demographic  or bloodP value from 0.35 to 0.11 in the case of SBP and from 0.23 to 0.08 in the case of DBP. Somewhat interestingly, the P values for the Medication-by-Week interaction effects actually increase by including the titration data. This increase could reflect the fact that, all else being equal, if there is, in fact, no Medication-by-Week interaction effect , adding data points is more likely to yield a result that reflects the true lack of a Medication-by-Week interaction effect. This, of course, reinforces the importance of correctly specifying the effect of interest.The protocol-specified statistical analysis Plan called for excluding the data collected during the one-week titration period. As stated above, deciding on the inclusion of the titration phase data needs to be based on information regarding the effective medication dose as well as the relative speed with which the medication manifests an effect on the outcome of interest. While we were not entirely certain of the effective dose of reserpine, as pertains to its hypothesized effects on cocaine use, we were certain that its effect would be fairly rapid see  above. FFor the reserpine trial, the analytic plan specified that the Medication-by-Week interaction effect, which provides information about the change in the treatment groups over time, was the effect of interest. The graphs of the SBP and DBP data can be found in P = 0.002) and DBP (P = 0.004).In addition, the analytic plan specified that baseline data not be included as a covariate unless the groups differed significantly at baseline. However, blood pressure has relatively high intrasubject variability and, thus, inclusion of baseline as a covariate can significantly reduce standard error even in cases where the groups do not differ significantly from one another during baseline. The impact of including baseline as a covariate and adding the Medication main effect as an effect of interest can be seen in P values for the Medication-by-Week interaction and Medication main effects as well as the standard error for the Medication main effect as a function of including the titration week and baseline as a covariate. The impact of some of the differences between the original statistical model, such as adding titration week and baseline as a covariate, is discussed above as well as in Tables P value from 0.35 to 0.098. The complete set of differences between the original statistical model and the version derived from a consideration of the pharmacodynamics of reserpine is illustrated in the results for SBP in This paper highlights the importance of considering a medication's pharmacodynamics when specifying the statistical model used to analyze pharmacotherapy trial data, with a specific focus on deciding whether to include titration data in the analysis, specifying the effect of interest, and specifying terms to include in the model. As demonstrated by the analysis of reserpine's effect on SBP and DBP, a statistical model that is specified without knowledge of the study medication's pharmacodynamics can lead to a type II error. This finding is consistent with Aalen's  observatThe primary potential limitation of the present analysis involves statistically, relative to clinically, significant effects. Specifically, this paper demonstrates that a statistically significant medication effect can be missed if the statistical model is not based on medication's pharmacodynamics but does not address the degree to which a clinically significant effect might be missed. In the present case, comparing the baseline-corrected final blood pressure readings for the reserpine and placebo groups revealed a difference of 3.0\u2009mm\u2009Hg for SBP and 6.9\u2009mm\u2009Hg for DBP. While these differences appear small, studies suggest that a 5-6\u2009mm\u2009Hg decrease in DBP is associated with a clinically significant reduction in stroke and coronary heart disease even among \u201cnormotensive\u201d individuals . This suWhile the focus of the present paper is somewhat circumspect, its implications are not: we cannot afford to conduct research in which there is a disconnect between the study investigators posing the general hypotheses to be tested and the statisticians who are defining, through the statistical models, very specific hypotheses to be tested that may or may not correspond to the hypotheses that should be tested based on knowledge of the medication's pharmacodynamics. It is therefore up to the investigator to provide this knowledge to the statistician and for the statistician to explain the statistical analysis to the investigator in order to ensure that he or she understands precisely what the analysis is testing and for this understanding to be reflected in the a priori analysis plan. Failure of the investigator and statistician to cooperate effectively could result in type II errors in cases, unlike reserpine's effect on blood pressure, where this error cannot be detected."}
+{"text": "Bioengineering and Translational Medicine, we are delighted to introduce Professor Francis (Frank) J. Doyle III, the Dean of Harvard's Paulson School of Engineering and Applied Sciences. He holds the John A. and Elizabeth S. Armstrong Professor of Engineering and Applied Sciences. Prof. Doyle received his BS degree in Chemical Engineering from Princeton University, his MS degree from Cambridge University (UK), and his PhD degree from the California Institute of Technology.In this issue of Prof. Doyle started his academic career in 1992 at Purdue University in the Department of Chemical Engineering. In 1997, Prof. Doyle moved to the Department of Chemical Engineering at the University of Delaware, initially as an associate professor, before becoming a full professor. Between September 2001 and June 2002, he was a Humboldt Research Fellow at the Institute for Systems Theory and Automatic Control at the University of Stuttgart, Germany. In 2002, Prof. Doyle moved to the University of California Santa Barbara (UCSB). At UCSB, Prof. Doyle held the Duncan and Suzanne Mellichamp Endowed Chair in Process Control. He also began a very fruitful partnership with the Sansum Diabetes Research Institute in Santa Barbara as an Adjunct Senior Investigator. His group was the first to demonstrate the operation of a fully closed and completely automated glucose feedback loop in a clinical trial in diabetic patients.In addition to research and teaching excellence, Prof. Doyle has had a passion for administrative leadership. At UCSB, he held several administration positions. He (co)directed the Institute for Collaborative Biotechnologies, a UCSB/MIT/Caltech Army sponsored University Affiliated Research Center. Under his leadership, ICB emerged as a globally leading research organization for biologically\u2010inspired technological innovation. Prof. Doyle also served as the chair of the Chemical Engineering department and Associate Dean of Research in the College of Engineering at UCSB. In the latter role, he championed building of UCSB's bioengineering research enterprise and infrastructure. In July 2015, Prof. Doyle moved to Harvard University as Dean of Paulson School of Engineering. Currently, Dean Doyle is realizing his vision of the new phase of Harvard's School of Engineering and Applied Sciences on the back of interdisciplinary bridges with Harvard's Business and Medical Schools.At the beginning of his career, Prof. Doyle's research focused primarily on the applications of advanced control schemes on nonlinear, multivariable, constrained industrial processes, with emphasis on particulate systems, and pulp and paper. At Purdue, he met and began a fruitful collaborative research project on glucose control for Type 1 Diabetes with Prof. Nicholas Peppas . While their paths later diverged, the collaboration seeded a transition in Prof. Doyle's research portfolio toward biology and biomedical applications. In the past 15 years, Prof. Doyle and his group have created and applied sophisticated tools from control systems theory to analyze the regulatory mechanisms of biological systems with the end goal of being able to manipulate their behaviors. The research has led to impactful advances in the understanding and control of biological systems, particularly in the regulation of blood glucose and circadian rhythms.Over the past two decades, the Doyle group has been at the forefront of the design and synthesis of control algorithms for the development of the artificial pancreas (AP) for type 1 diabetes treatment. In 1999, Prof. Doyle published a seminal paper on model predictive control (MPC) of blood glucose (BG) with his former PhD student, Prof. Robert Parker of U Pittsburgh, and Prof. Nicholas Peppas.In addition to diabetes, Prof. Doyle's contribution to circadian biology research has generated major insights on the design principles of the biological network behind the extremely robust body clock. In humans, the central pacemaker of the circadian rhythm comprises \u223c20,000 neurons in the suprachiasmatic nucleus (SCN) region of the hypothalamus, working in synchrony to generate a coherent circadian signal. Within each neuron, a gene regulatory network with interlocking feedback loops generates the endogenous oscillations of circadian protein expression. Using systems modeling and analysis of this network, Prof. Doyle and his coworkers were able to show how robustness in circadian periodicity and phase response is accomplished by allocating the fragility of the regulatory network to tightly regulated modules .Prof. Doyle's research has been disseminated in over 300 journal articles, book chapters and books, garnering over 18,500 citations. In recognition of his research achievements, Prof. Doyle has received numerous awards, including his election to the National Academy of Medicine (NAM) in 2016. He also received AAAC Control Engineering Practice award in 2015 and the Computing in Chemical Engineering award from AIChE in 2005. He is an elected Fellow of the American Association for the Advancement of Science (AAAS), the International Federation of Automatic Control (IFAC), the American Institute for Medical and Biological Engineering (AIMBE), and the Institute of Electrical and Electronics Engineers (IEEE). Prof. Doyle has also been an influential leader in many renowned scientific communities. He currently serves as vice president of IFAC, and previously served as President of IEEE Control Systems Society, President of Computer Aids in Chemical Engineering (CACHE), and Director and Chair of the Computing & Systems Technology Division within the American Institute of Chemical Engineers (AIChE).In addition to his commitment to research, Prof. Doyle is a passionate educator and has contributed greatly to the process control pedagogy of chemical engineers. He has published several books in the control field, and is a coauthor of the widely\u2010adopted Process Dynamics and Control textbook . His accomplishments as an educator have been recognized through the Ray Fahien award from the American Society for Engineering Education (ASEE) and the ASEE Section Outstanding Teacher Award (Illinois/Indiana).Beyond science, engineering, and medicine, Prof. Doyle invests time in three additional pillars: his family, soccer, and sailing. Prof. Doyle is a proud father and husband and enjoys spending time with his family exploring the outdoors. He is also certified as a professional soccer referee and dedicates many weekends on the field keeping up with college level athletes. Throughout his life, Prof. Doyle has embraced sailing both recreationally and competitively. One of his most memorable competitions included a regatta from Los Angeles to Hawaii (the TransPac), where he put to practice his expertise in circadian biology to maximize the performance and experience of his team around the clock and across time zones.As former members of the Doyle group, we benefitted tremendously from the collegial, collaborative and interdisciplinary research atmosphere that Prof. Doyle cultivated in his laboratory. His positive energy is quite infectious and brought out the best in us. Despite his busy schedule and countless commitments, Prof. Doyle was always eager to discuss research and champion each and every one of our projects. He has always had an open and positive outlook, embraced change, and served as an advocate for future generations of scientists. We feel most fortunate to have had the invaluable opportunity to work with, and learn from, Prof. Doyle as a mentor, teacher, role model, and colleague. His balance and leadership have always been an inspiration. We look forward to embracing and extending his teaching in our own academic pursuit.\u20031, Rudiyanto Gunawan2Neda Bagheri1Chemical and Biological Engineering, Northwestern University, 2145Sheridan Road, Evanston, IL 602082Department of Chemistry and Applied Biosciences, ETH Z\u00fcurich, 8093Z\u00fcurich Switzerland"}
+{"text": "The usable satellite spectrum is becoming scarce due to static spectrum allocation policies. Cognitive radio approaches have already demonstrated their potential towards spectral efficiency for providing more spectrum access opportunities to secondary user (SU) with sufficient protection to licensed primary user (PU). Hence, recent scientific literature has been focused on the tradeoff between spectrum reuse and PU protection within narrowband spectrum sensing (SS) in terrestrial wireless sensing networks. However, those narrowband SS techniques investigated in the context of terrestrial CR may not be applicable for detecting wideband satellite signals. In this paper, we mainly investigate the problem of joint designing sensing time and hard fusion scheme to maximize SU spectral efficiency in the scenario of low earth orbit (LEO) mobile satellite services based on wideband spectrum sensing. Compressed detection model is established to prove that there indeed exists one optimal sensing time achieving maximal spectral efficiency. Moreover, we propose novel wideband cooperative spectrum sensing (CSS) framework where each SU reporting duration can be utilized for its following SU sensing. The sensing performance benefits from the novel CSS framework because the equivalent sensing time is extended by making full use of reporting slot. Furthermore, in respect of time-varying channel, the spatiotemporal CSS (ST-CSS) is presented to attain space and time diversity gain simultaneously under hard decision fusion rule. Computer simulations show that the optimal sensing settings algorithm of joint optimization of sensing time, hard fusion rule and scheduling strategy achieves significant improvement in spectral efficiency. Additionally, the novel ST-CSS scheme performs much higher spectral efficiency than that of general CSS framework. Currently, mobile satellite systems (MSSs), adopting Geostationary (GEO) and Non-Geostationary (NGEO) orbits such as Low-altitude Earth Orbits (LEO) and Medium-altitude Earth Orbits (MEO), have attained a renewed R&D interest and market opportunities with extensive application in providing communication services to fixed or mobile users for a vast range of sectors , especially in the scenario of terrestrial communication infrastructure congested or gusty paralysis.Each orbit has some desirable properties as well as a set of drawbacks for efficient service delivery. Due to the concerns regarding the ease of use of large-sized terminals equipped with large antennas and power supplies as handsets, as well as the need for direct radio communications between the user located anywhere around the world, it leads to the idea of using low earth orbit (LEO) satellites at a height between 500 km and 2000 km of altitude rather than the geostationary orbit. Besides, in the case of delay sensitive application, LEO-based MSSs have a typical end-to-end propagation delay of 20\u201325 ms, which is comparable to the delay of a terrestrial link. On the other hand, considering the mobility issue and the number of LEO satellites required for global coverage, network management becomes more complex and more costly. According to the orbit types, Paolo Chini summarized some basic features of the operational GEO based MSSs and non-GEO based MSSs , Thuraya, ACeS (Asia Cellular System), Eutelsat & SES Astra , Hispasat, MSV (Mobile Satellite Ventures) and TerreStar ; the LEO-based systems of Iridium, Globalstar and Orbcom; MEO-based systems of ICO (Intermediate Circular Orbit)). Details of the satellite orbit, user link frequency bands, physical layer, multiple access, satellite features and ISL can be found in ,2,3.However, these systems are spectrum limited due to the static spectrum allocation policies at present. Frequency bands for MSSs are assigned at the World Radiocommunication Conferences (WRCs), which is organized by International Telecommunication Union-Radiocommunication sector (ITU-R), periodically. Although the rapid growth demands for the increasing wide band and data services have promoted the application of Ku/Ka band for MSSs , almost The statistical results of Cognitive radio (CR) proposed by Joseph Mitola is identified as the promising technology to improve the utilization efficiency of the available spectrum ,9. As thActually, wideband sensing techniques are required for detecting wideband satellite signals. The most representative solution is based on filter banks to actualize spectrum sensing in each narrowband at a time. However, this approach requires a large number of RF components and introduces large latency ,20. BesiThe sensing performance of a single node may degrade in wireless channels for several reasons such as the hidden node problem, shadowing, multipath fading, and interference/noise uncertainty. To address these issues, various collaborative spectrum sensing (CSS) schemes among multiple secondary users have been proposed ,28,29,30This paper mainly investigates the problem of joint designing the compressed sensing time and the parameters  of CSS scheme to maximize the spectral efficiency of SU, subject to adequate protection to PU. The main contributions of this paper are as follows. Firstly, we establish the wideband spectrum sensing model based on compressed detection. After compressed sensing, each SU makes local decision  and reports to the fusion center (FC), where counting fusion rule is employed to reach a global decision based on the sensing results. It is proven that SU spectral efficiency is unimodal convex function of compressed sensing time \u03c4. Moreover, inspired by the previous general CSS frame structure, we propose a novel wideband cooperative spectrum sensing (CSS) framework in which each SU reporting duration can be utilized for its following SU sensing. The sensing performance benefits from the novel CSS frame structure because the equivalent sensing time is extended by making full use of reporting time. Additionally, the spatiotemporal CSS (ST-CSS) scheme is designed to achieve space and time diversity gain simultaneously for time-varying channel without introducing additional time overhead. Finally, via computer simulations, it is shown that the proposed CSS framework with optimal settings of sensing time and fusion scheme significantly improves SU spectral efficiency.The rest of this paper is organized as follows. It is observed from the scenarios described in ,13 that We assume that all the SUs in the coverage of satellite can sense the PU. The signals received at SU may come from terrestrial or satellite-based primary users spreading over a wide band. The employed CS-based approach has been developed to detect PU using sub-Nyquist rate samples to reduce the burden on the ADC in wideband spectrum sensing (WSS). In the compressed settings, the problem of PU detection can be formulated as a testing of two hypotheses: one is Hypothesis According to the likelihood ratio test of Neyman\u2013Pearson (NP) optimal decision rule ,39:(4)\u039bAfter taking logarithms on both sides, we can obtain an equivalent test that simplifies as:We now define the output of the compressive detector as:\u03bd can be approximated expressed by Gaussian distribution as follows:Since Thus, the false alarm probability Firstly, considering a special case in which is an orthoprojector  lemma , then wiCombining Equations (13) and (14), the result follows. \u25a1It can be concluded that Equation (12) tells us how much information we lose via compressed samples in terms of our ability to solve our detection problem, precisely. Moreover, it is absolutely feasible to use Equation (15) to predict the probability of false alarm because Obviously, it is tightly concentrated around the expected performance curve as shown in Additionally, large numbers of trials of methods based on random orthoprojector, matrices with independent Gaussian entries and matrices with independent Bernoulli entries show that there is almost no difference among these three methods, and all perform highly consistent with expected. Actually, it is not necessary to limit i-th SU makes a binary decision  with probabilities of detection During local compressed sensing, the In general, the counting fusion rule is broadly classified as OR fusion rule (OFR), AND fusion rule (AFR) and MAJORITY fusion rule (MFR).For AFR, For OFR, For MFR, Due to the need for periodic spectrum sensing (PSS), a typical frame structure of a CR network will at least consist of a sensing slot and a data transmission slot, as depicted in i-th SU data transmission occurs in following two cases: correct detection absence of PU and missing detection presence of PU. When the former occurs, the amount of transmission data is i-th SU is derived as:It is assumed that I\u201d, which is applicable for all any SU. Obviously, for a given frame length As to be highlighted, Theorem\u00a02.is a decreasing function ofProof.For a given i-th SU. Then, we have:Obviously, Theorem\u00a03.For any target detection probabilitythere exists one optimal sensing timethat yields the maximal SU spectral efficiency.Proof.For a given Obviously,Because Equations (26) and (29) indicate that Eventually, we have In order to avoid interfering to PU, it necessitates an efficient and reliable detection method of PU. The scheme of CSS has been proposed to mitigate the impact of fading and shadowing. The secondary system consists of Cooperation among SU can greatly improve sensing performance, however, it also introduces additional overhead since the sensing results need to be reported to FC periodically. In the conventional frame structure, the SU can sense the PU before sensing results reporting and data transmission. A serious problem is that when the incumbent SU reports its local sensing results, other SU have no any manipulation until allocated reporting slot arrival. There is a waste of N frames for CSS frame structure with individual frame length of 160 ms [N is determined by the application scenarios. Thus, our proposed novel CSS frame structure can be applied to the scenario where the PU does not leave or arrive within one frame, which is the precondition of the proposed solution.Considering the effects of dynamic and time-varying spectrum environment on CSS performance, most recent works ,30,31,32f 160 ms , where ti-th SU reporting time can be utilized for its following SU sensing. In other words, for the i-th SU, the i-th SU is As depicted in i-th SU is denoted as:In summation, the equivalent compressed sensing time for the i-th SU is formulated as:The quantity of compressed samples at the i-th SU isTherefore, the equivalent compression ratio at the j-th SU is i-th SU transmission and the j-th SU sensing cannot happen at the same time. Thus, the signal that the j-th SU senses does not include the signal transmitted from i-th SU. Another situation to be highlighted is coincidence of the i-th SU reporting and the j-th SU sensing. According to the description in i-th SU reports a binary decision  in the reporting link, whereas the j-th SU senses in the sensing link. In this case, they are using different frequency channel. It is true that sensing link and reporting link are independent physical channels.As depicted in It can be seen clearly that each SU except SU 1 gets much longer compressed sensing time and higher compression ratio without additional time overhead compared with the general frame structure. Thus, SU spectral efficiency can be increased due to the reduced false alarm probability. In summation, it is the improvement of overall sensing performance that contributes to spectral efficiency, as demonstrated in aforementioned argument.i-th SU.In the proposed CSS framework, if sensing SNR In general, the optimal reporting scheduling strategy changes with the varied fusion rules. In this section, the optimal scheduling strategy for AFR, OFR and MFR are investigated. The sensing false alarm probability and detection probability dictate the feasibility of SU operation, and there is typically a tradeoff between the two. The PU would prefer a high detection probability to alleviate interference. However, the SU would prefer a low false alarm probability to use the PU\u2019s frequency band for secondary data transmission, thereby increasing wireless efficiency. Generally, the Neyman\u2013Pearson (NP) detector is the decision rule that maximizes detection probability ith SU is When AFR is applied at FC, according to Equation (17), the individual target detection probability at the Mathematically, the objective optimization function can be modeled as:Primarily, we take It can be seen from Equation (15) that individual Then, we haveIn the case of two SUs, when the sensing results of SU with higher received SNR is reported later, which gets more compressed samples under AFR, it will achieve better cooperative false alarm probability. The reporting turn of i-th SU is When OFR is used at FC, according to Equation (18), the individual target detection probability at the Similarly, we take two SUs to investigate the reporting scheduling strategy for the novel CSS framework. The cooperative false alarm probabilities of two SUs under OFR are, respectively, given as:Theoretically, Apparently, we haveIn the case of two SUs under OFR, it performs better in cooperative false alarm probability when higher SNR gets less compressed samples. Then we can attain When MFR is applied at the FC, the analysis process of optimal reporting scheduling strategy is similar to AFR and OFR above mentioned. For MFR, we consider the majority rule as i-th SU equivalent compressed sensing time amounts to l-th slot of i-th SU is given as: lth slot of i-th SU, l-th slot of i-th SU from PU and the signal power is constant over the lth slot of i-th SU and PU. The output of compressive detector is given as: i-th SU is obtained by summing As analyzed in the novel CSS scheme, the Note that i-th SU is given as:From now on, we call the above proposed scheme spatiotemporal CSS (ST-CSS), which combines the space diversity gain with time diversity gain. Thus, the false alarm and detection probabilities of the weighted ST-CSS at Combining Equation (49) with Equation (50), In order to achieve the maximal spectrum utilization efficiency for the proposed novel ST-CSS scheme, we seek to minimize For a given In order to minimize i-th SU, combining Equations (52) and (54), the minimum probability of false alarm Then, with a given With counting hard fusion rule deployed at FC, the final probabilities of detection and false alarm of the proposed ST-CSS scheme are given by:Thus, for the proposed novel ST-CSS scheme with hard decision fusion rule, similar to Equation (20), and the latency In the scenario of mobile satellite communication confronted with the problem of frequency scarcity, the degraded spectrum utilization will result in a greatly decreasing communication capacity or even interruption for mobile satellite service. In the proposed novel ST-CSS scheme, the interference to PU caused by the secondary network mainly results from missed detection of PU, a high probability of detection is required to ensure tolerable interference caused by SUs. For example, in IEEE 802.22 WRAN, the target detection probability is selected to be 90% for an SNR of \u221220 dB for the primary signal at SU . We focuTheorem\u00a04.is a decreasing function ofProof.For the given According to Equations (49) and (50), we have:Ultimately, we have The maximal spectral efficiency of the proposed novel ST-CSS scheme can be achieved if and only if when the following relationship establishes:Algorithm 1. Optimal sensing settings that maximize the spectral efficiency.Input:\u2003\u2003\u2003i-th SU, \u2003\u2003\u2003i-th SU from PU, \u2003\u2003\u2003ForInitiation:While: \u2003do\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u2003End While\u2003For:\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u2003Calculate End for\u2003End forCompare Output:In this section, we propose an algorithm based on Equations (55)\u2013(63) to find the maximal spectral efficiency of the proposed novel ST-CSS scheme with its corresponding optimal reporting scheduling strategy when N = 1000, and compressed domain sampling frequency is sf = 10 KHz. The PU\u2019s power is i-th SU g = 0 dB. For the novel CSS scheme, the frame duration is T = 200 ms, in which the single reporting duration rt = 1 ms when the information transmission rate from SU to FC is assumed to be 1 kbps. The chosen LEO height is h = 1000 km, and the feedback slot is approximately given as t0 = 10 ms taking space propagation delay and final decision-making delay into account.This section provides extensive computer simulations carried out jointly using STK and MATLAB to evaluate the performance of SU spectral efficiency for the proposed spectrum sensing scheme. For analysis, we have considered the number of cooperating SUs is \u22121\u00b7Hz\u22121 when target detection probability is 0.8, while it is only 0.8 Bits\u00b7s\u22121\u00b7Hz\u22121 and 0.5 Bits\u00b7s\u22121\u00b7Hz\u22121 when target detection probability is 0.9 and 0.95, respectively, which denotes that SU spectrum utilization decreases with debasement of the interference to PU. It can also be seen that optimal sensing time ms to achieve the maximal spectral efficiency; when SNR = 0 dB, ms to achieve the maximal spectral efficiency.i-th SU is set to be {\u221220 dB, \u221216 dB, \u221212 dB, \u22128 dB}. The first scheduling strategy is the descending sort of received In order to achieve a tradeoff between interference to PU and spectral efficiency, the target cooperative detection probability rt = 1 ms, and approximately 1.178 and 1.032 when rt = 2 ms. In the case of rt = 1 ms, and approximately 0.934 and 0.896 when rt = 2 ms, respectively. Thus, the novel ST-CSS scheme can achieve a significant improvement on spectral efficiency than that of the general CSS scheme. Additionally, it is clear that more time spending for reporting sensing results to FC predicts lower spectral efficiency, especially for the general CSS. This is because in the conventional CSS structure, there is less time for transmission when allocates more time for reporting for the fixed frame length. However, in the framework of the novel ST-CSS scheme, the sensing performance can be further enhanced with extended equivalent sensing duration, which can compensate the loss of average throughput resulted from the less transmission time. It is further summarized that the proposed ST-CSS scheme is less sensitive to reporting time and significantly improves SU spectral efficiency.In order to evaluate the energy consumption of the proposed ST-CSS method in scenario of energy limited CR system, In this paper, we consider the spectrum scarcity issue in the scenario of LEO-based mobile satellite systems and investigate the joint optimization of sensing time and hard counting fusion schemes to maximize the spectral efficiency subject to sufficient protection to PU. We formulate the wideband compressed detection model based on the allocation of L/S band for mobile satellite services promulgated by ITU-RR. It is proven that spectral efficiency is the unimodal function of sensing time, and there indeed exists one optimal sensing time that maximizes the spectral efficiency. In order to make full use of the reporting slot, we propose the novel wideband cooperative spectrum sensing (CSS) framework considering the particular characteristics of LEO systems in which each SU reporting duration has been utilized for its following SUs\u2019 sensing. On this basis, we derive the optimal reporting scheduling strategy of hard counting fusion rules with the consideration of different sensing SNR at SU in the novel CSS framework. Furthermore, the novel ST-CSS scheme is also presented for time-varying sensing environment to synchronously achieve time and space diversity gain. Analytical and simulation results provided the explicit impacts of target detection probability, number of cooperating SU, reporting time and the counting fusion rules on the spectral efficiency. The proposed scheme with optimal sensing settings achieved higher spectral efficiency compared to general CSS scheme and it is indeed needed in the scenario of spectrum scarce applications for LEO-based MSSs.The methods in this paper can be used in improving cognitive satellite systems in the aspect of spectral efficiency. However, some practical conditions, such as the imperfect reporting link, should be considered to make our work stronger. In addition, the tradeoff between spectral efficiency and latency will be another future work for the sake of PU\u2019s random arrival and departure within one frame length in the LEO-based mobile satellite systems with high traffic."}
+{"text": "Here, we employed a temporal reproduction task with a single motor response at a point in time with and without auditory feedback. This task limits participants to reducing errors by employing auditory feedback in a post hoc manner. Additionally, the participants were asked to judge the self-produced timing in this task. The results showed that, in the presence of auditory feedback, the participants exhibited smaller variability and bias in terms of temporal reproduction and the perception of self-produced time intervals in the subsecond range but not in the suprasecond range. Furthermore, in the presence of auditory feedback, the positive serial dependency of temporal reproduction, which is the tendency of reproduced intervals to be similar to those in adjacent trials, was reduced in the subsecond range but not in the suprasecond range. These results suggest that auditory feedback assists the post hoc error correction of temporal reproduction, and the perception of self-produced time intervals in the subsecond range.We examined whether auditory feedback assists the  Timing is essential during various activities, such as performing music and playing sports. In the timing literature, bias and variance in timing tasks have been utilized to construct models of temporal behavior . Therefore, unlike the temporal reproduction task used by post hoc error correction. If auditory feedback has a role in post hoc correction, the bias and/or variability in our temporal reproduction task would be reduced by the presence of auditory feedback.The remaining questions as regards the role of auditory feedback in temporal reproduction are whether immediate auditory feedback assists post hoc error correction.We also analyzed the lag-1 autocorrelation in the temporal reproduction task. This autocorrelation is the correlation between the produced intervals of trials and those of the next trials. A negative value means that the next produced interval of a larger  produced interval tends to be smaller (larger), suggesting overcorrection of error. Conversely, a positive value means that produced intervals tend to be similar in value to those in adjacent trials, suggesting memorized error and/or small correction. Therefore, this measure provides evidence as to whether or not auditory feedback affects the relationship between adjacent trials. In studies of repetitive tapping, it has been stated that auditory feedback negatively affects the lag-1 autocorrelation of produced intervals . Unlike Moreover, we examined whether the effect of auditory feedback is dependent on target duration in the subsecond and suprasecond ranges. Based on psychophysical, pharmacological, neuroimaging investigations it has been proposed that there are distinct timing mechanisms between these ranges e.g., . One claAdditionally, we measured the variability and bias of the perception of self-produced time intervals in a temporal reproduction task. Although the perception of self-produced time intervals will always accompany temporal reproduction, its properties have only been recently and as yet sparsely explored. Sixteen individuals  participated in the experiment. All participants had normal hearing. They gave written informed consent and were paid for their participation. This study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics and Safety Committees of NTT Communication Science Laboratories . The data for 2 of the 16 participants were excluded because of their reversed psychometric function . The data obtained from the remaining 14 participants  were analyzed in terms of perception performance.The experiment was conducted in a sound-insulated booth. Stimulus presentation and data acquisition were performed by a computer  using MATLAB 8.1 (The MathWorks) and Psychophysics Toolbox Version 3 . The stiFigure 1) \u00d7 2 . Participants were asked to listen to three successive tones  with two base intervals, and then immediately press a button (shift key) to create a subjectively isochronous interval between the button-press and the last tone of the three previously presented tones. In the auditory-feedback condition, a feedback tone was presented immediately when the participant pressed the button, whereas in the no-auditory-feedback condition, the feedback tone was not presented. The property of feedback tones was identical with the three successive tones. The participants were also asked to judge whether the produced timing was earlier or later than the subjectively isochronous timing. The judgment was indicated by pressing one of the two buttons . Except for the first trial of each session, each trial started 1 s after the judgment. The first trial of each session was initiated by the experimenter. The beginning of a trial was indicated by a tone . After a 6-s silent period from the beginning of a trial, the three successive tones that indicate the base interval were presented.The experiment was conducted under four conditions: 2  per condition were analyzed. All the sessions for a given condition were completed, and then the feedback condition was alternated. The base interval condition was alternated after the completion of all the sessions for the two feedback conditions of a base interval condition. The order of the feedback and base interval conditions were counterbalanced across the participants.First, we excluded the outliers of the produced intervals for each condition and participant. We defined the outliers as below two inter-quartile ranges (IQRs) from the first quartile or above two IQRs from the third quartile.To evaluate the reproduction performance, we calculated the SD, mean, and lag-1 autocorrelation of the produced intervals for each condition and participant. The autocorrelation was computed in each session, and then averaged across all sessions.To evaluate perception performance, we estimated the probability function of \u2018later\u2019 responses to a produced interval  in each condition and participant. To estimate the psychometric function, we used a logistic regression undertaken with the maximal likelihood method for the data of pair of judgment and produced interval. The produced interval at a 50% judgment rate was defined as a point of subjective equality (PSE), which indicates the criterion of judgments. Half of the difference between the produced intervals at judgment rates of 25 and 75% was defined as a just noticeable difference (JND), which indicates the variability of judgments. Two participants were excluded from the analysis because their psychometric functions were reversed in at least one condition.Figure 2A shows the standard deviations of produced intervals divided by their base interval. The results imply that motor variability for a 0.5-s base interval was less variable in the auditory-feedback condition than in the no-auditory-feedback condition , whereas that for the 3.2-s base interval was comparable in the feedback conditions . A two-way  repeated-measures ANOVA indicated a marginally significant main effect of the base interval . This result implies that temporal reproduction in a trial is performed with a produced interval memorized in the previous trial.We investigated whether auditory feedback affects the performance of temporal reproduction with a single motor response at a point in time, and the perception of self-produced time intervals in the subsecond and suprasecond ranges. The results indicated that auditory feedback improves both temporal reproduction and the perception of self-produced time intervals in the subsecond range but not in the suprasecond range. The results also indicated that auditory feedback reduces the serial dependency  of temporal reproduction in the subsecond range but not in the suprasecond range. Furthermore, we found that the serial dependency was significantly positive in all conditions except under the auditory-feedback and 0.5-s base interval conditions. In addition, the substantial deterioration in the perception of self-produced timing caused in absence of auditory feedback was limited to the subsecond range. This result clearly indicates the invalidity of the sensory attenuation explanation for the deteriorated perception of self-produced timing in the suprasecond range.The reduced variability and bias of temporal reproduction and the perception of self-produced time intervals by auditory feedback are consistent with previous studies indicating that the perception of intermodal intervals is less stable than that of intramodal intervals (for review see This improvement caused by auditory feedback in the subsecond range must be involved in the stabilization of the internal representation of self-produced time intervals by auditory feedback. There are several possible reasons for this stabilization. One possibility is the additional noise originating from the translation of a modal representation into an amodal representation to calculate the intermodal interval, when auditory feedback is not provided. This consideration is based on the notion that modality-specific and amodal mechanisms underlie the processing of subsecond timing. Another possibility is multisensory integration, which can be considered another aspect of amodal mechanisms. It has been suggested that human beings combine information from multisensory modalities to reduce the variability of estimates based on the reliabilities of each multisensory modality in time perception (for review see The comparable variability of temporal reproduction and the perception of self-produced time intervals in the feedback conditions in the suprasecond range does not necessarily mean that auditory feedback has no effect in the suprasecond range. It can be considered that there are duration-dependent and duration-independent noises for motor and perceptual timing . If the To reduce the variability and bias of temporal reproduction, the representation of self-produced time intervals must be stabilized and the temporal reproduction must be performed using the stabilized representation. The reduced lag-1 autocorrelation with auditory feedback in the subsecond range supports this idea. When participants know that a produced interval in a trial is overproduced, they will try to make the next produced interval shorter than the previous one. Therefore, error correction must lead to the reduction of the lag-1 autocorrelation of produced intervals.In addition, the positive lag-1 autocorrelation of produced intervals was found in all conditions except the auditory-feedback and subsecond conditions. This result implies that the self-produced time interval information is used not only when auditory feedback is available but also when it is not available. This idea is plausible because computing for temporal reproduction with the memory of previous intervals will lead to a greater reduction in variability and more computational loss than that without this memory. Consistent with this idea, post hoc error correction in temporal reproduction, and reduces the variability and bias of temporal reproduction and the perception of self-produced time intervals in the subsecond range. Our findings provide an insight how sensory feedback contributes to reducing the errors of motor and perceptual timing. We consider that they will contribute to the precise modeling of temporal processing.We examined the role of auditory feedback in temporal reproduction, and in the perception of self-produced intervals. The current study demonstrated that auditory feedback assists KM and MK conceived and designed the study. KM performed testing, data collection and data analysis. KM drafted the paper. MK provided critical revisions. KM and MK approved the final version of the paper for submission.MK is an employee of Communication Science Laboratories, which is a basic-science research section of Nippon Telegraph and Telephone Corporation. The other author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Improving transmission reliability is a crucial challenge for Wireless Body Area Networks (WBANs) because of the instability of channel conditions and the stringent Packet Loss Ratio (PLR) requirement for many WBANs applications. On the other hand, limited by the size of WBAN nodes, the energy consumption of WBAN nodes should be minimized. In this paper, we jointly consider transmission power control, dynamic slot scheduling and two-hop cooperative mechanism and propose an Autocorrelation-based Adaptive Transmission (AAT) scheme that achieves a better trade-off between transmission reliability and energy consumption for WBAN systems. The new scheme is designed to be compatible with IEEE 802.15.6. We evaluated the performance of the newly proposed scheme by importing the real channel datasets into our simulation model. Simulation results demonstrate that the AAT method can effectively improve the transmission reliability while reducing the energy consumption. We also provide the performance evaluation from three perspectives, namely packet error ratio, energy consumption and energy efficiency, and provide recommendations on the application of the two-hop cooperative mechanism associated with the proposed AAT in the contexts of WBANs. Wireless body area networks (WBANs) are radio networks of sensors and/or actuators, placed on, in or around the human body, and represent the latest generation of personal area networks . WBANs cOn the other hand, to ensure the comfortable and unobtrusive deployment of wireless devices in/on the human body, WBAN sensor or actuator nodes are required to have a small size, which obviously limits the battery capacity and the WBAN system lifespan. Meanwhile, in many WBAN applications, changing or replacing the battery is infeasible. For example, a pacemaker or a glucose monitor would require a lifetime lasting more than five years . For thiTPC refers to an adaptive method which optimizes the transmission power (Tx power) based on the channel conditions or QoS requirements. In fact, as a classical research topic, TPC has been extensively explored in the context of cellular networks and Wireless Sensor Networks (WSN). However, due to the presence of the human body, the extremely low power requirement and the need for high flexibility of transmission protocol designs, simple adoption of the TPC methods designed for other networks is not appropriate. For example, many existing TPC methods require the transmitting node to stay in active state to track the real-time channel condition to optimize the Tx power. However, in WBANs, the transmitter usually belongs to the sensor or actuator node, which is expected to be in sleep mode most of the time to save energy. Besides, to avoid the negative impact of electromagnetic radiation on the human body , the TxAmong the TPC methods proposed for WBANs, adjusting the Tx power based on the channel condition to achieve a better trade-off between energy consumption and transmission reliability is considered as one of the major categories. The work presented in  is consiposed in ,12,13,14posed in , the autposed in  exploredposed in , Yong etposed in , Wang etposed in  and Zhaoposed in  exploredposed in ,23,24,25posed in ,27. By tAlthough applying a TPC approach at the sensor side can reduce energy consumption, TPC alone may not be sufficient to meet the stringent transmission reliability requirement of some WBAN applications, e.g., m-medical or e-health WBAN systems. In recent years, there are some works exploring relay-aided TPC methods in WBANs to achieve a better balance between energy consumption and transmission reliability. Zhang and Zhang  proposedBesides, integrating Network Coding (NC) and cooperative communication in the context of WBANs is an emerging research topic. One category of these works only considers a two-hop star topology for data streaming from sensor or actuator nodes to the hub ,33,34,35In this paper, we focus on the adaptive transmission protocol in real daily life WBAN scenarios. Inspired by the autocorrelation analyses presented in the literature ,42,43, wMotivated by the significant autocorrelation feature of on-body channels, we propose an adaptive TPC algorithm. The new TPC method optimizes the transmission power level based on the autocorrelation characteristic between two consecutive superframes.Since DSS operations are carried out at the hub, a DSS mechanism is incorporated into the newly proposed TPC method to further optimize the transmission power.The combination of TPC and two-hop relay cooperation is explored. We detail the protocol implementation on the basis of IEEE 802.15.6, including relay node selection, relayed node selection and adaptive allocation of relay slots (these terms are explained below).The performance of the proposed protocol was evaluated using the measured channel data collected from the real daily scenarios. The evaluation results show that the newly proposed protocol achieves remarkably lower PLR (Packet Loss Ratio) while the energy consumption remains at a low level.The main contributions of this paper are summarized as follows.The rest of the paper is organized as follows. In In this section, the network architecture, channel model, and the energy consumption model are described.n on-body sensor nodes  to transmit data to the hub, and the selected relay node has its SRI  to transmit the packets. As for the exchange of management and control packets, the CSMA/CA access method is performed before the SUIs of sensor nodes. Specifically, we adopt the beacon mode with superframes in the IEEE 802.15.6 standard [As shown in It is well-known that the Packet Loss Ratio (PLR) in WBAN system is affected by many factors, including environment factors  and technical factors . However, as the focus of this paper is on the influence of WBAN channel, we employ a ceteris paribus assumption with respect to other technical factors that do not affect the channel. Besides, it is not realistic to consider all environment factors when we want to evaluate the quality of wireless channels. Hence, similar to Xiao et al. , we choonnel cf. . SpecifiDue to high variability of on-body channels, neither distance-based nor other formula-based methods seems to be sufficient to describe the on-body channel condition, especially in the dynamic scenarios. Therefore, adopting channel datasets collected from the real daily scenarios to model the on-body channel is a better choice. The portable wireless transceivers introduced in  are usedCorresponding to Our portable wireless transceivers work in a half-duplex mode, and the path losses of all direct channels are recorded by broadcasting sample packets from the hub to the sensors. The broadcasting method has the advantage that the path loss data for different links are synchronous, but it also means the path loss information between different sensors themselves is missing in the channel datasets. Accordingly, the relay channels are not represented in these datasets. In this paper, we mainly focus on the one-hop channel, i.e., the channel between the sensor and the hub, and the channel from one sensor to another sensor is considered only when the second sensor is chosen as the relay node. Therefore, we adopt a relatively straightforward method to represent the channel condition from the relayed node to the relay node. One parameter named There are a number of energy consumption models in the literature which can be applied to WBANs, e.g., the models used in ,54,55,56x to State y, and Because the MAC layer works in the beacon mode with superframes and the superframe length is defined as a fixed parameter in IEEE 802.15.6, taking the energy consumption in one superframe is enough to explain the energy consumption model. In each superframe, the energy consumption for one sensor node consists of four parts: the energy consumption in the Tx state, the energy consumption in the Rx state, the energy consumption in the sleep state, and the energy consumption used to complete the state transition. Hence, the energy consumption in one superframe for one sensor can be expressed as followsR (Kbps) refers to the transmission rate, which is assumed to be the same for all sensors. We first detail the calculation of If we denote the number of relaying packets as Next, we detail the calculation of Partitioned by the plus sign, Equation  has two Partitioned by the plus signs, the first two parts are similar to the Equation , which rNext, we calculate the energy consumption of the state transition. We first detail this kind of energy consumption for a sensor node, which can be expressed as followsPartitioned by the plus signs, the first and the last two parts in Equation  represenThe first eight lines of Equation  represenEquation .Finally, the energy consumption in the sleep state can be expressed asIn this study, we paper the energy consumption parameters from the CC2420 radio chip . The Tx The hub keeps track of the channel conditions from all sensor nodes, and then predicts the channel condition in the next TDMA round based on a temporal autocorrelation model.Based on these predicted channel conditions, the hub adjusts the transmission power and reschedules the SUI order for all sensors.After configuring the transmission power and SUI order, some channels may still be predicted to be in an outage state. In this case, the hub selects the relay and relayed nodes based on the predicted channel conditions, and then schedules the relay slots adaptively.Given that the hub is typically more powerful than the sensor nodes in terms of storage and computational resources, it is desirable to push more control and computational tasks to the hub. In the proposed AAT scheme, except for the ability of changing transmission power and optional relaying at the sensor side, the hub side implements all control and calculation operations, including channel condition prediction, transmission power level decision, adaptive relaying scheduling, etc. Specifically, AAT scheme consists of three main steps, which are summarized as follows:The temporal autocorrelation model used to predict the channel condition is firstly introduced. Suppose the sensor node S and Meanwhile, as proved in ,59,60,61roved in , the varNow, the expected value of the channel gain in the next superframe Equation  can be cN is the sample size. In fact, there exists a trade-off between accuracy and timeliness when choosing the sample size. On the one hand, with the increase of sample size, more data points are utilized to calculate the channel autocorrelations, which may appear to result in a more accurate estimation of autocorrelation. On the other hand, since the sampling frequency is fixed, a bigger sample size means these data points are collected from a wider time span. However, as demonstrated in [N consecutive superframes.To estimate the channel condition in the next superframe using Equation , the folrated in , on-bodyrated in , i.e., rAt the beginning of each superframe, the hub performs the calculations of Algorithm 1: Adaptive transmission power control method.n sensor nodes. n is the number of sensors.Algorithm 1 requires the following four inputs: the last known channel gain, autocorrelation coefficients between two consecutive superframes, the sample means, and the sample standard deviations. These inputs are used to schedule the SUI order and adapt the transmission power. Correspondingly, there are two output arrays: transmission power array  effectively could reduce unnecessary energy consumption. Second, relay node selection is of great importance in terms of the system lifetime. If the relay node is selected simply based on the channel condition or the distance to the hub node, some sensor nodes may run out of energy much more quickly than the other nodes due to their heavy traffic load and the whole system lifetime is shortened. Considering the aforementioned two aspects, the following two-hop cooperative mechanism is proposed.In short, at the start of each beacon period, the hub estimates the channel gains for each link based on the temporal autocorrelation model and performs Algorithm 1 to optimize the transmission power and SUI scheduling for the next superframe. Moreover, if the two-hop cooperative option is selected, the relay and relayed nodes are selected to further improve the transmission reliability. Then, all these configurations are inserted into the beacon packet, which is broadcast to all sensor nodes. Finally, the sensor nodes adapt the transmission parameters based on the configuration information in the beacon packet.Static scheme: The hub does not control the transmission power level of the sensor nodes. The transmission power is fixed at a predefined value, which is selected to be at 0 dBm. Besides, the static scheme does not change the slot scheduling of sensors. In other words, the permutation of SUIs is fixed after assigned randomly in the first superframe.Xiao\u2019s scheme: This TPC method, as proposed in [Conservative, Balance and Aggressive, are designed for different applications with different requirements in terms of PLR and energy consumption. Considering the significant autocorrelation of on-body channels, the Balance scheme with posed in , adapts AAT scheme: This is the adaptive transmission scheme proposed in this paper. Unlike Xiao\u2019s scheme, a temporal autocorrelation model is adopted to adjust the weight between the latest channel gain and the sample mean of historical channel gains. Moreover, the permutation of SUIs is also taken into account to optimize the transmission power margin. The two margin parameters, i.e., Ideal scheme: In the ideal scheme, the hub knows all the channel gain information for the next superframe. Accordingly, the transmission power could be set at the most suitable level, which not only avoids unnecessary energy wastage but also keeps the PLR at the lowest level. This scheme was considered to explore the upper bound of TPC approaches. Note that this method is infeasible for real WBAN systems because it assumes perfect prediction of future states.In this section, the performance of AAT is evaluated through simulations and compared with other transmission methods. To improve the authenticity of performance evaluation, the 16 channel datasets , energy consumption and energy efficiency. Specifically, the PLR presents the average PLR value of the five sensor nodes and energy consumption refers to the sum energy consumed by the five sensors. Energy efficiency (in KB/J) is defined as the ratio of data received in the hub side and the total power consumed by the five sensors. In our simulation model, the Rx sensitivity was configured as a variable value  to evaluate the performance of AAT in a range of Rx sensitivity values.We first considered the situations that the two-hop cooperative transmission is not used. The newly proposed AAT scheme was compared with the static or other TPC schemes by importing an example channel dataset, i.e., In All aforementioned figures are provided for the case in which only one channel dataset, i.e., As cooperative transmission and NC technologies are considered as promising methods for increasing the transmission reliability, we evaluated the performances when two-hop cooperation is considered. Specifically, we provide the performance comparison when two types of cooperative mechanisms are adopted. The first cooperative mechanism does not use NC, and is called non-NC cooperative mechanism. The sensor node(s) predicted to be in outage in the current beacon period is (are) selected as the relayed node(s), and one relay node is chosen randomly from the other sensor nodes. The second cooperative mechanism (denoted as NC cooperative mechanism) explores the RLNC (Random Linear Network Coding) technology in the relay node. Specifically, the hub selects one relay node, which stays in the Rx state during the whole DTP period to receive the data packets from all other sensor nodes. Then, the relay node performs the RLNC operation on all received packets to generate NC packets. These NC packets are sent to the hub in RTP. An advantage of the NC cooperative mechanism is that the hub does not specify the relayed node(s), and data packets from all sensor nodes have the chance to be recovered by the successful two-hop cooperative transmission. For instance, if the sensor node transmits two packets during its SUI and the RTP is configured as two times of the length of one SUI, then up to four packets in the DTP can be recovered by the successful transmission of four NC packets. However, the NC cooperative mechanism requires the relay node to stay in the active state during the entire DTP period, which increases its energy consumption.By importing the channel dataset Next, the energy consumption results are illustrated in Similar to the cases without two-hop cooperation, we also provide the overall energy efficiency results over all channel datasets collected from real WBAN scenarios, i.e., Based on the above simulation results, we have the following observations. Firstly, our newly proposed AAT scheme is capable of improving the transmission reliability and at the same time reducing the energy consumption. Compared to the Xiao\u2019s TPC scheme, AAT improves the performance in terms of PLR, energy consumption, and energy efficiency. Secondly, we found that, in the star topology network, using NC technology to assist the uplink two-hop transmission may not be a good choice. The newly proposed non-NC cooperative mechanism achieves a better PLR performance while consuming less energy.In this paper, we jointly consider transmission power control, dynamic slot scheduling and two-hop cooperative mechanism to achieve a better trade-off between transmission reliability and energy consumption. Motivated by the significant autocorrelation characteristic of on-body channels in the daily WBAN scenarios, we propose an Autocorrelation-based Adaptive Transmission (AAT) scheme that uses a temporal autocorrelation model to predict channel conditions. Then, the estimated channel conditions are used to optimize the transmission power level and the transmission order of all sensor nodes for the next superframe. AAT scheme is designed to be compatible with IEEE 802.15.6. We also evaluate the performance of the newly proposed scheme by importing the channel datasets collected from real WBAN daily scenarios into our simulation model. Simulation results demonstrate that the proposed method can effectively improve the transmission reliability while reducing the energy consumption. Moreover, we provide the performance evaluation when two-hop cooperative transmission is associated with the proposed AAT to further reduce the PLR. Two types of cooperative mechanisms are compared, i.e., the non-NC mechanism and the NC cooperative mechanism. Based on the evaluation results, the NC cooperative mechanism does not exhibit the advantage in all performance metrics, i.e., PLR, energy consumption, and energy efficiency. Hence, using NC technology to assist the uplink two-hop transmission may not be practical in the context of WBANs."}
+{"text": "Asterophila, a genus of endoparasitic gastropod in the family Eulimidae, forms cysts in the arms and central discs of asteroid sea stars. There are currently four known species in this genus, one of which has been described from the Antarctic Peninsula (A. perknasteri). This study employs molecular and morphological data to investigate the diversity of Asterophila in Antarctica and explore cophylogenetic patterns between host and parasite.Marine invertebrates are abundant and diverse on the continental shelf in Antarctica, but little is known about their parasitic counterparts. Endoparasites are especially understudied because they often possess highly modified body plans that pose problems for their identification. Asterophila and subsequent species-delimitation analysis uncovered nine well-supported putative species, eight of which are new to science. Most Asterophila species were found on a single host species, but four species were found on multiple hosts from one or two closely related genera, showing phylogenetic conservatism of host use. Both distance-based and event-based cophylogenetic analyses uncovered a strong signal of coevolution in this system, but most associations were explained by non-cospeciation events.A maximum-likelihood phylogeny of Asterophila and its asteroid hosts suggests that synchronous evolution may be rare even in obligate endoparasitic systems. The apparent restricted distribution of Asterophila from around the Scotia Arc may be an artefact of concentrated sampling in the area and a low obvious prevalence of infection. Given the richness of parasites on a global scale, their role in promoting host diversification, and the threat of their loss through coextinction, future work should continue to investigate parasite diversity and coevolution in vulnerable ecosystems.The prevalence of duplication and host-switching events in The online version of this article (10.1186/s12862-019-1499-8) contains supplementary material, which is available to authorized users. Antarctica is a unique geographic region that experiences extreme environmental conditions and ongoing threats from a changing climate, with some of the fastest warming waters globally e.g. , 2). His. His2]).Asterophila [A. japonica [A. perknasteri [A. rathbunasteri [A. japonica has been found in eight host species from four families and three orders [A. perknasteri has been found from three different Perknaster species [A. rathbunasteri has been found only from a single host  [erophila , a genusjaponica  from theknasteri  from Antunasteri  from Calunasteri . The leve orders  while A. species  and A. rornicus) . Becauseornicus) . Additioornicus) .Asterophila and asteroid hosts lends itself to exploring co-phylogenetic patterns and determining whether these groups are evolving in synchrony and which coevolutionary events are promoting diversification in this system. Early studies of coevolutionary biology found evidence for strict cospeciation between host and parasite  and it and lice , nematodand lice ), and soand lice ). Althouand lice , 26, mosand lice , 13).Asterophila, prompting us to explore diversity in this genus and investigate cophylogenetic patterns between parasites and hosts. This study employs molecular and morphological data for phylogenetic analysis and species delimitation. These phylogenetic methods shed light on an apparent radiation of Asterophila in Antarctica and aid in determining whether speciation in asteroid hosts has been paralleled by Asterophila. This work extends our knowledge of diversity in Asterophila and provides insight into the coevolutionary events promoting diversification in this system.Recent sampling in Antarctica uncovered a range of seastar hosts from different taxonomic groups that were parasitized by Asterophila parasites were recovered from 38 hosts forming large and noticeable cysts on the aboral side of the host in the arms or near the central disc. Asterophila was found between the epidermis and coelomic lining, with no affiliation to specific organs or body systems. This description is congruent with Sasaki et al. [Asterophila japonica in detail. Roughly 1500 potential hosts were collected in trawls on each cruise, and therefore an occurrence of only 38 infected hosts suggests the prevalence of parasitism in this group is low, though it may have been that only obvious infections were noted. Infected hosts and parasites were only recovered from near the Antarctic Peninsula, at Shetland Islands, Elephant Island, Shag Rocks and South Georgia  and 477 variable sites. The COI, 16S, and ANT markers were more variable among species than H3 and 28S. Despite some instances of low bootstrap support at interior nodes, there was still strong support for nine putative species-level entities within Asterophila (BS 93\u2013100), forming the PSH , while intraspecific distances ranged from 0 to 0.9%. An ancestral state reconstruction suggests that a Forcipulatida asteroid is the ancestral host for Asterophila (likelihood 68), with one transition to each of Paxillosida, Spinulosida, Valvatida and Velatida  the species-level identity of this host could not be validated. In all, we report Asterophila from five orders, seven families, ten genera, and 15 species of asteroid sea star.This study presents COI sequences for 35 out of 38 individual asteroid hosts with a final alignment of 700\u2009bp. Three hosts missing molecular data were excluded from cophylogenetic analyses, including two Asterophila species  and a total of nine larvae were examined and compared to larval morphologies in the existing literature  and measured 690\u2013720\u2009\u03bcm in A. perknasteri, 650\u2013690\u2009\u03bcm in A. sp. 3, and 540\u2013600\u2009\u03bcm in A. sp. 5. The shell width reported for A. perknasteri in this study corresponds to the size range reported in the original description of this species (650\u2013760\u2009\u03bcm) [A. perknasteri appears slightly deeper than both A. sp. 3 and A. sp. 5, although this character is not mentioned in the existing Asterophila descriptions. The larval shells of all species examined in this study appear smooth, with no sculpturing.Veliger larvae were recovered from individuals of three \u2013760\u2009\u03bcm) . MoreoveAsterophila was discovered in 38 hosts from a wide taxonomic breadth. Phylogenetic conservatism in host-use and host-specificity was observed in this system, with eight putative Asterophila species each discovered from a different host genus and one species discovered from two host genera  were found on multiple hosts, but these hosts were always closely related congeners  or confamilials  . Those species found parasitizing multiple hosts also had some of the largest sample sizes . The haplotype networks show some level of host partitioning for A. sp. 6 and A. perknasteri, demonstrated by a lack of haplotype sharing between individuals from different hosts and some clustering of haplotypes based on host species  Fig. a, confirAsterophila and 35 asteroid hosts were included in the cophylogenetic analysis, with the resulting cophylogenetic plot showing 57 individual associations  (Table P\u2009=\u20090.0001), rejecting the null hypothesis of random association (i.e. that hosts and parasites are evolving independently). A test of coevolution for the 57 associations with a Bonferroni correction revealed a total of 44 significant links (P\u2009<\u20090.05)  Table\u00a0.Fig. 5HAsterophila in Antarctica and increases the diversity of this group nearly tenfold, from one previously known species in the region (A. perknasteri) to a putative nine species. Although each of these putative nine species-level entities was strongly supported in the ML phylogeny, deeper relationships remain poorly supported. A lack of support in the Asterophila phylogeny, despite the use of both mitochondrial and nuclear markers that should be informative at both shallow and deep divergences, suggests that additional taxonomic and genomic sampling is required. Nevertheless, molecular data has been crucial for delimiting species boundaries in this genus. Each putative Asterophila species was recovered from a different host family, suggesting that host identity also serves as an informative character for species delimitation. The COI divergence between A. sp. 4 and A. sp. 5 is relatively low (1.9%) compared to interspecific distances for other molluscs , bu, buAsterAsterophila. These characters include an apical depression on the protoconch of A. japonica, rugose sculpture and a less depressed protoconch in A. rathbunasteri, and a large, smooth larval shell in A. perknasteri. The larval shells of two undescribed species examined in this study  appeared more similar in general shape to A. perknasteri than to either A. japonica or A. rathbunasteri but had a shallower umbilicus and appeared more depressed. Although larval shell size appeared to differ significantly between three Antarctic Asterophila species examined in this study, this variation in size may be related to differences in developmental stage and age of the larvae when they were retrieved from adult females. As such, there exists a deficit of informative morphological characters in this endoparasitic genus. Recent studies have also highlighted difficulties in using morphological characters for species delimitation in highly modified endoparasites , anAsterophila in the region. The retreat of populations into small, closely distributed glacial refugia around Antarctica would have facilitated host-switching events by bringing multiple potential hosts into proximity, as well as by driving some hosts to extinction [Some benthic marine invertebrate groups have radiated extensively in Antarctica, including isopods , dorid ntinction . It is aP\u2009<\u20090.05), with an increase to 44 following a Bonferroni correction. These results suggest a strong signal of coevolution, likely driven by host-switching speciation events that have produced congruent phylogenies, but the parasite phylogeny was poorly supported and the host phylogeny was based on a single mitochondrial marker. The different cost regimes in Jane produced different numbers of events, most notably ranging from zero cospeciation events in the first regime to 18 cospeciation events in Jane\u2019s default regime, suggesting that some regimes under-detect cospeciation events and that choosing a regime a priori may prove problematic. In any case, most events were explained by duplication and host-switching, suggesting that non-cospeciation events are primarily driving the coevolution of Asterophila and their hosts.Coevolution and cospeciation are often used synonymously in the literature, but the former simply refers to reciprocal evolution between two or more species and can involve several processes, mainly cospeciation, duplication, host-switching, loss of a lineage, or failure of a parasite to diverge . AlthougRecent work has highlighted the importance of non-cospeciation events in the diversification of endoparasites (e.g. endoparasitic nematodes and stick insect hosts ) and in Asterophila, including the presence of a planktonic larval phase, which results in greater dispersal potential and the ability to encounter new hosts [Asterophila co-occurring in Antarctica. The mechanism by which Asterophila larvae locate and occupy a host is currently unknown, but chemical cues from hosts induce settlement of planktonic larvae in other molluscs , with mibranchs ) and areon e.g. , 39, 41), 41AsterAsterophila species in this study were discovered from a single host genus, and always from a single host family, which differs from other endoparasites, including a species of splanchnotrophid copepod (Lomanoticola brevipes) that was recovered from five different nudibranch families [, A. sp. 1, 2, 4, 5, and 7 were all recovered from a single host species, while A. sp. 3, A. sp. 6, and A. perknasteri were recovered from two to four congeners, and A. sp. 8 was recovered from two genera in Solasteridae. The haplotype networks for A. sp. 6 and A. perknasteri show a complete lack of haplotype sharing between individuals from different hosts, indicating potential host partitioning and incipient host-shift speciation, but additional samples are needed to validate these patterns. In any case, multihost Asterophila species were recovered from phylogenetically-related hosts, as demonstrated by lower cophenetic distances between host pairs than between all other hosts, and these hosts often had the highest sample size, illustrating that the recovery of hosts, and ultimately parasite richness, may be linked to sampling effort ..AsterophAsterophila. Both the ML phylogeny and transformation suggest phylogenetic conservatism of host use, although further resolution of the Asterophila phylogeny, possibly through the use of a reduced representation library, will be crucial to better understanding these patterns. Phylogenetic conservatism of host use has been shown in other marine parasites, including in myzostomid annelids and their echinoderm hosts [Asterophila, but with only moderate support [Asterophila species, which will aid in clarifying their coevolutionary relationships. Future work should also look to sample from greater depths as Asterophila has yet to be reported from two orders of deep-sea asteroids. Lastly, previous studies have suggested that polyparasitism, where hosts are infected by multiple parasite species, is prevalent in natural populations [Asterophila species from each host individual.Poulin  suggestsrm hosts . An ance support . Continuulations \u201349, but A. perknasteri [Asterophila has now been reported from the South Shetland Islands, Elephant Islands, South Orkney Islands, South Georgia, and Shag Rocks, comprising the tip of the Antarctic Peninsula and parts of the Scotia Arc. Eight putative Asterophila species in this study were found co-distributed along the South Shetland Islands and Elephant Island, and a ninth putative species was found at Shag Rocks and South Georgia, but the absence of a single Asterophila species at both locations suggests their ranges are restricted. The octopus Pareledone charcoti also appears restricted to the South Shetland Islands and although this distribution may be due to deep-water and currents restricting gene flow in the area, it is also a consequence of a lack of a planktonic larval phase [Asterophila does possess. A single host with parasitic cyst was imaged from South Orkney but this specimen was not recovered from the collection for analysis. This host appears to be Diplasterias however, suggesting that the accompanying parasite could be Asterophila sp. 4. If so, this would extend the distribution of A. sp. 4 from the northern Shetland Islands and Elephant Island into South Orkney Islands, although additional samples and molecular analysis are needed to confirm this result. Moreover, some marine taxa show distinct genetic differences between populations at Shag Rocks and South Georgia , despittebrates , warrantAsterophila at many locations outside the Scotia Arc is surprising. Salmen et al. [Asterophila may also indicate that host-specific parasites are able to adapt to new hosts in sympatry, as suggested by Anton et al. [Asterophila in this study could also be an artefact of concentrated sampling around the Antarctic Peninsula and Scotia Arc, combined with a low prevalence of parasitism in this group. For instance, Sasaki et al. [A. japonica infection ranged from just 2.2 to 17.5% in Japan, and Schiaparelli et al. [Bathycrinola tumidula on the crinoid Notocrinus virilis in Antarctica. Given that hundreds of asteroids were examined in this study, and parasites were only discovered from 38 hosts, one could assume that the prevalence of parasitism in this system is comparably low and thus intensive sampling will be required for a more complete understanding of the distribution of this genus in Antarctica. Sampling effort at present has been focused around the Antarctic Peninsula and Scotia Arc, likely skewing the reported distribution of Asterophila in this study. In fact, specialist host-parasite systems rarely exhibit a broad distribution [Asterophila species. Alternatively, the use of different trawling equipment among research cruises may have negatively impacted host and parasite recovery in this study, ultimately skewing rates of parasitism. Lastly, although adult Asterophila form large, noticeable cysts in asteroid seastars, it is likely that recently settled juveniles would be less detectable in hosts. In any case, the marine fauna of the Antarctic Peninsula and surrounding subantarctic islands has been shown to be incredibly diverse , and thse (e.g. ) the conse , coupleAsterophila since the initial description in 1994, uncovering eight putative new species, adding several new host genera, and expanding its distribution from the tip of the Antarctic Peninsula to Shag Rocks and South Georgia. This work also explores cophylogenetic patterns between Asterophila and their hosts, demonstrating that non-cospeciation events are primarily driving diversification in this system. Four of nine Asterophila species were found on multiple hosts, but these hosts were always phylogenetically related, being from one or two genera from the same family. Relationships between Asterophila species remain poorly resolved and obtaining molecular data for other congeners will be essential to better understand interspecific relationships in this genus. Additionally, a more robust understanding of the life history of these animals is needed, particularly regarding larval development and dispersal potential as well as the biochemical pathway responsible for host recognition. The distribution of Asterophila reported in this study is likely not reflective of its true range, due to both under-sampling in most of Antarctica and a potentially low rate of infection. Intensive sampling is needed in east Antarctica and the deep sea, the latter of which is particularly interesting as Asterophila has never been recorded from two orders of deep-sea asteroids. Furthermore, future work should continue to investigate cophylogenetic patterns in marine invertebrates and their parasitic counterparts, as these relationships are currently underrepresented in the literature.This is the first study addressing Antarctic Asterophila specimens from 38 asteroid hosts were collected on three cruises  in Antarctica  and remaining specimens and tissue samples are housed at both the Benthic Invertebrate Collection at Scripps Institution of Oceanography (BIC-SIO) and the Western Australian Museum (WAM). Both host and parasite specimens were identified in the field and these identifications were later confirmed and corrected by comparing cytochrome c oxidase subunit I (COI) sequences against the Barcode of Life Data System (BOLD) and NCBI BLAST databases.A total of 61 Asterophila and tube feet from asteroid hosts were used for DNA extraction with DNeasy spin columns (Qiagen). Some molecular methods employed in this study have been previously described by [Preserved tissue from the pseudopalium of ribed by . PrimersNiso matsumotoi was selected as an outgroup for Asterophila phylogenetic analysis based on a larger phylogeny of Eulimidae , and these sequences were obtained from GenBank . The phylogenetic methods employed in this study have been previously described by [ribed by . A datasribed by  using a ribed by  was usedribed by  and Autoribed by  algorithribed by .p-values from the ParaFit analysis as two ParaFitLink tests were conducted for each comparison. The Jane and ParaFit analyses were chosen as they allow for uneven numbers of parasites and hosts, including multihost parasites. Lastly, for Asterophila species that were discovered from multiple hosts, TCS haplotype networks [Asterophila species were parasitizing phylogenetically related hosts, cophenetic distances between host pairs (hosts parasitized by the same Asterophila species) were compared to cophenetic distances between all other hosts using a Wilcoxon 2-sample rank sum test.Both distance-based and event-based cophylogenetic analyses were conducted on host and parasite phylogenies. A host phylogeny was generated using COI sequences from asteroids that were found with parasites. The ML tree was generated in RAxML  implemennetworks  with COInetworks  for explAsterophila species highlight only a few diagnostic morphological characters in this group, some of which are based on larval shell characters [Published descriptions of known aracters . As sucharacters  in RStudAdditional file 1:Figure S1. Primary species hypothesis. ML phylogeny (COI\u2009+\u200916S\u2009+\u2009H3\u2009+\u200928S\u2009+\u2009ANT) of Asterophila with highly supported least inclusive clades (and singletons) representing the PSH. Hash marks denote that the branch has been truncated to one half of its original length. (TIF 474 kb)Additional file 2:Figure S2. Host type. Ancestral state reconstruction (ML) for host type using asteroid order and an Mk1 likelihood model. Asterisks mark nodes with a likelihood of >\u200999%, with values less than this provided at nodes. (TIF 1268 kb)Additional file 3:Table S1. Specimen details. Asterophila specimens collected and analysed in this study, along with corresponding host details, locality information, and GenBank accession numbers. Hosts marked with an asterisk lack sequence data. (DOCX 37 kb)"}
+{"text": "The TFIIH subunit XPB is involved in combined Xeroderma Pigmentosum and Cockayne syndrome (XP-B/CS). Our analyses reveal that XPB interacts functionally with KAT2A, a histone acetyltransferase (HAT) that belongs to the hSAGA and hATAC complexes. XPB interacts with KAT2A-containing complexes on chromatin and an XP-B/CS mutation specifically elicits KAT2A-mediated large-scale chromatin decondensation. In XP-B/CS cells, the abnormal recruitment of TFIIH and KAT2A to chromatin causes inappropriate acetylation of histone H3K9, leading to aberrant formation of transcription initiation complexes on the promoters of several hundred genes and their subsequent overexpression. Significantly, this cascade of events is similarly sensitive to KAT2A HAT inhibition or to the rescue with wild-type XPB. In agreement, the XP-B/CS mutation increases KAT2A HAT activity in vitro. Our results unveil a tight connection between TFIIH and KAT2A that controls higher-order chromatin structure and gene expression and provide new insights into transcriptional misregulation in a cancer-prone DNA repair-deficient disorder. Chromatin structure plays a significant role in the regulation of gene expression. Here the authors show that TFIIH interacts with the histone acetyl transferase KAT2A and recruits the ATAC/hSAGA complexes to chromatin; and that loss of xeroderma pigmentosum group B (XPB) function results in chromatin decondensation and increased gene expression through activation of KAT2A. Inherited mutations in genes encoding three subunits of TFIIH lead to genetic disorders. Mutations in XPB trigger xeroderma pigmentosum (XP) combined with Cockayne syndrome (XP/CS) or trichothiodystrophy (TTD), mutations in XPD trigger XP alone, XP/CS or TTD and mutations in TTDA trigger only TTD4. These diseases have a broad spectrum of clinical features, including photosensitivity of the skin and high cancer predisposition mainly due to DNA repair deficiency and developmental and neurological defects likely related to transcriptional deregulation5. Consistent with the latter hypothesis, it has been shown in recent years that there are defects in several transcription activation pathways in XP/CS or TTD cells5.The transcription factor IIH (TFIIH) is composed of ten subunits; XPB, p62, p52, p44, p34, and p8/TTDA which form the core complex, cdk7, MAT1, and cyclin H which form the cdk-activating kinase (CAK) sub-complex, linked to the core by XPD. In addition to its role as a basal transcription factor involved in RNA polymerase (Pol) II-dependent gene expression, TFIIH has also been implicated in nucleotide excision repair (NER)8. XPB has two highly conserved core RecA-like helicase domains (HD1 and HD2), which are found in all SF2 members9. Eukaryotic XPB also contains N- and C-terminal domains (NTD and CTD) that flank the central HD1 and HD210. Interestingly, two of the three amino-acid substitutions in XPB found in XP/CS and TTD patients  are located in the NTD (from residues 1 to 320). XPB interacts with the p52 subunit of TFIIH through its NTD, resulting in an increase in its ATPase activity. The XP-B/CS F99S mutation weakens the XPB\u2013p52 interaction and reduces anchoring of TFIIH to damaged DNA, which would explain the NER defect in related patients11. Although the NTD of XPB is clearly implicated in two rare genetic disorders, its role and the impact of XP-B/CS and TTD mutations on its function have been insufficiently studied.XPB is a central TFIIH subunit that belongs to the SF2 helicase group, which is highly conserved in eukaryotes13, and analyzed chromatin structure using confocal microscopy and three-dimensional (3-D) reconstruction of the cell nucleus. We first showed that the deletion of XPB NTD induces large chromatin decondensation. We then demonstrated that the XP-B/CS mutation (F99S) mimicks the deletion of the NTD by inducing a similar chromatin decondensation, but the TTD mutation (T119D) does not. In order to address the mechanisms, we demonstrated that TFIIH/XPB interacts with KAT2A (GCN5), a histone acetyltransferase (HAT) that is a subunit of the Spt Ada Gcn5 acetyltransferase (hSAGA) and Ada two A-containing (hATAC) complexes16. Using an in vitro histone acetyltransferase assay, we observed that TFIIH-XPBF99S strongly enhances the enzymatic activity of KAT2A. Cells derived from the corresponding XP-B/CS patient have a global increase in H3K9 acetylation and a decrease of H3K9 methylation that trigger overexpression of several hundred genes. We further showed that co-recruitment of TFIIH-XPBF99S and KAT2A on chromatin results in the accumulation of the H3K9ac mark and the formation of Pol II initiation complexes at the promoters of overexpressed genes. We were able to restore the chromatin state, the promoter occupancy and the transcription program by expressing wild-type XPB or by inhibiting KAT2A HAT activity, highlighting the close relationship that exists between these two fundamental cellular actors.To better understand the role of the NTD of XPB and the impact of human XPB mutations on cellular homeostasis, we tethered several XPB mutants to chromatin using the lacO/LacR reporter system13. Constructs that express the lac repressor DNA binding domain (LacR) fused in frame to XPB and GFP were transfected into the human U2OS17 cells that have repetitive binding sites for lacO integrated in the genome17 , a substitution (XPBF99S) expressed in XP-B/CS patient-derived cells (XPCS1BA\u2009=\u2009XP-B/CSF99S) and a substitution (XPBT119P) expressed in TTD patient-derived cells (TTD6VI\u2009=\u2009TTD-XPBT119P). We also tested a large truncation of the C-terminus that deletes the CTD and HD2 (XPB1\u2013550) domains  tethering systeme17 Fig.\u00a0. GFP fac18 marked the lacO repeat clusters with small condensed dots  did not induce this decondensation 20 that we compared to XPCS1BA cells expressing wild-type GFP-tagged XPB (XP-B/CSF99S\u2009+\u2009XPBWT)21 PP lesions with kinetics similar to that of the control MRC5 wild-type fibroblasts , compared to stably transfected GFP-XPBWT expressing cells or wild-type fibroblasts , a small molecule that induces rapid degradation of XPB23. Interestingly, XPBF99S was more resistant than XPBWT to SP treatment, and only the amount of XPBWT decreased dramatically in XP-B/CSF99S\u2009+\u2009XPBWT cells  that reached the level observed in the parental XP-B/CSF99S cells . All together, these data demonstrate that XPBF99S impacts posttranslational modifications of histones and chromatin structure in cells derived from the XP-B/CSF99S patient.To further confirm that re-expression of XPBlls Fig.\u00a0. Therefo24, which are mutually exclusive subunits of the hATAC or hSAGA complexes16. We used GFP-Trap\u00ae to immunoprecipitate exogenous GFP-tagged XPBWT from nuclear extracts of XP-B/CSF99S\u2009+\u2009XPBWT cells. We observed that KAT2A, SUPT7L (a subunit of hSAGA) and WDR5 (a subunit of hATAC) were pulled-down with XPBWT, as well as additional subunits of the TFIIH complex   and KAT2B (PCAF)lex Fig.\u00a0. Intereslex Fig.\u00a0, suggestWT-LacR-GFP, XPBF99S-LacR-GFP, or XPB320\u2013782-LacR-GFP. We observed colocalization of KAT2A with the XPBWT and XPBF99S constructs on chromatin, which was not detected with LacR-GFP  expressed in insect cells11 and observed direct interaction with recombinant KAT2A (rKAT2A) in vitro 11 . After incubation with cIIH-XPBF99S, we observed a strong increase in KAT2A HAT activity that was not detected with cIIH-XPBWT RNA and detected a significant decrease in chromatin decondensation induced by the tethering of XPBBWT Fig.\u00a0.Fig. 5HiF99S was large enough to alter the size of the nucleus. We observed that the XPBF99S mutation induces a twofold increase in the size of XP-B/CSF99S nuclei compared to XP-B/CSF99S\u2009+\u2009XPBWT cells 26, arguing that it is directly linked to KAT2A activity   Fig.\u00a0, indicatlls Fig.\u00a0, implyinlls Fig.\u00a0. In contT2A Fig.\u00a0.F99S mutation was responsible for the overexpression of the representative genes that we selected to study (see above), we used a model system developed by Dr. Sarasin that can be used to correlate the relative expression levels of XPBF99S (XP/CS) and XPBT119P (TTD) with gene expression profiles27. In this system, the patient cell line XP-B/CSF99S was originally transfected with a plasmid expressing XPBT119P and two clones were isolated; clone 14 does not express the ectopic XPBT119P and therefore only expressed the endogenous XPBF99S while clone 5 only express the ectopic XPBT119P\u200927. We observed a decrease in the expression of the representative CYP26 gene with the expression of the mutant XPBT119P  on the promoters of the representative gene  in XP-B/CSene Fig.\u00a0. In XP-Bers Fig.\u00a0. In parater Fig.\u00a028 and obter Fig.\u00a0. The subter Fig.\u00a0 and the ter Fig.\u00a0 were obsers Fig.\u00a0.Fig. 7ChF99S cells  on one side and by 96 copies of the tetracycline response element on the other side (tetO). There are two insertions of LacO repeats in the U2OS17 cells, and consequently the following number of spots that can be observed depends on the cell cycle stage: two spots in G1, four spots in G2, three spots in the middle of S phase. Note also that in some cases the two spots are very closed to each other.The A0-3 cell line was cultured in DMEM/HAMF12 supplemented with 5% FCS and penicillin 100\u2009UI/ml and streptomycin 100\u2009\u03bcg/ml. The U2OS17 cell line was cultured in DMEM (4.5\u2009g/l Glucose) supplemented with 10% fetal calf serum (FCS) and Gentamycin (40\u2009\u03bcg/ml). The U2OS17 stable cell line is one of the clones generated by the team of Dr. SoutoglouWT or pEGFP-LacR-XPBF99S and selected with G418 500\u2009\u00b5g/ml and Hygromycin B 200\u2009\u00b5g/ml. Clones were selected and cultured in DMEM (4.5\u2009g/l glucose) supplemented with 10% fetal calf serum (FCS), Gentamycin (40\u2009mg/ml), IPTG 5\u2009mM, G418 500\u2009\u00b5g/ml, and Hygromycin B 200\u2009\u00b5g/ml.Stable U2OS17 cells expressing lacR fusion proteins were produced as follows. U2OS17 cells were stably transfected with pEGFP-LacR-XPBF99S cells (XPCS1BA-SV40)20 expressing ERCC3 c.296T>C mutant, pPhe99Ser (RefSeq accession number NM_000122.1) and the stably transfected XP-B/CSF99S\u2009+\u2009XPBWT cells are SV40-transformed human fibroblasts that were cultured in DMEM/HAMF10 supplemented with 10% FCS.XP-B/CS27. Clone 5 expresses only the XPBT119P mutant and clone 14 only XPBF99S.Clones 5 and 14 are XPCS1BA-SV40 cells that were stably transfected with a vector that expresses ERCC3 c.355A>C, pThr119P mutantG602D cells (XPCS2)40 are human primary fibroblasts that express ERCC2 c.1805G>A mutation (RefSeq NM_000400.3).XP-D/CS41 are SV40-transformed human fibroblasts that express CSB c.1088A>T mutation (RefSeq NM_001277058.1).CS-B cells (CS1ANSV)2 incubator.All these cells were cultured in DMEM/HamF10 (1:1) medium containing 10% FCS and 40\u2009mg/ml gentamycin at 37\u2009\u00b0C in a 5% COA complete list of reagents, oligonucleotides, siRNA, plasmids, and chemicals used in this manuscript is available in Supplementary Table\u00a0XPB::GFP cells expressing XPB-GFP from the XPB locus were prepared by genome editing with the CRISPR-Cas9 system, as follows. A guide RNA sequence targeting cleavage next to the STOP codon of the XPB gene was cloned into the pMLM3636 expression plasmid . A donor plasmid was constructed with eGFP-2A-puromycin coding sequences inserted at the position of the XPB STOP codon and flanked by the 5\u2032 homology arm chr2:127257599-127257872 (hg38) and 3\u2032 homology arm chr2:127257596-127256813 (hg38). Guide RNA, Cas9 and donor plasmids were electroporated into U2OS cells using the Amaxa nucleofector II with kit V and program X-001 . Clones of cells expressing XPB-GFP-2A-puromycin were selected with puromycin and targeted insertion was confirmed by PCR amplification of the expected junction fragments. A clone that expresses XPB-GFP but no longer expresses XPB  was used for further experiments. U2OS cells were purchased from ATCC and checked for the absence of mycoplasma contamination.U2OS42.Full-length XPB coding sequence was amplified and ligated into the EcoRI/BamHI restriction sites of the pEGFP-N1 vector (Clonetch) giving the pEGFP-XPB construct. To generate fusions between LacR, XPB, and GFP, the cDNA of LacR was amplified by PCR and cloned in pEGFP-XPB vector between GFP and XPB. The FLAG-KAT2A-expressing construct was described inp-values represent significant differences based on a Kruskal\u2013Wallis test.Staining was done following a standard IF protocol. Briefly, cells were washed with PBS, fixed in freshly prepared 4% paraformaldehyde (PFA) for 10\u2009min at RT and permeabilized with PBS 0.1% Triton X-100 for 3\u2009\u00d7\u20095\u2009min at RT. Blocking  and incubations with antibodies  were performed with 10% heat-inactivated FCS in PBS 0.1% Triton X-100 and washes were done with PBS 0.1% Triton X-100 at RT for 3\u2009\u00d7\u20095\u2009min. Nuclei were counterstained with freshly prepared 1\u2009\u03bcg/mL DAPI in PBS for 2\u2009min at RT and cells were mounted using the ProLong Gold antifade reagent of Molecular Probes. Confocal microscopy pictures were taken with a Leica SP2 microscope; Z stack width was usually 0.5\u2009\u03bcm. For counting colocalised signals, at least 100 cells were analyzed for each condition. For 3D reconstructions, stacks were captured using a Leica DM6000 microscope with a Leica CSU22 spinning disc and an Andor Ixon 897 camera, the step size was 0.2\u2009\u03bcm. For reconstruction of the images and the quantification of the volume filled by the signal, Imaris Software (Bitplane) was used. Results are shown in box and whisker plots. 2) for the indicated period of time at 37\u2009\u00b0C, 5% CO2 and recovered in fresh medium. Immuno-labeling of (6-4)PP was performed using the mouse 64M-2 antibody following the IF protocol. A step of DNA denaturation was added after permeabilization where cells were treated with freshly prepared 2M HCl for 30\u2009min at RT. Cells were washed with PBS for 5\u2009min and blocked. (6-4)PP lesions were quantified using an IN Cell Analyser 1000 imaging system  and the amount of (6-4)PP removal was determined.Five thousand cells were plated in 96-well plates . 24\u2009h later, cells were UV-irradiated with a UV-C lamp , 1\u2009mM EDTA, 0.5\u2009mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140\u2009mM NaCl for 30\u2009min at 4\u2009\u00b0C and centrifuged at 18\u2009kg for 20\u2009min at 4\u2009\u00b0C. Fifteen micrograms of each extract was loaded on an SDS-PAGE gel.2) containing 0.5\u2009M KCl and twice with IP buffer containing 100\u2009mM KCl. After washing, proteins were eluted with a 1000\u2009\u00d7 excess of the flag epitope peptide. The reconstituted human HAT module of the ATAC complex (rHAT-ATAC) containing the KAT2A, ADA2a, ADA3, and SGF29 subunits was expressed and purified from insect cells25.Recombinant FLAG tagged KAT2A was purified using anti-FLAG M2-agarose beads (Sigma). M2-agarose-bound protein complexes were washed three times with immunoprecipitation (IP) buffer  glycerol, 0.1% Nonidet P-40, 0.5\u2009mM DTT, 5\u2009mM MgCl25. Recombinant histones H3.3 were incubated with rKAT2A or the rHAT-ATAC module in the presence of acetyl-CoA in HAT buffer  for 1\u2009h at 30\u2009\u00b0C. The reaction was then analyzed by western blotting with specific antibodies (H3 and H3K9ac).The HAT activity of rKAT2A or rHAT-ATAC was measured using histone acetyltransferase assayXPB was expressed in baculovirus and incubated for 4\u2009h at 4\u2009\u00b0C with anti-XPB covered beads in lysis buffer . Following three washes with lysis buffer, beads were further incubated at 4\u2009\u00b0C for 1\u2009h with 500\u2009ng of purified Flag-KAT2A in lysis buffer. Pull downs were washed with RIPA buffer and analyzed by western blotting.GAPDH mRNA. Sequences of primers are available in Supplementary Table\u00a0Total RNA was isolated using TRI REAGENT (MRC) and purified by phenol-chloroform extraction. RNA was reverse transcribed with SuperScript IV reverse transcriptase (Invitrogen) and Oligo d(T). Quantitative PCR was done using the SYBR Green Master Mix in a Lightcycler 480 (Roche). mRNA levels were normalized against the housekeeping WT cells were extracted using GenElute Mammalian Total RNA Miniprep Kit (Sigma). Libraries were prepared with TruSeq Stranded mRNA Sample Preparation Kit following guide instructions and subsequently analyzed on an Illumina Hiseq 4000 as single-end 50 base reads following Illumina\u2019s instructions. Image analysis and base calling were performed using RTA 2.7.3 and bcl2fastq 2.17.1.14. Reads were mapped onto the hg19 assembly of the human genome. Read counting was performed with HOMER v4.8.3(65) and expression was estimated with EdgeR. Genome ontology was performed with The Database for Annotation and Integrated Discovery (DAVID) v6.7 (https://david.ncifcrf.gov).Total RNAs from XP-B/CS and XP-B/CS\u2009+\u2009XPB28. Protein G Sepharose beads (Upstate) were prepared by incubation with antibodies at 4\u2009\u00b0C for 2\u2009h and incubated with samples at 4\u2009\u00b0C overnight. After washing, protein-DNA complexes were eluted and decrosslinked. DNA fragments were purified using Qiaquick PCR purification Kits (QIAGEN) and analyzed by qPCR using the set of primers indicated in Supplementary Table\u00a0Cells were cross-linked at room-temperature (RT) for 10\u2009min with 1% formaldehyde. Chromatin was prepared and sonicated on ice for 30\u2009min using a sonicator The ON-TARGET plus smart pool siRNA control or human KAT2A targeting siRNAs were purchased from Dharmacon and transfected at a final concentration of 100\u2009nM using the Lipofectamine RNAiMax reagent (Invitrogen) following the manufacturer\u2019s protocol.t test for ChIP and RT-PCR, and a Kruskal\u2013Wallis test for 3D reconstruction analysis. Results are presented as mean\u2009\u00b1\u2009standard deviation (SD). p-values are shown.Statistical analysis of experimental data was performed using a Student\u2019s paired Further information on experimental design is available in the\u00a0Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting SummarySource Data"}
+{"text": "Nitric oxide (NOx) is an important biomolecule which interacts with other molecules including 25(OH)D to mediate various metabolic pathways. Interactions and associations of NOx with 25(OH)D have been well studied both A total of 740 (245 boys and 495 girls) apparently healthy Saudi adolescents aged 10-17 years were included in this cross-sectional study, to determine the associations of NOx with 25(OH)D and other biomarkers in Saudi adolescents. Serum NOx, 25(OH)D, and other biochemical and anthropometric parameters were measured following standard protocols and manufacturers' guidelines.p<0.001). In all subjects, NOx showed a significant inverse correlation with 25(OH)D. After stratification according to sex however this significant association was observed only in boys and not in girls. NOx was also significantly associated with BMI, serum triglycerides, and systolic blood pressure in all subjects. NOx level was significantly higher in boys than girls ( The significantly inverse association of NOx and 25(OH)D among apparently healthy adolescents is influenced by sex and further strengthens the extraskeletal role of 25(OH)D in maintaining endothelial homeostasis in this age group, particularly in boys. Whether vitamin D correction can influence NOx production over time among adolescents remains to be proven. Nitric oxide (NOx) is an important molecule produced by endothelium which plays a variety of vital roles in humans such as reproduction, inflammation, vasodilation, cardiac function, oxidative stress, gene transcription, translation, and posttranslational modifications of the proteins \u20136. Exerc2 and \u226530kg/m2 were considered overweight and obese, respectively. We excluded subjects who were underweight, overweight, and obese, based on their BMI z-scores. We recruited only apparently normal individuals for current study.A total of 740 (245 boys and 495 girls) Saudi adolescents 10-17 years) were included in this cross-sectional study. Subjects' information regarding vitamin D was taken from the school project master database of the Prince Mutaib Chair for the Biomarkers of Osteoporosis (PMCO) in King Saud University (KSU)  years we. In brieA total 5-10 CC fasting venous blood samples were collected from all subjects. Part of the blood samples were centrifuged for serum isolation while remaining blood samples were transferred to EDTA tubes for other future analyses. Blood and serum samples were immediately delivered and stored at \u221280\u00b0C in PMCO, KSU, Riyadh, KSA.Fasting lipid profile and blood glucose in all recruited individuals were determined using a chemical analyzer . COBAS e-411 automated analyzer  was used for measuring serum 25(OH)D. The inter- and intra-assay were applied for estimation of serum 25(OH)D; coefficients of variation (CV) were taken as 8.0 and 5.6%, respectively, with a lower detection limit (LOD) of 7.5nmol/ml .\u03bcL serum samples and 50\u2009\u03bcL of distilled water (blank) were added to microplate wells, and 50\u2009\u03bcL sulfanilamide (1% in 5% H3PO4) was added to samples. Samples were incubated for 10\u2009min at 37\u00b0C. 50\u2009\u03bcL of [N-(1-naphthyl) ethylendiamine dihydrochloride (0.1%) in distilled water] was then added to both samples and blank and reincubated for 5\u2009min at 37\u00b0C. Absorbance was taken at 540\u2009nm using the enzyme-linked immunosorbent assay (ELISA) reader . Serum NOx concentration was determined from the linear standard curve established using 0-50\u03bcM sodium nitrate. Inter- and intra-assay coefficients of variations were set at 5.2% and 4.4%, respectively, while the recovery of the assay was 93\u00b11.5%.Usually, NOx can be estimated from determining the concentrations of nitrite and nitrate end products. The measurement of nitrate/nitrite concentration or of total nitrate and nitrite concentration is routinely used as an index of NOx production . The con p value <0.05 was considered statistically significant.SPSS  was used to analyze the data. Continuous data were presented as mean \u00b1 standard deviation (SD) for normal variables and non-Gaussian variables were presented in median (1st and 3rd) percentiles. Categorical data were presented as frequencies and percentages (%). All continuous variables were checked for normality using Kolmogorov-Smirnov test. Non-Gaussian variables were log-transformed prior to parametric analysis. Correlations between variables were done using Pearson's correlation analysis. Independent T-test was used to compare mean differences. Ap<0.001) while girls had significantly higher WHR and waist circumference (p<0.001). Girls also had significantly higher systolic blood pressure while boys had significantly higher diastolic blood pressure. Girls showed significantly higher levels of serum total cholesterol and 25(OH)D while the boys had significantly higher levels of triglycerides and NOx (p<0.001) , whereas the vitamin D sufficient group had significantly higher mean values of WHR, total cholesterol, and vitamin D  were recruited to analyze the biological relation of NOx with vitamin D and other serological parameters. Initially, the general characteristics of subjects were studied and no significant differences were found between the mean age and triglycerides. Significantly higher BMI and hip circumference were noted in boys (p<0.001) . The sexp<0.001) . The difitamin D . Vitaminitamin D . For conitamin D .Vitamin D and NOx are important molecules that may have a functional association as observed in different cell lines, animal models, and humans. Different studies showed different associations, so this ambiguity has been a hot topic that remains unanswered. NOx has a role in the pathogenicity of several illnesses like neurodegenerative diseases , CVD, arthritis, and infertility , 22\u201325. It has been observed that 1,25(OH)2D3 reduces PFF-induced NOx response in primary human osteoblasts while 25(OH)D3 did not show significant effects on NOx response in primary human osteoblasts . WithoutAlthough our findings are similar to previous studies and the relatively large sample size had sufficient statistical power to explain the relationship between NOx and other parameters, the study still has some limitations. One is its cross-sectional design, which precludes our ability to make causal inferences about the observed associations. We also did not measure levels of lactate and other inflammatory markers which may give additional insights into the significant associations elicited. These markers can be recommended for further investigations. Furthermore since our subjects were adolescents, we did not study the involvement of growth hormones and sex steroids, which are suggested to have influence on NOx and 25(OH)D levels. Finally, since the associations observed do not prove causality, a sufficient vitamin D status may, at best, serve as a \u201cmarker\u201d for other conditions, e.g., for good health or for UV exposure. In consequence, it can be speculated whether the associations observed may be caused by other, unknown factors that are associated, e.g., with good health or UV exposure.In summary, the associations of NOx with 25(OH)D and other cardiometabolic parameters were studied in 740 Saudi adolescents. Serum 25(OH)D and NOx showed a significant inverse association in all subjects, more so in boys, and NOx was positively associated with triglycerides and systolic blood pressure. The association between the NOx and the parameters measured are significantly influenced by sex and age. A large scale study on people of different ages and ethnicity would help in better understanding of the NOx's association pattern with metabolic and clinical factors."}
+{"text": "Hargeria was considered a monospecific leptocheliid genus, with the species Hargeria rapax considered a taxon with a wide distribution, from the Northwestern Atlantic to the Mexican Caribbean. Herein, after a detailed revision of type and topotype materials and specimens collected from the Mexican Caribbean, a new species H. chetumalensis sp. nov. is described, and the redescription of H. rapax is provided. Also, we found a significant genetic divergence between the two species based on the nucleotide sequences of cytochrome oxidase subunit I, which support the morphological data. The morphological features used to recognize both species are also adequate to link males, females, and juvenile stages, although these species have a high intraspecific polymorphism.Until now,  Leptochelia rapax; unfortunately, the description lacks illustrations. The females of this species were distinguished from other leptocheliids by the last segment of the antennule, which is scarcely longer than the preceding one; while the males, by having the elongate and slender antennules and chelipeds ; also, specimens identified as H. rapax deposited in the Reference Collection of Benthos (ECOSUR) of El Colegio de la Frontera Sur, Chetumal, Mexico were reviewed.We studied type and topotypical specimens of The tanaids were examined under a stereomicroscope Carl Zeiss SV6. The total length of specimens was measured from the anterior end of the cephalothorax to the posterior margin of the pleotelson. Dissection of appendages was generally performed on the right side of the body; the pieces were mounted in glycerol and sealed with transparent nail varnish. Drawings of taxonomically features were made with a camera lucida at 4\u201340\u00d7. In the description section, we follow H. rapax, L. forresti (L. longichelipes (Chondrochelia dubia (L. dubia) were obtained from the GenBank database , following the standard protocols of the program \u201cBarcode of the Life.\u201d Cytochrome oxidase subunit I (COI) nucleotide sequences between 579\u2013658 bp were amplified using M13F (5\u2032-TGTAAAACGACGGCCAGT-3\u2032) and M13R (5\u2032-CAGGAAACAGCTATGAC-3\u2032) primers . Sequencforresti , L. longchelipes , and Choia dubia   with five rate categories and by assuming that a certain fraction of sites is evolutionarily invariable (+I) as model to construct a tree using the maximum likelihood analysis. Furthermore, we used the Kimura 2-Parameter model to estimate the average evolutionary divergence over sequence pairs within and between species. All analyses were carried out with Mega 7 .http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:B55A046E-55AA-491A-ABED-C9BA3D7300DE. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central, and CLOCKSS.The electronic version of this article in portable document format will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix Superorder Peracarida Calman, 1904Order Tanaidacea Dana, 1849Suborder Tanaidomorpha Sieg, 1980Family Leptocheliidae Lang, 1973Subfamily Leptocheliinae Lang, 1973HargeriaLang, 1973Genus Hargeria Lang, 1939: 225.Type species.Leptochelia rapax Harger, 1879, by original designation . Pereopod 1 with one dorsodistal setae and two ventrodistal setae on merus.Description. Adult male. Topotype USNM 65779, 2.5 mm .Hargeria rapax, as noted above, was regarded as a junior synonym of Leptochelia based on phylogenetic studies where the sister relationships between H. rapax and L. dubia (currently C. dubia) was observed. Recently, Hargeria is mainly characterized by having an anal plate, which is absent in Leptochelia and ChondrocheliaHargeria has a small proximal process and a large distal process on cutting edge, whereas in Leptochelia can be observed only a medial process and in Chondrochelia one large proximal process and one small distal process. Moreover, Hargeria has uropodal exopod uniarticulate, while in Leptochelia it is biarticulate and in Chondrochelia uni- or biarticulate. In Hargeria the length of cheliped carpus is up to 8.0 times as long as broad, whereas in Leptochelia the carpus is more than eight times as long as broad; Hargeria has uropodal endopod five-articulate and the first two articles without long setae, while Leptochelia has uropodal endopod six-articulate and the first two articles with long setae.Hargeria has three flat spines ; while Leptochelia only has two flat spines and Chondrochelia three to five flat spines. Other differences between these genera can be seen in propodus of Pereopods 2 and 3. For example, although Hargeria and Leptochelia have the same setation on propodus of Pereopod 2 , they differ on propodus of Pereopod 3, Hargeria has two dorsodistal setae and one ventrodistal spine, whereas Leptochelia has one dorsodistal seta and one ventrodistal spine. A similar condition occurs when compare Hargeria with Chondrochelia; both genera have two dorsodistal setae and one ventrodistal spine on propodus of Pereopod 3, but they differ because Hargeria has two dorsodistal setae and one ventrodistal spine on propodus of Pereopod 2, while Chondrochelia has three dorsodistal setae and one ventrodistal spine. Furthermore, these morphological differences are supported by the results of the molecular analysis (see below).Regarding females, the maxillipedal endite of Hargeria chetumalensis sp. nov.urn:lsid:zoobank.org:act:A8D8DCA7-E71C-4124-83B7-F5E66CDA31A1\u201314Hargeria rapax: Type material. Holotype adult male, ECOSUR 0209, 2.9 mm, Raudales, 18\u00b042\u203225.2\u2033N 88\u00b015\u203218\u2033W, Quintana Roo, Mexico, in Bathophora sp. on rock, 0.5 m, May 28, 2015. Paratypes: 13 ovigerous females ECOSUR 0210, 44 non-ovigerous females ECOSUR 0211, 17 adult males ECOSUR 0212, same data as holotype.Additional material. ECOSUR-C1079, five ovigerous females, 10 non-ovigerous female, three males, Calderitas, 18\u00b029\u203213.2\u2033N 87\u00b045\u203225.2\u2033W, Chetumal Bay, Quintana Roo, Mexico, in Bathophora sp. on rocks, June 28, 2016. ECOSUR-C1080, 20 ovigerous females, 32 non-ovigerous females, nine males, mouth of Guerrero Lagoon, 18\u00b042\u203230.4\u2033N 88\u00b009\u203205.2\u2033W, Quintana Roo, Mexico, in Bathophora sp. on rocks, April 23, 2016.Molecular material. ECOSUR TANAI104-15, TANAI105-15, TANAI106-15, TANAI107-15, TANAI109-15 : Guerrero Lagoon, 18\u00b041\u203219.3\u2033N 88\u00b015\u203250.4\u2033W, Quintana Roo, Mexico, in Bathophora sp. on rocks, 0.5 m, May 28, 2015. ECOSUR TANAI071-15, TANAI072-15, TANAI073-15, TANAI074-15, TANAI075-15, TANAI076-15, TANAI077-15, TANAI078-15, TANAI079-15, TANAI110-15, TANAI111-15, TANAI112-15, TANAI113-15, TANAI115-15, TANAI116-15, TANAI117-15 : Raudales, 18\u00b042\u203225.2\u2033N 88\u00b015\u203218\u2033W, Quintana Roo, Mexico, in Bathophora sp. on rocks, 0.5 m, May 28, 2015.Diagnosis. Male. Rostrum pointed, narrow, not extending beyond eyes. Anal plate tongue-shaped, more than times as long as broad, extending beyond length of peduncle and first article of uropodal endopod combined. Cheliped with carpus about eight times as long as broad; fixed finger with blunt proximal process on cutting edge. Pereopod 1 with one small ventrodistal serrate seta on merus; with three distal serrate setae on carpus. Pereopod 4 with two long distal serrate setae extending beyond the median length of propodus. Female. Right mandible with incisor crenulate and slightly bifurcate apex. Maxillipedal endite with three flat spines . Pereopod 1 with one small ventrodistal serrate seta on merus.Etymology. The specific epithet is derived from the locality name of the Chetumal Bay System, where the species was found.Description. Adult male. Holotype ECOSUR0209, 2.9 mm  and the anal plate. For example, males of H. chetumalensis have a pointed, narrow rostrum that does not extend beyond the eyes; in contrast, H. rapax has a convex and broad rostrum that does extend beyond eyes. H. chetumalensis has the first peduncle article of the antennule more than eight times as long as broad, while in H. rapax it is up to seven times as long as broad; H. chetumalensis has the carpus of cheliped about eight times as long as broad, whereas in H. rapax it is five to seven times as long as broad. Also, in H. chetumalensis the two long distal setae on merus of pereopod 4 exceed the median length of propodus, while in H. rapax such setae do not. Furthermore, H. chetumalensis has a tongue-shaped anal plate, which is more than three times as long as broad, whereas H. rapax has a capitate anal plate that is less than three times as long as broad , while in Mexico  it was 0.2% (n = 21). It is probable that the larger genetic divergence between the populations of Florida and Maryland is due to greater geographic distance between them than those found in the Chetumal Bay and Guerrero Lagoon System.The morphological differences found between  the COI . Our resHargeria species and species of the other genera analyzed, both species of Hargeria have a genetic divergence higher to 40% concerning C. dubia and 35\u201338% with Leptochelia species. These values are higher than those considered for intrageneric divergence on Crustacea (19\u201332%)  . Our molH. chetumalensis. With regards to female and manca, The SEM analysis showed that the cuticle of all structures, as well as setae of pereopods and mouthparts are more ornate than they can appear using the optical microscope, where they may appear to be simple; however, they comprise scales, pores, and small setae, giving the \u201cserrated\u201d appearance on such features. For example, It is the first time that this type of ornamentation of the cuticle and setae is reported for species belonging to the Leptocheliidae. Therefore, we consider that a more detailed examination of these structures will allow us a better characterization of all structures. With this kind of information, we will have the robustness to clarify the morphological differences of the supposed species complexes; furthermore, we will obtain more information for phylogenetic studies.H. chetumalensis sp. nov. and H. rapax, the data allowed us to confirm that the morphological features used for the recognition of both species were adequate to link males, females, and juvenile stages, although these species have a strong polymorphism  along the extensive coastline varies considerably, it would be unlikely that an estuarine species could have a wide distribution. A detailed revision of the other specimens reported in this region as H. rapax need to be revaluated to corroborate this idea; however, it is beyond the scope of this study.As we noted above, Hargeria as a valid genus, different from Chondrochelia and Leptochelia. Besides, we redescribed H. rapax based upon type and topotype materials; also, we described the second species belonging to Hargeria, which is the first tanaidacean described from the Mexican Caribbean. We consider that the information provided in this work will help as the baseline to clarify the taxonomic status of the specimens that have been reported under the name of H. rapax within the wide geographical distribution that was attributed to the species.The taxonomy of tanaidaceans represents a great challenge, since their small size (one to three mm on average), high intraspecific polymorphisms, and the lack of specialized regional literature have increased the degree of complexity for the recognition of genera and species. In recent years, the use of morphology, genetic, and morphometric data have allowed to detect and solve some taxonomic problems related to cryptic species of leptocheliids. Herein, based on morphological and molecular data, we give new evidence to support the recognition of 10.7717/peerj.7472/supp-1Supplemental Information 1Click here for additional data file."}
+{"text": "Y-box-binding protein-1, YB-1, plays an important role in regulating the cell cycle, although precisely how it does the is unknown. Using live cell imaging, we show that YB-1 is essential for initiating the last step of cell division (cytokinesis), required for creation of two daughter cells. Using confocal microscopy we showed that YB-1 regulates the spatial distribution of key proteins essential for cytokinesis to occur and that this required YB-1 to be phosphorylated on several residues. In-silico modeling demonstrated that modifications at these residues resulted in conformational changes in YB-1 protein allowing it to interact with proteins essential for cytokinesis. As many cancers have high levels YB-1 and these are associated with poor prognosis, our data suggest developing small molecule inhibitors to block YB-1 phosphorylation could be a novel approach to cancer therapy.High levels of the cold shock protein Y-box-binding protein-1, YB-1, are tightly correlated with increased cell proliferation and progression. However, the precise mechanism by which YB-1 regulates proliferation is unknown. Here, we found that YB-1 depletion in several cancer cell lines and in immortalized fibroblasts resulted in cytokinesis failure and consequent multinucleation. Rescue experiments indicated that YB-1 was required for completion of cytokinesis. Using confocal imaging we found that YB-1 was essential for orchestrating the spatio-temporal distribution of the microtubules, \u03b2-actin and the chromosome passenger complex (CPC) to define the cleavage plane. We show that phosphorylation at six serine residues was essential for cytokinesis, of which novel sites were identified using mass spectrometry. Using atomistic modelling we show how phosphorylation at multiple sites alters YB-1 conformation, allowing it to interact with protein partners. Our results establish phosphorylated YB-1 as a critical regulator of cytokinesis, defining precisely how YB-1 regulates cell division. YBX1 mRNA or protein are tightly associated with patient relapse and poor prognosis in multiple cancer types 2SO). The cells were diluted to 1 \u00d7 104 cells per \u00b5L in PBS and the 25 mM DSP solution was added to the cell suspension at a ratio of 1:25. Cross-linking took place with gentle mixing at room-temperature for 45 min. The cells were spun and the supernatant removed. The remaining cross-linker was then quenched by the addition of 100 mM Tris in PBS which was added in a stoichiometry of at least 1:1 with the cross-linking reagent dithiobis-succinimidyl propionate (DSP) and incubated at room temperature for 10 min. This step was repeated once before the cells were lysed at 1 \u00d7 104 cells per \u00b5L in low-detergent RIPA, snap frozen, and stored at \u221220 \u00b0C. The Protein G Mag Sepharose beads were used at a concentration of 75 \u00b5L for every 50 \u00b5g sheep-anti-YB-1. For these IPs, the antibody and Protein G Mag Sepharose beads were complexed together in 500 \u00b5L of low-detergent RIPA for 30 min, immobilized, and then rinsed once with another 500 \u00b5L of soft buffer. The lysate was then added to the immobilized antibody plus Protein G Mag Sepharose beads and rotated for 1 h at 4 \u00b0C. The interaction of the antibody with the protein G beads was disrupted using competitive elution. The peptide to which NYB1 was raised was added to each sample at a concentration of 100-fold to native PAGE buffer (Life Technologies) with 1% Digitonin (Sigma), 1 \u00d7 phosSTOP (Roche) and 1 \u00d7 complete EDTA- (Roche). The samples were incubated at room temperature for 2 h with gentle mixing. Following this, the samples were concentrated using centrifugation at 15,000 g at room temperature in a Vivaspin 500, 30 000 MWCO column (Sartorius) until their volume was 25 \u00b5L.Proteins were cross-linked in vivo by treating cells with dithiobis[succinimidyl] propionate (DSP) buffered in PBS. Cells were trypsinised, counted, and rinsed twice with PBS. Immediately prior to its use, a 25 mM DSP solution was prepared in DMSO ([CHw/v) (NH4)2SO4, 3% phosphoric acid, 0.1% Coomassie G-250 ). Protein bands and fractions were excised and subjected to in-gel digestion with trypsin using the automated in-gel digestion method [2 affinity chromatography [LC-MS/MS was used to identify the proteins that copurified with immunoprecipitated YB-1. The immunopurified proteins were separated using SDS-PAGE and the gel was stained using colloidal coomassie staining .For targeted detection of phosphorylated YB-1 peptides, phospho-enriched peptide fractions were prepared from whole cells. Peptides were generated from 500 \u00b5g of protein from whole cell lysates by tryptic protein digestion using the filter-aided sample preparation method . Phosphov/v) acetonitrile, 0.2% (v/v) formic acid in ultrapure H2O, and analysed on three different LC-coupled linear ion trap/orbitrap instruments located at the Centre for Protein Research , the Children\u2019s Medical Research Institute , and the Bioanalytical Mass Spectrometry Facility . For targeted analysis of YB-1 phosphorylation enriched phosphorylated peptides for targeted analysis of YB-1 phosphorylation were re-solubilised in 2% (v/v) acetonitrile, 0.2% (v/v) formic acid in ultrapure H2O and separated using an LC-coupled TripleTOF 5600+ . The method used at the Centre for Protein Research is here described as a representative instrument method. Peptides were separated on emitter-tip columns . The gradient for liquid chromatography was modified depending on the complexity of the fraction or band. Generally, the gradient developed from 1% (v/v) acetonitrile, 0.2% (v/v) formic acid to 80% (v/v) acetonitrile, 0.2% (v/v) formic acid in ultrapure H2O at a flow rate of 400 nL/min.All samples except those for targeted sequencing were re-solubilized in 1%  in the LTQ ion trap at a normalized collision energy of 35% using an AGC target of 1 \u00d7 105 and two microscans. Dynamic exclusion was enabled with two repeat counts during 30 s (sec) and an exclusion period of 180 s. The exclusion mass width was set to 0.01.Full MS (MS1) in a mass range between http://www.matrixscience.com) and Sequest (ThermoFisher Scientific) search engines. The search was set up for full tryptic peptides with a maximum of three missed cleavage sites. Carboxyamidomethyl cysteine, oxidized methionine, deamidation , and phosphorylation  were included as variable modifications. The CAMthiopropanoyl (K) modification, which corresponds to a DSP adduct that has been reduced and alkylated, was added to searches of LC-MS/MS data from cross-linked samples. The precursor mass tolerance threshold was 10 ppm and the maximum fragment mass error 0.8 Da. Peptides were accepted as identified if their false discovery rate adjusted scores were above the score threshold at a false discovery rate of \u2265 1%, as assessed by the Percolator algorithm [Peaklists and MS/MS data were generated using the Proteome Discoverer (version 1.4) software using default settings and searched against the Human Swiss-Prot amino acid sequence database using both the Mascot  method on a Triple TOF 5600+ mass spectrometer. The data from targeted LC-MS/MS and SWATH were analysed using Mascot (http://www.matrixscience.com) and Skyline (v4.2.0). Quantification of phosphorylation at different sites on YB-1 was performed using the peak areas from fragment ions unique to individual phosphorylation sites.Phosphorylated YB-1 peptides using targeted mass were detected using multi-reaction monitoring mode where the precursor The automated I-TASSER pipeline  which gepmemd.CUDA module of the program Amber14 [Xleap module of Amber14 was used to prepare the system for the MD simulations. All the simulation systems were neutralized with appropriate numbers of counterions. Each neutralized system was solvated in an octahedral box with TIP3P [\u22121 \u00c5\u22122) on the protein over a period of 0.25 ns allowing water molecules and ions to move freely. During an additional 0.25 ns, the positional restraints were gradually reduced followed by a 2 ns unrestrained MD simulation to equilibrate all the atoms. For each system, three independent MD simulations  were carried out for 250 ns with conformations saved every 10 ps. Simulation trajectories were visualized using VMD [The three-dimensional model of full length YB-1 was subject to molecular dynamics (MD) simulations in its phosphorylated form , unphosphorylated form and of mutant forms, S167A and S176A (where the two mutant forms had the other 5 serines phosphorylated) using the  Amber14 . The all Amber14  was used Amber14 . For theth TIP3P  water moth TIP3P  method uth TIP3P  algorithsing VMD  and figusing VMD .One tailed Student\u2019s t-test was performed to assess the significant differences between control and treated cells. Mann-Whitney U test was used to determine the significance of the difference in distribution between cell cycle phases of control and treated cells. Chi-square and Fisher\u2019s Exact test was used to assess significant differences in the distribution of number of twisted furrows between control and YB-1 depleted cells. One-way ANOVA with Tukey\u2019s multiple comparison test was used to assess the significance of difference in percentage of multinucleate cells in RSK and AURKB treated cells with and without YB-1 overexpression. All statistical analyses were performed using GraphPad Prism software version 7.03 .In summary, we define precisely how YB-1 regulates cell division. We provide unequivocal evidence that defects in YB-1 result in cytokinesis failure and multinucleation in cancer cells. We demonstrate that YB-1 is essential for microtubule organization and facilitates the specification of the cleavage plane in vitro and in vivo, thus establishing its physiological function. We show that RSK phosphorylates multiple sites on YB-1 and highlight the importance of the YB-1/RSK axis during cytokinesis. Finally, using molecular modelling we demonstrate the importance of structural rearrangement regulated by simultaneous phosphorylation for YB-1 function during cytokinesis. Our work conclusively shows phosphorylated YB-1 is critical for assembly of microtubules, specification of the cleavage plane and localization of the CPC during cytokinesis."}
+{"text": "Antibiotic resistance is a growing problem that can be ameliorated by the discovery of novel drug candidates. Bacterial associates are often the source of pharmaceutically active natural products isolated from marine invertebrates, and thus, important targets for drug discovery. While the microbiomes of many marine organisms have been extensively studied, microbial communities from chemically-rich nudibranchs, marine invertebrates that often possess chemical defences, are relatively unknown.We applied both culture-dependent and independent approaches to better understand the biochemical potential of microbial communities associated with nudibranchs. Gram-positive microorganisms isolated from nudibranchs collected in the Red Sea were screened for antibacterial and antitumor activity. To assess their biochemical potential, the isolates were screened for the presence of natural product biosynthetic gene clusters, including polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes, using PCR. The microbiomes of the nudibranchs were investigated by high-throughput sequencing of 16S rRNA amplicons.In screens against five model microorganisms, 51% of extracts displayed antimicrobial activity against more than one organism, and 19% exhibited antitumor activity against Ehrlich\u2019s ascites carcinoma. Sixty-four percent of isolates contained PKS and NRPS genes, suggesting their genomes contain gene clusters for natural product biosynthesis. Thirty-five percent were positive for more than one class of biosynthetic gene. These strains were identified as belonging to the Firmicutes and Actinobacteria phyla via 16S rRNA gene sequencing. In addition, 16S rRNA community amplicon sequencing revealed all bacterial isolates were present in the uncultured host-associated microbiome, although they were a very small percentage of the total community. Taken together, these results indicate that bacteria associated with marine nudibranchs are potentially a rich source of bioactive compounds and natural product biosynthetic genes. Ecteinascidia turbinata  and Escherichia coli O-19592 (EHEC) respectively. Another study on Indonesian nudibranchs found that two bacterial isolates had anti-MRSA activity  at water depths between one and eight meters . Five animals belonging to different species  were hanQuick\u2014DNA Fungal/Bacterial Miniprep Kit . Partial mitochondrial cytochrome oxidase I (COI) gene sequences were amplified using LCO1490-JJ and HCO2198-JJ primers  collected in November 2015. After it was noted that a high proportion of the extracts from the isolates had bioactivity, additional nudibranch specimens were collected  in May 2017 to produce more isolates. Each nudibranch was rinsed three times with filter-sterilized natural sea water (NSW) to remove debris and unattached bacteria. Nudibranchs were cut into pieces with a sterile razor blade and then homogenized in a blender. The homogenate was serially diluted with NSW and spread (100 \u03bcL) on solid Marine Agar media, R2A, Actinomycetes Isolation Agar (AIA), starch casein agar, ISP2 agar and M1 agar. All media were prepared using NSW except Marine Agar, which was prepared with distilled water and 20% NaCl. All media were supplemented with 100 \u03bcg/mL cyclohexamide and 25 \u03bcg/mL nystatin to suppress fungal growth, and 25 \u03bcg/ml nalidixic acid to inhibit the growth of aggressive Gram-negative bacteria , and transferred to a separatory funnel to separate the organic phase. This ethyl acetate extraction was performed three times. The organic phase solvent was evaporated and the residue was weighed and dissolved in DMSO to achieve a concentration of 5 mg/mL.S. aureus ATCC 25923, Escherichia coli ATCC 25922, Bacillus subtilis ATCC 6633, and the fungus Candida albicans ATCC 10231. Equivalent volumes of DMSO and ethyl acetate were used as negative controls. Following incubation at 37 \u00b0C overnight, resultant zones of inhibition were measured.The first group of bacterial isolates (100) were screened for antimicrobial activity using the standard well diffusion assay . Crude m6 cells/ml phosphate buffer) were treated with bacterial metabolic extracts (500 \u00b5g/mL) in DMSO. After 120 min of incubation at 37 \u00b0C, 50% trypan blue was added to an aliquot of the EAC mixture. The number of cells that showed signs of damage by stain penetration was counted using a hemocytometer with light microscope. The bacterial metabolic extracts that exhibited antitumor activity at 500 \u00b5g/mL were also tested at 50 and 250 \u00b5g/mL in DMSO using the same protocol.Ehrlich\u2019s ascites carcinoma (EAC) cells collected from donor female mice   were suspended in sterile isotonic saline (0.9%). The viability of the cells was 99% as determined by the trypan blue assay, according to the method described in . EAC celQuick\u2014DNA Fungal/Bacterial Miniprep Kit . A single colony was inoculated into five mL of R2A broth and incubated in a shaker 150 rpm at 30 \u00b0C for 2\u20135 days depending on culture turbidity. Pelleted cells were resuspended in the recommended volume of Bashing Bead Buffer. Proteinase K was added and the cells incubated at 65\u00b0C for 1 h to increase cell lysis. Thereafter, the DNA extraction was carried out according to the manufacturer\u2019s protocol for actinomycetes. Aliquots of the isolated DNA were visualized after gel electrophoresis and stored at \u221220 \u00b0C.Genomic DNA of the bacterial isolates  was extracted using the I amplification), but with DMSO instead of BSA. The thermocycling conditions were as follows: an initial denaturation at 98 \u00b0C for 30 s followed by 35 cycles of denaturation 98 \u00b0C for 5 s, annealing Tm and time depending on the primer sequence, extension at 72 \u00b0C for 10 s, followed by a final extension at 72 \u00b0C for 2 min. The amplicons were visualized following gel electrophoresis in 1% agarose.The bacterial isolates were screened for genes involved in the biosynthesis of secondary metabolites, specifically polyketide synthase  and non-ribosomal peptide synthetase (NRPS). Genomic DNA extracted from each isolate (20\u201350 ng/\u03bcL) was used as template for the amplification of the PKS-I, PKS-II and NRPS biosynthetic genes by PCR. Six sets of degenerate primers  were useEscherichia coli DH5\u03b1. Plasmids were isolated from overnight cultures of colonies using the PureYield Miniprep kit . The insert was Sanger sequenced from both directions using the T7 forward and M13 reverse primers. The full 16S rRNA gene was assembled in Geneious Prime 2019.0.4 (USA). The taxonomy of the strains was determined using BLAST against the NCBI database based on the top BLAST hit. The partial 16S rRNA gene sequences for isolated bacteria have been submitted to the NCBI under accession numbers MT393617\u2013MT393684.Sixty-eight bacterial isolates  tested positive for more than one type of biosynthetic gene . These 68 isolates were identified based on their 16S rRNA gene sequence. Genomic DNA was used as a template to amplify approximately 1,400 base pairs of the 16S rRNA gene using the universal bacterial primers 27f and 1492r . All PCRChromodoris africana, Chormodoris quadricolor, G. annulatus and J. funebris. In addition, Chromodoris africana was not collected in initial sampling expeditions, and therefore, there are no microbial isolations from it. A specimen collected later in September 2017, however, was used for the microbial community analysis as a comparison to its congener, Chromodoris quadricolor. The microbial community analysis was not performed on Ceratosoma trilobatum due to limited sample material. Each animal was cut longitudinally into two pieces and the skin and gut were separated for each half, resulting in four samples from each animal. The tissues were homogenized in liquid nitrogen, and genomic DNA was extracted according to the C-TAB protocol  with Earth Microbiome Project primers 515F (Parada) and 806R (Apprill) appended with Illumina-specific adapters  were visualized after gel electrophoresis in 2% agarose. PCR amplicons of the correct size were purified using the DNA Clean & Concentrator-5 kit  and sequenced in both directions. Sequences were aligned with nudibranch-associated microbiome sequences using Geneious followed by BLASTn searches against the NCBI nr database. In order to obtain longer sequences for more accurate identification, the specific primers were used in PCRs with universal bacterial 16S rRNA gene primers (331F and 797R) and nudibranch metagenomic DNA. Resulting PCR amplicons were sequenced in both directions. The taxonomy of the three individual SVs were determined using BLAST against the NCBI database based on the top BLAST hit.Specific primers were designed for unidentified sequences from several individual SVs grouped under these identifiers that could not be classified beyond the domain level as \u201cBacteria\u201d  to confiCOI gene sequences , another 96 isolates were obtained from homogenates of J. funebris, Chormodoris quadricolor and G. annulatus. Chormodoris quadricolor yielded the highest number of isolates. Most of the isolates were obtained on Marine Agar (32%), followed by R2A (27%), and M1 (17%). Nine percent were isolated on SCA and ISP2 and six percent from AIA.A total of 196 bacterial isolates differentiated by colony morphology were obtained from the four nudibranch species in two rounds of isolation on six media types. The first isolation from S. aureus ATCC 25923, Escherichia coli ATCC 25922, B. subtilis ATCC 6633), and one fungus . Fifty-one isolates exhibited antimicrobial activity against at least one tested organism and 19 isolates displayed antimicrobial activity against two or more organisms  had intermediate antitumor activity against EAC (IC50 less than 600 \u00b5g/mL).The metabolic extracts of the first 100 isolates (obtained in round 1) were tested for inhibitory activity against three model bacteria  were identified by their 16S rRNA gene sequence . The isoChormodoris quadricolor, G. annulatus, Chromodoris africana and J. funebris) were assessed by high-throughput amplicon sequencing of 16S rRNA gene fragments. Alpha diversity analyses suggested that the nudibranch microbiomes were largely captured by the sequencing depth  of two sequences with no close relatives. These sequences shared ~92% identity unidentified Bacteria and unidentified Gammaproteobacteria, and were found in the microbiome sequences from most of the nudibranch tissue samples. The presence of these sequences in holobiont DNA was confirmed by PCR with specific primers, followed by Sanger sequencing (Endozoicomonadaceae (Gammaproteobacteria), Enterobacteriaceae (Gammaproteobacteria) and Bacillaceae (Firmicutes) (Bacillaceae and Enterobacteriaceae) varied significantly among nudibranch species. The family Rhizobiaceae and an unidentified alphaproteobacterial taxon were represented only in J. funebris, while the Mycoplasmataceae and Arcobacteraceae were highest in Chormodoris quadricolor and absent in J. funebris. Cyanobacteria were higher in J. funebris and G. annulatus than in Chormodoris quadricolor and absent in Chormodoris africana. Fourteen Gram-positive genera were present in microbiome data, while only seven were isolated from homogenized animal tissues. Interestingly, while Rhodococcus and Staphylococcus represent a high number of SVs , they were a small proportion of the bioactive cultured isolates, one and ten isolates respectively. Similarly, although Nocardiopsis represented a small number of SVs (28), they represented 10% of identified cultured isolates (seven isolates).The skin and gut microbiomes of four nudibranchs  . ArchaeaThe increasing frequency of antibiotic resistant bacterial infections necessitates the discovery of novel antibiotics to counteract pathogen resistance. Currently, cultivation and fermentation of microorganisms isolated from the marine environment is one of the most important sources for novel pharmaceuticals . ExploriCeratosoma trilobatum, Chormodoris quadricolor, Chormodoris africana and G. annulatus) and the Discodorididae (J. funebris). There are relatively few studies regarding the natural products from these organisms. Australian specimens of Ceratosoma trilobatum were shown to possess different furanosesquiterpenes in the mantle compared to the viscera  and Actinobacteria  . As manKocuria) . Streptoompounds  suggestsBacillus spp. may represent a particularly fruitful source of bioactive compounds. Cultured Bacillus represent 2.5% of the total bacilli SVs in the microbiome analysis. Interestingly, while Bacillus represented a small proportion of the community, they were a high proportion of the bioactive isolates (38%) from Chormodoris quadricolor. The genus Bacillus currently includes more than 200 described species and many strains are known as producers of antimicrobial compounds, including peptides and lipopeptides (Bacillus siamensis strain ICMP 20282. Another B. siamensis strain (JFL15) was recently shown to produce cyclic lipopeptides with antimicrobial activity against multidrug resistant pathogens (Associated peptides . All isoathogens , suggestStreptomyces are prolific producers of secondary metabolites, including antivirals, insecticides, pesticides, and herbicides (Streptomyces isolates exhibited antimicrobial activity and contained PKS and NRPS biosynthetic gene clusters. While a few Streptomyces have been shown to associate with invertebrates, especially insects (Streptomyces spp. are not highly represented in either the isolates or the community analysis, their ability to produce natural products with diverse ecological activity including allelopathy (Nocardiopsis is also well known for its ecological versatility and production of a variety of bioactive compounds such as antimicrobial agents, anticancer substances, tumor inducers, toxins, and immune modulators, and has been shown to associate with a variety of invertebrate and vertebrate animals from marine and terrestrial environment (Nocardiopsis isolates in this study exhibit antimicrobial activity and harbor PKS and NRPS biosynthetic gene clusters. Their representation in both the isolates and microbial community, coupled with their ability to produce natural products with diverse ecological activity including antimicrobial (Other taxa known for producing bioactive natural products were represented in the isolates and the microbial community. rbicides . All Str insects , most inelopathy , anti-prelopathy , and antelopathy , suggestironment . All of icrobial , neuroacicrobial , portendChromodoris species suggest there is little consistency in microbiome composition at the animal genus level. Moreover, most samples featured high levels of a SV classified only to the domain Bacteria, suggesting important novel diversity within the nudibranch microbiome (Endozoicomonadaceae and Arcobacteraceae, both of which are often host-associated microbes. In particular, Endozoicomonads are extremely prevalent in marine invertebrate microbiomes, particularly those of coral (To better understand the bacterial community associated with nudibranchs, we coupled traditional cultivation with next generation sequencing. Our analyses suggested that the communities associated with the four nudibranchs were different, even though they were collected from the same environment. In addition, not surprisingly, the 16S rRNA sequences indicate that the animals possess a more diverse Gram-positive community compared to the cultured isolates. For instance, only four genera of actinomycetes were isolated while nine genera were identified though microbial community analysis. Further, 297 actinomycete SVs were identified in the community analysis, but only 20 were isolated. The microbial communities in the nudibranchs displayed higher alpha diversity of the skin microbiome relative to the gut microbiome , possiblcrobiome . Other pof coral . Sequencof coral . These rof coral . CombineMarine bacteria are an important source for the discovery of unique natural products with novel bioactivity. While considerable effort has gone into the study of many marine invertebrates and their bacterial associations, much less attention has been paid to the microbiomes of nudibranchs, prolific sources of natural products. In this study, Gram-positive bacteria associated with nudibranchs were screened for antimicrobial and antitumor activity, as well as the presence natural product biosynthesis genes. We found that a majority of the isolates tested had bioactivity and possessed natural product biosynthetic gene fragments. Many isolates with multiple types of biosynthetic genes were identified as Firmicutes and Actinobacteria, frequent producers of bioactive compounds. It is not known if these isolates produce compounds that contribute to nudibranch defense, but 16S rRNA community amplicon sequencing indicated all bacterial isolates were present in the uncultured host-associated microbiome, suggesting that they may be ecologically relevant. Most nudibranch specimens featured high levels of a SV classified only to the domain Bacteria, suggesting important novel diversity within the nudibranch microbiome. As nudibranch-associated bacteria have the potential to produce bioactive compounds, future metagenomics and metabolomics studies of these understudied systems may reveal novel biosynthetic gene clusters and natural products to help augment the natural product preclinical pipeline.10.7717/peerj.10525/supp-1Supplemental Information 1Click here for additional data file."}
+{"text": "Background and objectives: The impact of cesarean and vaginal delivery on cognitive development was analyzed in 5 year old children. Materials and Methods: Two cohorts of 5 year old children born in the years 2013 and 2014 in Karvina (Northern Moravia) and Ceske Budejovice (Southern Bohemia) were studied for their cognitive development related to vaginal (n = 117) and cesarean types of delivery (n = 51). The Bender Visual Motor Gestalt Test (BG test) and the Raven Colored Progressive Matrices (RCPM test) were used as psychological tests. Results: In the comparison of vaginal delivery vs. cesarean section, the children delivered by cesarean section scored lower and, therefore, achieved poorer performance in cognitive tests compared to those born by vaginal delivery, as shown in the RCPM (p < 0.001) and in the BG test (p < 0.001). When mothers\u2019 education level was considered, the children whose mothers achieved a university degree scored higher in both the RCPM test (p < 0.001) and the BG test (p < 0.01) compared to the children of mothers with lower secondary education. When comparing mothers with a university degree to those with higher secondary education, there was a significant correlation between level of education and score achieved in the RCPM test (p < 0.001), but not in the BG test. Conclusions: According to our findings, the mode of delivery seems to have a significant influence on performance in psychological cognitive tests in 5 year old children in favor of those who were born by vaginal delivery. Since cesarean-born children scored notably below vaginally born children, it appears possible that cesarean delivery may have a convincingly adverse effect on children\u2019s further cognitive development. In recent years, a possible impact of the type of delivery on the neurobehavioral functions of children has been discussed. Despite the recommendation of the World Health Organization suggesting that the average percentage of cesarean section deliveries should not be higher than 15% of all deliveries, the rate in developed countries has now reached now 25% [Cesarean delivery requested by mothers without any maternal or fetal indication seems to be rising . As a suSo far, there is little knowledge regarding the known effects of the mode of delivery on neurodevelopmental trajectories in children. Measured intelligence is perceived as a strong predictor of further personal development in life such as physical and mental health and mortality .Khadem and Khadivzadeh  in Iran n = 3666). The authors found that, across several measures, only cesarean-born children at the age of 8\u20139 performed significantly below vaginally born children, by up to one-tenth of a standard deviation in national numeracy test scores.In the Longitudinal Study of Australian Children, Polidano et al.  measuredn = 43,927) vs. elective CS (n = 9460). Parents evaluated their children\u2019s behavioral, cognitive, and motor developmental outcomes at the age of 9 months using the Ages and Stages Questionnaire (ASQ), and at the age of 3 years using the Strengths and Difficulties Questionnaire (SDQ). At the age of 9 months, elective CS affected scores for personal social skills (odds ratio (OR) = 1.41, 95% confidence interval (CI) 1.19\u20131.66, p < 0.05), problem solving , and gross motor function . At the age of 3 years, no increased risk in SDQ scores was observed. According to the conclusions from this study, children who were born by elective cesarean section might face a higher risk of neurodevelopmental delay.Between December 2007 and March 2008, Khalaf et al. [n = 650) vs. vaginal birth (n = 2959) in women with a previous cesarean delivery. The assessment included reading, writing, spelling, grammar, and numeracy. Analyses suggest that previous cesarean section did not increase the risk of poor school achievement of children at the age of 8 years.In Australia, Smithers et al.  analyzedn = 1,489,925), with 1,036,424 children born by unassisted vaginal delivery (VD) and 42,107 by elective cesarean section (CS). Poor school performance was observed in 138,202 unassisted VD vs. 7064 elective CS, OR = 1.06 (95% CI 1.03\u20131.09). Therefore, this study did find an association between delivery by CS and school performance.Curran et al.  assessedn = 84) and cesarean delivery (n = 40) in 6 year old children born in twin births. Children were evaluated in their first year of primary school, using the Child Neuropsychological Maturity Questionnaire and Kaufman\u2019s Intelligence Test. Significant differences were found for verbal development, nonverbal development, global development, and general intelligence. Cesarean delivery was found as a possible risk factor for neuropsychological development and intelligence in twin births.The impact of the two different modes of delivery on neuropsychological development was also investigated by Gonzales-Valenzuela et al.  who compn = 401) and vaginally delivered (n = 1354) children at the age of 1\u201359 months using the ASQ. They did not observe any risk in the ASQ domains of communication, fine motor, gross motor, problem solving, personal, and social. Their findings suggest that cesarean delivery does not increase or decrease the risk of developmental delay in children when compared to vaginal delivery.Zhou et al.  measuredWhile some of these studies indicated an impact of cesarean delivery on cognitive development in children, other studies did not observe any difference between vaginal and cesarean deliveries. Nevertheless, according to a systematic review and meta-analysis performed by Zhang et al. , birth bThe children were selected from our cohorts in two districts which differ by the level of air pollution: the higher polluted district of Karvina (Northern Moravia) and the control district of Ceske Budejovice (Southern Bohemia) .The cohorts were created in the summer of 2013 and winter of 2014 from newborns born at the Ceske Budejovice Hospital, Department of Obstetrics and Gynecology and Department of Neonatology, and at the Karvina Hospital, Department of Obstetrics and Gynecology and Department of Neonatology. Newborns were selected from full-term pregnancies with physiologic course (38th\u201341st week) of nonsmoking mothers who signed a written consent form. Cohorts included 99 newborns (summer) and 100 newborns (winter) in Ceske Budejovice and 71 newborns (summer) and 74 newborns (winter) in Karvina. Vaginal deliveries did not include any instrumental deliveries (the frequency of vacuum extraction (VEX) was around 2%, while that of forceps was around 0.9%; therefore, they were not observed due to the size of our cohorts). Cesarean sections were mostly planned. Indications for Cesarian sections were as follows: breech presentation of the fetus when mother did not accept vaginal delivery (approximately 40%), elective repeat C-section (ERCS) in mothers with previous C-section in cases when mother did not accept VBAC  or there was another medical indication for the C-section  (approximately 40%), cephalopelvic disproportion (CPD) (approximately 10%), and other reasons such as primary neurologic indication, primary ophthalmologic indication, or primary orthopedic indication (approximately 10%).The study was approved by the Ethics Committee of both hospitals and the Institute of Experimental Medicine CAS in Prague.Between November 2018 and November 2019, 199 mothers from the Ceske Budejovice district and 143 from the Karvina district who provided blood and urine samples from their newborns in 2013 and 2014 were approached to take part in psychological testing. Out of the total number of 342 potential subjects, 5 years after joining the study, 140 refused to take part in the psychological examination and 31 were impossible to contact. In the present study, the data were collected from 99 children from Ceske Budejovice and 70 children from Karvina. The presented study, therefore, includes 169 children. The data collected included a questionnaire, nonverbal intelligence test, and visual motor functioning test. The relationship between the type of delivery and psychological tests was analyzed in these children.This study was approved by the Ethical Committee of the Faculty of Health and Social Sciences, University of South Bohemia, on 30 June 2017.We asked mothers who were engaged in our study to fill out a questionnaire that consisted of 11 categories: personal data, social environment, mother\u2019s level of education , cigarette smoking, breastfeeding, the child\u2019s kindergarten attendance, allergic diseases of the tested child, symptoms of a disease of tested child within the last year, eating habits, presence of domestic animals, and household conditions. When analyzing the results, we examined information gathered in relation to the psychological test results.Breastfeeding was adopted as a potential confounder from the pediatric questionnaire in form of the full breastfeeding in months, and the continuous parameter was transformed into a binary scale using a cutoff point of six months.Father and sibling interactions were not evaluated.The Bender Visual Motor Gestalt Test (BG test)  is a psyIn the present study, a psychologist presented each figure in sequence to the test subject and asked the subject to copy it onto a sheet of paper, while asking the child to make the best copy possible. This test has no time limit and its administration usually took around 10 min. After the test completion, the results were scored on the basis of accuracy and organization. In total, 168 children at the age of 5 years completed the test.After completing the Bender Visual Motor Gestalt Test (BG test), the children were assessed by the culture-free nonverbal Raven Colored Progressive Matrices (RCPM test) , which iAccording to our experience, 5 year old children are not always ready to complete complex IQ tests upon their first visit. Therefore, more sessions are often required so that the child adapts to the test situation and successfully completes the test. The given tests (the RCPM test and the BG test) were chosen bearing in mind the fact that they are usually well accepted by 5 year old children which would facilitate adaptation to the test situation. Both the psychometric qualities and the time effectiveness of the chosen tests were considered as significant advantages, and mothers were found to be more willing to take part in the study. Out of all tested children, only one was not able to undergo the assessment, and one child was only able to complete the BG test. Both tests were administered by a trained psychologist in a quiet place provided by the hospitals in Ceske Budejovice and in Karvina. The psychologist was blinded to the type of delivery . The exact age at the day of measurement was calculated by from the difference between the date of birth and the date of psychological testing. When collecting the data, the education level of mothers was also taken into consideration.We used two statistical methods for the evaluation of differences in the cohorts. The Mann\u2013Whitney U-test (Wilcoxon rank-sum test) was used for direct comparison of the RCPM test and BG test results between cohorts.Logistic regression was used for the purpose of estimating the impact of the type of delivery on the scores of the RCPM test and BG test as dependent values. A necessary conversion of rough scores of the test values into binary scale was done by dividing medians of the appropriate group distribution. Logistic regression was used for the quantification of impact intensity to calculate the odds ratio (OR), thus estimating the strength of the association between vaginal delivery and cesarean delivery when achieving a testing score above the median of the group distribution.The odds ratio is expressed as the ratio of the odds of event A in the presence of event B and the odds of event A in the absence of event B. An OR equal to 1 suggests no association, an OR above 1 denotes a positive association, and an OR below 1 denotes a negative association of events. The OR value also quantifies how often an event is associated . CalculaTo be able to exclude all other possible confounders of estimated impacts, multiple other parameters were tested with respect to health and social status of mothers, mostly related to the maternal questionnaire, such as maternal age, maternal ETS , various maternal health status parameters, children birth parameters and birth procedures, and quantified child illness by categories in the period from birth to 2 years of age. No other statistically significant impact was found this way.p < 0.001) and the BG test (p < 0.001). When we compared both types of delivery in the less polluted Ceske Budejovice, vaginally born children achieved better results in both tests (p < 0.05), while, in the more polluted Karvina, a higher level of differences was observed in the RCPM test (p < 0.01) compared to the BG test (p < 0.05). When we analyzed this effect according to gender, statistically significant differences between vaginal type and cesarean section were seen in boys in both the RCPM test (p < 0.001) and the BG test (p < 0.001). Those differences were not statistically significant in girls.The results of psychological tests related to the type of delivery are listed in p < 0.001) born to mothers with higher education. Similar results were observed for the subsets of Ceske Budejovice (p < 0.01), Karvina (p < 0.001), boys (p < 0.001), and girls (p < 0.001). In a similar comparison using the BG test, better results were detected in all children (p < 0.01), as well as in the subsets of Karvina (p < 0.01) and boys (p < 0.05), but not in the Ceske Budejovice group and in girls. Comparing university level vs. higher secondary education, a higher score in test performance was only achieved in the RCPM test in all children (p < 0.001), as well as in the subsets of Ceske Budejovice (p < 0.05), Karvina (p < 0.01), boys (p < 0.05), and girls (p < 0.05)  .The impact of breastfeeding was also analyzed. Initially, only the simple \u201cbreastfeeding\u2014yes/no\u201d parameter was assessed. Since the number of non-breastfeeding mothers was too small, no significant impact on the psychological test results was observed. Since the parameter mentioned above was found to be insufficient for a more detailed analysis, we adopted another two parameters from the questionnaires: full breastfeeding and partial breastfeeding lengths (both in months). We split the breastfeeding length values into a binary scale of less or more than 6 months. Non-breastfeeding mothers were assigned to the first group (less than 6 months).Cross-checking tests showed that there were no statistically significant associations between the type of delivery and breastfeeding; however, a strong positive association between breastfeeding and maternal university degree was found.p < 0.01) and a highly significant impact of mothers\u2019 university education in the Karvina and boys groups . On the contrary, multivariate analysis showed that the above-median BG test results were significantly more dependent on the delivery type in both cohorts and for boys .The results for the vaginal type of delivery vs. cesarean section, mothers\u2019 university education vs. other education levels, and breastfeeding above 6 months vs. below were slightly different using the RCPM and the BG tests . The logThe analysis of the simultaneous impact on psychological tests using the multivariate model of the same factors did not show any substantial change in the results related to the type of delivery and breastfeeding length ; thus, tp < 0.01 and 0.19 \u00b1 0.39 vs. 0.06 \u00b1 0.24, p < 0.01, respectively). Several confounders significantly differed in Karvina vs. Ceske Budejovice; the gestation age was longer , the birth length was shorter , the Apgar 5\u2032 score was higher , and the TBC primovaccination was higher . The incidence of some children diseases was also higher in Karvina, such as GIS   and viral diseases . However, those tested confounders did not affect the results of both psychological tests.Results obtained from the maternal questionnaires are presented in We tested the hypothesis of whether there was a significant correlation between the mode of delivery and the performance in cognitive tests in 5 year old children. In our study, we observed a very significant positive impact of the vaginal type of delivery on the psychological cognitive test results in 5 year old children, which was more pronounced in boys. The observed results were not affected by any of the tested confounders. These outcomes correspond to those from studies by Polidano et al. , Curran The IQ score of children, assessed on the basis of the performance in the intelligence test, correlated positively with the level of maternal education. These findings are in accordance with the common paradigm regarding the heritability of general cognitive abilities , which sThe mechanisms of the negative impact of cesarean section on children\u2019s neurobehavioral functions may be explained by several factors. When a child is born by cesarean section, the child is not exposed to the mother\u2019s vaginal microbiota at birth . ChangesAnother possible explanation for the differences in test performance could have been the result of the influence of the environment. Even though we included many parameters such as sex, socioeconomic parameters, morbidity, air pollution, and others, there remain other variables such as father and sibling interactions, childhood diet, way of upbringing, and social enrichment . Other vA limitation of our research was the total number of participants. By presenting this evidence, we would like to motivate more research within this topic. A larger number of participants, as well as methods and diagnostic tests, may help us to understand these problems in further detail.According to our findings, birth by cesarean section may present a higher risk related to psychological development of children compared to vaginal birth, especially in boys. The cesarean section is one of the most frequently performed surgical procedures in the world, and the rates of the cesarean delivery seem to be rising. We believe there is a need for a better understanding of all the potential effects and impacts of this method of delivery to gain more detailed insight into an informed decision-making process in order to be able to consider all the benefits and risks of each type of delivery.We analyzed the impact of cesarean delivery and vaginal delivery on the cognitive development of 5 year old children, using the Bender Visual Motor Gestalt Test (BG test) and the Raven Colored Progressive Matrices (RCPM test). Children born by cesarean section achieved poorer performance in both tests compared to children born by vaginal delivery. This effect was more pronounced in boys. The results of cognitive tests were also influenced by mothers\u2019 education level. Our findings support the hypothesis that cesarean section may have an adverse effect on children\u2019s further cognitive development."}
+{"text": "This study explores whether semantic processing in parafoveal reading in the Italian language is modulated by the perceptual and lexical features of stimuli by analyzing the results of the rapid parallel visual presentation (RPVP) paradigm experiment, which simultaneously presented two words, with one in the fovea and one in the parafovea. The words were randomly sampled from a set of semantically related and semantically unrelated pairs. The accuracy and reaction times in reading the words were measured as a function of the stimulus length and written word frequency. Fewer errors were observed in reading parafoveal words when they were semantically related to the foveal ones, and a larger semantic facilitatory effect was observed when the foveal word was highly frequent and the parafoveal word was short. Analysis of the reaction times suggests that the semantic relation between the two words sped up the naming of the foveal word when both words were short and highly frequent. Altogether, these results add further evidence in favor of the semantic processing of words in the parafovea during reading, modulated by the orthographic and lexical features of the stimuli. The results are discussed within the context of the most prominent models of word processing and eye movement controls in reading. An interesting debate in the literature on reading concerns the nature of the information extracted from words located in the parafovea. It is well known that the foveal region  is the region with the highest visual acuity, generally comprising 6\u20138 characters, and that it is clearly distinguished from the parafoveal region , which extends beyond the foveal region for up to approximately 15\u201320 characters , it has The PPE is generally tested by using a display change paradigm such as the boundary paradigm , in whicHowever, it is still a matter of debate whether, and to what extent, parafoveal information may be deeply processed up to the semantic level. For example, Rayner, Balota and Pollatsek  and RaynThe PoF effect refers to the possibility that parafoveal word processing can influence foveal word processing during reading ,26,27,28A larger set of studies using a display change paradigm (such as the boundary paradigm) for sentence reading tasks has, however, failed to report systematic semantic parafoveal-on-foveal effects ,31,32. AEvidence of semantic processing have been shown in German ,33 and CMore recently, Snell, Declerck and Grainger  directlyIn this study, we explore parafoveal semantic processing using the rapid parallel visual presentation paradigm (RPVP), whereby two words are briefly and simultaneously presented , with onOverall, the aim was to investigate the existence of semantic parafoveal processing and, by recording eye movements and vocal reaction times, how it may be modulated by perceptual and lexical variables, such as word length and written word frequency, as well as exploring the preview benefit effect by analyzing the correct naming percentage of parafoveal words and the parafoveal-on-foveal effect by analyzing foveal word reaction times as a function of different parafoveal stimulus features. We thus orthogonally manipulated the stimulus length, written word frequency and the semantic relationship between the two stimuli. We hypothesized that if the parafoveal processing reaches a semantic stage, the parafoveal word should achieve higher accuracy when it is semantically related to the foveal word, as opposed to when the two stimuli are not semantically related. We also hypothesized that the semantic preview effect should be modulated by the length and written frequency of both words, with a higher accuracy attained when both words are short  and hThirty undergraduate students from Libera Universit\u00e0 Maria Ss. Assunta (LUMSA) University gave written informed consent for their participation in the study. The average age of the participants was 24 years old . All participants reported themselves to be non-dyslexic and had normal or corrected-to-normal sight. The participants were naive with regard to the final purpose of the experiment.One hundred twenty-eight pairs of words, selected from the LEXVAR database , were up > 0.05). Each list was composed of pairs of words labeled w1 for the first word presented in the fovea and w2 for the second word presented in the parafovea. Half of w1 and w2 were short (N = 32 pairs) and half were long (N = 32 pairs). Half of w1 and w2 were of a high frequency (N = 32 pairs) and half were of a low frequency (N = 32 pairs). All possible combinations among word lengths and frequencies were used. The lengths in letters and the written word frequencies of the stimuli are reported in p < 0.001. Synonyms and antonyms were excluded from the set of stimuli pairs.Two lists were created, one semantically related (SR) and one semantically unrelated (SU), each consisting of 64 pairs of words. The two lists of words were matched by age of acquisition, familiarity, imaginability, concreteness, adult written frequency, number of orthographic neighbors, bigram frequency, number of syllables and length in letters  was used for programming and running the experiment. Monocular eye movements were recorded in binocular vision via an SR Research Ltd. Eye Link 1000 eye-tracker, sampling at 1000 Hz in order to ensure the fixation stability and retinal position of the stimuli prior to their presentation on the screen. Participants were seated at a 60 cm distance from the display, with their heads held firm by the use of a chin rest. Stimuli were presented on a 17 inch LED screen . Participants\u2019 voices were recorded via a one-way microphone connected to an external sound card (M-track 2 \u00d7 2).A nine-point calibration and validation procedure and a drift correction to ensure fixation stability were performed before each individual trial. Subsequently, a fixation cross  was then presented on the left-hand side of the screen. The onset of the stimuli was then triggered by steady fixation from the participant on the fixation cross which, positioned between the second and the third letters of the foveal word , remain2(1) = 295.9, p < 0.0001), when w2 was of a high frequency rather than a low frequency  = 16.8, p < 0.0001) and when there was a semantic relation between the two words  = 95.5, p < 0.0001). Moreover, the three-way interactions of w1 length, w2 frequency and semantic relation (\u03c72(1) = 22.3, p < 0.0001) and w2 length, w1 frequency and semantic relation (\u03c72(1) = 12.3, p < 0.0005) were statistically significant. Crucially, the four-way interaction of w1 length, w2 length, w1 frequency and semantic relation (\u03c72(1) = 3.8, p < 0.05) was statistically significant. As shown in p < 0.001) for both the semantically related and semantically unrelated words, only in the semantically related condition did the word frequency affect the accuracy. Indeed, in the semantically related condition, when w1 was a long, low-frequency word, the accuracy was low regardless of the length of w2 (p < 0.001). Conversely, when w1 was a long, high-frequency word, an increase in accuracy was observed only for a short w2 (p < 0.001).On average, the accuracy was 100% on w1 and 56% on w2 (range = 25\u201389%). Thus, no further analysis was performed on the accuracy of w1. In order to investigate the proportion of errors in the different experimental conditions, a logistic regression analysis was run on the binary accuracy data, with participants as repeated measures. The length (short or long), frequency (low or high) and semantic relationship  between the two words represented the independent variables. A higher accuracy for w2 was observed when w1 was short than when it was long  were measured in milliseconds as the interval between the onset of the two stimuli on the screen and the onset of the vocal response of the participant. The participants named w1 and w2, in this order, and the RT was measured on w1. Only the RTs of trials in which the participant was accurate, concerning both w1 and w2, were included in the analysis. Trials in which the response time was beyond 2.5 standard deviations from the mean were discarded . A factorial ANOVA was run with the reaction times as dependent variables and the length, frequency and semantic relationship between the two words as independent variables.p < 0.0005, \u03b7p2 = 0.007). The significant interaction of the w1 length, w2 length and semantic relation  = 4.8, p < 0.05, \u03b7p2 = 0.002) indicated that shorter RTs were observed when both words were short, but only when semantically related. Moreover, the significant interaction of the w1 frequency, w2 frequency and semantic relation  = 3.8, p < 0.05, \u03b7p2 = 0.002) indicated that shorter RTs were observed when both words were of a high frequency, but only when semantically related  = 12.6, p = 0.00001, \u03b7p2 = 0.007), indicating significantly shorter reaction times in the case of correct readings of w2  versus no responses  and misread words . Therefore, the correct identification of the parafoveal word appeared to speed up the RTs measured for w1.In order to investigate whether the performance for w2  modulated w1 reaction times, a one-way ANOVA variance analysis was applied, with the reaction times for w1 as the dependent variable and the accuracy of reading w2  as grouping variables. This revealed significant differences among the conditions  = 11.7, N = 1101) of the total being no responses and 29.9% (N = 470) being misread words. The distribution of the errors (no responses and misread words) according to the different experimental conditions is represented in The total number of w2 reading errors was 1571 (44% of response), with 70.1% (N = 470) were therefore found to be distributed as follows: 64% perceptual (N = 302), 3.4% semantic (N = 16) and 26.8% a combination of both (N = 126). The remaining 26 errors (5.5%) were other types of errors not falling the three categories .We divided the misread errors into three major categories: perceptual, semantic and a combination of the two. The errors were perceptual if (1) it had the first letter in common with the target word ; (2) it shared important perceptual features, such as a double consonant ; (3) it had the same length in letters ; (4) it consisted of a letter transposition ; and (5) it consisted of a letter substitution . The errors were semantic if they were semantically related to the words actually presented . Finally, a proportion of the errors had both a perceptual and a semantic component . The misread errors . Indeed, the presence of a semantic relation between the two linguistic stimuli was observed to speed up the naming of the foveal word when both words were short and highly frequent.Altogether, these results provide evidence in favor of the semantic processing of words in the parafovea region during reading ,14,15,40The parallel model of word recognition features the simultaneous processing of not only perceptual and lexical information, but also high-level semantic information from parafoveal vision, as in the OB1-Reader model . The modHowever, the large amount of errors in naming the parafoveal word is not fully justified by parallel models, pushing toward a serial model of word processing. In fact, our results are also in accordance with the hybrid mechanism of the saccade triggering model , based oWhile the present study\u2019s qualitative error analysis suggests a strong influence from low-level perceptual stimuli features, the results on reading speed provide strong evidence in favor of possible semantic processing in the parafovea region, highlighting the presence of both a semantic parafoveal preview benefit and a semantic parafoveal-on-foveal effect during reading that demands further investigation. Indeed, this study indicates that a limited capacity mechanism that decodes only partial information might be sufficient to trigger high-level semantic word processing.Despite the already mentioned advantages of the RPVP paradigm, as also clearly showed by Snell et al. , one limIn this study, we investigated the parafoveal semantic advantage netted by other linguistic factors that contribute to functional reading. Thus, future studies are required to further investigate how readers extract word meaning from the parafovea region during natural reading."}
+{"text": "The activity of novel therapies that utilize patient\u2019s own T-cells to induce remission of B-cell non-Hodgkin lymphoma (B-NHL), including chronic lymphocytic leukemia (CLL), is still suboptimal. In this review, we summarize the clinical efficacy of T-cell-based therapies in B-NHL and provide a biologic rationale for the observed (lack of) responses. We describe and compare the acquired T-cell dysfunctions that occur in the different subtypes of B-NHL. Furthermore, we discuss new insights that could enhance the efficacy of T-cell-based therapies for B-NHL and CLL.The next frontier towards a cure for B-cell non-Hodgkin lymphomas (B-NHL) is autologous cellular immunotherapy such as immune checkpoint blockade (ICB), bispecific antibodies (BsAbs) and chimeric antigen receptor (CAR) T-cells. While highly successful in various solid malignancies and in aggressive B-cell leukemia, this clinical success is often not matched in B-NHL. T-cell subset skewing, exhaustion, expansion of regulatory T-cell subsets, or other yet to be defined mechanisms may underlie the lack of efficacy of these treatment modalities. In this review, a systematic overview of results from clinical trials is given and is accompanied by reported data on T-cell dysfunction. From these results, we distill the underlying pathways that might be responsible for the observed differences in clinical responses towards autologous T-cell-based cellular immunotherapy modalities between diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), and marginal zone lymphoma (MZL). By integration of the clinical and biological findings, we postulate strategies that might enhance the efficacy of autologous-based cellular immunotherapy for the treatment of B-NHL. B-cell non-Hodgkin lymphomas (B-NHL) arise during different stages of B-cell development, which is reflected by differences in biological and clinical characteristics. This also explains their distinct sensitivity to therapeutic modalities. Current treatment of B-NHL includes DNA crosslinking agents such as cyclophosphamide, bendamustine, and purine analogs such as fludarabine in combination with CD20 targeting antibodies. In more aggressive tumor types, inhibitors of DNA synthesis such as doxorubicin are added. Our increased understanding of oncogenesis and the relevance of crosstalk between immune cells and cancer cells in the tumor microenvironment (TME) has led to the development of targeted therapies in B-NHL, such as inhibitors of the B-cell receptor signaling pathway, and inhibitors of apoptosis regulating proteins such as the Bcl-2 binding drug venetoclax ,2. FurthICB reverses inhibitory interactions between the T-cell and cancer cell, thereby improving cytotoxicity of tumor recognizing T-cells and inducing cancer cell lysis. Within the tumor microenvironment, chronic stimulation of tumor infiltrating lymphocytes (TILs) leads to T-cell exhaustion, which hampers proper anti-tumor responses. Blocking of inhibitory receptors, such as programmed death-1 (PD-1) or cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), on T-cells, or their ligands on tumor or adjacent T-cells, potentiates anti-tumor responses. Investigations of these antibodies in hematological malignancies were prompted by the success of ICB in solid tumors such as melanoma and non-small cell lung cancer ,9 and leIn contrast to ICB, BsAbs induce tumor cell killing without the need for specific tumor cell recognition by autologous T-cells. BsAbs tether T-cells and cancer cells via CD3 on T-cells and a tumor-associated target antigen on cancer cells resulting in direct tumor cell killing. Blinatumumab was the first Food and Drug Administration (FDA)-approved CD3xCD19 bispecific T-cell engager (BiTE). This BsAb is currently only approved for relapsed/refractory acute lymphoblastic leukemia (ALL), in which trials have shown complete remission (CR) rates of around 33% ,12.Similar to BsAbs, CAR T-cells act through direct recognition of cancer cells. T-cells acquire CARs through retroviral or lentiviral vectors, which requires ex vivo transduction, activation, and expansion. First generation CARs consisted of an antigen binding moiety (ScFv) covalently linked to CD3 zeta, allowing tumor antigen recognition and subsequent T-cell activation ,14. SecoResponses to aforementioned immune therapies are still suboptimal in B-NHL, including chronic lymphocytic leukemia (CLL). By exploring clinical data and connecting them to ex vivo and in vitro observations on T-cell (dys)function, we propose underlying mechanisms of treatment failure for the most common types of B-NHL, including diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), and marginal zone lymphoma (MZL). These data are integrated in order to suggest possible solutions to the current hurdles of autologous-based T-cell therapies in B-NHL.n = 34) was only 3% [Although response rates were modest in early trials, patients that responded to ICB were reported to be in remission for over a year ,21,22. T only 3% . Primary only 3% .These trials indicate that ICB monotherapy is not sufficient to elicit significant responses in B-NHL. Therefore, research has focused on combining ICB antibodies or adding other treatment modalities to enhance efficacy. Unfortunately, combining ipilimumab and nivolumab in B-NHL patients, FL or DLBCL, resulted in a modest partial response (PR) of three out of 15 patients (ORR 20%) . In contCombinations of ICB and lenalidomide have also been studied. Preliminary data from a phase I trial combining nivolumab with lenalidomide in 10 B-cell lymphoma patients  showed an ORR of 30% . AnotherThe combination of nivolumab with the Bruton\u2019s tyrosine kinase (BTK) inhibitor ibrutinib was tested in a large phase I/II study. ORR rates in CLL/small lymphocytic leukemia (SLL) (36 patients), CLL with RT (20 patients), FL (40 patients) and DLBCL 45 patients) were 61%, 65%, 33% and 36%, respectively . Except  patientsAgain, relatively high response rates are seen in PBMCL patients treated with a combination of ICB (nivolumab) with the anti-CD30 antibody-drug conjugate brentuximab vedotin (ORR of 73%) with CR in 11 patients (35%) .Together, these results indicate that, unlike in HL, results for ICB in B-NHL are thus far disappointing, except for PMBCL. Although combinations, such as with rituximab or lenalidomide, seem to increase response rates, it remains to be elucidated whether a true synergistic, or at least additive, effect exists.n = 34 of 41 included patients) [Multiple studies have investigated BsAbs in B-NHL . The firatients) . Althougatients) . This coatients) . Blinatuatients) .Treatment with mosunetuzumab, a CD3xCD20 BsAb, resulted in an ORR of 64% and CR of 42% in 64 patients with indolent B-NHL, mainly consisting of FL patients . In contIn general, these studies show that reasonable responses against B-NHL can be elicited by BsAb monotherapy. Although patients with indolent lymphomas such as MCL and MZL are currently underrepresented, enhanced efficacy of BsAbs towards indolent lymphomas compared to DLBCL is observed . For CLLn = 108) treated with 28z CAR, a CR rate of 58% was observed. The DOR was 11 months (PFS of 5\u20139 months) [Virtually all CAR construct studies in trials thus far are directed against the pan B-cell marker CD19 and contain either the intracellular domain of the co-stimulatory receptor CD28 (28z) or 4-1BB (BBz). Since 28z and BBz CARs are functionally different, we will discuss them separately . Current months) . Similar months) . In anot months) .Early and small trials (that recruited \u2264 50 patients) using BBz CAR T-cells yielded CRs of 19.5\u201387.5% ,78,79,80To our knowledge, only one study has directly compared the clinical efficacy of 28z and BBz CARs. Three patients received 28z CARs and six patients received BBz CARs. Unfortunately, the 28z CAR was not well tolerated. Eight patients were evaluable for response, of which one had MZL (received BBz CAR), three DLBCL (two BBz and one 28z), and five FL (three BBz and two 28z). Eighty-seven percent of the patients had CR with an ORR of 87% after 3 months .In an effort to search for better responses to CAR T-cell therapy, multiple studies have investigated combinations of CAR with ibrutinib or anti-PD-1. For example, 22 patients with CLL who had received ibrutinib as a prior therapy received BBz CARs, which resulted in CR in 45.5% . ImproveBased on the clinical results described above, it becomes clear that CAR T-cell therapy can yield impressive and lasting responses in B-NHL patients. Especially in DLBCL, FL, MCL, and MZL, CAR T-cell therapy yields high CR rates but still falls well below of what is observed in ALL, with the exception of very few clinical trials . The mosFrom the clinical observations, it follows that efficacy of autologous T-cell-based cellular therapies differs between lymphoma subtypes and between treatment modalities, with the lowest responses for ICB, and that in general, such treatments are less effective in the discussed lymphoma entities than in HL (ICB) and ALL (BsABs and CAR T-cells). In recent years, it has become apparent that patients with B-NHL acquire alterations in T-cell differentiation and function. These T-cell abnormalities affect responses to cellular therapies but are not similar among the different lymphoma subtypes. In this section, we predominantly focus on changes in phenotype and function of T-cells in B-NHL.Successful ICB depends on recognition of tumor cells by immune effector cells such as T-cells. These T-cells often recognize antigens that arise due to mutations within tumor cells. Indeed, a clear correlation exists between tumor mutational load and efficacy of ICB ,90. Alth+/CD27+/CCR7+), memory ), effector memory (CD45RA\u2212/CD27\u2212/CCR7\u2212) T-cells, and terminally differentiated effector T-cells (CD45RA+/CD27\u2212/CCR7\u2212) [T-cells can be subdivided into naive (CD45RA\u2212/CCR7\u2212) ,95. Of t\u2212/CCR7\u2212) . Skewing+ and CD8+ numbers are increased [+ and CD8+ effector memory T-cells are expanded at the cost of naive T-cells [+ T-cells produce interleukin (IL) 4 is described [+ T-cells produce more interferon y (IFNy), possibly contributing to an anti-tumor response [In PB of CLL, absolute CD4ncreased ,99,100, ncreased ,102. In  T-cells ,102,103. T-cells . Both T escribed ,106,107,response .+ tumor-infiltrating T helper cells from FL patients showed a skewing towards an effector memory phenotype, while naive and central memory cells were decreased [+ T-cells were found in PB of FL patients, attributable to a decrease in naive T-cells [+ numbers were normal, a skewing towards T effector memory cells re-expressing CD45RA (Temra) cells could be observed within the CD8+ compartment [Th2 and Th17 cells are increased in LN of FL patients ,110. Alt T-cells . Althougpartment .+ T-cells were observed as the main infiltrating T-cell type [+ T-cells were found in extranodal MZL with predominance of the na\u00efve phenotype [+ T-cells in MZL had a terminally differentiated phenotype [T-cell skewing is observed in MZL as well. T-cell infiltrates in mucosa-associated lymphoid tissue (MALT) lymphoma predominantly have a Th1 phenotype, while in some cases CD8ell type ,114,115.henotype . Similarhenotype .It thus seems that T-cells from B-NHL patients are skewed towards a more antigen-experienced phenotype, and have a disturbed Th1/Th2 balance, although the latter varies between different B-NHL subtypes.+/CD27+/CD45RO\u2212, were found in the product in CLL patients that do respond to therapy [+ CAR T-cells negative for PD-1 correlated with improved clinical outcome [Skewing of T-cells can greatly influence T-cell therapy. Since memory T-cells are the driving force of T-cell persistence in vivo, and their presence is correlated with highly durable clinical remissions in CAR T-cell therapy, it is plausible that the composition of the patients\u2019 T-cell subsets may influence the therapy outcome. For CAR T-cells, indeed, persistence and peak expansion of CAR T-cells in vivo are predictors of favorable clinical outcome in B-NHL, coinciding with the presence of memory-like T-cells ,78,116.  therapy . Further outcome . For BsA outcome ; howeverChronic stimulation and inflammatory signals can induce exhaustion of T-cells leading to loss of effector function. Multiple exhaustion markers have been extensively studied, including PD-1, T-cell immunoglobulin, and mucin domain-containing protein 3 (TIM3), T-cell immunoglobulin and ITIM domain (TIGIT), CTLA-4, and lymphocyte activation gene-3 (LAG3), and negatively regulate T-cells via different mechanisms as extensively reviewed in . Ligands+TIM3+ and CD8+TIM3+ T-cells were observed [In DLBCL, an estimated 11\u201324% of all patients show high expression of PD-L1 on tumor cells and high PD-1 expression on T-cells, which are associated with poor patient survival ,123,124.observed , correlaobserved . Furtherobserved . Immunomobserved . Interesobserved .Additionally, in CLL, exhaustion may play a role. T-cells derived from these patients display increased expression of PD-1, BTLA, CTLA-4, TIGIT, CD160 and CD244 ,132,133.In MCL, intratumoral T-cells display significantly higher expression of TIGIT, CD244, and LAG3 compared to controls, especially in the effector-memory compartment . Only a In MZL, PD-1 and PD-L1 also might play a role. PD-L1 was expressed by splenic MZL cells , and itsDespite high expression of exhaustion markers on B-NHL-derived T-cells, the efficacy of ICB is disappointing, which may not only be explained by a relative low tumor mutational load see  but alsoT-cell exhaustion can influence the efficacy of CAR T-cell therapy as well. For example, five patients with the highest PD-1/PD-L1 interactions showed no response to therapy, or quickly relapsed in a CAR T-cell trial for DLBCL patients . In addiIt is currently unclear what role exhaustion plays in the treatment of patients with BsAbs since this has not been investigated extensively to our knowledge.Chronic stimulation of T-cells by tumors can lead to functional impairments such as defective cytokine production, activation, synapse formation and cytotoxic potential. For example, impaired formation of the immunological synapse impairs CAR T-cell efficacy, and therapy effectiveness can even be predicted by assessing the quality of synapse formation between the CAR T-cell and target cells . The funIn CLL, T-cells retain cytokine production, but have impaired synapse formation proliferation, activation, and cytotoxicity ,151,152.+ T-cells in FL samples compared to reactive lymph nodes [In FL, similar defects have been reported. Upon T-cell receptor (TCR) triggering, TILs in FL have a defective proliferative capacity ,140, impph nodes . These Tph nodes .Helicobacter pylori may impair T-cell functionality demonstrated by reduced killing of autologous Epstein\u2013Barr virus B-cells [In MZL, induction of MALT lymphoma by  B-cells .It can thus be concluded that different B-NHL subtypes harbor different functional defects, although most seem to have acquired defective cytotoxic and proliferative capacities as well as impaired synapse formation. Nevertheless, it remains to be elucidated whether this is also the case for other B-NHL subtypes.It has been widely accepted that T-cell metabolism dictates function and development as extensively reviewed previously ,152. In + T-cells that express forkhead box protein P3 (FOXP3), play a major role in preventing autoimmunity by dampening immune responses. However, Tregs have conversely been implicated in hindering effective anti-tumor immunity as well [Regulatory T-cells (Tregs), a subset of CD4 as well . Tregs hAlthough Tregs are classified as immunosuppressive, increased numbers of Tregs correlate with favorable prognosis in DLBCL and FL, where low Treg numbers can predict transformation of FL to DLBCL ,169,170.+CXCR5+PD-1+, can play a role in support of the TME and are well studied in DLBCL and FL. For DLBCL, Tfh cells seem to promote DLBCL cell growth and survival, via IL-10 secretion [+CXCR5+Foxp3+, and may regulate follicular T helper cells (Tfh) and B-cells (reviewed in [T follicular helper cells (Tfh), characterized as CD4ecretion . These Decretion , and theecretion . Anotheriewed in ). In DLBiewed in . Tfr celiewed in . Additioiewed in . Furtheriewed in ,183,184.The multiple levels of T-cell dysfunction that we describe here could serve as novel targets to improve the efficacy of T-cell-based therapies. Of the many approaches that have been described, we will focus on three possible solutions that could already be tested within clinical trials. The first is the addition of agents that target T-cells to improve their efficacy, the second is timing of autologous T-cell-based therapy and the third are specific solutions to improve CAR T efficacy either by adjusting the CAR T-cell design itself, or the CAR T cell production process.Lenalidomide is an immunomodulatory drug that has been approved for treatment of multiple myeloma, MCL, and myelodysplastic syndromes. Lenalidomide was shown to bind to the E3 ubiquitin ligase cereblon, thereby inducing degradation of the transcription factors Aiolos and Ikaros ,186. SinSince T-cells from patients with B-cell malignancies might benefit from lenalidomide treatment, combination strategies involving T-cell therapy might therefore be feasible. These types of agents have already been tested in a clinical setting. For DLBCL, a phase II trial was started to evaluate the efficacy of CD19 CAR in combination with either lenalidomide or rituximab NCT04002401). The studies on the combination of lenalidomide with either ICB or BsAbs in B-NHL are still limited but do seem to show enhanced effects ,39,40,5102401. Th+ T-cells together with decreased IFNy production and degranulation in the E\u00b5-TCL1 mouse model [ClinicalTrials.gov Identifier: NCT02332980). In DLBCL, PBMC stimulation in the presence of idelalisib also resulted in less production of IFNy, but more IL-2 and a less differentiated phenotype as measured by CD27 and CD28 expression [Results with lenalidomide combinations show that targeted drugs can potentiate T-cells and thereby improve T-cell-based therapy. However, due to toxicities, other options in addition to lenalidomide need to be considered. Idelalisib might be a feasible candidate. Especially for CLL, reports have been published regarding this phosphoinositide 3-kinase (PI3K) \u03b4 inhibitor. Recently, it has been shown that idelalisib inhibits IFNy production and skews HD T-cells towards more effector differentiation upon stimulation . In CLL,se model . Whetherpression , which mpression .+ T-cells resulting in impaired cytotoxicity [Additionally, ibrutinib has gained much interest with regard to its use in combination with autologous T-cell therapy. In addition to targeting BTK, ibrutinib also inhibits IL-2-inducible T-cell kinase (ITK) expressed by T-cells . ITK inhtoxicity . It thustoxicity ,202,203.toxicity ,62. Effitoxicity . In additoxicity . Additiotoxicity . Despitetoxicity . Similartoxicity .Long-term treatment with ibrutinib, idelalisib or lenalidomide can lead to the development of severe adverse events ,208. TheIn conclusion, T-cell modulatory agents are readily available and may be able to enhance T-cell therapy in a combination setting, in which timing could also play a role in the future. Lenalidomide trials, especially, indicate that they improve T-cell-based therapy, but future studies are needed to confirm this.+ T-cells, indicating a reversion of T-cell dysfunction and recovery of T-cell skewing [As discussed above, suppression of T-cell function can be the result of overwhelming numbers of tumor cells causing metabolic pressure in addition to restriction of T-cell function by expression of inhibitory ligands on tumor cells. Therefore, it may be beneficial to debulk tumor cells prior to T-cell-based therapies as it may create a window of opportunity in which T-cells recover  their functions. This was perhaps unintentionally demonstrated in the first CAR T-cell trial where two patients who achieved CR after cytoreductive chemotherapy sustained that CR after subsequently receiving CAR T-cells . Additio skewing . Blinatu skewing , which i skewing , althougPersistence and peak expansion of CAR T-cells are predictors of favorable clinical outcome and can greatly influence CAR T-cell efficacy ,78,210. The type of costimulatory ligand in CAR can greatly impact CAR T cell function and phenotype. For example, differences in phenotype between 28z and BBz have been demonstrated in animal models, which show that the 4-1BB signaling domain enhanced CAR T-cell memory development and persistence, and promoted oxidative phosphorylation and mitochondrial biogenesis, while the CD28 signaling domain pushed towards an effector phenotype and glycolysis ,215. Sel+CCR7+ subset, and simultaneously reduce expression of inhibitory receptors PD-1 and TIM-3 [The most evident method to obtain persisting CAR T cells may be through generating CAR T cells from a pool of purified memory T-cells. Wang et al. treated eight patients with first generation central memory-derived CAR T-cell infusions, and eight more patients received second generation central memory-derived 28z CAR T-cells . The CR nd TIM-3 . Idelalind TIM-3 . Improvind TIM-3 . Preventnd TIM-3 . Furthernd TIM-3 . These mPreventing T-cell exhaustion either as a result of chronic stimulation, or indirectly induced by cancer cells, may improve CAR T-cell therapy as well. Overexpression of c-Jun, a component of the AP-1 transcription factor, can protect CAR T-cells from exhaustion and greatly enhance proliferation compared to a control CAR . AdditioThis review comprehensively shows that the efficacy of autologous-based T-cell therapy differs among B-NHL subtypes as well as per treatment modality. ICB has shown very discouraging results, and even in combination strategies, efficacy does not seem to improve and ICB is therefore likely not a feasible treatment modality in B-NHL including CLL. This is in contrast to BsAbs and CAR T-cells, which do elicit responses, although not to the extent of those observed in ALL. These diminished responses are likely due to different aspects of T-cell dysfunction that have been described in B-NHL subtypes. The recent advantages made in CAR T-cell production, and the development of immunomodulatory agents can be used to overcome T-cell dysfunction in these malignancies and improve T-cell mediated therapy for B-NHL."}
+{"text": "Thymbra capitata (L.) Cav., Mentha \u00d7 piperita L. and Santolina chamaecyparissus L. essential oils (EOs) on Avena fatua L., Echinochloa crus-galli (L.) P. Beauv, Portulaca oleracea L. and Amaranthus retroflexus L. and their effects on soil microorganisms. A pot experiment was set up and three EOs at three doses were applied by irrigation. Efficacy and effects of EOs on weed growth were determined. Soil microbial biomass carbon and nitrogen, microbial respiration, and the main microbial groups were determined at days 7, 28 and 56. EOs demonstrated herbicidal activity, increasing their toxicity with the dose. T. capitata was the most effective against all weeds at the maximum dose. P. oleracea was the most resistant weed. Soil microorganisms, after a transient upheaval period induced by the addition of EOs, recovered their initial function and biomass. T. capitata EO at the highest dose did not allow soil microorganisms to recover their initial functionality. EOs exhibited great potential as natural herbicides but the optimum dose of application must be identified to control weeds and not negatively affect soil microorganisms.Essential oils (EOs), extracted from aromatic plants, have been proposed as candidates to develop natural herbicides. This study aimed to evaluate the herbicidal potential of  One of the main challenges of the Agriculture of the 21st century is to increase crop production in a sustainable way, e.g., minimizing the use of pesticides . FurtherPlant secondary metabolites, such as essential oils (EOs), include allelochemicals compounds which have been proved to inhibit seed germination and seedling growth . EOs areMentha \u00d7 piperita L. (Peppermint) and Thymbra capitata L. (Cav) (synonym Thymus capitatus (L.) Hoffmanns. & Link) (Thyme)  nonane (8.2%) as main chemicals, showed a moderate phytotoxicity against the leaf growth of L. perenne, but did not show negative effects against L. sativa seeds  orchard  never treated with synthetic chemicals was used. Its main characteristics were: sand 64.9%, clay 15.9%, organic carbon 2.3%, pH 7.0, electric conductivity 0.1 dS m\u22121, and total nitrogen 1.2 g kg\u22121. After sampling, the soil was air-dried and sieved at 2 mm. Aliquots of 350 g of soil were placed in 1L plastic bottles and moistened with only water up to 2/3 of 50% of its WHC. Then, a volume of EO emulsion was added thus reaching the 50% of its WHC. The amounts of EO added were 31 (THY1), 62 (THY2) and 93 \u03bcL 100 g\u22121 (THY3) of soil for T. capitata treatment, and 93, 123 and 153 \u03bcL 100 g\u22121 of soil for both M. piperita  and S. chamaecyparissus  treatments. Two controls were also prepared: The first with only water (Cw) and the second with water and fitoil (Cf) at a concentration of 0.05% (v/v) to moisten the soil. Four replicates per treatment were run. After the EOs addition, plastic bottles were incubated in the dark, at constant temperature (25.0 \u00b1 0.5 \u00b0C), for 56 days. During the incubation, water loss was monitored by weighing the bottles and eventually watering them with only water to maintain the soil WHC at 50%.To test the effects of EOs on soil microorganisms, a short-term laboratory incubation experiment was set-up. The topsoil (0\u201315 cm) of a citrus [2SO4, at a ratio of 1:4 (w/v). Total organic C in soil extracts was determined by hot digestion-oxidation (sulphuric acid-dichromate mixture). MBC was estimated as the difference between the organic C held in fumigated extract and that in non-fumigated extract, multiplied by a conversion factor (kEC) of 2.64. The K2SO4-extractable C of not fumigated soil was assumed as a proxy of the readily available C pool [2SO4 extracts, respectively, according to Joergensen and Brookes [2 accumulated in the headspace of the glass jars at days 1, 4, 7, 10, 17, 23, 31, 39, and 53 was assessed by a gas chromatograph equipped with a thermal conductivity detector. At each CO2 determination, jars were ventilated with fresh air for 30 min and then sealed again, after possible replenishment of lost soil moisture by distilled water. The C mineralization rate, expressed as mg CO2\u2013C kg\u22121 dry soil day\u22121, was fitted to the following first order decay function:0 is the biologically available C (mg kg\u22121) at time zero , k is the decay rate constant, and t is the sampling incubation day.At days 7, 28 and 56, soils were analyzed to determine some biochemical properties. The fumigation\u2013extraction method  was usede C pool . Microbi Brookes . Concurr2\u2013C mineralized over 59 days of incubation was calculated according to Ioppolo et al. [i and i + 1 are the first and the last of two close CO2\u2013C rate measurements; n is the last day of measurement of CO2\u2013C rate; r is the CO2\u2013C rate expressed as mg CO2\u2013C kg\u22121 dry soil day\u22121; and d is the number of days between the two consecutive CO2 rate measurements.The total COo et al.  by the l2), i.e., the amount of CO2 emitted per unit of MBC per time unit, was calculated as mg CO2\u2013C g\u22121 MBC h\u22121.The specific respiration rate, or metabolic quotient (qCO\u22121 in a constant flow mode was used as a carrier. The injected volume was 1 \u03bcL (50:1 split ratio). Nonadecanoic acid methyl ester  was used as internal standard for the quantification of FAMEs. Peak identification was done by comparing the retention times of each FAMEs to known standards . Fatty acids with less than 14 carbon atoms or more than 20 carbon atoms were excluded as considered originating from non-microbial sources. The FAs i15:0, a15:0, 15:0, i16:0, i17:0, 17:0, cy17:0,18:1\u03c97, and cy19:0 were used to represent bacterial biomass while using 18:2\u03c96,9 for fungal biomass. The fungal-to-bacterial ratio (F/B), i.e., a measure of what proportion of the microbial community is bacteria compared to the proportion of the microbial community that is fungi, was calculated. The FAs i15:0, a15:0, i16:0, and i17:0 were chosen to represent Gram-positive bacteria (BacG+) while 16:1\u03c97, 18:1\u03c97, cy17:0 and cy19:0 for Gram-negative bacteria (BacG\u2212) [Fatty acids (FAs) were extracted from soils according to the modified Bligh and Dyer method . The fat (BacG\u2212) ,45. Fattp < 0.05) for the separation of the means in each species. A multifactor analysis of variance (ANOVA) was performed on efficacy including species and treatment as effects.The plant experiment was carried out in a completely randomized design because our unit of experimentation was the pot (10 repetitions for each treatment), and the treatments were assigned to the pots in a random way. After the application of the treatments, the pots with the same treatment were placed in a tray in order to avoid the loss of the treatments by lixiviation when the pots were irrigated. The pots were considered always as singular units and all the variables were measured individually for each pot. Biometric plant variables  and DL data were evaluated for normality and variance homogeneity and then subjected to one-way ANOVA, followed by Fisher\u2019s multiple comparison test  post-hoc test. For both biometric plant and soil variables, ANOVA was carried out without any transformation of the data. All analyses were performed by Statgraphics Centurion version XVII.Reported soil data, referred to as oven-dry soil (105 \u00b0C) weight, are the arithmetic means of four replicates. Before performing parametric statistical analyses, normal distribution and variance homogeneity of the data were checked by Kolmogorov\u2013Smirnoff goodness-of-fit and Levene\u2019s tests, respectively. Within each EO treatment, soil data were subjected to two-way ANOVA with EO dose  and incubation day  as factors. Within each EO type , significant differences at T. capitata, M. piperita and S. chamaecyparissus against targeted weeds and soil microorganisms have been studied with a more practical approach, i.e., in vivo conditions, monitoring their effects in order to know their real potential as an alternative to synthetic chemicals, within a strategy of Integrated Weed Management and analyzing the benefits or disadvantages derived from their employment.Several studies have been carried out on the phytotoxic activity of EOs against weeds and on their potential use as natural herbicides. The majority of these works have been performed in vitro experiments and not in microcosms that try to mimic the natural conditions. Moreover, in vitro approaches seeds and/or seedlings are directly exposed to the EOs in sterile conditions, i.e., strongly reducing and/or retarding EOs transformation/degradation normally mediated by soil microorganisms. To our knowledge, this is the first time that the effects of essential oils from T. capitata was the most effective, followed by M. piperita. Both EOs showed a broad spectrum of activity, with T. capitata at the highest doses applied (12 \u03bcL mL\u22121) killing plants of all weed species (100 efficacy), except for P. oleracea (90 efficacy). M. piperita at the highest dose (20 \u03bcL mL\u22121) controlled completely (100 efficacy) A. retroflexus and A. fatua plants but showed 90 and 40 efficacy on P. oleracea and E. crus-galli, respectively. Although S. chamaecyparissus EO was less active compared with the other EOs, it displayed a very remarkable selective activity, being highly effective against A. retroflexus . It could be interesting to study it more profoundly as a selective herbicide, while T. capitata and M. piperita could have a wider action, exhibiting excellent potential for the development of broad-spectrum herbicides. A good natural herbicide, besides being effective, at the same time should not have side effects on soil microorganisms. Here, results clearly demonstrated that, except for T. capitata EO at the highest concentration, which significantly increased the specific respiration rate, the other EOs generally stimulated soil biochemical properties, or their effect on them was transient. Furthermore, even when changes in the main microbial groups persisted, soil microbial activity was not irredeemably affected, suggesting that essential oils did not compromise the functional redundancy.Results clearly demonstrated that tested EOs, to a different extent, were significantly effective against weeds, killing them completely or reducing significantly their growth parameters. Among them, Since EOs are able to decrease the weed growth parameters by reducing their fitness and competitiveness, another advantage in using these EOs, from a conservationist point of view in agro-ecosystems, could be that to maintain a high biodiversity by not completely eradicating the weeds, instead giving the crop an opportunity to outcompete them."}
+{"text": "Papillary muscle rupture is a rare condition. Its clinical presentation, diagnosis and management can be very challenging for the clinician.A 73-year-old woman with hypertension presented with chest pain, ST-segment changes, and elevated serum troponin levels. Coronary angiography was normal. Echocardiography revealed normal ventricular function, flail posterior mitral leaflet, and severe mitral regurgitation. She underwent emergent mitral valve replacement.The diagnostic and management strategies of this uncommon presentation are discussed.The online version contains supplementary material available at 10.1186/s12872-022-02570-4. Papillary muscle rupture (PMR) is itself uncommon, with incidence rates of 0.029% in patients presenting with MI, and few case reports of non-MI etiology . CompareA 73-year-old woman with a medical history of hypertension presented to the emergency department after 30\u00a0min of severe pressure-like substernal chest pain that radiated to her left arm and jaw at rest. The pain began 30\u00a0min prior to presentation. Her review of systems was positive for nausea and acute shortness of breath at rest. Pertinent negatives included any history of these symptoms, dyspnea on exertion at baseline, absence of trauma, initiation of new medications, exposure to sick contacts, or recent travel.Patient's past medical history was significant for hypertension, generalized anxiety disorder, major depressive disorder, hypertension, and severe osteoarthritis. Patient was noted to have atypical polymyalgia rheumatica for which she was placed on 6\u00a0months taper course of steroids. She has been under the care of rheumatology with autoimmune and inflammatory markers all negative, indicative of osteoarthritis. Patient was on buspirone, duloxetine, hydrochlorothiazide, valsartan and meloxicam as needed. Patient's family history was significant for a father with sudden cardiac death due to dilated cardiomyopathy. No other risk factors or high-risk behaviors were reported.On physical examination, the patient was normothermic and tachycardic with a rate of 112 beats per minute. Her blood pressure was 88/55\u00a0mm of Mercury (mmHg) and peripheral oxygen saturation was 88% on 5 L of supplemental oxygen via nasal cannula. She was in acute distress, diaphoretic, with jugular venous distension to the mid-neck, and bibasilar crackles. Normal S1 and decreased S2 were noted, with a holosystolic murmur radiating to her axilla; well-perfused extremities with\u2009+\u20091 lower extremity edema to the knees were also noted.Initial electrocardiogram was indicative of diffuse ST-segment depressions and ST-segment elevation in lead aVR Fig.\u00a0. InitialAn intra-aortic balloon pump (IABP) was placed in the cardiac catheterization laboratory and patient was admitted to the cardiac intensive unit for diuresis. She was intubated in the cath lab due to severe acute hypoxemic respiratory failure. She improved with diuresis and hemodynamic support. She was taken for operative repair on hospital day 3. Intraoperative transesophageal echocardiogram (TEE) revealed severe eccentric MR . Given patient's presentation consistent with acute myocardial ischemia, myocardial infarction (MI) complicated by PMR was higher in our differential. Nonetheless, coronary angiogram showed no evidence of coronary artery disease. Coronary spasm, myocardial infraction without coronary artery occlusion (MINOCA) or acute coronary artery thrombosis followed by spontaneous recanalization could not be ruled out at this point. The patient\u2019s unstable hemodynamic status did not allow for a magnetic resonance imaging (MRI) prior to surgery. A diagnosis of idiopathic papillary muscle rupture leading to severe acute mitral regurgitation was made.The patient had a prolonged postoperative hospitalization complicated by respiratory failure and ventricular cataplexy requiring prolonged support with IABP and milrinone. Cardiac output and cardiac index did improve to normal, she was weaned off all support and was discharged from the hospital. There was no MR or decreased LVEF on follow up transthoracic echo.We present a rare case of idiopathic papillary muscle rupture causing severe MR requiring urgent surgical correction with good outcome. Spontaneous PMR in the absence of coronary artery stenosis is rarely reported in the literature \u20135, howevInitial medical management of PMR includes diuretics, and oxygenation delivered with non-invasive and invasive mechanical ventilation. Mechanical support with IABP should be considered in patients presenting with cardiogenic shock . NeverthAcute mitral regurgitation as a complication of myocardial infraction has a very poor prognosis if left untreated. We present a rare case of idiopathic papillary muscle rupture causing severe MR requiring urgent surgical correction with good outcome.Additional file 1: Video 1. RAO CRAN view of Right Coronary Artery; Less than 30% occlusionAdditional file 2: Video 2. RAO Caudal view of Left Main, Circumflex and Anterior Descending Arteries; less than 30% occlusion in all territoriesAdditional file 3: Video 3. Ventriculogram of left ventricle showing\u2009+\u20094 regurgitation jet in a severely dilated left atriumAdditional file 4: Video 4. Posterior Long Axis View, Transthoracic ECHO; Flail posteromedial mitral valve cusp, eccentric regurgitant jet, dilated left atriumAdditional file 5: Video 5. Transesophageal ECHO, midesophageal 3 chamber view; dilated LA, eccentric regurgitant jet, vena contracta 0.6\u00a0cmAdditional file 6: Video 6. Intraoperative Transesophageal ECHO Post Mitral Valve Replacement, midesophageal 4 chamber view; 31\u00a0mm Epic bioprosthetic valve, no regurgitation"}
+{"text": "No significant differences were reported for the CAIT or PACES-8. This study supports the feasibility and safety of stability-based training with VIS in those with CAI. The enhanced performance outcome on the SEBT suggests VIS may enhance stability-based training.Chronic ankle instability (CAI) is associated with recurring symptoms that inhibit daily activity. Stability-based rehabilitative training is recommended for CAI. Visualisation (VIS) produces real-time feedback using motion capture and virtual reality. This pilot study aimed to determine the feasibility, adherence, safety, and efficacy of incorporating VIS into stability training for people with CAI. Efficacy was examined through effect of VIS training on dynamic stability, perception of stability, and rehabilitative experience. Individuals with CAI completed a 4-week stability-based training programme with VIS, or without visualisation (NO-VIS). Participants completed the Star Excursion Balance Test (SEBT) and Cumberland Ankle Instability Tool (CAIT) prior to, and after training. Enjoyment of training was recorded using the Physical Activity Enjoyment Scale (PACES-8). Of 17 participants , there were 2 drop outs . No adverse events were reported, and participant drop-out was due to injury unrelated to the study. The VIS group showed a significantly greater increase in average SEBT reach distance ( Chronic ankle instability (CAI) is a complicated multi-faceted clinical condition affecting 20\u201370% of those who have experienced an ankle sprain , 2. CAI Stability-based rehabilitative training is the most recommended rehabilitation strategy for people with CAI , 9. StabVirtual reality (VR) presents an enhanced opportunity for interactive simulation using computer software to provide feedback of movement and performance. For stability-based training, this facilitates practice with externally focussed augmented feedback incorporating motor and cognitive manipulation in a safe environment , 14, 15.VR training enhances stimulation and engagement and has been associated with greater satisfaction and enjoyment of training \u201319. For Visualisation is an emerging technique that connects biomechanical analysis and VR. Visualisation produces real-time feedback by accurately monitoring movement and progress in a diverse, challenging, and controllable environment, representative of real-world situations. Communication could therefore be improved between patient and specialist by making tasks easier to understand, promoting ownership of rehabilitation and intrinsic motivation, whilst enabling objective monitoring of progress .The aim of this pilot study was to determine the feasibility, adherence, safety, and efficacy of incorporating visualisation into stability training for people with CAI. Efficacy was examined through the effect of visualisation on dynamic stability, perception of stability, and rehabilitative experience.A pilot randomised-controlled trial was conducted to assess the feasibility of a stability-based training programme using visualisation for people with CAI. The study was approved by the University of Strathclyde and Deakin University ethics committee (DEC 2018.243) and received NHS R&D approval for testing on an NHS site (IRAS project ID 247615).2,1\u2009=\u20090.96) [Volunteers from local universities and surrounding communities were recruited via poster and social media advertisements and screened for CAI using the International Ankle Consortium guidelines . Partici\u2009=\u20090.96) . The CAIAll testing and training sessions were supervised and completed in one of three laboratories. The three sites used were (i) Human Performance Laboratory at Glasgow Royal Infirmary, Scotland, UK; (ii) SportScotland Institute of Sport, Scotland, UK; and (iii) Biomechanics laboratory at Deakin University, Geelong, Australia. Testing sessions were conducted the week prior to, and the week following completion of the training block. The training programme was completed biweekly over a 4-week period with visualisation (VIS) or training without visualisation (NO-VIS).At the pre-training testing session, participant\u2019s age, mass, leg dominance, physical activity levels, sport participation, and ankle injury history were collected.Each of the testing sites had motion capture systems for measuring biomechanical data . Data was live-streamed into D-Flow software for the visualisation . Body segments were determined using pointer calibration at 16 pre-determined anatomical landmarks and segment clusters (Strathclyde Cluster Model) which allowed for an avatar to be generated that showed the participants\u2019 pose in real time . The researcher was aware of the potential for performance bias and remained as objective as possible so as not to treat the groups differently during training.Each exercise was performed at every training session at light-moderate intensity with an expected rate of perceived exertion (RPE) of 10\u201312 , 38, 39.Participants wore comfortable clothing that was suitable for rehabilitative practice, and all testing procedures and training exercises were performed barefoot.Feasibility was recorded as rate of recruitment, retention, and adherence to the training intervention. Safety was recorded as the number of adverse events during testing and/or training. Risks included ankle or other musculoskeletal injury from physical activity related to the stability training or falls during training in the laboratory. As in current clinical practice, the training was designed to be progressive enabling participants to stay within their capabilities, and participants were instructed to not perform exercises at levels beyond their ability. Falls risk was mitigated by ensuring the testing, and training space was kept clear of hazards.Efficacy was reported through the effect of visualisation on dynamic stability, perception of stability, and rehabilitative experience.At baseline, participants performed the Star Excursion Balance Test (SEBT) and CAIT. At the post-training testing session, the SEBT was reassessed, and the CAIT and Physical Activity Enjoyment Scale (PACES-8) questionnaire was completed.The SEBT is an accepted and reliable method adopted in clinical practice to assess the outcome of stability-related interventions , 44\u201348.While maintaining unilateral stance, eight maximal reaches were performed Fig.\u00a0 with eacThe change in maximal reach distance from pre- to post-test for the eight reach directions, and average of the eight, was analysed. In cases of bilateral CAI, the ankle perceived as more unstable was analysed. A recent meta-analysis reported a minimal detectable change (MDC) of greater than 8.15% in the PM direction to identify success of a 4-week balance rehabilitation programme . No studThe CAIT quantified perception of stability. The minimal detectable change and minimal clinically important difference of the CAIT score is\u2009\u2265\u20093 .The PACES-8 was used to quantify user experience. This measure has been frequently used in rehabilitation research into VR and balance \u201357, howeThe eight-item questionnaire used a five-point Likert Scale to evaluate the participants\u2019 level of enjoyment \u2014 1 being \u2018strongly disagree\u2019 and 5 being \u2018strongly agree\u2019, giving a total score out of 40. A high overall score signified high enjoyment of the training.Participants were randomly assigned to the NO-VIS or VIS training group using a random number generator . This waParticipants and tester remained blind to group allocation until after the pre-training test. Thereafter, neither was blind to the intervention group.All outcomes relating to the feasibility of the study are descriptively presented. Group data are presented as group means and standard deviation.Statistical analysis was performed using SPSS . Shapiro\u2013Wilk normality tests were conducted to test for the assumption of normality.t-test. All tests were analysed at a 0.05 level of significance. The magnitude of the effects was calculated and interpreted using Cohen\u2019s effect size recommendations (d), i.e. small: 0.3\u20130.49; medium: 0.5\u20130.79; large\u2009\u2265\u20090.8 [To test if the visualisation improved performance more than stability training alone, the SEBT performance and CAIT were compared using an ANCOVA. The dependent variable was the post-test scores and the independent variable the groups . The pre-test scores acted as a covariate to control for any differences pre-training. The PACES-8 questionnaire was analysed using a 2-sample ge\u2009\u2265\u20090.8 .From the 129 people assessed for eligibility of inclusion criteria over 4\u00a0months in the UK and 1.5\u00a0months in Australia, 17 were recruited for the study. This equated to 0.38 and 1.83 participants per week for each site, respectively. The main reason for not participating in the study were people not responding to correspondence after receiving the participant information sheet. Secondary to this was the study requiring a larger time commitment than could be given, no reimbursement for participation, and not meeting the inclusion criteria. This included revealing instability around ankle but no previous ankle injuries, lower limb dislocation, breaks, fractures, and/or surgeries, and recent significant ankle sprains. There were two dropouts during the study \u2014 an Achilles injury (VIS group) and an acute injury to the unstable ankle (NO-VIS group). Both were unrelated to the study and prevented continuation of participation. There were no adverse events related to the treatment allocations. The final analysis included 15 participants \u2014 an 88% retention rate .Descriptive statistics are presented in Table d\u2009=\u20091.5\u20131.8, Table d\u2009=\u20091.7, p\u2009=\u20090.02, Table The results of the SEBT showed a greater increase in performance for the VIS group in the posterior-lateral (PL), and lateral (L) directions with large effect   Fig.\u00a0. The CAId\u2009=\u20090.6, p\u2009=\u20090.26) , and strength has been shown to significantly effect SEBT performance \u201368. SpecRehabilitation programs require adherence to be effective , which iFor both groups, a large majority of participants (\u2265\u200950%) were previously unsatisfied with their rehabilitation. In the current study, participants in both groups reported clinically meaningful improvements in perceived stability, and this may have increased confidence and trust in the program. This would satisfy both the need for competence and relatedness. In turn, this may have increased motivation in both groups, leading to increased effort and engagement , 24. AltThere are limitations to this study. Firstly, despite the sample population representing a diverse group, the sample size was small, and the randomization of participants led to unequal numbers in each of the groups. Therefore, any inferences from the results should be interpreted with caution. Participant recruitment was impacted by the multi-centre nature of the study. Given the study was conducted internationally across three sites, a new participant recruitment protocol was required each time. However, this is believed to strengthen the study, despite the small sample size, since the protocol was completed in three different locations with no defining impact on retention, adherence, and safety. Given the limited research regarding chronic ankle instability and use of visualisation, the study was designed to assess initial feasibility to establish whether further study would be warranted with a larger sample size, and it is believed this has been achieved. It is important for future work to consider the recruitment rate. Primarily, this should include increased follow up of people who have expressed interest in the study. Further, researchers could consider providing reimbursement to participants. Reducing the time commitment may also be considered, but this may influence efficacy, and the protocol used in this study aimed to represent current clinical practice and research \u201336, 39.After allocating the participants to the VIS or NO-VIS group following baseline testing, the participants were no longer blinded as to the training group they were part of. Due to the nature of the study this was not possible but is a limitation presenting possible bias. For the testing and training, the lead researcher was the only tester, subjecting the study to further potential bias. However, having only one tester allowed for consistent practice throughout testing and training as would occur in rehabilitative practice.Due to the nature of the study, supervised laboratory visits were required for study completion. Participants attended all training sessions, but future studies could aim to monitor adherence to visualisation during unsupervised or home-based training scenarios, if facilities and equipment permit. Further to this, future research should use a specific measure for motivation to analyse the specific type of motivation the training may have created relative to the self-determination theory  continuuThis study was sufficiently powered for the SEBT. Based on the results of this study, an estimated sample size of 16 per group would be required for future work to assess enjoyment with visualisations included in the training programme and for the CAIT 62 per group. This was based on the results of the current study at a power of 0.80 and alpha level of 0.05. To be adequately powered for all outcomes, the minimum sample size would be 62, not accounting for dropouts. Future studies could also examine additional or alternative outcome methods of perceived stability, such as the Foot and Ankle Ability Measure , as wellThe results of this pilot study support the feasibility and safety of stability-based training with visualisation in those with CAI. We found enhanced performed on the SEBT when training with visualisation which suggests this could be an effective approach to stability-based training. Further investigation using a larger sample and additional subjective measures is needed to thoroughly assess stability and enjoyment when visualisation is incorporate into training."}
+{"text": "Perioperative respiratory adverse events were defined as the occurrence of any episode of single/combination of coughing, breath holding, hypoxemia, laryngospasm and bronchospasm. Bivariate and multivariate binary logistic regression analyses were performed and variables with The prevalence of PRAEs was 26.2% (CI: 20.5\u201330.9%). A total of 129 episodes of PRAEs were occurred and of them, 89 (69.0%) were occurred in the postoperative period. Desaturation was the predominant adverse event which was observed 61 (47.3%) times. Age <1 year , ASA \u2265 3 , upper respiratory tract infections (URTIs) , secretions in the upper airway  and airway related surgery  were significantly associated with PRAEs.Prevalence of PRAEs was high among pediatric surgical patients; the postoperative period was the most critical time for the occurrence of PRAEs and desaturation was the commonest PRAE. Age <1 year, URTIs (recent or active), secretions in the upper airways, ASA \u2265 3 and airway related surgery were significantly associated with PRAEs. Clinicians should perform effective risk assessment, preoperative optimization and preparation for the management of PRAEs. Preoperative identification of those children at high risk is a challenging process  and Tibebe-Ghion Specialized Hospital (TGSH). The hospitals are located at Gondar and Bahirdar towns respectively in the Northwest Ethiopia. The source population was all pediatric surgical patients (0\u201312 years of age) that underwent surgery under general anesthesia and the study population was all pediatric surgical patients that underwent surgery under general anesthesia at UoGCSH and TGSH during the study period. All pediatric surgical patients whose parents/legal care-givers were volunteer were included and patients who had severe head injury, intubated patient, acute respiratory distress, hypoxia requiring mechanical ventilation in the preoperative period, operated for more than once, and transferred to intensive care unit for mechanical ventilation after operation were excluded.Sample size was calculated by using single population proportion formula with 50% proportion, maximum acceptable difference (d) = 5%, and 95% confidence interval and found 385. We used a reduction formula to determine achievable sample size as the surgical registries showed that only 780 and 660 pediatrics surgical procedures were performed annually at UGCSH and TGCSH respectively. In-addition, due to COVID-19 pandemic, the flow of pediatric surgical patients was reduced. After reduction, the sample size became 195 and 15% non-response rate was added and the final sample size was 225.All eligible pediatric surgical patients who received general anesthesia were included in the study. The dependent variables were PRAEs which were measured in terms of any episode of either coughing, breath holding, hypoxemia, laryngospasm, or bronchospasm. The independent variables were patient factors , anesthetic factors , and surgical aspects .Perioperative respiratory adverse event: any episode of single/combination of coughing, breathe holding, hypoxemia, laryngospasm and bronchospasm  <95% more than 30 seconds measured by pulse oximetry regardless administrations of 100% Oxygen or SpO2 <90% in atmospheric air. Oxygen saturation was documented when pulse oximetry showed consistent readings with no artifacts  (aseline) .Oropharyngeal secretions: the presence of secretion that requires suctioning of more than once  to 4 (serious complication). For patients who received anesthesia with endotracheal tubes or laryngeal mask airways, scores were multiplied by a constant factor of five. When facemask was used, scores were multiplied by a constant factor of three. Patients received a composite score of 5\u201320 for ETT or LMA and 3\u201312 facemask for each adverse event  23). Th. Th23). Reference No: PGC/587/07/2012) was obtained from the Ethical Review Committee of School of Medicine, University of Gondar. Written informed consent was obtained from parents/legal care-givers of each child. When clinically significant PRAEs were noticed, data collectors reported for clinicians  to provide appropriate management. A pre-test was conducted on 20 (8%) patients whose data were not included in the main study. The data were analyzed by using SPSS version 20 (IBM Corporate). The normality was checked by using Shapiro-Wilk normality test. The Chi-squared or Fisher's exact tests were used when appropriate. The Hosmer-Lemeshow test was used to assess model fitness. The variance inflation factors and tolerance were used to diagnose multicollinearity. The associations between variables were determined by using bivariate and multivariate binary logistic regression. The cut-point of statistical significance was p < 0.2 for bivariate and <0.05 for multivariate regression at 95% confidence interval. Odds Ratio were used to describe the strength of associations.Ethical approval (Two hundred ten (210) pediatric surgical patients who received general anesthesia were included in this study. The response rate was 93.3% and data from 15 patients were excluded due to incompleteness. The majority of the patients 115 (54.8%) were males. The median age (inter-quartile range) was 4.0 (1.1\u20138.0) years. Most of the patients 189 (90%) were classified under ASA 1 and 2 while the rest were ASA 3 and above. The 23 (11%) patients had URTIs. Eighty-nine (42.4%) patients had low hemoglobin levels (<11 g/dl) preoperative. The mean duration of surgery was 82 \u00b1 41.5 min .Out of 210 pediatric patients, 55  had developed PRAEs. We have observed 129 episodes of PRAEs and desaturation was the commonest adverse event which occurred 61 (47.3%) times; followed by partial upper airway obstruction 21 (16.3%), breath holding 17 (13.2%), persistent coughing 15 (11.6%), laryngospasm 11 (8.5%) and bronchospasm 2 (1.6%). Of 129 episodes of PRAEs, 89 (69.0%) occurred in the postoperative period while 23 (17.8) were occurred during induction of anesthesia, and 17 (13.2%) during the maintenance phase .2 <80%) and 1 (0.5%) patient had continuous coughing, 3 (1.4%) patients had complete airway obstruction which required muscle relaxant, and 9 (4.3%) patients had laryngospasm which was effectively managed by the applications of simple airway maneuvers and positive airway pressure , light anesthesia, perioperative opioid use and duration of surgery \u2265 60 min were found associated with PRAEs. The final multivariate binary logistic regression analysis demonstrated that age <1 year, URTIs (recent or active), secretions in the upper airways, ASA \u2265 3 and airway related surgery were associated with PRAEs.p: 0.029). Pediatric surgical patients who underwent airway related procedures had developed PRAEs more frequently compared to their counterparts . Having moderate to copious oropharyngeal secretions was found associated with the occurrence of PRAEs . The results of this study claimed that prematurity, premedication, induction agents, types of airway devices and experiences of the anesthetists were not associated with PRAEs . The likelihood of PRAEs to occur in infants was 3.6 times than older pediatric surgical patients . Interestingly, repeated attempts of tracheal intubation were noticed among infants . ASA physical status of three and above increases the occurrence of PRAEs by more than 5 folds . This result was congruent with a study done by Mamie et al. in which it was 21% 6.2%. Thi. The difPrevalence of PRAEs was higher in our study compared to previous study in which it was 15% \u201329. HighOxygen desaturation was occurred in 50 (23.8%) patients which was the commonest PRAE, particularly among patients who had URTIs and airway secretions. This finding was similar with a multicenter study in terms of the overall occurrence. However, it was more frequent intraoperatively compared to emergence . In our Persistent coughing and upper airway obstruction were occurred in 15 (7.1%) and 21 (10%) patients respectively. In a previous study, the prevalence of upper airway obstruction was 3.6\u20139% , 29.Breathe holding was occurred in 16 (7.6%) patients and it was higher compared to a study done by Tait et al. . A largeThe prevalence of laryngospasm was 12 (5.7%) and consistent with a systematic review which claimed 0.1\u201316%. Particularly, it was 4% in general pediatric population , 32. Famp: 0.34). The occurrence of PRAEs was significantly higher immediately after tracheal extubation than induction and maintenance phases (Children that presented with recent or active sign and symptoms of URTIs were found to have increased risk of developing PRAEs 73.9%). The higher risk of PRAEs during URTIs can be justified by the morphologic damage to the epithelium and mucosa of the respiratory tract after infections and make the airway sensitive to potentially irritant anesthetic gases and secretions that result in activation of irritant receptors and contractions of airway smooth muscles . A study.9%. The e phases .p: 0.008). A previous study concluded that multiple intubation attempts were associated with PRAEs  and this is supported by previous studies , 11. In The risk of developing PRAEs was increased by 6-folds when a surgical procedure involves the airway. Airway related procedures were associated with frequent desaturation and upper airway obstruction. Our finding was supported by previous multicenter prospective studies , 5, 36. A multicenter study done by Habre et al. including 261 hospitals across Europe has concluded that the experience of the team of anesthesia providers was a very important factor that determines the safety and the rate of adverse events in the pediatric anesthesia practices . Our stuThe study was the first for its type in our country and we believe that it can be a foundation for future studies in the field; especially in resource-limited settings. Hypoventilation and residual neuromuscular blockade were not measured due to the lack of the devices in the post-anesthesia care units of the hospitals. Smaller sample size due to COVID-19 pandemic is another limitation of this study.There was high prevalence of perioperative respiratory adverse events among pediatric surgical patients. The postoperative period was the most critical time for the occurrence of PRAEs and desaturation was the commonest adverse event. Age <1 year, URTIs (recent or active), secretions in the upper airways, ASA \u2265 3 and airway surgery were significantly associated with adverse events. Clinicians should perform effective risk assessment, optimization and preparation for the management of perioperative respiratory adverse events.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by University of Gondar. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.DW and YWB have conceptualized the study, objectives, and manuscript preparation. DW has developed the proposal. YWB, WC, HA, and MW have criticized the proposal. All authors had participated in the data and statistical analyses and read and approved the final manuscript.This work was supported by University of Gondar and Health Bureau of Amhara National Regional State, Ethiopia.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Acute myocardial infarction (AMI) case ascertainment improves for the UK general population using linked health data sets. Because care pathways for people with chronic kidney disease (CKD) change based on disease severity, AMI case ascertainment for these people may differ compared with the general population. We aimed to determine the association between CKD severity and AMI case ascertainment in two secondary care data sets, and the agreement in estimated glomerular filtration rate (eGFR) between the same data sets.2, and mild CKD or at risk of CKD was defined as eGFR \u226560\u2009mL/min/1.73 m2 or eGFR missing. CKD stages were grouped as (1) At risk of CKD and Stages 1\u20132 (eGFR missing or \u226560\u2009mL/min/1.73 m2), (2) Stage 3a (eGFR 45\u201359\u2009mL/min/1.73 m2), (3) Stage 3b (eGFR 30\u201344\u2009mL/min/1.73 m2) and (4) Stages 4\u20135 (eGFR <30\u2009mL/min/1.73 m2).We used a cohort study design. Primary care records for people with CKD or risk factors for CKD, identified using the National CKD Audit (2015\u20132017), were linked to the Myocardial Ischaemia National Audit Project  and Hospital Episode Statistics  secondary care registries. People with an AMI recorded in either MINAP, HES or both were included in the study cohort. CKD status was defined using eGFR, derived from the most recent serum creatinine value recorded in primary care. Moderate\u2013severe CKD was defined as eGFR <60\u2009mL/min/1.73 mWe identified 6748 AMIs: 23% were recorded in both MINAP and HES, 66% in HES only and 11% in MINAP only. Compared with people at risk of CKD or with mild CKD, AMIs in people with moderate\u2013severe CKD were more likely to be recorded in both MINAP and HES , or MINAP only (22% vs 5%), and less likely to be recorded in HES only (36% vs 84%). People with AMIs recorded in HES only or MINAP only had increased odds of death during hospitalisation compared with those recorded in both . Agreement between eGFR at AMI admission (MINAP) and in primary care was poor (kappa (K) 0.42, SE 0.012).AMI case ascertainment is incomplete in both MINAP and HES, and is associated with CKD severity. Our study includes a large sample size of 6748 acute myocardial infarction (AMI) events.We have assessed the completeness of AMI hospitalisations recorded in two healthcare data sets widely used in observational research in England.We evaluated, for the first time, the validity of using serum creatinine recorded in secondary care at the time of an AMI to estimate pre-AMI chronic kidney disease (CKD) stage.Generalisability to the general population is limited as the National Chronic Kidney Disease Audit only included people with CKD and/or risk factors for CKD.Prognosis following acute myocardial infarction (AMI) has improved considerably over the past 50 years such that 85% of individuals now live longer than 1\u2009year post-AMI.4Most major RCTs investigating AMI interventions excluded patients with advanced CKD.In the UK, data on AMI treatment and outcomes is collected in unlinked, disease-specific registries or in broad registration databases. While there are known differences in the reliability and validity of AMI case ascertainment using these resources in the general population,16In this study we linked records from the National Chronic Kidney Disease Audit (NCKDA) to the Myocardial Ischaemia National Audit Project (MINAP) and Hospital Episode Statistics (HES) to determine the reliability of these data sources to investigate cardiovascular disease comorbidity and outcomes in people with or at risk of CKD in England. Our objectives were to: (1) Compare case ascertainment of AMI hospitalisations in secondary care data sets (MINAP and HES); (2) determine if MINAP and/or HES case ascertainment defines populations of patients with CKD with different risks of death during and after AMI; and (3) compare CKD stage classification using admission SCr recorded in secondary care (MINAP) versus primary care (NCKDA).Data from all sources were restricted to patients treated in England. People with or at risk of CKD were identified using primary care data from the NCKDA.th Edition (ICD-10) codes.21The population identified from NCKDA was linked with Office of National Statistics (ONS) data, January 1998 to September 2019, as well as secondary care data from HES Admitted Patient Care (APC) and MINAP, April 2007 to April 2017. HES APC includes hospital admission data for National Health Service-treated patients in England, including admission and discharge dates, and diagnoses recorded using International Classification of Diseases 10Cohort study.We included people in the NCKDA registered with a GP in England, with one or more AMI hospitalisation recorded in HES or MINAP. People in each NCKDA extract must have been alive according to GP records at the time of that extract. We therefore included people with an AMI hospitalisation recorded in MINAP or HES only after the date of their GP\u2019s final NCKDA extract. People with an AMI hospitalisation that started prior to the extract date and ended after the extract date were added to the cohort, since they were at risk of death (n=183). In addition, people with an ONS death date indicating death during an AMI hospitalisation that occurred within 90 days prior to the NCKDA extract date were included (n=96), since they were likely misclassified as alive at the time of the extract because of delays in updating the death date in the GP systems. People with an ONS death date earlier than 90 days prior to the extract were excluded (n=4).2, CKD stages 3\u20135), defined using the most recent eGFR recorded in primary care (NCKDA data) prior to the AMI hospitalisation. People with no eGFR recorded in primary care or an eGFR \u226560\u2009mL/min/1.73 m2 were categorised as at risk of CKD or having mild CKD, respectively. We assumed people with no eGFR recorded in primary care did not have moderate to severe CKD since these people are much less likely to have CKD than those with eGFR recorded.23 24Our main exposure variable was moderate to severe CKD (eGFR <60\u2009mL/min/1.73 m2), (2) Stage 3a (eGFR 45\u201359\u2009mL/min/1.73 m2), (3) Stage 3b (eGFR 30\u201344\u2009mL/min/1.73 m2) and (4) Stages 4\u20135 (eGFR <30\u2009mL/min/1.73 m2).Our secondary exposure was CKD stage, defined by the Kidney Disease Improving Global Outcomes CKD staging, based on a single eGFR record without the requirement for two measures 3\u2009months apart.We used the latest eGFR recorded prior to the AMI hospitalisation to categorise people with a history of kidney transplant into the primary and secondary exposure groups. We categorised people with a history of dialysis prior to the AMI hospitalisation as moderate to severe CKD for the main exposure and CKD stages 4\u20135 for the secondary exposure, even if the latest eGFR did not agree.As the use of a single SCr test at the time of AMI hospitalisation to determine CKD stage has not previously been validated, we have used the term \u2018eGFR stage\u2019 in place of CKD stage to refer to the eGFR level calculated from this test.The primary outcome was AMI case ascertainment, defined as the data set(s) in which the AMI hospitalisation was recorded. We defined an AMI as being recorded in both HES and MINAP if an AMI hospitalisation in HES was within 30 days of an AMI hospitalisation in MINAP. Where multiple HES AMI hospitalisations fell within 30 days of a MINAP AMI hospitalisation, the HES AMI hospitalisation closest in time to the MINAP AMI admission was selected as the single matched event. AMI hospitalisations without a match were categorised as HES or MINAP only. Study participants could contribute multiple AMI hospitalisations.We defined an AMI in HES data using ICD-10 codes I.21, I.22 or I.23 in the primary admission diagnosis field (first diagnostic position in the first episode of an admission).1210.1136/bmjopen-2021-057909.supp1Supplementary dataWe investigated in-hospital mortality during each person\u2019s first AMI hospitalisation within the study period. In those who survived and were discharged from their first AMI hospitalisation, we also investigated post-discharge mortality using the ONS death date (up to 15 September 2019). Variables in HES, MINAP and ONS used to define death are described in We investigated the agreement between CKD stage derived from the most recent primary care SCr test (NCKDA data) and eGFR stage derived from the secondary care SCr test conducted within 24 hours of AMI hospitalisation (MINAP data). We used the same methods to determine eGFR stage in MINAP data as we did for NCKDA data.We described age at AMI admission , sex, ethnicity (white or other), index of multiple deprivation quintiles  and relevant comorbidities including angina, cerebrovascular disease, chronic obstruction pulmonary disease (COPD), diabetes mellitus (type 1 and 2), heart failure, hypertension, previous myocardial infarction and peripheral vascular disease. We also described dialysis and transplant status, and smoking status. Data sources for each key covariate are described in We summarised key covariates by CKD status. We used Venn diagrams to describe AMI case ascertainment overall and stratified by CKD status (at risk of CKD or mild CKD vs moderate to severe CKD). We used multinomial, multivariable logistic regression to quantify the association between CKD stage and AMI case ascertainment. We used the \u2018HES and MINAP\u2019 category as the base outcome and reported crude and adjusted relative risk ratios (RRR) and 95% CIs, using \u2018At risk of CKD and Stages 1\u20132\u2019 as the reference exposure category. We adjusted for sex, age category, ethnicity, IMD quintile, previous AMI, heart failure, COPD, diabetes mellitus and clustering by participant (using cluster-robust standard errors (SEs)). We used a complete case analysis since we could not assume that missing values for ethnicity were missing at random. In a secondary analysis, we stratified these regressions by AMI subtype (STEMI and NSTEMI).We used multivariable logistic regression to calculate the odds of death in hospital during each person\u2019s first AMI hospitalisation in people with AMI recorded in MINAP only or HES only, relative to MINAP and HES. After confirming the proportional hazards assumption with a Schoenfeld Residuals test on the full multivariable model (p=0.35), we used multivariable Cox regression to estimate HRs for death during total follow-up in those who survived their first AMI hospitalisation with AMI recorded in MINAP only or HES only, relative to MINAP and HES.2 in either NCKDA or MINAP as these are unlikely to be true values. We drew a Bland-Altman plot to describe differences in the distribution of eGFR measures in primary and secondary care2) in primary care and grouped by time between the most recent primary care eGFR measure and the AMI hospitalisation .Finally, to assess the validity of using MINAP-recorded eGFR at AMI admission as a proxy for pre-admission CKD status, we compared eGFR and its corresponding eGFR stage within 24 hours of AMI admission (MINAP data) to the most recent eGFR and its corresponding CKD stage in primary care (NCKDA data). In this analysis, we excluded people with eGFR measures greater than 120\u2009mL/min/1.73 mWe repeated the main analyses for AMI events occurring prior to the study start date (the latest NCKDA extract). People who experienced AMI hospitalisation before the study start were survivors, since only people alive at the time of the NCKDA were included in the study.In addition, we re-drew the Venn diagrams after including HES AMI hospitalisations recorded in both the first and second diagnostic positions of the first episode to include AMIs recorded as co-primary diagnoses.We did a complete case analysis when building our multivariable models. People with missing ethnicity (~1%) and IMD data (<1%) were excluded prior to building our unadjusted, partially adjusted and adjusted multinomial models.We used discharge dates to help re-categorise people who were in-hospital at the time of the NCKDA extract into the cohort, as well as to determine death in hospital and the start of follow-up in those who survived their first AMI hospitalisation. Discharge date was missing in 19% and 1% of the MINAP and HES data sets, respectively. We assumed these dates were missing at random and used the median length of admissions in those without missing admission and discharge dates  to impute the missing discharge dates.https://www.kidneycareuk.org/) supported the research questions, grant applications and the related record linkage application for section 251 permissions critical to the development of the NCKDA. Patient members of the UK Renal Registry Patient Council  reviewed the study results. Their feedback supported a further planned record linkage of renal and cardiac data to look at patient outcomes.The Kidney Care UK patient organisation  people with or at risk of CKD who experienced 6748 AMIs between the final NCKDA extract and 1 April 2017 . BaselinOverall, 23% of AMI hospitalisations were captured by both MINAP and HES data sets  . There wCrude and adjusted RRRs and 95% CIs describing the association between CKD stage and AMI case ascertainment are presented in Of those with a first AMI recorded in both HES and MINAP, 209 people (15%) died during the AMI hospitalisation, compared with 151 (23%) with a first AMI recorded in MINAP only and 579 (15%) recorded in HES only . After aMean follow-up among people who survived a first AMI hospitalisation was 2.4 years. The rate of death per 100 person-years during complete follow-up was 18.0 (95% CI 16.4 to 19.7) for AMI recorded in MINAP and HES, 23.3 (95% CI 20.6 to 26.5) for AMI recorded in MINAP only and 10.3 (95% CI 9.61 to 11.0) for AMI recorded in HES only . After a2 (IQR 33.5\u201361.6). The Bland-Altman plot comparing the primary care eGFR and the secondary care eGFR indicated a negligible mean difference but wide variation   had SCr recorded within 24 hours of AMI admission . Median to 30.1) .The per cent agreements and kappa statistics between eGFR stage derived from MINAP eGFR at AMI admission and the CKD stage derived from NCKDA using primary care data are shown in When stratifying by months between the primary and secondary care eGFR measures, we observed the best agreement in staging within a 0\u20135\u2009month gap between the primary and secondary care eGFR measures: 61.0%, K 0.48 (SE 0.03) . AgreemeAMI case ascertainment in MINAP and HES was similar in AMI hospitalisations recorded prior to the study start  compared with after the study start  . There wAfter expanding the AMI definition in HES to include any hospitalisations with AMI coded in the second diagnostic position as well as the first, the proportion of AMI hospitalisations captured in both HES and MINAP decreased slightly . After cFinally, the 10 most common diagnoses in HES matching with the MINAP only AMI hospitalisations from the main analysis are shown in We compared recording of AMI hospitalisations for people with CKD between two large secondary healthcare data sets in England. In a cohort of 6042 people, we found that both HES and MINAP missed a significant proportion of AMI hospitalisations. CKD stage influenced likelihood of AMI recording by data set: AMI hospitalisations in people with moderate to severe CKD were more likely to be recorded in MINAP compared with people at risk of CKD or with mild CKD. We found an association between AMI hospitalisation recording by data set and in-hospital mortality. There was marked variation between eGFR at AMI admission and preceding eGFR measurements in primary care, but no obvious systematic bias in terms of over/underestimation of eGFR at AMI admission.et alet alOur results agree with previous research demonstrating incomplete capture of AMI events by individual healthcare data sets in the overall English population and extend them to a population with CKD. Herrett et alet al29In contrast to both studies, we found significantly worse agreement in case ascertainment for AMI hospitalisations between MINAP and HES for people at risk of CKD or with mild CKD. Torabi The high prevalence of CKD risk factors in people at risk of or with mild CKD could put them at greater risk of type 2 AMI; a mismatch of myocardial oxygen supply and demand in the absence of the \u2018classical\u2019 coronary artery plaque rupture with thrombosis reflective of type 1 AMI.et alPeople with AMI recorded in both MINAP and HES had lower in-hospital mortality compared with those with AMI recorded in either MINAP or HES only. Our findings agree with Herrett 2, binary classification between individuals with CKD stages 3\u20135 and those with stages 1\u20132 is more reliable than classification by CKD stage. These findings suggest that although previous researchAcross all levels of eGFR, we found significant variation between eGFR stage derived from SCr taken within 24 hours of AMI admission (recorded in MINAP) and that derived from SCr in primary care, which is in line with reported variability of eGFR in validation studies.The NCKDA only included people with CKD and/or risk factors for CKD; therefore, we cannot generalise our results to people without risk factors for CKD. We may have incorrectly misclassified people who have no documented tests for CKD in primary care as having risk factors for CKD only; however, previous work has shown this group of people are much less likely to have CKD than those who do have CKD tests recorded in primary care.32This study demonstrates how AMI case ascertainment in England can be improved by using linked healthcare data sets. Further research investigating cardiovascular and kidney disease incidence, prevalence and outcomes should follow this approach. Other countries with similarly rich, yet fragmented healthcare data sets would benefit from applying similar methods to evaluate the validity and completeness of cardiovascular and kidney disease capture in similar data. Optimising data quality in healthcare data sets and simplifying the process of data linkage would facilitate high-quality observational research to inform the design of future RCTs and provide estimated treatment effects where RCT data are lacking.The use of linked healthcare data sets should be prioritised in observational research investigating multimorbidity."}
+{"text": "Competition for resources shapes ecological and evolutionary relationships. Physiological capacities such as in locomotor performance can influence the fitness of individuals by increasing competitive success. Social hierarchy too can affect outcomes of competition by altering locomotor behaviour or because higher ranking individuals monopolize resources. Here, we tested the hypotheses that competitive success is determined by sprint performance or by social status. We show that sprint performance of individuals measured during escape responses (fast start) or in an accelerated sprint test did not correlate with realized sprint speed while competing for food within a social group of five fish; fast start and accelerated sprint speed were higher than realized speed. Social status within the group was the best predictor of competitive success, followed by realized speed. Social hierarchies in zebrafish are established within 7 days of their first encounter, and interestingly, there was a positive correlation between social status and realized speed 1 and 4 days after fish were placed in a group, but not after 7 days. These data indicate that physiological performance decreases in importance as social relationships are established. Also, maximal physiological capacities were not important for competitive success, but swimming speed changed with social context. The mecSprint performance is a repeatable trait that is thought to be under selection because of the advantages it confers for predator escape, prey capture, territorial defence and survival . Sprintsper se takes precedence over locomotor capacity in determining competitive success. In zebrafish, a social hierarchy among unfamiliar animals is established within 7 days [Social relationships may influence locomotor behaviour , and then 7 days . This es. 2. 2.1Danio rerio, form social hierarchies that are stable [www.opensourcephysics.org).All animal handling and experimental procedures were conducted with the approval of the University of Sydney Animal Ethics Committee . Zebrafish, e stable  and there stable . All fis. 2.2\u22121 , and a submerged 300 mm ruler served as a scale. We analysed videos  using the fish centre of mass as the tracking point. Centre of mass was defined as the location at 0.35 body lengths from the tip to the snout [We measured swimming performance in all experimental fish . There are different measurement techniques of sprint speed in the literature , and to he snout . The purhe snout ,19. Esca10) in a 10 s constant acceleration trial in a cylindrical, Blazka-style swimming flume (21 mm diameter \u00d7 150 mm length) [\u22121 for 30 s, after which water flow was continuously increased by an average of 0.05 m s\u22121 every 1 s until fish could no longer maintain their position in the water column and fell back against a mesh net. The maximum flow speed was recorded when the fish was still holding its position in the water column and used as maximum sprint performance.The second method consisted of determining the maximum attained speed U in a 10 . 2.3n = 10 groups) of five fish each. Fish within groups were size matched so that they were within 2 mm standard length of each other, and individuals had not been in contact with each other before the assembly of the groups. Each group was housed in a separate tank (0.30 \u00d7 0.21 \u00d7 0.18 m). For trials, all five fish per tank were placed underneath a perforated 1 l cylindrical container, which allowed visual inspection of the tank but prevented egress from the container. A piece of fish flake food  was then placed approximately 20 cm away from the perforated container. After 5 s, the container was removed allowing the fish to move freely around the tank. Fish were filmed  from just before the container was lifted, and the identity of the fish that obtained the food was recorded. This procedure was repeated 10 times consecutively per day for 7 days. In preliminary trials, we determined the best size of the food flake so that it was visible to the fish, but small enough for only one fish to obtain it. During filmed trials, water depth was lowered to 7 cm, and between trials fish groups remained in their tanks and water levels were increased to 15 cm when fish were not filmed. After the food trials each day fish were fed to satiation. From the videos, we also determined the speed at which fish travelled towards the food . We defined realized speed as the maximum speed calculated across two video frames while a fish was moving after the container was lifted and until the food item was eaten by any fish. Note that the maximum speed attained during competitive trials was significantly correlated with the average speed , and this relationship did not differ between days . We used maximum speed as a representation of realized speed because it provides a better comparison with sprint speed, which represents maximum physiological capacity.To measure competitive success and social interactions, we assembled experimental groups (. 2.4\u22121) moving freely and undisturbed in their home tank for 1 min; the first footage was taken immediately after fish were assembled into experimental groups on the first day. From videos, we recorded aggressive interactions between individuals. An aggressive interaction was defined as a charge by an individual towards another fish, or a nip towards another individual that elicited a response in the receiving individual. We used the method described in [Before each competitive trial, we filmed fish .We analysed all data with permutational analyses in the R  package umptions . The p-vatistics . The samp > 0.1). To test the relationship between sprint speed and realized speed, we used realized speed as the dependent variable and fast start or U10 speed and \u2018day\u2019 as the dependent variables. We used a fast start or U10 speed, realized speed and social score as independent variables to test their influence on competitive success. Lastly, we tested whether realized speed influenced social score by using the social score as the dependent variable and realized speed and \u2018day\u2019 as independent variables. When \u2018day\u2019 was significant in the whole dataset, we repeated the analysis using subsets of data for each day to test on which days there were significant effects. We analysed data from days 1, 4 and 7 only. Effect sizes of different predictors on competitive success were calculated as Cohen's d, and 95% confidence intervals were determined by bootstrapping in the R package \u2018boot\u2019 [In all models, we used 'tank' (=group) as a random blocking factor. In the event, 'tank' was not significant in any of the comparisons  was lower than fast start (a) and U10 speed , and there was no statistically significant association between realized speed and fast start speed  or U10 speed . However, between days 1 and 7 realized speed increased significantly by 0.19 m s\u22121 on average . Additionally, fish with greater fast start speed reduced their realized speed to a disproportionally greater extent than fish with slower fast start speed , and this pattern was more pronounced on day 1 than on days 4 and 7 .Realized speed of individuals while moving towards the food within the group of five fish  and social score  had a positive effect on competitive success, but fast start speed  and U10 speed  did not influence competitive success .Realized speed , and there was a significant effect of day   and on day 4 , but not on day 7 .There was a significant positive relationship between social score and realized speed ( 0.0001) . The ass. 4We have shown that position within a social hierarchy is the most important determinant of competitive success, although realized speed while competing does contribute to success. However, maximal physiological capacities did not lead to a competitive advantage, and realized speed while competing in a group was less than maximal performance.Group living is advantageous because it protects individuals from predation, and information transfer between individuals in the group increases access to resources ,27. GrouThe consequences of social hierarchies are particularly important when competing for limited resources such as food, which can lead to differences in fitness. However, even though subordinate individuals experience restricted access to resources, they remain within the group because of the benefits of group living . It is tIt is interesting that animals rarely perform maximally under natural or naturalistic situations ,12, whicClick here for additional data file."}
+{"text": "There are few studies regarding Internet use behaviors of Chinese rural adolescents based on behavioral theory. The aim of this study is to examine the applicability and effectiveness of the health action process approach model (HAPA) in the intervention of excessive Internet use behaviors among rural adolescents in China.Three hundred twenty-seven participants who met the excessive Internet use criteria were involved in this study. Four interventions based on the HAPA model were conducted during 2015\u20132017. The structural equation model (SEM) was applied to fit the HAPA model.P\u2009<\u20090.001). The rate of daily game time more than 2 h decreased from 51.1 to 35.2% (P\u2009<\u20090.001). The result of SEM showed that both the applicability and effectiveness of the HAPA model were well in the intervention of excessive Internet use behaviors with good fitted indicators . The direct and indirect effects of the main pathways in the HAPA model were statistically significant (P\u2009<\u20090.05). The comparison analysis of HAPA model variables identified that outcome expectancy, intention, maintenance self-efficacy had been improved significantly after interventions.The rate of average daily time spent online on weekends more than 4 h dropped from 57.2 to 39.1% (The intervention measures based on the HAPA model can effectively reduce excessive Internet use behaviors of Chinese rural adolescents, mainly through strengthen outcome expectancy, intention, and maintenance self-efficacy.The online version contains supplementary material available at 10.1186/s12889-021-10999-z. The Internet increasingly has become one of the ways for adolescents to learn, as well as to entertain . AccordiExploring the influencing factors of adolescents\u2019 Internet use behaviors is crucial for the development of targeted interventions. Previous studies have found that age , 11, 12,Some evidence suggests that behavior interventions based on theoretical frameworks are more effective than interventions that lack a theoretical basis . Social-The HAPA model has been used to study the determinants of behavior, as well as for the development of behavior change interventions in many studies; examples are studies with regard to physical activities and eating behaviors . StudiesTo date, only very few studies addressed the determinants of excessive Internet use by adolescents in rural areas , 25, 26;The participants were derived from the first, third, and fifth survey data of a 2-year longitudinal study for rural adolescents in Sichuan Province (2015\u20132017). Typical sampling and cluster sampling were used to select the study areas and participants in November 2015. In the first stage, a typical sampling method was used to select Zizhong County as the study area, representing rural areas in Sichuan. In the second stage, cluster sampling was used to randomly select two middle schools in the study area, one as the control school and the other as the experimental school. According to the characteristics of the Chinese middle school system and the 2-year longitudinal intervention design, only seventh grade and tenth grade students could meet the requirements. Therefore, all students in seventh grade and tenth grade in both schools were included in this study. After the first baseline survey, a follow-up survey was conducted every 6 months with a total of five surveys. In the fifth survey, the samples were in the nine-grade and twelve-grade. 1044, 973, and 874 samples were obtained in the experimental school in the first, third, and fifth survey, respectively, and 1399, 1777, 1583 samples respectively in the control school.According to the baseline survey, samples who met one of the inclusion criteria were considered to have excessive use of the Internet and were involved in this study. The inclusion criteria were 1) average daily online time from Monday to Friday\u22652\u2009h (hour); (2) average daily online time on weekends\u22654\u2009h; (3) usually daily game time\u00a0\u2265\u00a02\u2009h; (4) being online overnight at least once in the past 30\u2009days. There were 327 eligible participants from the experimental school and 448 from the control school, divided into the experimental group and the control group respectively. The experimental and the control group were homogenous in sociodemographic characteristics  \u201cYour average daily Internet time from Monday to Friday (included smartphone and computer)\u201d, the answers were scored 1\u20135, representing \u201c\u22654\u2009h\u201d, \u201c3\u2009h\u201d, \u201c2\u2009h\u201d, \u201c1\u2009h\u201d, and \u201calmost not\u201d, respectively; (2) \u201cYour average daily Internet time on weekends (included smartphone and computer)\u201d, the answers were scored 1\u20135, representing \u201c\u22659\u2009h\u201d, \u201c7\u20138\u2009h\u201d, \u201c4\u20136\u2009h\u201d, \u201c2\u20133\u2009h\u201d, and \u201c\u22641\u2009h\u201d, respectively; (3) \u201cYour usually daily game time \u201d, the answers were ranked 1\u20136, representing \u201c\u22654\u2009h\u201d, \u201c3\u2009h\u201d, \u201c2\u2009h\u201d, \u201c1\u2009h\u201d, \u201c<1\u2009h\u201d, and \u201cnever\u201d, respectively; (4) \u201cNumbers of online overnight in the past thirty days\u201d, the answers were scored 1\u20135, representing \u201c\u22654 times\u201d, \u201c3 times\u201d, \u201c2 times\u201d, \u201c1 time\u201d and \u201cnever\u201d, respectively. Cronbach\u2019s alpha was 0.662.The items of the HAPA model variables were measured and modified in accordance with the compilation principles of Schwarzer Ralf, and related behaviors , 33.Risk perception was assessed by three items . Items were scored with 5 Likert scales . Cronbach\u2019s alpha range was 0.737\u20130.809.Outcome expectancy was examined by five items . Items were scored with 6 Likert scales . Cronbach\u2019s alpha range was 0.863\u20130.879.Action self-efficacy was estimated by five items . Items were scored with 5 Likert scales . Cronbach\u2019s alpha range was 0.809\u20130.876.Intention was assessed by three items . The item was scored with 6 Likert scales . Cronbach\u2019s alpha range was 0.896\u20130.912.Planning was determined by three items . Items were scored with 6 Likert scales . Cronbach\u2019s alpha range was 0.932\u20130.940.Maintenance self-efficacy was assessed by two items  Items were scored with 6 Likert scales . Cronbach\u2019s alpha was 0.833\u20130.896.Sociodemographic characteristics and excessive Internet use behaviors were derived from the baseline survey. When fitting the HAPA model, we used risk perception, outcome expectancy, action self-efficacy, and intention from the third survey, and obtained planning, maintenance self-efficacy, and Internet use behaviors from the fifth survey.We used unified questionnaires and survey process in the investigations. Before the interventions and surveys, we conducted uniform training for investigators to standardize the intervention and investigation process. Self-administered questionnaires were used to obtain the data of participants. After each investigation, interactive inspections were executed in investigators to find and rectify mistakes such as misfiled, omissions, and logic errors. To ensure the accuracy of the data, double entry was used for questionnaire data entry.P-value <\u20090.05 was considered statistically significant.Epidata\u00a03.1 was used to establish the database and data entry. SPSS 24.0 was used to sort and analyze the data. AMOS 21.0 was used to fit the SEM. The Maximum Likelihood (ML) method was used for parameter estimation, and the Bootstrap method was used synchronously when fitting the model. 2/df\u00a0\u2264\u00a05.0, GFI>0.90, CFI>0.90, TLI>0.90, NFI>0.90, RMSEA<0.08. The SEM can analyze the direct and indirect relationships between latent variables and observed variables in the longitudinal design, which fits the analysis framework of the applicability of the HAPA model. Therefore, SEM was used to explore the applicability of the HAPA model in excessive Internet use behaviors. If the result of the SEM shows good applicability, then the effectiveness of the HAPA interventions will be discussed further. In addition, the path coefficients of variables in the SEM could help to find the key variables in behavior change, and as the basis to make interventions. The Chi-square test and the t-test were used to compare the differences of HAPA variables and Internet use behaviors before and after the interventions.In the descriptive analysis, the mean\u2009\u00b1\u2009standard deviation (M\u2009\u00b1\u2009SD) was used to describe quantitative variables and the frequency (%) was used to describe qualitative variables. The bivariate analysis was adopted to analyze the correlations between the variables of the HAPA model. The SEM was constructed based on the HAPA model and previous research results, and the model fitting was evaluated according to the criteria of \u03c7P < 0.05). Compared to low grade, senior students had a better performance in planning making and Internet use behaviors (P < 0.05). However, there was no statistical difference between left-behind and non-left-behind adolescents in the scores of the HAPA model variables.In the baseline survey, the mean (SD) age of the participants was (15.37\u00a0\u00b1\u00a01.31) years old, 168 (51.4%) were boys, 279 (85.3%) were in tenth grade, and 217 (66.4%) were left-behind adolescents. Table\u00a0P < 0.05) (Table\u00a0P < 0.01). The correlation coefficients between other variables were positive (P < 0.05).The results of the correlation analysis showed that there was no statistical correlation between outcome expectancy and risk perception, maintenance self-efficacy, as well as Internet use behaviors, while other variables were all correlated (5) Table\u00a0. Risk pe2/df\u00a0=\u00a02.066, GFI\u00a0=\u00a00.889, CFI\u00a0=\u00a00.938, TLI\u00a0=\u00a00.928, IFI\u00a0=\u00a00.938, RMSEA\u00a0=\u00a00.057, indicating that the model was acceptable. The results of group analysis of SEM illustrated that model 1 was applicable to the Internet use behaviors of rural adolescents with different gender, grade, and left-behind status.The initial model was obtained by adjusting action self-efficacy and risk perception, outcome expectancy, as well as maintenance self-efficacy, all of which were found relevant in previous studies , 34. TheP < 0.05). The indirect effects of model 1 were presented in Table\u00a0The path coefficients of model 1 showed that except for the path from risk perception to intention and the path from maintenance self-efficacy to Internet use behaviors, other paths were statistically significant (The results in Table\u00a0The results of this study illustrated the HAPA model has good applicability in Internet use behaviors, moreover, interventions based on the HAPA model can effectively improve social-cognitive factors of the excessive Internet use behaviors of Chinese rural adolescents. After the interventions, the rate of excessive Internet users reduced by 30%. Among participants, the rate of average daily Internet time on weekends\u22654\u2009h dropped from 57.2 to 39.1%, and the rate of usually daily game time\u00a0\u2265\u00a02\u2009h reduced from 51.1 to 35.2%. Although no improvement was observed in average daily Internet time on weekdays\u22652\u2009h and online overnight in the past 30 days, the rates after intervention did not increase. Meanwhile, the interventions improved outcome expectancy, intention, and maintenance self-efficacy of rural adolescents.The results of SEM identified that the HAPA model has good applicability in rural adolescents\u2019 Internet use behaviors. In model 1, most paths had been effectively verified, apart from risk perception to intention, and maintenance self-efficacy to Internet use behaviors. We found that there was only an indirect impact between maintenance self-efficacy and Internet use behaviors, while the direct effect was not observed. It demonstrated that for Internet use behaviors of adolescents, the role of maintaining self-efficacy was to promote planning convert into behaviors and to maintain the implementation of planning. Further, the strong correlation between action self-efficacy and maintenance self-efficacy had been verified .Different from the HAPA hypothesis, this study did not find the association between risk perception and intention similarly to other studies \u201337. It mPrevious school-based studies were designed to improved Internet use behaviors of urban adolescents. The studies focused on health education courses have positive effects on game time, while no impact on daily online time , 40. DifIn this study, the changes in outcome expectancy, intention, and maintenance self-efficacy were key indicators to the reduction of Internet use. Previous studies have claimed that outcome expectancy is the strongest predictor of intention , 41, 42.Meanwhile, intention is the most predictable factor for future behaviors . The genUnexpectedly, there was no significant improvement in action self-efficacy and planning. One possible explanation may be the interventions focused on health education were low-intensity for action self-efficacy and planning . MoreoveOne of the interventions in this study was to increase physical exercise time by providing sports equipment, thereby reducing online time. Although there was no investigation on the specific use of sports equipment, through qualitative interviews with school teachers, it was found that these sports equipment were popular among participants, and often used after classes. This showed that increasing physical exercise time can indirectly intervene with excessive Internet use behaviors.It is worth mentioning that the ideal results of interventions should be obtained from the comparison between the experimental school and the control school. However, because of the heavy academic pressure, the control school had added a strict system of residence in the research process, and forbidden students to bring mobile phones and any other electronic products into campus. And students can go home for only 2-day holidays each month. While the experimental school didn\u2019t have such rules. Therefore, it is impossible to directly compare the change degree of excessive Internet use in the experimental school with the control school. Nevertheless, it could be seen that the social-cognitive factors from HAPA in the experimental school were improved more than those in the control school.There were some limitations to this study. First, the data came from the self-reports of the participants, and there might be reporting bias. Second, previous studies confirmed that planning could be divided into action planning and coping planning. Action planning is important for the beginning of behaviors, and coping planning is crucial to the maintenance of behaviors . HoweverDespite these limitations, the strengths of this study could not be ignored. This study applied the HAPA model to intervene in adolescents\u2019 excessive Internet use behaviors, which could provide new ideas for understanding the cognitive process of Internet use behaviors in adolescents. Further, longitudinal data in this study were used for analysis to better illustrate the continuous process of cognitive, intention, and behavioral change. Since the transformation of intention, planning and behaviors takes time, the longitudinal design was able to describe the transformation process. Besides, targeted intervention measures were formulated based on the HAPA model, and a positive effect had been obtained in the Internet use behaviors of adolescents. This study not only verified the effectiveness of the HAPA model in guiding behavioral interventions but also proposed several effective measures to reduce rural adolescents\u2019 excessive Internet use behaviors.This study applied the HAPA model as the theoretical framework to examine its applicability and effectiveness for reducing excessive Internet use behaviors in Chinese rural adolescents. The results of confirmatory research revealed that the HAPA model was effectively verified in Internet use behaviors in rural adolescents. The results of intervention research suggested that interventions based on the HAPA model could effectively reduce excessive Internet use behaviors in rural adolescents, mainly through the enhancement of outcome expectancy, intention, and maintenance self-efficacy.Additional file 1: Appendix table 1. The comparison between the control group and the experiment group before interventions."}
+{"text": "We perform simulations of a system containing simple model proteins and a polymer representing chromatin. We study the interplay between protein-protein and protein-chromatin interactions, and the resulting condensates that arise due to liquid-liquid phase separation, or a via a \u201cbridging-induced attraction\u201d mechanism. For proteins that interact multivalently, we obtain a phase diagram which includes liquid-like droplets, droplets with absorbed polymer, and coated polymer regimes. Of particular interest is a regime where protein droplets only form due to interaction with the polymer; here, unlike a standard phase separating system, droplet density rather than size varies with the overall protein concentration. We also observe that protein dynamics within droplets slow down as chromatin is absorbed. If the protein-protein interactions have a strictly limited valence, fractal or gel-like condensates are instead observed. A specific example that inspired our model is heterochromatin protein 1, or HP1. Recent in\u00a0vivo experiments have shown that HP1 exhibits similar droplet size buffering behavior as our simulations. Overall, our results provide biologically relevant insights into the general nature of protein-chromatin condensates in living cells. Liquid-liquid phase separation has been much discussed as a mechanism for protein droplet formation in the cell nucleus. Yet how this can drive gene-regulatory protein clustering only at specific chromatin sites remains unknown. Here, we study the physics of clustering of chromatin binding proteins using simple simulation models. We focus on the interplay between protein self-interactions and protein-chromatin bridging. Our results on cluster structure, dynamics, and polymer compaction, have strong implications for our understanding of the role these processes play in\u00a0vivo. Particularly, we find that chromatin acts to slow down protein dynamics within droplets, and we uncover a regime where varying the overall protein concentration alters the concentration within and outside the droplet, strikingly different to standard phase separation.The cell nucleus is a highly structured organelle, which contains much of an organism\u2019s genetic material . This maThere has been much recent interest in how protein foci form in the nucleus, and whether a liquid-liquid phase separation (LLPS) mechanism plays a role. A common notion is that flexible, low complexity, and intrinsically disordered protein (IDP) domains facilitate LLPS . IDPs ofFor the case of chromatin binding proteins, which we consider here, another mechanism that can lead to protein phase separation is the \u201cbridging-induced attraction\u201d (BIA). This was first uncovered in simulations studying how protein-chromatin interactions can drive chromosome organization , 17, andThe idea that LLPS is involved in genome regulation gained popularity after it was shown that heterochromatin protein 1 (HP1), one of the chief constituents of heterochromatin, was found to undergo phase separation in\u00a0vitro , 23. HP1In this paper we study the interplay between LLPS and BIPS, considering how they could drive protein-chromatin foci localization and compaction in\u00a0vivo. Inspired by work on patchy particles , 31, 32,In this work we use coarse-grained Langevin dynamics simulations to study the behavior of a system of simple HP1-inspired model proteins interacting with a model chromatin fiber.For chromatin we use a common coarse-grained polymer model where the fiber is represented as a chain of beads of diameter a shows a schematic representation of the domains of HP1  are represented in our simulations. They are modeled as rigid bodies made up from seven spheres, each representing a different domain . Thn, e.g.,  it was smultivalent interactions (b), using a longer range interaction potential between the spheres representing the hinge and NTE, such that several NTEs can simultaneously interact with a hinge and vice versa (determined by the geometry and steric hindrance). Second, we consider limited valence interactions (c), using a shorter-ranged potential such that at most one hinge and one NTE can interact at a time. Since an HP1 dimer has two hinges and two NTEs, in the limited valence model a given dimer can bind to at most four others at once. Importantly, the interaction between the proteins and chromatin is identical for the two models. The strength of attractive interactions between HP1s, and between HP1s and chromatin, are given by the energies As noted above, we study two versions of the model that differ in their protein-protein interactions. First, we consider ractions b, usingractions c, using\u03c4 is a simulation time unit. All results are obtained by averaging of at least four simulations of at least The dynamics of the polymer beads and HP1s (rigid body translation and rotation) are governed by a Langevin equation; we perform extensive simulations using the LAMMPS molecular dynamics software . The dynBelow we present simulations of a system containing a\u2013c.With this version of the model, when the HP1-chromatin interaction energy, . Whdense droplet regime. By measuring the density \u03c1 of HP1s inside and outside of the droplet, we can also map out the phase diagram on the \u03c1-ctop, see When a and b). In this sense there is a phase separation; however, this regime is profoundly different from the dense droplet phase: a significant fraction of the proteins remain unbound, while the rest tend to \u201ccoat\u201d the polymer. Hence, we refer to it as the coating regime.For small HP1-HP1 interaction energies, blue region in a and b) a protein droplet forms, but now the polymer is also absorbed into it. Or in other words, the droplet compacts the polymer. We call this the absorbing droplet regime. Interestingly, the polymer is absorbed to a different degree depending on the precise values of the interaction energies . As before, measurements of HP1 density inside and outside of the droplet\u00a0allow us to construct the \u03c1-cbottom, and see When both b), we measure the local protein density by splitting the simulation box into ith sub-box, the local density is d shows how dinset), and we observe a sharp crossover . For To characterize these regimes more quantitatively . First, an HP1 could bind through only one of the CDs; we call this \u201cdangling,\u201d since it leaves one free CD. Second, the CDs could both bind to the chromatin at adjacent (We now consider the nature of the interactions between the HP1 dimers and the chromatin. Since each model HP1 dimer can interact with the polymer via two distinct domains (the CDs), they can bind in three different modes a. Firstailed in , the shaailed in  and detaailed in  have indb we plot the fraction of bridging, coating, and dangling proteins as a function of b). This could indicate the presence of a first-order phase transition in the thermodynamic limit. Within the absorbing droplet regime we also observe that the fraction of bridging proteins increases with c). As detailed further in b.In ed again c. As deith chromatin bead, and a and b show how c and d)). The reason for this nonmonotonicity is strikingly apparent in the top row of snapshots in a: in the leftmost snapshot the polymer is swollen, in the second from the left it is fully absorbed into a protein droplet .One proposed function of HP1 in\u00a0vivo is to compact heterochromatin. The ability of our model proteins to compact the chromatin can be probed by measuring its radius of gyration, defined aseins, fc , c and db moves to the left, so droplets can form at if the protein-chromatin interaction energy is large enough. In other words, HP1-chromatin attraction promotes protein aggregation. This is reminiscent of the scaffold-client systems studied via simulations of multivalent patchy particles .Finally in this section, we consider intermediate values of the HP1-HP1 interaction strength, articles , 13, 14,N (and further increase of the droplet size) does not lead to a swelling of the polymer: the ratio N (e). When N increases, the total number of proteins bound to chromatin increases and the ratios of binding in the different modes changes slightly; strikingly, this does not lead to further polymer compaction . This can be rationalized as follows. For small N a protein droplet forms on the polymer via the BIA. This droplet is rather \u201cloose\u201d and, as N increases, more space within the droplet becomes filled with proteins and the density . This has important implications for protein-chromatin interaction in\u00a0vivo . This is similar to classic low-valence patchy particles, which have been studied extensively using both simulations (\u03c4 is the simulation time unit); after this time the measured quantities . For c), behavior that is not observed in the multivalent model. This arises because, while clusters do form, there are many of them; they are also highly dynamic, continually forming, dissolving, and merging and breaking apart , and the radius of gyration of the polymer (c and d)) as a function of the two interaction energies. The behavior is broadly similar to the multivalent model, but the limited valence proteins are less able to compact the polymer, and the chromatin \u201clooping out\u201d  does not tend to occur here. Instead, most of the polymer is associated with the irregularly shaped cluster.We again measure the fraction of proteins bound in different modes , the fra polymer , c and da. We observe that, for small So far we have considered structural properties of the protein clusters for each of the two models. Here, we consider protein dynamics. This is often studied in\u00a0vivo using fluorescence recovery after photobleaching (FRAP) experiments: the timescale of fluorescence recovery of a protein droplet gives a measure of how quickly proteins are exchanged between the droplet and the soluble (unbleached) pool. The internal dynamics of a droplet can also be probed by photobleaching half of the droplet: tracking fluorescence in the bleached and unbleached halves gives information on the relative timescales of mixing within the droplet and exchange with the soluble pool . A simili and j are interacting at time t, and 0 otherwise (proteins are said to interact if a hinge or NTE domain on protein i is within the interaction range from an NTE or hinge on protein j). Angle brackets denote an average over time t, repeat simulations, and all possible More quantitatively, we can measure how the proteins change their binding partners during a given time interval, b we plot bleft) there is a clear step change in a, the presence of chromatin within the droplet leads to a dramatic slow-down of protein dynamics (bleft).In ncreased bleft tThere are two main physical reasons for the slow-down upon polymer absorption. First, since polymer bead motion is constrained by their connection to neighboring beads in the chain, the motion of proteins bound to these beads becomes similarly constrained (observing that faster protein dynamics is restored if polymer bonds are \u201ccut\u201d confirms this). Second, if protein-chromatin bonds are effectively stronger than protein-protein bonds, this will also lead to a step-like slow down when the polymer becomes absorbed.c) show similar behavior. The decorrelation time c (right) shows that again The limited valence proteins c show sIn this paper we have studied the behavior of simple model proteins interacting with a bead-and-spring polymer model for chromatin. We considered rigid bodies composed of spheres that represent different protein domains that interact attractively with each other or with chromatin. The domain structure was based on that of HP1, but our goal was to obtain insight on the interplay between protein-protein and protein-chromatin interactions in general.HP1 has been shown to undergo LLPS in\u00a0vitro , 23. Thaudied in ). The re\u03c1, which is fundamentally different to standard model B phase separation. The density of proteins within and outside the droplet depends on \u03c1, and the droplet volume grows sublinearly as \u03c1 increases. This behavior originates from the formation of a loose protein cluster on the chromatin for small \u03c1, which can \u201cfill up\u201d as proteins are added to the system; at larger \u03c1, sites on the chromatin become saturated; so, as more proteins are added these instead remain unbound (increasing the density in the protein poor region). This is reminiscent of recent work showing that varying the overall concentration of the nucleophosmin protein  leads to variation in its density both inside and outside the nucleolus .To understand how the behaviors observed in our simulations might play a role in\u00a0vivo, it is useful to discuss how model parameters map to physical quantities. For the attraction strengths, 0\u2013250\u00a0ms ; while tar range . In any does not lead to an increase in the size of foci, but instead the protein density within the foci increases  is therble pool  also shoble pool  has sugge of DNA , has mule of DNA .The limited valence model showed similar regimes to the multivalent case but, instead of a spherical droplet, the proteins formed fractal clusters . ThWhile above we have highlighted clear similarities between our simulations and previous experimental observations, we hope our results will prompt new experiments to mechanistically test whether these regimes are realizable for HP1 and other proteins. For example, one might consider an in\u00a0vitro setup with purified protein and long reconstituted chromatin fibers where concentrations could be precisely controlled. Then, spatially varying density and dynamical properties could be measured via fluorescence correlation spectroscopy (FCS), FRAP, or microrheology . Or, to Finally, we note that our model proteins are \u201cpoor bridgers,\u201d which tend to coat the chromatin . It woulhttps://doi.org/10.7488/ds/3474.All simulation and figure data arising from this work are\u00a0available via the Edinburgh DataShare repository at M.A. and C.A.B. performed and designed the research and wrote the paper."}
+{"text": "Background and Objectives: Little information is available on the role of Vitamin D as a micro-nutrient deficiency with masticatory muscle efficiency and its effect on the function of removable prosthesis. The aim of this study was to evaluate the role of vitamin D on masticatory muscle activity among completely edentulous patients and its effect on the retention of removable complete dentures (RCDs). Materials and Methods: A non-randomized clinical control trial was conducted on completely edentulous patients (60.53 \u00b1 7.01 years) in the Indian population between 2017 and 2019. Subjects were evaluated for temporomandibular disorders according to the Diagnostic Criteria for Temporomandibular Disorders (DC/TMD). Serum Vitamin D (S Vit D) levels, Ultrasonography (USG), and surface Electromyography (sEMG) readings of the masseter muscle were recorded at enrolment (Level 0), after 3 months of Vitamin D therapy (Level 3), and after consecutive 3 months of maintenance therapy, i.e., after 6 months from baseline (Level 6). The fabrication of new RCDs was done for all after the enrolment, and the retention of RCDs was assessed by asking a question regarding denture retention and asking respondents to mark their satisfaction on a 5-point Likert scale. Data were analysed using ANOVA, Paired\u2019-test and Pearson correlation coefficients. A p-value less than 0.05 indicated a statistically significant association. Results: Between enrolment and a six-month follow-up, S Vit D levels showed an increase from 16.03 \u00b1 5.68 ng/mL to 31.35 \u00b1 9.28 ng/mL, showing an increase of 15.32 \u00b1 9.38 ng/mL (95.57% rise). Statistically significant values were observed for USG and sEMG. Conclusions: Results showed that S Vit D affects masticatory muscle activity by improving its thickness and boosting its tonicity. Healthy muscles assist in the retention of RCDs, consequently aiding in mastication, speech, and phonetics, hence improving patient satisfaction. Clinical implication: Acknowledging the fact that the prevalence of Vitamin D deficiency is worldwide. We suggest Vitamin D therapy as a nutritional intervention among the elderly completely edentulous population, following dietary counselling, and consider Vitamin D therapy to be an adjunct to nutritional counselling for improving masticatory muscle activity and efficiency, which aids in RCD retention and stability. Consequently, improving oral health-related quality of life for individuals. Vitamin D is the hormone that regulates calcium phosphate homeostasis and mineral bone metabolism. Different tissues have varied receptors to absorb the micronutrient; vitamin D receptors are important as they are responsible for the biological effects of vitamin D ,3,4,5,6.Vitamin D is either consumed in the form of food or synthesized in the body when exposed to sunlight (accounting for 80\u201390% of the vitamin D body stores). Cholecalciferol (vitamin D3) is produced in the skin and found in fatty fish and mammals, while ergocalciferol (vitamin D2) is obtained from yeasts and plants. Vitamin D supplements contain vitamins D2 or D3, and recent studies have proved the superiority of vitamin D3 in the treatment of vitamin D insufficiency .Generally, patients with vitamin D deficiency present with muscle pain and weakness ,7,9. AccEvidence shows that good muscle strength is a key to success in improving the retention of RCDs ,19,20. PEven in the modern era of dental implantology, a majority of edentulous patients still opt for RCD to improve oral health-related quality of life (OHRQoL) ,24,25. AIn the present study, the role of vitamin D on muscle activity was evaluated through surface electromyography (sEMG) ,34,35,36A double-blinded, interventional, non-randomized control trial was conducted in the Aligarh Province  between 2017 and 2019. This study aimed to evaluate the role of vitamin D on masticatory muscle activity among completely edentulous patients and its effect on the retention of dentures as perceived by patients. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Institute. JNMC, AMU, Aligarh, India [JNMC-AMU/ECL/22-2013-14]. The study protocol was developed, and all subjects gave their written informed consent for inclusion before they participated in the study.A total of 130 completely edentulous patients between the ages of 38 and 75 were recruited for the study. Subjects were evaluated for temporomandibular disorders according to the Diagnostic Criteria for Temporomandibular Disorders (DC/TMD) . InclusiIn the present study, the assessments were done at three points in time: at 0 months (T0); 3 months (T3) and 6 months (T6).Pre-intervention\u2014At T0-S Vit D, USG and sEMG values were recorded for each enrolled subject. Based on S Vit D levels, only treatment group subjects were recommended the Vitamin D oral supplements.Intervention: For treatment group subjects, the intervention of vitamin D was done orally following the recommendations of Holicks , Grant eAt T3 and T6 the S Vit D levels, USG-measured muscle thickness and sEMG-measured muscle activity were recorded as compared with baseline (pre-intervention) T0. As far as treatment groups are concerned, they were formulated only to assess the need for vitamin D supplementation. The collected data comprised categorical variables as well as continuous variables. Therefore, the statistical tests were performed based on these two types of variables. The association of different clinico-demographic factors with S Vit D levels was analysed . The colThe data for S Vit D, USG-measured muscle thickness, and sEMG-measured muscle activity were collected at three different points in time: T 0 was the baseline after intervention, T3 was at 3 months, and lastly, the values of these three variables were collected at T6, which was at 6 months. The three values of S Vit D, USG measured muscle thickness, and sEMG measured muscle activity were paired values as they are the before and after values of an intervention.At T0, even if the old dentures were present, for each subject they were removed for 1 week, and the recording of USG and EMG occlusal bite records were made (by the chief researcher and trained technician who was blinded from the study) for each patient, and the averaging of the EMG and USG values of masseter were completed in the resting position and during maximum bite clenching. After the recordings, the RCD fabrication and insertion were done for both groups of patients, following the normal conventional procedure for removable complete denture fabrication. In this procedure, initially, the primary impression was taken, and the primary cast was fabricated. Furthermore, after making a special tray, the secondary impression was made, and the master cast was poured with type IV die stone , to be used in further steps. Clinical steps involved were primary impressions, border molding with definitive impressions, jaw relations using a face bow [Hanau Spring-Bow ] and inter-occlusal bite record with polyether bite registration material , teeth selection, wax trial placement, denture adjustment , and insertion. Laboratory steps were master cast and special tray fabrication, mounting on a semi-adjustable articulator , teeth arrangement, and fabrication of CDs with conventional compression molding technique, lost wax technique, and a long polymerization cycle (9 h in a water bath at 73_C \u00b1 1_C followed by 1/2 h in boiling water as recommended by the manufacturer) using heat-polymerizing acrylic resin . The heat-polymerized CDs were finished and polished before the insertion visit ,18,19,26At T3, after completing 3 months of drug regime and dentures use, all three readings  were recorded again for both group subjects. After assessing the values of S Vit D, the intervention of Vitamin D was continued as a maintenance dose for the next 3 months in treatment group subjects. All subjects were asked to continue denture use regularly. Furthermore, the same question regarding denture retention was asked, and the response was recorded on the same scale.At T6, three months after T3, both the group control and treatment group subjects were recalled, and readings were taken for the same variables  and the same question\u2019 response was recorded.Measurement of S Vit DThe institute\u2019s medical pathology laboratory support was taken for the test. The trained technician blinded from the study collected the blood sample from each subject. The liquid chromatography-tandem mass spectroscopy (LC-MS) was applied for the direct measurement of 25(OH)D [25-hydroxyvitamin D] in the serum. The Vit-D levels were expressed in ng/mL ,33.sEMG of masseter musclesEMG of the masseter muscle was done with surface electrodes, using surface electromyography apparatus  available in the institute pathology department. Surface electromyography (sEMG) is a form of measurement of the masticatory muscles\u2019 functionality, able to identify variations of the electric potential of the muscles during each performed contraction, both in chewing and swallowing . The tecAll sEMG tests were performed by the same technician as the chief researcher both of them had been calibrated for the same. Prior to each sampling, site friction with non-sterile gauze soaked in 70% alcohol was performed in order to minimize artifacts and improve signal capture. The reference electrode (earth) was placed in the front portion of the patient\u2019s head. Data were obtained on the electrical activity of the masseter muscle group during the tasks of rest and maximum clenching . The records were collected by MBF, maintained for 5 s, and repeated three times with a 1 min interval addition to rest between each collection. The average was used for signal normalization, equivalent to 100% of the electrical activity. The signals collected during mastication were analysed by Root Mean Square (RMS) and expressed in microvolts (uV) ,35,36.USG of Masseter muscleAll scans were carried out in the ultrasonography department. Each subject was examined by the same operator using the Thei Style Ultrasound system with a 7.5\u20139.0 MHz broadband transducer. A line was drawn joining the lateral commissure of the mouth to the intertragic notch (space that separates the tragus from the antitragus in the outer ear) of the ear, crossing the masseter muscle. A generous amount of water-soluble conductive gel was applied evenly on the muscle area on the cheeks using a gauze pad. The ultrasound probe was placed on the line with a feather-like pressure. The angle of the probe was adjusted to produce the strongest echo from the mandibular ramus, which was achieved when the scan plane was perpendicular to its surface. The imaging and measurements were performed bilaterally with the subjects in a supine position under two different conditions: when the teeth were gently occluding with the muscle in a relaxed position and during maximal clenching, with the masseter muscle contracted. The measurements were made directly from the image at the time of scanning .Each measurement was recorded twice at an interval of 1 h and the average value was considered for analysis. The three levels of S Vit D, USG measured muscle thickness, and sEMG measured muscle activity was paired values as they are the before and after values of an intervention. A thorough analysis was conducted.As far as the use of a repeated measures mixed model is concerned, we would like to admit that S Vit D levels over a period of 6 months are not dependent upon the vitamin D supplementation alone but are also dependent on a host of other factors such as season, dietary intake, level of sun exposure, etc., and hence it is not desirable to study this multifactorial model without considering all these factors.t-test and Mann-Whitney tests were used depending on the normality assumption of the variables. For more than two categories, ANOVA with Scheffe post hoc and Kruskal-Wallis tests were applied to determine the statistical significance of the continuous variables between the groups of discrete variables. For pairwise comparisons after a significant Kruskal-Wallis test, the Mann-Whitney test was used with a Bonferroni correction. The three paired levels of serum D, USG-measured muscle thickness, and sEMG-measured muscle activity were analysed for significant differences using the Friedman test. The pairwise comparison after a significant Friedman test was made using a Wilcoxon signed rank test with a Bonferroni correction. The level of significance was fixed at 5%. The implemented statistical tests were said to be significant if the p-value was less than or equal to the level of significance. IBM-SPSS version 20 was used for conducting all the statistical analysis.Descriptive characteristics were determined for each variable. The mean \u00b1 standard deviation (sd) was reported for continuous variables, while the total frequency (percentage) was written for categorical variables. Pearson correlation and Spearman correlation coefficients were calculated for continuous and categorical variables. The correlation of S Vit D was estimated with each continuous and categorical variable. Apart from this, each level of S Vit D was tested for correlation with each level of USG-measured muscle thickness and sEMG-measured muscle activity. S Vit D was tested for significant differences with respect to the categorical variables gender, occupation, sun exposure, eating habit, address, old denture, and S Vit D status. Normality was tested for all continuous variables by using the Shapiro-Wilk test. If there were two categories, then independent samples A total sample of 130 patients was collected for the study, of whom 93 (71.5%) were males and 37 (28.5%) were females. The average age was 60.62 \u00b1 6.94 years. The ratio of sun exposure was about the same, with the yes and no categories having 68 (52.3%) and 62 (47.7%) patients. Average S Vit D was 16.09 \u00b1 5.62 (ng/mL) at level 0, 33.47 \u00b1 10.83 (ng/mL) at level 3 and 31.31 \u00b1 9.12 (ng/mL) at level 6. It is to be noted that the average values of S Vit D increased due to the intervention from level 0 to levels 3 and 6. Looking at the S Vit D status variable, it was found that there were no severely deficient patients at the 3-month and 6-month time points. The number of insufficient S Vit D individuals also reduced largely at 3 months and 6 months. From 3 months to 6 months, deficient S Vit D individuals increased. However, the number of patients slightly decreased in the sufficient S Vit D category. The Pearson correlation coefficient between level 0 S Vit D and level 0 USG measured muscle thickness was estimated at 0.611; between level 3 S Vit D and level 3 USG measured muscle thickness was 0.313; and between level 6 S Vit D and level 6 USG measured muscle thickness was 0.419. The highest correlation between S Vit D and sEMG-measured muscle activity was found between the two level 0 values (rp = 0.778). The descriptive characteristics in the form of mean \u00b1 sd, number (percentage), Pearson, and Spearman correlation are shown in p value < 0.05) but level 6 values followed normal distribution . The average value of S Vit D at level 0 in males was 16.91 \u00b1 5.58 (ng/mL) while in females it was 14.09 \u00b1 5.23 (ng/mL). S Vit D at level 0 was statistically significant  between males and females. At level 6, the average value of S Vit D went up to 33.94 \u00b1 10.09 (ng/mL) in males and 29.95 \u00b1 9.74 (ng/mL) in females, and the results were statistically non-significant  between males and males. The occupation variable had 5 categories; therefore, Kruskal-Wallis and ANOVA tests were used. Only level 0 vitamin D values were significant . The significant difference was observed between farmer/labourer/field and housewife/household  and it was also observed between farmer/labourer/field and shopkeeper/Service/business . There was a significant difference  between the three levels of S Vit D continuous values and the three status variables of vitamin D. The remaining S Vit D characteristics based on categorical variables are presented in The characteristics of S Vit D based on all categorical variables are represented in p values were obtained using a two-sample independent t test and the Mann Whitney test. These were followed by the application of the Bonferroni correction. At 3 months and 6 months\u2019 time points, there were no severely deficient cases. Thus, no p values were estimated for such cases, and this is portrayed in The two-sample analysis required to test the significance between the three levels of S Vit D continuous values and the three status variables of S Vit D is described in p values are not written. All three variables resulted in a significant p value  and it was concluded that there is a significant difference between the three levels of vitamin D. Similarly, it is concluded that a significant difference was determined between the three levels of USG-measured muscle thickness and between the three levels of sEMG-measured muscle activity.p values are presented in To find out which paired levels had a significant difference, a pairwise comparison after the Friedman test was performed by using Wilcoxon signed rank test followed up with a Bonferroni correction. The resulting In the present study, it was found that demographic and occupational variations have an impact on S Vit D levels . AmongstAt enrolment, S Vit D levels ranged from 5.5 to 41.5 ng/mL in the study population. After intervention at 3 months, S Vit D levels range improved from 12.5 to 75.80 ng/mL. Initially, only 3 (2.3%) had S Vit D sufficiency, but after administrating Vitamin D therapy, we found the majority (63.1%) were vitamin D sufficient. After the intervention of 3 months, none of the patients had very severe S Vit D deficiency . After aIn the present study, improvement was observed in the values of S Vit D, USG, and sEMG measured masseter muscle thickness and activity after 3-month intervention among group B subjects, which was in line with the study conducted by CegliaL  where ViIn vitamin D insufficiency, the effect on muscle function and physical function mostly appeared before the clinical signs of bone disease were evident ; it had Regarding the association between S Vit D status and masticatory musculature efficiency at all three levels, after the intervention, both masseter muscle thickness and sEMG values showed a significant increase during the period , thus inThe present study shows vitamin D as one of the most important factors affecting masticatory muscle activity, thus showing the need to include vitamin D assessment and supplementation as an important measure while planning the prosthodontic rehabilitation of the edentulous patient. However, it is the need of the hour to systematically evaluate the role of various other micro-nutrients on the various oral health-related issue at a global platform.The present study had certain limitations, which include the short duration of the study. Furthermore, S Vit D levels are not only dependent upon Vitamin D supplementation only but are also dependent on a host of other factors such as season, dietary intake, level of sun exposure, etc. Similarly, the retention of dentures may also be affected by other factors which were not considered in the study, such as muscle activity and the quality of old dentures. The time and cause of tooth loss were not recorded, which might have affected the bony contour of the edentulous ridges. Furthermore, due to some reasons, muscle insertion might vary in a few patients, so it can be considered a physiological limitation. The effect of old dentures on ridges, soft tissues, and muscle activity was not evaluated in the study so as to simplify the study and establish a relationship between S Vit D levels and muscle thickness and activity, which play an important role in prosthodontics rehabilitation by RCDs. Thus, further studies are recommended to assess the need for long-term requirements of vitamin D therapy during the maintenance phase of deficiency-related ailments. Furthermore, the effect of various factors affecting RCDs including parameters such as edentulous ridge height, other nutritional deficiency, eating habits, education levels, etc., should be correlated in future studies for a better understanding of the role of vitamin D in denture retention. Along with all these, to avoid the Hawthorne effect in further studies, it is recommended to use either placebo tablets in the control group or administer vitamin D in the treatment group confined to dietary essentials.Based on the findings of this non-randomized clinical trial, the following conclusions were drawn. The present study observed S Vit D deficiency irrespective of age, gender, or dietary habits. However, occupation, sun exposure, and a non-urbanization lifestyle had an impact on S Vit D levels. Only 2.3% of S Vit D-sufficient people were present in the study population, making it another nutritional pandemic. After administrating recommended Vitamin D therapy for three months, the majority (62.3%) became vitamin D sufficient. Vitamin D therapy has increased S Vit D levels resulting in improved USG and sEMG values of the masseter muscle, enhancing muscle thickness and boosting muscle activity, respectively. Acknowledging the fact that the prevalence of Vitamin D deficiency is worldwide. We strongly suggest vitamin D therapy as a nutritional intervention among the elderly completely edentulous population, following dietary counselling."}
+{"text": "Fusarium oxysporum is a soilborne fungal pathogen that causes Fusarium wilt, one of the most important diseases on tomato. During the infection of F.\u00a0oxysporum, some proteins are secreted that modulate host plant immunity and promote pathogen invasion. In this study, we identify an RNase, FoRnt2, from the F.\u00a0oxysporum secretome that belongs to the ribonuclease T2 family. FoRnt2 possesses an N\u2010terminal signal peptide and can be secreted from F.\u00a0oxysporum. FoRnt2 exhibited ribonuclease activity and was able to degrade the host plant total RNA in vitro dependent on the active site residues H80 and H142. Deletion of the FoRnt2 gene reduced fungal virulence but had no obvious effect on mycelial growth and conidial production. The expression of FoRnt2 in tomato significantly enhanced plant susceptibility to pathogens. These data indicate that FoRnt2 is an important contributor to the virulence of F.\u00a0oxysporum, possibly through the degradation of plant RNA.Secreted RNase proteins have been reported from only a few pathogens, and relatively little is known about their biological functions.  Fusarium oxysporum, is able to degrade host RNA and represents an important contributor to the virulence of the soilborne fungal pathogen.FoRnt2, a member of the ribonuclease T2 family secreted from  Secreted RNases have been reported in many pathogens and play crucial roles in plant\u2013pathogen interactions. For example, Nuc1 and Nuc2, which are secreted from F.\u00a0oxysporum is an important soilborne fungal pathogen that has a broad host range, causes root rot and wilting disease in the plant vascular system, and results in economic loss worldwide . This pathogen has many different formae speciales that can infect only one or a few host species. F.\u00a0oxysporum can infect the vascular bundle of host plants, leading to clogged vessels, yellowing of leaves, wilting and finally death of the whole plant. The germination of dormant spores in soil results in invasion of plant roots by fungal hyphae. The movement of hyphae from root cortex to the xylem, where it produces and disseminates microconidia, is critical for disease progression. It is difficult to efficiently control this pathogen because F.\u00a0oxysporum is soilborne and can produce chlamydospores in the soil. Chlamydospores are resistant to different environments, such as high temperature and drought. To control F.\u00a0oxysporum, chemical fungicides are widely used. However, the resistance of this pathogen to fungicides is becoming increasingly important. To date, no effective methods to control the disease have been used in the field  proteins, which play important roles in host specificity. Six1 is required for full virulence and is responsible for avirulence in tomato plants carrying the resistance (R) gene I\u20103. The effector Six6 is essential for the virulence of F.\u00a0oxysporum and suppresses host cell death  in this study. In addition, the results from SignalP 5.0 predicted that FoRnt2 possesses a secretion signal peptide domain in residues 1\u201319 in the N\u2010terminal region and a cleavage site between residues 19 and 20 . The two catalytic sites of the FoRnt2 fusion protein were also mutated to other amino acids, and the FoRnt2M2 recombinant protein was then expressed under the same conditions  tag in  Figures\u00a0 and S2.M2 recombinant proteins was tested by incubation with tomato total RNA. FoRnt2 could significantly degrade the tomato total RNA, while the FoRnt2M2 proteins could not degrade the RNA under the same conditions, indicating loss of RNase activity  strain  and Congo red (CR). Surprisingly, we found that compared with those of the WT and \u2206FoRnt2\u2010C strains, the growth rate of the \u2206FoRnt2 strain showed no difference under any of the stresses on PDA, implying that deletion of the FoRnt2 gene did not affect cell wall integrity or osmotic regulation  localizes in the nucleus and was used to verify the nuclear localization of N.\u00a0benthamiana  alone or FoRnt2 fused with GFP at its C\u2010terminus  in a Figure\u00a0. To furts Figure\u00a0, which f2.6GFP or FoRnt2\u2010GFP transgenic tomato plants. Microscopic observation showed that green fluorescence signals were distributed in the tomato cells of the two transgenic plant lines  was used to analyse the differentially expressed genes (DEGs) in s Figure\u00a0. The RNAs Figure\u00a0. This res Figure\u00a0. The tops Figure\u00a0. All 12 s Figure\u00a0, suggest2.7Agrobacterium\u2010mediated transgenic tomato seedlings were used in this assay. Immunoblot assays showed that GFP or FoRnt2 was expressed successfully in the stable Sl:GFP or Sl:FoRnt2 transformant plants, respectively  were cultured in lysogeny broth liquid or solid medium at 37\u00b0C for vector construction and protein expression. A.\u00a0tumefaciens GV3101 was used to transiently express target proteins in plants. N.\u00a0benthamiana and tomato (Solanum lycopersicum \u2018Ailsa Craig\u2019) were grown at 25\u00b0C under a 16\u2009h light and 8\u2009h dark photoperiod in an artificially controlled growth room.The WT 4.2FoRnt2 gene deletion mutant . All the transformant strains were purified by single\u2010spore isolation and then stored at \u221280\u00b0C in 30% glycerine. The primers used in the construction of different strains are listed in Table\u00a0The split\u2010marker approach was used in this study to generate a gene replacement for the 4.3The cDNA sequences of the signal peptide regions of FoRnt2 and PsAvr1b were cloned into the pSUC2 vector, which carries a truncated invertase gene lacking both the initiation residue Met and a signal peptide  and anti\u2010actin antibodies .To test the secretion of the FoRnt2 protein, the FoRnt2\u2010FFLAG strain was cultured in potato dextrose broth to harvest the conidia. Then, the conidia were transferred into 10% YEPD liquid medium with the tomato roots and cultured at 25\u00b0C and 180\u2009rpm for 16\u2009h. The culture supernatants were collected after centrifugation. The total proteins in the culture supernatants were precipitated by adding 20% acetone (wt/vol) and stored at \u221280\u00b0C overnight. The solution was centrifuged at 13,800\u00a0\u00d7 4.5FoRnt2 gene constructs were transformed into A.\u00a0tumefaciens GV3101 through heat shock treatment. The correct A.\u00a0tumefaciens clone was cultured overnight in LB medium  at 28\u00b0C. The A.\u00a0tumefaciens cells were collected and resuspended in infiltration buffer at an OD600 of 0.4. This experiment was carried out on 3\u2010week\u2010old N.\u00a0benthamiana leaves using needleless syringes. At the same time, the same A.\u00a0tumefaciens strain or cells harbouring the empty GFP vector were used as the negative control and BAX was used as a positive control. For subcellular localization, fluorescence observation was carried out after treatment for 48\u2009h. For fluorescence observation after plasmolysis, the leaves of N.\u00a0benthamiana after Agrobacterium injection were treated with 0.8\u2009M NaCl under static conditions. The inoculation for P.\u00a0capsici was performed by the same method as described previously. The total proteins in the leaves of N.\u00a0benthamiana were extracted with protein extraction buffer and the proteins were detected by SDS\u2010PAGE or western blotting.The 4.6FoRnt2 or GFP transgenic tomato plants, the FoRnt2 gene was cloned into a GFP construct with the CaMV 35S promoter. The resulting constructs and the 35S::GFP empty vector were transformed into WT tomato plants separately using Agrobacterium\u2010mediated transformation as described previously  and active site mutant sequences were cloned into the expression vector pMAL\u2010c2x. The recombinant vectors or empty vector were transformed into E.\u00a0coli BL21 (DE3) and the cells were cultured in LB liquid medium at 37\u00b0C. Then, the cells were diluted into fresh LB grown to a final OD of 0.6. IPTG (0.1\u2009mM) was used to induce the expression of the recombinant proteins for 16\u2009h at 16\u00b0C. The cells were harvested by centrifugation at 9,600\u00a0\u00d7 g and then sonicated at 300\u2009W for 10\u2009min. Recombinant proteins were purified using amylose resin (BioLabs) and the protein concentration was determined using a BCA Protein Assay Kit (Solarbio). The primers used for the expression constructs are listed in Table\u00a0The coding sequence of the 4.9M2 (MBP\u2010FoRnt2H80F/H142R) proteins at 25\u00b0C for 30\u2009min. Five micrograms of RNase A with equal amounts of total RNA was used as a positive control in this experiment. All samples were mixed with 1\u00d7 loading buffer and then run in a 0.1% agarose gel.The enzymatic activity of FoRnt2 was tested on total tomato RNA in an in vitro assay  according the manufacturer's instructions. Reverse transcription was performed using the All\u2010In\u2010One 5\u00d7 RT MasterMix Kit (Abm). HiPer SYBR Premix EsTaq  was used for qPCR to analyse the expression pattern. The relative expression of each gene was calculated using the 2Ct\u2212\u2206\u2206 method as previously described. All experiments were repeated three times. The primers used in RT\u2010qPCR are listed in Table\u00a0Total RNA was extracted from the 4.11http://www.cbs.dtu.dk/services/SignalP/). The conserved domains of the proteins were identified using the Pfam database (http://pfam.xfam.org/).The homologous protein sequences of FoRnt2 in different pathogens were identified by querying the FoRnt2 protein sequence against the NCBI database using the BLAST tool. The protein sequence alignment of the FoRnt2 protein and other ribonuclease T2 proteins was performed using the Clustal W2 program. Phylogenetic dendrograms of FoRnt2 and its homologous proteins were generated using MEGA 5. The signal peptides of each protein\u00a0sequence were predicted by the SignalP 5.0 server . The crude samples were centrifuged at 4\u00b0C for 15\u2009min at 13,800\u00a0\u00d7 Figure S1 Western blotting confirmation of FoRnt2\u2010overexpressing strains using anti\u2010FLAG antibodies. The targeted protein was detected in the #1 and #2 strainsClick here for additional data file.Figure S2 SDS\u2013PAGE analysis of the FoRnt2 recombinant proteins used in this study. The proteins were expressed in Escherichia\u00a0coli BL21 (DE3) and purified using amylose resin (BioLabs). The protein concentration was determined by a BCA kitClick here for additional data file.Figure S3 Targeted gene knockout of FoRnt2 in Fusarium oxysporum. (a) Schematic diagram of the gene deletion strategy for FoRnt2 gene in F.\u00a0oxysporum using the split\u2010marker method. (b) PCR products of each fragment and fusion amplification of split\u2010marker PCR. (c) Agarose gel of PCR products from the wild type (WT) and deletion strain genomic DNA. The F.\u00a0oxysporum WT strain was used as a positive control and water was used as a negative control. M represents the molecular markers of DNA fragment size; In represents the FoRnt2 gene; Out represents whether the hph gene is correctly embedded. (d) PCR confirmation of the \u2206FoRnt2\u2010C strain. FoRnt2 genes of the same size were detected in the WT and \u2206FoRnt2\u2010C strains but not in the \u2206FoRnt2 strainClick here for additional data file.Figure S4 The numbers of differentially expressed genes (DEGs) in the FoRnt2 transgenic tomato plants. The x axis shows the difference between green fluorescent protein (GFP) and FoRnt2\u2010GFP expressed in tomato. The y axis represents the number of DEGs. Red and blue colours represent up\u2010regulated and down\u2010regulated DEGs, respectivelyClick here for additional data file.Figure S5 The morphology of 4\u2010week\u2010old green fluorescent protein (GFP) or FoRnt2\u2010GFP transgenic tomato and wild\u2010type tomato. All tomato seedlings were grown at 25\u00b0C under a 16\u2009h light and 8\u2009h dark photoperiod in an artificially controlled growth roomClick here for additional data file.Figure S6 FoRnt2 could not induce cell death in Nicotiana\u00a0benthamiana. (a) The cell death\u2010inducing ability of FoRnt2 was determined in 3\u2010week\u2010old N.\u00a0benthamiana leaves infiltrated with Agrobacterium\u00a0tumefaciens carrying the target gene. BAX was used as a positive control and green fluorescent protein (GFP) was used as a negative control. The photographs were taken 4\u2009days post\u2010agroinfiltration. (b) Immunoblot analysis showed the protein expression levels of GFP or GFP\u2010tagged protein in N.\u00a0benthamiana leaves. Equal amounts of protein were confirmed by Ponceau S staining on the membraneClick here for additional data file.Table S1FoRnt2\u2010GFP The unique differentially expressed genes identified in FoRnt2\u2010GFP expression tomato plantsClick here for additional data file.Table S2 KEGG enrichment analysis of the down\u2010regulated genes in tomato plantsClick here for additional data file.Table S3 Primers used in this studyClick here for additional data file."}
+{"text": "In addition, we performed EWASs in five brain regions belonging to the neurocircuitry of addiction: anterior cingulate cortex (ACC), Brodmann Area 9, caudate nucleus, putamen, and ventral striatum (n = 38\u201372). (3) Results: cg15925993 within the LOC339975 gene was epigenome-wide significant in the ACC. Of 16 identified differentially methylated regions, two (PRSS50 and LINC00612/A2M-AS1) overlapped between multiple brain regions. Functional enrichment was detected for biological processes related to neuronal development, inflammatory signaling and immune cell migration. Additionally, our results indicate the association of the well-known AHRR CpG site cg05575921 with smoking in the brain. (4) Conclusion: The present study provides further evidence of the strong relationship between aberrant DNAm and smoking.(1) Background: Epigenome-wide association studies (EWAS) in peripheral blood have repeatedly found associations between tobacco smoking and aberrant DNA methylation (DNAm), but little is known about DNAm signatures of smoking in the human brain, which may contribute to the pathophysiology of addictive behavior observed in chronic smokers. (2) Methods: We investigated the similarity of DNAm signatures in matched blood and postmortem brain samples ( Whilen = 221) using postmortem tissue, finding seven CpG sites to be associated with smoking at epigenome-wide significance [Findings from blood have been partially replicated in other tissues such as the lungs ,7 and adificance . None ofificance . DecipheIn substance use disorders (SUDs), a whole neurocircuitry of addiction consisting of multiple cortical, striatal, and limbic brain regions exhibits functional changes . CorticaTo investigate differential methylation in the context of smoking, methylation changes within brain regions which are part of the neurocircuitry of addiction need to be assessed. In the present study, we investigated the tissue-specificity of smoking-associated methylation signatures by performing EWASs of smoking and evaluating the similarity of smoking-associated methylation patterns between blood and multiple brain regions , CN, PUT and the VS). Based on the results, we performed downstream analyses including the assessment of differentially methylated regions (DMRs), gene ontology (GO) enrichment analysis, and GWAS enrichment analysis.n = 1), Black (n = 1), Hispanic (n = 2), and White (n = 8) ethnicities. For 10 of the 12 subjects, matched blood samples were available. Total sample sizes of different brain regions ranged from 38 to 72 , as part of a previous study on alcohol use disorder (AUD) . Informa38 to 72 . InclusiExtraction of DNA, sample randomization, epigenome-wide DNAm analysis, and quality control of methylation data was performed as described in Zillich et al. . In brieAll statistical analyses were performed using R version 3.6.1 . A schemn = 10) was determined using the Pearson correlation method.M-values of methylation were generated by logit-transformation of \u03b2-values as described by Du, Zhang . For eacn = 38 (ACC), n = 65 (VS), n = 68 (CN and PUT), and n = 72 (BA9). Prior to EWAS, a variance inflation factor (VIF) analysis was performed to investigate multicollinearity in the linear model. If a VIF larger than 10 was detected, one of the correlated covariates was removed in a way so that the final models for each brain region were most comparable to each other . The EWASs were restricted to samples originating from donors with European American ancestry to reduce confounding by ancestry. A linear regression model with M-values specified as the dependent variable and smoking status as a predictor was used. As covariates, sex, age, postmortem interval (PMI), AUD status, batch, and the first 10 principal components of control probes (PCcp) were included. Neuronal cell fractions were estimated using the Houseman approach  with thech other . p-valuerocedure .p-values < 0.01 for a minimum number of two CpG sites within a 500 bp genomic window. Correction for multiple testing was performed using the S\u012dda\u00e1k method as implemented in comb-p.Differentially methylated regions were identified using the comb-p software (v. 0.50.2) . The folhttp://webdata.illumina.com.s3-website-us-east-1.amazonaws.com/downloads/productfiles/methylationEPIC/infinium-methylationepic-v-1-0-b4-manifest-file-csv.zip) as downloaded on 10 August 2018. Analysis was restricted to CpG sites with association p-values < 0.001.Functional enrichment of genes harboring smoking-associated CpG sites within GO terms was investigated using missMethyl  (v. 1.20p < 0.05) association with smoking were annotated to genes. Enrichment of these gene sets within GWAS signals of different smoking phenotypes and SUDs was investigated using Multi-marker Analysis of GenoMic Annotation  [n = 313,959) [n = 37,003) [n = 374,287) [n = 435,563) [n = 8, p < 6.25 \u00d7 10\u22123).To investigate a potential overlap between polygenic and polyepigenetic signal, we performed GWAS enrichment analysis. Gene sets were derived from EWAS results for each of the five brain regions. For this, CpG sites exhibiting a nominally significant (. 1.08b) . Summary. 1.08b) : (1) smo313,959) , opioid  37,003) , and can374,287)  were tes435,563)  were alsWhile sample sizes ranged between 38 and 72 per brain region, tissue of 92 donors was included in the present study. Sample characteristics are displayed in n = 632,086) revealed a clear distinction based on tissue of origin . This CpG site was annotated to the long non-coding RNA (lncRNA) LOC339975. No epigenome-wide significant CpG sites were identified in the other brain regions. The strongest association in BA9 was observed for cg22909901  which was annotated to MCF2L2/B3GNT5. In the ventral striatum, cg09139806 in RNF220 was most significant . In the dorsal striatum, the strongest associations with smoking were observed for cg07022048 in KRT7 for the caudate nucleus  and for cg18712580 in STK38L  in the putamen. A summary table including EWAS sample sizes, genomic inflation factors and resulting FDR-thresholds is provided in To expand the analysis of methylation signatures of smoking to the neurocircuitry of addiction, we performed EWASs of smoking in five brain regions. In the ACC, one CpG site (cg152925993) was differentially methylated at epigenome-wide significance between smokers and non-smokers . DMRs for each brain region are summarized in We identified a total of 16 DMRs in the five brain regions that were associated with tobacco smoking. Two DMRs were observed in multiple brain regions. One was a region consisting of 5 probes in In the ACC, smoking-associated CpG sites were enriched for GO terms related to neurodevelopment, cell growth, and morphogenesis. Enriched GO terms in BA9 were related to dendritic spine development and chromatin modification. In the VS, neuron-specific pathways were enriched, as well as processes associated with the regulation of vessel development. In both dorsal striatal regions, genes harboring differentially methylated CpG sites were enriched for GO terms related to immune pathways. After correction for multiple testing, no GO terms remained statistically significant. Result tables for the top 10 associated GO terms are shown in Gene sets consisting of smoking-associated CpG sites were overrepresented in GWAS summary statistics of smoking traits, such as smoking initiation, age of initiation, and cigarettes per day. Furthermore, significant enrichment for other SUDs, such as cannabis-, alcohol-, and opioid use disorder was observed. Statistical significance of enrichment for all tested gene-sets is displayed in AHRR CpG site cg05575921, even used as a single-marker smoking status predictor, was significantly associated with smoking in the ACC (p = 0.038) and PUT (p = 0.033). cg21566642, an intergenic CpG site, was associated with smoking in BA9 (p = 0.018). Full results of the consistency analysis are listed in We examined differential methylation of CpG sites in the brain, which have previously been suggested to predict smoking status in peripheral blood. When we investigated the nine available CpG sites from the prediction model by Maas et al. , we founIn the present study, we investigated the consistency of smoking-associated methylation signatures in blood and brain, examined differential methylation in the brain associated with smoking status, and performed several EWAS downstream analyses in five brain regions related to the neurocircuitry of addiction. The strong overall correlation of methylation levels in matched blood and brain samples is in line with previous findings of high cross-tissue correlation coefficients ,30.LOC339975. We identified a total of 16 differentially methylated regions associated with smoking status. A DMR in PRSS50 was shared between the ACC and the VS. A functional role of PRSS50 in the brain has not been systematically evaluated so far. However, in cancer cells, knockdown of PRSS50 resulted in impaired cell proliferation and increased levels of apoptosis [PRSS50 was detected in an EWAS of age-related macular degeneration (AMD) in blood and retinal tissue [PRSS50 DMR in the ACC and the VS. The second smoking-associated DMR shared between two regions of the brain (CN and PUT) was annotated to LINC00612/A2M-AS1. Both lncRNAs, LINC00612 and A2M-AS1, are involved in inflammatory processes. An anti-inflammatory function disrupted by smoking has been discovered for LINC00612 in the lungs [A2M-AS1 has been linked to interleukin 1 receptor signaling in cardiomyocytes [For the EWAS of smoking in the ACC, one epigenome-wide significant CpG site was observed within poptosis . Also, pl tissue . In the myocytes . Furthermyocytes  and neurmyocytes , resultsGWAS enrichment analysis revealed significant overrepresentation of EWAS-derived gene-sets within GWAS signals of several smoking phenotypes and SUDs. This points towards a fraction of genes detected by GWAS and EWAS contributing to the development and maintenance of tobacco smoking and SUDs. Further research needs to uncover how genetic and epigenetic mechanisms collectively contribute to the disease course in SUDs.AHRR CpG site cg05575921 was associated with smoking in the ACC and PUT and was consistently hypomethylated in all investigated brain regions. As hypomethylation and significant association with smoking has previously also been detected for cg05575921 in blood [The well-known in blood  and in ain blood , it may In contrast to the strong general correlation of methylation levels between tissues, associations with smoking were heterogenous between tissues. Blood samples might thus be limited in the extent to which they can function as a proxy for smoking-associated differential methylation in the brain. Concurrently, a potential specificity of associations implies that specific methylation signatures of smoking in the brain could contribute to the pathophysiology of TUD. This is also supported by the overlap of polygenic and poly-epigenetic signals identified for smoking phenotypes. Nevertheless, smoking could also have a non-specific effect on the entire organism and further studies are needed to investigate the functional relevance of smoking-associated differential methylation within the neurocircuitry of addiction and its contribution to the development and maintenance of TUD.N = 72, the EWAS of smoking in the brain is still insufficiently powered. At the same time, postmortem brain tissue is scarce which makes it challenging to obtain enough samples.Several limitations need to be addressed. First, a large fraction of tissue donors was diagnosed with AUD prior to death. Despite adjustment for AUD in the linear model, we cannot rule out residual confounding by alcohol consumption and future studies should investigate smoking independent of other SUDs. However, a recent Mendelian randomization study has shown that a smoking-specific risk on other disorders remains after correcting for the genetic risk for alcohol consumption . Second,The present study identified smoking-associated DNAm changes in the neurocircuitry of addiction related to immunological and neurodevelopmental processes. However, certain DNAm signatures might represent a predisposition to tobacco smoking rather than a consequence of it. Follow-up studies should thus investigate tissues of donors deceased at different stages of TUD, which may enable the identification of changes in DNAm levels during the disease course and differentiate between predispositions and consequences of smoking. The functional consequences of smoking-associated changes in DNAm levels should also be addressed in a more comprehensive design, for example using a multi-omics approach integrating methylation and transcriptomic data. Ultimately, deeper insight into methylation changes within the neurocircuitry of addiction could lay the foundation for better understanding of the pathomechanisms of tobacco use disorder."}
+{"text": "Ceraphronoidea (Hymenoptera) is a group of parasitoid wasps for which a barcoding approach could be of great help, if it were not for the very poor results. The inability to obtain barcodes for the majority of treated ceraphronoids halts progress on the taxonomy of this hyperdiverse parasitoid group. We here present a working protocol for the barcoding of ceraphronoid wasps which yields a first-time over 90% success rate.DNA barcodes provide a reliable and efficient solution to resolving cryptic species complexes and accelerate species discoveries. The superfamily  Ceraphronoidea is still severely understudied. Only a fraction of their species diversity has been described and any large-scale biodiversity assessment that involved ceraphronoid wasps (Ceraphronidae and Megaspilidae) were not very successful . One of the reasons behind this is the poor results obtained by molecular approaches using standard protocols. Ceraphronoidea stands out amongst all hyperdiverse lineages of Microhymenoptera due to their low DNA barcoding success rate. This is reflected in the extremely low number of barcodes available in BOLD (26 species out of the ~ 660 described and over 1200 unassociated BINs). Additionally, in the two previous phases of the German Barcode of Life (GBOL) Project, only 28.5% of all extractions carried out for Ceraphronoidea resulted in DNA barcodes.Despite being one of the most abundant groups of microhymenoptera recovered by various collecting efforts, be it Malaise trap or sweep netting , CeraphrCeraphronoidea, as barcoding would be essential for species delimitation in this group. External morphology alone is not sufficient to diagnose species as characters tend to be monotonous throughout the superfamily and often affected by allometry. The two taxonomically useful morphological characteristics of this group are the male genitalia .In this publication, we provide a step-by-step solution and present an optimised barcoding protocol for the previously \"unbarcodable\" Non-destructive DNA extraction was performed either by the use of the Qiagen Blood & Tissue Kit following manufacturer\u2019s protocol with minor alterations as in Ceraphronidae and Megaspilidae both. DNA quantity in the eluate was checked prior to PCR reactions using an Implen NanoPhotometer N60. Amplification of the mitochondrial COI was attempted by the use of several primer pairs ; unfortunately, four out of the six samples sequenced turned out to be of Wolbachia sp., one of them beings a cecidomyiid (Diptera) DNA and just one sequence matched with sequences of unidentified Ceraphronidae. The trace files of the non-ceraphronoid sequences showed no indication of the presence of another organism\u2019s genome in the eluate (i.e. the traces were clean and peaks were unique). This leads to the conclusion that the primers were very unlikely to attach to the DNA strands of the wasp, if there were any in the extract.In the test batch of twelve specimens, the DNA concentration was between 2.65 and 10.9 ng/\u00b5l . This attempt resulted in very good PCR  and sequencing results . According to BLASTn, all of the obtained sequences matched unidentified species of Ceraphronidae or Megaspilidae. By ruling out the option of the absence of any parasitoid DNA in the extracts, the only reasonable explanation for the failed attempts at barcoding was that the DNA was either in an advanced state of fragmentation or there was an issue at the binding site of the forward LCO1490 and LepF primers. The first hypothesis was tested by pairing the COI_pF2 primer with the COI_2437d reverse one which should result in the amplification of the slightly longer, ~ 870 bp fragment. We obtained a 91.6% sequencing success rate in the test batch and the same approach was subsequently used on a higher number of samples (n = 46). The PCR reaction resulted in the amplification of 41 samples out of 46 attempted, producing 33 clean sequences belonging to Ceraphronidae (the sequencing failed in six reactions and showed contamination in two others). Nonetheless, a 71.7% success rate was significantly better than anything obtained in the previous stages of the GBOL project. The only downside of this workflow was that it produced sequences overlapping with the standard barcode region only on ~ 450 nucleotides, as the COI_pF2 primer was placed ~ 200 nucleotides downstream from the LCO1490/LepF binding site.We decided to target a shorter region of the barcode, in case the main reason of consequent failures was the state of fragmentation of the DNA strands. For this purpose, the COI_pF2 primer was paired with the reverse HCO2198 primer which should produce a sequence with a length of ~ 450 nucleotides. This protocol is usually used as a last resort in The Research Group of Invertebrate Diversity and Phylogeny in Ia\u0219i, Romania (first author\u2019s former lab) in cases where DNA fragmentation is presumed to be the reasoning behind poor results and has been successfully tested in Ceraphronoidea. Using sequences available from Genbank, we designed a new forward primer Cer_COI_F: GSTTTATGAGCHGGAATANTAGG positioned downstream from the classic forward primers. The optimal annealing temperature was determined to be 53\u00b0C by temperature gradient PCR. By pairing the newly-designed primer with the reverse HCO2198, we achieved a 100% PCR and sequencing success in our test batch. The new forward primer was ultimately tested on a larger batch of samples (n = 140) and the barcoding success rate dropped to 82.1%. It is also notable to mention that the quality estimate exceeded 95% in all obtained sequences but nine . Unfortunately, the use of the newly-designed forward primer, paired with the HCO2198, shortens the barcodes to a final length of 617 nucleotides, but considering the significant rise in efficiency, from ca. 30% to over 80% success rate  and the binding site in at least some In the world of standard operating procedure and pipeline workflows, it is important to remember that a successful protocol for one group or another does not equate to a universal procedure and might not yield similar or any results at all when applied somewhere else. We were reminded through the course of our work here that a protocol might need to be tailored for the specifics of a group. Additionally, even though a pipeline approach is more appealing, at least from the time investment point of view, it is clear that sometimes the benefit might be overruled by the poor success rate. The protocol we provide is functional for the molecular treatment of ceraphronoids. We hope that by providing a solution to an anecdotally \"unbarcodable\" taxon, we will accelerate species discovery and aid further exploration of ceraphronoid wasps.C74BAFAF-D1F7-5E02-8733-5D294C9B8B9710.3897/BDJ.10.e84860.suppl1Supplementary material 1Supplementary dataData typePCR results and barcode qualityBrief descriptionDetails regarding the extraction method and PCR primers used on every specimen, including the quality of the obtained sequences, with notes on failed reactions.File: oo_663357.xlsxhttps://binary.pensoft.net/file/663357Cristina Vasilita"}
+{"text": "The rate of Cytomegalovirus (CMV)-induced SNHL is not well documented in developing countries, such as Iran. Therefore, this prospective follow-up study aimed to evaluate this rate among neonates with cCMV infection in Iran.Congenital Cytomegalovirus Neonates with cCMV infection admitted to neonatal intensive care units and neonates with CMV infection identified in two other prospective screening studies in Tehran, Iran, were enrolled in this study. Audiological assessments, including otoacoustic emission and auditory brainstem response tests, were performed for all the cases. Antiviral therapy was administered for the newborns in case of having severe symptoms.A total of 22 neonates with cCMV infection were entered into the study, of whom 8 and 14 subjects had symptomatic and asymptomatic cCMV infection, respectively. In total, 3 of 22 newborns had SNHL , 2 of 8 cases with symptomatic cCMV infection  and 1 of 14 cases with asymptomatic cCMV infection . No association was observed between SNHL and CMV-related risk factors in newborns.The findings of this study revealed that the rate of cCMV-induced SNHL is high among neonates born in Tehran. The severe sequelae of cCMV infection indicate the need for screening for CMV infection at birth to reduce the risk of CMV complications and the financial load of treatment imposed on healthcare and treatment systems in Iran. Congenital Cytomegalovirus(cCMV) is the leading cause of congenital anomalies and neurological damage in children worldwide . The annGenerally, more than 90% of newborns with cCMV infection are asymptomatic at birth, and of the remaining 10% infected neonates, numerous cases go unidentified due to the lack of specific symptoms or signs of CMV infection . UnfortuMoreover, it is estimated that approximately 9.3-17% of neonates with cCMV infection will develop SNHL during childhood . It is iBased on published investigations, the awareness of the epidemiology of cCMV infection and its role as a cause of HL in neonates born to mothers with high CMV seroprevalence is of considerable importance. Recently, some studies have demonstrated that HL occurs at a similar range in neonates born to mothers with non primary CMV infection and women with primary CMV infection , 11. HowIn this prospective follow-up study, neonates with cCMV infection admitted to the neonatal intensive care units (NICUs) of university-affiliated hospitals in Tehran, Iran, and CMV-infected neonates identified in two prospective screening studies of 1398 neonates born in six cities of Tehran province  were enrolled within April-September in 2017, 13.The inclusion criterion for cases was the detection of CMV deoxyribonucleic acid(DNA) in the urine or dried blood spot (DBS) samples of neonates within the first 3 weeks of life (<21 days of birth). The exclusion criteria were defined as the inability of parents to sign an informed consent form, unwillingness to continue the study, and/or absence in scheduled follow-up visits twice.This study was approved by the Ethics Committee affiliated with Iran University of Medical Sciences, Tehran, Iran, and the study followed the principles outlined in the declaration of Helsinki. Written informed consent was obtained from the parents of all infected neonates for participating in this study. The clinical characteristics and demographic data of neonates and related maternal factors were collected by questionnaires or documented from their medical records. Educational brochures containing data about CMV infection, transmission, and prevention were distributed between newborns\u2019 parents.Total DNA was isolated from the DBS samples in triplicate using heat shock assay according to a modified protocol. BrieflyAll neonates with cCMV infection were examined by two pediatricians and underwent a clinical evaluation, laboratory tests, ophthalmological examination, liver function test, and computed tomography (CT) scan of the brain, if necessary. Antiviral drugs were administered for the newborns in case of having severe symptoms and based on physician decisions.The results of the hearing screening test of newborns consisting of transient otoacoustic emission (OAE) testing at the birth time were received from their parents and documented as \u201cnormal\u201d or \u201cfail\u201d results. The infected neonates with impaired OAE were referred to an otolaryngologist in the Department of Head, Neck, and Ear Surgery in Hazrate Rasool hospital in Tehran for treatment. The otoscopic examination was performed for the infected neonates to diagnose any middle-ear disorders. Then, an auditory brainstem response (ABR) test using the threshold method was performed for all the cases with cCMV infection within 3 months of life and subsequently every 6 months up to the age of 24 months . The ABRThe data were collected and entered into the datasheet and analyzed using MedCalc software for Windows . Univariate analyses were performed by Fisher\u2019s exact test and Chi-square test to compare the clinical findings and demographic characteristics between neonates with and without SNHL. A p-value of less than 0.05 was considered statistically significant.Of 23 eligible newborns with confirmed cCMV infection, 1caserelocated to another city and was excluded from the study. Moreover, 9 and 13 of 22 neonates were female and male, respectively. The mean age of neonates at the screening time was 16 days. The clinical symptoms and signs consistent with symptomatic cCMV infection were observed in eight neonates (at least one specific symptom) five of whom had multisystem diseases and underwent antiviral therapy , based on the pediatric decision. The remaining 14 neonates had no specific symptoms related to cCMV infection and were classified under asymptomatic CMV infection.Clinical FindingsIn this study, 14 of 22 infected neonates presented jaundice after birth, of whom 10 subjects did not require phototherapy; however, 4 subjects had severe jaundice and underwent phototherapy. The peripheral blood smear of seven neonates revealed few erythroblasts and pancytopenia. Germinal matrix hemorrhage was observed in the brain ultrasound of one infected newborn. In two neonates, the abdominal ultrasound scan revealed hepatomegaly and splenomegaly; nevertheless, the kidney size was normal. Respiratory distress syndrome was observed in one infected neonate; nonetheless, the chest X-ray was normal. The brain CT scan in one newborn revealed mild hydrocephaly with no signs of calcification. When this neonate was treated with ganciclovir, a significant reduction was observed in spleen size; however, no increase was observed in head circumference. In ophthalmologic assessment, retinal hemorrhage without the inflammation of systemic deficits was detected in one neonate. Additionally, chorioretinitis was reported in one infected newborn after 9 months of birth.Hearing Status of Neonates and Audiological Follow-upAll infected neonates completed a follow-up study for 24 months of age and underwent at least one OAE test and five ABR tests. Accordingly, 19out of 22 newborns with cCMV infection initially passed the OAE test as normal; nevertheless, the OAE test was abnormal for three subjects. One of the three newborns with failed OAE tests during initial hearing screening revealed normal response in subsequent assessments by OAE and ABR tests. On the other hand, a child with a normal OAE test at initial hearing screening developed SNHL during the follow-up period at 18 months of age (this neonate was born to a mother with primary CMV infection). Therefore, of 22 newborns undergoing audiological evaluation by the ABR test during 24 months, 3 subjects were demonstrated to have SNHL , 2 of 8 cases with symptomatic cCMV infection  and 1 of 14 cases with asymptomatic cCMV infection . Two of the three neonates had moderate unilateral HL (>45-70 dB), and the remaining neonate had a profound bilateral HL (>90dB). Maternal FactorsThe results of CMV serologic status during the first trimester of pregnancy were available for 17 of 22 mothers with CMV infected neonates, of whom11 subjects were previously infected with CMV and classified as women with non primary CMV infection (based on the presence of CMV immunoglobulin G antibody in their serum samples) and 6 subjects were infected newly with CMV and classified as mothers with primary CMV infection. One of the paramount etiologies of pediatric HL is cCMV infection . AccordiTo the best of our knowledge, this has been the first study to investigate the rate of SNHL among neonates with cCMV infection in Iran. The current study was conducted on newborns with cCMV infection identified through screening newborns within the first 3 weeks of life . The obtained results revealed that the frequency of CMV-related SNHL in this region of Iran is about 13.6% (3 of 22 subjects). This result is consistent with those reported from studies performed in developed countries with moderate to low maternal CMV seroprevalence, in which 9.3-17% of neonates with cCMV infection are reported to have SNHL (The above-mentioned data confirm the findings of this study in which 25% and 7.1% of CMV infected neonates with SNHL were among symptomatic and asymptomatic cases, respectively. Although this rate should be lower in Iran due to high maternal CMV seroprevalence, differences in study enrollment criteria between the present survey and the above-mentioned investigations conducted in developed countries with low CMV seroprevalence probably justify the present study\u2019s findings. It is important to note that 8 of 22 neonates with cCMV infection in the current study were enrolled from newborns admitted to the NICU of the hospitals who generally have more chances to develop HL.The present study\u2019s findings demonstrated that the rate of CMV-induced SNHL in Tehran is about 2 per 22 cases. In addition, based on the previous data from Mashhad and Yazd located in the northeast and center of Iran, the incidence of HL in Iran is reported within the range of3.5-6.5 per 1000 live births, 20. AltIn the present study, 4.5% of neonates who failed in initial hearing screening by the OAE test were infected with cCMV. This finding supports the findings of studies that indicated cCMV infection accounted for about 6% of neonates with HL . Other rIn the current study, 9% and 16.6% of infected newborns with SNHL were born to mothers with non primary and primary CMV infection, respectively. This result is consistent with recently published reports of studies in which 7-10% and 11-15% of infected neonates born to mothers with non primary and primary CMV infection had SNHL, respectively(The data of the present study revealed that no parents in this study had previously heard about CMV or CMV-associated symptoms. Although an efficient vaccine for cCMV infection is not available at present, the risk of CMV infection among populations can be reduced by enhancing CMV awareness level about prevention-based strategies, such as further attention to hygiene practices and routine hand-washing with soap .The current study\u2019s results should be considered in light of some limitations. Firstly, due to the small sample size of 22 infected neonates with cCMV infection, the statistical data appear to be insufficient to properly calculate the SNHL associated risk factors. Secondly, the participants were mainly selected from the infected neonates who were hospitalized or from admitted neonates to NICUs that might affect the outcome analyses. Finally, the study site was located in one region; therefore, the findings might not be generalizable to all regions of Iran. The findings of this study demonstrate that cCMV infection is one of the most important infectious factors inducing SNHL in neonates, and neonates with symptomatic CMV infection are more prone to develop HL. The results also indicate that neonatal screening for cCMV infection at birth and further assessment of infected newborns even with normal hearing status is necessary to identify the late-onset HL in children and start antiviral therapy to prevent disease progression. Considering other serious consequences of cCMV infection in addition to HL, it would be of interest to clarify different effects of this infection on neonates and determine their incidence in this population; therefore, future screening tests can be programmed similar to those implemented for metabolic diseases.Samileh Noorbakhsh: Thesis Supervisor, designed the study, acquired and interpreted the data and critically revised the manuscript; Mohammad Taghi Joghataei: Thesis Supervisor, acquired and interpreted the data and critically revised the manuscript; Mohammad Farhadi: Thesis Supervisor, acquired and interpreted the data and monitored audiological assessments; Faezeh Haghighi: Collected and analyzed the data; Hesamaldin Emamjomeh: Carried out the audiological tests; Morteza Haghighi Hasanabad: Collected and tested the samples, and was involved in all the steps of the experiments, drafted and critically revised the study manuscript. All the authors have read and approved the final manuscript prior to submission.The authors declare that they have no conflict of interest."}
+{"text": "This study demonstrates an accurate method for determining the moisture content in flavor microcapsules using headspace gas chromatography. The method involves measuring the gas chromatography signals of water from vapor in a headspace vial containing flavor microcapsules at a temperature of 125 \u00b0C. The measurements were recorded over four headspace extractions, from which the moisture content in the microcapsule samples was extrapolated via simple vapor-phase calibration. The results revealed that the proposed method demonstrated good precision (a relative standard deviation of <3.11%) and accuracy. The proposed method is accurate, highly sensitive, automated, and suitable for testing the moisture content of flavor microcapsules and related products. Microcapsules, with sizes ranging between 1 and 1000 \u03bcm, are composed of a core  and shell  . The phy2 or I2 in CH3OH and pyridine or imidazole buffer medium) and water in the sample. This method demonstrates better reproducibility and is sensitive to the moisture content in the sample compared to the oven-drying method. However, in addition to the requirements of a complex titrator, expensive reagents, and time-consuming procedures complicating this method, certain compounds containing carbonyl groups (commonly present in flavorings) in the matrices can react with the Karl Fisher reagent, affecting measurement accuracy [Conventionally, the water content in flavor microcapsules is determined using the direct oven-drying method , in whicaccuracy . The lowaccuracy . In thisaccuracy . Herein,Headspace gas chromatography (HS-GC), which is different from the conventional GC technique, is an effective tool to detect volatile analytes in solid or liquid complex matrices ,21. WhenThe objective of this study was to develop an accurate method for the determination of moisture content in flavor microcapsule samples using MHE-GC. The major focus areas involved the establishment of a methodology and determination of the effects of GC conditions, equilibration time, temperature, sample size, and extraction number on the measurement of moisture content. To evaluate the accuracy of this method, microcapsule samples with different flavors were analyzed using MHE-GC, and the results were compared with those obtained using reference methods.Flavor microcapsule samples were collected from EnDian Science and Technology Development of Yunnan Co., Ltd. . The polymer shell materials were prepared using carboxymethyl chitosan and sodium alginate. The core materials were composed of flavor components and octyl and decyl glycerates. Distilled water was obtained from a laboratory device. All flavor microcapsule samples were stored in sealed bags prior to the analysis.The HS-GC measurements were performed using an automated headspace sampler  and GC system  equipped with a TCD and capillary column , J&W Scientific, Folsom, CA, USA). The volumes of the headspace vials and sample loop were 21.6 and 3 mL, respectively. The MHE-GC measurement conditions were as follows: the temperatures of the oven, sample loop, and transportation line were 125, 130, and 135 \u00b0C, respectively; both the injector and detector temperatures were 250 \u00b0C; the GC injection mode was split, and the split ratio was 10:1; the carrier gas was nitrogen with a flow rate of 25 mL/min; the vial pressurization time was 0.2 min; the loop equilibration time was 0.05 min, and the sample-loop time was 0.2 min; the carrier gas pressure and pressurization were 1.5 and 2.00 bar, respectively; the headspace vial was strongly shaken during equilibration.Approximately 0.20 g of the microcapsule sample was added to a dried and empty headspace vial, as presented in R) was calculated using the following Equation (1):m0 denotes the weight of the microcapsule sample added to the dish, and m1 denotes the weight of the sample after reaching a constant weight.The water content in the microcapsule sample was determined using the low-temperature dry nitrogen purge method. The procedure was as follows: the temperature and relative humidity in the chamber were (40 \u00b1 0.1) \u00b0C and below 1.0%, respectively. Approximately 5 g of the sample was added to a dish, and the water in the sample was eliminated by injecting heated absolute dry nitrogen. The weight was recorded every hour until the ratio of the mass change to the initial sample mass was less than 0.04%. The water content in the microcapsules (R) was calculated using the following Equation (2):m0, m1, and m2 denote the weights of the microcapsule sample, dish after reaching a constant weight, and the total weight of the dish and microcapsule sample after reaching a constant weight, respectively.The moisture content of the microcapsule samples was also determined using the oven-drying method. In this method, the test was conducted as follows: approximately 5 g of the sample was added to a dish and placed in a drying oven at 105 \u00b0C for approximately 4 h. The dish was then placed in a dryer and allowed to cool to room temperature. This procedure was repeated until a constant weight was obtained. The moisture content in the microcapsules , water co-existed in the solid and gas phases. Using the MHE-GC technique, consecutive analyses were conducted on the same sample vial, from which a part of the water in the gas phase could be extracted. As illustrated in nA and n can be described as Equation (3):nA and A0 denote the GC signals at n and 0 extractions, respectively, and b denotes the slope obtained from the linear fit. Therefore, the integrated GC signal (tA) can be expressed as Equation (4):tA denotes the sum of the peak areas, and A1 indicates the GC signal of the first extraction.According to MHE-GC theory , the reltm) is linearly proportional to tA in Equation (5):K can be obtained using the calibration method.The total mass of water in the gas phase  and (5), the tracer water content in the microcapsule sample can be calculated as Equation (6):Effective GC separation for water and other volatile substances is required to determine the water content of the microcapsule samples. The MHE is based on the gradual removal of water from microcapsules. Therefore, the equilibrium temperature should be higher than the boiling point of water (>100 \u00b0C). However, if an extremely high equilibration temperature is applied during the test, it may lead to a high pressure in the sealed headspace vial and increase the risk of leakage ,29. TherBecause the present method is based on the equilibrium of water partitioning between the vapor and solid phases, the time required for equilibration before each headspace extraction must be determined. A1 is crucial for calculating the water content in the microcapsule samples. A1 value was lower than the actual value, resulting in an error in the test results. This outcome is because the total gas pressure in the headspace bottle is very high, resulting in pressure effects [In MHE-GC measurements, a large sample weight is helpful for improving the sensitivity of the method . However effects ,31. TherAs mentioned in The HS-GC method was calibrated using a vapor phase calibration technique . CalibraA = a(\u0394a) + s(\u0394s) \u00d7 m, where A represents the integrated GC peak areas of water in the MHE-GC measurements, and m denotes the mass (mg) of distilled water added to the headspace vial. s, a, and \u0394a represent the slope, intercept, and uncertainty, respectively, of the intercept of Equation (7).In other words, LOQ) of the method was 0.0461 mg, which was calculated using Equation (8) [LOQ was approximately 0.0231%. Clearly, the MHE-GC method is highly sensitive and meets the requirements for moisture content testing in the production of flavor microcapsules and related products.The limit of quantitation (tion (8) . If 0.2 The reproducibility of the MHE-GC method was investigated by testing three different microcapsule samples in triplicates. As presented in To verify the performance of the present MHE-GC method, seven different particle sizes and types of flavor microcapsules were analyzed using the traditional oven-drying method , low-temAn accurate method for determining the moisture content of flavor microcapsule samples was introduced based on the multiple headspace extraction gas chromatography technique. Compared with the traditional oven drying method, the significant advantage of the MHE-GC method is that it is extremely accurate, as it can effectively eliminate the interference caused by the loss of volatile organic compounds in the sample at high temperatures. The results revealed that the proposed method has good precision (a relative standard deviation of <3.11%) and accuracy. The present method is simple and automated and can serve as a reliable tool for testing the moisture content of flavor microcapsule samples and related products."}
+{"text": "With the access-to-medicines conundrum facing its populations, the East African Community has adopted a policy framework which promotes a collective approach to resolving the access gap in the region. To this end, crucial policy documents on the implementation of TRIPS obligations, harmonisation of drug regulation and boosting regional manufacturing capacity have been adopted. This paper is a case study of the regional policy on the implementation of TRIPS obligations, specifically examining the extent to which partner states\u2019 implementation of TRIPS obligations mirrors the regional recommendations. The paper finds that, while many partner states follow the regional recommendations on implementing TRIPS obligations, coherence remains a big challenge. This, the paper concludes, may affect the overall usefulness of a regional approach to solving the access conundrum. The COVID-19 pandemic\u00a0has laid bare the inefficiency of the existing arrangement for meeting access-to-medicines needs in low- and middle-income countries (LMICs) \u2013 substantially relying on goodwill donations from high-income countries.Bolar exception, test data protection, disclosure requirement, opposition procedure, parallel importation, and compulsory licence. Cumulatively, these recommendations are aimed at expanding available policy spaces within the region with the expected long-term benefit being the enhancement of regional pharmaceutical production capacity. The gravamina of these recommendations have been extensively critiqued elsewhere.10The EAC has seven partner states overall, five of which  are long-time members, while two (South Sudan \u2013 September 2016 and Democratic Republic of Congo \u2013 July 2022) have recently joined. Similarly, all partner states (except South Sudan)sinequanon for the success of any regional approach. As already mentioned, both South Sudan and the Democratic Republic of Congo have\u00a0only recently joined the EAC; hence, their exclusion. To achieve its aim, this paper employs both doctrinal and interviewing research methods \u2013 the former in analysing relevant primary and secondary source materials; the latter in bridging the gap between theoretical policy statements and actual practices. The interview data used were part of a bigger pool of interview data collected by the author in 2018 as part of his PhD fieldwork.12This paper aims at examining the extent to which actual implementation in leading EAC partner states  reflects the regional recommendations expressed in the policy and the Protocol. This exercise is particularly germane since a harmonised approach to implementation is a It should be noted that while the analysis in this paper relies on the intellectual property laws of five partner states, interviews were only conducted in four  \u2013 Burundi was left out because it was engulfed in political upheaval at the time of interview. Similarly noteworthy is that, even though five partner states are considered, six IP laws are evaluated. This is occasioned by the peculiar constitutional arrangement in Tanzania, where IP law is not a Union  matter, thereby resulting in both Tanzania-Mainland and Tanzania-Zanzibar having separate IP laws.13Following from the foregoing background, Section\u00a0The EAC policy/protocol on TRIPS flexibilities principally aims at charting a coherent access-to-medicines-friendly interpretative course for partner states to adapt and adopt while incorporating TRIPS obligations. This section assesses the extent to which partner states reflect regional recommendations in their implementation. Each of the ten flexible obligations under the EAC policy/protocol is analysed for this purpose.Transition period means the time allowance available to different categories of WTO members  to implement TRIPS obligations domestically.Reference in the above recommendations to \u201clater extensions\u201d is very crucial as it ensures that, like other WTO LDCs, the IP laws of EAC LDCs are well-positioned to take advantage of any subsequent extensions that may be approved by the Council for TRIPS.16So, what does the incorporation of the transition period look like among partner states? An overview of the national responses indicates that no partner state completely complies with these recommendations: Starting with Uganda and Zanzibar, patent protection for pharmaceutical products is excluded until 1 January 2016 or any other date as may subsequently be applicable \u2013 both in line with EAC recommendations.19The responses of Rwanda and Burundi are similar: both, in partial compliance with the EAC recommendation, exclude patent protection for pharmaceutical products.On other recommendations, since process patent protection is not expressly excluded, it is impliedly available for pharmaceuticals in Rwanda and Burundi; in the same way, non-implementation of the mailbox system is a clear indication of the non-availability of the system in both partner states. Tanzania-Mainland, the last partner state, complies with the EAC recommendation on mailbox applications, although it ignores others by making patent protection available for pharmaceutical products and processes.Partner states\u2019 responses are tabulated below.Table 24Finally, it is pertinent that partner states adopt, as much as possible, a harmonised approach to implementing this flexibility so that technological development can occur across the region at a relatively equal pace. According to TRIPS, a transitional period is conceded to WTO LDC members to assist them overcome \u201ceconomic, financial and administrative constraints\u201d, and create a \u201c\u2026 viable technological base.\u201dinteralia three omnibus criteria of novelty, inventive step, and industrial application.Under TRIPS, whether an invention is patentable is assessed using The flexibility that patentability requirements provide could be used by all partner states, notwithstanding the fact that most EAC LDCs do not offer patent protection for pharmaceutical products. This is more so since most EAC LDCs offer protection for pharmaceutical processes; hence, they can construe patentability criteria strictly to exclude processes which are not deserving of protection. Additionally, extending this flexibility to EAC LDCs will serve a futuristic purpose since the transition period which currently exempts patent protection for pharmaceuticals is time-bound and may cease anytime. Table In implementing the patentability criteria, partner states have largely followed the regional recommendations. As such, all partner states construe \u201cnovelty\u201d strictly by testing claimed inventions against worldwide prior arts;One missing implementation from Table GuidelinestoPatenting document and, according to these Guidelines, a person skilled in the art \u201cshould be presumed to be an ordinary practitioner aware of what was common general knowledge in the art at the relevant date\u00a0\u2026 .\u201dIt is equally interesting that most partner states prefer to assess \u201cinventive step\u201d using the \u201cordinarily skilled\u201d standard as opposed to the regionally recommended \u201chighly skilled\u201d standard. Apart from the regional approach failing to provide any guideline on what capability is to be ascribed to this nominal person, assessing \u201cinventive step\u201d using such a standard is unrealistic.The use of this obligation as a national policy tool derives from TRIPS\u2019s non-mandatory provisions on excludable subject-matter.In response to the category one recommendation, partner states have excluded mostly identical subject-matter, the majority of which originates from TRIPS Art. 27(2) and (3).46However, viewed from the perspective of a region mostly comprised of LDCs, it is possible to argue that this approach is TRIPS-consistent, especially given that EAC LDCs are entitled to the pharmaceutical exemption, which allows them to exclude patents for pharmaceuticals until January 2033.Partner states have implemented the category two recommendations as follows: Uganda, Rwanda, Zanzibar and Burundi exclude natural substances in the exact manner recommended by the EAC;Although other partner states implement the exclusion for new uses and derivatives of known substances, they do not draw a distinction between the two. Thus, Rwanda excludes \u201cknown substances for which a new use has been discovered\u201d, but permits patentability if the resulting new use satisfies a strict patentability test.Table any molecule or other substances useful for treating or preventing any disease designated by the Minister as a serious health hazard or as being life threatening.55Another issue worthy of comment from Table Overall, there is a need to tidy up these conflicting provisions, as failing to do so may frustrate the purpose intended to be served by the flexibility.Like other flexibilities, the research exception \u2013 a narrowed TRIPS-sanctioned permission to use a patented invention for research purposes without the patentee\u2019s authorisationon patented articles for non-commercial purposes,on patented articles for commercial purposes, but they both disregard the balancing requirement that such use must be for generating new knowledge.As usually the case, partner states have responded differently. In compliance with the regional advice, all partner states exempt research activities carried out Few observations could be made on partner states\u2019 implementation choices. In failing to exempt commercial research, partner states seem to favour the US approach where any inkling of a commercial link defeats the research exception claim.Secondly, the approach adopted in Uganda and Zanzibar is equally flawed. Both partner states provide a blanket exemption for commercial research, regardless of whether it is intended to advance knowledge or not. In addition to encouraging free riding, this implementation will disadvantage patentees of research tools by exposing them to infringement without compensation. By their nature, research tools are mainly used complementarily in conducting other research, including commercial experimental research.Bolar exception applies narrowly to uses reasonably related to obtaining marketing approval for generic or originator pharmaceutical products or, sometimes, products generally.Unlike the research exception, which any willing WTO member may broadly implement to exempt both infringing commercial and non-commercial research \u201con\u201d patented articles, the Bolar exception as regionally recommended, i.e. by exempting otherwise infringing uses of patented pharmaceutical products prior to patent term expiry provided the purpose of such use is reasonably related to generating dossiers for obtaining marketing authorisation for new or generic products under domestic or foreign law. A perusal of national responses shows a contrasting approach. All EAC LDCs (except Tanzania-Mainland), on the one hand, implement the Bolar exception in these partner states may not have a negative impact for access since pharmaceutical production capacity in the region is largely focused on basic generic formulations anyway. This notwithstanding, a harmonised approach to implementing this exception could benefit the region in the nearest future when manufacturing capacity eventually peaks , the IP laws in both countries postpone the commencement of all the above provisions to a later date. However, while the provision adopted in Zanzibar successfully achieves this purpose, the same cannot be said for Burundi. For instance, in Zanzibar, these provisions will only enter into force after expiry of the country\u2019s transition period (currently January 2033 or any subsequent extensions approved by the TRIPS Council).77Pondering over the convoluted regime favoured in Zanzibar and Burundi, one cannot but wonder what two EAC LDCs, expressly exempted from test data obligations, stand to gain by enacting comprehensive and confusing provisions on test data.80Kenya, Tanzania-Mainland and Rwanda are in group two \u2013 the trio have no provision on test data protection. The absence of a legal provision in Kenya notwithstanding, an officer of the Poison and Pharmacy Board (PPB) (Kenyan drug regulatory body) told the author that the PPB often relies on dossiers submitted by applicants and that whether separate test data submission would be required would depend on the historical origin of the medical products involved.84In the last group is Uganda, which protects test data against unfair commercial use and disclosure using a trade secret law.86In sum, while these varied approaches \u2013 especially in Zanzibar and Burundi \u2013 should generate concerns, the reality is that test data provisions, including the pro-patent owners\u2019 approach embraced in two EAC LDCs, are not likely to have any practical effect in the region for some time. The reason for this is not far-fetched: the leading pharmaceutical firms and importers in the region deal almost entirely in generic products, which do not require the submission of new test data, as already indicated. As a matter of fact, neither Zanzibar nor Burundi has a vibrant pharmaceutical industry, and they both rely heavily on importation within and outside the region.quidproquo arrangement under which, in return for a promise of limited monopoly rights, patent applicants undertake to fully and sufficiently disclose to the public how to work their inventions.The entire patent system is based on a The regional recommendations on this requirement having failed to stress the importance of a \u201csufficiently clear and complete\u201d disclosure, most partner states fill this lacuna (using different wordings with similar effect) in their implementation. This is in addition to requiring the disclosure of the \u201cbest mode\u201d as the EAC recommends. Both Tanzania-Mainland and Zanzibar require disclosure \u201cin a manner sufficiently clear and complete\u201d; patent applicants must also \u201cindicate the best mode for carrying out the invention\u201d.95In Rwanda, the preferred language is disclosure in a \u201cmanner sufficiently clear, complete and intelligible\u201d, and an additional requirement is that an applicant indicates \u201cthe best way\u201d of using the invention.One identified shortcoming of the EAC approach is its failure to provide a harmonised guide for partner states on implementing the core principle of the disclosure requirement, namely, the need for disclosure to be made in a sufficient and clear manner.Opposition procedure is another optional TRIPS obligationHere is what implementation among partner states looks like: three partner states which have existing obligations to offer patent protection either for both pharmaceutical products and processes (Kenya and Tanzania-Mainland) or for pharmaceutical processes only (Rwanda), fail to provide for either a pre-grant or post-grant opposition procedure. These partner states only provide for the revocation or invalidation of patents through the conventional court system.106As can be seen from Table 111exhausted\u201d and subsequent non-authorised sales by others stop being infringing \u2013 provided of course that the original sale is by the patentee or their authorised agent.TRIPS Art. 6 makes it crystal clear that WTO members are in absolute control vis-\u00e0-vis choice of exhaustion regime. Exhaustion (of patent rights) defines the point when a patentee\u2019s right of first sale becomes \u201cunconditionally.Three implementation groups could be identified from partner states\u2019 patent laws. In the first group are Kenya, Uganda and Zanzibar, all of whom follow the EAC advice by exempting \u201cacts in respect of articles which have been put on the market in Kenya [Uganda or Zanzibar] or in any other country or imported into Kenya [Uganda or Zanzibar].\u201dsuomotu or at the request of an interested party.119Burundi is in the second group: \u201cacts relating to goods placed on sale in Burundi or in any other country by the patent holder or with his consent\u00a0\u2026\u201d are exempted from infringement.Rwanda and Tanzania-Mainland are in group three. Both apply a national exhaustion regime which generally precludes parallel importation.A few points can be deduced from the data in Table The above responses may be contrasted with those of Kenya, Zanzibar and Uganda, where no limit is imposed on the use of parallel importation. It is doubtful that these countries can derive any practical benefit from an unregulated use of parallel importation for reasons canvassed elsewhere \u2013 a regional exhaustion regime with adequate regulatory framework to prevent re-exportation appears to offer a better solution.125bis) enumerates the conditions with which WTO members must comply to use a compulsory licence either for local production or import/export.bis, to issue a compulsory licence which a recipient local pharmaceutical firm can use to import essential medicines from an overseas pharmaceutical firm, the latter firm having also been issued with a compulsory licence by its home government \u2013 this has been tagged compulsory-licence-for-export.TRIPS Art. 31  between patentees and the person seeking a compulsory licence must have failed before the latter can bring an application for a compulsory licence. Comparing partner states\u2019 responses  where the original patent term is 20 years from filing date.It is quite difficult to fathom what the rationale is for incorporating TRIPS-plus obligations by partner states in a region which seeks to optimise the use of TRIPS flexibilities in strengthening pharmaceutical manufacturing capacity and improving access to medicines. Supposedly, partner states must have known that adopting these excessive, patentee-friendly obligations will complicate the achievement of the regional objective. Take patent linkage for example: not only will it empower a patentee to challenge a drug\u00a0registration application on the ground that the products covered by the application infringe patent rights, but also such intervention puts the consideration of the application on hold until the patent challenge has been resolved. Without a linkage system, the application for marketing approval and a challenge to patent rights are treated as two distinct events, so that the occurrence of one does not automatically affect the other. Patent term adjustment also constitutes a clog in the wheel of progress towards the regional goal since its enactment adds an extra layer of monopoly to patentees, thereby potentially delaying the early entry of generic drugs into the regional markets.163Given the relatively small population of each EAC partner state,Focusing on ten TRIPS obligations, the paper found that not only have partner states failed to implement all the regional recommendations, but also there is a lack of coherence in the implementation of some crucial obligations. Specific examples discussed above include Tanzania-Mainland, which offers patent protection for pharmaceutical products when it could have deployed the transition period flexibility to exclude such protection; Burundi and Zanzibar, which both enact a suspended data exclusivity regime, thereby adding another layer of exclusivity to patentees\u2019 rights; and Kenya, Tanzania-Mainland and Rwanda, which have failed to legislate any opposition procedure. These dark spots of incoherent implementation could potentially have a defeating effect on the overall regional goal of enhancing pharmaceutical manufacturing capacity and ultimately boosting access to essential medicines. Equally defeating, according to the paper\u2019s finding, is the implementation of two TRIPS-plus obligations \u2013 patent linkage in Kenya and Uganda and patent term adjustment in Zanzibar and Burundi. Rather than facilitating the early entry of generic pharmaceutical products into the EAC market as envisioned under the regional policy, these obligations will delay such entry and by implication deprive EAC populations of the opportunity for timely access to essential pharmaceutical products.All hope is, however, not lost! The above analysis also revealed that, in addition to many regional recommendations which are implemented to the letter by partner states, some of the non-complying national implementation options are more suited to achieving the regional goal of improved access than the original regional recommendations. One example is the favoured implementation approach on the disclosure requirement: while the regional recommendation omitted the need for disclosure to be sufficiently clear and complete, the legislation in partner states fill in this salient requirement. Many EAC LDCs have also disregarded the regional recommendation against offering patent protection for pharmaceutical processes \u2013 an approach that the paper argued could help improve the research skill of local innovators. Yet another instance is the preferred definition of a person skilled in the art among partner states. Contrary to the regional recommendation (highly skilled), many partner states define this person as someone of ordinary skill. Lastly, some partner states\u2019 approach to the exhaustion regime deviates from the regional recommendation by opting for national  exhaustion, with the possibility of using parallel importation in exceptional circumstances. As contended in the paper, this is more aligned to the regional goal of enhancing pharmaceutical manufacturing capacity because it ensures that parallel importation will only be used sparingly.Going forward, for this and other regional policies on improving access to medicines to be effective, partner states must show more commitment in implementing obligations willingly assumed when acting as part of the region. This is of utmost importance in view of the centrality of coherent implementation to the success of the bigger regional plan, since the EAC framework on TRIPS flexibilities is just a piece in a bigger regional policy puzzle intended to achieve a gradual self-sufficiency in pharmaceutical production. As for the bigger policy puzzle, this is laid out in the EAC Pharmaceutical Manufacturing Plan of Action166"}
+{"text": "N = 10 590 participants which tested for associations between depressive symptoms and 55 biomarkers of inflammation and related processes in participants living with HIV. Formal meta-analyses were not possible as statistical reporting in the field was highly variable; future studies must fully report test statistics and effect size estimates. The majority of included studies were carried out in the United States, with samples that were primarily older and primarily men. Substantial further work is necessary to diversify the geographical, age, and sex distribution of samples in the field. This review finds that alterations in concentrations of certain biomarkers of neuroinflammation  may influence the association between HIV and depression. Equally, the chemokines monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) or the metabolic index kynurenine:tryptophan (Kyn:Trp), which have been the focus of several studies, do not appear to be associated with depressive symptoms amongst people living with HIV, as all (MCP-1) or most (IL-8 and Kyn:Trp) available studies of these biomarkers reported non-significant associations. We propose a biomarker-driven hypothesis of the neuroimmunometabolic mechanisms that may precipitate the increased risk of depression among people with HIV. Chronically activated microglia, which trigger key neuroinflammatory cascades shown to be upregulated in people with HIV, may be the central link connecting HIV infection in the central nervous system with depressive symptoms. Findings from this review may inform research design in future studies of HIV-associated depression and enable concerted efforts towards biomarker discovery.People with HIV are at increased risk for depression, though the neurobiological mechanisms underlying this are unclear. In the last decade, there has been a substantial rise in interest in the contribution of (neuro)inflammation to depression, coupled with rapid advancements in the resolution and sensitivity of biomarker assays such as Luminex, single molecular array and newly developed positron emission tomography radioligands. Numerous pre-clinical and clinical studies have recently leveraged these next-generation immunoassays to identify biomarkers that may be associated with HIV and depression (separately), though few studies have explored these biomarkers in co-occurring HIV and depression. Using a systematic search, we detected 33 publications involving a cumulative  Inflammation may be a central link explaining the high risk for depression among people living with HIV. In a scoping review, Mudra Rakshasa\u2013Loots highlights biomarkers of inflammation and related processes that are associated with depressive symptoms in people living with HIV and offers suggestions for future directions of inquiry.  In addition, needing regular medical check-ups, adhering to drug regimens (with varied side-effects), and managing co-morbid conditions represent heavy personal and economic burdens for many people with HIV. Together with widespread stigma and marginalization, these factors contribute to the high risk for depression amongst people with HIV, especially for women with HIV and those in the Global South.5 However, as the brain is a key reservoir of HIV infection, it is likely that neurobiological processes also contribute to this high risk for depression. Identifying the precise mechanisms by which HIV leads to depression is crucial as it may enable us to identify individuals at greatest risk for developing depression, tailor the HIV care continuum, and deliver effective pharmacological or psychosocial interventions.People with HIV experience depression at a rate at least twice as high as the general population.6 In this review, the authors hypothesized that HIV infection interacts with neurobiological and psychosocial factors to give rise to depression. Specifically, HIV infection in the brain results in sustained microglial activation, elevations in circulating cytokines, depletion of monoamines, and neurotoxicity . Cerebral elevations in inflammatory neurometabolites and white matter abnormalities arising due to HIV infection persist despite viral suppression.7 These neurobiological consequences of central nervous system (CNS) infection may result in specific components of depression, such as cognitive loss , chronic fatigue (TNF-\u03b1 elevations), and sickness behaviour (cytokine storms). HIV infection in the CNS may elicit a chronic neuroinflammatory response. Neuroinflammation is, in turn, associated with depressive symptoms in the general population. Thus, it is possible that neuroinflammation contributes to the increased risk for depressive symptoms among people living with HIV.8The last substantial review of the possible neurobiological mechanisms underlying HIV-associated depression was published by Del Guerra and colleagues in 2013.et al.\u2019s review, there has been continued interest in the contribution of (neuro)inflammation to depression, coupled with rapid advancements in the resolution and sensitivity of biomarker assays. Luminex, single molecular array, and similar next-generation multiplex assays have enabled the quantification of tens of biomarkers from single samples with enhanced sensitivity compared to enzyme-linked immunosorbent assays.10 These technologies have been leveraged in several recent investigations of biomarkers that may influence the association between HIV and depression.In the intervening decade since the publication of Del Guerra To translate these findings into clinical practice, we must identify biomarkers that are reliably associated with depressive symptoms in people living with HIV. Depending on the study design and statistical methods, at least three questions may thus be investigated:predictive biomarkers);Is the onset of depressive symptoms in people living with HIV preceded by elevations in concentrations of certain biomarkers ; andAre depressive symptoms associated with certain biomarkers therapeutic targets).Is the severity of depressive symptoms in people living with HIV mediated by concentrations of certain biomarkers; that is, are these biomarkers at least partly mechanistically involved in the pathogenesis of symptoms and can therefore be targeted by antidepressant interventions , and facilitating the translation of new and improved tools for stratification in the treatment of depression .By answering these questions, we may identify biomarkers that enable early risk stratification, phenotyping, and individualized interventions for depression in people living with HIV. Crucially, the utility of these biomarkers may extend beyond the context of HIV. Many inflammatory insults, including clinical conditions such as rheumatoid arthritis and psychosocial stressors such as early life adversity, result in an increased risk for depression.13 This review aims to address this gap in the literature. Our objective was to synthesize recent evidence on associations between depressive symptoms and biomarkers of neuroinflammation or related biological processes in people living with HIV. Notably, in this scoping exercise, we did not seek to elucidate the mechanisms behind these associations but synthesize the reported associations themselves.The biomarkers tested in existing studies of HIV-associated depression are highly variable, in part due to the diversity and customizability of available biomarker panels. Recent reviews have narratively surveyed the literature on the potential role of inflammatory biomarkers in HIV-associated depression, but no attempt has been made to systematically capture and synergize these findings.https://syrf.org.uk/), we carried out a systematic search aimed at capturing all recent studies of people living with HIV which reported a measure of depression and at least one inflammatory biomarker. PubMed and Web of Science (Core Collection) were searched using detailed search terms, which are available in in vitro studies, and genetic studies were also excluded. The review protocol was not pre-registered. Study screening was carried out by a single reviewer.Using the Systematic Review Facility (14 Data collection was carried out by a single reviewer. We first recorded whether each study reported a significant association between a biomarker of interest and depression or depressive symptoms (+1 score), which was our primary outcome of interest. Notably, we included studies that assessed depression as a clinical diagnosis  and those that measured depressive symptoms using screening tools such as the Beck Depression Inventory. Diagnostic or screening tools used in each included study are listed in In order to assess the clinical utility and strength of evidence for each inflammatory biomarker, available evidence from each included study was scored against the validation criteria in without HIV, may thus represent an HIV-specific risk factor for depressive symptoms and thus at least partly explain the greater prevalence of depression in this population.For studies reporting a significant association, we recorded the direction of association (positive/negative) and whether this association was significant only amongst participants living with HIV (+1 score). An HIV status-dependent association would be important for a truly useful predictive biomarker of HIV-associated depression. Although neuroinflammation is linked to depressive symptoms in the general population as well, it may be possible that certain inflammatory processes that are heightened during HIV infection  may exacerbate the risk for depressive symptoms amongst people living with HIV. Biomarkers which are found to be associated with depressive symptoms in people living with HIV, but not in those Finally, for studies that reported a significant association only amongst people living with HIV, we recorded whether there was a reported correlation between the biomarker concentration and a continuous scale of depressive symptom severity (+1 score). Correlations with depressive symptom severity may suggest a dose response and thus, indicate greater utility and sensitivity of the biomarker in community-based samples.N), number of participants with HIV, mean [standard deviation (SD)] age of participants, % men in studies, recruitment country, % participants on ART, and % participants reported as virally suppressed. Where variables were reported as medians and interquartile ranges (IQR), means and SDs were imputed using equations (14) and (17) in Wan et al.15 Where variables were reported only for subgroups, mean (SD) for the full sample was imputed using equations in Table 6.5.a of the Cochrane Handbook.16 For biomarkers that were assessed in at least five publications, we extracted the type of screening or diagnostic tool used to measure depression or depressive symptoms, type of test statistic, and value of the effect size estimate. Data collection was carried out by a single reviewer.For all studies, we recorded all biomarkers assessed (including those which were reported only as part of a cluster of biomarkers), biofluids in which biomarkers were assessed, total number of participants  recruited participants from the US. Five studies  involved participants who were not virally suppressed, whereas six studies did not report viral suppression status for their samples. At least a proportion of participants in all remaining studies were virally suppressed; median (IQR) % of virally suppressed participants overall was 79% . Active HIV replication results in elevated inflammation, which may in turn worsen depressive symptoms.50 However, given that many participants in the included studies were on ART and virally suppressed, inferences about the impact of untreated HIV infection on inflammation and depression cannot be made.Of 165 records identified from the two databases, three records identified from manual citation searching, and one record identified based on reviewer recommendation, a total of 33 studies (representing 28 unique samples and a cumulative 10 590 participants) were eligible for inclusion in this review .17\u201349 Ke51 which suggests that care must be taken to choose a biofluid that is appropriate for the study design and with consideration for acceptability and scalability. Seven studies measured a single biomarker;52 all other included studies measured more than one biomarker. Three studies only reported associations involving a cluster or composite score of multiple biomarkers, such as effects for individual biomarkers could not be parsed out.40 Included biomarkers were broadly categorized based on their primary putative function in inflammation (or related processes) as follows: neuroinflammation, chemotaxis, systemic inflammation, monocyte activation, immunometabolism, coagulation, and neurogenesis. Note that biomarkers may be involved in multiple processes; for instance, many biomarkers that were categorized as neuroinflammatory in this review likely also play a role in systemic inflammation.Collectively, the included studies tested associations of depression or depressive symptoms with 55 biomarkers of inflammation and related processes, of which IL-6, TNF-\u03b1, C-reactive protein (CRP) and MCP-1/CCL2 were assessed most frequently . Biomark33 another as a composite of \u2018depression or PTSD\u2019,52 and another as an interaction of biomarkers with depression and neurocognition.The majority of included studies used a screening tool to measure depressive symptoms, rather than a diagnostic interview to establish a clinical diagnosis of depression . Three sn = 6, 5.2%) or scaled intensity  which could not reliably be converted to a meta-analysable statistic. Therefore, at least 41.7% of reported associations were not meta-analysable. Given these issues, an assessment of publication bias or a meta-analysis of the strength of the associations between biomarkers and depressive symptoms was not possible.Formal meta-analyses were not possible in this review for a number of reasons. First, there was substantial heterogeneity in the scales used to measure depressive symptoms and the biofluids in which biomarkers were measured. Of 55 included biomarkers, 30 (54.5%) were assessed in only one publication, and a further 9 (16.4%) were assessed in two publications. We extracted effect size estimates for biomarkers which were assessed in at least five publications . Reporti18 some adjusted for confounding variables statistically,21 while others combined both approaches.44 Where available, we extracted data for associations from statistical models adjusted for relevant covariates.53 Four included studies reported an active hepatitis C co-infection in at least a proportion of their participant samples,49 whereas one study specifically excluded participants with co-infection during recruitment.36 Active co-infections were not reported in the remaining studies. Several studies also reported psychosocial factors that may influence inflammation. In particular, a number of included studies focused on participants living with HIV who had experienced sexual violence in their lifetime.46 Jones et al.29 specifically attempted to assess depression as one component of \u2018syndemic burden\u2019, which was defined as incidence of depression, cigarette use, low education, or obesity. They found that syndemic burden was associated with increased viral load, but not inflammatory biomarkers, in women living with HIV. Overall, only a proportion of included studies reported measurement of and statistical adjustment for potential clinical or psychosocial confounders.Included studies adopted varied approaches to accounting for confounding demographic or clinical variables. Some studies included demographically and clinically comparable controls,Proportion of studies reporting a significant association and direction of those associations are summarized in Cytokines are biomolecules produced by immune cells to activate, promote, or regulate the (neuro)inflammatory response. Concentrations of these biomarkers can be measured in biofluids including CSF, blood plasma or serum, saliva, or cervico-vaginal lavage. Included studies that assessed the relationship between neuroinflammatory cytokines and depressive symptoms are summarized in 54\u201361 A recently published meta-analysis involving a cumulative 3075 participants also indicated that blood neopterin (regardless of type of blood sample) is significantly higher in participants with depression.62 The current review detected 17 publications that tested the relationship between IL-6 and depression or depressive symptoms, of which seven reported a significant positive association such that increased IL-6 was associated with greater risk for depression or depressive symptoms.39 Similarly, a proportion of studies exploring the relationship between TNF-\u03b148 or neopterin46 and depressive symptoms observed significant positive associations. None of the studies reporting a significant association for IL-6 or TNF-\u03b1 included participants without HIV. Saloner et al.42 observed a correlation between neopterin and depressive symptoms, which was significant only amongst people living with HIV, indicating that neopterin may be a valuable biomarker for HIV-associated depression. Together, these findings suggest that increased IL-6, TNF-\u03b1 and neopterin may be associated with increased depression, including in people living with HIV. However, further studies that include appropriate controls are necessary to determine whether these cytokines  mediate the risk for depressive symptoms in a manner explicitly dependent on HIV status: that is, whether these associations are more pronounced in people living with HIV compared to those without HIV.The most frequently assessed neuroinflammatory cytokines in included studies were interleukin-6 (IL-6), tumour necrosis factor alpha (TNF-\u03b1), neopterin, interleukin-1 beta (IL-1\u03b2) and soluble tumor necrosis factor receptor-II (sTNFR-II). Numerous studies have found an association between circulating IL-6 or TNF-\u03b1 concentrations and depressive symptoms in the general population, albeit with varied strengths.33 though Lu et al. only reported this association for sTNFR-II as part of a cluster of biomarkers [\u2018exploratory factor analysis-identified inflammatory processes\u2019 (EIP)]. Similarly, Lu et al.31 also reported a significant association with depressive symptoms for soluble interleukin-2 receptor alpha (sIL2R\u03b1), soluble interleukin-6 receptor (sIL6R) and soluble glycoprotein 130 (sGP130) but only as part of an EIP. Thus, no inferences can yet be drawn regarding the contributions of these individual biomarkers to HIV-associated depression.In line with findings related to TNF-\u03b1, two studies reported a significant association between increased serum concentrations of sTNFR-II and greater depressive symptoms,49 No significant associations with depression or depressive symptoms were observed in the included studies for the cytokines interleukin-1 alpha (IL-1\u03b1), soluble tumor necrosis factor receptor I (sTNFR-I), transforming growth factor beta (TGF-\u03b2), cathepsinB, interferon alpha (IFN-\u03b1), interleukin 10 or 12 , matrix metallopeptidase 9 (MMP9) or soluble interleukin-1 receptor II.Other neuroinflammatory cytokines for which a significant association with depressive symptoms was reported were interferon gamma  and interleukin-12 (IL-12), interleukin-15 (IL-15) and interleukin-18 (IL-18), though these studies did not include participants without HIV and the findings from the overlapping sample in particular warrant further replication in independent samples.Chemokines are biomolecules primarily involved in the recruitment of immune cells to locations of immunological challenges. Expression of these biomarkers is often regulated by neuroinflammatory cytokines such as TNF-\u03b1 and IL-1\u03b2. Studies that assessed relationships between chemokines and depression in people living with HIV are summarized in et al.63 found that animals who exhibited depression-like behaviour had increased concentration of MCP-1 in the hippocampus, but treatment with meloxicam did not rescue depression-like behaviour in these animals, despite successfully inhibiting Mcp-1 gene expression. Therefore, although MCP-1 has been implicated in the risk for depression in the general population,65 the evidence suggests that this chemokine may not play a significant role in depression among people living with HIV specifically.Monocyte chemoattractant protein-1 (MCP-1/CCL2) was the most frequently assessed chemokine in included studies. Of 11 studies, none reported a significant association between MCP-1 concentration and depression or depressive symptoms among people living with HIV. These findings are compatible with pre-clinical studies using a transgenic rat model of HIV, where Nemeth et al.18 reported that increased plasma IP-10 was linked to depressive symptoms amongst people living with HIV but not controls. Three other studies also observed a significant positive association between IP-10 and depressive symptoms, but findings were limited only to men,39 in a small sample (n = 23),49 or reported only as part of a cluster of biomarkers.66 One study found that IL-8 concentrations were significantly higher amongst people with HIV and remitted depression compared to people with HIV and no depression,39 though several other studies with larger sample sizes did not find a significant association for IL-8. No notable associations were reported between depression or depressive symptoms in people living with HIV and the chemokines monokine induced by gamma interferon (MIG/CXCL9), macrophage inflammatory protein-1 alpha (MIP-1\u03b1/CCL3) macrophage inflammatory protein-3 alpha (MIP-3\u03b1/CCL20), macrophage inflammatory protein-1 beta (MIP-1\u03b2), eotaxin, or monocyte chemoattractant protein-4 (MCP-4/CCL13).Interferon gamma-induced protein 10 (IP-10/CXCL10) and interleukin-8 (IL-8/CXCL8) are closely related chemokines belonging to the C-X-C family. Cassol Evidence for associations between depression or depressive symptoms in people living with HIV and biomarkers of systemic inflammation or monocyte activation are summarized in 67 During an inflammatory response, CRP is induced by inflammatory cytokines such as IL-6, TNF-\u03b1 and IL-1\u03b2, which promote its production in the liver and secretion into plasma.68 CRP is elevated in people with HIV70 and people with depression,54 although the role of CRP in depression is unresolved due to contradictory findings. This review detected 11 studies which assessed CRP concentrations in people living with HIV, of which only four reported a significant association with depression or depressive symptoms.41 Notably, Rubin et al.39 only saw this association in women living with HIV and Saloner et al.41 only reported this association as an interaction between HIV, depressive symptoms, and neurocognition. None of these four studies reported that the association between CRP and depression was only significant for participants with HIV and not for those without HIV. Together with the existing contradictions in the literature on the role of CRP in depression, these findings suggest that CRP may not reliably influence HIV-associated depression.C-reactive protein is an acute phase reactant which promotes phagocytosis and macrophage proliferation.38 The authors of this study speculate that albumin may be protective against depression, though such a claim would require substantial experimental work to verify. Furthermore, this study did not include participants without HIV and thus no conclusions can be drawn whether this effect may be influenced by HIV status. Other biomarkers of systemic inflammation assessed in the included studies were B cell activating factor , glycoprotein acetylation marker (GlycA), urokinase plasminogen activator surface receptor (uPAR/CD87), and vascular endothelial growth factor (VEGF), though no significant associations were reported for any except BAFF.Serum albumin was assessed in two studies, of which one study reported a negative association with depressive symptoms.71 Included studies assessed four different biomarkers which are released by monocytes when they are activated: soluble cluster of differentiation 14, 163, 27, and 40 Ligand . Of the studies Investigating sCD14, Stewart et al.44 reported a significant correlation between somatic depressive symptoms and serum sCD14 concentrations amongst people living with HIV but not in people without HIV, which offers support for the validity of sCD14 as a biomarker for HIV-associated depression, though the difference in effect size for both groups was small. Similarly, Anderson et al.17 observed that higher plasma sCD163 was associated with increased odds for severe depressive symptoms only amongst people living with HIV. Lu et al.66 reported a significant association for sCD14 and sCD27 only as part of an EIP, whereas other studies captured in this review did not report a significant association between these biomarkers and depression. In all, sCD14 and sCD163 may drive the risk for depressive symptoms in people living with HIV, though further work is necessary to confirm these results.Biomarkers of monocyte activation are expressed on the surfaces of monocytes and released in soluble form when these cells are activated. Neuroinflammatory cytokines such as IL-6 or IL-1\u03b2 are involved in the induction of these markers.72 Ellis et al.73 recently investigated associations between CSF concentrations of GFAP with depressive symptoms in a sample of n = 212 people living with HIV in the US, with the majority (82.1%) of participants being men. They observed a statistically significant association between GFAP concentrations and total BDI-II scores with higher CSF GFAP correlating with higher BDI-II scores, suggesting that astrocytic activation may be associated with depressive symptoms in people living with HIV. These early findings indicate that GFAP may be a useful biomarker for depressive symptoms in this population. Further research into the function of astrocytes in neuroinflammation and depression in the context of HIV is warranted. Notably, this study was published in December 2022, outside the stated date range for our systematic searches, and was, therefore, not included in reported analyses in the rest of this review.Glial fibrillary acidic protein (GFAP) is an intermediate filament protein which is constitutively expressed in astrocytes, and thus, serves as a marker of astrocyte activation and CNS pathologies.Several included studies measured biomarkers of physiological processes that overlap with neuroinflammation. Findings from these studies are summarized in 74 Under certain conditions, such as an inflammatory response, immune cells (including microglia and astrocytes) can instead metabolize tryptophan into kynurenine , kynurenic acid (KynA) and quinolinic acid (Quin), all metabolites which form part of the kynurenine pathway.76 The ratio of kynurenine to tryptophan (Kyn:Trp) is an index of IDO-1 activity, which is triggered by key neuroinflammatory cytokines such as IL-6, TNF-\u03b1, IL-1\u03b2 and IFN-\u03b1.78The kynurenine pathway of tryptophan metabolism sits at the intersection of neuroinflammation and metabolism. Typically, tryptophan (Trp) is catabolized by tryptophan hydroxylase into 5-hydroxytryptophan, the neurotransmitter otherwise known as serotonin and heavily implicated in the pathogenesis of depression.79 HIV viral proteins can activate the kynurenine pathway, in addition to inducing the release of pro-inflammatory cytokines which further promote this activation, leading to a feedforward loop of sustained tryptophan depletion.81Activation of the kynurenine pathway depletes available tryptophan for serotonin production and thus is considered a possible mechanism by which immune responses interact with neurotransmitter dysfunction to give rise to depressive symptoms. The kynurenine pathway has particular clinical relevance to HIV infection, which has been reviewed elsewhere.et al.32 and Vadaq et al.45 both observed that lower plasma tryptophan was significantly associated with greater depressive symptoms, though their samples did not include people without HIV. Finally, although the Kyn:Trp ratio was assessed in several studies, only Martinez et al.32 observed a statistically significant association between higher plasma Kyn:Trp ratio and greater depressive symptoms in people living with HIV, and the effect size of this association was small (+0.01 HSCL-D score for each 10\u2005nM/\u00b5M increase in Kyn:Trp ratio). Conversely, Keegan et al.30 saw a trend towards lower plasma Kyn:Trp weakly associating with depression in aviremic people with HIV (compared to viremic people with HIV or people without HIV), though this difference was not statistically significant after Bonferroni correction. Taken together, these findings suggest that lower plasma Trp may be a useful biomarker of the risk for depression in HIV, but further work is necessary to resolve contradictory findings on the association between Kyn:Trp ratio and depression.Several included studies tested for associations between Kyn or KynA and depressive symptoms, with none reporting a significant association. Five studies investigated associations between Trp in plasma or CSF with depressive symptoms. Of these, Martinez 77 Absolute concentrations of Quin are increased in untreated depression and correlate with depression severity.82 Furthermore, antidepressant behavioural activation therapy leading to reduction in depressive symptoms is significantly associated with decreased serum Quin.83 Crucially, this relationship has also been observed in the specific context of HIV. Drivsholm et al.23 saw that higher Quin:KynA ratio (but not absolute KynA concentrations) and higher absolute concentrations of Quin were both significantly associated with depression in a large sample of people with HIV. Given these findings, Quin may be a reliable biomarker of depression in people living with HIV.Quinolinic acid is considered a neurotoxic metabolite, leading to the hypothesis that increased production of Quin may be a driver of an inflammatory subtype of depression.30 Two publications from an overlapping participant sample34 found that monoamine metabolites (phenylacetate and 4-hydroxyphenylacetate), acylcarnitine metabolites , and neuroactive steroids  were decreased in people living with HIV who experience depression. These findings suggest that these metabolites may be protective against depressive symptoms, though replication of these results in independent samples is warranted. Notably, Cassol et al.18 observed the effect of monoamines and acylcarnitines amongst participants without HIV as well, suggesting that these relationships are not dependent on HIV status.Studies included in this review did not find significant associations of tyrosine (Tyr), phenylalanine (Phe), Phe:Tyr ratio, or Phe:Trp ratio with depressive symptoms.84 Increased d-dimer concentration is independently linked to serious HIV-related conditions such as cardiovascular disease or immune reconstitution inflammatory syndrome (IRIS) and with depressive symptoms.85\u201387 The current review detected five studies that tested for associations between d-dimer and depressive symptoms, of which two reported a significant association.44 Notably, Stewart et al.44 observed that this association was only significant for people with HIV (and not for those without HIV), and that serum concentration of d-dimer was positively correlated with somatic symptoms of depression. Each SD increase in PHQ-9 somatic score was associated with a 6% increase in d-dimer concentration in people living with HIV, but no increase in people without HIV. These studies offer early evidence that d-dimer may be associated with depressive symptoms in a manner dependent on HIV status.D-dimer is a biomarker of coagulation which is also elevated in systemic inflammation.88 BDNF binds to the receptor tropomycin receptor kinase B (TrkB) to promote neuronal and synaptic growth and survival. Given this, reduction in BDNF is associated with neuronal death and synaptodendritic injury. Decreased BDNF has been linked with depression in rigorous meta-analyses, though the effect size of this association is relatively small.89Brain-derived neurotrophic factor (BDNF) is a regulator of neurogenesis and synaptic plasticity, with additional functions in apoptosis and T cell survival.90 Concentrations of BDNF are elevated in CSF following CNS infections, suggesting that neuroinflammation influences BDNF expression.92 Furthermore, there is some evidence that BDNF signalling may exhibit cross-talk with the kynurenine pathway and associated immuno-metabolic dysfunctions.93 Pre-clinical studies show that induction of depression-like behaviours in rats was accompanied by decreased BDNF expression and increased IDO-1, Kyn, and Trp production, whereas inhibition of the kynurenine pathway significantly increased cortical BDNF levels.95 Therefore, BDNF signalling is influenced by neuroimmunometabolic responses known to be induced by HIV infection.BDNF is not a marker of neuroinflammation, but it does interact with HIV-induced neuroinflammatory processes in ways which are relevant to the current discussion. Notably, HIV infection in the brain leads to production of neuroinflammatory cytokines such as TNF-\u03b1 and IL-1\u03b2, which have recently been shown to inhibit expression (TNF-\u03b1), retrograde transport (IL-1\u03b2) and anterograde transport (TNF-\u03b1) of BDNF.et al.47 recently explored this question in a sample of older adults with and without HIV. In this study, the authors found that lower plasma BDNF concentrations were associated with higher scores on specific dimensions of mood disorders, notably Depression-Dejection . This association was only significant for participants with HIV, crucially demonstrating that the effect of BDNF on depressive symptoms may be dependent on HIV status. This study offers promising (though early) evidence that BDNF may be a useful biomarker of HIV-associated depression, with further work necessary to replicate this finding in diverse cohorts.Given this evidence, it is possible that BDNF may be associated with the risk for depression in HIV. Woods 96 For this reason, glial cell activation is widely considered to be the key characteristic of neuroinflammation. However, measuring glial cell activation directly in humans is challenging, as this requires access to brain tissue via biopsies or autopsies.As the resident immune cells in the CNS, microglia are essential to the neuroinflammatory response. Under homeostatic conditions, microglia are highly ramified, whereas \u2018activated\u2019 microglia reacting to immunological challenges are amoeboid. These activated microglia\u2014and, to some extent, astrocytes\u2014perform key immune functions in the brain, such as phagocytosing cellular debris and triggering immunological cascades.in vivo assessment of certain biomarkers which are considered indirect measures of glial cell activation, namely, 18\u2005kDa translocator protein, myo-inositol and choline. However, despite explicitly including search terms for TSPO, myo-inositol and choline, our systematic search did not detect any studies published since 2013 which examined changes in these neuroimaging biomarkers directly in relation to depression or depressive symptoms among people living with HIV. Newly developed radiotracers for PET ligands offer greater capacity to measure microglial activation in vivo.97 The leading-edge imaging modality diffusion-weighted MRS has recently been shown to be sensitive to an experimental model of neuroinflammation in humans.98 Thus, it will be useful for future research to test the associations of these rapidly developing neuroimaging biomarkers with depressive symptoms amongst people living with HIV.Positron emission tomography (PET) and magnetic resonance spectroscopy (MRS) allow for non-invasive 100 Participants in included studies also tended to be older, with none of the included studies investigating biomarkers of depression in children or adolescents living with HIV. More people live with HIV in the Global South, and the majority of these are women.102 Biomarkers of depression have been shown to significantly differ based on sex or gender,103 with different biomarkers found to be elevated in men with depression compared to women with depression.104 Additionally, women living with HIV exhibit greater systemic inflammation compared to men living with HIV.105 These demographic factors\u2014age, sex and gender, and geography\u2014interact significantly such that the prevalence of depression is higher in young women in the Global South.106 Therefore, the participant samples in included studies do not reflect the global epidemiology of HIV or depression. Effective translation of biomarker discovery into predictive or therapeutic tools will require careful consideration of differences in immune activation by factors such as age, sex, gender, and geography. Equitable cross-border collaborations are necessary to ensure greater inclusion in future research, as recommended elsewhere.107In a scoping review of the field, it is useful to consider which communities and lived experiences are not yet represented in the literature. This review found that the majority (70%) of all eligible studies investigating associations between inflammatory markers and depression in people living with HIV were carried out in the US. There was also a clear skew towards a greater proportion of men in participant samples . StudiesThis review aimed to identify studies investigating associations between depression or depressive symptoms and biomarkers of neuroinflammation and related processes in people living with HIV. Eligible studies utilized a variety of depressive symptom scales and assessed 55 biomarkers across neuroinflammation, chemotaxis, systemic inflammation, monocyte activation, immunometabolism, coagulation and neurogenesis. A key limitation of this review was that statistical reporting was highly variable, with many studies not reporting any measures of effect size at all. As a result, we could not conduct a meta-analysis of effect size estimates or assess whether studies in the field showed evidence of publication bias. Few included studies met our validation criteria for a grade of 3, which represented a biomarker which was significantly associated with depression only in people living with HIV (not in controls) and correlated with depressive symptom severity. Given this, for many biomarkers in this review, it is difficult to ascertain whether the significant associations observed in included studies are reliably dependent on HIV status and offer clinical utility. Finally, study screening and data extraction were carried out by a single reviewer, and the search was restricted to studies published in English, which are notable limitations to the methodology of this review.Regardless of these limitations, this review identified promising evidence that increased concentrations of IL-6, TNF-\u03b1, neopterin, IP-10, sCD14, sCD163 and d-dimer may be linked to greater depressive symptoms among people living with HIV. Conversely, higher concentrations of tryptophan and BDNF may be protective, since early evidence indicates significant negative associations between these biomarkers and depressive symptoms in people living with HIV. The biomarkers MCP-1 and IL-8 as well as the Kyn:Trp ratio were assessed in several studies, but no significant associations with depression were observed in the majority of these studies, indicating that these biomarkers may not substantially influence HIV-associated depression. The remaining biomarkers in the included studies were only assessed in a few studies, with little evidence for a significant association with depression in people living with HIV.108 The cytokines TNF-\u03b1 and IL-1\u03b2 regulate the production of IL-6, all of which in turn induce the release of CRP.109 Soluble monocyte activation markers such as sCD14 can induce the production of these cytokines.110 These inflammatory biomarkers may also trigger the kynurenine pathway of tryptophan metabolism, thus contributing to immuno-metabolic dysfunction, and inhibit the production of BDNF, thus impairing neurogenesis and synaptic plasticity. Kynurenine and other immunometabolites may similarly contribute to the release of chemokines such as MCP-1 and IL-8.111 Concentrations of MCP-1 and IP-10 are correlated with lower homovanillic acid, a dopamine metabolite, which in turn is correlated with depressive symptoms.40 Alterations in inflammatory biomarkers may thus also interact with dopaminergic signalling deficits to produce depressive symptoms.112 Together, these biomarkers thus represent feed-forward loops of neuroinflammation and related physiological processes which may drive the pathogenesis of depressive symptoms as a result of CNS HIV infection.These biomarkers are known to interact with each other as part of the (neuro)inflammatory response. For instance, TNF-\u03b1 regulates the release of chemokines such as MCP-1 and IP-10.et al.6 The evidence identified in this review implicates neuroinflammatory and metabolic biomarkers in HIV-associated depression. Given this, we hypothesize that chronically activated microglia, which trigger these neuroimmunometabolic cascades, are the central link connecting HIV infection in the CNS with the elevated risk for depressive symptoms  and induced depressive-like behaviour, whereas mice with deleted ACC microglia did not display depressive-like behaviour in response to experimentally induced inflammation.116 Crucially, the microglial activation marker TSPO is elevated in the ACC of people experiencing moderate or severe depressive episodes as well.117 Furthermore, novel human brain organoid models with co-cultured microglia have directly demonstrated that microglia release TNF-\u03b1 and IL-1\u03b2 in response to HIV infection, which are key cytokines frequently associated with depression.118 Taken together, these results strongly suggest that microglia may play a critical role in the pathogenesis of HIV-associated depression.Microglial activation due to inflammatory challenges such as infections is involved in the pathogenesis of depression. Evidence supporting the role of microglia in inflammation and depression has been extensively reviewed elsewhere.119 One recent study73 specifically explored associations between depressive symptoms and an astrocytic activation marker in people living with HIV. Replication and further investigation of such associations are necessary to determine whether astrocytic markers may be valuable predictive biomarkers or therapeutic targets for HIV-associated depression. Glial cell functions in neuroinflammation do overlap: in an experimental model, both microglia and astrocytes express inflammatory cytokines corresponding to sickness behaviours, but microglial responses precede astrocytic responses.120 This temporal distinction may implicate microglia as \u2018first responders\u2019 to inflammatory challenges, and therefore, an important target for anti-inflammatory interventions to treat depression.Although we focus on chronically activated microglia for this discussion, the role of astrocytes in HIV-associated depression also merits investigation. Pre-clinical evidence suggests that astrocytes are important regulators of neuroinflammation and may produce neuroprotective responses to cytokines.It is crucial to recognize that neurobiological mechanisms such as those outlined above can only explain in part, if at all, the elevated risk for depression amongst people living with HIV. The pathogenesis of depression in this population is likely driven by multiple contributing factors, including:\u2013121psychosocial and structural factors such as social isolation, racism, or transphobia, which impact many of the communities disproportionately affected by HIV;\u2013122socioeconomic stressors, including unemployment and food or housing insecurity;\u201352 orantiretroviral medication, some of which lead to depressive symptoms as a side-effect and may thus confound the measurement of depression in virally suppressed people living with HIV;\u2013123managing other co-morbid chronic health conditions unrelated to HIV, such as diabetes.Tackling the challenge of depression within this population will therefore require concerted efforts to systemically improve quality of life and social support, combat discrimination and stigma, and expand access to psychosocial services alongside biomedical interventions.124 Notably, depression has been linked to gut microbiome dysbiosis in people living with HIV.125 These associations may be explored further in future evidence-synthesis exercises.This review revealed evidence from the past decade identifying associations of key neuroinflammatory cytokines, chemokines, and monocyte activation markers with depressive symptoms among people living with HIV. Future research may focus on testing the association between biomarkers such as IL-6 and TNF-\u03b1 in samples that include participants without HIV, in order to establish whether the effects of these biomarkers are reliably influenced by HIV status. Leveraging developing techniques, especially neuroimaging biomarkers, may also support further investigation of neuroinflammation in HIV-associated depression. Although discussion of the gut microbiome was outside the scope of the present review, it must be noted that the composition of the gut microbiome may also modulate the impact of inflammation on depression.P values, for all biomarkers, including those which do not show statistically significant associations. Reporting effect size estimates such as Odds Ratios (for dichotomous depression/no depression) or correlation coefficients  will enable a robust meta-analysis of the strength of associations between various biomarkers and depressive symptoms.Future studies must endeavour to fully report test statistics and effect size estimates, not just Findings from this scoping review may support the translation of existing findings into clinical care for depression in people living with HIV. Biomarkers highlighted in this review may be further investigated in the following ways for appropriate translation:Validating predictive biomarkers, by employing machine learning prediction techniques to large, diverse cohorts of people living with HIV for whom inflammatory biomarkers and depressive symptoms have been measured at multiple time-points.Validating diagnostic biomarkers, by comparing concentrations of biomarkers in large, diverse cohorts of people living with HIV and demographically comparable people without HIV;Validating potential therapeutic interventions, by directly assessing mediating effects of biomarkers  and developing new therapies or re-purposing existing therapies which target these biomarkers.Going forward, a strategic balance will be necessary between exploring biomarkers which have shown promise in several studies (as highlighted in this review) and employing broad panels of biomarkers which have not yet been widely assessed. In doing so, future studies can consolidate efforts for biomarker discovery while remaining receptive to serendipitous discoveries. Identifying robust and reliable biomarkers which are correlated with depressive symptoms in people living with HIV may enable the application of immunotherapies to target specific features of depression within this population. These interventions may then be delivered using a precision medicine approach to people living with HIV who exhibit an elevated immune profile alongside depressive symptoms. Together with socioeconomic and psychosocial support interventions, these immunotherapies may thus help reduce the burden of depression among people living with HIV.For the purpose of open access, the author has applied a CC-BY public copyright licence to any Author Accepted Manuscript version arising from this submission.fcad231_Supplementary_DataClick here for additional data file."}
+{"text": "Reduced hemispheric asymmetry has been identified as a potential risk factor for schizophrenia, characterized by diminished brain lateralization and a lack of dominance in the left hemisphere. Moreover, there is growing evidence of disrupted connectivity between various cortical regions. This study aimed to investigate gender differences in left-footedness as a potential biological marker for neuronal dysontogenesis in individuals with schizophrenia and control subjects.A New Combined Foot Dominance Scale (14 foot tests), comprising a Modified Chapman & Chapman Subscale (10 foot tests) and a Complex Tasks Subscale (four foot tests) was administered as performance tasks in 180 subjects [98 schizophrenia patients with mean age 34.45\u2009years  for men and 42.20\u2009years  for women and 82 controls with a mean age 34.70\u2009years  for men and 44.50\u2009years ]. As our data are not continuous and lacks normal distribution, the non-parametric Mann\u2013Whitney test was used for comparing categorical data.The mean left-footedness, as assessed by the New Combined Foot Dominance Scale, is significantly higher in individuals with schizophrenia compared to control subjects. Our findings from inter-gender comparisons reveal that female schizophrenia patients exhibit a significantly greater average left-footedness than female control subjects, while in males no such a statistical significant difference is detected.Left foot dominance is higher in patients with schizophrenia than in control subjects and women contribute significantly more to this difference. The concept of hemispheric lateralization began to play an increasingly important role in the neuropsychological and pathophysiological models of schizophrenia . The facIn recent years, the hypothesis that schizophrenia is linked to abnormal cerebral lateralization has gained support by studies demonstrating anomalies in hand, eye, and foot preferences. Brain asymmetry has been observed at the structural level in the fetal brain of both humans and nonhuman primates  and appeFootedness refers to the dominant or preferred foot in a context that necessitates choosing one of the feet for performing manipulative or mobilizing actions. Footedness is less influenced by cultural norms or social teaching compared to handedness for which influences of these factors have been documented repeatedly \u20138.A recent meta-analysis confirms previous studies, which indicated that non-right-footedness prevalence was higher in males than in females in general population , 10. AnoAs far as we know there are few studies investigating gender differences in foot dominance in schizophrenia patients. In a prospective study of children, who develop schizophrenia spectrum disorders and those who did not develop, was found that males and females did not significantly differ in rates of left or mixed-handedness, footedness, and eye dominance.This study is part of a larger investigation project on the intriguing relations between six groups of markers of neuronal dysontogenesis\u2014left-handedness, left-footedness, left-eyedness, minor physical anomalies, digit ratio, and cognitive (attention and memory) deficit. The aim of the current article is to investigate gender differences in foot dominance as biological markers of neuronal dysontogenesis in schizophrenia patients and control subjects.The study was conducted in the Clinic of Psychiatry at the University Hospital in Sofia and at the State Psychiatric Hospital in Radnevo. The sample included 98  consecutively admitted inpatients with schizophrenia with mean age 34.45\u2009years  for men and 42.20\u2009years  for women.The patients satisfied the Diagnostic and Statistical Manual of Mental Disorders (DSM-V) criteria for a diagnosis of schizophrenia on the basis of case records review, DSM-V based semi-structured interview and information obtained from relatives for stronger validation of the diagnosis. In order to enhance the homogeneity of the schizophrenia group, potential subjects were excluded if they had a history of drug or alcohol abuse, identifiable neurological disorder , any signs of intellectual disability or somatic disorder with neurological components. Exclusion criteria were also any previous or present lower limb disorders hampering the performance of the foot tasks and any disease which affects balance or coordination.The control group consisted of 82 subjects  with a mean age 34.70\u2009years  for men and 44.50\u2009years  for women. Normality was defined as the absence of a previous or present psychiatric disorder. Controls satisfied exclusion criteria similar to those applied to the schizophrenia patients. In addition, for better separation between the control and the schizophrenia group, we implemented another exclusion criterion for controls\u2014having a first-degree relative with a history of a psychotic disorder, major affective disorder, or suicide.To avoid potential confound due to ethnic and racial differences related to lateralization both schizophrenia patients and controls were of Bulgarian origin. Individuals were excluded if their parental or grandparental ethnic group was other than Bulgarian.The refusal rate of potential participants was insignificant [below 5%], excluding selectivity bias.The study was approved by the Local Ethics Committee and all subjects gave written informed consent prior to participation.It includes two subscales:Among the foot dominance instruments in the literature available to us, the most popular is the Chapman & Chapman questionnaire . It inclTwo modifications were made to the subscale:The task \u201cTapping out a rhythm of Yankee Doodle,\u201d since this melody is unknown to Bulgarians, has been reformulated to \u201cTapping out the rhythm of a favorite song.\u201dThe task \u201cGoing through cubes with a ball\u201d was excluded due to poor informativeness. The participants (mainly schizophrenic patients) could not complete the task by choosing only one foot to lead the ball and used both feet. This led to the false overuse of the category \u201cBoth equally,\u201d which however did not reflect the actual dominant foot of the subject for this task.It includes four foot dominance tests, measuring more complex tasks.Step up on a chair (assess spontaneity).Pick up object with toe (assess precision).Push shovel into the ground (assess strength).Standing on one foot .Measuring these additional complex tasks allows for a subtle and comprehensive assessment of foot dominance.The New Combined Foot Dominance Scale includes a total of 14 tasks, administered as performance assessments, not as preference questionnaires. Each test was performed twice and the subject was asked to perform the test again if there was any inconsistency in the preference.Each foot test is rated: 0\u2014Preference of right foot; 1\u2014No preference ; and 2\u2014Preference of left foot. Each test score ranges from 0 to 2; the total score of the New Combined Foot Dominance Scale ranges from 0 to 28.The refusal rate of potential participants was insignificant [below 5%], excluding selectivity bias.All assessments were performed by the same examiner (KA).Data were analyzed with SPSS 25.0.Parametric statistics (mean and standard deviation) were used for descriptive purposes.As our data are not continuous and lack normal distribution, the non-parametric Mann\u2013Whitney test for means difference between two independent groups was used.The categories of the foot tests can be treated as ordinal data\u2014graded, in ascending order of leftedness: 0\u2014Preference of right foot; 1\u2014No preference; and 2\u2014Preference of left foot. Ordinal data allow calculating the mean left-footedness for every single foot test. A variable mean is usually more sensitive than its categories in detecting a difference between groups. Furthermore, it enables comparison of the important differences of the mean sum of left-footedness of the Modified Chapman & Chapman Subscale (10 foot tests), Complex Tasks Subscale (four foot tests), and the New Combined Foot Dominance Scale (14 foot tests) between schizophrenic and control subjects.p\u2009<\u20090.05; two-tailed.Statistical significance was defined as Combined Foot Dominance Scale are higher in patients with schizophrenia vs. control subjects:The mean left-footedness of all the foot tests (except arrange cubes) of the Chapman & Chapman subscale is significantly higher in patients with schizophrenia vs. control subjects .The sum mean left-footedness of the Complex Task Subscale is also significantly higher in patients with schizophrenia than in control subjects .The sum mean left-footedness of the New Combined Foot Dominance Scale is strongly significantly higher in patients with schizophrenia than in controls .Importantly, the cumulative sum mean left-footedness of the This shows that the overall mean left-footedness is higher in patients with schizophrenia compared to control subjects.However, when examining the mean left-footedness between patients with schizophrenia and control subjects, a notable difference emerges when considering gender. The comparison reveals significant distinctions between men and women.Analyzing the breakdown by gender of the contribution  to the pChapman & Chapman Subscale and Step up on a chair, Pick up object with toe, and Standing on one foot from the Complex Task Subscale , nor the subtotal mean left-footedness of the Complex Task Subscale (p\u2009=\u20090.817) is statistically significantly higher in male patients with schizophrenia than the same-gender control subjects.Neither the subtotal mean left-footedness of New Combined Foot Dominance Scale is not statistically significantly higher in male patients with schizophrenia than in the same-gender control subjects (p\u2009=\u20090.279).It should be emphasized that the total mean left-footedness of the In contrast to male subjects, there is a difference in female subjects.Chapman & Chapman Subscale and Step up on a chair, Pick up object with toe, Push shovel into the ground, and Standing on one foot from the Complex Task Subscale , Balancing a rod on the thigh (p\u2009=\u20090.011) and Hopping on one foot (p\u2009=\u20090.011) from the Chapman & Chapman Subscale and Step up on a chair (p\u2009=\u20090.011) from the Complex Task Subscale.Importantly, four of these differences are statistically significant at Chapman & Chapman Subscale . Arrange cubes from the Chapman & Chapman Subscale has equal means (0.38) in male patients with schizophrenia and same-gender controls (p\u2009=\u20090.991).In contrast to males, a very slight opposite trend of higher mean left-footedness in controls is displayed in merely only one foot test\u2014Write your initials on the sand from the p\u2009=\u20090.003, representing an increase of over 1.8 times). Additionally, the subtotal mean left-footedness of the Complex Task Subscale also shows significant differences  at a significance level of p\u2009<\u20090.05.Opposite to males, where neither subtotal mean exhibits statistically significant differences, a distinct pattern emerges in females. In female subjects, both subtotal mean left-footedness values reveal statistically significant differences. Specifically, the subtotal mean left-footedness of the Chapman & Chapman Subscale demonstrates a significantly higher value .Most importantly, the total mean left-footedness of the p\u2009<\u20090.004 in the control group.p\u2009<\u20090.05) between them and avoid Type 1 error, i.e., finding difference between groups when there is not. The need for ANOVA arises from the error of alpha level inflation, which increases Type 1 error probability  and is caused by multiple comparisons.In ANOVA analysis due to the statistical rules that the more groups compared, the greater the differences between them must be in order to reach statistical significance , which approaches p\u2009<\u20090.05.We think that although none of our three combined total scores of mean left-footedness reach statistical significance of p\u2009=\u20090.070, the strongest difference of the three means.The most impressive difference is very logically for the ANOVA model showing that the differences between the four groups\u2014Females-Controls, Males-Controls, Males-Schizophrenia, and Females-Schizophrenia in the Subtotal mean left-footedness of four foot tests for Complex Task p\u2009<\u20090.094 in the context of one-tailed testing as statistically significant interaction at p\u2009<\u20090.1. It is noteworthy to stress that studying the role of two factors in the variation of a certain variable, interpreting their interaction is obligatory and indispensable. In our case, the weakest statistical significance for the role of gender per se is even for the \u201cSubtotal mean left-footedness of four foot tests for Complex Task\u201d in comparison with the other two (If we do not use the general alternative hypothesis, positing that the interaction Gender*Group may be possible in both way, i.e., stronger in men or stronger in women, and thus use two-tailed testing and postulate the specific alternative hypothesis that the interaction Gender*Group is logically higher in women than in men due to the fact that in the literature , female ther two . The lacOur results show that patients with schizophrenia have markedly higher mean left-footedness than normal controls when not considering gender. However, the gender comparison reveals significant difference between females and males across different areas shown in the result section. The greatest proof of the female vs. male contribution to the overall higher difference in patients with schizophrenia vs. control subjects is given by comparing the subtotals mean left-footednesses and the total mean left-footedness by gender. In sharp contrast to females, neither the subtotals mean left-footednesses, nor the total mean left-footedness of the New Combined Foot Dominance Scale are statistically significantly higher in male schizophrenia patients vs. same-gender controls. Taken together, when considering the cumulative effect of all the aforementioned differences observed in the areas of comparison, shown in the results, it leads to the conclusion that only the females make a significant contribution to the overall higher mean left-footedness difference between patients with schizophrenia and control subjects. Thus, in terms of laterality schizophrenic dysontogenesis makes female patients with schizophrenia more lateralized than normally, matching normal males in that regard. Our findings of intra-gender difference in the mean left-footedness between female schizophrenia patients and control subjects in almost all tests correlate with the findings from magnetoencephalography-based studies also suggest anomalous cerebral lateralization in schizophrenia. In their study, Martin Reite et al.  found thFootedness can be identified with a combination of manipulation and stability tasks. The use of familiar tasks is more likely to elicit a stronger, more lateralized response. There is coherence between preference and performance across the stability modes, but preference for manipulation is substantially lateralized relative to performance . This coBecause of abnormal brain asymmetry and failure of the left-hemisphere dominance in schizophrenia, lateralized functions are compromised in schizophrenia. Indeed, different studies have revealed abnormal patterns of connectivity in the left hemisphere in relation to specific psychotic domains \u201329. LeftIn conclusion, the lateralized function in schizophrenia are compromised as a result of abnormal brain asymmetry and failure of the left-hemisphere dominance. Schizophrenia patients have markedly higher mean left-footedness than normal controls with a clear gender-specific effect, only notably pronounced in women not in men. Left foot dominance emerges as a promising indicator of altered hemispheric lateralization, as it is minimally influenced by cultural and traditional factors and reveals notable gender-specific differences.The study is clinic-based and cross-sectional and not community-based, so there is certain selectivity bias in some Schizophrenia characteristics, as some representative demographics, severity of Schizophrenia process, but all such studies we encountered in literature are clinic-based and cross-sectional, because of exclusive difficulties of organizing such study as community-based and especially longitudinal.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving humans were approved by Medical University of Sofia, Department of Psychiatry and Medical Psychology. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.KA: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing \u2013 original draft, Writing \u2013 review & editing."}
+{"text": "Herein, we reporta new application of \u03b2-nitroenonesas valuablebuilding blocks for the preparation of polyfunctionalized homoallylicalcohols; they can be used as key precursors of conjugated nitrotrienesystems. The synthesis of homoallylic alcohols was performed exploitingthe chemoselective addition of metal allylating agents to the ketonemoiety vs the nitroalkenyl group. The conversion of alcohols intonitrotrienes was achieved under Lewis-acid-promoted conditions. Bothclasses of compounds were obtained in good to excellent yields. The juxtapositionof these functionalities makes 1 highly reactive speciestoward a plethora of nucleophiles, and in particular, they chemoselectivelyreact at position 2, behaving as excellent Michael acceptors  and DMF. The reactiongave a modest result, and 3a was isolated in 56% yield.No improvement was observed by changing the solvent  iodide. To our delight,the reaction provided 3a in 94% yield  halides, which however were less effectivethan InI in terms of both yield and reaction time due to the lowerthermodynamic stability, according to what was previously reported.9 Finally, the reaction was attempted using allylmagnesiumchloride 2d without any appreciable result  iodide and the scalabilityof our protocol exploring the conversion of 1a into 2a on 5 mmol scale. Pleasingly, both studies provided excellentresults. In fact, it was seen that the reaction demonstrated goodgenerality, affording the target homoallylic alcohols 3a\u2013o in very good yields (3a in 95% yield (see SI).Next, we explored boththe substrate generality forthe allylationof \u03b2-nitroenones d yields 3, and th3 as valuable building blocks for the possiblesynthesis of conjugatednitrotriene systems 4. This hitherto unknown class ofnitro derivatives may open new scenarios in the synthesis of functionalizedmolecules exploiting the chemistry of trienes.10 In order to find the best reaction conditions and followingthe available literature,11 we initiallyexplored the conversion of 3a into 4a using p-toluenesulfonic acid in toluene under reflux conditions,which however provided a complex mixture of inseparable compounds.Conversely, at room temperature, the reaction was completely ineffective.Then, we tested the dehydration reaction utilizing boron trifluoridediethyl etherate in dichloromethane  by working at \u221210 \u00b0C and in the presenceof 1.5 equiv of BF3\u00b7Et2O. Noteworthy, theformation of the other regioisomer 5a was limited toless than 10%. The increase of the observed regioselectivity at lowertemperature is probably the result of better kinetic control in theformation of compounds 4. Moreover, we attempted to convert 4a into 5a with the aim to obtain a single regioisomer.With this scope, the reaction was stirred at room temperature for24 h; however, we observed extensive degradation of the product withoutany change concerning the 4a:5a regioisomeric ratio and 22%  yield, respectively. The reaction promoted by BBr3 at \u221210 \u00b0C for 6 h led to 4a in just41% conversion, while warming to room temperature resulted in completedegradation of the expected product. Finally, use of ethylaluminumdichloride and the heterogeneous BF3 and AlCl3 was completely ineffective.The reaction was also investigated using a differentsolvent suchas 1,2-dichloroethane (DCE) and additional Lewis acids including AlCl3a to demonstrate the largerscale viability of the protocol. Evenunder these conditions the process was very efficient; in fact, theregioisomeric mixture of 4a and 5a (90:10)was isolated in 69% yield (see SI), a valuecomparable to that obtained on a smaller scale . Compounds 1a\u2013o were prepared starting from alkyl- and arylglyoxals andnitro compounds by following reported procedures;141a was a known compound,2b while compounds 1b\u2013o werenew compounds. Allyltributyltin 2a was purchased fromSigma-Aldrich (code 271411) and used as received. Allyl bromide 2b was purchased from Sigma-Aldrich (code A29585) and usedas received. Allylboronic acid pinacol ester 2c was purchasedfrom Sigma-Aldrich (code 324647) and used as received. Allylmagnesiumchloride 2d was purchased from Sigma-Aldrich (code 225908)and used as received. Boron trifluoride diethyl etherate was purchasedfrom Sigma-Aldrich (code 216607) and used as received. Tetrahydrofuranwas purchased from Sigma-Aldrich (code 87368) and distilled over sodiumbefore usage. Dichloromethane (stabilized with amylene) was purchasedfrom Carlo Erba (code 463314) and used as received. Indium(I) iodidewas prepared according to the literature.13 Heating for synthesizing compounds 3a\u2013o was accomplished by means of a heating magnetic stirrerequipped with an aluminum heating block. Configuration assignmentof compound 4a was made with additional information fromthe NOESY experiment.1 (0.5 mmol), dry THF (2.5 mL), InI, and the allylboronic acid pinacol ester 2c . The resulting mixture was vigorouslystirred at 40 \u00b0C for the appropriate time (see 3 (10 mL). After phase separation, the aqueous phase was extractedwith dichloromethane (2 \u00d7 20 mL), and the combined organic layerswere dried with dry Na2SO4. Finally, the solutionwas filtered and concentrated in vacuo to give the crude products 3, which were purified by flash column chromatography (hexane/ethylacetate).An oven-dried round-bottom flaskwith a magnetic stir bar,maintained under inert atmosphere, was charged with the appropriate\u03b2-nitroenones time see 3, dilute3a  as apale yellow oil. IR : 703, 924, 1334,1520, 1634, 3392. 1H NMR  \u03b4:7.47\u20137.42 , 7.40\u20137.34 , 7.31\u20137.25, 5.73\u20135.60 , 5.32\u20135.24 , 2.78, 2.76 , 2.50 , 0.92 . 13C{1H} NMR \u03b4: 155.8, 144.3, 138.3, 131.7, 128.9, 127.9, 125.3, 122.3, 74.5,49.1, 20.8, 12.5. GC-MS (70 eV): m/z 206 (59), 159 (100), 131 (12), 105 (37), 77 (32). Anal. Calcd forC14H17NO3 (247.29): C, 68.00; H,6.93; N, 5.66. Found: C, 68.05; H, 6.96; N, 5.69.Flash chromatography on silica gel using hexane/EtOAc= 95:5 as eluent yielded 3b  as apale yellow oil. IR : 832, 926, 1034,1176, 1248, 1334, 1513, 1608, 3464. 1H NMR  \u03b4: 7.38\u20137.32 , 6.89 , 5.74\u20135.60 , 5.29 , 5.27\u20135.23, 3.81 , 2.81\u20132.71 , 2.38 ,0.95 . 13C{1H} NMR  \u03b4: 159.2, 155.4, 138.3,136.2, 131.9, 126.6, 122.1, 114.2, 74.3, 55.6, 48.9, 20.7, 12.6. GC-MS(70 eV): m/z 236 (73), 189 (100),135 (42), 81 (15), 77 (13). Anal. Calcd for C15H19NO4 (277.32): C, 64.97; H, 6.91; N, 5.05. Found: C, 65.02;H, 6.94; N, 5.02.Flash chromatography on silica gel using hexane/EtOAc= 95:5 as eluent yielded 3c  as apale yellow oil. IR : 709, 931, 1331,1434, 1525, 1609, 3528. 1H NMR  \u03b4: 7.27 , 7.24, 7.00\u20136.96 , 5.82\u20135.68 , 5.33\u20135.31, 5.30\u20135.26 , 2.92\u20132.76 , 2.65, 1.04 . 13C{1H} NMR  \u03b4: 155.3,148.7, 136.8, 131.4, 127.4, 125.6, 124.0, 122.5, 73.9, 49.2, 20.7,12.9. GC-MS (70 eV): m/z 212 (61),165 (100), 111 (47), 81 (19), 39 (17). Anal. Calcd for C12H15NO3S (253.32): C, 56.90; H, 5.97; N, 5.53;S, 12.66. Found: C, 56.93; H, 6.00; N, 5.56; S, 12.70.Flash chromatography on silica gel using hexane/EtOAc= 95:5 as eluent yielded 3d  as apale yellow oil. IR : 739, 833, 920,1035, 1177, 1252, 1334, 1508, 1607, 3539. 1H NMR  \u03b4: 7.35 ,7.33 , 6.89 , 5.73\u20135.61, 5.29 , 5.27\u20135.23 , 3.81 , 2.78\u20132.70, 2.37 , 1.39\u20131.15 , 0.83 . 13C{1H} NMR  \u03b4: 159.2, 154.5, 138.4, 136.3, 131.9, 126.6,122.1, 114.1, 74.3, 55.5, 48.9, 26.9, 30.2, 22.9, 13.9. GC-MS (70eV): m/z 264 (67), 243 (12), 217(100), 175 (13), 135 (91), 77 (21). Anal. Calcd for C17H23NO4 (305.37): C, 66.86; H, 7.59; N, 4.56.Found: C, 66.91; H, 7.63; N, 4.59.Flash chromatography on silica gel using hexane/EtOAc= 95:5 as eluent yielded 3e  as apale yellow oil. IR : 748, 823, 1331,1426, 1517, 1607, 1635, 3062, 3545. 1H NMR  \u03b4: 7.94 , 7.88\u20137.81, 7.54\u20137.48 , 7.47 , 5.74\u20135.62, 5.35\u20135.27 , 2.88 , 2.74 , 2.54, 1.08\u20131.37 , 0.74 . 13C{1H} NMR  \u03b4: 155.2, 141.5, 137.9, 133.3, 132.8, 131.7, 128.8,128.4, 127.8, 126.8, 126.6, 124.1, 123.5, 122.4, 74.6, 49.0, 30.0,27.0, 22.8, 13.8. GC-MS (70 eV): m/z 278 (15), 263 (64), 237 (17), 207 (17), 155 (100), 127 (76), 77(12). Anal. Calcd for C20H23NO3 (325.41):C, 73.82; H, 7.12; N, 4.30. Found: C, 73.78; H, 7.09; N, 4.27.Flash chromatography on silica gel using hexane/EtOAc= 95:5 as eluent yielded 3f . IR : 698, 724, 987, 1326, 1519, 1608, 1640, 3062,3537. 1H NMR  \u03b4: 7.47\u20137.45, 7.44 , 7.40\u20137.35 , 7.32\u20137.27, 5.74\u20135.61 , 5.33\u20135.24 , 2.79, 2.46 , 2.24 . 13C{1H} NMR  \u03b4:150.3, 143.8, 138.3, 131.5, 128.7, 127.7, 125.1, 122.0, 74.2, 48.7,13.6. GC-MS (70 eV): m/z 192 (60),145 (100), 105 (29), 77 (31), 67 (32). Anal. Calcd for C13H15NO3 (233.27): C, 66.94; H, 6.48; N, 6.00.Found: C, 66.99; H, 6.52; N, 5.97.Flash chromatography on silica gel using hexane/EtOAc= 95:5 as eluent yielded 3g  as apale yellow oil. IR : 698, 1042, 1247,1328, 1432, 1520, 1583, 1600, 1640, 2249, 3460. 1H NMR \u03b4: 7.40 , 7.30 , 7.03\u20136.96 , 6.87\u20136.81, 5.70\u20135.57 , 5.32 , 5.30\u20135.26 , 3.82 , 2.86\u20132.69, 2.52 , 2.27 ,1.65\u20131.34. 13C{1H} NMR  \u03b4: 159.9, 152.9, 145.5, 138.9, 131.2, 129.9, 122.4, 119.5,117.4, 112.5, 111.4, 74.2, 55.3, 48.7, 26.9, 26.0, 25.0, 16.7. GC-MS(70 eV): m/z 284 (25), 268 (93),229 (30), 187 (16), 135 (100), 107 (27), 77 (34). Anal. Calcd forC18H22N2O4 (330.38): C,65.44; H, 6.71; N, 8.48. Found: C, 65.49; H, 6.74; N, 8.51.Flash chromatography on silica gel using hexane/EtOAc= 95:5 as eluent yielded 3h  as apale yellow oil. IR : 699, 912, 994,1331, 1521, 1640, 3540. 1H NMR  \u03b4: 7.47\u20137.42 , 7.40\u20137.34 , 7.32\u20137.26, 5.80\u20135.59 , 5.30 , 5.28\u20135.24 , 5.00\u20134.88 , 2.78, 2.76\u20132.70 , 2.41, 1.96 , 1.14\u20131.12. 13C{1H} NMR  \u03b4: 154.4, 144.0, 138.5, 138.1, 131.5, 128.6, 127.7, 125.1,122.1, 114.5, 74.2, 48.8, 33.3, 28.6, 27.2, 26.8. GC-MS (70 eV): m/z 260 (7), 213 (11), 186 (18), 105 (100),91 (10), 77 (26), 41 (14). Anal. Calcd for C18H23NO3 (301.39): C, 71.73; H, 7.69; N, 4.65. Found: C, 71.77;H, 7.73; N, 4.62.Flash chromatography on silica gel using hexane/EtOAc= 95:5 as eluent yielded 3i  as apale yellow oil. IR : 699, 733, 1326,1453, 1521, 1603, 1640, 3540. 1H NMR  \u03b4: 7.46 , 7.34\u20137.29 , 7.26\u20137.18, 7.17\u20137.12 , 5.65\u20135.54 , 5.27\u20135.20, 3.20\u20133.06 , 2.79\u20132.60 , 2.38, 2.05 . 13C{1H} NMR  \u03b4: 152.5, 140.8, 140.7, 139.5, 137.5, 131.5,129.4, 128.7, 128.5, 126.3, 125.0, 121.6, 74.4, 48.5, 33.9, 29.1,21.0. GC-MS (70 eV): m/z 290 (19),275 (48), 248 (9), 199 (12), 119 (100), 91 (82), 65 (24). Anal. Calcdfor C21H23NO3 (337.42): C, 74.75;H, 6.87; N, 4.15. Found: C, 74.79; H, 6.90; N, 4.12.Flashchromatography on silica gel using hexane/EtOAc= 95:5 as eluent yielded 3j  as apale yellow oil. IR : 476, 734, 748,1326, 1521, 1600, 1639, 3027, 3537. 1H NMR  \u03b4: 7.94\u20137.85 , 7.59 , 7.58\u20137.52, 7.50 , 7.29\u20137.17, 7.06 , 5.67\u20135.56, 5.33\u20135.22 , 3.21\u20133.07 , 2.87\u20132.69, 2.65\u20132.55 , 2.20 . 13C{1H} NMR  \u03b4: 152.9, 141.0,140.6, 139.2, 133.1, 132.6, 131.3, 128.7, 128.6, 128.5, 128.2, 127.6,126.6, 126.4, 126.3, 123.9, 123.2, 122.0, 74.5, 48.5, 33.8, 29.1.GC-MS (70 eV): m/z 326 (34), 311(34), 284 (59), 207 (29), 155 (100), 127 (90), 91 (34), 77 (17). Anal.Calcd for C24H23NO3 (373.45): C,77.19; H, 6.21; N, 3.75. Found: C, 77.24; H, 6.18; N, 3.78.Flash chromatography on silica gel using hexane/EtOAc= 95:5 as eluent yielded 3k  as apale yellow solid. Mp: 134\u2013176 \u00b0C. IR : 701, 716, 935, 1331, 1398, 1517, 1703, 1770, 3073, 3513. 1H NMR  \u03b4: 7.80\u20137.77, 7.72\u20137.68 , 7.50 , 7.28\u20137.11, 5.49\u20135.37 , 5.08 , 4.94 ,3.99\u20133.84 , 3.28\u20133.22 , 2.74 , 2.67 , 2.51 . 13C{1H} NMR  \u03b4: 168.5, 149.4, 143.4, 141.2, 134.1,132.4, 131.3, 128.9, 127.9, 125.0, 123.5, 122.2, 74.8, 48.5, 36.0,26.5. GC-MS (70 eV): m/z 345 (35),330 (38), 183 (99), 171 (47), 160 (65), 105 (100), 77 (80). Anal.Calcd for C22H20N2O5 (392.41):C, 67.34; H, 5.14; N, 7.14. Found: C, 67.38; H, 5.17; N, 7.11.Flash chromatography on silica gel using hexane/EtOAc= 95:5 as eluent yielded 3l  as ayellow oil. IR : 699, 1331, 1520, 3537. 1H NMR  \u03b4: 7.47\u20137.35, 7.32\u20137.27 , 5.72\u20135.59 , 5.33\u20135.25, 3.44 , 2.81\u20132.74, 2.47 , 1.73\u20131.63 , 1.60\u20131.46, 1.44\u20131.31 . 13C{1H} NMR \u03b4: 153.5, 143.9, 138.7, 131.3, 128.7,127.8, 125.0, 122.3, 74.3, 48.7, 44.4, 32.2, 26.1, 25.2. GC-MS (70eV): m/z 268 (43), 221 (81), 105(100), 77 (54), 41 (17). Anal. Calcd for C16H20ClNO3 (309.79): C, 62.03; H, 6.51; N, 4.52. Found: C,62.08; H, 6.48; N, 4.55.Flash chromatography on silica gel using hexane/EtOAc= 95:5 as eluent yielded 3m  as ayellow oil. IR : 1329, 1522, 3558. 1H NMR  \u03b4: 7.10 , 5.80\u20135.69, 5.27 , 5.23 , 3.06\u20132.98 , 2.84\u20132.76, 2.61 , 2.34 , 1.80 , 1.56\u20131.47, 1.43\u20131.21 , 1.04 , 0.90 . 13C{1H} NMR  \u03b4: 154.6, 136.7, 133.2, 121.0, 78.8, 41.3,39.2, 31.7, 29.6, 28.9, 28.6, 26.4, 25.4, 22.6, 14.0. GC-MS (70 eV): m/z 256 (24), 209 (30), 139 (15), 109 (14),95 (18), 81 (25), 69 (100), 57 (90), 41 (74). Anal. Calcd for C17H31NO3 (297.44): C, 68.65; H, 10.51;N, 4.71. Found: C, 68.60; H, 10.54; N, 4.74.Flashchromatography on silica gel using hexane/EtOAc= 95:5 as eluent yielded 3n  as apale yellow oil. IR : 699, 1331, 1520,1732, 3484. 1H NMR  \u03b4:7.46\u20137.24 , 5.72\u20135.58 , 5.31\u20135.23, 3.65 , 2.78 ,2.73 , 2.57 ,2.23 , 1.59\u20131.47 ,1.43\u20131.11 . 13C{1H} NMR  \u03b4: 174.2, 154.2, 144.0, 138.3, 131.5, 128.6,127.7, 125.1, 122.0, 74.3, 51.5, 48.8, 33.8, 28.7, 27.2, 26.6, 24.3.GC-MS (70 eV): m/z 300 (6), 285(13), 199 (22), 197 (18), 105 (100), 77 (41). Anal. Calcd for C19H25NO5 (347.41): C, 65.69; H, 7.25;N, 4.03. Found: C, 65.73; H, 7.28; N, 4.07.Flash chromatography on silica gel using hexane/EtOAc= 95:5 as eluent yielded 3o  as apale yellow oil. IR : 735, 921, 1334,1457, 1519, 1641, 3533. 1H NMR  \u03b4: 7.03 , 5.89\u20135.76 , 5.33\u20135.19, 2.95 , 2.52\u20132.46, 2.42\u20132.35 , 1.94 , 1.45 ,1.13 . 13C{1H} NMR  \u03b4: 154.6, 138.4, 132.0,121.1, 71.7, 47.8, 28.4, 20.1, 13.2. GC-MS (70 eV): m/z 144 (45), 97 (100), 43 (58). Anal. Calcd forC9H15NO3 (185.22): C, 58.36; H, 8.16;N, 7.56. Found: C, 58.40; H, 8.19; N, 7.58.Flash chromatography on silica gel using hexane/EtOAc= 90:10 as eluent yielded 3\u00b7Et2O was added dropwise at \u221210 \u00b0C to a stirred solution ofthe appropriate homoallylic alcohol 3 (0.5 mmol) in dichloromethane(5 mL). The reaction was stirred at the same temperature (\u22125\u00b0C for compound 3e) for the appropriate time (see 3 (10 mL). After phase separation, the aqueous phase was extractedwith dichloromethane (2 \u00d7 20 mL), and the combined organic layerswere dried with dry Na2SO4. Finally, the solutionwas filtered and concentrated in vacuo to give the crude regioisomericproducts 4 and 5, which were purified byflash column chromatography (hexane/ethyl acetate).BFtime see 4, dilute4a and 5a  as a pale yellowoil. Major regioisomer 4a. IR : 695, 760, 1333, 1520, 1632. 1H NMR  \u03b4: 7.81 , 7.44\u20137.29 ,6.73\u20136.48 , 5.51 ,5.39 , 2.42 , 0.96 . 13C{1H} NMR  \u03b4: 155.7,138.8, 133.8, 133.1, 132.7, 130.1, 128.5, 128.4, 126.7, 122.0, 21.1,11.5. GC-MS (70 eV): m/z 229 , 168 (100), 152 (59), 128 (32), 115 (36), 91 (29),77 (24). Anal. Calcd for C14H15NO2 (229.28): C, 73.34; H, 6.59; N, 6.11. Found: C, 73.38; H, 6.63;N, 6.14.Flash chromatography on silicagel using hexane/EtOAc = 98:2 as eluent yielded a 90:10 mixture of 4b and 5b  as a pale yellowoil. Major regioisomer 4b. IR : 832, 1030, 1175, 1244, 1333, 1511, 1604. 1H NMR \u03b4: 7.76 , 7.28 , 6.89 ,6.63\u20136.44 , 5.45 , 5.33 , 3.82 , 2.43 , 0.97 . 13C{1H} NMR  \u03b4: 160.0, 155.8, 133.5, 131.3, 131.1, 130.7,128.7, 128.1, 121.3, 114.4, 55.6, 21.4, 11.8. GC-MS (70 eV): m/z 259 , 215 (51),198 (100), 183 (47), 65 (27), 153 (31), 128 (24), 115 (26). Anal.Calcd for C15H17NO3 (259.31): C,69.48; H, 6.61; N, 5.40. Found: C, 69.52; H, 6.57; N, 5.42.Flash chromatography on silicagel using hexane/EtOAc = 98:2 as eluent yielded a 90:10 mixture of 4c and 5c  as a pale yellowoil. Major regioisomer 4c. IR : 694, 828, 909, 1329, 1523, 1612, 1656. 1H NMR \u03b4: 7.65 , 7.25 , 7.01\u20136.97 , 6.94 , 6.68 , 6.47\u20136.33 , 5.48 , 5.33 , 2.53, 1.05 . 13C{1H} NMR  \u03b4: 156.4, 142.2, 132.8, 131.2, 129.3, 129.0, 127.8,125.6, 125.5, 121.4, 21.2, 11.8. GC-MS (70 eV): m/z 235 , 174 (100), 147 (26),128 (35), 115 (33), 97 (24), 77 (19). Anal. Calcd for C12H13NO2S (235.30): C, 61.25; H, 5.57; N, 5.95;S, 13.63. Found: C, 61.29; H, 5.60; N, 5.89; S, 13.67.Flash chromatography on silicagel using hexane/EtOAc = 98:2 as eluent yielded a 90:10 mixture of 4e and 5e  as a pale yellowoil. Major regioisomer 4e. IR : 464, 751, 812, 1325, 1430, 1467, 1519, 3054. 1H NMR  \u03b4: 7.96 , 7.89\u20137.83, 7.76 , 7.58\u20137.49, 6.84 , 6.70\u20136.58, 5.58 , 5.45 , 2.45\u20132.38 , 1.42\u20131.33, 1.15\u20131.05 , 0.70 . 13C{1H} NMR  \u03b4: 154.9, 136.0, 133.6, 133.3, 133.2, 133.1, 130.5,128.8, 128.5, 128.2, 127.6, 126.7, 126.5, 126.1, 124.2, 122.2, 29.1,27.4, 22.3, 13.4. GC-MS (70 eV): m/z 307 , 264 (37), 218 (100), 202 (92), 165 (14),41 (14). Anal. Calcd for C20H21NO2 (307.39): C, 78.15; H, 6.89; N, 4.56. Found: C, 78.19; H, 6.92;N, 4.59.Flash chromatography on silicagel using hexane/EtOAc = 98:2 as eluent yielded a 90:10 mixture of 4i  as a paleyellow oil. IR : 699, 821, 915, 1330,1520, 3025. 1H NMR  \u03b4:7.89 , 7.29\u20137.07 , 6.93\u20136.89 ,6.62\u20136.45 , 5.47 , 5.37 , 2.66 , 2.37 . 13C{1H} NMR  \u03b4: 153.1, 140.1, 138.4, 136.0, 133.6, 133.0, 132.9,131.8, 129.5, 128.4, 128.3, 126.8, 126.3, 122.0, 33.0, 29.9, 21.2.GC-MS (70 eV): m/z 319 , 227 (100), 182 (92), 165 (50), 152 (17), 91 (58). Anal. Calcdfor C21H21NO2 (319.40): C, 78.97;H, 6.63; N, 4.39. Found: C, 79.01; H, 6.60; N, 4.42.Flash chromatography on silica gel using hexane/EtOAc= 98:2 as eluent yielded 4l and 5l  as a pale yellowoil. Major regioisomer 4l. IR : 700, 1326, 1519, 1640. 1H NMR  \u03b4: 7.93 , 7.43\u20137.27 , 6.70\u20136.51, 5.52 , 5.42 , 3.34 , 2.40\u20132.34 , 1.62\u20131.44 . 13C{1H} NMR  \u03b4:153.7, 138.8, 133.7, 133.6, 132.8, 131.0, 128.9, 128.5, 126.8, 122.6,44.1, 32.0, 26.9, 24.4. GC-MS (70 eV): m/z 291 (29), 214 (43), 168 (100), 167 (70), 165 (53), 153(32), 152 (59), 115 (22), 91 (20), 41 (21). Anal. Calcd for C16H18ClNO2 (291.77): C, 65.86; H, 6.22;N, 4.80. Found: C, 65.91; H, 6.19; N, 4.83.Flash chromatography on silicagel using hexane/EtOAc = 98:2 as eluent yielded a 90:10 mixture of 4n and 5n  as a yellow oil.Major regioisomer 4n IR :697, 732, 1331, 1521, 1734. 1H NMR  \u03b4: 7.87 , 7.40\u20137.27 , 6.68\u20136.49, 5.50 , 5.39 , 3.63 , 2.37\u20132.31, 2.16 , 1.49\u20131.30, 1.15\u20131.04 . 13C{1H} NMR \u03b4: 174.0, 154.2, 138.8, 133.7, 133.2,132.9, 130.7, 128.8, 128.4, 126.8, 122.3, 51.5, 33.7, 28.7, 27.4,26.6, 24.2. GC-MS (70 eV): m/z 294(10), 220 (81), 210 (80), 168 (100), 167 (94), 115 (40), 87 (41),55 (35). Anal. Calcd for C19H23NO4 (329.40): C, 69.28; H, 7.04; N, 4.25. Found: C, 69.33; H, 7.07;N, 4.21.Flash chromatography on silicagel using hexane/EtOAc = 98:2 as eluent yielded a 90:10 mixture of"
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