{"text": "In this report we re-examine some recent experiments with digital organisms to test some predictions of quasispecies theory. These experiments revealed that under high mutation rates populations of less fit organisms previously adapted to such high mutation rates were able to outcompete organisms with higher average fitness but adapted to low mutation rates.We have verified that these results do hold in the original conditions and, by extending the set of initial parameters, we have also detected that the critical mutation rate was independent of population size, a result that we have found to be dependent on a different, contingent factor, the initial fitness vector. Furthermore, in all but one case, the critical mutation rate is higher than the error threshold, a key parameter in quasispecies theory, which prevents its extrapolation to natural viral populations.From these results we conclude that digital organisms are useful tools for investigating evolutionary patterns and processes including some predictions from the quasispecies theory. RNA viruses are among the most infective pathogens affecting plants, animals and humans. Several of their features such as their reduced genomes, high genetic heterogeneity, large population sizes, short generation times and fast evolutionary rates place them among the best models for evolutionary and population genetic studies ,2. TheseAs a consequence, selection, that translates in competition with the host and among viral variants, usually results in the persistence of the most infective, pathogenic or more persistent variants. The molecular bases for this genetic variability are three mechanisms differentially used by each kind of virus: mutation, homologous and non-homologous recombination and genome rearrangement .10individuals in short times), high mutation rates  derived from lack of proof-read correction in the polymerase), and small genome sizes (ranging from 3 to 30 kilobases).Attempts to model the evolutionary dynamics of RNA viruses incorporate their most relevant features, such as large population sizes .- Genetic drift has no relevant effects: their tiny genomes, large population sizes and high mutation rates allow the exploration of all the neutral space around the master sequence.- The average consensus sequence remains stable along quasispecies evolution.This model contrasts sharply with conventional population genetic models in which the existence of a large number of neutral mutations would lead to genetic drift of the population and the individual is the unit of selection rather than a cloud of related variants . This laThe difficulty in experimentally testing some predictions of quasispecies theory has led to the search of alternative systems. In this work we have used digital organisms as an approximation to the population dynamics of RNA viruses. Digital organisms are self-replicating entities and compete for access to resources, in this case CPU cycles, as implemented in the AVIDA platform ,15.Avidians are programs (genomes) composed by arrays of logical instructions (genes) that allow them obtaining CPU cycles. There are 28 possible instructions. The number of instructions in a digital organism is equivalent to the genome size in a biological organism ,17.Many studies have been done using the AVIDA platform. The possibility that genome sizes change during the course of evolution and the rewards that they can obtain by the combination of functions have allowed investigations about the evolution of genomic complexity . Furthersurvival of the flattest and not survival of the fittest.In this work we have focused in a recent study by Wilke et al.  with digHere we have extended the original experimental conditions in order to study the effect and interaction between three of the key factors in the quasispecies model: population and genome sizes and mutation rates. Our results indicate that chance events in the form of historical contingency play an important role in the evolution of these populations. Moreover we have established a new, corrected mutation rate necessary for quasispecies formation with a higher value than the original one. The implications of this correction are discussed.We considered three factors affecting the critical mutation rate in our digital organisms: genome size, population size and the influence of the initial fitness vector. This is a vector of randomly assigned priorities for the first time evaluation of each organism fitness, incorporated to prevent the system from collapsing if all organisms simultaneously try to enter the CPU, and can be interpreted as a historical, contingent factor in evolution. The range of genome sizes studied varied from 54 to 272 instructions Table . Our expTable As expected from our preliminary results, the use of an initial random vector for each experiment and not for each organism resulted in clear differences with the results encountered by Wilke et al. . These dNevertheless, despite these differences between organisms we cannot conclude that there is a globally significant effect of population size on critical mutation rate. Using Bonferroni's correction for multiple comparisons we obtained a new significance level \u03b1' = 0.0045. Therefore only organism C148 has a significant, positive correlation . But the differences in the use of a random or fixed initial fitness vector are clear and can be observed by comparing Figures Table The quasispecies model requires a series of conditions to be fulfilled. These requirements are related to four key factors: population size, mutation rate, genome size and neutrality ,23. In o1) The use of different initial fitness vectors for otherwise identical experiments results in unpredictable effects on critical mutation rates for different population sizes. These effects were not detected in the original experiments by Wilke et al.  and can 2) There is no significant correlation between genome size and critical mutation rate.3) The originally calculated critical mutation rates underestimate their real values.Despite the lack of a general correlation between critical mutation rate and population size, the comparison of Figures Another key factor in the quasispecies model is genome size. The establishment of the quasispecies is easier in populations with small genomes, as in these the number of neutral sites is reduced and therefore the neutral space is also smaller. However the relationship between critical mutation rate and genomic size to be expected is somewhat contradictory. On the one hand, larger genomes need higher mutation rates because the neutral and adaptive landscapes are larger. On the other hand, it is well known that large genomes require a stability not supplied by high mutation rates, hence the existence of an error threshold that will be discussed later. In fact Eigen  proposedOur correction to the critical mutation rates estimated in the original paper relates directly to the limits imposed by the mutation rate. In most cases Wilke et al.  obtainedgag region in isolates from different patients, an indication of the limited adaptive solutions able to produce escape mutants to the immune response of cytotoxic T lymphocytes [In conclusion, although some predictions from quasispecies theory are not fulfilled in our experiments, we do have observed the principal prediction that lower fitness competitors can win the competition to high fitness ones, but only under very high mutation rates. Recently, several papers ,23,30 haphocytes . Furtherphocytes .Analogies are very useful in science, but they have to be used cautiously. Similar features and dynamics between digital organisms and RNA viruses are tempting and usually lead to conclude that both kinds of entities are governed by the same laws. This is not necessarily the case, as practitioners of the comparative method know. In any case, digital organisms are an extraordinary system to experiment with controlled, repeatable evolution conditions and further work with them is necessary to ascertain which evolution features are of their own and which are of common application to other evolving entities.The project was started with the twelve pairs of organisms generated in a previous experiment  that hadWith these twelve pairs we followed the same experimental procedure designed by Wilke et al. . BasicalIn AVIDA organisms occupy the limiting environmental resource, the computer CPU, depending on their \"fitness\". In order to prevent the collapse of the system when all the competing organisms simultaneously try to use the CPU, there exists one feature designed to prevent the simultaneous replication of all organisms at the start of the competition, when all the organisms might be equally fit since they have not been tested yet in the environment. This is achieved by asynchronously introducing organisms in the competition system by assigning an initial fitness to each organism that introduces a small time lag in the accession to the CPU. For this, Wilke et al.  generateDuring exploratory experiments we noticed that this vector could play a decisive influence in the result of the competition. In consequence, we divided the study into two parts. In the first one we kept the vector assigned by Wilke et al.  to each The critical mutation rate is \"the midpoint between the highest rate at where A prevailed and the lowest rate where B prevailed\" . It reprIt is necessary to clarify the conceptual difference between the critical mutation rate and the error threshold. The first one is the rate at which the prediction of quasispecies theory that organisms with lower fitness can win the competition is fulfilled. However the error threshold is the genomic mutation rate beyond which the information in the molecules that compose the quasispecies loses sense due to mutational degeneracy . In pracWe used versions 1.4 and 1.6 of the AVIDA program. Basically, digital organisms are chains of instructions that act over the CPU with the objective of reproducing as fast as possible. In this manner the CPU time becomes the limiting resource in their evolution. During replication their genomes can mutate and, as a result, a system with variation and therefore with selection and evolution is obtained. Genome sizes of the twelve pairs of digital organisms varied between 54 and 272 instructions . Generally 50 generations were enough for the fixation of the A or B organism in the population. Configuration files used in these experiments are available from the authors web site .Each mutation rate-population size combination was replicated six times. For the verification of the relation between the population size and the critical mutation rate we used Pearson's correlation coefficient and a significance level of 5% using Bonferroni's correction . The samIC performed all the simulations, made the statistical analyses and wrote the first draft of the manuscript. AM contributed to the design of the experiments and the discussion of the results. FGC designed and supervised the experiments, discussed the results and their analyses and wrote the final version of the manuscript. All the authors read and approved the final manuscript."}
{"text": "When Darwin proposed the interplay between variation and natural selection as the driving force of evolution, he had no idea what material produced that variation. Ninety-one years later, Hershey and Chase's famous blender experiment identified the source of variation as DNA. Today, biologists are still struggling to elucidate the details of that interplay.Natural selection works on genetic variations that produce physical changes in an organism. Think of an organism as a collection of genes (its genotype), and its physical characteristics (its phenotype) as its genotype interfacing with the environment, a natural laboratory that saves or discards a genotype based on the performance of its phenotype. Assuming that a population of genotypes is well adapted to its environment, most mutations are likely to reduce performance in that environment. Thus, populations at equilibrium should experience selection for mechanisms that protect the phenotype against mounting deleterious mutations\u2014a phenomenon called mutational (or genetic) robustness. Most of the evidence that robustness arises from selection rather than chance comes from theoretical studies and from studies of \u201cdigital organisms\u201d\u2014computer programs that self-replicate, mutate, and evolve\u2014and has proved difficult to establish in the lab. In a new study, Rebecca Montville, Paul Turner, and their colleagues provide experimental evidence for adaptive genetic robustness by working with a mutation-prone virus that infects bacteria, called RNA phage \u03c86.  Though theoretical predictions for mutational robustness assume that phenotype expression results solely from the underlying genotype, many viruses can overcome their own mutational deficiencies by co-opting the proteins produced by more fit viruses co-infecting the same host, a feature called complementation.In a previous study, the authors demonstrated for RNA phage \u03c86 that complementation buffers less fit viruses against the harmful effects of mutations. They then created six replicate populations of phages and allowed them to adapt to their bacterial host over hundreds of virus generations; three populations evolved at a low ratio of infecting viruses to bacteria  and three at a high MOI. Populations at high MOI experienced higher rates of co-infection. In this study, the authors investigated the evolutionary consequences of this phenomenon with the hypothesis that selection for mutational robustness should be relaxed for co-infecting phages, since phenotype constancy is bolstered by co-infection with their fitter viral companions.Using clones from their six replicate populations, Montville et al. generated 60 new lineages and subjected them to a mutation accumulation experiment under conditions that allowed mutations to accumulate at roughly the same probability in high and low MOI lineages. The authors then evaluated the fitness consequences of mutation accumulation on the lineages by comparing their growth rate in the bacteria before and after mutation accumulation. The authors found greater variance in fitness change for the high co-infection lineages compared to the low-infection lineages, supporting the hypothesis that selection for mutational robustness is stronger in the absence of co-infection.The authors go on to rule out the notion that different fitness effects were produced by different mutation rates in the lineages. Interestingly, the less robust viral genomes were copied more accurately than their more robust counterparts; why less accurate genome replication might accompany the evolution of robustness is a question for future study.Liza GrossWhile complementation appears to buffer the damage of mutational onslaughts in the short-term, this benefit of co-infection eventually disappears because the buffer slows the rate that harmful mutations are culled from the virus population. The authors highlight an additional cost of co-infection: by weakening selection for robustness, co-infection may favor the evolution of genomes that are more vulnerable to the harmful effects of mutation. Future work is needed to examine this seeming tug-of-war between short-term and long-term consequences of co-infection, and its impact on the evolution of virus traits. \u2014"}
{"text": "Mutational robustness is defined as the constancy of a phenotype in the face of deleterious mutations. Whether robustness can be directly favored by natural selection remains controversial. Theory and in silico experiments predict that, at high mutation rates, slow-replicating genotypes can potentially outcompete faster counterparts if they benefit from a higher robustness. Here, we experimentally validate this hypothesis, dubbed the \u201csurvival of the flattest,\u201d using two populations of the vesicular stomatitis RNA virus. Characterization of fitness distributions and genetic variability indicated that one population showed a higher replication rate, whereas the other was more robust to mutation. The faster replicator outgrew its robust counterpart in standard competition assays, but the outcome was reversed in the presence of chemical mutagens. These results show that selection can directly favor mutational robustness and reveal a novel viral resistance mechanism against treatment by lethal mutagenesis. Understanding the conditions that favor the constancy of phenotypes in the face of deleterious mutation pressure\u2014mutational robustness\u2014is an outstanding question in evolutionary biology. Theoretical and in silico studies utilizing digital organisms predict that slow-replicating populations can outcompete those with higher individual fitness if the former show greater robustness. This \u201csurvival of the flattest\u201d hypothesis sits in contrast to most models of natural selection based on individual fitness, and hence challenges the \u201csurvival of the fittest\u201d paradigm. In this work, the authors use experimental populations of the rapidly evolving vesicular stomatitis RNA virus to provide the first evidence of natural selection for mutational robustness. Based on the analysis of fitness distributions, genetic variability, and the ability to tolerate mutation accumulation, two populations with different levels of robustness were characterized. At artificially enhanced mutation rates following the application of mutagens, the more robust viral population outcompeted the other population despite having a lower replication rate. This study has important implications for lethal mutagenesis\u2014an antiviral strategy that consists of increasing viral mutation rates through the use of mutagenic drugs\u2014since selectively favored mutational robustness may allow RNA viruses to evolve resistance to this form of treatment. Lethal mutagenesis consists of overwhelming viral populations with an excessive number of deleterious mutations and has been proposed as a candidate therapeutic strategy against RNA viruses \u20134. SeverRobustness to mutation determines the phenotypic expression of genetic variation and thus should be central to many evolutionary processes . InsofarThe conditions for the evolution of robustness have been experimentally explored in silico \u201317. SuchDue to their high spontaneous mutation rates , RNA virRhabdoviridae family. We characterized two populations adapted to a common environment but with different evolutionary histories. We studied the genetic variability and the individual fitness distribution of each population near the mutation-selection balance, and we estimated mutation rates and selection coefficients for deleterious mutations. This allowed us to conclude that one population was replicating near a high fitness peak, but was also less robust to mutation than the other. Whereas at spontaneous mutation rates, the fitter population was the best competitor, the addition of mutagens provided an advantage to the more robust, flatter, population.Herein, we provide evidence for the survival of the flattest using experimental populations of VSV, a lytic negative-stranded RNA virus of the 21) . The expected genetic variance for log-fitness among lineages is \u03c3G2(logtW) \u2248 dtEU[(log(1 \u2212 s))2], and the total variance is \u03c3T2(logW) = \u03c3E2(logW) + \u03c3G2(logW) where sub-index E refers to environmental variance. Since genetic variance is null for the ancestor and all fitness assays were performed in the same environmental conditions, \u03c3E2(logtW) = \u03c3E2(logW0) = \u03c3T2(logW0), and therefore \u03c3G2(logtW) = \u03c3T2(logtW) \u2212 \u03c3T2(logW0) = \u0394\u03c3T2(logW). It follows that\u03b8 is the coefficient of variation (standard deviation to mean ratio) of log W associated to single deleterious mutations. (1 + \u03b82)E[log(1 \u2212 s)] and dU/(1 + \u03b82) were directly estimated from the data.The expected change in log-fitness after \u03b8 = 1. Nonetheless, the actual distribution of mutational effects might have a heavier tail and thus a \u03b8 > 1. A previous fitness dataset of 28 nonlethal random mutants of VSV  and then, of s. For A, \u03b8 = 1.598 should be an accurate estimation of the true \u03b8-value, because it was obtained for the above-mentioned full-length infectious cDNA clone. It is possible, however, that, due to its higher mutational robustness, B showed a lower \u03b8-value. This potential bias could account for the slightly higher dU estimate in B, but it could not account for the nearly one order of magnitude difference in s-values .For an exponential distribution (a simple and relatively accurate model for describing fitness effects associated to single mutations), s of VSV  gave thedU and s between A and B were statistically significant, we generated 1,000 bootstrap pseudo-replicates from both the 24 averaged fitness values of the derived clones and the six averaged fitness values of the ancestors. After obtaining the corresponding 1,000 pseudo-replicates of dU, we counted the number of times dU was larger for A versus B, and the same for s.To address whether differences between dU and s were also estimated using the maximum likelihood approach implemented in the program MLGENOMEU (http://homepages.ed.ac.uk/eang33/mlgenomeu/mlginstructions.html), in which a Gamma distribution of the form Ga(s) = \u03b1\u03b2s\u03b2\u2212 1e \u2212 s\u03b1/\u0393(\u03b2) is used to describe the distribution of mutational effects (s =\u03b2/\u03b1) [dU and \u03b1 at fixed \u03b2-values varying from 99 to 0.01. The log likelihood monotonically increased as \u03b2 decreased, yielding increasingly higher \u03b1-values (s \u2192 \u221e and dU \u2192 0).Parameters (s =\u03b2/\u03b1) . Followihttp://www.microsoft.com) and the SPSS 12.0 package (http://www.spss.com). Resamplings and simulations were done using a Perl script.Statistics were done with MS Excel (http://www.ebi.ac.uk/cgi-bin/emblfetch) database accession numbers for the A and B evolved populations are AM690336 and AM690337, respectively. For nonmutagenized populations, accession numbers are AM689332\u2013AM689519 and for 5-FU mutagenized populations, accession numbers are AM689705\u2013AM689876.The European Molecular Biology Laboratory (EMBL) ("}
{"text": "The most consistent result in more than two decades of experimental evolution is that the fitness of populations adapting to a constant environment does not increase indefinitely, but reaches a plateau. Using experimental evolution with bacteriophage, we show here that the converse is also true. In populations small enough such that drift overwhelms selection and causes fitness to decrease, fitness declines down to a plateau. We demonstrate theoretically that both of these phenomena must be due either to changes in the ratio of beneficial to deleterious mutations, the size of mutational effects, or both. We use mutation accumulation experiments and molecular data from experimental evolution to show that the most significant change in mutational effects is a drastic increase in the rate of beneficial mutation as fitness decreases. In contrast, the size of mutational effects changes little even as organismal fitness changes over several orders of magnitude. These findings have significant implications for the dynamics of adaptation. In any population, two factors determine whether the average fitness of individuals will increase (adaptation) or decrease: the size of the population and the distribution of mutational effects . Although it is relatively simple to get quantitative information on population size, it is much harder to gain insight into the distribution of mutational effects. Very little information exists on the relative rates of beneficial versus deleterious effects, on the shapes of mutational distributions, or on whether the distributions change over time. Thus, it remains difficult to even speculate whether a given population will adapt over time. Here, we use laboratory evolution of a bacterial virus to quantify the distribution of mutational effects. Our results reveal that the average impact of a mutation is approximately constant with respect to fitness, that most mutations have small effects, and that the rate of beneficial mutation depends on the fitness of the organism. Our study demonstrates the simple, but perhaps underappreciated fact that mutational effects are dynamic. It also proposes and tests an explicit model of adaptation in which organismal fitness specifies both the rate and distribution of deleterious and beneficial mutations, and it presents specific and testable predictions of the circumstances under which populations will adapt. Experimental evolution of bacteriophage reveals that mutational effects are dynamic and dependent on genetic background, thus providing fundamental and testable insights into the nature of adaptation. The process of adaptation in finite populations is determined by the distribution of mutational effects on fitness and the size of the population. In large populations, beneficial mutations are frequently fixed, whereas deleterious mutations will only be fixed if they are of vanishingly small effect size (approximately the inverse of the effective population size ). In smaAn accurate description of the distribution of mutational effects is thus fundamental to understanding how adaptive changes occur in biological populations. Experimental manipulation of organisms in the laboratory  \u201315 and gImportantly, the rate and effect size of beneficial and deleterious mutations are not constant. These quantities change if the fitness of the evolving organism changes. One manifestation of this effect is the fitness plateaus reached by populations adapting to constant environments ,32,33. TThere is an analogous process that occurs in populations that experience decreases in fitness. This process is equally important for evolutionary dynamics, but has received less attention. When populations are so small that drift overwhelms selection, deleterious mutations frequently fix ,22,23, cWe used experimental evolution of a bacteriophage to investigate how the distribution of beneficial and deleterious mutations changes  when fitness changes in the course of evolution, and how this impacts adaptation. We corroborate the finding that in large populations, fitness increases to a plateau. We also report the complementary effect that in small populations, fitness does not decrease indefinitely, but reaches a lower plateau. The equilibrium level of fitness is thus largely determined by the effective population size, and we show that this is a consequence of simple changes in mutational effects that cause qualitative changes in the processes of selection and drift.To study the impact of population size on adaptation, we used experimental evolution in the bacteriophage \u03c6X174, an organism in which population sizes can be controlled by adjusting the bottleneck size at transfer. We used phage populations to investigate two related questions: First, do populations evolve to a fitness equilibrium? Second, do populations that differ in effective population size reach different fitness equilibria? As a measure for fitness, we used competition with a reference strain . Because4). Two lower fitness ancestors were derived by serially bottlenecking this clone and selecting small plaques. This procedure resulted in two strains that had low fitness in competition with the ancestral strain.To identify fitness equilibria, we initiated experimental populations with either an ancestor of high fitness or an ancestor of low fitness. We then investigated whether the populations converged toward the same fitness value, irrespective of their starting condition. If populations that were initialized with high- or low-fitness ancestors converged in fitness during evolution, the evolved fitness levels of these populations were concluded to bracket a fitness equilibrium. The high-fitness ancestor was a clone derived from a phage line that had been passaged in our laboratory for 100 transfers at a large population size , two maintained almost constant fitness (Ne = 10), and one declined only slightly in fitness (Ne = 3) (Ne = 30 and Ne = 100), five of the six evolved to a mean fitness above that of the high-fitness ancestor. It was thus difficult to ascertain whether these populations were evolving towards an equilibrium fitness. To establish whether these larger populations were evolving toward an equilibrium, we took the Ne = 100 population that had evolved the highest fitness and propagated it at an expanded population size of Ne = 250 for an additional 55 transfers (275 generations). Three Ne = 250 population replicates were propagated, and none changed significantly in fitness  . In the  fitness . This laAlthough care was taken to minimize the opportunity for cross-contamination between populations, it was important to directly rule out that contamination was responsible for the fitness convergence. To ascertain this, we sequenced the complete genome of one clone from each evolved population. Convergence of substitutions at a large fraction of sites would imply contamination. However, the number of sites at which convergence occurred was not significantly greater than expected by chance see , so crosThe sequence data revealed that the fitness convergence was achieved despite little indication of genetic convergence. Hence our system reproduced one previously observed phenomenon in experimental evolution: fitness in a large, adapting population does reach a plateau with different starting genotypes. Interestingly, our results show that this effect can be extended to small population sizes: small populations converge to low-fitness equilibria without genetic convergence.compensatory) when organismal fitness decreases  change as fitness changes. For simplicity, we illustrate the case in which the effect on fitness of all beneficial mutations is identical, as is the effect on fitness of all deleterious mutations . The substitution rates of deleterious and beneficial mutations are thus:Ne is the population size, ud is the deleterious mutation rate, ub is the beneficial mutation rate, pd = (1 \u2212 es2)/(1 \u2212 eNs2) [sd, and pb = (1 \u2212 es\u22122)/(1 \u2212 eNs\u22122) is the probability of fixation of a beneficial mutation of effect size sb. If a population is reduced in size such that fitness begins to decline, by definition,We focus on testing these two models here, by using an explicit model to describe how epistatic changes affect fitness convergence and equilibria. In this model, the deleterious and beneficial substitution rates  =  \u2212 e2s/. This required determining the ratio of beneficial and deleterious mutations, and the distribution of the effects of these mutations. To estimate these quantities, we used MA. In an MA experiment, mutations are allowed to accumulate by randomly collecting the progeny of a parental phage with no bias against low-fitness progeny. By measuring the fitness of a number of parents and a number of randomly chosen progeny, it is possible to estimate the fitness effects of mutations that occur in a single generation. We determined the fitness of 50 parents and 50 progeny for three high-fitness populations and three low-fitness populations . If negas, and the proportion of beneficial mutations, which we refer to as B, and define as ub/(ub + ud). Importantly, this analysis assumes that the distribution of mutational effects can be modeled as a reflected gamma distribution. The gamma distribution is used because it has great flexibility in terms of the shapes it can assume, with shapes ranging from highly leptokurtic to exponential to log-normal to Gaussian (We used maximum-likelihood (ML) analysis  to estimGaussian . Howevers and B requires an estimate of the mutation rate (U). We derived this estimate from sequence data rather than the ML analysis to avoid conflating estimates of s and U [Ne = 3 and Ne = 10 to estimate the neutral mutation rate.  The average rate of synonymous substitution in these lineages was 0.18 per transfer. We extrapolated this rate to a genomic rate by multiplying by the ratio of total sites to synonymous sites in the ancestral genome while accounting for a proportion of lethal mutations . Although the estimate was slightly lower in the high-fitness populations (0.29 as compared to 0.40), this difference was not significantly different between the two groups . The joint ML estimate of B for all low-fitness populations was 16%, and was not significantly different between these populations  A. The esB for all the evolved populations. For a certain Ne and a specified distribution of s, it is possible to use numerical simulations to find the value of B at which mean population fitness no longer increases or decreases. Using the above estimate for the shape of the distribution of mutational effects, we could thus obtain an estimate of B at which each of the evolved populations should not increase or decrease in fitness , suggested that B must increase to approximately 18% to maintain fitness equilibrium in the Ne = 3 populations. We used additional simulations to derive the value of B required for all other population sizes to maintain fitness equilibria. We then used the median values of the experimentally determined equilibrium fitness values at each Ne  allows low-fitness populations to reach a fitness equilibrium. There are two different ways in which this ratio can increase, either through an increase in ub, the rate of beneficial mutations, or through a decrease in ud, the rate of deleterious mutations. The two alternatives have different biological meanings. An increase in ub with decreasing fitness would correspond to the hypothesis of compensatory epistasis. A decrease in ud, the rate of deleterious mutations, could result if a large fraction of deleterious mutations become lethal in less fit genotypes. In our experimental regimes, the number of lethal mutations was not directly measured, and it was thus important to test for such an increase directly.The results of the MA experiment and the above analysis suggest that an increase in the ratio between the rates of beneficial and deleterious mutations U, which is the sum of ub and ud. We derived an independent measure of U based on the total number of nucleotide substitutions that occurred in all lines; we used a ML analysis to determine the U that best fit the observed number of substitutions in each population . There is thus no evidence that low-fitness populations have lower U, as would be expected if the proportion of lethal mutations had increased in these populations. The new joint ML estimate of U using the nucleotide substitution data was 0.64. This closely matched our first estimate of 0.56, which relied only upon the number of synonymous substitutions observed in the Ne = 3 and Ne = 10 lines. Again, this suggests that our results are robust and internally consistent, and that increases in ub, and not a change in the genomic mutation rate, accounted for the observed changes in B . Conversely, if a population is decreasing in fitness, to stop this decrease, one or more of the following must occur: the mean effect size of beneficial mutations must increase; the rate of beneficial mutations must increase (and consequently the rate of deleterious mutations must decrease); or the effect size of deleterious mutations must increase.This distribution of mutational effects has significant implications for adaptation, because the direction and rate of fitness change in any population is governed by four quantities intrinsic to the organism: the rate and mutational distribution of beneficial mutations and the rate and mutational distribution of deleterious mutations. As shown above, that populations at a specific effective population size converge toward a single level of fitness necessarily constrains the values and dynamics of these quantities. In a population in which mean individual fitness is increasing, the rate of fixation of beneficial mutations multiplied by the mean effect size must outweigh this analogous quantity for deleterious mutations. In order for the rate of adaptation to decrease as fitness increases, one or more of the following must occur: the mean effect size of beneficial mutations must decrease; the rate of beneficial mutations must decrease (and consequently the rate of deleterious mutations must increase); or the effect size of deleterious mutations must decrease . Although the distribution differs slightly from others that have been recently proposed, it concurs well with others [To untangle the different possibilities, we performed MA experiments on lines differing by approximately 300-fold in fitness to characterize the distribution of deleterious mutation effects on fitness . This dih others . We alsoThe deleterious mutation distributions found do not provide significant support for the hypothesis that the effect size of deleterious mutations changes with fitness  , despiteB, the proportion in beneficial mutations, is not driven by a decrease in the deleterious mutation rate . This is shown through an analysis of the molecular data and is also suggested by the magnitude of change: it is likely that the beneficial mutation rate changes by much greater than 10-fold between the high- and low-fitness lines. A comparable change in the overall mutation rate would be easily detected; indeed, it is very unlikely that the rate of lethal mutation in the low-fitness lines increased more than 2-fold  and log-fitness is noteworthy. Because the shape of the mutational distribution does not change with fitness, log-fitness should, on average, scale linearly with the number of deleterious mutations that an individual has. Under the simplest mutational model, deleterious mutations interact multiplicatively i), and each deleterious mutation would result in a single beneficial mutation becoming available\u2014the back mutation. Because the expected value of log-fitness and the number of deleterious mutations scales linearly (log(W) = i*log(1 \u2212 s)), and the number of deleterious mutations scales linearly with the number of beneficial mutations (in a 1:1 manner), then log-fitness should also scale linearly with the beneficial mutation rate. This is true under any model in which each deleterious mutation results in a constant average number of newly available beneficial mutations. The nonlinearity of the relationship between B and fitness thus implies that the number of beneficial (compensatory) mutations that become available in the context of each additional deleterious mutation increases as fitness decreases. This suggests that there may be changes in the number of loci involved in compensatory mechanisms as fitness declines. One interpretation of this is that pleiotropic interactions between genetic loci (functions) increase as fitness decreases. Although a negative relationship between the rate of compensatory mutation and fitness has been predicted by modeling, little has been predicted concerning pleiotropy. We hope that our results will spur new interest in this topic.The nonlinear relationship that we observed between This study is the first to propose and test an explicit model of adaptation in which organismal fitness specifies both the rate and distribution of deleterious and beneficial mutations. Although the model necessarily relies upon an idealized form of the genotypic landscape, it presents specific and testable predictions of the circumstances under which populations will increase or decrease in fitness.Additionally, the model offers a simple mechanistic explanation for the diminishing rate of fitness increase observed within large populations adapting in a constant environment , as wellEscherichia coli (mutT), obtained from D. Bregon at INSERM.The original \u03c6X174 bacteriophage used in this study was derived from a clone kindly provided by B. Fane. This clone was passaged for 100 transfers at 32 \u00b0C to allow general adaptation to laboratory conditions. The deleterious mutants were obtained through serial bottlenecks with mutagenesis (see below), during which small plaques were purposefully chosen. The host was E. coli (mutT), and then grown overnight at 32 \u00b0C. From these plates, a number of plaques equal to the bottleneck size were randomly selected and diluted into culture tubes containing 3 ml of 1 mM EDTA. These tubes were vortexed and centrifuged, after which 0.5 ml was removed to a fresh Eppendorf tube. Chloroform was added, the tubes were vortexed and centrifuged, and 0.3 ml was removed. This was used for further mutagenesis, and the remainder of the stock was stored in 40% glycerol at \u221220 \u00b0C.During passaging, phage were mutagenized in 250 mM hydroxylamine (HA) , 1 mM EDCompetition experiments were designed and performed as described previously in Burch and Chao . BrieflyNe = 100 populations  as a reflected gamma distribution [U, the distribution of s, and B, we fixed U based on the estimate from the synonymous substitution rates in the small bottleneck lines (see below). This independent estimate of U allowed greater confidence in the estimates of the distribution of s and B. The likelihood analysis assumes a normally distributed measurement error; the estimate of this error was allowed to vary between experiments, because all six MA experiments were not conducted simultaneously . For the analysis, log-transformed values of fitness were used, because without this transformation, error was not normally distributed. Additionally, the means of the ancestral  generation were set to one  for the analysis. After the analysis, the parameters were used to calculate a gamma distribution, and this distribution was then re-transformed to obtain the standard mutational distribution (transformation: s = 1 \u2212 10s\u2212). The maximum likelihood shape and size parameters for the mutational distribution values were 5.3 and 1.0, respectively.The ML model used models the distribution of selective coefficients ribution ,45. AlthB, 0 in the high-fitness lines and 0.16 in the low-fitness lines).The likelihood surfaces were generated by calculating the likelihood values over a grid of parameter values while maintaining the other parameters at their ML-estimated values  and the amount of over-dispersion of substitutions to infer an S, of the synonymous cytosine \u2192 thymine transitions result in a nonviable phenotype, the over-dispersion is decreased. We fit the \u03bbS parameter by minimizing the \u03c72 goodness-of-fit statistic; the value of this statistic is approximately equal to the probability of observing the data. We estimate \u03bbS = 0.14, with 95% confidence limits ranging from \u03bbS = 0 to \u03bbS = 0.40. A similar analysis may be done with non-synonymous missense sites. During experimental evolution, a total of 141 missense cytosine \u2192 thymine substitutions at 120 different sites occurred. We removed one of these sites from the analysis, because the substitution occurred in more than four independent lines, and was thus likely subject to strong positive selection. The estimate of \u03bbM was 0.37, with 95% confidence limits ranging from \u03bbM = 0.01 to \u03bbM = 0.61. Finally, we examine nonsense mutations. Of the 560 substitutions observed over all lines during the period of evolution, only one was a nonsense mutation, although the expected frequency is 9%. We thus estimate that \u03bbN is essentially 1. All of these estimates are similar to previously estimates values in other viruses, with \u03bbS estimated at 0.11 and \u03bbM at 0.40 [We base the estimate of the mutation rate on the substitutions observed in the populations propagated at the smallest effective population sizes, because mutations appearing in these lineages should be least subject to selection. Within these lineages, a total of 162 synonymous or intergenic cytosine \u2192 thymine substitutions occurred at 117 different sites. These substitutions are slightly over-dispersed when compared to the random expectation, although not significantly. If we assume that some proportion, \u03bbS = 0.14, this suggests that substitutions can occur at 265 of these sites. Similarly, with 736 cytosines at which cytosine \u2192 thymine substitutions are non-synonymous, and an estimated \u03bbN = 0.37, this suggests that substitutions can occur at 466 of these sites. The ratio of total substitutable cytosine sites to substitutable synonymous sites is 2.76, implying a genomic cytosine \u2192 thymine mutation rate of 0.50 per transfer. Finally, because cytosine \u2192 thymine substitutions account for only 89% of all substitutions, we estimate the total effective genomic mutation rate as 0.56 per transfer.We calculated the effective mutation rate using these parameters. The genome of the ancestral \u03c6X174 clone contains 1,151 cytosines; at 309 (27%) of these residues, a transition from cytosine to thymine is synonymous or intergenic; at 736 (64%), a transition is non-synonymous; and at 106 (9%), a transition results in a change to or from a stop codon. The observed rate of synonymous cytosine \u2192 thymine substitution was 0.18 per transfer. With a total of 309 cytosines at which this transition is synonymous or intergenic, and an estimated \u03bbS includes zero.)If cross-contamination occurred, one would expect that two lines would share substitutions at a large number of sites (thus significantly increasing over-dispersion). This is clearly not the case for synonymous substitutions. . Thus the bottleneck population sizes are an accurate characterization of the effective population size of the phage populations. Secondly, even if individual phage were tracked, this would give rise to similar rates of fixation , and the following equation was numerically solved for each population size:B is the fraction of beneficial mutations, u(s) is the ML gamma distribution of mutational effects, and pfix is the probability of fixing a mutation of effect s in a haploid population of size N: (1 \u2212 es\u22122)/(1 \u2212 eNs\u22122) [We modified an individual-based model of adaptation  for the tion see . The two size N:  \u2212 e\u22122s/ Confidence limits for the ML parameter values of the gamma distribution in the low-fitness clones. The proportion of beneficial mutations, B, was left at its ML value of zero.(B) Confidence limits for the ML parameter values of the gamma distribution in the high-fitness lines. The proportion of beneficial mutations, B, was left at its ML value (0.16 or 0). The joint log-likelihood value is indicated to the right of each panel; the ellipses indicate the 50%, 80%, 90%, and 95% confidence limits. The y-axis shows the estimated mean of the distribution, while the x-axis is the coefficient of variation (1/\u221a\u03b2). The estimated mean of the distribution is relatively constant for the two groups, providing little support for any hypothesis under which the mean value of the selection coefficient changes with fitness. However, there is less confidence in the shape of the distribution (beta).(C) Confidence limits for the ML parameter values of the gamma distribution in all MA lines. The proportion of beneficial mutations, (146 KB PDF)Click here for additional data file.Figure S2(A) Confidence limits for the ML parameter values of the gamma distribution in the low-fitness clones. The value of beta was kept constant, at one.y-axis shows the estimated mean of the distribution, while the x-axis indicates the estimated proportion of beneficial mutations (B).(B) Confidence limits for the maximum likelihood parameter values of the gamma distribution in the high-fitness lines. Again, the value of beta was kept constant, at one. The (100 KB PDF)Click here for additional data file.Text S1(26 KB DOC)Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank) accession numbers for the genome sequences of the bacteriophage \u03c6X174 lineages discussed in this paper are EF380009\u2014EF380032.The GenBank ("}
{"text": "Escherichia coli and measured its effect on adaptation at high and low mutation rates. Recombination increased the rate of adaptation \u223c3-fold more in the high mutation rate treatment, where beneficial mutations had to compete for fixation. Sequencing of candidate loci revealed the presence of a beneficial mutation in six high mutation rate lines. In the absence of recombination, this mutation took longer to fix and, over the course of its substitution, conferred a reduced competitive advantage, indicating interference between competing beneficial mutations. Together, these results provide experimental support for the Fisher\u2013Muller model and demonstrate that plasmid-mediated gene transfer can accelerate bacterial adaptation.Identification of the selective forces contributing to the origin and maintenance of sex is a fundamental problem in biology. The Fisher\u2013Muller model proposes that sex is advantageous because it allows beneficial mutations that arise in different lineages to recombine, thereby reducing clonal interference and speeding adaptation. I used the F plasmid to mediate recombination in the bacterium  Why have sex? One explanation is that sex is good because it allows beneficial mutations from different lineages to recombine. This reduces competition between mutations in a population and can increase the speed with which the population can adapt to environmental change. This explanation, known as the Fisher\u2013Muller model, has extensive theoretical support; however, it is difficult to test experimentally. Using a simple microbial system I showed that recombination increased the rate of fitness improvement when beneficial mutations were common in the population and had to compete for fixation, but had little effect when mutations occurred rarely. Sequencing of candidate genes revealed the presence of the same beneficial mutation in a number of replicate populations. In the absence of recombination, this mutation took longer to spread and conferred a lower overall competitive advantage, indicating interference between competing beneficial mutations. Together, these results provide direct experimental support for the Fisher\u2013Muller model. Why there is sexual reproduction is an important question. Here the authors show that making bacteria have sex allows faster accumulation and fixation of beneficial mutations. Understanding the factors that contribute to the origin and maintenance of sex is an important unanswered problem in evolutionary biology \u20134. A larThe Fisher\u2013Muller (FM) model proposes that the advantage of sex results from recombining competing beneficial mutations into one lineage ,14. In tDespite extensive theoretical support for the FM model, the ability of the model to predict the effect of recombination in biological systems is subject to at least two caveats. First, an advantage of recombination depends on competition between beneficial mutations arising in different lineages being strong enough that the fixation of the higher fitness mutation is appreciably slowed. If that is not the case, competition will not effectively limit the rate of adaptation, and recombination will have little effect . ResultsAn ideal experimental test of the FM model would compare the effect of recombination on the rate of adaptation in treatments that differ solely in the extent of competition between beneficial mutations. In practice this ideal has been hard to achieve. A number of experimental studies have examined aspects of the relationship between recombination and environment \u201312. ThesmutS, was shown to control the rate of adaptation in evolving populations of the bacterium Escherichia coli by increasing the rate at which new mutations were produced by \u223c30-fold. By using the same strains and environmental conditions, I define high and low mutation rate treatments that differ only in the supply of new mutations and thus the degree of competition between beneficial mutations. All other aspects of the environment, including population size, remain constant. To permit recombination I use recombination proficient (rec+) strains made by introducing the F plasmid into the ancestral strains used by de Visser et al.  \u00d7 34.9 \u00d7 [4.64 \u00d7 106 bp])\u2014well short of the genomic mutation rate of \u223c1 required by the mutational deterministic model to explain an advantage to recombination. Also, there is no general tendency for deleterious mutations to interact synergistically in this strain, a second prerequisite of the mutational deterministic model [The results presented above are consistent with the FM model, whereby recombination provides an advantage when multiple beneficial mutations compete for fixation by combining them in the same lineage. However, a possible alternative explanation is that recombination was advantageous because it separated beneficial mutations from linked deleterious mutations ,33\u201337. Bckground ,35. Thisckground . Thus, tneration . Multiplic model .In the light of the alternative mutation load explanation for the observed benefit of recombination, I sought to directly examine the prediction made by the FM model that recombination reduces competition between beneficial mutations. To do this, I compared the dynamics of a focal beneficial mutation as it rose in frequency and ultimately fixed in rec+ and rec\u2212 populations. A decrease in the fitness conferred by a focal beneficial mutation, relative to contemporary clones that did not have this mutation, would be consistent with the spread of alternative beneficial mutations in competing lineages.spoT, that contributed to adaptation of E. coli to an environment nearly identical to the one used here [spoT in three randomly chosen clones isolated from each of the 16 populations in the high mutation rate treatment and found mutations in four rec+ and two rec\u2212 lines. The temporal dynamics of each mutation were examined by screening clones at regular intervals from stored samples of the evolved lines. spoT (spoTEv) alleles were very different in the rec+ and rec\u2212 lines. The time between detection and fixation averaged 300 generations in rec+ lines and 900 generations in rec\u2212 lines . Thus, a beneficial mutation in the same gene took longer to fix in the absence of recombination.A previous study found a mutation in a regulatory gene, sed here . This gespoTEv alleles to fix in rec+ and rec\u2212 lines was due to an increase in competition, the selective advantage conferred by each allele was measured at different time points during its substitution. I isolated two clones that had spoTEv alleles from three of the rec+ and two rec\u2212 lines that fixed a mutation in this gene , and estimated their fitness relative to five contemporary clones that were isolated from the same line and time point, but that did not have the focal mutation   , the average advantage conferred by focal = 0.878) . To testh slower . Here, the other ; Table 2= 0.322) . No signmutation . TherefospoTEv mutations became negative, such that additional beneficial mutations must have occurred on the same background in order for the focal mutation to fix. The possible existence of linked deleterious mutations cannot explain this result by itself, because the deleterious mutations would impose a constant cost to the focal mutation. By contrast, in the rec+ lines, the relative advantage conferred by the spoTEv mutations showed no overall change, indicating that recombination effectively reduced the effect of competition between beneficial mutations. This reduction in competition is consistent with recombination bringing competing beneficial mutations together into one lineage, the mechanism proposed by the FM model.The findings presented above indicate that multiple beneficial mutations were present in the evolving populations and that, as required by the FM model, in the absence of recombination, competition between these mutations was associated with slower fixation times. Indeed, this competition was so strong that in the rec\u2212 lines, the relative effect of the focal spoTEv beneficial mutation in one rec\u2212 line, consistent with at least one additional beneficial mutation having arisen and reached an appreciable frequency within this lineage  . Third, the advantage conferred by spoTEv alleles in the competitions carried out against contemporary clones is substantially lower than the advantage of \u223c9.4% seen in competition with the ancestral strain (cf. [s = \u22120.014) [Three additional lines of evidence support the interpretation that competition between beneficial mutations was a significant factor in the adaptation of the high mutation rate lines. First, there was a significant difference in relative fitness between the two clones containing a fixation   .w(gen), prevailing in the rec+ lines at the time when the focal mutations arose. For deleterious mutations to play an influential role in limiting the spread of these mutations, the benefit they confer must be small relative to this variance (s \u226a 6\u03c3w(gen) [w(gen) in the rec+ lines, based on those clones that did not have the focal spoTEv mutation, when this mutation was at a low frequency. This measure is conservative because it combines differences between clones due to beneficial as well as deleterious mutations. I found that the genetic variance in fitness was clearly sufficient to influence the spread of the focal mutation in only one of the three rec+ populations  ). Removiulations . TherefoThe results reported in this study have important implications for bacterial adaptation. Competition between different lineages of the same species may be common in certain environments, for example, in clinical settings where adaptation to novel hosts can select for strains with high mutation rates ,43. In tIn summary, I found that recombination and mutation rate interact with each other in determining the speed of adaptive evolution. This finding supports the FM model for the evolution of sex, demonstrating that recombination increases the rate of adaptation only when competing beneficial mutations are present in the population. Also, I was able to identify a beneficial mutation that contributed to adaptation in a number of evolved populations. Comparing the dynamics of this mutation across recombination treatments allowed me to demonstrate directly that recombination reduced the amount of interference between a focal beneficial mutation and other competing beneficial mutations. This reduction in competition shortened the time needed for the focal mutation to fix in the population, providing an explanation for the observed benefit of recombination.E. coli genome encompassing the lac operon. This plasmid was chosen because this region of homology increases the rate at which the plasmid recombines into the host chromosome and therefore the frequency of chromosomal gene transfer. To make a rec\u2212 derivative of this plasmid, a non-polar traD deletion was introduced using a PCR-based approach [4-fold and reduced the frequency of chromosomal gene transfer to undetectable levels [traD deletion affected this cost, I performed a competition assay (see below) to measure the relative fitness of identical host cells carrying either the F plasmid or the F \u0394traD derivative. This assay found that the cost of carriage was reduced slightly, but not significantly, by the traD mutation .An F plasmid was obtained from the Coli Genetic Stock Center , as an isolate from strain K603 (CGSC#6451). This plasmid, designated F1\u201310, encodes resistance to tetracycline and contains a portion of the approach . This mue levels . This gee levels  and has e levels . The F pe levels . To testE. coli B, REL606, was used as the host bacterium in the low mutation rate lines [mutS, mutS::Tn5, into REL606 [mutS allele does not have any measurable effect on fitness [mutS::Tn5 were made by using standard methods to separately introduce F and F\u0394 traD plasmids into both backgrounds to create the four ancestral strains used to found the evolution experiment. REL607, a spontaneous mutant of REL606 that is able to utilise arabinose, and a spontaneous nalidixic acid (Nx) resistant mutant of REL606 were obtained and used to allow selection of different strains in control assays designed to measure recombination rates (see below). Control experiments found that the rate of recombination was not affected by the mutS::Tn5 allele.A previously described strain of te lines . This sto REL606 . Disrupt fitness . Rec+ anhttp://www.qiagen.com/) with shaking at 150 rpm. Each well was filled with 1 ml of medium. LB broth was used to grow cells from \u221280 \u00b0C freezer stocks. Davis minimal medium supplemented with glucose at 25 mg/l (DM25) was used at all other times.All incubations were carried out at 37 \u00b0C in 96 \u00d7 2-ml blocks . These strains were inoculated from frozen stocks into LB medium and incubated for 24 h. Strains were then diluted 100-fold and 5 \u03bcl inoculated separately into DM25. Following a further 24 h of incubation, the two strains were mixed 1:1 and diluted 1:100 into fresh DM25 medium. After 24 h of co-incubation, cells were plated on minimal arabinose plates supplemented with Nx. Only cells that had recombined the Nx resistant and Ara+ markers could grow on this plate. To estimate the total amount of horizontal gene transfer, the number of recombinant cells was first multiplied by the number of genes separating the two markers. Assuming that only half of these transmitted genes become incorporated into the recipient cell's chromosome  and thatmutation ,38. It itraD) , REL606 mutS::Tn5 (F) , and REL606 mutS::Tn5 (F \u0394traD) . All lines were propagated in 1 ml of DM25 medium in 96 \u00d7 2-ml blocks. Each day 5 \u03bcl of culture was transferred to 1 ml of fresh medium, allowing \u223c7.64 generations per day (= log2 200). This environment and strain combination corresponds to a relative mutation supply  of \u223c2.9 in the low mutation rate lines and \u223c101.1 in the high mutation rate lines with respect to The evolution experiment consisted of eight replicate lines started with each of four ancestral strains: REL606 (F) , REL606 /NE(0))/ln(NA(1)/NA(0)), where NE(0) and NA(0) represent the initial densities of the evolved and ancestral strains, respectively, and NE(1) and NA(1) represent corresponding densities at the end of the competition. All competitions between ancestral and evolved clones were carried out with 10-fold replication.The fitness of evolved strains relative to the ancestor was assayed by competitions carried out in the same conditions prevailing during the evolution experiment. All evolved strains were Ara\u2212, to allow these strains to be distinguished from their ancestors; derivatives of the ancestral strains were selected that were Ara+. These two marker types can be distinguished by plating on tetrazolium arabinose indicator medium. On this medium, cells that can utilise arabinose form white colonies, whereas cells that cannot use arabinose form red colonies. The arabinose marker is selectively neutral in the evolution environment . Before spoTEv allele were performed similarly. All clones were initially Ara\u2212. To allow the two types to be distinguished from one another, spontaneous Ara+ revertants were selected from those clones that had the spoTEv allele and used in competitions. These competitions were carried out over two or four transfer cycles to increase the precision of fitness estimates. Competitions were carried out with 3-fold replication.Competitions involving evolved clones that did and did not have the spoT including upstream regulatory regions. Purified PCR products were sequenced on an Applied Biosystems (http://www.appliedbiosystems.com/) 3130XL capillary sequencer. Three clones were assayed at the final time point in each high mutation rate line. I sequenced spoT in three randomly chosen clones isolated from each of the 32 lines in the high and low mutation rate treatments and found mutations in four rec+ and two rec\u2212 lines in the high mutation rate treatment and one rec\u2212 line in the low mutation rate treatment. In one high mutation rate treatment rec\u2212 line, the spoTEv allele had not fixed by 1,000 generations. This line was continued for a further 300 generations, after which time the line was screened again and the allele was found to have fixed. The mutations found all caused amino acid substitutions in a region of SpoT involved in the synthesis of the \u201calarmone\u201d molecule (p)ppGpp [Primers were designed to amplify overlapping fragments of the gene (p)ppGpp , but no spoTEv allele isolated from early time points and (ii) there was no effect of sample time (early versus late) on the relative fitness of clones having a spoTEv allele in the absence of recombination. In all cases denominator degrees of freedom was estimated using a Satterthwaite approximation.Mixed models were run to test the interaction between recombination and mutation rate, with replicate evolved line nested within recombination and mutation rate treatments. Mixed models were also used to test the null hypotheses that (i) there was no effect of recombination treatment on the relative fitness of clones having a Table S1(30 KB DOC)Click here for additional data file.Table S2(30 KB DOC)Click here for additional data file.Table S3(29 KB DOC)Click here for additional data file."}
{"text": "In the 1930s, a flock was relocated to Hirta, an adjacent island in the St. Kilda archipelago, after villagers evacuated to the mainland. Since then, the sheep have been left mostly to their own devices, save for ongoing encounters with scientists keen to test a range of evolutionary hypotheses on this unique population.No one knows for sure when or how they got there, but Soay sheepMark-and-recapture studies have revealed important clues to the ecology of the population, which periodically crashes, typically when a severe winter follows a robust reproductive season. These studies have also generated a wealth of information on birth and death rates, reproduction, pedigree , and phenotype (such as birth weight).Alastair Wilson, Loeske Kruuk, and colleagues took advantage of a 20-year study of birth weight in Soay sheep\u2014which covered nine generations and significant climatic fluctuations\u2014to examine the impact of environmental quality on the strength of selection and the amount of genetic variation associated with a fitness-related trait. Evolution should occur when selection acts on a trait, such as birth weight, that has a genetic basis. Birth weight is closely linked to juvenile mortality, with larger lambs having higher survival and higher fitness. The first six months of life are critical; lamb deaths during this crucial window account for nearly a quarter of all deaths.Even though birth weight has all the raw materials for evolution\u2014a positive correlation between birth weight and survival, and a heritable basis for variation, attributed to maternal genetic effects\u2014previous studies found no evidence for evolution of the trait. Since annual birth weight had not increased over the 20 years, the researchers hypothesized that a variable environment could limit its micro-evolution by limiting heritability, selection, or both. Wilson et al. found evidence that that this is indeed the case.To study the micro-evolutionary dynamics of a wild population, they adapted a theoretical model long used by animal breeders. Quantitative genetic models measure the genetic contribution  to variation in a given trait, such as milk production in dairy cows. With these models, plant and animal breeders maintain or develop desired traits by predicting an evolutionary path based on the strength of selection and the amount of genetic variance underlying the trait. Some of these models assume a constant environment, which is fine for breeders, who can artificially control their subjects' environment, but proves problematic for evolutionary studies of wild populations.To overcome these limitations, the researchers incorporated a statistical tool called random regression analysis. With this approach, they could not only model the effects of environmental variation on selection and heritability but also maximize the statistical power of their dataset. This is a particularly effective technique for natural populations, for which the data are inevitably limited. It also allowed the researchers to model the genetic variance underlying lamb birth weight as a function of environmental quality , creating a more realistic evolutionary picture. The most powerful modeling result showed that the extent to which birth weight is heritable\u2014that is, the variance caused by maternal genetic effects\u2014increased along with environmental quality. But when Wilson et al. modeled the relationship between birth weight and fitness  across environments, they found that the strength of selection followed a different pattern. Fitness increased with birth weight, and with environmental quality, but the positive relationship between fitness and birth weight\u2014indicating the strength of selection\u2014became weaker in better environments.Environmental quality shapes the trajectories for both genetic variance and the strength of selection. Lambs are not growing bigger and bigger because there's a lack of heritable variation for selection to act on in harsh environments and there's a lack of selection to act on higher genetic variation during favorable conditions. These results emphasize the importance of using biologically realistic models to predict the evolution of traits in wild populations\u2014an important component of genetic restoration for endangered species, for example. They also confirm the value of long-term studies on wild populations to better detect patterns of selection and genetic variation in the natural world."}
{"text": "Female only unisexual vertebrates that reproduce by hybridogenesis show an unusual genetic composition. They are of hybrid origin but show no recombination between the genomes of their parental species. Instead, the paternal genome is discarded from the germline prior to meiosis, and gametes (eggs only) contain solely unrecombined maternal genomes. Hence hybridogens only transmit maternally inherited mutations. Hybridity is restored each generation by backcrossing with males of the sexual parental species whose genome was eliminated. In contrast, recombining sexual species propagate an intermixed pool of mutations derived from the maternal and paternal parts of the genome. If mutation rates are lower in female gametes than males, it raises the possibility for lower mutation accumulation in a hybridogenetic population, and consequently, higher population fitness than its sexual counterpart.We show through Monte-Carlo simulations that at higher male to female mutation ratios, and sufficiently large population sizes, hybridogenetic populations can carry a lower mutation load than sexual species. This effect is more pronounced with synergistic forms of epistasis. Mutations accumulate faster on the sexual part of the genome, and with the purifying effects of epistasis, it makes it more difficult for mutations to be transmitted on the clonal part of the genome. In smaller populations, the same mechanism reduces the speed of Muller's Ratchet and the number of fixed mutations compared to similar asexual species.Since mutation accumulation can be less pronounced in hybridogenetic populations, the question arises why hybridogenetic organisms are so scarce compared to sexual species. In considering this, it is likely that comparison of population fitnesses is not sufficient. Despite competition with the sexual parental species, hybrid populations are dependent on the maintenance of \u2013 and contact with \u2013 their sexual counterpart. Other problems may involve too little genetic diversity to respond to changing environments and problems in becoming hybridogenetic (e.g. disruption of meiosis and subsequent infertility or sterility). Yet, lower mutation accumulation in hybridogenetic populations opens the possibility that hybridogenetic species can develop into new sexual species once recombination is re-established and reproductive isolation from sexual ancestors has occurred. Finding explanations for the evolution and maintenance of sexual reproduction is a long-running research problem in evolutionary biology. A large number of scenarios and models have been developed under which either sexual or asexual reproduction could have advantages or disadvantages . In the Whereas the theories of mutation accumulation and selection against deleterious mutations are well established for asexual and sexual species, they are not for unisexual vertebrates that reproduce by hybridogenesis, a reproductive mode that is intermediate between sexual and asexual reproduction. Figure Poeciliopsis with supposed hybridogenetic ancestry.The long time evolutionary perspectives of such hybridogenetic unisexuals have been questioned by several authors e.g. -12] base base12]]Such speciation events can only be successful if the newly arising species has not accumulated too many deleterious mutations during its hybridogenetic history. In evaluating the risk of mutation accumulation in hybridogens, as compared to other reproductive modes, one has to consider that parthenogenetic (including gynogenetic) and hybridogenetic vertebrates are all-female species (with the exception of hybridogenetic water frogs), whereas sexual species consist of males and females. This becomes important when mutation rates are sex-specific. Starting with Haldane ,15 a numet al.  but  but 22]]The fitness effects of deleterious mutation accumulation in hybridogens are not simply at an intermediate level between sexual and asexual populations. The latter comparison depends on the ratio of male to female mutation rates \u03b1. If \u03b1 = 1 in a large population with synergistic epistasis, conventional expectations hold and the average fitness of a hybridogenetic population is indeed intermediate between sexuals and asexuals. This intermediate position is due to the property that mutation accumulation is slower within the chromosomes derived from the sexually reproducing parental species (where mutations are more effectively purified due to synergistic epistasis and recombination). However, the situation starts to change as \u03b1 becomes larger than a value of one. In the latter case, sexually reproducing species not only face the often cited twofold costs of reproduction , but alsTo understand the simulation results presented here, it is necessary not only to consider the mechanistic differences in modes of genetic transmission, as represented by the three reproductive systems discussed here. It is also necessary to understand the interplay of population dynamics with these mechanisms. This becomes apparent when one considers mutation accumulation in the clonal part of the hybrid genome. As seen in the cases with quadratic and truncation selection Figure , as \u03b1 beThis is not because mutations do not occur on the clonal part, but rather that they accumulate much faster on the sexual part of the genome, and hence the quadratic and truncation selection effects allow for no more mutations to accumulate on the whole genome. Selection is too effective in purging the \"late arriving\" mutants on the clonal part of the genome. In other words, if all mutations that a hybridogen carries affect fitness, regardless whether they reside on the sexually or clonally transmitted part of the genome, selection against all newly acquired mutations is strong if mutations interact synergistically, because of the \"borrowed\" high mutational load on the paternal genome. Since only the maternal genome can be transmitted (which has been under strong selection against new mutations), the clonally transmitted part of the genomes of hybridogenetic organisms stays remarkably  free ofA challenge for the persistence of asexually and hemiclonally reproducing organisms are drift effects in small or fragmented populations . The comAlthough population size had little influence on the mutational load and on the number of fixed alleles in the size-limited populations with epistatic mutation interaction, hybridogenetically reproducing populations were generally less prone to the accumulation of deleterious mutations than asexual populations. The selective forces against deleterious mutations seemed to be sufficiently effective to prevent a substantial drop in average population fitness , as the mutation rate on the clonally transmitted genome is reduced relative to that on the sexual genome.U are still debated , and, andU arRedfield . At highc models ,28.The simulation results in this work indicate that with male to female mutation rates higher than one (\u03b1 > 1), hybridogenetic populations can be less prone to the accumulation of deleterious mutations, and hence have a higher population fitness than their sexual counterpart. As \u03b1 becomes larger, mutation accumulation in the part of the genome derived form the sexual species is faster, and these mutations make it more difficult for new mutations to accumulate on the clonal part of the genome (which has a slower mutation rate). This effect becomes more pronounced with synergistic forms of epistasis (quadratic and truncation). Furthermore, in cases with epistasis and \u03b1 = 1, although sexual populations have a higher fitness than the hybrids, the hybrids in turn do better than the asexuals, because mutations are purged more effectively in the sexually derived part of their genome.These results lead to the question why hybridogenetic organisms are so scarce compared to sexual species. At the genetic level, one reason could be that, in the real world, mutation interactions are far more complicated and diverse than the ones used in this model ,30. If wFurthermore, one cannot consider the evolution of hybridogenesis by only looking at genetic mechanisms but has to include ecology as well. The survival of the hybrid population totally depends on the continued contact with the sexual parental population which serves as an indispensable sperm donor. Their sperm-dependence prohibits the hybridogens to occupy niches which are markedly different from the niche of the sexual parent and does not allow out-competing the parental species and leading it to extinction. Hence, hybridogenesis can only evolve and persist under ecological conditions that allow stable populations of both, the sexual parasite (i.e. the hybridogens) and their sexual hosts . This ecological issue was not considered in the present work, but has been dealt with in previous publications -35Our result, that clonal or hemiclonal polymorphism can not be maintained with the used model framework, suggests that the often observed diversity in sympatric clones or hemiclones either originates from subsequent recruitments of new clones or hemiclones through repeated primary hybridization, or that diversity in clones is maintained through the occupancy of different microhabitats or \"frozen niches\" -39Although mutation accumulation is less of a threat to hybridogenetic populations  under several of the conditions used in this model, there are other factors inherent to asexual or hybridogenetic reproduction which affect the long time success of such populations: the response to rapidly changing environments or to parasites is still best ensured through sexual reproduction ,41. But Here, we describe the models for infinite und finite populations, and how the respective simulations were implemented. The features common to all model populations have been mentioned in the Background section.In our simulations, we used three different types of selection against mutations, each represented by a fitness function with a specific form of gene interaction : namely independent, quadratic, and truncation interaction Figure . The funi be the relative fitness (in terms of the number of gametes it is able to produce) of individuals with i mutations compared to an individual with no mutations. With independent fitness effects,Let Ws is the selection coefficient for each new mutation. For our simulations, we chose an s of 0.1, so the relative fitness of an individual with i mutations is Wi= (0.9)iwhere i be the relative fitness of individuals with i mutations compared to an individual with no mutations. Based on Kimura and Maruyama [Again, let WMaruyama , and RedMaruyama  the syneh1 and h2 are constants. The third term in this equation is the nonlinear term that produces the synergistic effects as i becomes larger. In our simulations, h1 = 0.014 and h2 = 0.0112 [Wi \u2264 0, we set the value of Wi to zero.where = 0.0112 . For valWi= 1 if i < = 7, else Wi= 0.Under truncation interaction, accumulated mutations do not have a negative effect on fitness until the number of mutations reaches a threshold. Beyond this threshold, fitness drops to zero, thus U, the genomic mutation rate per individual and generation. Hence the probability P(j) that an individual acquires j mutations is given by (j) = UjeU-(j!)-1. Let us assume that all individuals reproduce at the same time and that generations do not overlap and that there are no back mutations. The class of individuals with i mutations at the beginning of their lifespan originates from a set of parents who could be sequentially ordered into classes , composed of parents with 0 to i mutations. Individuals in each parental class ci-j, would have to produce j mutations to produce progeny in the class ci. Hence, the number of individuals in class ci is the sum of the progeny from each class ci-j with j mutations, where 0 \u2264 j \u2264 i. If Wi is the relative fitness of individuals in the class with i mutations compared to the class with no mutations and fitness is expressed in the number of gametes an individual can produce, the frequency ci of the class with i mutations in the generation t+1 then calculates asAccording to Kimura & Maruyama  the mutaWi-j is the fitness of individuals with i-j mutations. If ci(t+1) = ci(t) for all i, the population has reached a stable state between mutation pressure and selection against deleterious mutations. The fitness of such a population in mutation-selection balance, relative to a mutation free population, is then where ci(t+1) by c'i(t) in equation 1. The term c'i(t) now describes the frequencies of mutation classes after the new mutations in the same generation have occurred, and ci(t) the distribution before the acquisition of new mutations. If we apply the modified equation 1 separately for both sexes with different values for U, we will get two distributions, c'\u039ei(t) and c'Xi(t), for all i. For convenience, we assume here that genomes recombine freely and that none of the mutations occur in homozygous state (i.e. mutations do not coincide at the same locus). An individual bearing a total of i mutations on its entire genome can produce gametes containing 0 to i mutations. We model gamete production as a Bernoulli trials process with i chance experiments, with the probability of passing on a mutant allele for each of the i heterozygous loci being 1/2. Hence the relative frequencies pi(k) of gametes containing k mutations produced by an individual with i mutations follows a binomial distribution withFor the sexual populations we add an additional stage to the asexual model. The individuals in the population are first subject to mutation and selection, following equation (1), and then, subsequent to recombination, are used as a source to form the male and female gamete pool for the next generation. Individuals of the next generation are formed by randomly combining gametes from the latter pool. Infinite sexual populations can be modeled if we replace t for the male gametes mt(g), and for the female gametes ft(g), calculate as:If we pool all male gametes and all female gametes produced by the entire population, the mutation class distribution functions at time g indicates the number of mutations in the specific gamete class.where i mutations in the new generation before mutation accumulation then calculate asBy randomly combining gametes from the two distributions in equation 2, we can build the new generation of individuals. The frequencies of the male and female classes with c\u039ei(t+1) and cXi(t+1) again in equation 1 the values for c'\u039ei(t+1) and c'Xi(t+1) can be calculated with the respective mutation rates.Using z new mutations in the time span between birth and reproduction, 0 to z of these mutations may end up on the clonally transmitted part of the genome. The probability qz(x) that x of these z mutations end up on the clonally transmitted part of the genome follows again a binomial distribution with If a hybrid acquires fn(t+1) of eggs containing n mutations at time t+1 produced by the hybridogens can be calculated asUsing again equation 1, the frequency U being the genomic mutation rate for the hybrid females. The first term in equation (4) comes from equation (1), delineating mutation and selection. The second term gives the frequency of individuals with i-j mutations, having y mutations on the egg and i-j-y mutations on the sperm. The third term gives the probability that of the j mutations occurring in an individual of class ci-j, n-y of those mutations (where n-y \u2264 j) occur on an egg with y mutations to give an egg with n mutations. We assume that the distribution of mutation classes within sperms ml is constant over time since the sperms originate from males of the sexual population in mutation selection balance.with ci of hybrids with i mutations in the next generation before mutation accumulation is again The new generation of hybridogens is then again built by randomly combining eggs with sperms. Thus the frequency For the finite populations, we used a Monte Carlo approach to simulate the effect of stochasticity and to be able to model effects of genetic drift and Muller's ratchet. A total of 1000 loci susceptible to deleterious mutations is assumed for all individuals of the different reproductive types. We further assume that initially all genomes are free of deleterious mutations and that back-mutations do not occur. All simulations were programmed in Pascal or C++ using Metrowerks CodeWarrior 5 on an IBM-compatible PC.At the end of one generation all offspring produced by the individuals are pooled. The offspring inherits all mutations (the inherited and the newly acquired ones) from its parent. The relative contribution of an individual's offspring to this pool corresponds to its fitness compared to the fitness of the other individuals. The fitness is determined by the number of deleterious mutations on an individual's genome and the chosen mutation interaction for the simulation . From this pool of offspring, random individuals are drawn to build the new generation until a preset maximum population size is reached.In sexual populations, simulation starts with randomly choosing two parents. The likelihood of a parent to sire offspring is proportional to its fitness compared to the rest of the population of the same sex. Once a pair is determined, it produces a single offspring. For convenience, we assume that all mutations in the parent's genome reside on separate chromosomes. The offspring then inherits each of its parent's heterozygous mutations with a probability of 0.5. All homozygous mutations present in the parents are transferred to the offspring. After the process, the individuals are put back into the pool of parents. The whole process is repeated until the maximum population size is reached. After the new generation is established, each individual undergoes mutation accumulation. Note that males and females can have different average mutation rates and that the number of new mutations per individuals follows again a Poisson distribution with the sex specific genomic mutation rate as the mean.U. A newly acquired mutation occurs with the same probability on the hybrid parents' part of the genome as on the sexual parent's part. The fitness of a hybrid is determined by the total number of mutations in its soma . All mutations affect the hybrid's fitness equally, regardless where they reside. At reproduction, the number of gametes produced by the hybrids corresponds to their relative fitness compared to the other hybrids but the gametes contain only the mutations that resided on the clonally transmitted part of the parents' genome. For the next generation random gametes from this pool are drawn and combined with sperms from the infinite sexual population. The frequencies of mutation classes in the sexual parent population remain constant.The simulation starts with a hybridogenetic subpopulation where no mutations are present on the clonally transmitted part of the hybrid's genome . The sympatric sexual population on the other hand shows the stable distribution of mutation classes, determined with the model for infinite sexual populations. The reproductive cycle starts by randomly combining sperms from males from the sexual population and eggs from the all female hybrid population. As in the previous models, the all-female hybrids acquire a random number of mutations with a probability that follows a Poisson distribution with the mean of the female specific Wherever applicable, the same parameter sets were used as in the model of Redfield . Genomicfi is the fitness of the class with i mutations and nj the frequency of the clonally transmitted haploid set with j mutations in the hybrid population.We furthermore tested the average health of the clonally transmitted part of the genomes of hybridogenetically reproducing populations. This was achieved by calculating the mutation load of a hypothetical diploid population built by combining the clonally transmitted haploid sets of the hybrids with the formula CS came up with idea for this study, developed the model and wrote the initial version of the manuscript as part of his PhD thesis. H-UR wrote the research proposal that lead to this study, obtained the funding and drafted the present version of the paper. HB helped in rewriting the present version of the manuscript. All authors read and approved of the final manuscript."}
{"text": "Various definitions of biological complexity have been proposed: the number of genes, cell types, or metabolic processes within an organism. As knowledge of biological systems has increased, it has become apparent that these metrics are often incongruent.Here we propose an alternative complexity metric based on the number of genetically uncorrelated phenotypic traits contributing to an organism's fitness. This metric, phenotypic complexity, is more objective than previous suggestions, as complexity is measured from a fundamental biological perspective, that of natural selection. We utilize a model linking the equilibrium fitness (drift load) of a population to phenotypic complexity. We then use results from viral evolution experiments to compare the phenotypic complexities of two viruses, the bacteriophage X174 and vesicular stomatitis virus, and to illustrate the consistency of our approach and its applicability.Because Darwinian evolution through natural selection is the fundamental element unifying all biological organisms, we propose that our metric of complexity is potentially a more relevant metric than others, based on the count of artificially defined set of objects. A persistent question in biology is how organismal complexity changes through the course of evolution Phenotypic complexity quantifies the number of genetically uncorrelated phenotypic traits contributing to an organism's fitness. A phenotypic trait contributes to an organism's fitness only to the extent that natural selection acts upon that trait. Thus an organismal phenotype that is no longer under selection , although expressed by the organism, contributes nothing to organismal complexity. Secondly, if two phenotypes contribute to complexity, they must be genetically separable: some mutations must exist that affect one phenotype but not the other. If no such mutations exist, then although we may perceive two phenotypes under selection, these phenotypes contribute only a single trait toward determining phenotypic complexity. As an example consider the affinity of an enzyme for a substrate, and the rate at which that substrate is converted to product. If there are no mutations that affect one of these traits but not the other, then these two phenotypes are considered one, until the organism gains the genetic complexity to generate variation in one phenotype without affecting the second, for example by evolving functionally separate domains in the enzyme. Phenotypic complexity is thus a combined description of how natural selection perceives organismal phenotypes and how phenotypic variation is generated by the organism. This concept was first articulated by Orr, and followed later by others An important aspect of measuring complexity in this manner is that both the organism and the environment affect the metric. An organism with many phenotypes, but living in simple environment could thus be just as complex as a simpler organism in the same environment. For example, if one organism is capable of metabolizing both lactose and glucose, while second can metabolize only glucose, the first organism will only be designated as more complex when there is a possibility that lactose will be present in the environment.Recent population genetic theory The phenotypic model used to link drift load to phenotypic complexity was first formalized by R. A. Fisher A population of individuals can be represented as a collection of points in FGM and the phenotypic values of each point allow ascertainment of the fitness of each individual. Individual fitness then determines the probability of each individual surviving and reproducing the next generation. Evolution is thus described in FGM by following the collection of points over many generations. To generate novel genetic variation, mutations are drawn from an assumed distribution that is centered on the phenotypic position of each individual . By neveFGM makes a set of theoretical predictions about how adaptation tends to occur, and many of these have been corroborated by experimental results. The greater frequency of small-sized beneficial mutations The utility of FGM lies in the fact that it does not require any particular assumptions about the map between phenotype and genotype, and that the specific predictions about how fitness changes during evolution appear to be robust. In the present paper, we further refine previous predictions derived from FGM The link between drift load and phenotypic complexity under FGM was first investigated by Hartl and Taubes e\u22121 in the diploid case and 2\u22c5Ne\u22122 in the haploid, and \u03c1(f), the density function, i.e. the size of fitness f isocline, is dependent on the number of dimensions and of the fitness function used.In which \u03bd\u200a=\u200a2\u22c5Neq is the equilibrium fitness (drift load) expressed as a fraction of the maximum attainable fitness of the organism, ne is the effective number of dimensions of the phenotypic space , and Ne is the effective population size. This confirms the results obtained by Poon and Otto who approximated Feq as 2Ne/(2Ne+ne) If fitness is assumed to be a linearly decreasing function of the phenotypic distance to the optimum, then we find that the average fitness is given by :in the dAlthough earlier studies on FGM model have used such linear fitness functions , recent experimental studies do not seem to support the use of such a function Q)), in which fitness is an exponentially decaying function of the distance to the optimum to the power of Q. Q is a parameter that modifies the concavity of the fitness decline. As organisms move away from the optimum the effect of the mutation tend to have bigger effect if Q>1 and smaller effect if Q<1. In such a case the fitness equilibriums are \u200a=\u200aexp (ixiQ), where X\u200a=\u200a is the coordinate of an individual in FGM, and \u03b1i are positive parameters  suggests, the fitness equilibrium is independent of the mutational properties. As long as mutation is assumed to be isotropic, only the convergence time to equilibrium, and not the equilibrium fitness value, is affected by the distribution of mutational effects (data not shown). Second, although the results depend on the shape of the fitness function , they are independent of the slope: equilibrium values will be the same if f(d)\u200a=\u200aexp(\u2212\u03b1dQ) . Third, rameters . FinallyThe equilibrium drift load seems to be a robust property of FGM that is determined by the number of dimensions of phenotypic space, the population size and the fitness function . An accurate estimate of phenotypic complexity can thus be obtained if it is possible to estimate equilibrium fitness values (drift load) for several population sizes, as well as the amount of curvature in the fitness function.Escherichia coli C. We increased the mutation rate of the phage to hasten convergence to fitness equilibrium with the use of hydroxylamine; this resulted in a mean mutation rate of 0.1 per genome per generation through the 450 generations of evolution. Population size-dependent fitness equilibriums were observed, confirming that evolution occurred in a manner compatible with the assumptions of FGM. From these populations we obtained 21 measures of equilibrium fitness at five different population sizes. Every transfer corresponded roughly to five phage generations and effective population size was then approximated to five times the number of plaques transferred (the harmonic mean).We used two sets of evolution experiments in which both fitness equilibrium values and fitness curvature have been investigated . In the To estimate the curvature of the fitness function we performed a mutation accumulation analysis for high and low fitness clones and showed that the distribution of deleterious mutations was similar at both ends of the fitness range spanning a 300-fold difference. This suggests that there is very little curvature of the fitness function.We used a second set of data from the literature, in which populations were evolved for 20 transfers at different effective sizes. Novella et al. Additionally, an impressive set of data using site directed mutagenesis in VSV suggests that the concavity of fitness function is slightly upward e/Q, fref), in which ne is the phenotypic complexity, Q a parameter of the curvature of the fitness surface, and fref the maximum attainable fitness. We also take into account the noise in our experimental assessment of fitness values; especially for high fitness populations, noise in the estimates of fitness can alter the estimation of fref, as this parameter is by definition higher than all fitness measures. Thus rather than using the probability of the point estimate of fitness, we integrated the probability between plus (f+) and minus (f\u2212) one standard deviation of the point estimate. In Appendix C we show that We wish to use the previous mathematical results to estimate phenotypic complexity from experimental data. However, there are two unknown parameters in the experimental system that affect the equilibrium drift load in a population: phenotypic complexity and the maximum attainable fitness that can be reached by the viruses in the laboratory environment . Using methods from statistical physics, we can find the distribution of population fitness at equilibrium (shown above), and thus derive a likelihood model that gives the probability of the observed data for each couplet (ne/Q\u200a=\u200a45 (42\u221249), and fref\u200a=\u200a1.245 (1.23\u22121.26), whereas for VSV we found ne/Q\u200a=\u200a10 (8\u221212) and fref\u200a=\u200a1.98 (1.94\u22122.05) , which we list here in parentheses. For \u03a6X174 we found n94\u22122.05) Fig. 3. e). We have quantified this metric in the viruses \u03a6X174 and VSV by utilizing a population genetic model that describes how phenotypic complexity affects the drift load that a population experiences.To understand how biological complexity changes during the course of evolution, a metric is needed. Previously, measures such as the number of genes, cell types or metabolic processes have been proposed, but they often lead to incongruent results. Organisms with more cell types do not necessarily have more genes. Here we suggest that a metric unifying biological systems has not been appropriately identified. To circumvent this problem, we have developed a metric of biological complexity termed phenotypic complexity (ne) is a measure of the number of genetically uncorrelated phenotypes that are acted upon by natural selection. Because Darwinian evolution through natural selection is the fundamental element unifying all biological organisms, we propose that ne is potentially a more relevant metric than those previously suggested.Phenotypic complexity  on the effective population size and phenotypic complexity. The linear fitness function that has been employed previously to simplify the mathematical analyses is no longer necessary. Such a function makes strong assumptions about the form of the fitness landscape; specifically, mutational effects become very large as fitness is reduced. Thus at low fitness most mutations are either lethal or of very large effect, a scenario which is incompatible with what we have previously observed Q)), and found that Feq\u200a=\u200a(1\u2212(2\u22c5Ne\u22121)\u22121)(ne/Q). It appears that this equation remains valid over a much wider range of conditions than those used in the canonical FGM, in which mutations are required to be isotropic and fitness isoclines are symmetric about the origin. An interesting feature of this formula is that it does not require a model in which mutations can affect all phenotypic traits simultaneously. In the initial formulation of FGM, all phenotypic axes intersect at the origin of each axis. This original FGM can be modified slightly such that some phenotypes are grouped into separate phenotypic modules, and within a module, all phenotypes again intersect at each other's origin. Any mutation that occurs within a module can affect only other phenotypes within that module, and none that lie outside of it \u200a=\u200aexp predicted a 0.002 mean fitness effect per mutation, while more recent and accurate estimates  brought the estimate it to 14% (a 70-fold difference). Secondly, neutral mutations are not considered in the model developed by Martin and Lenormand. However, part of the mutation produced by transposable elements might affect genes useless in laboratory conditions and will therefore be taken into account in the calculation of mean fitness effects of mutations even if they do not affect any phenotype in the laboratory environment. Third, contrary to our model, if phenotypes are organized in different modules (as many data suggest), their model will provide different estimates; in other words, their model requires that all traits can be simultaneously affected by a single mutations. All these considerations suggest that a second independent model should be used to estimate phenotypic complexity.Recently, another theoretical study developed a framework to estimate phenotypic complexity One of the central FGM hypotheses that we have so far not addressed is the single-peaked nature of the landscape. Although FGM contains few assumptions about the nature of the genotypic landscape, the model explicitly requires a phenotypic landscape containing a single peak; without this, then the fitness function, f(d), cannot be described by a decreasing function. However, recent experimental evidence over large evolutionary time scales strongly suggests that while the genotypic landscape may contain multiple peaks, the phenotypic landscape is generally much less complex. Several experimental studies using microbes have shown that a considerable amount of phenotypic convergence occurs during evolution e is the number of genetically uncorrelated phenotypes that are influenced by the action of natural selection. The dimensions enumerated by ne are thus genetically orthogonal to each other, and analogous to the axes needed to describe the variation among multiple phenotypes measured on a collection of individuals and mutants in a principal component analysis. However, the number of axes enumerated by ne is filtered by natural selection, while in a PCA analysis the number of axes is limited only by the number of independent phenotypes that are measured. Because each phenotype is optimized at a value determined by each organism's ecological environment, there is a dependence of phenotypic complexity on the complexity of the ecological niche experienced by each organism; if natural selection does not act on a phenotype, then that phenotype does not contribute to the complexity metric. Finally, although the estimates of ne arise from an idealized model of phenotypic evolution; as Orr suggested previously, estimates of phenotypic complexity using FGM can be viewed as \u201ceffective\u201d estimates of phenotypic complexity e, in which two populations with different numbers of individuals and different sex ratios might have the same effective population size and therefore respond similarly to the different population genetic forces. Thus two organisms, although they may differ in both the underlying genetic mechanisms and in the complexity of the environment in which they live, may have similar phenotypic complexities. The utility of the concept lies not in the implications it makes about specific phenotypes or genetic details, but in that it enables a general quantification of how an organism is affected by natural selection , and furthermore, how this action affects the evolutionary dynamics of that organism As discussed previously, the quantity denoted by ni.e. they are genetically separable phenotypes), it is clearly unlikely that each nucleotide or amino acid is independently acted up by natural selection. Secondly, each estimate of complexity is greater than the number of genes encoded by each virus . The presence of multiple functional domains within a single protein is consistent with an estimate of complexity that is greater than the number of encoded proteins.Unsurprisingly, our estimates of phenotypic complexity are orders of magnitude smaller than either the number of nucleotides or even the number of amino acids encoded by the genomes of these organisms . This agrees with the concept of phenotypic complexity that we have defined. Although mutations that occur at one nucleotide or amino acid do not affect those at another , and a quantitative level (that the number of phenotypic dimensions are reasonable) that the predictions from FGM theory are borne out. An assessment of drift load and phenotypic complexity in a greater number of organisms is needed before further conclusions can be drawn.Although the theory presented here appears to be quite robust, it is too early to conclude that it is an accurate reflection of the underlying biology. To be studied in an FGM framework, organisms need to present at least one phenotypic property to selection. Additionally, populations, even those of very small size, should evolve towards a fitness equilibrium that is explicitly dependent on population size. We found data in the literature consistent with this expectation for one organism, VSV. We now provide further support for population size-dependent fitness equilibria by evolving populations of the bacteriophage \u03a6X174. Together, these two data sets suggest that evolutionary analyses using an FGM framework are a valid approach. Moreover, the use of very simple organisms such as viruses is useful for gaining insight into metrics of complexity, as for such simple organisms, gene number is likely to be a very good correlate of organismal complexity, and this should be reflected by the metric. Although our observations are currently limited to two viral species, it is clear that from both a qualitative level . Two important conceptual differences separate these two approaches. Most importantly, phenotypic complexity is dependent on both the organism and the environmental context. An organism is not complex because it has many measurable phenotypes; it is complex because it has many phenotypes on which natural selection acts. Secondly, phenotypic complexity does not rely on artificially constructed concepts such as genes However, phenotypic complexity remains an inherently abstract metric. It cannot aid in identifying the specific characteristics contributing to the complexity of an organism. Instead, it addresses the complexity with which natural selection views an organism, and the complexity with which an organism is capable of generating novel phenotypic variation. For this reason, testing how phenotypic complexity compares to more traditional metrics of complexity  may provide significant insight into biological systems. Finally, phenotypic complexity (and the resulting equilibrium drift load) affords a unique opportunity to contrast the action of natural selection between different organisms or different environments in a very general and unconstrained manner.The details of experimental evolution of \u03a6X174 have been described previously e\u22121 in the diploid case and 2Ne\u22122 in the haploid, and \u03c1(f) is the density function of fitness value f.Sella and Hirsh f) df is derived from the hyper-sphere surface of radius x, with f\u200a=\u200af(x), f(x) being the fitness function describing the dependency of fitness on the distance to the optimum, x. The surface of the hyper-sphere of radius x is \u03a9(n)xn\u22121) is the unit radius hyper-sphere surface, \u03a9(n)\u200a=\u200a2\u22c5\u03c0n/2)(/\u0393(n/2). We have therefore \u03c1(f)df\u200a=\u200a\u03a9(n) xn\u22121)\u200a=\u200a1\u2212x, then \u03a9(n) xn\u22121)( dx\u200a=\u200a\u2212\u03a9(n) (1\u2212f)n\u22121 df andIf fitness is defined as f(x)\u200a=\u200aexp(\u2212xQ) we have If fitness is defined as: f\u200a=\u200aexp(\u2212\u03b1 xQ)Note that if f(RQ) where R is defined by x\u200a=\u200a is the position in the n-dimensional space and \u03b1i are positive numbers. We then have ellipsoidal fitness isoclines of semi-axes R/\u03b1i. As the volume of such an ellipsoid is f) df\u200a=\u200adV(f) df is similar to the one found in the previous case Let us assume that f\u200a=\u200aexp\u200a=\u200aexp(\u2212xQ), we find the probability that f lies between a and b isUsing the previous derivations with f(ref, we must estimate it and therefore fitness b and a be used relative to fref.Because we do not know maximum fitness f"}
{"text": "Mutational (genetic) robustness is phenotypic constancy in the face of mutational changes to the genome. Robustness is critical to the understanding of evolution because phenotypically expressed genetic variation is the fuel of natural selection. Nonetheless, the evidence for adaptive evolution of mutational robustness in biological populations is controversial. Robustness should be selectively favored when mutation rates are high, a common feature of RNA viruses. However, selection for robustness may be relaxed under virus co-infection because complementation between virus genotypes can buffer mutational effects. We therefore hypothesized that selection for genetic robustness in viruses will be weakened with increasing frequency of co-infection. To test this idea, we used populations of RNA phage \u03c66 that were experimentally evolved at low and high levels of co-infection and subjected lineages of these viruses to mutation accumulation through population bottlenecking. The data demonstrate that viruses evolved under high co-infection show relatively greater mean magnitude and variance in the fitness changes generated by addition of random mutations, confirming our hypothesis that they experience weakened selection for robustness. Our study further suggests that co-infection of host cells may be advantageous to RNA viruses only in the short term. In addition, we observed higher mutation frequencies in the more robust viruses, indicating that evolution of robustness might foster less-accurate genome replication in RNA viruses. RNA phage viruses evolved under high co-infection of host cells are less robust to mutations than those propagated under low co-infection, suggesting that co-infection may be advantageous to RNA viruses only in the short term. Mutational (genetic) robustness can be defined as constancy of phenotype in the face of mutational perturbation . GeneticHigh mutation rate is perhaps the most important prerequisite for adaptive genetic robustness , so mutaPseudomonas syringae pathovar (pv) phaseolicola bacteria for hundreds of virus generations. Three of the populations were evolved at low multiplicity of infection , and three at high MOI. Co-infection level was controlled by mixing viruses and bacteria in liquid medium at a given MOI, allowing sufficient time for virus attachment to cells, and then plating a dilution of the mixture onto agar with a superabundance of cells. During overnight incubation, viruses formed distinct plaques in the lawn, which resulted from lysis of infected cells and the release of viral progeny that infected neighboring cells. The passage cycle was repeated by harvesting plaques, removing the bacteria by filtration, and mixing viruses and naive bacteria at the controlled MOI. A total of 60 passage cycles were conducted, which equaled roughly 300 viral generations  = e\u2212MOI \u00d7 MOI. Therefore, at MOI = 0.002, only approximately 0.1% of all infected (n > 0) cells are co-infected and clonal infections predominate. In contrast, approximately 97% of infected cells should be multiply infected at MOI = 5, and co-infection predominates, generally with two to three viruses . Every ormation , the proon in \u03c66 ) infection in \u03c66 .) Thus, on in \u03c66 .Here we examined whether evolution of mutational robustness occurs differently for viruses evolved at low and high levels of co-infection. We tested the hypothesis by randomly isolating clones from each of the six previously evolved populations, and using these clones to found lineages that were subjected to a mutation accumulation experiment \u201319. MutaP. phaseolicola for 20 d (see n = 3) fitness assays against a common virus competitor for the focal genotypes  of each lineage. In this way, we were able to measure the mean change in log10 fitness (\u0394log10W) for each lineage as a result of mutation accumulation; \u0394log10W = log10Wpost-bottleneck \u2212 log10Wpre-bottleneck. This design resulted in 360 total fitness estimates , in which focal genotypes of a lineage were always assayed within the same temporal block.We isolated ten clones at random from each replicate population in the low co-infection and high co-infection level treatments at generation 300, and used these to found 60 independent virus lineages (ten clones \u00d7 three populations \u00d7 two co-infection treatments). We then conducted a mutation accumulation experiment ,19, in w20 d see . Plaque 10W values estimate the fitness effect of adding roughly one random non-lethal mutation to the founding genotype of a lineage. Mutational robustness is defined as decreased phenotypic variability in the face of mutational change. Thus, one set of genotypes can be considered more robust than another collection of genotypes if the first group has a significantly lower variance in the fitness change brought on by addition of mutation(s) to the genome  = 0.00, p = 0.9940; P. tomato: F = 0.17, p = 0.7175). This result is intriguing because the robust viruses seem to feature higher mutation frequencies on average. In turn, the data suggest that evolution of mutational robustness (whatever the underlying molecular mechanism) allows RNA viruses to tolerate less-accurate genome replication, perhaps explaining why these viruses remain highly mutable. Unraveling the exact molecular mechanism(s) for these results would be of great interest. Furthermore, our study implies a need for population genetic models that consider the impact of adaptive robustness on the evolution of replication fidelity, theory that has not been previously explored.One possibility is that the rare occurrence of complementation at low co-infection caused these viruses to adapt by evolving greater robustness than the wild-type \u03c66 ancestor; but this is unknown. If so, our data suggest it is unlikely that this greater robustness was accompanied by more accurate RNA replication; ANOVA shows mutation frequencies of the low co-infection genotypes do not differ from the ancestor on either host . Most important, we found that mutation frequency in these assays was significantly lower in the high co-infection viruses , which again argues that our robustness conclusions are not confounded by elevated mutation rates in these viruses. Overall, our data suggest that mutation rate governed by the accuracy of the viral replicase is a trait under selection, especially in the viruses evolved at high co-infection. It is unclear why viruses that frequently experience complementation would evolve higher fidelity of replication, and this may be a pleiotropic effect of selection occurring elsewhere in the genome. The possible adaptive significance of this result is unclear and merits future exploration.For the wild-type \u03c66 ancestor and evolved genotypes in our study, mutation frequencies were lower than expected given the ease with which host-range\u2013marked mutants of \u03c66 were obtained in previous studies. That is, host-range mutants occurred at frequencies ranging between 10aligenes . To explosts see . The datosts see  supporteF = 0.05, p = 0.84). Thus, we rejected the possibility that diminishing-returns epistasis, or any argument that hinged on differing fitness among the starting clones, provided an alternative explanation for our results.We considered the potential relevance of two other possible confounding factors in our study. First, we examined whether the two groups of viruses differed in fitness prior to mutation accumulation; the alternative explanation is that the low co-infection viruses were already of low fitness (in comparison to the high co-infection strains), and that fixation of one or more additional mutations via bottlenecking did not lead to a further reduction in their fitness. That is, lower fitness genotypes of \u03c66 are shown to be less affected by addition of further deleterious mutations, a result demonstrating that diminishing-returns epistasis can operate in phage \u03c66 . This phWe also considered that erroneous conclusions may be drawn from mutation accumulation experiments if conditions impose a change in selective environment for only a subset of test lineages. A similarity between the mutation accumulation experiment and the low MOI treatment is that viruses must infect cells alone, whereas in every fifth generation of the high MOI treatment viruses are forced to undergo co-infection. Thus, it might be argued that the high co-infection lineages (but not low co-infection lines) experienced a change in growth conditions, and that the magnitude and variability in their performance in the bottlenecking experiment results from adaptation to the new conditions. However, we believe that this could not have been a confounding factor in our study for several reasons.First, the pre-bottleneck clones drawn from both MOI treatments performed equally well prior to mutation accumulation (see above), as measured using fitness assays conducted at low MOI. These data may seem surprising, given that at generation 200 the treatment populations showed significantly different fitness under low-MOI conditions . HoweverSecond, the mutation accumulation experimental conditions can be considered novel for all test lineages. In particular, the experimental evolution necessitated that viruses attached to host cells in liquid medium prior to overnight growth on agar plates, whereas the mutation accumulation habitat did not. Even if phage \u03c66 attachment to cells in liquid versus on agar surfaces is fundamentally the same (this is not well described), it is plausible that these two habitats differ substantially in the dispersal of phages between infected cells .8 virus particles within a plaque. However, the mutation accumulation design prevents virus adaptation because the action of genetic drift overwhelms that of natural selection; i.e., lineages evolve at an effective population size of N \u224d 2, the harmonic mean of the serial passage fluctuating between 1 and 108 viruses.Third, it is highly unlikely that the test lineages could have adapted to the mutation accumulation conditions at all. Plaque formation on agar necessarily involves expansion of the bottlenecked population to large size, which allows for some positive selection. That is, a plaque forms from a single virus experiencing five generations of growth on the plate, where the average number of progeny made by an infecting virus is 100 particles, yielding ~10Our study suggests several intriguing possibilities for future research in \u03c66 and other RNA viruses. Identifying whether the low co-infection populations, the high co-infection populations, or both have changed in robustness relative to their common ancestor could help shed light on the genetic mechanism(s) underlying this difference in the \u03c66-derived viruses.  The 300-generation experiment  that preP. phaseolicola was purchased from American Type Culture Collection . L. Mindich  kindly provided host strain P. pseudoalcaligenes ERA (East River isolate A); G. Martin  kindly providedP. syringae pv.tomato and P. syringae pv.atrofaciens. Bacterial stocks were stored in 4:6 glycerol/LC (v/v) at \u221280 \u00b0C. Viruses were grown on lawns made from overnight bacterial cultures. Agar concentrations in plates were 1.5% and 0.7% for bottom and top LC agar, respectively. Plates contained 3 ml of top agar and a 200 \u03bcl bacterial lawn. Phage lysates were prepared by growing viruses on a P. phaseolicola lawn for 24 h; plaques were then collected and filtered  to remove bacteria. Phage lysates were stored at \u221220 \u00b0C in 4:6 glycerol/LC (v/v).Phages and bacteria were cultured at 25 \u00b0C in LC medium, Luria-Bertani broth (pH 7.5) . OvernigP. phaseolicola cells in a test tube containing liquid LC medium, incubated for 40 min at 25 \u00b0C. These infected cells were then diluted and plated on a P. phaseolicola lawn on which viruses form distinct plaques. Population size of N \u224d 500 was held constant across treatment populations by controlling the number of harvested plaques [N \u224d 500 was controlled by harvesting 500 plaques from a low MOI population and 100 plaques from a high MOI population. After the experimental evolution, ten clones were chosen at random from each population. Each of the 60 clones was then used to found a single lineage subjected to 20 d of mutation accumulation via bottlenecking [P. phaseolicola lawn, placing it in sterile liquid medium, and vortexing gently to disperse the progeny viruses contained within the plaque. A sterile platinum loop was then used to streak the viruses onto agar containing a naive host lawn. After 24-h incubation, the viruses formed distinct plaques, and the process was repeated for 20 consecutive days. Because each plaque grows from a single virus, daily passage caused a test lineage to be forced through an extreme bottleneck , in which the intense drift allows fixation of non-lethal mutations, the majority of which are presumed to be deleterious. Because the phage expand to approximately 108 phage in five generations within a plaque, this creates the opportunity for selection to operate within a population propagated by such one-plaque transfers. Thus, positive selection on the plate should bias against fixation of highly deleterious mutations, and it is possible to observe the fixation of a rare beneficial mutation in a bottlenecked lineage [P. phaseolicola lawn containing 8 \u00d7 108 cells; MOI on the plate of 5 \u00d7 10\u22127).Virus populations were experimentally evolved at MOI of 0.002 or 5 for 250 generations , followe plaques . On averenecking ,19. Bott lineage . However lineage \u201319. AlsoP. phaseolicola of a focal genotype relative to a common competitor of \u03c66 bearing a genetic marker  and in the harvested lysate (R1) were obtained by plating on mixed lawns of P. phaseolicola and P. pseudoalcaligenes (200:1 mixture) on which ordinary and host-range genotypes form turbid and clear plaques, respectively. Fitness (W) was defined as the relative change in ratio of ordinary to host-range virus, or W =R1/R0.Fitness assays consisted of paired-growth experiments , which cbacteria ). The coP. tomato or P. atrofaciens. A high-titer lysate  of a virus genotype was grown and titered on P. phaseolicola, and a sample of the lysate was then screened on a selective plate. Mutant frequency was calculated as the number of plaque-forming mutants per viruses in the inoculum; when no mutants were found, we used the limit of detection as a conservative estimate. Acquisition of a second host-range mutation was examined by rearing phage mutants on P. atrofaciens, followed by selective plating on P. tomato.We measured the appearance of host-range mutants formed on selective plates: bacterial lawns of A mixed general linear model controlling for experimental assay day was run , testingTable S1(91 KB DOC).Click here for additional data file.Table S2(26 KB DOC).Click here for additional data file."}
{"text": "Can a single unifying mathematical framework help to explain robustness - the ability of organisms to persist in the face of changing conditions - at all biological scales, from biochemical to ecological? One of the most important features of biology is the ability of organisms to persist in the face of changing conditions. Consider the remarkable fact that every organism alive today is the product of billions of generations in which its progenitors, without fail, managed to produce progeny that survived to reproduce. To achieve this consistency, organisms must have a balance between robustness and evolvability, that is, between resisting and allowing change in their own internal states . MoreoveExamples of robust biological systems are found at many scales, from biochemical to ecological. At each scale, robustness may reflect the properties of individual elements or, alternatively, the dynamic feedbacks between interacting elements. The expression of some metabolic function, for example, may be robust in the face of temperature change, because an enzyme maintains its shape and specificity across a range of temperatures or because an interconnected network of reactions sustains the supply of product, even when some enzyme fails. A genome may be robust because it encodes proofreading and repair systems that reduce replication errors or because it is organized such that many mutations have little effect on its phenotype. An ecosystem might be robust if it resists the extinction of some keystone species or, if extinction does occur, because surviving species can compensate over physiological, demographic, or evolutionary time scales.One important question is whether there exists a single unifying mathematical framework that can encompass such diverse examples of biological robustness. Might new insights come from such a conceptual unification, or will future understanding require detailed analyses of specific cases? Across the different scales, recurring mechanisms for achieving robustness\u2014including redundancy of component parts and negative feedbacks\u2014might serve as organizing principles. Yet, similarities in mechanism could mask important differences in the evolutionary origins of those mechanisms. At the level of genes in genomes or of cells in multicellular organisms, it is reasonable to suggest that redundancy evolved by natural selection to maintain some functional capacity in the face of perturbation . But wheAnd if robustness has evolved to maintain performance, what prevents systems from becoming ever more robust? We will focus on genomic robustness to mutations, because it provides a concrete example, although many ideas are speculative and much work is needed to formalize and test them. Two mechanisms that could make a genome more robust are genetic redundancy, so that many otherwise deleterious mutations are masked, and proofreading during replication, so that fewer mutations occur. Redundancy imposes a cost of replicating the additional gene copies , whereasMutational robustness can also arise in more subtle ways. Populations evolving at high mutation rates may settle in regions of genotypic space where mutations are less deleterious, on average, than those regions that attract populations that experience low mutation rates. The idea is that evolution at low mutation rates favors populations that achieve high fitness peaks, even if they are surrounded by steep cliffs, because mutations that push progeny off those cliffs are rare. By contrast, at high mutation rates, most offspring carry mutations, and selection favors populations that find lower fitness peaks surrounded by less precipitous mutational chasms. Experiments with digital organisms (self-replicating computer programs) provide direct support for \u201csurvival of the flattest\u201d at high mutation rates . RNA virBut generalizing to other organisms presents some difficulties. The strength of selection for robustness should be weaker in larger genomes if the advantage to a mutation that increases robustness locally is correspondingly smaller. According to one alternate hypothesis, mutational robustness is not so much a directly evolved property as it is a correlated benefit of selection for robustness in the face of variable environments . The essTwo recent studies with evolving computational systems have shown, unexpectedly, that sexual reproduction promotes the evolution of mutational robustness ,14. The Another important issue revolves around the tension between robustness and evolvability. Are genomes that are more robust to mutations less evolvable in the face of changing environments? In other words, does canalizing the phenotype to minimize perturbations\u2014including biochemical and environmental as well as mutational\u2014lead to an evolutionary conservatism that inhibits the discovery of new adaptive solutions? Some mechanisms of robustness, such as proofreading and repair, must inhibit evolvability because they reduce the production of new beneficial mutations. But are robustness and evolvability inversely correlated more generally? In the case of redundancy, the presence of multiple gene copies might mask the beneficial effects of some new mutations, thus suppressing evolvability. But redundancy can also promote adaptation by allowing duplicated genes to evolve distinct functions ,17.Evolving populations can also become robust by finding regions of genotypic space that are flat because they contain a high proportion of neutral mutations . As showTheoretical population genetics has historically emphasized models with one or two loci, whereas quantitative genetics has relied on a sort of statistical mechanics that ignores underlying detail. Richer mathematical representations of genotypic spaces and fitness landscapes may be required to understand the balance between robustness and evolvability. Meanwhile, empiricists must push ahead to obtain data about evolvability and robustness. Experimental evolution, in which populations are monitored while they evolve under defined conditions, offers the potential to observe changes in these properties as a function of environmental and genetic manipulations . For exa"}
{"text": "After 20 cycles of random substitution at sites 12\u2013130 of the initial random polypeptide and selection for infectivity, the selected phage showed a 1.7\u00d7104-fold increase in infectivity, defined as the number of infected cells per ml of phage suspension. Fitness was defined as the logarithm of infectivity, and we analyzed (1) the dependence of stationary fitness on library size, which increased gradually, and (2) the time course of changes in fitness in transitional phases, based on an original theory regarding the evolutionary dynamics in Kauffman's n-k fitness landscape model. In the landscape model, single mutations at single sites among n sites affect the contribution of k other sites to fitness. Based on the results of these analyses, k was estimated to be 18\u201324. According to the estimated parameters, the landscape was plotted as a smooth surface up to a relative fitness of 0.4 of the global peak, whereas the landscape had a highly rugged surface with many local peaks above this relative fitness value. Based on the landscapes of these two different surfaces, it appears possible for adaptive walks with only random substitutions to climb with relative ease up to the middle region of the fitness landscape from any primordial or random sequence, whereas an enormous range of sequence diversity is required to climb further up the rugged surface above the middle region.The fitness landscape in sequence space determines the process of biomolecular evolution. To plot the fitness landscape of protein function, we carried out  In vitro molecular evolution can be considered an adaptive walk on a fitness landscape in sequence space, where \u201cfitness\u201d is a quantitative measure of a certain physicochemical property of a biopolymer, such as thermostability or enzymatic activity n-k landscape model, in which substitutions occurring on one of n sites affect the contribution of residues at k other sites to fitness, was proposed as a model of the fitness landscape k and the difference in altitude between the global peak and the foot, defined as the region in the sequence space where random sequences are located. If k\u200a=\u200a0, all amino acid sites are independent, and so the effects of substitutions on fitness are additive. As fitness changes gradually with substitutions, the landscape is smooth with a single global peak, which is referred to as a \u201cMt. Fuji-type\u201d profile. In this case, adaptive walks of the search with single substitutions gradually reach the global peak. On the other hand, larger values of k are associated with more rugged landscapes. If substitutions at a single amino acid site affect residues on k other sites, the effects of double substitutions on two different sites may not be equal to the sum of the effects of the two independent single substitutions k is an essential determinant for the fitness landscape structure. There have been a number of theoretical studies of evolutionary dynamics on both smooth and rugged landscapes n-k model to affinity maturation of the immunoglobulin V region; based on the number of steps in the adaptive walk up to the local optima, the value of k was estimated to be about 40 in this case The e.g., 10 different random sequence for esterase activity, are sufficient to allow Darwinian selection of random polypeptides composed of about 140 amino acid residues Escherichia coli by the coliphage fd is mediated by the minor coat protein g3p via flexible glycine-rich linker sequences Although a great deal is known about the landscape structure near the fitness peaks of native proteins in vitro molecular evolution by increasing the library size gradually from 102 to 106. By applying the experimental data to an original theory of the adaptive walk on the n-k fitness landscape model k value and other parameters to plot a protein fitness landscape and discussed its implications regarding the primordial stages of protein evolution and in vitro evolutionary molecular engineering.Although it was shown to be possible for a single arbitrarily chosen polypeptide to evolve infectivity, the evolution stagnated after the 7th generation, which was probably due to the small mutant library size at each generation. Therefore, we have extended in vitro molecular evolution, which we previously carried out up to the 7th generation, with the addition of an enrichment process by which the fittest phage clone(s) becomes dominant through several cycles of infection and growth in E. coli. As stagnation occurred due to small library size in our previous experiments, we gradually increased the library size N if the time course of infectivity reached a plateau  fitness in the stationary phase and the library size, and (2) the time course of changes in fitness in the transitional phase toward the stationary phase. The method applied here was based on the findings of theoretical studies and its applicability to the experimental data and more detailed analyses are described elsewhere .The parameters determining the structure of the N\u200a=\u200a10, 103, 105, and 106, respectively. These stagnations can be explained by the balance between mutation, selection, and random drift due to the limited library size. If a mutant clone with greater fitness than the parent clone appears with a limited library size N, it has a high chance of increasing the fitness of the parent clone for the next generation. On the other hand, if most but not all of N mutants have lower fitness than the parent clone, one of these mutants can be selected by chance as only a limited number of cells are chosen at random after fitness-dependent growth. Thus, the fitness of the parent clone for the next generation can decrease. Therefore, with a limited library size, N, there will be a certain fitness value of the parent clone at stationary phase, given by Eqn. (11) (d is the mutation rate (2.4 per generation) and n is the length of the amino acid sequence subjected to mutation (119), which corresponds to amino acid residues 12\u2013130 of RP3-42. Note that n\u200a=\u200a119 implicitly assumes that the region contributes to phage infectivity with little epistatic effect with other regions of the phage genome. Using the mean fitness in the stationary phase for each N value in the evolutionary dynamics shown in W* at each stationary phase followed Eqn. (11) (N\u200a=\u200a1 as the random sequence RP3-42 in fd-RP was assumed to be \u201cselected\u201d among N\u200a=\u200a1 arbitrarily chosen sequences. This assumption was confirmed by the observation that the infectivity of fd-RP was comparable to that of the deletion mutant phage lacking the D2 domain. Fitting Eqn. (11) to the plot of the W* values against the respective values of N (O+\u03b5 as 6.0 and 4\u03bdn/d(1+k) as 5.8. We adopted the estimated values of W* given above, and Eqn. (13) was fitted to the plot of the time course of changes in fitness for the 1st\u20137th and 8\u2032th generations and for 8th\u201313th generations, as shown in d(1+k)/n\u200a=\u200a0.5 for N\u200a=\u200a10 and 0.39 for N\u200a=\u200a103, and with n\u200a=\u200a119 and d\u200a=\u200a2.4, we estimated the values of k as 24 and 18, respectively. Thus, we adopted an average of k\u200a=\u200a21 for the fitness landscape of phage infectivity. By combining the values determined above and Eqns. (4) and (5) with k\u200a=\u200a21, we estimated the following parameters: O\u200a=\u200a21.5, \u03b5\u200a=\u200a\u221215.5, \u03bd\u200a=\u200a0.64. It should be noted that the fitness of the wild-type fd-tet phage, which was not used for determination of the parameters, was as high as the global peak.We assumed that the adaptive walk shown in qn. (11)  where d qn. (11) . Note thues of N , we obtan-k model with the parameters estimated as described above, we depicted the structure of the fitness landscape of the g3p minor coat protein D2 domain for phage infectivity as follows. First, the frequency of sequences that take the fitness values of W obeys the Gaussian distribution given in Eqn. (3) with average O+\u03b5\u200a=\u200a6.0 and variance \u03bd\u200a=\u200a0.64 2/2\u03bd).Based on the \u03bd\u200a=\u200a0.64 . The regr-th order as a function of fitness r-th order that takes r-fold point mutants around it have fitness values lower than r+1-fold point mutants has a value greater than r\u200a=\u200a1, 2, 3, 4, 5, 6). The vertical broken lines represent the fitness values, the relative fitness of W* (N with N(d)all in Eqn. (11), where the mutation-selection-random drift balance sets when all conceivable d-fold point mutants of N(d)all , given by Eqn. (14), are explored in each generation. In the range from i.e., they have at least one ascending path. The fraction of the local optima in the first order (r\u200a=\u200a1) rises above (1)all of all single point mutants will find only lower fitness, whereas around the latter sequences, the search will find at least one with higher fitness. Thus, the adaptive walk with N(1)all of all single point mutants will lead to small fluctuations around r\u200a=\u200a1) reach their maximal frequency of 60% at (2)all of all double point mutants ride and fluctuate due to the mutation-selection-random drift balance. With further increases in fitness, the local optima tend to have a larger basin size and the frequency of sequences located on the slopes decreases.We found that there are no local optima between the foot and the middle altitude on the landscape, whereas above the middle region the number of local optima is very large. Hereafter, we indicate the altitude on the landscape by the relative fitness, .40 A in . At \u200a=\u200a0.47 B in , about 4.47 B in . That is.49 C in . At \u200a=\u200a00.5 D in , the loc4-fold increase in infectivity as compared to the original phage carrying a random polypeptide of 139 amino acids in place of the D2 domain of the g3p minor coat protein. We applied the fitness data of selected clones at each generation to our theory regarding adaptive walking on the n-k landscape model to estimate several parameters of the model and calculate the frequencies of local optima depending on their fitness values.We have extended our previous experimental evolution based on phage infectivity, which showed stagnation after the 7th generation. Increasing the library size overcame the stagnations to some extent and resulted in a 1.7\u00d710k, representing the number of amino acid sites the contributions of which to fitness are affected by a single substitution on a given site, was 21 indicating that an arbitrary residue interacts with about 21 amino acid residues through its mutational effects. The k value around the level of fitness for native proteins was estimated to be 40 for immunoglobulin k of 40 is around the native protein on the fitness landscape and is not necessarily close to our value of 21, but may be an overestimate because two assumptions were made in computer simulations to estimate the k value: a relatively small population was used, particularly at the beginning, and the cloned immunoglobulins were assumed to be at local peaks. If these assumptions are not true, the estimate of k for the native protein will be smaller and may be closer to approximately 20. The interaction of amino acid residues with approximately 20 other residues through mutational effects suggests that a single amino acid residue may interact with 20 other residues through direct or indirect contacts. Clustering the interactions by a size of 20 may allow the clusters to evolve as \u201cmodules\u201d The estimated value of the epistasis parameter (1)all For example, a local peak at relative fitness r, the ridge declines down to the fitness given by Eqns. (9) and (10) with N(1)all recursively r times before meeting ridges of other peaks. All ridges meet with others above (1)all.The fitness landscape was depicted schematically but semi-quantitatively based on the frequency of local optima calculated from the experimental data . The lanMore than one such mountain exists in the fitness landscape of the function for the D2 domain in phage infectivity. The sequence selected finally at the 20th generation has The landscape structure has a number of implications for initial functional evolution of proteins and for molecular evolutionary engineering. First, the smooth surface of the mountainous structure from the foot to at least a relative fitness of 0.4 means that it is possible for most random or primordial sequences to evolve with relative ease up to the middle region of the fitness landscape by adaptive walking with only single substitutions. In fact, in addition to infectivity, we have succeeded in evolving esterase activity from ten arbitrarily chosen initial random sequences d)/F\u2032 traD36 + lacIproABqlacZ\u0394M15], JM109 [recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi\u0394(lac-proAB)/F\u2032 traD36 proAB+lacIqlacZM15], and HB2151 [\u0394(lac-proAB) aranalrthi/F\u2032 traD36 proAB+lacIqlacZ\u0394M15], . The fd-tet The SfiI fragment of the fd-7 genome encoding the target sequence corresponding to the D2 domain of the g3p minor coat protein as follows. The SfiI fragments of phage genomes in the library obtained after the enrichment process of the previous generation were amplified under error-prone PCR conditions SfiI, and the SfiI fragments were cloned into the corresponding region of the fresh fd-RP vector digested with SfiI. Fresh fd-RP vector is the shorter vector prepared by first digesting the fd-RP DNA with BamHI and ligating the resultant vector fragment, yielding a construct with a truncated SfiI fragment E. coli JM109 cells by electroporation E. coli HB2151 was used instead of E. coli JM109.One generation of our evolutionary study consisted of one cycle of mutation and enrichment processes. Random mutations were introduced in the region of the g for 10 minutes to remove the bacterial cells, and the supernatants containing the phage particles were filtered through Dismic 0.45 \u00b5m membranes  to ensure the elimination of any remaining bacterial cells. The filtrate containing the phage particles was stored at 4\u00b0C as the phage library of the generation for the enrichment process and the phage infectivity assay described below.Aliquots of 10 \u00b5l of the mutant library cell suspension described above were dispensed into 10 ml of 2\u00d7YT medium containing 20 \u00b5g/ml tetracycline and grown at 37\u00b0C overnight. The cultures were centrifuged at 6000\u00d7E. coli JM109 cells at OD600 0.8\u20130.9 (900 \u00b5l) for 40 minutes at 37\u00b0C. The bacteria-phage mixtures were spread onto 2\u00d7YT agar medium containing 40 \u00b5g/ml tetracycline by gently swirling the plates. Cells in the colonies grown on the plates after incubation overnight at 37\u00b0C were collected as described above and stored at \u221280\u00b0C as the 1st-round enriched library of this generation. The enrichment was repeated until the infectivity of the phage library stopped increasing. Aliquots of 10 \u00b5l of the last-round enriched library were dispensed into 10 ml of 2\u00d7YT medium containing 20 \u00b5g/ml tetracycline and grown at 37\u00b0C overnight. The phage genomes in the cells collected by centrifugation of the cultures were purified as the replication form for generating the mutant library in the next generation.The enrichment process consisted of several rounds of phage preparation and infection. The phage particles contained in aliquots of 100 \u00b5l of the phage library suspension prepared as described above were allowed to infect freshly grown E. coli with phage particles contained in the library was used as a proxy measure of infectivity, as described previously E. coli JM109 cells as described previously E. coli cells was about threefold greater than that of fd-RP phage, although the major change in CFU was attributed to the change in infectivity The number of tetracycline-resistant colonies that grew after infection of W, was defined here as ln(CFU) under the assumption that CFU is approximately proportional to exp(\u2212\u0394G/kT), where \u0394G is the free energy change of phage infection. Thus, W can be handled as an apparent energy as described below in the n-k fitness landscape. Actually, this definition of W satisfied some theoretical predictions on the n-k landscape . Experimentally, it has been shown that the mutational effect on protein properties can be described well by taking the logarithmic scale of the equilibrium constant or rate constant Fitness was originally defined as the relative growth rate for predicting population dynamics E. coli for evaluation of infectivity in the selection process: TG1 for generations 0\u20137, JM109 for generations 8\u201315, and HB2151 for generations 16\u201320. For systematic analysis of the lineage through generations 0\u201320, we used the CFU values for the JM109 strain as the standard measure of infectivity. Then, the infectivity of the best phage clone or clones selected in each of the 0th\u201320th generations was evaluated simultaneously with the CFU values for JM109. A strong correlation was found between the CFU values for JM109 and those for the other strains, with differences of less than one order of magnitude. The landscape climbed is the infectivity landscape for TG1 strain through generations 0\u20137 or HB2151 strain through generations 16\u201320. We converted the time series of the CFU values for TG1 through generations 0\u20137 or for HB2151 through generations 16\u201320 to the time series of the CFU values for JM109 as follows:XXX denotes the CFU value for E. coli strain \u201cXXX.\u201d These converted values were then used in the analysis. The fitness, W, was defined as ln(CFUJM109) for all phages.We used three different strains of n be the number of amino acid residues in a variable region subjected to random mutagenesis through error-prone PCR. The fitness, W, for a given amino acid sequence, \u201c\u03b11\u03b12\u2026\u03b1n,\u201d is defined by:i.e., the fitness contribution from a particular amino acid residue, j\u03b1, at the j-th site when the k sites {j1, j2,\u2026,kj} are occupied by particular residues {j\u03b11, j\u03b12,\u2026,jk\u03b1}. The k sites {j1, j2,\u2026,kj} are chosen randomly from all n-1 sites except the j-th site. The site-fitness of an arbitrary amino acid residue, \u03b1 , at each site with a given set {j\u03b11, j\u03b12,\u2026,jk\u03b1} is assigned randomly from the following set of 20 values, but degeneracy of assignment is not allowed: Let \u03c32 is approximately \u03b52/3. As the first term on the right-hand side of Eqn. (1) is \u223c0 for the globally optimal sequence , the second term O is determined as the fitness for the global peak. The fitness landscape resulting from this model is called the \u201cn-k landscape.\u201d Note that the original n-k landscape proposed by Kauffman et al. is slightly different from the model defined above \u03b5(\u22640) is a negative constant equivalent to the mean of the site-fitness over all available amino acid residues. Note that there is no significant effect on the theoretical conclusion when the \u03b5 value is different by sites n-k landscape. \u03b5(\u22640) is defined as the expectation of the first term in Eqn. (1) and represents the difference in fitness from the peak to the foot of the landscape, where the \u201cfoot\u201d is the region in which random sequences are located in the sequence space. Then, O+\u03b5 is the fitness at the foot of the landscape, and corresponds to the expected fitness of an arbitrarily generated random sequence. \u03bd is the variance of fitness over all possible sequences in the whole sequence space. The probability density of the fitness over all possible sequences in the whole sequence space approximately follows the Gaussian distribution:n-k fitness landscape, with its precise derivation and justification reported elsewhere . We consider the following rule of the adaptive walk: the clone(s) with the highest fitness in the previous generation generate N mutants in the t-th generation, and subsequently the fittest clone(s) among the N clones will become new parental clone(s) in the t+1-th generation. N is the \u201clibrary size\u201d of mutants to be screened for the next generation. The Hamming distance between a parent and each of its children or mutation rate is d. In the n-k model described above, d-fold point substitutions cause changes in site-fitness at about d(k+1) sites, because a single mutation causes site-fitness changes on the mutated site and k other sites. Let tW be the fitness of the parent in the t-th generation. In addition, let \u0394W be the fitness change from the parent to an arbitrary mutant in the mutant population. The probability density of \u0394W with tW fixed is described by: Here, we present the essence of the theory regarding evolutionary dynamics on the et al., in preparation). Using extremal statistics of normal distribution, the expectation of the change in fitness from the parent to a new parent after a single generation is given as follows:\u03b6 is defined as the expectation of the greatest value among the N random numbers from the standard Gaussian probability density, \u03b6 is approximately given by transforming N via:The average and variance of the fitness over the generated mutants were roughly consistent with Eqn. (7) and Eqn. (8), respectively. In addition, the validity of Eqn. (9) was confirmed experimentally. The details are reported elsewhere  indicates that the evolution rate increases with increases in N. Substituting Eqns. (7) and (8) into Eqn. (9), we find that, as the adaptive walker climbs the fitness landscape, the evolution rate decreases gradually and finally becomes zero. By solving Eqn. (9) under E[tW+1]\u2212tW\u200a=\u200a0, we obtained the fitness value in the stationary phase, W*, as follows:The change in fitness, WN, was confirmed experimentally, as described elsewhere .This stationary phase is caused by the mutation-selection-random drift balance W*, Eqn. (9) can be rewritten as follows:Using the stationary fitness value, t can be obtained approximately by the following function of t:W0 is the fitness of the initial sequence.Then, the expectation of the fitness in generation Nd), the probability that all conceivable d -fold point mutants take fitness values less than W is given by:Next, we refer to the existence of local optima. Let r-fold point mutants take fitness values less than W but at least one of the r+1-fold point mutants takes a fitness value greater than W, then the parent sequence is designated the \u201clocal optimum in the r-th order.\u201d That is, r represents the basin size for the local optimum. The probability that a parent with fitness W is the local optimum in the r-th order is given by:Here, we define the local optima as follows. If all conceivable single point mutants, double point mutants,\u2026,"}
{"text": "As the mutation rate was increased within a moderate range, deleterious mutation accumulation and mean fitness improvement both increased. The fixation rates were higher than predicted by many population-genetic models. This seemingly paradoxical result was resolved in part by the observation that, during the time to fixation, the selection coefficient (s) of initially deleterious mutations reversed to confer a selective advantage. Significantly, more than half of the fixations of initially deleterious mutations involved fitness reversals. These fitness reversals had a substantial effect on the total fitness of the genome and thus contributed to its success in the population. Despite the relative importance of fitness reversals, however, the probabilities of fixation for both initially beneficial and initially deleterious mutations were exceedingly small .Deleterious mutations are considered a major impediment to adaptation, and there are straightforward expectations for the rate at which they accumulate as a function of population size and mutation rate. In a simulation model of an evolving population of asexually replicating RNA molecules, initially deleterious mutations accumulated at rates nearly equal to that of initially beneficial mutations,  Mutations are the fuel of natural selection. It is widely believed that most mutations are deleterious, that is, they harm the organisms in which they occur. Thus, biologists would like to understand how deleterious mutations impact evolution. Most of the theoretical work on this problem makes an important assumption: mutations that start bad stay bad. It may be possible, however, for an initially bad mutation to become good  by interacting with subsequent mutations. In this study, Cowperthwaite, Bull, and Meyers show that such \u201cfitness reversals\u201d are surprisingly common and can lead to the fixation of initially deleterious mutations. Perhaps mutations that undergo such changes serve as stepping stones for greater evolutionary progress. Modern evolutionary theory recognizes that deleterious mutations may reduce fitness and retard adaptation \u20135. AccumIn the standard infinite population experiencing a combination of natural selection and random mutation, deleterious mutations should not fix, but accumulate to a level perfectly balanced by mutation and selection. Some processes can lead to deleterious mutations fixing in infinite populations, however. For example, in Eigen's quasispecies model, high rates of mutation can overwhelm selection and shift the mutation\u2013selection balance such that deleterious mutations accumulate to exceedingly high levels ,8. In fiThe fixation of deleterious mutations certainly reduces the fitness of populations. It may be possible, however, for the fitness effect of an initially deleterious mutation to change over time. In particular, compensatory mutations may evolve that reduce the negative impact of deleterious mutations or, in extreme cases, the resulting fitness may be even higher than the fitness of the ancestor in which the deleterious mutation arose . Such coEvolutionary geneticists have long considered mutations that ameliorate or compensate for the deleterious effect of a prior mutation. The literature on this subject, however, focuses almost exclusively on compensatory mutations occurring after the fixation of the initial deleterious mutation, and therefore does not address the likelihood that the initial mutation will fix in the first place \u201315. One If the mutation rate is relatively large, however, additional mutations may arise in the genome carrying the initially deleterious mutation before it fixes or is lost. Such secondary mutations change the genetic background and thus potentially change the fitness effect of the initial deleterious mutation. The background selection literature has frequently considered the scenario in which a good mutation is driven to extinction by bad mutations  mutations.We used a computational simulation of a population of replicating and evolving RNA molecules. Similar simulation models have been extensively used in previous studies of evolutionary dynamics \u201324. The L = 76 nucleotides, which is similar in size to a typical tRNA molecule. The focal phenotype is RNA secondary structure , which provides the scaffold for functional tertiary structure and has been highly conserved during evolution . A comparison of the observed and expected rates of fixation suggests that, under U = 0.32, fitness reversals may lead to rates of deleterious mutation fixation that are higher than expected by drift alone, while under U = 0.08 the rates of deleterious fixation do not exceed the expected rates from Kimura's model. We stress, however, that our populations are significantly different from the idealized ones Kimura envisioned and thus there may be multiple reasons for the observed discrepancies.We finally ask whether the number of fixation events that we attribute to fitness-effect reversals and hitchhiking might be within the range predicted to occur by drift alone. Populations experiencing genomic mutation rates of ectively . Kimura'U = 0.01 to U = 0.08 to U = 0.32), perhaps contributing to the evolution of higher mean fitnesses across these mutation rates. Fitness-effect reversals are only part of the story, however, as the initial deleterious effects of fixed deleterious mutations were much larger in the U = 0.32 populations than in the U = 0.08 populations.In summary, a complicated mix of forces allowed initially deleterious mutations to occasionally rise to fixation. Hitchhiking may work in concert with fitness-effect reversals, and therefore our estimates of the contributions of these two processes may be low . FurtherIn this study, we offer a new perspective on the effect and role of deleterious mutations in adaptation. We simulated the adaptation of asexual populations of 1,000 individual RNA genomes that each coded for a phenotype, which consisted of a set of thermodynamically probable secondary structures. In turn, fitness depended on the overall similarity of a molecule's phenotype to a target shape. The effect of a mutation was determined by measuring its impact on the shape of the molecule (its phenotype), and thus the distribution of fitness effects behaved as might be expected of a biological system.The novel result is that nearly one-third of the mutations that evolve in the MRCA lineage  arise with deleterious effects, yet this apparent load of deleterious mutations does not impede adaptation. This can be explained by the frequent occurrence of fitness reversals, that is, more than half of these deleterious mutations do not stay deleterious, but become neutral or beneficial through interactions with compensatory mutations. Importantly, the compensatory mutation(s) arise and reverse the deleterious effect well before the deleterious mutation fixes, and the beneficial combination of mutations then ascends together to fixation.s. He showed that under continuous mutation pressure, the double mutant can fix relatively rapidly, even in large populations. The fixation time for the double mutant was not unreasonably long, being slightly longer than the fixation time for a pair of neutral mutations and much shorter than the fixation time for a pair of unconditionally deleterious mutations.Kimura described a special case of our process in a model of neutral compensatory mutations . He deriA major question is whether this process occurs in nature. There is abundant experimental evidence that the fitness effect of a mutation can depend on genetic background \u201339. TherTwo factors may be necessary for this process to occur: a high mutation rate and epistasis. The mutation rate must be high enough that a second, interacting mutation arises in the genome before the first mutation is lost or fixed. While background selection typically refers to pairs of mutations  that have net negative fitness effects and no epistatic interactions, here we focus on pairs of mutations (with at least one deleterious) that epistatically interact to yield net positive fitness effects. We conjecture that, to the extent background selection is occurring, fitness reversals may likewise be important to the evolutionary dynamics. Furthermore, the mutation rate of interacting sites must be high enough to have a reasonable probability of creating the right combinations. Some natural systems are characterized by high mutation rates, including RNA viruses. Additionally, there is a sense that the self-replicating molecules present at the origin of life may have had high error rates, and so may fit this model. In the early stages of the process, a small population size may be important to the extent that it affects the rate of drift.Ascent via fitness reversals also requires a rugged (epistatic) fitness landscape. Although epistasis is widely recognized in genetics and evolution, the process described here requires an extreme form of it: the fitness effect of a mutation actually reverses (from bad to good) in the presence of a second mutation. Most studies of epistasis focus on the weaker form in which the fitness effect of a first mutation undergoes small changes in response to a second interacting mutation.Recent theoretical and experimental efforts, however, are beginning to elucidate additional details of these stronger epistatic interactions  ,39. For Other studies suggest that compensatory mutations occur at relatively high frequencies ,43. For prior to fixation (or loss) of the deleterious mutation, and thus have a fundamentally different evolutionary implication: they alter the fitness effect of a deleterious mutation sufficiently early to sway its ultimate evolutionary fate.Our results are a natural extension of previous work examining compensatory evolution in viruses and bacteria. As noted above, those studies almost exclusively considered compensatory beneficial mutations appearing after the fixation of a deleterious mutation and demonstrated that the compensatory effect depends on the presence of the initial deleterious mutation \u201315,44. TWhile it is widely recognized that asexuality poses several problems to adaptation through processes such as clonal interference, background selection, and Muller's ratchet , the relIn our study, deleterious mutations accumulated rapidly without impeding adaptation\u2014a result counter to most theoretical predictions. We attribute our results, at least in part, to the fact that the fitness effect of a mutation can change dramatically and rapidly upon additional mutations. It remains unclear whether these reversions are sufficient not only to ensure fixation of the original mutation, but also to constitute major adaptive steps."}
{"text": "A number of recent papers have cast doubt on the applicability of the quasispecies concept to virus evolution, and have argued that population genetics is a more appropriate framework to describe virus evolution than quasispecies theory.I review the pertinent literature, and demonstrate for a number of cases that the quasispecies concept is equivalent to the concept of mutation-selection balance developed in population genetics, and that there is no disagreement between the population genetics of haploid, asexually-replicating organisms and quasispecies theory.Since quasispecies theory and mutation-selection balance are two sides of the same medal, the discussion about which is more appropriate to describe virus evolution is moot. In future work on virus evolution, we would do good to focus on the important questions, such as whether we can develop accurate, quantitative models of virus evolution, and to leave aside discussions about the relative merits of perfectly equivalent concepts. I findquasispecies in this 1971 paper; he coined this term in a later paper coauthored with Peter Schuster [Quasispecies theory has its origin in a seminal paper written by Eigen in 1971 , in whicSchuster . These eSchuster ) were soSchuster ,12-14.s, and all other sequences have inferior fitness 1. As a result, much of the generality of Eigen's original work, as well as its connection to population genetics, have been obscured, and the conclusions of these special-case studies are frequently taken to be general predictions of quasispecies theory.Eigen's papers also generated substantial interest among theoreticians (mostly physicists), who found the description of highly error-prone replication an interesting theoretical challenge. Unfortunately, much of the theoretical follow-up work -21 has fBecause of the development of quasispecies theory independently from population genetics, and because of the widespread emphasis on a single fitness landscape in quasispecies theory, many authors now hold a set of beliefs about quasispecies theory that do not correspond to the actual predictions of the theory. These beliefs are:1. Quasispecies theory is at odds with population genetics.2. Quasispecies theory is inapplicable if populations are finite and there is neutral drift.3. Quasispecies theory predicts an error threshold.In the next three sections, I will address each of these points in detail. However, first I have to define what exactly I mean by quasispecies theory.Throughout this paper, by quasispecies theory I mean specifically Eq. (6) in Ref. ,xi(t) is the concentration of sequence i, Wij = AjQij is the product of the replication rate (fitness) Aj of sequence j and the mutation probability Qij from sequence j to i, and E(t) is the total production of new sequences,where In my definition of quasispecies theory, I also include straightforward generalizations of the above equation that have been used in the quasispecies literature, such as the discrete-time quasispecies equation, which can be written as ,23Wij. In both cases, the steady-state solution is given by the dominant eigenvector of Wij.and leads to the same steady-state solution as Eq. (1). Both Eqs. (1) and (3) can be mapped onto linear equations, and then solved by diagonalizing the matrix Wij, which consist of the fitness landscape (as given by the Aj) and the mutation landscape (as given by the Qij), are constants. In the most general case, fitness will depend on the mutant frequencies xi, as different mutants may make use of different resources, and the relative resource concentrations change as the mutant frequencies change. It turns out that the mapping onto a linear system is still valid if resource abundances change due to external factors [xi. In this latter case, which corresponds to frequency-dependent selection, the conclusions drawn from quasispecies theory do not apply.The mapping onto a linear system assumes that the  factors , but notSeveral recent papers present quasispecies theory as a theory that is alternative to (and maybe even contradictory to) standard population genetics ,7. Is ths over the a allele. Further, assume that allele a mutates into allele A, and likewise allele A into allele a, with probability \u03bc. Then, in Eq. (1), we have WAA = (1 + s)(1 - \u03bc), WaA = (1 + s)\u03bc, WAa = \u03bc, Waa = 1 - \u03bc, and hence (note that xa(t) = 1 - xA(t))Let us investigate what form the quasispecies equations take in a simple example. Consider a single locus with two alleles a and A, and assume that the A allele has a selective advantage \u03bc to zero, then this equation turns intoIf we set the mutation rate , and findthat is, into the standard logistic equation that describes the rise of a beneficial allele in an otherwise homogeneous population. Thus, we can recover standard population dynamics from the quasispecies equations. Now, let us calculate the steady state solution of Eq. (4) for an arbitrary mutation rate. We set xa = 1 - xA. For \u03bc = 0, this expression becomes xA = 1, which simply means that the A allele will reach fixation in the absence of any mutation pressure. As \u03bc increases, xA decreases, and xa increases. For a positive \u03bc, even though the a allele is removed from the population by selection, it is constantly regenerated from the A allele by mutation pressure, and thus reaches a positive equilibrium frequency. If the mutation rate is sufficiently high, then the equilibrium frequency of the a allele, maintained by the balance of selection and mutation pressure, can be substantial. In summary, we find that for the case of a single locus with two alleles, the quasispecies model predicts logistic growth of the beneficial allele in the absence of mutations, and mutation-selection balance in the presence of mutations.and of course xi of a sequence with i mutations changes from one generation to the next according to (Eq. (3.1) in Ref. [Now consider the multi-locus case. A classic paper on mutation-selection balance is the one by Kimura and Maruyama, written in 1966 . In this in Ref. ):wi is the fitness of a sequence with i mutations, , and \u03bc is the mutation rate (note that Kimura and Maruyama use fi instead of xi and 2M instead of \u03bc). Now, define the mutation matrix Qij aswhere Wij in Eq. (3) as Wij = wjQij. Then, we see that E(t) as defined in Eq. (2) becomes . Furthermore, the sum \u2211jWijxj in Eq. (3) runs from j = 0 to j = i, since Qij = 0 for i <j. After introducing a new index k = i - j, we can rewrite the sum asand write the matrix L of a population described by the quasispecies model is in many  cases approximately given by L= 1 - e\u03bc-.  follows directly from the quasispecies equation Eq. (3). As a consequence, the quasispecies model is in agreement with the Haldane-Muller principle , which mutations ,28.)Now that we have seen that quasispecies theory and mutation-selection balance are equivalent, the question remains whether Eigen just reinvented parts of population genetics, or actually contributed to the development of the field. While Eigen was not the first to consider mutation-selection balance (this concept goes back to Wright and Fisher in the early 20th century), by studying multi-locus mutation-selection equations at arbitrary mutation rate he was certainly at the forefront of theoretical population genetics in the late 1970s and early 1980s. The first analytic solutions to equations of the form Eq. (1) were found by Thompson and McBride in 1974  and indeWij, is a stable mixture of closely related mutants. This mixture of mutants, also called a mutant cloud or quasispecies, does not necessarily have to contain the fastest-replicating individual sequence that exists in the fitness landscape. In other words, sequences with high fitness can lose out against sequences with lower fitness that have better support from their mutational neighbors [survival of the flattest  that the equilibrium state of the population, which is given by the dominant eigenvector of the matrix eighbors ,33, an et Figure .It is important to understand that the emergence of a quasispecies is not something that has been put into the model ad hoc, but is a necessary consequence of the mutation-selection equations. We see in Eqs. (1) and (3) that the model is built on reproduction of individual sequences, but that mutations provide coupling between the different sequence types. When the mutation rate is low, then the quasispecies model predicts that the fastest-replicating sequence takes over the population, as we witness from the emergence of the logistic equation Eq. (5) at zero mutation rate. However, when the mutation rate is high, then the coupling between sequences caused by mutations can become stronger than the individual selection coefficients, and a quasispecies forms. Note that this effect will arise in any model of mutation-selection balance that correctly takes into account the coupling of different mutants at high mutation rate.quasispecies effects, I mean that the population behaves in a way that can only be explained through strong mutational coupling between genotypes. An example would be the observation of the survival of the flattest effect.)In the previous section, I have established that the quasispecies model is equivalent to the theory of mutation-selection balance in an infinite, haploid, asexual population. However, this equivalence does not necessarily imply that the quasispecies model applies to populations of RNA viruses, because these populations are finite. Jenkins et al.  argue thFirst, let us have a look at some theoretical studies of finite populations that have been carried out within the context of the quasispecies model ,34-38. IHowever, one could still object that these models may be describing idealized and simplified situations that differ too much from the reality of RNA viruses to be of any relevance. To counter this argument, we have to ask whether there is a more general way to determine the relevance of quasispecies theory to finite populations of RNA viruses. The hallmark of quasispecies theory  is that for a sufficiently high mutation rate, we must take into account the formation of a quasispecies to obtain a faithful description of the population dynamics. Therefore, the question is under what conditions does mutational coupling become so strong that we can observe quasispecies effects in a finite population. Can theory help us to address this question?evolution of mutational robustness [\u03bc and effective population size Ne was significantly larger than one, \u03bcNe \u226b 1 [In the formation of a quasispecies, the population minimizes the mutational load by accumulating sequences that have a reduced probability to suffer from deleterious mutations ,28,39,40bustness . Van Nim \u03bcNe \u226b 1 . I have  \u03bcNe \u226b 1 . What is \u03bcNe \u226b 1 . These r \u03bcNe \u226b 1 ,43. Note \u03bcNe \u226b 1 ,45.47 and 1068 sequences large. Clearly, a population of size 250  cannot even come close to complete coverage of such a huge sequence space.Finally, the recent paper by Comas et al.  also proThe previous paragraphs show that on purely theoretical grounds, there is no reason to assume that quasispecies effects cannot play a role in finite populations of RNA viruses. Nevertheless, to date we have no experimental evidence that unequivocally demonstrates such effects in a specific experimental system. On the other hand, selection for specific, individual mutants is common (see e.g. Ref. ). What dIn summary, we currently have no evidence  that contradicts the existence of quasispecies effects in finite populations of RNA viruses, but we also have no experimental evidence in favor of it. Theoretical studies and computer simulations indicate that quasispecies effects should become important when the product of effective population size and genomic mutation rate is significantly larger than one. Since for many RNA viruses the genomic mutation rate is already on the order of one ,49, evenThe error threshold is probably one of the most misunderstood concepts of quasispecies theory. Eigen described the error threshold in his 1971 paper as a limit to the amount of information a genome can store at a given mutation rate . If the The first important point to understand about the error threshold is that it is a deterministic effect. This means that the position of the error threshold depends only weakly on the population size, and that even in an infinite population the error threshold occurs at a finite mutation rate ,35. In tper se makes no statements about population extinction. The alternative model is hard selection, where the population size will decline if the mean fitness of the population is too low. Extinction due to mutation pressure can occur under hard selection, and is usually called mutational meltdown [There is certainly no lack of lethal mutants in viruses ,58, and meltdown -65. Mutaerror cascade [Finally, it is interesting to note that in certain fitness landscapes, several error-threshold-like transitions can occur one after the other as the mutation rate increases . At each cascade , and in  cascade ,33,46 caTo summarize, I have provided arguments for the following conclusions: Quasispecies theory is in perfect agreement with population genetics, it can make usefull predictions for finite populations if the product of population size and mutation rate is large, and it predicts an error threshold only for fitness landscapes that lack lethal mutants, and which therefore have little relevance for virus evolution.However, these arguments do not imply that quasispecies theory is the final answer to all questions of virus evolution. Quasispecies theory has its short-comings that need to be addressed in future modeling work. Ironically, the biggest shortcoming of quasispecies theory, as far as I can see, does not have its origin in quasispecies theory being at odds with population genetics, but rather in quasispecies theory being too similar to the population genetics theory of asexual, haploid organisms. Viruses differ from other forms of life in that they don't have a well-defined ploidy. When a single virus particle infects a cell, the virus can be considered a haploid organism, and indeed the quasispecies model makes this assumption. However, frequently several virus particles coinfect the same cell, in which case the ploidy is given by the number of coinfecting particles. Standard population genetics has no model for such a variable-ploidy organism, and only a handful of authors have considered the theoretical implications of viral coinfection in detail -81.COW carried out all aspects of this work."}
{"text": "Phenotype of biological systems needs to be robust against mutation in order to sustain themselves between generations. On the other hand, phenotype of an individual also needs to be robust against fluctuations of both internal and external origins that are encountered during growth and development. Is there a relationship between these two types of robustness, one during a single generation and the other during evolution? Could stochasticity in gene expression have any relevance to the evolution of these types of robustness? Robustness can be defined by the sharpness of the distribution of phenotype; the variance of phenotype distribution due to genetic variation gives a measure of \u2018genetic robustness\u2019, while that of isogenic individuals gives a measure of \u2018developmental robustness\u2019. Through simulations of a simple stochastic gene expression network that undergoes mutation and selection, we show that in order for the network to acquire both types of robustness, the phenotypic variance induced by mutations must be smaller than that observed in an isogenic population. As the latter originates from noise in gene expression, this signifies that the genetic robustness evolves only when the noise strength in gene expression is larger than some threshold. In such a case, the two variances decrease throughout the evolutionary time course, indicating increase in robustness. The results reveal how noise that cells encounter during growth and development shapes networks' robustness to stochasticity in gene expression, which in turn shapes networks' robustness to mutation. The necessary condition for evolution of robustness, as well as the relationship between genetic and developmental robustness, is derived quantitatively through the variance of phenotypic fluctuations, which are directly measurable experimentally. Robustness is ability to function against changes in the parameter of a systemIndeed, phenotype of isogenic individual organisms is not necessarily identical. ChemotaxisNoise, in general, can be an obstacle in tuning a system to the fittest state and maintaining it there. Phenotype of an organism is often reproducible even under fluctuating environment or under molecular fluctuationsWhether these two types of robustness emerge under natural selection have long been debated in the context of developmental dynamics and evolution theoryipV, gives an index for robustness to noise in developmental dynamics. On the other hand, robustness to mutation is measured from the fitness distribution over individuals with different genotypes. An index for it is given by variance of phenotypic fluctuation arising from diversity of genotypes in a populationgV. This variance gV increases as the fraction of low-fitness mutants increases.Under the presence of noise in gene expression, phenotype as well as fitness, of isogenic organisms is distributed, usually following a bell-shaped probability function. When the phenotype is less robust to noise, this distribution is broader. Hence, the variance of this distribution, i.e., variance of isogenic phenotypic fluctuation denoted as ipV\u2265gV is satisfied. Since the isogenic phenotypic fluctuation ipV increases with noise, this means that evolution of robustness is possible only when the amplitude of phenotypic noise is larger than some critical value as derived by ipV\u2265gV, implying a positive role of noise to evolution. We demonstrate that both the two variances ipV and gV decrease in the course of evolution, while keeping the proportionality between the two. This proportionality is consistent with an observation in a bacterial evolution experiment Here we show that evolution to increase both types of robustness is possible only when the inequality We explain the origin of the critical noise strength, by noting that smooth dynamical behavior free from a rugged potential landscape evolves as a result of phenotypic noise. When the noise amplitude is smaller than the threshold, we observe that low-fitness mutants are accumulated, so that robustness to mutation is not achieved. Generality and relevance of our results to biological evolution are briefly discussed.P where x is a variable characterizing a phenotype and a is that for the corresponding genotypex is responsible for the fitness of an individual, and the selection depending on x is considered as an evolutionary process. Since the phenotype differs even among isogenic individuals, the distribution P for a fixed genotype a0 has some variance. This isogenic phenotypic variance ipV, defined as the variance over clones, is written as a, namely ipV works as a measure of robustness of the phenotype against noise.In natural population, both the phenotype and genotype differ among individuals. Let us consider population distribution gV here, is then defined as the variance of the average P(a) is the distribution of the genotype a and <x\u0305 > is the average of a. While ipV is defined as variance over clones, i.e., individuals with the same genotype, gV comes from those with different genotypes. As gV is smaller, the phenotypic change by genetic variation is smaller. Hence gV gives a measure of robustness of the phenotype against mutation.On the other hand, the standard evolutionary genetics ip>VgV between the two variances, by assuming evolutionary stability of the population distribution P, that is preservation of single-peakedness through the course of evolution ipV approaches gV, where the distribution is extended to very low values of x (fitness). In other words, error catastrophe occurs at g\u2248VipV;  is based on the existence of two-variable distribution function P, and the postulate that single-peaked distribution is maintained throughout evolution, which is not trivial. Hence the above relationships need to be examined by some models for evolution. In addition, ip is smaller than Vgwhy does the population distribution extend to low-fitness values when the phenotypic fluctuation V? Or, put it another way, why do systems with small phenotypic noise run into \u201cerror catastrophe\u201d? In fact, the emergence of error catastrophe as a result of decreasing isogenic phenotypic fluctuation below gV may look rather counter-intuitive, since in general one expects fluctuation to perturb a system from the fittest state. The necessity of fluctuation for evolution to increase robustness to noise and to mutation needs theoretical examination.Note, however, that the derivation of these relationships  from a given initial state. In general, this dynamic process is complex so that the process may not reach the identical phenotype due to the noise through this developmental process. This leads to the isogenic variance of the phenotype ipV. On the other hand, the expression pattern varies by mutation in the network, and gives rise to variation in the average fitness, resulting in gV.We consider a simple model to satisfy the requirement on \u2018development\u2019 above. It consists of a complex dynamic process to reach a target phenotype under a noise which may alter the final phenotypic state. We do not choose a biologically realistic model that describes a specific developmental process, but instead take a model as simple as possible, to satisfy a minimal requirement for our study. Here we take a simplified model, borrowed from a gene regulatory network, where expression of a gene activates or inhibits expression of other genes under noise. These interactions between genes are determined by the network. The expression profile changes in time, and eventually reaches a stationary pattern. This gene expression pattern determines fitness. Selection occurs after introduction of mutation at each generation in the gene network. Among the mutated networks, we select a network with a higher fitness value. Since there is a noise term in the gene expression dynamics, fitness fluctuates even among the individuals with an identical gene network, which leads to the isogenic fluctuation ix is described byijJ\u200a=\u200a\u22121,1,0, and \u03b7(t) is Gaussian white noise given by <\u03b7(t)\u03b7(t\u2032)>\u200a=\u200a\u03b4(t\u2212t\u2032). M is the total number of genes, and k is the number of output genes that are responsible for fitness to be determined. The value of \u03c3 represents noise strength that determines stochasticity in gene expression . By following a sigmoid function tanh, ix has a tendency to approach either 1 or \u22121, which is regarded as \u201con\u201d or \u201coff\u201d of gene expression. Even though x is defined over , it is attracted to the range  (or slightly above or below the range due to the noise term). We consider a developmental process leading to a matured phenotype from a fixed initial state, which is given by ; i.e., all genes are off, unless noted otherwise. .This simplified gene expression follows a typical switch-like dynamics with a sigmoid input-output behavior ix) for genes i\u200a=\u200a1,2,\u2026,k(<M) would reach an \u201con\u201d state, i.e., ix>0. The fitness is maximum if all k genes are on after a transient time span iniT, and minimum if all are off. To be specific, we define the fitness function byS(x)\u200a=\u200a1 for x>0, and 0 otherwise, [\u2026]temp is time average between init\u200a=\u200aT and ft\u200a=\u200aT . Adoption of the value (S(jx)\u22121) after initial time iniT leads to the same result  is always \u201con\u201d and takes the minimum (F\u200a=\u200a\u2212k) when all k genes are always off. Note that fitness is calculated only after time iniT, which is chosen sufficiently large so that the temporal average can be computed after the gene expression dynamics has fallen on an attractor. This initial time can be considered as the time required for developmental dynamics.Let us define a fitness function so that gene expression levels , for a given network \u201cg\u201d, whose variance gives isogenic phenotypic fluctuation. At each generation, we compute the fitness F over L runs, to obtain the average fitness value F\u0305 of a given network.As the model contains a noise term, fitness fluctuates at each run, which leads to the distribution in ijJ which determines the \u2018rule\u2019 of the dynamics, it is natural to treat ijJ as a measure of genotype. Individuals with different genotype have a different set of ijJ At each generation there are N individuals with different sets of ijJ For each individual network, we compute the average fitness F\u0305. Then we select the top sN(<N) networks that have higher fitness values. .Now we consider the evolutionary process of the network. Since the network is governed by ijJ at a given mutation rate \u00b5. We rewire the network at a given rate so that changes in ijJ produce N new networks. At each generation, mutation changes the network, i.e., changes sN/N mutants from each of the top sN networks, so that there will be N networks again for the next generation. From this population of networks we repeat the process of the developmental dynamics, fitness calculation, selection, and mutation .Here we make ijJ are nonzero). At each generation, the N individuals have slightly different networks ijJ, so that the values of F\u0305 are different. We denote the fitness distribution over individuals with different genotype as P(F\u0305 ). On the other hand, the fitness distribution for an identical network (\u201cg\u201d) is computed to obtain p.Simulations start from a population of random networks with a given fraction of paths  are invariant.Developmental dynamics and selection process in our model are too simple. Still, this model is relevant to examine general statement on phenotypic fluctuations, as the model at least captures complex dynamics giving rise to a phenotype, stochasticity in dynamics, mutation, and selection according to a given phenotype. Indeed, real gene expression dynamics depend on environmental conditions, and the fitness is defined as expression patterns to adapt each environmental condition. We have also carried out some simulations by imposing such fitness but the results to be discussed  increases, so that the top of F\u0305 in the population approaches the highest value 0. Indeed, in all cases, the top group quickly evolves to the highest fitness state F\u0305\u200a=\u200a0 . The time necessary for the system to reach this state becomes shorter as the phenotypic noise decreases (see P(F\u0305) depends drastically on the noise strength \u03c3. When \u03c3 is small, the distribution is broad and the existing individual with the lowest F\u0305 remains at the low fitness state, while for large \u03c3, even the individuals with the lowest fitness approach F\u0305\u200a=\u200a0 (see c\u03c3(\u22480.02), below which the distribution P(F\u0305) is broadened, as is discernible in the data of the variance of the distribution, gV in F\u0305> and the lowest fitness over individuals F\u0305min, after a sufficiently large number of generations, are plotted against \u03c3 in c\u03c3, below which low-fitness mutants always remain in the distribution. For c\u03c3>\u03c3, the distribution P(F\u0305) takes a sharp peak at F\u0305 \u223c0, where the variance is rather small. Distribution below and above c\u03c3 are displayed in c\u03c3>\u03c3).Let us first see how the evolutionary process changes as a function of the noise strength ases see . On the \u200a=\u200a0 see . There igV and ipV Here ipV is defined as variance from the distribution p, i.e., over individuals with the same genotype. As the distribution p depends on each individual with different genotype, the variance changes accordingly. Naturally, the top individual has a smaller variance, and the individual with lower fitness has a larger variance. As a measure of ipV, we used either the average of the variance over all individuals or the variance of phenotype from a gene network that is located closest to the peak in the distribution P(F\u0305). Both estimates of ipV do not differ much, and the following conclusion is drawn in both cases. gV, on the other hand, is simply the variance of the distribution P(F\u0305), i.e., over individuals having different genotypes present.Let us study the relationship between gV and ipV thus evaluated is plotted in c\u03c3>\u03c3, where we note:The relationship between ipV>gV for c\u03c3>\u03c3.(i) gV\u221dipV during the evolutionary time course under a fixed noise strength \u03c3(>c\u03c3) and a fixed mutation rate. As \u03c3 is lowered toward c\u03c3, gV increases so that it approaches ipV.(ii) gV\u2248ipV at c\u03c3\u2248\u03c3, where error catastrophe occurs.(iii) ipV>gV is satisfied. As \u03c3 is decreased to c\u03c3, ipV approaches gV, error catastrophe occurs and a considerable fraction of low-fitness mutants accumulates. Hence, the relationships proposed theoretically assuming evolutionary stability of a two-variable distribution function P is confirmed. Here, without introducing phenomenological assumptions, the three relationships are observed in a general stochastic gene-network model.In other words, the fittest networks maintaining a sharp distribution around the peak dominate only when c\u03c3<\u03c3? To study the difference in dynamics evolved for different values of \u03c3, we choose the top individual (network) that has evolved at \u03c3\u200a=\u200a\u03c30, and place it under a different noise strength \u03c3\u200a=\u200a\u03c3\u2032. In F\u200a=\u200a0 under such circumstance. As shown, the successful fraction decreases when \u03c3\u2032 goes beyond \u03c30. In other words, the network evolved under a given noise strength successfully reaches the target gene expression up to that critical noise level, while it begins to fail doing so at a higher noise strength. Accordingly, the distribution p extends to lower values in fitness, when a network evolved under small phenotypic noise is developed under a higher noise level. On the other hand, the network evolved under high level noise maintains a high fitness value, even when the noise is lowered.Why didn't the system maintain the highest fitness state under small phenotypic noise ix>0 for i\u200a=\u200a1,\u2026,k). Hence the basin of attraction for this target attractor is expected to be larger for the dynamics evolved under higher level noise. We have simulated the dynamics (1) for the evolved fittest network under zero noise, starting from a variety of initial conditions over the entire phase space, and measured the distribution of F at each attractor. The distribution is shown in jx\u200a=\u200a1\u2194jx\u200a=\u200a\u22121 in the model, the distribution is symmetric around F\u200a=\u200a\u2212k/2 when all initial conditions are taken. In fact, by starting from ix\u200a=\u200a1 for all i, the orbit reaches an attractor jx<0 for j\u200a=\u200a1,\u2026,k, resulting in F\u200a=\u200a\u2212k). For the network evolved under c\u03c3>\u03c3, the distribution has a sharp peak at F\u200a=\u200a0 (and F\u200a=\u200a\u2212k due to the symmetry), with more than 40% attraction to each. On the other hand, for the networks evolved under c\u03c3<\u03c3, the peak height at F\u223c0 is very small, i.e., the basin for the attractor with F\u200a=\u200a0 is tiny. There exist many small peaks corresponding to attractors with \u2212k<F<0, having similar sizes of basin volumes. In fact, the basin volumes for attractors with \u2212k<F<0 grow as \u03c3 is decreased, and are dominant for c.\u03c3<\u03c3Next we study the basin structure of attractors in the present system. Note that an orbit of the network with the highest fitness, starting from the prescribed initial condition, is within the basin of attraction for an attractor corresponding to the target state  which is defined by \u0394 here. The distance \u0394 remains small for the dynamics evolved under a low noise strength c\u03c3<\u03c3, and increases for those evolved under a higher noise. It is interesting to note that evolution influences the basin structure globally over the phase space, although the fitness condition is applied locally to an orbit starting from a specific initial condition.The difference in the basin structure suggested by jx for j\u200a=\u200a1,\u2026,k changes its sign. Recall that the fixed point solution ix\u200a=\u200atanh(\u03a3jJijxj) changes its sign when \u03a3jJijxj in the sigmoid function changes its sign. This change may occur during the developmental dynamics by noise, and we call such points in the phase space \u2018turning points\u2019. When an orbit of eq.(1) passes over turning points, jx takes a negative value for some j (for 1\u2264i\u2264k) at the attractor  changes its sign at several points in the phase space {jx} generally. Hence there can be many turning points in the phase space. The fittest network with F\u0305\u22480 chooses such orbits having no turning points within the noise range from the original orbit. An orbit from the fittest individual evolved under low-level noise encounters many turning points when subjected to noisy environment.The results in The average distance between the turning points and an orbit that has reached the target gene expression pattern is estimated by the distance \u0394 defined above. Recall that the distance \u0394 is small for the dynamics evolved under a low noise strength. Such dynamics, if perturbed by a higher level of noise, are easily caught in the turning points, which explains the behavior shown in gV and ipV. Noise disturbs an orbit so that it may go across the basin boundary (turning points) with some probability. We denote the standard deviation of the location of the orbit due to noise as p\u03b4, which is proportional to the noise strength \u03c3. Since the distance between the orbit and the basin boundary is deviated by p\u03b4, and the fitness value drops when the orbit crosses the basin boundary, the variance ipV is estimated to be proportional to (p\u03b4/\u0394)2.Let us now discuss the relationship between ijJ, there exists a variation of the order of g\u03b4, which increases with the mutation rate. The variance gV is estimated to be proportional to (g\u03b4/\u0394)2, with the same proportion coefficient as that between ipV and (g\u03b4/\u0394)2.Next, we discuss how the mutation in the network influences the dynamics. When the network is altered, i.e., a path is added or removed as a result of mutation in p\u03b4 from the \u201ctarget\u201d orbit. This leads to an increase in \u0394. However, if p\u03b4 is larger than p\u03b4, the memory of this distance between the target and the boundaries will not be propagated to the next generation, due to large perturbation to the original network by the mutation. Hence increase in \u0394  is expected only if p>\u03b4g\u03b4. Since p\u03b4 and g\u03b4 increase with the noise strength \u03c3 and the mutation rate \u00b5 respectively, there exists a critical noise strength c\u03c3 beyond which this inequality is satisfied. From the relationship between p,g\u03b4 and ip,gV, the condition for robust evolution is rewritten as ip>VgV.Under the presence of noise, there is a selective pressure to avoid the turning points (basin boundaries) that exist within the distance ip>VgV  is satisfied, the system increases \u0394 during evolution. We have computed the temporal evolution of basin distribution. With generations, the distribution evolves from the pattern at a low level noise in \u03c3 characterized by enhanced peak at F\u200a=\u200a0. Accordingly \u0394 increases with generations. Recall that ipV\u223c(p\u03b4/\u0394)2, and gV\u223c(g\u03b4/\u0394)2, both variances decrease with generations, while ip/VgV is kept constant.When the condition gV and ipV, through numerical evolution experiment of a gene network. As phenotypic noise is decreased and the inequality ip>VgV is broken, low-fitness mutants are no longer eliminated. This is because the mutants fail to reach the target gene expression pattern, by crossing the boundary of the basin of attraction to the target. When the amplitude of the noise is larger, on the other hand, the networks of the dynamics with a large basin volume for the target attractor are selected and thus mutants with lower fitness are removed successively through the selection process. Hence noise increases developmental robustness through evolution, together with genetic robustness.We have demonstrated the inequality and proportionality between ipV and gV and the error catastrophe, we expect this relationship to be generally applicable to systems satisfying the following conditions:Although we used a specific example to demonstrate the relationship between (i) Fitness is determined through developmental dynamics.(ii) Developmental dynamics is sufficiently complex so that its orbit, when deviated by noise, may fail to reach the state with the highest fitness.(iii) There is effective equivalence between mutation and noise in the developmental dynamics with regards to phenotype change.Note that the present system as well as the previous cell modelg<VipV must be satisfied in order for the low-fitness mutants to be eliminated. Violation of the inequality leads to accumulation of low-fitness (or deleterious) mutants, a phenomenon known as error catastrophegV of given phenotype in mutants, and comparing it with that of isogenic bacteria, ipV, one can examine the validity of our conclusion between gV and ipV.When developmental process fails due to phenotypic noise, the fitness function takes a low value. Evolution under noise acts to prevent such failure within the range of noise. On the other hand, due to the condition (iii), mutation may also lead to such lethality. When the effect of mutation goes beyond the range given by the phenotypic noise, mutants with very low fitness values begin to accumulate. Hence there appears a threshold level of phenotypic noise below which low-fitness (or deleterious) mutants accumulate (or threshold mutation rate beyond which such mutants accumulate). In this sense, we expect that for robust evolution, the inequality It is worthwhile to mention that in a class of theoretical models, fitness landscape is given as an explicit continuous function of a gene sequence  is formed through a mutation and selection process. As a result, dynamics over the entire phase space are simplified to those having only a few attractors, even though the fitness function is given locally without scanning over the entire phase space. When the time-course is represented as a motion along a potential surface, our results suggest that the potential landscape becomes smoother and simpler through evolution, and loses ruggedness after generations. Indeed, existence of such global attraction in an actual gene network has recently been reported in yeast cell-cycleijJ between spins (corresponding to gV) and the temperature  is formulated. Such relationship may be relevant to understand the relationships between gV and ipV in our study.Such smooth landscape was also studied in protein foldinggVipV and gV then suggests that the evolution speed is proportional to the isogenic phenotypic fluctuation, as is also supported by an experiment on bacterial evolution in a laboratoryAccording to established Fisher's theorem on natural selection, evolution speed of phenotype is proportional to the phenotypic variance by genetic variation, Isogenic phenotypic fluctuation is related to phenotypic plasticity, which is a degree of phenotype change in a different environment. Positive roles of phenotypic plasticity in evolution have been discussedText S1Derivation of General Relationship on Fluctuations. Mathematical derivation on general relationships on phenotypic variances is presented.(0.05 MB PDF)Click here for additional data file."}
{"text": "Using a transparent mathematical model, we illustrate the concept of genetic potential and show that as environmental variability decreases, the evolving population reaches three distinct steady state conditions: (1) organismal flexibility, (2) genetic potential, and (3) genetic robustness. As a specific example of this concept we examine fluctuating selection for hydrophobicity in a single amino acid. We see the same three stages, suggesting that environmental fluctuations can produce allele distributions that are distinct not only from those found under constant conditions, but also from the transient allele distributions that arise under isolated selective sweeps.Organisms employ a multitude of strategies to cope with the dynamical environments in which they live. Homeostasis and physiological plasticity buffer changes within the lifetime of an organism, while stochastic developmental programs and hypermutability track changes on longer timescales. An alternative long-term mechanism is \u201cgenetic potential\u201d Variation is the fuel of natural selection. Understanding the mutational processes that underlie evolution has long been a central objective of population genetics. Today, amidst a computational revolution in biology, such understanding is pivotal to progress in many biological disciplines. For example, neutral mutations make the molecular clock tick, and this clock is fundamental to reconstructing phylogenies, measuring recombination rates, and detecting genetic functionality. In this manuscript, the researchers provide an original perspective on a long-standing question in evolutionary biology: to what extent do mutation rates evolve? They argue that to cope with environmental fluctuation, populations can evolve their phenotypic mutation rate without changing their genetic mutation rate. That is, populations can evolve \u201cgenetic potential\u201d\u2014a heightened sensitivity to the effects of mutation. The researchers use a simple mathematical model of amino acid evolution to illustrate the evolution of genetic potential, and show that as environmental variability decreases, evolving populations reach three distinct states. In a rapidly fluctuating environment, organisms evolve the flexibility to cope with variation within an individual lifetime; in moderately variable environments, populations evolve the ability to evolve rapidly; and in fairly constant environments, populations evolve robustness against the adverse effects of mutation. Recent work in evolutionary biology has highlighted the degeneracy of the relationship between genes and traits . For anyA separate body of evolutionary theory addresses adaptation under fluctuating conditions ,8. The rIn this paper we show that genetic degeneracy may give rise to an alternative outcome under fluctuating conditions: the evolution of genotypes with heightened sensitivity to mutation. We introduce the term \u201cgenetic potential\u201d to describe this state. Metaphorically, populations with genetic potential lie near the edge of neutral plateaus. Although the rate of mutation is unchanged, the likelihood that mutations produce beneficial variation increases. Heightened sensitivity to mutations has been recognized as a critical and transient phase of adaptive evolution ,15,16. HWe then present a more biological example of this phenomenon using a model of amino acid evolution. There is evidence that, within viral pathogens, the physiochemical properties of amino acids found within epitopes\u2014regions of proteins that directly interact with the host immune system\u2014can rapidly evolve ,18. LikeUnder infrequent environmental change, populations evolve from one mutationally robust phenotype to another, briefly passing through genotypes that can easily mutate to either state. One might therefore be tempted to equate genetic potential with confinement to the intermediate steps on a path from robustness for one phenotype to robustness for another . While tA and EB), spending exactly \u03bb generations per state between shifts. The simple model includes three phenotypes\u2014one optimal phenotype for each of the two environments (A and B) and a third that has intermediate quality in both environments (V)\u2014and a minimal amount of degeneracy in the relationship between the genotype and the phenotype. In particular, there is a single genetic locus, and five allelic possibilities at that locus  mod 5( or gi + 1) mod 5 in its preferred environment, and 0 \u2264 k \u2264 1 determines the intermediacy of the V phenotype.where We can write the full model as a set of difference equationsi \u2208 {0,1,2,3,4}, where \u03bc is the mutation rate and tw denotes the fitness in the current environment , \u03a6i is the set of three transition point mutations of codon i, and \u0393i is the set of six transversion point mutations of codon i.for 1 \u2264 A, the equilibrium relative frequencies of g0, g1, g2, g3, and g4 are 0.291, 0.412, 0.292, 0.003, and 0.002, respectively, and in environment EB, they are 0.005, 0.000, 0.000, 0.010, and 0.985, respectively. When there is degeneracy, as there is for phenotype A, the populations evolve genetic robustness, that is, more mutationally protected genotypes appear in higher frequency. In particular, g1, which lies in the center of the three genotypes that code for A, appears in higher frequency than either genotype on the edge of the neutral network for A (g0 and g2) at equilibrium in EA. In the absence of degeneracy (phenotype B), we observe a mutation\u2013selection balance around the single optimal genotype. These findings are consistent with and provide a transparent example of the extensive theory on mutation\u2013selection balance, quasi-species, and the evolution of genetic robustness in neutral networks [We provide an intuitive perspective on evolution in fluctuating environments using the simple model and then demonstrate the generality of the results in the codon model. The first results assume a mutation rate \u03bc = 0.01, and fitnesses 1, 1.5, and 2 for the unfavored, intermediate, and favored phenotypes, respectively. In a constant environment, a population will equilibrate on genotypes that encode the optimal phenotype. In environment Enetworks \u201324.A and EB, thereby alternating between genetic robustness for A and a mutation\u2013selection balance around the single allele for B. At intermediate rates of fluctuation, populations hover near g4 and g0, where the genotypes for A abut the genotype for B. Thus, mutation between the two phenotypes occurs frequently. We call this outcome genetic potential because of the enhanced potential for mutations to give rise to novel  phenotypes. Finally, for highly variable environments, the populations converge on the phenotype V, which has unchanging, intermediate fitness in both environments. Phenotype V corresponds to organismal flexibility\u2014individual organisms tolerate both conditions, but neither one exceptionally well. There are a variety of mechanisms that can give rise to an intermediate phenotype including homeostasis, somatic evolution, physiological plasticity, and behavioral plasticity [Under rapid environmental fluctuations, populations do not have time to reach a stable allele distribution. As the environment becomes more variable, the distributions of alleles go through three distinct phases. asticity ,8. As orasticity , the asc\u22125, and fitnesses 1, 1.5, and 2 for the unfavored, intermediate, and favored phenotypes, respectively. (We address the impact of mutation rate in the Discussion.) Whereas in the simple model only one of the three phenotypes had multiple genotypes, in this model all three phenotypic classes have genetic degeneracy, and thus can evolve genetic robustness  if and when the environment reverts. For populations that have equilibrated in a constant environment and have recently experienced an environmental shift, genetic potential will decrease as the population becomes increasingly robust to the effects of mutation . For pop2 generations. A and EB. We then compared these two distributions to the evolving amino acid distribution in a population that equilibrates in one of the two environments and then faces an environmental shift. This difference also appears in the distributions of amino acids. We calculated the genetic potential in each generation of a population experiencing fluctuations every \u03bb = 10\u22125 in the codon model, populations evolve genetic potential when environment varies at rates of 101 < \u03bb < 106 generations, approximately K [aK/sK, such genes should employ a distinct set of codons with high genetic potential. Note that this type of genetic potential is not equivalent to codon bias, but rather implies changes in the actual distribution of amino acids.A long-standing technique for identifying selected genes is to compare the frequencies of nonsynonymous and synonymous substitutions (Ka/Ks) . Genes e(Ka/Ks) ,41 or ge(Ka/Ks) . In the A similar argument also underlies the recent use of codon distributions for detecting genetic loci under directional selection . Codon vaK/sK ratios. Such sites have been identified, for example, in human class I MHC genes, the HIV envelop gene, and a gene from a human T cell lymphotropic virus (HTLV-1) [We would like to emphasize that the goal of this study was not to develop a new method for detecting positive (or fluctuating) selection, but rather to develop a theoretical framework for considering the multiple outcomes of evolution under fluctuating conditions. We conclude by suggesting an empirical method to identify loci that have evolved genetic potential under such conditions as distinct from those that have experienced a recent selective sweep. Suppose that a gene experiences fluctuations at a characteristic rate across many species. Furthermore, suppose that multiple sites within the gene are influenced by such fluctuations. For example, there may be fluctuating selection for molecular hydropathy, charge, size, or polarity, and several sites within the gene may contribute to these properties. Such sites should evolve in tandem and equilibrate on similar levels of genetic potential, and thus exhibit similar codon (and amino acid) distributions across species. In contrast, if a gene experiences isolated selective sweeps, then the variation at all sites should correspond to both the history of selective events and the species phylogeny, and the amino acid distributions at sites should correlate only when sites functionally mirror each other. Thus, one can seek evidence for the evolution of genetic potential as follows. First, identify genes that are rapidly evolving, perhaps by calculating (HTLV-1) ,32. WithMA and MB be the normalized transition matrices that govern changes in the allele frequencies in EA and EB epochs, respectively. The entries in these matrices are defined by equations 2 and 4. The left leading eigenvectors for MA and MB give the equilibrium frequency distributions of alleles in each of the two constant environments, respectively. Under fluctuating conditions with epoch duration of \u03bb generations, we iteratively apply the matrices, and then compute the left leading eigenvector of vB, gives the allele frequency distribution at the end of an EA epoch followed by an EB epoch.For the two models, we calculate the deterministic, infinite population allele frequency distributions in constant and fluctuating environments. Let A epoch byWe are interested not only in the final allele distributions, but also in the dynamics throughout each epoch. Thus, we calculate the average frequency of each allele across a single EG is the total number of alleles in the model (G = 5 for the simple model and G = 64 for the codon model) and the subscript k indicates the kth entry in the vector. Similarly, the average distribution across an EB epoch is given bywhere vA is the allele frequency distribution at the end of an EB epoch followed by an EA epoch and is equal to the left leading eigenvalue of where For the codon model, we compare these calculations that assume a regularly fluctuating environment to numerical simulations that assume a Poisson distribution of epoch lengths. In each generation of the simulations, the environmental state switches with probability 1/\u03bb and the codon frequencies are then multiplied by the appropriate transition matrix.We use an elementary group theoretic result known as Burnside's Lemma  to proveX be the set of all pentagons with vertices labeled {A, B, V} having at least one A vertex and at least one B vertex. The size of X is the number of all pentagons with labels {A, B, V} minus the number of pentagons with labels {A, V} or {B, V}, that is, |X| = 35 \u2212 (2 \u00b7 25 \u2212 1) = 180.Let G of all actions on X that produce equivalent pentagons (as specified above). G is made up of (1) the identity, (2) the four rotations and five reflections of the pentagon, (3) interchanging all As and Bs, and (4) all the combinations of the above actions. Thus G is equal to the 20-member group {\u03b9, \u03c1, \u03c12, \u03c13, \u03c14, \u03c30, \u03c31, \u03c32, \u03c33, \u03c34, \u03b1, \u03b1\u03c1, \u03b1\u03c12, \u03b1\u03c13, \u03b1\u03c14, \u03b1\u03c30, \u03b1\u03c31, \u03b1\u03c32, \u03b1\u03c33, \u03b1\u03c34} where \u03b9 is the identity, \u03c1 is a single (72\u00b0) rotation, \u03c3i is a reflection through vertex i, and \u03b1 is replacement of all As with Bs and all Bs with As. We define the group G on X. Burnside's Lemma tells us that this number isThe number of distinct mutational networks is equal to the number of orbits of F(g) = {x \u2208 X | gx = x} is the set of fixed points of g. For each of the twenty elements of G, we exhaustively count F(g).where X, that is, F(\u03b9) = X. Each of the various rotations of a pentagon  has the property that its iterations move a given vertex to every other vertex of the pentagon without changing the letter assigned to that vertex. The same is true of the square of the product of any rotation and an A\u2013B flip. Hence, any fixed point of one of these elements of the group G would necessarily have the same label at each vertex of the pentagon. Since every labeled pentagon in X has at least one A label and at least one B label, then no element of X has the same label at each vertex. Thus, the fixed point set of every rotation and of every product of a rotation and an A\u2013B flip must be empty, that is, F(\u03c1n) = F(\u03b1\u03c1n) = \u2205\ufe00 for all n. By a similar argument, the simple A\u2013B flip also has no fixed points. Every reflection fixes 12 elements of X, for example,The identity fixes all elements of X, for example,and every product of a reflection and an A\u2013B flip fixes eight elements of X, all five reflections fix 12 elements of X, and all five combinations of a reflection and an A\u2013B exchange fix eight elements of X. Thus,In sum, all eight group elements that involve rotations fix no elements of"}
{"text": "Quasispecies are clouds of genotypes that appear in a population at mutation\u2013selection balance. This concept has recently attracted the attention of virologists, because many RNA viruses appear to generate high levels of genetic variation that may enhance the evolution of drug resistance and immune escape. The literature on these important evolutionary processes is, however, quite challenging. Here we use simple models to link mutation\u2013selection balance theory to the most novel property of quasispecies: the error threshold\u2014a mutation rate below which populations equilibrate in a traditional mutation\u2013selection balance and above which the population experiences an error catastrophe, that is, the loss of the favored genotype through frequent deleterious mutations. These models show that a single fitness landscape may contain multiple, hierarchically organized error thresholds and that an error threshold is affected by the extent of back mutation and redundancy in the genotype-to-phenotype map. Importantly, an error threshold is distinct from an extinction threshold, which is the complete loss of the population through lethal mutations. Based on this framework, we argue that the lethal mutagenesis of a viral infection by mutation-inducing drugs is not a true error catastophe, but is an extinction catastrophe. The concept of a mutation\u2013selection balance is one of the oldest and most fundamental pillars of population genetics: natural selection increases the frequency of fit variants while mutations introduce unfit variants, giving rise to an equilibrium distribution balanced between these two effects. Mutation\u2013selection balance has been invoked to explain the persistence of undesirable genes, for example, those underlying inbreeding depression, genetic diseases, and even senescence. Despite the long history of the concept, some of its consequences were only realized in 1971, when Manfred Eigen studied mutation\u2013selection dynamics in long genomes . This coThe error catastrophe has been applied liberally as a metaphor for complications of high mutation rates, as likely plagued primordial life  and currEigen's insights were developed in the context of genomes with many loci, each of which suffered mutation. Appropriately, the quasispecies has since been considered in this large-genome context. Yet many of its concepts are easily illustrated in the much simpler case of few genotypes, which is our approach here. Our results are not new, per se, but our models should convey quasispecies and error-catastrophe concepts to a broad audience and correct some common misunderstandings.A1 has fitness w1, and of those w1 offspring a fraction 1 \u2212 \u03bc1 retain the A1 genotype  or the mutation-free fraction of offspring (1 \u2212 \u03bc1) of A1 relative to that of A2, then the equilibrium distribution will move to progressively lower values on the vertical axis, until it lacks A1 entirely. This brings us to the error catastrophe.If a mutation rate or fitness is changed, the equilibrium distribution changes. Thus, the analogous figure for a different equilibrium would have the curves converging to a different value on the vertical axis. In particular, if we continually lower the fitness (w1(1 \u2212 \u03bc1) is the number of A1 offspring of an A1 parent; we will refer to this product as the replacement rate of the genotype\u2014the product of fitness times the mutation-free fraction of offspring. For mathematical simplicity, we assume that this number is an absolute quantity greater than one and that it does not change with population density. Hence, the population is forever expanding in unlimited space (this assumption is convenient but not necessary). For convenience, we will continue to treat A1 and A2 as single genotypes, but later in the paper, we will explain how they can be treated as sets of genotypes and how additional types can be included .Error thresholds and error catastrophes are properties of mutation\u2013selection equilibria, and an error catastrophe is a specific type of change in the equilibria. Consider again the genotypes and fitnesses illustrated in A1 has a higher fitness than A2, it follows that, for small mutation rates \u03bc1, the replacement rate of A1 is higher than the replacement rate of A2, or w1 (1 \u2212 \u03bc1) > w2 (1 \u2212 \u03bc2). As long as the replacement rate of A1 exceeds that of A2, both genotypes will be maintained at equilibrium: A1 because of its higher replacement rate, and A2 because of mutation from A1. If the mutation rate of A1 increases such that the mutation-free fraction of A1 drops relative to that of A2, however, the replacement rate inequality will eventually reverse, yielding a simple error catastrophe: A1 is no longer maintained. The error threshold is the point at which both genotypes have identical replacement rates. Beyond this point, A2 has the higher replacement rate, and A1 is absent at all equilibria\u2014the lack of back mutations ensures that it is not recreated from A2.Since A1 and A2 replacement rate functions approaching each other as the mutation rate increases toward the error threshold. The functions cross at the error threshold, giving rise to an error catastrophe (complete loss of A1) for higher mutation rates. The right part of the figure illustrates that, despite the suggestive terminology (threshold and catastrophe), an error catastrophe is the culmination of a gradual decrease in the equilibrium frequency of A1 rather than a dramatic one. An apt analogy is the geological transition from mountains to plains: starting from the mountains, the plains are approached gradually until they are reached, at which point the plains continue in the absence of any trace of mountains.  TheA1 experiences a potentially much greater mutational loss than A2. Beyond the error threshold this deleterious effect of mutation is retarded: by replacing a mutation-sensitive genotype with a mutation-robust genotype, the error catastrophe reduces the speed at which the replacement rate drops in response to increases in the mutation rate  < 1 for all genotypes). The most trivial example of the distinction between an error threshold and an extinction threshold is the case in which only one genotype exists, and all mutations are lethal. No error catastrophe is possible because no other genotypes exist, but extinction is obviously possible. Our model is one genotype more complicated: mutant offspring of A1 survive as A2, but mutants of A2 are inviable. Thus, there is not an additional error threshold beyond A2, because the model does not allow a third inferior genotype. An A2 population, however, will go extinct if its net growth rate drops below one  can disappear through either an error catastrophe or a population extinction, but A2 can disappear only through extinction. Depending on the values of w1, w2, and \u03bc2, the rate of mutation \u03bc1 at which A1 would be lost to A2 in an error catastrophe may be less than or greater than the value of \u03bc1 at which the population would go extinct. If the error threshold lies beyond the extinction value, then as \u03bc1 increases, the A1 + A2 population will go extinct of its own accord before a true error catastrophe can happen, because the population has disappeared before the mutation rate rises high enough for the error catastrophe . The interplay between extinction catastrophes and error catastrophes can likewise add interesting complexity to the model.The model implies that multiple error thresholds may exist in a fitness landscape. For example, a fitness landscape may include multiple i and \u03bci . With annd so on . It is and so on . For exaA1 and A2 or to A2 exclusively. This equilibration strictly requires an infinite amount of time in an infinite population. In a finite population, as long as the effective population size times the mutation rate is significantly bigger than one (N\u03bc \u226b 1), the finite population behaves similarly to the infinite population, except that the error threshold is not as sharp. In particular, the lack of back mutations ensures that random events will eventually cause the loss of genotype A1 from the population, so that only genotype A2 remains. If A2 represents multiple types of mutants, then this random loss of A1 is Muller's ratchet [A2 to A1, as described in Our model assumes deterministic dynamics and ignores the stochastic effects of finite population sizes. We have argued that populations will equilibrate either to a combination of  ratchet ,15. BeyoA1 could not be regenerated once lost from the population . Two equations describe the system:At this point we use simple matrix algebra to explore the problem. Assuming discrete time steps, let 3, which allows for back mutation and cannot exceed \u03bc2. If we set \u03bc3 = 0, then these equations describe our earlier model exactly (in n1(t) and n2(t) as t becomes arbitrarily large; that is, we calculate \u2009tM for large t. The standard method for evaluating long-term behavior in such a linear system is to consider the eigenvalues of the system, found as the values of \u03bb satisfyingso that actly in . To eval3 = 0). Equation 4 then yields the solutionsTo establish a baseline, we first assume no back mutation  and y = w2(1 \u2212 \u03bc2).where 1 to \u03bb2 as the dominant eigenvalue occurred at x = y in the absence of back mutation, now \u03bb1 remains superior at all values of x and y. As \u03bc3 approaches arbitrarily close to zero, the eigenvalues approach each other but do not cross. Thus, there is no strict error threshold unless \u03bc3 equals zero exactly. Yet as \u03bc3 approaches zero, the system appears more and more like that with no back mutation  leads to a higher net replacement rate of the phenotype.Error thresholds require that some genotypes or phenotypes are more sensitive to mutation than others. Biologically, many phenotypes have the property that they can be produced by multiple, mutationally related genotypes, and mutational robustness is known to vary among protein and nucleic acid sequences ,18\u201322. THere we illustrate how the breadth of a neutral network\u2014the number of and mutational connectivity among genotypes contained within it\u2014affects the competitiveness of the phenotype. In particular, \u201csurvival of the flattest\u201d refers to the ability of inferior phenotypes with large neutral networks to displace superior phenotypes with smaller neutral networks. This phenomenon was recently demonstrated in digital life simulations and simulated RNA molecules ,24.A2 (3 = 0), the error threshold is unaffected by the \u201cnetwork\u201d and is the same as with a single A2 genotype: A2 here has exactly the same properties as the A2 in A1/A2 error threshold is the same as in A2 genotype (A2). Back mutation provides a mutational \u201cconnectivity\u201d among both genotypes in the neutral network and renders the network more competitive against A1. This connectivity serves to bolster the frequency of the left A2 genotype at the expense of the right A2. Since only the right peak experiences mutations off the neutral network (at rate \u03bc2), this shift increases the overall replacement rate of A2, allowing A2 to more easily supplant A1. Higher rates of back mutation increase this effect We explore the impact of neutrality on the error threshold with a simple extension of our previous model, this time allowing two genotypes to have the phenotype A2 . This crs effect , top andA2 genotypes into the middle of the neutral network, each with forward mutation rate \u03bc2 and back mutation rate \u03bc3. A2 network also increases, thus shifting the error threshold in favor of A2 and buffering A2 against extinction.This model can also be extended to demonstrate the impact of neutral network size on the error threshold. In this case, we add In summary, the error threshold decreases as the fitness landscape around the inferior phenotype broadens, either via the extension of the neutral network to include additional genotypes or via a net decrease in mutation off the network. The error threshold above which a particular wild-type is lost will depend on its fitness relative to the fitness and breadth of the inferior network. The broader and more mutationally connected the inferior neutral network, the greater its tolerance for mutation and the more likely an error catastrophe that tips the population in its favor. Nonetheless, a sufficiently high fitness can compensate for a high rate of mutational loss.These trade-offs also apply to more complex fitness landscapes consisting of multiple neutral networks with differing fitnesses. In this case, the sequence of error thresholds becomes difficult to intuit and has not previously been addressed quantitatively. L loci with a per locus mutation rate of \u03bd. A single mutant-free, wild-type genotype A1 has highest fitness w1, and all other genotypes with one or more mutations fall into the same neutral network in which each genotype has the same fitness w2. Eigen's classic error threshold result is that, depending on L and w1/w2, there is a specific per base mutation rate \u03bd beyond which the wild-type is lost or maintained only through back mutation and the w2 neutral network prevails, as in our For those familiar with Eigen's original quasispecies model . Base analogs are incorporated into the viral genome as it is being copied from the parent strand. In general, incorporation of a base analog can have two types of effects: chain termination, resulting in immediate death of the progeny strand, or mutation, whereby the base analog is later miscopied when the genome gets replicated. The latter mechanism underlies the antiviral activity of the drug ribavirin for some RNA viruses [Quasispecies error thresholds have been invoked in an important medical application, the extinction of RNA virus populations by lethal mutagenesis\u2014elevating mutation rates with base analog drugs to the point that the virus population disappears. In 2005, an entire issue of  viruses ,29,30. I viruses ,5,30\u201332)volume 10, issue 2From a practical perspective, it does not matter whether lethal mutagenesis is referred to as an error catastrophe or extinction catastrophe. There may be a benefit, however, to distinguishing between these two concepts and, more generally, to clarifying the concept of a quasispecies. For example, consider a protocol of sublethal mutagenesis, whereby a virus mutation rate is artificially elevated close to an extinction threshold but the virus population persists. Conventional wisdom suggests that such a strategy should be disastrous from a host health perspective, because the increased mutation rate may accelerate viral adaptation. Yet two components of the quasispecies framework suggest that a permanently elevated mutation rate may be deleterious to the virus even when extinction is not achieved. First, a higher mutation rate reduces mean fitness around any local fitness peak. Second, a high mutation rate \u201cflattens\u201d the fitness landscape, so that some narrow peaks cannot be attained. Thus, a sustained high mutation rate that does not drive the virus to extinction may enforce a low mean fitness. Holmes  has likeIn light of this potential impact of high mutation rates, it seems paradoxical that a new class of drugs is being touted for its ability to reduce mutation rates, and thereby block the evolution of resistance to other drugs . Yet mutWe have used a variety of simplifications to explain and depict error catastrophes from a mathematical perspective. Yet it may not be obvious how an error catastrophe would manifest itself in a population, especially how it would appear in a sample of genome sequences taken before and after crossing the error threshold. One possible scenario is similar to that of A second scenario is that of a population in which each individual's genome belongs to either of two \u201cdisjoint\u201d neutral networks from very different parts of the fitness landscape. This type of polymorphism could arise if one type was present in the starting population and the other was introduced from a different population through migration. For this scenario, we assume that the population size is sufficiently small relative to the mutational distance between the two neutral networks, so that it is essentially impossible for the population to reach one network from the other via mutation . If one of the networks is more mutationally robust but of lower fitness than the other, an error catastrophe can cause the loss of the higher-fitness network. This type of error catastrophe might reveal itself in genomic data as the loss of polymorphism through the independent mutational meltdown and subsequent disappearance of one of the original types. Back mutation complicates the previous scenario by continually re-introducing the fitter nucleotides, which are then maintained at only a slightly higher level than neutral nucleotides. Back mutation is not a factor in the disjoint network model because the first network to disappear is exceedingly unlikely to be re-introduced via point mutations from the remaining population.Given the difficulty of demonstrating the relatively simple properties of quasispecies dynamics and equilibrium properties, it is not surprising that the empirical demonstration of an error threshold/catastrophe has not been achieved. An interesting step in this direction was provided recently in a study that estimated the error threshold mutation rate that might apply to a primitive organism consisting of RNA molecules\u2014a riboorganism . The stuThe systems offering the best hope for empirically demonstrating an error threshold are in fact naked RNA molecules evolved by directed evolution: ribozymes  and aptamers (binding molecules) evolved in vitro from initial pools of random-sequence molecules. In this work, mutational robustness is recognized as a key property of molecules affecting evolution , but thiw) than a short motif. By virtue of differences in mutational robustness among the different motifs, there should be an error threshold mutation rate, above which the long motifs will be displaced by the short motifs. If one knows the fitnesses i), than the class II and class III ligases . KinetiThe ligase ribozymes will permit an easier demonstration of an error threshold than most other empirical systems, because the alternative functional networks have been identified and can be seeded into the experiment. We thus know in advance what to compare. If, instead, we have information about only the wild-type but not the suboptimal type that dominates beyond the error threshold (as would be the case when working with viruses or bacteria), then it becomes far more complicated to identify an error threshold empirically. Even with the ligase ribozymes, if a population was initiated with only the class I molecule, the class II or III molecules might not evolve at high mutation rates because their sequences might be too distant in sequence space to arise in a quasispecies of class I molecules. All three classes arose in the initial experiment because they were selected from a random sequence pool rather than evolved from a single type.Identification of an error threshold is certainly facilitated by use of a previously characterized genotype\u2013fitness map, but that kind of information is not usually available. In ignorance of the alternative neutral networks for a phenotype, the obvious protocol to use in blindly testing for an error catastrophe is to impose stabilizing selection for the wild-type trait while progressively increasing the mutation rate. One would then test for characteristics of an error catastrophe, such as a population shift in genotype space or increased mutational robustness of the genotypes evolved at high mutation rate. The key difficulty in demonstrating an error catastrophe without a priori knowledge of alternative networks is that there are population genetic processes not considered in the simple quasispecies models that give rise to superficially similar outcomes. Thus, under our suggested experimental protocol, shifts in genotype space may stem from (i) an error catastrophe\u2014the deterministic fixation of deleterious mutations, (ii) stochastic fixation of deleterious and neutral mutations, (iii) the higher mutation rate allowing the population to find higher adaptive peaks, or (iv) adaptive evolution because mutagenesis has altered the selective environment (other than through higher mutation rate). Likewise, increases in mutational robustness may, in fact, suggest an error catastrophe, or may simply be a by-product of a general decline in fitness under high mutation rates. Recent work suggests that genotypes of reduced fitness generally experience a higher fraction of mutations with beneficial effect than genotypes of high fitness ,41. ThisThe theory of quasispecies error thresholds appears sound. The major avenues for new developments are twofold: (i) obtaining empirical support and (ii) incorporating additional realism into the basic models. There are several papers that make progress on this second point, by including recombination , stochas"}
{"text": "E. coli may have the key.Why have sex? It seems that genetic studies in the normally non-sexual The evolution of organisms that mix their genes during reproduction is considered one of the major transitions in evolution , becauseNineteenth-century German biologist August Weismann  was the Drosophila melanogaster that could not recombine and hence evolved asexually. This trick allowed the authors to study the effect of recombination on the fate of an introduced beneficial mutation without the obscuring effects of differences in initial fitness or environmental conditions used to induce sex in some populations. In another recent attempt, Goddard et al. [Several recent laboratory evolution experiments have found support for Weismann's proposal that sex facilitates the response to selection . Howeverd et al.  construcd et al.  to compaPLoS Biology, Tim Cooper [Escherichia coli\u2014which, like all bacteria, reproduce asexually\u2014to test the relative contribution of the models mentioned above. The choice of E. coli may seem odd but is an excellent one for several reasons. First, by introducing the F plasmid carrying the genes for conjugation , Cooper could study the consequence of sex\u2014i.e., genetic recombination\u2014while excluding obscuring effects from changed conditions or fitness. Second, by manipulating the mutation rate, he could create conditions where competition among different beneficial mutations is known to limit the rate of adaptation [In this issue of m Cooper  takes thaptation , a prereaptation , Cooper spoT), which was found in rec+ and rec\u2212 lines alike. This allowed him to test two specific predictions of the FM model. First, competition with other clones carrying beneficial mutations should slow down the fixation of the focal mutation in rec\u2212 lines, but not in rec+ lines. Second, this same interference should lower the fitness of clones carrying the focal mutation relative to contemporary clones in rec\u2212 lines, but not in rec+ lines, once competing clones reach appreciable frequencies  as primary explanation, and recombining (rec+) lines  had improved more than nonrecombining (rec\u2212) lines. To distinguish between the explanations offered by the FM model  and the ML model , Cooper looked at the competitive dynamics of a focal beneficial mutation  and from the more efficient removal of multiple deleterious mutations (MD model) may have contributed to the higher fitness of the rec+ populations. Another complicating factor is that the support for the FM model based on the dynamics of the focal mutation depends on the assumption that beneficial mutations go to fixation one-by-one in the rec\u2212 populations. A recent study of asexual adaptation in yeast [spoT mutations from rec\u2212 and rec+ populations relative to the ancestor and to contemporary clones (along the lines suggested in How strongly do these data reject alternate explanations for the evolutionary maintenance of sex? Parasites or otherwise-fluctuating environmental conditions can be ruled out from the experimental conditions set during evolution. Similarly, the faster removal of deleterious mutations, proposed by the MD model, is unlikely to have been very important, because both of its prerequisites\u2014a rate of deleterious mutations of at least one per genome per generation and prevailing synergistic interactions among mutations\u2014are not met by in yeast  indicatein yeast . A systeHow relevant are these bacterial results for understanding the evolutionary maintenance of sex in higher organisms? In other words, does the bacterial support for the FM model mean that recombination between beneficial mutations is the universally best explanation for the existence of sex and recombination? Not necessarily. The benefits of sex depend on factors that are likely to differ among species, including the variability of environmental conditions, the population size, and the rate, fitness effects, and interactions of mutations. We are gathering information on some of these factors for micro-organisms, but they are hardly known for higher organisms. Only the patient collection of such data from a variety of different organisms may ultimately reveal whether organisms share general unifying principles that allow a universal explanation for sex, or alternatively whether different explanations are required for different species . PossiblUndoubtedly, the study by Cooper  will sti"}
{"text": "Understanding interactions between mutations and how they affect fitness is a central problem in evolutionary biology that bears on such fundamental issues as the structure of fitness landscapes and the evolution of sex. To date, analyses of fitness landscapes have focused either on the overall directional curvature of the fitness landscape or on the distribution of pairwise interactions. In this paper, we propose and employ a new mathematical approach that allows a more complete description of multi-way interactions and provides new insights into the structure of fitness landscapes.Escherichia coli obtained by Elena and Lenski. The genotypes were constructed by introducing nine mutations into a wild-type strain and constructing a restricted set of 27 double mutants. Despite the absence of mutants higher than second order, our analysis of this genotypic space points to previously unappreciated gene interactions, in addition to the standard pairwise epistasis. Our analysis confirms Elena and Lenski's inference that the fitness landscape is complex, so that an overall measure of curvature obscures a diversity of interaction types. We also demonstrate that some mutations contribute disproportionately to this complexity. In particular, some mutations are systematically better than others at mixing with other mutations. We also find a strong correlation between epistasis and the average fitness loss caused by deleterious mutations. In particular, the epistatic deviations from multiplicative expectations tend toward more positive values in the context of more deleterious mutations, emphasizing that pairwise epistasis is a local property of the fitness landscape. Finally, we determine the geometry of the fitness landscape, which reflects many of these biologically interesting features.We apply the mathematical theory of gene interactions developed by Beerenwinkel et al. to a fitness landscape for A full description of complex fitness landscapes requires more information than the average curvature or the distribution of independent pairwise interactions. We have proposed a mathematical approach that, in principle, allows a complete description and, in practice, can suggest new insights into the structure of real fitness landscapes. Our analysis emphasizes the value of non-independent genotypes for these inferences. The image of populations evolving on fitness landscapes traces to Sewall Wright's seminal work in the thirties . Since tIf all mutations were strictly additive or multiplicative in their effects on fitness, then it would be rather easy to describe the structure of fitness landscapes and understand the resulting dynamics of adaptation by natural selection. However, many mutations interact with one another in complex ways. For example, two or more mutations may interact such that their combined effect on fitness is much greater or much less than predicted from their individual effects; their combined effect may even be opposite in sign to the expectation based on their individual effects. These deviations from simple expectations are called epistasis ,9, and tE. coli that was obtained and first analyzed by Elena and Lenski [Over the last decade, several studies have sought to examine the form and prevalence of epistatic interactions by measuring the fitness effects of numerous mutations alone and in combination in viruses, bacteria, fungi, and animals ,27-31. Ta, b, c), , and , where all genotypes in each of the three groups share the same antibiotic-resistance markers. Notice that double mutants, each denoted by a pair of letters, were produced except for those consisting of pairs from the same set of three adjacent letters. For example, a was paired with r, s, t, x, y, and z, but not with either b or c. Table We organized the 37 genotypes in the symmetric 10-by-10 matrix with missing values as shown in Table genotopes of all three-locus subsystems of the nine-locus system. A genotope is the set of all possible allele frequencies for a collection of genotypes. As a reference point, Figure a, r, and x, induces the genotope in Figure a, b, and r, further prunes the cube to a triangular prism as in Figure a, b, and c, results in a tetrahedron such as the one shown in Figure The experimental design is illustrated geometrically in Figure E. coli fitness landscape obtained by Elena and Lenski [w\u00b7ar - a\u00b7r, where w is the fitness of the wild-type, ar is the fitness of a double mutant, and a and r are fitness values for each associated single mutant. They found many significant deviations from zero, including several cases of both positive and negative epistasis. The key messages of our paper are that additional types of gene interactions exist even within this fitness landscape, and that geometric methods can be used to describe and analyze the system more exhaustively.The goal of our analysis is to describe the geometry of the d Lenski . The fitOur analysis of this landscape is based on the approach developed by Beerenwinkel et al. . We idenw\u00b7ar - a\u00b7r compares two genotypes having zero and two mutations with two others each having one mutation. The minimal Markov basis  of the space of tables of the type displayed in Table ar\u00b7bs - as\u00b7br. Second, there are another 108 orthogonal \"single-double\" tests that compare one single mutant and one double mutant with another single mutant and another double mutant, again holding constant the allele frequencies, for example, a\u00b7br - b\u00b7ar. For both types of non-standard tests, the two genotypes on the right-hand side can be regarded as the products of recombination between the two genotypes on the left-hand side. Notice that the same relationship also holds true for the standard tests. Our first point is that the genotype space in Table Experimental biologists (including two authors of this paper) are likely to raise three concerns about these non-standard tests. What biological insights can non-standard tests provide beyond those obtained using standard tests? Are these additional tests independent of the standard tests? What computational tools are available to perform such tests on other datasets?As we show in the sections that follow, the non-standard tests are potentially useful in at least three respects.  First, they allow one to focus attention on features of epistasis that are not quantifiable by the standard tests.  For example, we perform non-standard tests of the \"single-double\" type to explore whether some mutations are better overall mixers than others.  Second, non-standard tests span greater genetic distances than do pairwise tests, allowing more powerful analyses of the structure of fitness landscapes.  For example, we use the \"double-double\" tests to test curvature at genetic distances of four, whereas standard tests allow curvature to be examined only at distances of two.  Third, non-standard tests are an integral part of the complete geometric description of a fitness landscape.  While this high-dimensional geometry is abstract and even foreign, we describe how biological features of gene interactions, such as mixing ability, are embedded in the geometric shape of the landscape.    The non-standard tests are not independent, in a statistical sense, of the pairwise tests or of one another because all the tests are calculated from the same underlying data.  Nonetheless, this complication can be addressed by employing appropriate statistical methods  to ensure that significance levels reflect the data structure.  Regarding the availability of computational tools, we provide references to programs that automate the calculations of the Markov basis and perform the triangulations necessary to describe the geometry of landscapes, and these tools can be applied to other datasets.  In supplementary material, we illustrate the use of these computational tools and show their output .Figure w\u00b7ar - a\u00b7r) the average fitness decrement \u0394 = 1 - (a + r)/2 of the two single mutants relative to the wild-type. We then plot the epistasis values as a function of \u0394 for all 27 equations. In this way, we can test the hypothesis that epistatic deviations tend more toward positive values when mutations in the wild-type background are more deleterious (large \u0394) than when mutations are less deleterious . The scope of this analysis can be extended substantially by also including the non-standard \"double-double\" epistatic equations (such as ar\u00b7bs - as\u00b7br). We anchored each double-double test with the fittest genotype (say ar) on the left-hand side, and calculated the average fitness decrement, \u0394 = ar - (as + br)/2, of the two genotypes that appear on the right-hand side of the equation. These tests ask more generally for the relationship between epistasis and the average fitness loss relative to any local fitness peak rather than only the one centred on the wild-type. Moreover, these non-standard tests may have greater power because their \"reach\" extends over mutational distances of four, rather than the two allowed by standard tests. .To test this prediction for the fitness peak at the wild-type, we calculated for each standard test of epistasis , but highly significant for the larger set of 108 double-double tests , if each of the deviations is viewed as independent. However, these values all rest on 37 genotypes, whose fitness values were estimated with error , and hence the errors are not independent for those epistatic terms that share a genotype. To take this complication into account, we performed Tukey's jackknife test [r = 0.440, ts = 3.761, 26 d.f., one-tailed p = 0.0004) and non-standard tests . It is important to understand that this inference concerns the relationship between average fitness decrements and the form of epistasis around any local peak within the particular landscape represented by these 37 genotypes. In other words, it is an inference about a restricted region of the complete E. coli genotypic space.Figure E. coli genome. In that case, the unit of independent observation becomes the nine single mutations, and the resulting inference concerns the landscape of all possible double mutations derived from the same parental strain. We again applied Tukey's jackknife, this time eliminating in turn all tests containing a particular mutation in any of the four double mutants, rather than eliminating a particular genotype as before. In this case, the association is only marginally significant , but still supports the trend for increasingly positive epistasis with increasing average fitness loss. Given that these tests aim to infer a complex genome-wide relationship between average mutational effects and the form of epistasis from a small sample of mutations, it is encouraging to find such a strong trend.If we want to make the same type of inference about the complete genotype space, rather than the specific subset sampled by Elena and Lenski, we can apply a similar, but not identical, test. More precisely, we want to investigate the correlation between average fitness decrease and epistasis among any single and double mutants of the We present these alternative analyses to emphasize the subtly different hypotheses that can be addressed by using our mathematical approach. Comparing the last two analyses suggests that individual mutations might have pervasive effects on the shape of the local landscape. While pervasive effects of certain mutations can make it more difficult to test broader generalizations, the precise nature of these pervasive effects is of biological interest. In the next section, we follow the same general approach, but focusing on a different set of epistatic interactions, to examine differences between individual mutations in greater detail.a\u00b7br - b\u00b7ar. This test asks, in effect, whether mutation a or b mixes better with a third mutation r. An individual test of this form might be interesting when one has specific knowledge about the identity of the three mutated genes and the position of their products in a metabolic pathway, for example. By contrast, Elena and Lenski emphasized the statistical properties of epistatic interactions. In this context, we can examine related sets of these single-double equations to ask whether one mutation is a better mixer than another in the context of the sample of mutations with which they were each tested.In this section, we use non-standard tests of the \"single-double\" type to explore a particular aspect of the fitness landscape, specifically whether certain mutations are better mixers than others. The mixing ability of any particular mutation indicates whether its epistatic interactions with other mutations tend to be positive or negative. We can then measure the relative mixing ability of two mutations by holding constant the identity of other mutations with which the two of interest are mixed. Consider the polynomial a, b, c}, {r, s, t}, or {x, y, z}) was tested with the exact same set of six mutations belonging to the other two sets. For example, the following six equations examine the relative mixing ability of \"focal mutations\" a and b with respect to \"tester mutations\" r, s, t, x, y, and z: a\u00b7br - b\u00b7ar; a\u00b7bs - b\u00b7as; a\u00b7bt - b\u00b7at; a\u00b7bx - b\u00b7ax; a\u00b7by - b\u00b7ay; and a\u00b7bz - b\u00b7az. All in all, there are nine groups of six equations each that compare the general mixing abilities of two focal mutations from the same set: a versus b, a versus c, b versus c, r versus s, r versus t, s versus t, x versus y, x versus z, and y versus z. There are another 18 groups of three equations each that compare the mixing abilities of two focal mutations from different sets. Any pair of mutations belonging to the same set of three  of focal mutations, a was the better mixer with two tester mutations whereas b mixed better with four others. For each focal pair, we performed a t-test to ask whether the average epistatic deviation was significantly different from zero. The focal pairs  and  were both significant (p \u2264 0.027), with y being the better mixer in both cases. Two other focal pairs,  and , were marginally non-significant with p = 0.083 and p = 0.068, respectively, and b was the better mixer in both of these cases. The  comparison survives even a stringent Bonferroni correction that accounts for the multiplicity of related tests (p = 0.0054\u00b79 < 0.05). Therefore, we can conclude that mutations are variable in their general mixing ability. The molecular identities of mutations were not identified by Elena and Lenski [In our analysis, we focus on the nine comparisons of mixing ability that each involves six tester mutations, because these provide more statistical power that might reveal differences between focal mutations. Figure t p \u2264 0.07, with yOur final set of results is concerned with the geometric shape of the fitness landscape. Since fitness landscapes are high-dimensional and complicated objects, it is desirable to classify them into a finite set of distinct shapes; with the general idea that fitness landscapes with the same shape are likely to share biological properties. This approach generalizes the classification of bi-allelic two-locus landscapes into those with positive epistasis versus those with negative epistasis. This appealing binary classification has been linked, for example, to the advantage of sex, but it does not extend to higher-dimensional genotype spaces. We present here a notion of the shape of a fitness landscapes for any genotypic space. This concept is intimately related to the interaction tests discussed so far, because the shape is determined by a certain subset of the gene interactions that includes the Markov basis. Thus, the proposed classification of landscapes into shapes can be regarded as a formal summary of all the various standard and non-standard tests.E. coli genotypes and their fitness values as shown in Table The fitness landscape studied in this paper consists of the 37 w, a, r, and ar.  Its genotope is the unit square in -space, and a generic fitness landscape has exactly one of two possible shapes corresponding to either negative or positive epistasis, i.e., to w  ar - a r being either negative or positive.  Negative epistasis induces the triangulation of the square consisting of the two triangles {w, a, r} and {a, r, ar}, whereas positive epistasis results in the other possible triangulation with {w, a, ar} and {w, r, ar}.Consider the bi-allelic two-locus system with genotypes E. coli fitness landscape investigated in this paper.  The general idea is to investigate and describe the interaction space.  This space has finite dimension, but infinitely many elements, and each element of this space represents one interaction.  There are many different ways of extracting potentially interesting subsets of the interaction space.  We suggest looking at the circuits, which generate the space but are not linearly independent.  The signs of the circuits determine the triangulation of the genotope and thus the shape of the fitness landscape.  However, the number of circuits grows fast with the size of the fitness landscape; the present E. coli landscape has 772,731 circuits.  Therefore, we restrict our attention to the much smaller subset provided by the Markov basis.  The circuits and the Markov basis provide a natural generalization of the concept of pairwise epistasis.For larger genetic systems, the role of the triangles is played by simplices.  The shape of the present fitness landscape on 37 genotypes is a triangulation of the genotope into 362 nine-dimensional simplices . This graph provides the basis for statistical inference about the three-way interactions in the given fitness landscape. We find the following shapes among all 27 of the three-dimensional genotopes of type (b) that appear as subsets of the complete dataset: shape 56 (frequency: 6), 7 (5), 25 (4), 8 (3), 52 (3), 21 (2), 23 (2), 2 (1), and 20 (1). A closer inspection of this shape distribution reveals many deviations from linearity in the fitness landscape, but no single dominating shape. This result therefore confirms and extends the earlier finding of Elena and Lenski [The three-locus subsystems that occur in this dataset are represented in Figure The analogous graph of possible shapes for the three-locus subsystem corresponding to the triangular prism Figure  is a hexw, a, b, r, ar, br}, is determined by the signs of the three tests w\u00b7ar - a\u00b7r, w\u00b7br - b\u00b7r, and b\u00b7ar - a\u00b7br. Therefore, the shape summarizes the information about the standard pairwise interactions between mutations a and r and between b and r, as well as the relative mixing ability of a and b with respect to r. For example, five of the six subsystems of type (c) involving mutations y and z have shape 21, while the subspace {w, y, z, b, by, bz} has shape 52. Shape 21 is defined by a negative sign for the second of the three tests above and a positive sign for the other two, whereas shape 52 is defined by positive signs for all three tests. Hence, these shapes reflect positive epistasis involving y (6/6 tests), positive epistasis involving z (5/6 tests), and superior mixing ability of y over z (6/6 tests). As such, the geometric shapes provide more details about the form of mutational interactions than the average values of the tests analyzed in the previous section. On the other hand, these shapes summarize the data by classifying continuous fitness values into discrete shape classes which reflect the sign pattern of the interaction tests.The shape of fitness landscapes on type (c) spaces, such as {Epistasis occurs whenever mutations interact non-linearly with one another, and it represents a major challenge in describing the mathematical structure of real fitness landscapes. With epistatic interactions, the combined effect of two or more mutations on fitness may be greater than, less than, or opposite in sign to expectations obtained by combining their separate effects. A growing body of empirical research indicates that epistasis is very common in nature ,23,26-34To date, two different aspects of epistasis have served as summary statistics of these interactions. First, studies have used the overall directional curvature of mean fitness as a function of the number of random mutations introduced into the genome of some wild-type organism ,27-31. AThe objective of this paper is to introduce biologists to a new mathematical framework for characterizing epistatic interactions between mutations, which goes beyond both overall directional curvature and pairwise interactions by providing a complete geometrical description of the epistatic interactions that define an empirically determined fitness landscape. To that end, we have re-analyzed the dataset from the pairwise experimental design performed by Elena and Lenski  using thE. coli that were constructed by introducing nine mutations into a wild-type strain and constructing a restricted set of 27 double mutants. Despite the absence of any triple or other higher-order mutants in the dataset, our analysis reveals complex new epistatic interactions, beyond the pairwise interactions reported previously. First, our analysis confirms and extends Elena and Lenski's inference that the fitness landscape is complex, such that an overall measure of curvature obscures a complex admixture of interaction types, some with positive and others with negative effects on fitness  on G. The genotope \u03a0G \u2282 R9 is the set of all allele frequencies that can be realized by populations on G. The genotope is a nine-dimensional polytope, defined as the convex hull of the 37 genotypes, i.e., as the set of all convex combinations \u03bbg \u2265 0 and G, all of which are contained in \u03a0G .Our analysis is based on the mathematical framework presented by Beerenwinkel et al. . The datG \u2192 \u03a0G that assigns to each population its allele frequency spectrum. The kernel of this map defines the interaction space . The el\u03c1. The kernel of \u03c1 is the space of integer tables of the same format with zero margins (row sums). A canonical set of generators for this kernel is the minimal Markov basis [\u03c1  affinely independent points in Rn [n + 1) points span an affine space of dimension n. For example, Figure w, a, b, and c. By a triangulation of a polytope we mean its decomposition into a set of simplices. A fitness landscape on G gives rise to a triangulation of the genotope \u03a0G  by algebraic computations.  However, there is no simple recipe for writing out the Markov basis.  We computed the Markov basis independently with the computer algebra systems Macaulay 2  and SingN.B., L.P., and B.S. are responsible for the development of the mathematical theory. S.F.E. and R.E.L. designed and performed the experiments with bacteria. N.B. and R.E.L. formulated the specific analyses reported here and wrote the paper. All the authors contributed to editing the paper and approve of its final form.Instructions for calculating the Markov basis and triangulation of a dataset with Macaulay 2.Click here for fileMacaulay 2 file for computing Markov bases of interaction spaces and triangulations of genotopes. Click here for fileExamples of fitness landscape files to be processed with the program fitness.m2 .Click here for fileMinimal Markov basis of the interaction space.Click here for fileGeometry of the fitness landscape.Click here for file"}
{"text": "In any natural population, mutation is the primary source of genetic variation required for evolutionary novelty and adaptation. Nevertheless, most mutations, especially those with phenotypic effects, are harmful and are consequently removed by natural selection. For this reason, under natural selection, an organism will evolve to a lower mutation rate. Overall, the action of natural selection on mutation rate is related to population size and mutation effects. Although theoretical work has intensively investigated the relationship between natural selection and mutation rate, most of these studies have focused on individual competition within a population, rather than on competition among populations. The aim of the present study was to use computer simulations to investigate how natural selection adjusts mutation rate among asexually reproducing subpopulations with different mutation rates.The competition results for the different subpopulations showed that a population could evolve to an \"optimum\" mutation rate during long-term evolution, and that this rate was modulated by both population size and mutation effects. A larger population could evolve to a higher optimum mutation rate than could a smaller population. The optimum mutation rate depended on both the fraction and the effects of beneficial mutations, rather than on the effects of deleterious ones. The optimum mutation rate increased with either the fraction or the effects of beneficial mutations. When strongly favored mutations appeared, the optimum mutation rate was elevated to a much higher level. The competition time among the subpopulations also substantially shortened.Competition at the population level revealed that the evolution of the mutation rate in asexual populations was determined by both population size and mutation effects. The most striking finding was that beneficial mutations, rather than deleterious mutations, were the leading force that modulated the optimum mutation rate. The initial configuration of the population appeared to have no effect on these conclusions, confirming the robustness of the simulation method developed in the present study. These findings might further explain the lower mutation rates observed in most asexual organisms, as well as the higher mutation rates in some viruses. Understanding the genetic structure of populations requires knowledge of the mutation rate, an important parameter of evolution. One of the essential problems in population genetics is determining how natural selection acts on the mutation rate of an organism during long-term evolution. Although mutation provides the ultimate source of genetic variation, it typically leads to decreased fitness. Even when a population is in the process of adaptation, the majority of its mutations are still deleterious and will ultimately be eliminated by selection. This type of selection pressure was first observed by Sturtevant , who queU) in DNA-based microbes was about 0.0034 per generation despite a wide variation in genome size. This relatively constant observed value indicates that the genome mutation rate in microbes has evolved perfectly to fit the pace of environmental changes through natural selection. Several theoretical methods have the potential to explain Drake's observation from different perspectives [Since Sturtevant's pioneering work, the evolution of mutation rate has been researched by many evolutionary biologists and our understanding of this question has been improved in many respects. At present, several methods have been proposed for characterization of the evolution of mutation rate, including direct estimates from mutation accumulation experiments -5, indirpectives ,12,14,15pectives . He descIn any finite population, the process of evolution is well known to be influenced by population size and mutation effects . BeneficWhen both deleterious and beneficial mutations are present, it is necessary to explore whether an organism could evolve to an \"optimum\" mutation rate under these two opposing forces. The nature of the dominating factors that shape the optimum mutation rate also needs to be determined. In the present paper, we have developed a simulation method based on competition among subpopulations with different mutation rates to examine how selection may impact the evolution of genome mutation rate. Our results indicate that a larger population could tolerate a higher mutation rate than could a smaller one. The optimum mutation rate depends almost exclusively on the effects of beneficial mutations regardless of the extent of deleterious mutation effects. Possible reasons for these findings are discussed in comparison with previous studies.N) that comprises 10 subpopulations, each of which has N/10 individuals and a different mutation rate, with everything else equal. The rationale of the method is that these subpopulations compete for existence under natural selection and random drift. At the end of a simulation, only one subpopulation remains and the rest are extinct. The mutation rate of the remaining population becomes the \"fixed\" mutation rate in that competition. By simulating the process many times, we can define the most frequently fixed mutation rate as the \"optimum\" mutation rate.We consider a finite strictly asexual haploid population (with constant population size b pand d p(i.e. 1- bp), respectively. The effects (selection coefficients) of both beneficial and deleterious mutations are drawn from continuous probability distributions. We denote b sas the effects of beneficial mutations (in which case fitness w is increased by a factor 1+ bs), while d srepresents the effects of deleterious mutations (in which case fitness w is decreased by a factor 1- ds)[Each of the ten subpopulations is assigned with a distinct mutation rate per genome per generation (see parameters). Both deleterious and beneficial mutations occur in each subpopulation with fractions for beneficial and deleterious mutations represented by or 1- sd).b sfollows an exponential distribution:d sin analytical calculations; however, empirical studies support a gamma distribution with shape parameter smaller than one (other distributions are not necessarily excluded)[d sfollows a skewed gamma distribution We assume that s models -24. The excluded),26. In tst cases ,28.U)) is roughly uniformly distributed between -4 and -1. In addition, we adopt several ranges consisting of different mutation rates, which are shown in Table In our simulations, the sizes of fractions and effects of both beneficial and deleterious mutations are the most important quantitative parameters. Numerous experimental studies on microbes have shed some light on this area and some estimates of these parameters are summarized in Table m) appearing in an individual belonging to the i-th subpopulation is drawn from a Poisson distribution i Uis the genome mutation rate of the i-th subpopulation. The deleterious mutation rate is then given by iU\u00d7d pand the beneficial mutation rate is iU\u00d7bp. Given that a deleterious  mutation occurs, the fitness w of the individual is decreased (or increased) by 1- d s(or 1+ bs), where d s(or bs) is randomly drawn from a gamma  distribution. Here, we assume that no epistasis occurs; therefore, all mutations have independent effects on fitness and act multiplicatively. It is possible that an individual may carry multiple mutations within a single generation. In this case, the fitness of an individual in the n-th generation (nw) is a function of the mutation numbers the individual carries (m), their mutation effects (js), and the fitness of its parent in the (n-1)-th generation (n-1w). This function can be described asThroughout the study, we assume that generations are discrete and non-overlapping. In each generation, the number of new mutations . Some initial conditions of the population are also relaxed to test the robustness of the method (see Discussion).We trace the numbers of individuals of each subpopulation until the population size of one subpopulation reaches opt Uas the optimum mutation rate and G as the mean number of generations required for competition in one simulation. We also used u Rto represent which group of mutation rates was adopted in Table Our extensive simulations were designed to test whether natural selection could shape the optimum mutation rate, given the initial configuration of the population. Each curve represents one simulation result with 300 competitions among all the figures and each point represents the frequency of being fixed of the corresponding mutation rate. For convenience of description, we used the symbol N). Clearly, N had a significant effect on the frequencies of fixed mutation rates. With all other factors held constant, increasing in N caused the curve to shift to the right, which demonstrated that a larger population tolerated a higher mutation rate. Large populations could benefit from relatively higher mutation rates because beneficial mutations appeared more frequently and selection was more efficient in removing deleterious mutations in large populations than in small ones [4 to 107.Figure all ones . As Figubp), while holding the effects of both deleterious and beneficial mutations constant. The curve was shifted to the right with increasing bp, indicating that a population could evolve to a higher mutation rate when beneficial mutations appeared more frequently. As Figure b pvaried from 1% to 10%.To investigate the influence of the relative fraction of deleterious and beneficial mutations, and the combined effects of both, on the optimum mutation rate, we ran simulations where one factor was controlled and the other was varied. In Figure \u03b2) of the gamma distribution for describing deleterious mutations effects, while holding the shape parameter and the effects of beneficial mutations constant. The frequencies of the fixed mutation rates varied only slightly and the optimum mutation rate held constant when \u03b2 was varied, although the mean effects of deleterious mutations changed substantially. Evaluation of how the shape parameter (\u03b1) of the gamma distribution influenced the results was also important. In Figure \u03b1, while holding the scale parameter and the effects of beneficial mutations constant. As \u03b1 decreased, the deleterious mutations that had small effects increased while those with large effects decreased, shifting the curve slightly to the right. Varying the shape parameter therefore had more effect on the frequencies of fixed mutation rates than did varying the scale parameter. Nevertheless, the optimum mutation rate remained constant in this case, indicating that deleterious mutations were effectively eliminated by relentless selection and that their effects had little influence on the optimum mutation rate in long-term evolution. Only when the deleterious mutation effects became small enough to compensate for the beneficial mutation effects could the optimum mutation rate evolve to a higher level (data not shown).In Figure \u03bb) of exponential distribution, while holding the effects of deleterious mutations constant. In contrast to the very small impact seen in response to varying the deleterious mutation effects, the beneficial mutation effects contributed significantly to the optimum mutation rate. The curve was substantially shifted to the right with increasing G = 210) shortened sharply compared to Finally, in Figure In this study, we developed a simulation method based on competition among subpopulations in order to explore the pattern of evolution of mutation rate in large asexual populations. Our simulation results showed that populations tended to form an optimum mutation rate based on their initial configuration. This optimum mutation rate depended on the influx of favorable mutations as well as on their corresponding effects. Below, we first discuss the influence of the initial configuration of the population. We then discuss why beneficial mutations are important in asexual populations. Finally, we compare the present results to previous studies about mutators.To assess whether different mutation ranges and different initial fitness influenced the optimum mutation rate, we performed new simulations, yielding the following results. In Figure We also assigned rugged initial fitness to replace the assumption that all individuals had unified initial fitness value of 1.0. In Figure Finally, we show the influence of organism's fertility limitation on the optimum mutation rate in Figure To summarize, the initial configuration of the population had little influence on the optimum mutation rate, demonstrating the robustness of the developed method based on competition among subpopulations.In this study, we have made three assumptions about mutation effects: first, the mean effects of deleterious mutations are much larger than those of beneficial ones; second, beneficial mutation effects are exponentially distributed; and finally, deleterious mutations effects follow a gamma distribution. However, our simulation results hinge mainly on the first two assumptions. The first assumption is likely to be biologically realistic in many cases, although surely not universally true. Indeed, theory analysis ,28 and ein silico system to show that beneficial mutations with small effects have always existed in the process of evolution. Although beneficial mutations are much rarer compared to deleterious mutations, they supply the driving force for adaptive evolution and contribute to survival of populations in tough environments [bp) and effects (bs) [In general, organisms are well adapted to their living environments, so only a few changes lead to fitness increases and these beneficial mutations have very small effects ,34,35,37ronments . As showronments -44. How cts (sb) ,43.bp) is relatively high, the clonal interference becomes important. However, in this case, there will also be more chances for multiple beneficial mutations to occur in the same genetic background. Whenever clonal interference is important, so are multiple mutations. As Desai and Fisher showed, evolution in asexual budding yeast populations was dominated by the accumulation of multiple mutations of moderate effect [If the fraction of beneficial mutations (e effect . Individbs) are small, these mutations need more generations to be fixed and remain at low frequency in the population for quite a long time [b sincreased. As Wilke pointed out, in the presence of clonal interference, adaptation speed in asexuals still continued to grow with the mean beneficial mutation effects [b smay further increase the adaptation rate of populations considerably. In this case, the population favored a much higher mutation rate. Our simulation results indicated that if strong beneficial mutations (Uopt = 0.05).On the other hand, if the effects of the majority of new arising beneficial mutations (ong time . This prong time . By contong time ,45,47. T effects , althougThis might provide an alternate explanation for why viruses are capable of evolving to a much higher mutation rate  under thThe action of selection on mutation rate can be classified as either direct or indirect: direct action is dependent on the effects of modifier alleles on fitness, while indirect action is dependent on the \"linkage disequilibrium\" between modifier alleles and alleles at other loci affecting fitness . Strong Nevertheless, it should be noticed that in all of the previous studies, high genome mutation rates were generally disfavored in asexual populations except when organisms were under extreme conditions. Gerrish et al. suggested that in the case of complete linkage, the mutation rate would continue to increase until it reached an intolerable level and then lead to organism extinction, rather than elevate without a ceiling . The intOther studies involving modifiers also suggested that even if a high mutation rate increased the rate of adaptation in the short term, due to deleterious mutations, selection would be likely to decrease the mutation rate in the long term evolution ,52-54. TBased on competition among subpopulations with different mutation rates, we investigated the evolution of mutation rates in finite asexual populations. The efficiency of natural selection on mutation rate was shown to depend on population size and mutation effects. Large populations tend to have high mutation rates. The optimum rate is also the result of a balance between two opposing forces: a decreasing rate caused by deleterious mutations and adaptation caused by beneficial mutations. However, the influx of favorable mutations is the critical factor and largely determines the optimum mutation rate in large asexual populations. Contrary to our intuition, the effects of deleterious mutations have little impact on this rate as long as there is no an abundance of deleterious mutations with tiny effects. We hope this simulation method and these findings provide useful inspiration for further modeling of the evolution of mutation rates in asexual populations.XJ carried out the simulation experiments, analyzed the data, and drafted the manuscript. BM and ZH participated in writing the computer programs. MZ and XW proposed important suggestions for conceiving the simulation process. ST conceived of the study and supported the research. All authors have read and approved the final version of the manuscript."}
{"text": "A large number of medically important viruses, including HIV, hepatitis C virus, and influenza, have RNA genomes. These viruses replicate with extremely high mutation rates and exhibit significant genetic diversity. This diversity allows a viral population to rapidly adapt to dynamic environments and evolve resistance to vaccines and antiviral drugs. For the last 30 years, quasispecies theory has provided a population-based framework for understanding RNA viral evolution. A quasispecies is a cloud of diverse variants that are genetically linked through mutation, interact cooperatively on a functional level, and collectively contribute to the characteristics of the population. Many predictions of quasispecies theory run counter to traditional views of microbial behavior and evolution and have profound implications for our understanding of viral disease. Here, we discuss basic principles of quasispecies theory and describe its relevance for our understanding of viral fitness, virulence, and antiviral therapeutic strategy. The rapid evolution of RNA viruses complicates the management of chronic infections and the control of emerging infectious agents The evolutionary dynamics of RNA viruses are complex and their high mutation rates, rapid replication kinetics, and large population sizes present a challenge to traditional population genetics Here we explain basic aspects of quasispecies theory, describe key experiments that define \u201cquasispecies effects,\u201d and highlight how these results may shape our view of viral pathogenesis, antiviral drug development, and vaccine design. We will stress three clinically relevant principles. First, the fitness of a particular virus sequence may be determined more by its freedom to mutate into related sequences than by its own replicative capacity. Second, many viruses operate near a threshold of \u201cerror catastrophe\u201d and may be combated by increasing their replication error rates. Third, increasing the fidelity of genome replication may paradoxically attenuate viruses.\u22124 mutations per nucleotide copied, which is orders of magnitude greater than those of nearly all DNA-based viruses and organisms Most viruses encode enzymes responsible for replicating their DNA or RNA genomes. The intrinsic error rate, or fidelity, of the replicase determines the mutation rate for that virus and the range of genetic variation upon which natural selection can act. Viral RNA polymerases exhibit characteristically low fidelity with measured mutation rates of roughly 10The genetic organization of populations is often depicted using the concept of sequence space, a geometric representation of all possible sequences where physical distance reflects genetic similarity. For example, a given viral genome will undergo replication and generate hundreds of progeny that differ at roughly one position . SubsequViral populations evolve within a fitness landscape where the \u201cground level\u201d is a representation of the range of genotypes in sequence space. The \u201caltitude\u201d at any given location is the fitness associated with that particular genotype. The environment and its selective pressures determine the contours of the corresponding landscape, and adaptation to an environment involves a mutational walk from one point in the fitness landscape to another . In quasA viral quasispecies, then, is a cloud of diverse variants that are genetically linked through mutation, interact cooperatively on a functional level, and collectively contribute to the characteristics of the population. The unit of selection is the population as a whole, and the nature of the functional interactions among genetically distinct variants is therefore of critical importance to pathogenesis in infected hosts. These effects and their biomedical implications are described below.Mutation and selection are the most fundamental processes in evolution. In Darwinian evolution, natural selection acts on existing genetic variation to optimize fitness. Conceptually, fitness refers to how well a given organism \u201cfits\u201d into its environment, often reflected in how well it survives and reproduces Measuring the fitness of individual variants within a population may misrepresent the fitness of a quasispecies. Early experiments with vesicular stomatitis virus (VSV) established that high fitness variants could be suppressed to low levels within a complex population A flat quasispecies with an expansive mutant repertoire can explore vast regions of sequence space without consequence and is poised to adapt to rapid environmental change. This framework may explain many observed phenomena with direct clinical relevance. Arboviruses must successfully adapt to insect and mammalian hosts and their associated fitness landscapes. A quasispecies that occupies a broad, flat region of sequence space could access neutral networks and local fitness peaks in either environment. Similarly, retrospective studies of primary HIV isolates suggest that HIV could be moving to a flatter and less fit region of sequence space Given their high mutation rates, it is not surprising that many discussions of RNA virus evolution focus on the relationship between genetic diversity and adaptability. While it is clear that RNA viruses have the capacity to quickly explore large regions of sequence space, genome size and selective constraints place significant limitations on the amount of diversity that is actually expressed Quasispecies theory accepts this trade-off, and proposes an upper limit to mutation rate, the \u201cerror threshold\u201d . AccordiRecent work on host cell restriction of retroviral infection suggests that lethal mutagenesis is a natural form of antiviral defense These observations suggest that lethal mutagenesis could be an effective therapeutic strategy for RNA virus infections While these results are encouraging, much work needs to be done before lethal mutagenesis can be considered as a quasispecies-inspired therapeutic strategy. Some have argued for a distinction between lethal mutagenesis and error catastrophe, and have suggested, on theoretical grounds, that the above experiments do not show a true phase transition with loss of a master sequence. Indeed, careful studies of lethal mutagenesis by Lowenstein and colleagues have shown an imperfect correlation between mutational load and population extinction The potential for mutagen resistance is another important concern. In principle, a virus could evolve biochemical resistance to nucleosides either by excluding the drug from its active site or by lowering its intrinsic error rate (see below). Quasispecies theory suggests that viruses could also achieve resistance by moving to flatter regions of the fitness landscape, where the density of neutral mutations is higher. Combination therapy with mutagens and traditional antivirals may minimize the probability of mutagenic escape, and work in the FMDV system is encouraging Evolutionary theory predicts that high mutation rates are favored in a dynamic environment, and viral error rates may have been optimized by natural selection G64S) Recent work by two groups in the poliovirus system provides experimental support for this model. Drawing on experience with ribavirin and lethal mutagenesis, they hypothesized that a mutant with a low mutation rate would be less sensitive to lethal mutagenesis and resistant to ribavirin. Both groups sought to isolate ribavirin resistant mutants from a poliovirus quasispecies and recovered a variant with a single amino acid substitution in the viral polymerase , the site of disease This work shows how quasispecies theory and its predictions can lead to the rational development of live, attenuated vaccines. Because variants with low mutation rates would be less likely to revert to wild type in a vaccinee, attenuation should be a stable phenotype and increase the safety of candidate vaccines. Caution is warranted, however, since the error rates of the high fidelity variants are still orders of magnitude greater than those of DNA viruses. It is also unclear whether reducing mutation rate will similarly affect the adaptability and virulence of other RNA viruses. Further work in other systems is clearly needed before this work can be translated into new vaccines Quasispecies theory has had a profound influence on virology, and experiments with model RNA viruses have validated many of its predictions. Considerably less is known about how its population-based models apply to the evolutionary behavior of RNA viruses in infected hosts, and it will be challenging to translate them to the complex reality of viral infection in patients. The initial studies pose as many questions as they answer. What is the best measure of viral fitness in a dynamic population? How can we use a fitness landscape model to understand selection pressure in vivo, where many such landscapes coexist? Do the principles of mutational robustness and survival of the flattest determine the behavior of populations in the host ecosystem? How does population diversity influence pathogenesis? What are the mechanisms by which variants or subpopulations cooperate, and does this have implications for coinfection?While early forays into complex experimental systems have provided tantalizing results, much more work is needed in this area if quasispecies theory is to be translated from bench to bedside. Improved assays for characterizing viral populations are critical, and the advent of ultra high throughput, or \u201cdeep\u201d sequencing, is particularly exciting. Several groups have used this technology to explore the complexity of mutant spectra in clinical samples from HIV- and HBV-infected patients, although the sequencing error rate presents certain limitations for studies of RNA as opposed to DNA viruses"}
{"text": "The rate of mutation is central to evolution. Mutations are required for adaptation, yet most mutations with phenotypic effects are deleterious. As a consequence, the mutation rate that maximizes adaptation will be some intermediate value. Here, we used digital organisms to investigate the ability of natural selection to adjust and optimize mutation rates. We assessed the optimal mutation rate by empirically determining what mutation rate produced the highest rate of adaptation. Then, we allowed mutation rates to evolve, and we evaluated the proximity to the optimum. Although we chose conditions favorable for mutation rate optimization, the evolved rates were invariably far below the optimum across a wide range of experimental parameter settings. We hypothesized that the reason that mutation rates evolved to be suboptimal was the ruggedness of fitness landscapes. To test this hypothesis, we created a simplified landscape without any fitness valleys and found that, in such conditions, populations evolved near-optimal mutation rates. In contrast, when fitness valleys were added to this simple landscape, the ability of evolving populations to find the optimal mutation rate was lost. We conclude that rugged fitness landscapes can prevent the evolution of mutation rates that are optimal for long-term adaptation. This finding has important implications for applied evolutionary research in both biological and computational realms. Natural selection is shortsighted and therefore does not necessarily drive populations toward improved long-term performance. Some traits may evolve because they provide immediate gains, even though they are less successful in the long run than some alternatives. Here, we use digital organisms to analyze the ability of evolving populations to optimize their mutation rate, a fundamental evolutionary parameter. We show that when the mutation rate is constrained to be high, populations adapt considerably faster over the long term than when the mutation rate is allowed to evolve. By varying the fitness landscape, we show that natural selection tends to reduce the mutation rate on rugged landscapes (but not on smooth ones) so as to avoid the production of harmful mutations, even though this short-term benefit limits adaptation over the long term. The mechanisms of DNA replication and repair are themselves genetically encoded and variable Experiments have shown that genotypes with increased mutation rates can be favored by selection if they face novel or changing environments These limitations can be overcome using evolution with digital organisms owing to the speed and ease of data collection. Digital organisms are self-replicating computer programs that inhabit a virtual world where they reproduce, mutate, compete for resources, and evolve according to the same fundamental processes as biological organisms \u22125 to 10 mutations per genome per generation). The final fitness values confirmed that there was an optimal mutation rate at an intermediate value, with optU\u22484.641 (\u22125) produced the highest fitness values, whereas for generations 50\u2013230 a mutation rate of 2.2 gave the highest fitness values.We studied the evolution of mutation rates using the Avida digital evolution platform pt\u22484.641 . An analoptU, we ran additional experiments in which mutation rates were allowed to change (see \u22123) or above (10) the optimum. Strikingly, mutation rates evolved to levels far below the long-term optU, regardless of the starting value  for the populations starting at high and low initial rates, respectively. Both of these values are significantly lower than the 14.45\u00b10.64 obtained for populations evolved at optU . The fitness advantage for optU is also clear for nearly all intermediate time points . We conclude, therefore, that selection fails to optimize mutation rates for long-term adaptation in a broad range of experimental conditions.The finding that mutation rates evolved to be suboptimal was robust to diverse and substantial changes in the experimental conditions. First, we tested whether our results depended on the particular ancestral organism used. In the original experiments, the ancestor was a default, hand-coded organism. To assess whether this condition substantively influenced our results, we let a population founded by this organism adapt for 50,000 updates to an environment without any rewarded functions, using optU is that selection favored those genotypes that minimized the short-term fitness costs caused by deleterious mutations. This explanation is supported by the observations that, during the earliest generations of the evolution experiments, the lowest mutation rate yielded the highest fitness values. To test whether short-term selection would favor low mutation rates, we performed competition experiments between two kinds of organisms, designated A and B. These organisms were identical except for their mutation rate, which was set to optU for A and 0 for B; neither mutation rate was allowed to change during the competition. All competitions were conducted with the same environmental configurations as in the main experiments. In all of 50 runs, B drove A extinct in fewer than 40 generations. Competitions were also performed using U\u200a=\u200a1.0 and U\u200a=\u200a2.154 for B in order to address whether selection would also favor less extreme reductions in mutation rate. In both treatments, B drove A extinct in all 50 trials in fewer than 800 generations. These experiments confirm our hypothesis that natural selection was shortsighted and favored low mutation rates, even when such low rates precluded further adaptation.A possible explanation for why mutation rates evolved to be much lower than We conclude from the results presented thus far that the failure of the evolving populations to achieve or even maintain the mutation rates that maximize long-term adaptation reflect the conflict between the short-term cost of deleterious mutations and the long-term potential for adaptive evolution. We further hypothesize that the resolution of this tension may depend on the topology of the fitness landscape on which evolution occurs. In a rugged fitness landscape, where there are multiple peaks separated by maladaptive valleys \u22125) or above (1) the long-term optimum. Near-optimal values were efficiently selected in those landscapes without a fitness valley or with a narrow valley (optU (optU (P<0.001 in both cases), which indicates a small benefit of adjusting mutation rates during evolution optU (P<0.001 in both cases), confirming that the evolved mutation rates were suboptimal on the these rugged landscapes.A quantitative investigation of mutation rates spanning orders of magnitude revealed, once again, that intermediate mutation rates were optimal over the long-term . We thenw valley . Howeverey , which we call \u03a0, affects the evolutionarily stable mutation rate. Our intuition was that lower values of \u03a0 would make contests between lineages with different mutation rates less frequent, but that the long-term results of many such contests would remain the same. To test this prediction we again used the explicit landscape with a valley size of three. Even when \u03a0 varied over four orders of magnitude, it did not affect the final mutation rate that was reached . Hence, We have shown that mutation rates evolve to near-optimal levels on extremely smooth fitness landscapes. However, if fitness landscapes are rugged, and the maladapted valleys between nearby fitness peaks are wide, then the scarcity of immediately accessible beneficial mutations tips the scale such that short-term selection favors mutation rates that are far below the optimum that would produce the fastest long-term adaptation. Moreover, this process is self-reinforcing because the lower the mutation rate, the less likely it becomes to produce a genotype on the other side of the fitness valley, thereby effectively widening the valley. The digital organisms in the standard Avida configuration used in our first set of experiments exhibit extensive and variable genetic interactions, making the fitness landscape rugged Our experiments were performed under conditions that were favorable for the optimization of mutation rates. First, the organisms reproduced asexually. Both theoretical The inability of evolving populations to optimize their mutation rates for long-term adaptation, even with such favorable conditions, indicates that mutation rates will be suboptimal under a wide range of circumstances, at least when fitness landscapes are rugged and populations are far from a global fitness peak. While novel environments can promote increases in the mutation rate if many beneficial mutations become accessible Recent theoretical work by Gerrish et al. Beyond their implications for understanding nature, our findings are also relevant for applied fields that use evolution to improve the performance of biological and computational systems, from molecular and microbial engineering to robotics and evolutionary computation In summary, natural selection is not universally effective at optimizing mutation rates for long-term adaptation; in fact, it is very poor in this respect for populations that evolve on complex fitness landscapes. Also, our results caution against making generalizations based on analyses of simple fitness landscapes, whether one is studying natural systems or using evolution for engineering. As we have shown, the mere inclusion of fitness valleys\u2014which are presumably common to the vast majority of fitness landscapes\u2014can yield radically different conclusions from those based on smooth fitness landscapes.A general description of the Avida software can be found elsewhere \u03bc, the genomic mutation rate being U\u200a=\u200a100\u00d7\u03bc. All instructions were equally likely to result from any given mutation. The mutation rate was held constant in some experiments, while in others the rate could change by evolving over time. In treatments where the mutation rate could change, \u03bc had a constant and high probability \u03a0 of changing by a small amount during any replication cycle. The magnitude of any resulting change was obtained by drawing log2(\u03bcoffspring/\u03bcparent) values from a Gaussian distribution . For the experiments in which mutation rates were more likely to increase than to decrease, we drew log2(\u03bcoffspring/\u03bcparent) from a Gaussian , where b controls the upward bias, and tested values such that mutation rates were up to \u223c1.6 times more likely to increase than decrease .During replication, each genomic instruction could mutate to another with probability Organisms died when another organism's offspring replaced them or when they executed 2,000 instructions without producing an offspring of their own. All experiments using the standard configuration lasted 150,000 updates. Updates are an arbitrary unit of time in Avida; they represent the time during which each organism, on average, executes 30 instructions 10 transformed, and finally averaged over all replicate populations  in an experimental treatment.Each organism's phenotype depended on the complex rules that governed how its genomic program was executed, and its fitness depended on the interaction between the resulting phenotype and its environment To perform logic functions, organisms used inputs consisting of three randomly generated 32-bit strings, which they manipulated to produce an output. The manipulation of these numbers occurred as organisms moved them on and off stacks or between registers by executing instructions such as push, pop, add (combines the numbers in the two specified registers and places the result in a third), shift-r (bit shift right), and so on. A function was rewarded only if the input to output conversion conformed to one of the 77 canonical one-, two- or three-input logic operations. For example, the two-input EQU  function requires inputting two strings and outputting a third string that had a 1 for each of the 32 bits where both inputs had the same value and a 0 where they differed.Avida runs are inherently stochastic with respect to mutation and death. Therefore, we performed 50 replicate runs for each treatment. Those replicates had identical initial conditions except for a random number seed. That seed affects the outcome of all subsequent stochastic events.The standard and the explicit Avida configurations differed in the instruction set, the fitness calculation and the mode of replication. We modified Avida to mimic a two-allele, 10-locus bit-string model used in a previous study In the standard configuration, digital organisms had to copy their genomic instructions in order to replicate, and their fitness depended on their speed of replication as well as any rewards they obtained for performing computational functions. Under this alternative configuration, the organisms did not copy themselves, and only the number of A or B instructions mattered to their fitness. The rest of the setup, such as population size, was identical to the standard configuration.http://devolab.cse.msu.edu/software/avida. Default settings were used unless otherwise indicated.All experiments were performed with the Avida software, which can be downloaded for free at"}
{"text": "Pseudomonas aeruginosa as a model system, we investigate the underlying distribution of fitness effects of beneficial mutations on which natural selection acts. Consistent with theory, the effects of beneficial mutations are exponentially distributed where the fitness of the wild type is moderate to high. However, when the fitness of the wild type is low, the data no longer follow an exponential distribution, because many beneficial mutations have large effects on fitness. There is no existing population genetic theory to explain this bias towards mutations of large effects, but it can be readily explained by the underlying biochemistry of rifampicin\u2013RNA polymerase interactions. These results demonstrate the limitations of current population genetic theory for predicting adaptation to severe sources of stress, such as antibiotics, and they highlight the utility of integrating statistical and biophysical approaches to adaptation.Understanding how beneficial mutations affect fitness is crucial to our understanding of adaptation by natural selection. Here, using adaptation to the antibiotic rifampicin in the opportunistic pathogen  P. aeruginosa. When the fitness of the wild type is high, most beneficial mutations have small effects. This finding is consistent with existing population genetic models of adaptation based on statistical theory. When the fitness of the wild type is low, most beneficial mutations have large effects. This distribution cannot be explained by population genetic theory, but it can be readily understood by considering the biochemical basis of resistance. This study confirms an important prediction of population genetic theory, and it highlights the need to integrate statistical and biochemical approaches to adaptation in order to understand evolution in stressful environments, such as those provided by antibiotics.Adaptation by natural selection depends on the spread of novel beneficial mutations, and one of the most important challenges in our understanding of adaptation is to be able to predict how beneficial mutations impact fitness. Here, we investigate the underlying distribution of fitness effects of beneficial mutations that natural selection acts on during the evolution of antibiotic resistance in the opportunistic human pathogen  Adaptation by natural selection ultimately depends on the spread of novel beneficial mutations that increase fitness. Can we predict the fitness effects of beneficial mutations? GillespiePseudomonas aeruginosa as a model system. Experimental studies of the underlying distribution of fitness effects of beneficial mutationsPseudomonas aeruginosa with mutations in the \u03b2-subunit of RNA polymerase (rpoB) that are beneficial in the presence of the drug rifampicin rpoB of the mutants. To test the hypothesis that the fitness effects of beneficial mutations are exponentially distributed, we used log-likelihood tests that have been specifically developed to test this hypothesis using this experimental designIn this paper, we investigate how the distribution of fitness effects of beneficial mutations changes with the fitness of the wildtype population using the evolution of antibiotic resistance in the opportunistic pathogen Using this approach, we show that the distribution of fitness effects of beneficial mutations is variable: under conditions where the fitness of the wild-type is high, the fitness effects of beneficial mutations are exponentially distributed, as predicted by theory. However, when the fitness of the wildtype is low, the data may no longer fit an exponential distribution because many beneficial mutations have large effects on fitness. We show that this non-exponential distribution of fitness effects emerges as a direct consequence of the molecular interactions that are under selection in this system and we argue that existing theory on the fitness effects of beneficial mutations cannot be applied to understand adaptation to novel stressful environments, such as those provided by antibiotics.To investigate how the distribution of fitness effects of beneficial mutations changes with the fitness of the wild-type, we measured the fitness of beneficial mutations isolated at a high concentration of rifampicin  and the One limitation of this study is that our power to test the null hypothesis is weakest under conditions where the fitness of the wild-type is high, because only half of the mutants that we isolated increase fitness at low concentrations of rifampicin. Given that we had to sample 80 mutants in order to identify 15 beneficial mutations , it unlikely that increasing the sample size of our study would have substantially increased the power of our analysis. It is also important to note that this limitation is not unique to this study: beneficial mutations are rare events, and all other comparable experimental evolution studies are based on a similar sample size of beneficial mutationsr\u200a=\u200a.86\u2013.9, P<.0001). The genetic variation in growth rate in the absence of antibiotics generated by spontaneous mutation is normally distributed , hence the fitness effects of beneficial mutations are exponentially distributed because only mutations in the right tail of the distribution . Residues that surround the binding pocket interact only indirectly with rifampicin, and it has been argued that resistance arises at these residues due to amino acid changes that alter the folding of the protein in the binding pocket. We identified only a small number of beneficial mutations (n\u200a=\u200a4) in residues that are involved in indirect Rif-RNAP interactions and mutations at these residues give rise to intermediate levels of rifampicin resistance . This biophysical approach to understanding the effects of beneficial mutations suggests that the data may no longer fit an exponential distribution because of the high specificity of interactions between rifampicin and RNA polymerase: changes to the majority of amino acids that are involved in rifampicin-RNAP interactions results in large increases in resistance and, therefore, large increases in fitness at high concentrations of rifampicin. To test this hypothesis further, we assayed fitness in the presence of sorangicinP\u200a=\u200a.09).The large effect of beneficial mutations on resistance is consistent with the molecular interactions that occur between rifampicin and RNA polymerase. Structural studies have shown that rifampicin binds to a small, highly conserved pocket of the \u03b2-subunit of RNAP and only 12 amino acid residues are involved in direct interactions with rifampicinIt is important to note that these results do not necessarily imply that the distribution of fitness effects of mutations that are beneficial in the presence of sorangicin is universally exponential. Most mutations that increase sorangicin resistance do so by altering membrane permeability, instead of altering the structure of RNAPet al.The variability in the distribution of fitness effects of beneficial mutations in this study is consistent with population genetic theory. When the fitness of the wild-type is high, beneficial mutations can be viewed from a statistical perspective as representing draws from the extreme tail of the distribution of fitness effects of mutations, hence the fitness effects of beneficial mutations will be exponentially distributed. However, EVT does not specify how high fitness has to be in order for this theory to apply. In our experimental system, this distribution held over a wide range of parameter space: we failed to detect significant deviations from the exponential distribution when the fitness of the wild-type was reduced by 20\u201330%. When the fitness of the wild-type is low, statistical theory does not make any predictions regarding the form of the distribution of fitness effects of beneficial mutations and hence there is no reason to expect an exponential distribution. Comparable experimental studies in viral systems are in agreement with this idea: Sanjuan and colleagues a priori). Specifically, the distribution is biased towards mutations of large effect as a result of the high specificity of interactions between rifampicin and RNA polymerase that arises from the low conformational flexibility of rifampicin. Antibacterial and antiviral drugs are usually involved in highly specific interactions with their target proteins Despite our lack of certainty of the statistical explanation for the observed distribution when the fitness of the wild-type is low, we have a good molecular mechanistic explanation . This overnight culture was diluted down 10\u22126 into fresh M9KB and 120 uL aliquots of this diluted culture were used to setup 480 cultures on 5 96 well microplates. These cultures were incubated overnight at 37 C without shaking. To isolate beneficial mutations, 5 uL of each of the 480 overnight cultures was plated out on M9KB supplemented with 62.11 ug/mL of rifampicin, the minimal concentration required for complete inhibition of growth of the wildtype strain. To isolate beneficial mutations, we isolated a single colony from each of the first 80 cultures that gave samples containing exactly 1 colony on agar plate containing rifampicin.A single clone of rpoB gene in each of the 80 colonies that we isolated in our fluctuation test. Genomic DNA was isolated from each colony using a Wizard Genomic DNA extraction kit  as per the manufacturer's instructions. Our sequencing strategy was to first sequence a highly-conserved domain of rpoB that is known to be important for rifampicin resistance in all 80 clones. This region was amplified with primers rpoB_fwd (5\u2032-GTTCTTCAGCGCCGAGCG-3\u2032) and rpoB_rev (5\u2032-GCGATGACGTGGTCGGC-3\u2032) that amplify the region of the rpoB gene between nucleotides 1178 and 1864. Reaction mixtures consisted of BIOTAQ polymerase , 1 mM dNTPs, 16 nM (NH4)2SO4, 62.5 mM Tris-HCL (pH 8.8), .01% Tween 20, 2 mM MgCl2, and each primer at a concentration of .2pM. Amplification reactions were carried out as follows: 94 C for 5 minutes, followed by 35 cycles of 94 C for 30 seconds, 60 C for 30 seconds, and 72 C for 1 minute, followed by a final incubation at 72 C for 10 minutes. PCR products were purified using a MultiScreen PCR96 filter plate  as per the manufacturer's instructions. Purified PCR products were sequenced with both forward and reverse primers using BigDye 3.1 sequencing  followed by ethanol/EDTA precipitation of sequencing products. In all cases, this strategy identified either a single mutation in this region of rpoB or no mutations.To determine the mutations underlying adaptation, we sequenced the rpoB were subsequently sequenced for a second region that has previously been implicated in rifampicin resistance spanning nucleotides 1 to 1012 of the rpoB gene. This region was amplified and sequenced with primers rpoB_up (5\u2032\u2013ATGGCTTACTCATACACTGAG-3\u2032) and rpo_B1 (5\u2032-CTCGATGCG CACGACCTG-3\u2032). The protocol was the same as described above, except that the annealing temperature used in the PCR reactions was 54 C instead of 60 C. We idenfied a single mutation in this region in all of the clones that did not contain a mutation in the highly conserved domain of rpoB. As a further control, we sequenced the entire rpoB gene in six randomly chosen clones. We failed to detect any second site mutations in rpoB using this approach.Clones that lacked a mutation in the highly conserved domain of r, of each beneficial mutation and wildtype PAO1 at 4 different concentrations of rifampicin . Pre-assay overnight cultures of each mutant were prepared by growth in M9KB. These cultures were then diluted 100 fold into fresh culture medium and we measured the growth rate of each culture using an automated microplate reader by taking hourly measurements of optical density at 600 nm (OD600) over a period of approximately 12 hours. All incubations were carried out at 37 C. Assays at low concentrations of rifampicin  were carried out with 12 fold replication and assays at high concentrations of rifampicin (64 mg/L) were carried out with 18 fold replication. A further assay was carried out using the same method to measure fitness in the presence of sorangicin (20 ug/mL). OD600 is proportional to the log of cell density, and the slope of OD600 against time (mOD/min) in exponential growth phase therefore provides an estimate of r, the growth rate of the bacterial clone, such that i,jr is the growth rate to the ith genotype at the jth concentration of rifampicin.To assay fitness, we estimated the growth rate, i,jw\u200a=\u200ai,jr\u22121,jr where i,jw is the fitness of the ith beneficial mutation in the jth environment, i,jr is the growth rate of the ith beneficial mutation in the jth environment, and 1,jr is the growth rate of the least fit beneficial mutation in the the jth environment .To test the hypothesis that the fitness effects of beneficial mutations are exponentially distributed, we used a likelihood ratio test developed by Beisel and colleagues 0:\u03ba\u200a=\u200a0, HA: \u03ba\u22600). P values were calculated by performing 10000 parametric bootstrap replicates using a software package (EVDA) developed for R (software available at http://www.webpages.uidaho.edu/\u02dcjoyce/Lab20Page/Computer-Programs.html). This test is potentially sensitive to measurement error, but the accuracy of our fitness measurements was high enough (Average CV\u200a=\u200a19.8%) that measurement error should not inflate the probability of making a type I error The likelihood ratio test developed by Beisel and colleagues calculates \u22122log\u039b, negative twice the difference in log-likelihood between two statistical models, one model in which the shape parameter of the GPD is set to 0 and the other in which the scale parameter of the GPD is free to vary : 0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, and 1000. Assay cultures were incubated at 37 C and we measured the optical density of cultures after exactly 24 hours of incubation (+/\u221210 minutes) at 600 nM using an automated microplate reader. We assayed the resistance of 12 cultures of each of the rpoB mutants that we identified and 12 replicates of PAO1. Resistance was calculated as IC50, the concentration of rifampicin necessary to cause a 50% reduction in optical density, using the following regression model: y is optical density, measured in absorbance units, x is the concentration of rifampicin, measured in ug/mL, and H is parameter that estimates the rate of decay in optical density with increasing rifampicin concentration.To measure the resistance conferred by"}
{"text": "Recent outbreaks of novel infectious diseases  have highlighted the threat of cross-species pathogen transmission. When first introduced to a population, a pathogen is often poorly adapted to its new host and must evolve in order to escape extinction. Theoretical arguments and empirical studies have suggested various factors to explain why some pathogens emerge and others do not, including host contact structure, pathogen adaptive pathways and mutation rates. Using a multi-type branching process, we model the spread of an introduced pathogen evolving through several strains. Extending previous models, we use a network-based approach to separate host contact patterns from pathogen transmissibility. We also allow for arbitrary adaptive pathways. These generalizations lead to novel predictions regarding the impact of hypothesized risk factors. Pathogen fitness depends on the host population in which it circulates, and the \u2018riskiest\u2019 contact distribution and adaptive pathway depend on initial transmissibility. Emergence probability is sensitive to mutation probabilities and number of adaptive steps required, with the possibility of large adaptive steps (e.g. simultaneous point mutations or recombination) having a dramatic effect. In most situations, increasing overall mutation probability increases the risk of emergence; however, notable exceptions arise when deleterious mutations are available. Such \u2018sA number of risk factors have been proposed to explain why some pathogens emerge and others do not. These include the breadth of the pathogen's host range , susceptThe association between transmissibility and likelihood of emergence is, perhaps, not surprising. Any factor that increases the expected number of transmitted infections, such as higher initial transmissibility between members of the new host species, means that the pathogen can circulate for longer after the cross-species jump, and thus has greater potential for ultimate evolutionary adaptation . A high mutation rate might lead to a greater likelihood of the appropriate adaptive mutations occurring, implying greater evolutionary potential . Indeed,In this paper, we investigate the above factors through mathematical modelling. We ask two main questions: (i) what types of host contact structure lead to the greatest risk of evolutionary emergence? and (ii) how do patterns of mutation affect the risk of evolutionary emergence? We address these questions using a branching process model that tracks the number of infected hosts as a newly introduced pathogen spreads and evolves. There have been two main approaches to using such models in the epidemiological literature, a network-based approach and a phenomenological approach. The former explicitly considers the patterns of contact among individuals in the population and the probability of transmission through any given contact e.g. , but has2.In order to develop a unified modelling framework that encompasses both the network-based and phenomenological approaches, it is useful first to focus on single-strain models that ignore evolution.2.1.We model in discrete time, using a Galton\u2013Watson process. To define the branching process, we require a distribution for the number of \u2018offspring\u2019 produced by each individual. In the present context, infected hosts are the individuals of interest, and an \u2018offspring\u2019 is viewed as a contact to whom the infection is transmitted. The network-based approach and the phenomenological approach arrive at this offspring distribution in different ways. The network-based approach builds up the offspring distribution from underlying assumptions about the processes of host\u2013host contact and pathogen transmission. The phenomenological approach, on the other hand, simply specifies this offspring distribution directly, without explicit consideration of the underlying contact and transmission processes through which it might arise.n infective  are considered generation n + 1 infectives, and \u2018later-generation\u2019 infectives (i.e. generation \u2265 1) receive their infections from other individuals within the population.The network-based approach represents the host population by a graph: each vertex or node corresponds to an individual, and an edge between two nodes signifies that the two individuals are acquaintances, i.e. can contact and potentially transmit disease to one another. We assume that this network is static, and restrict attention to random graphs in the limit of infinite population size. In this situation, the network becomes a tree graph, with the number of \u2018branches\u2019 from each node drawn independently from the arbitrary degree, or contact, distribution p\u0303d} given by the PGFd\u232a = g\u2032(1) denotes the expected value of degree d.Following the derivation presented by independently with probability T, called the transmissibility, taken to be a constant. That is, if d is the number of still-susceptible contacts, then the number of infections transmitted is distributed as Binomial. For the initial infective, who has been infected by a source outside the population, all contacts (given by the degree) remain susceptible. Thus, the PGF \u03b3(s) for the number of infections transmitted by a randomly chosen initial infective is (\u0393(s) for the number of infections transmitted by a later-generation infective isR0, can then be defined as the mean number of infections transmitted by one typical (later-generation) infective:G\u2032(1)) and pathogen transmission (represented by T). R0 is an appropriate measure of the fitness of a single strain of pathogen.An infective is assumed to transmit infection to each still-susceptible contact ctive is A laterX, produced by an infected individual. The approach typically uses a Poisson distribution of infections, implicitly assuming homogeneous mixing in the population, but sometimes incorporates individual heterogeneity by allowing the mean of the distribution itself to be a random variable. For example, \u03bd, from a Gamma distribution with mean R0 and dispersion parameter \u03b2; then X has a negative binomial distribution with mean R0 and dispersion \u03b2. As special cases, \u03b2 = 1 corresponds to \u03bd \u223c Exp(R0) and X \u223c Geometric(R0), while \u03b2 \u2192 \u221e corresponds to \u03bd = R0  and X \u223c Poisson(R0). In any case, the key difference from the network-based approach is that \u0393(s) is effectively specified directly. As before, one then defines the basic reproductive number as R0 = \u0393\u2032(1). Notice, however, that unlike the network-based approach, no distinction is made here between generation 0 infectives (i.e. those who receive the infection from an outside source) and later-generation infectives.  If we consider only later-generation infectives, the phenomenological approach can be obtained via the network-based approach, provided that the contact distribution in the latter is chosen to obtain the same end result for the offspring distribution (appendix A.3).The phenomenological approach (e.g. \u03bb and dispersion \u03b2 (appendix A.1), i.e. G(z) = (1 + (\u03bb/\u03b2)(1 \u2212 z))\u03b2\u2212 (\u03bbT and dispersion \u03b2: \u0393(s) = G(1 \u2212 T + Ts) = (1 + (\u03bbT/\u03b2) (1 \u2212 s))\u03b2\u2212. Since R0 = \u03bbT, this gives exact correspondence with the phenomenological approach of For example, if the contact distribution is such that the number of still-susceptible contacts has a negative binomial distribution with mean extinction probabilityq of the process\u2014that is, the probability that the disease outbreak eventually ends, infecting only a finite number of people\u2014is the smallest non-negative solution of the equation \u0393(s) = s. The epidemic is guaranteed to go extinct if R0 \u2264 1, but persists with non-zero probability if R0 > 1 (g(1 \u2212 T + Tq) \u2261 \u03b3(q)  \u2261 \u03b3(q) .2.2.et al.A phenomenological approach has also been used to explore how pathogen evolution affects disease emergence by accounting for multiple pathogen strains et al.. In the i  denotes which one of the m possible pathogen strains is infecting that host. We allow for arbitrary contact distribution and assume that the contact network is determined by the host, independently of the pathogen strain. Thus, as before, degree is described by the PGF g(z) and the excess degree by G(z) for every individual, regardless of type. We retain the assumption that transmissions occur independently to each contact, and further assume that transmissibility T is strain-specific, denoted Ti for strain i. Thus, the probability of making an infectious contact depends only on the current infective's strain type. However, a different strain may be transmitted as a result of pathogen mutation. Specifically, given that a type i infective makes an infectious contact, strain j is transmitted with probability \u03bcij  allowed in the evolution of the pathogen and their probabilities.We account for pathogen evolution using a multi-type branching process. An individual's type within individual hosts, but we account for this in a phenomenological way. The most accurate interpretation of our approach would be that a host currently infected with strain i transmits only strain i, but that each infective produced in the next generation has probability \u03bcij of converting to strain j over the course of infection, before any further transmission occurs. In this approach, \u03bcij is an \u2018effective conversion rate\u2019 from strain i to strain j within one host, summarizing the results of within-host dynamics (\u00a74.3). This is a common type of simplification involving a separation of the time scales on which within- and between-host processes occur. We use the word \u2018mutation\u2019 loosely to mean any processes resulting in a change in the strain identified as a host's type. These processes actually occur i infective with d susceptible contacts now has distribution Multinomial, where Ui\u00b7 is the ith row of U and the probability vector is given in the order: no transmission, transmit strain 1, \u2026 , transmit strain m. The corresponding PGF for the number of transmitted infections of each type, , given d susceptible contacts, isi isReturning to our multi-type process, the number of transmissions of each type made by a type i, denoted qi, is obtained as the smallest non-negative root of the equation qi = \u0393i, solved simultaneously for all i (appendix D). More compactly, we can write this as a vector fixed-point equation: i, chosen uniformly at random, the overall probability of extinction is then given by g(1 \u2212 Ti + Ti \u2211 j=1m\u03bcijqj) \u2261 \u03b3ii is introduced to a randomly chosen individual, is to allow the initial infective to be type j with probability \u03bcij. We generally assume that strain 1 is, by definition, initially introduced to the host population.The probability of extinction starting from one later-generation infective of type i\u2019, denoted Ri0, and defined as the expected total number of infectious contacts made by a typical (later-generation) type i infective. Since this number is distributed as Binomial, we havei were the only strain present, with no possibility of mutation, Ri0, would be the value of its basic reproductive number in the single-strain model.In what follows, it is sometimes useful to refer to the \u2018basic reproductive number of strain Ri0, > 1. Probability of emergence is thus the complement of probability of extinction. We present numerical results for the probability of emergence beginning from one later-generation infective of type 1, 1 \u2212 q1, but these results may be extended to account for generation 0 and/or type conversion as described above. Note that the branching process may be either indecomposable or decomposable, depending on the mutational scheme. Thus, if the epidemic persists, the complement of strains that will be present must be treated on a case-by-case basis (appendix D).We define emergence as the situation in which the pathogen escapes extinction, which necessarily requires evolution to a strain having a priori restrictions on the choices of contact distribution, transmissibilities and mutation probabilities. However, in the numerical results to follow, we limit ourselves to specific examples.Our derivation so far is quite general, imposing no Contact distributions. We consider the following contact distributions, each with mean \u03bb .Deterministic. Every individual has exactly \u03bb contacts.\u2014\u2003Mixed deterministic. There are n types of individuals, where the kth type occurs in proportion pk and has exactly \u03bbk contacts.\u2014\u2003Poisson.\u2014\u2003Mixed Poisson. There are n types of individuals, where the kth type occurs in proportion pk and has a Poisson(\u03bbk) distribution of contacts.\u2014\u2003Negative binomial, with dispersion \u03b2.\u2014\u2003Mutational schemes. We consider two broad types of mutational schemes: \u2018linear\u2019 and \u2018hub-and-spoke\u2019 . In all cases, we assume that strain m is well adapted to the host; that is, Tm is chosen such that Rm0, > 1. On the other hand, any other strain i is poorly adapted to the host  unless otherwise specified.m \u2212 1 are intermediates between strains 1 and m. We consider the following possibilities.One-step irreversible. The pathogen must acquire m \u2212 1 point mutations, one at a time and in a fixed order. Thus, mutation occurs only from strain i to i + 1. This scheme is presented by \u2014\u2003Multi-step irreversible. Again the pathogen must acquire m \u2212 1 point mutations in a fixed order, but now possibly simultaneously, implying that mutations can occur from strain i to any strain j > i, though with diminishing probabilities. Essentially, higher order terms are being included where they were neglected in the previous scheme. This scheme is presented by \u2014\u2003Interchangeable and irreversible. This scheme, also presented by i in our model representing any \u2018real\u2019 strain having i \u2212 1 of the required mutations. Our model thus implicitly assumes that any collection of i \u2212 1 mutations yields the same transmissibility, Ti.\u2014\u2003Point mutation and recombination. One-step point mutation occurs as in scheme 1, but we simplistically model an additional mechanism of more extensive genetic change .\u2014\u2003One-step irreversible mutations, coupled with a Poisson(\u03bb) distribution of contacts, corresponds to the model of Ri0, = \u03bbTi.Linear mutational schemes represent single directions through strain space, where strains 2, \u2026 , m \u2212 1 no longer represent intermediates on the path to strain m. For simplicity, in our results we consider only paths of length 1 , with equal probability of proceeding along any path. However, this set-up could obviously be extended to incorporate pathways of various lengths and mutation probabilities. We consider two possible scenarios.One-step irreversible.\u2014\u2003One-step reversible.\u2014\u2003Finally, we must specify the transmissibility of each strain, m \u2212 1 have identical transmissibility, and hence the same value of Ri0, < 1. Other biologically interesting possibilities include a fitness valley, where intermediate strains have lower fitness than strain 1, or an additive model, where fitness increases linearly with strain number . Though still numbered sequentially for convenience, strains 2, \u2026 , 3.3.1.R0, is widely used as a predictor of when a disease has epidemic potential, as well as a descriptor of disease spread T. Using the relationship G(z) = g\u2032(z)/g\u2032(1) gives R0 = (g\u2033(1)/g\u2032(1))T. Then substituting g\u2033(1) = \u03c32 \u2212 g\u2032(1) + (g\u2032(1))2, where \u03c32 is the variance of the contact distribution, yieldsc = \u03c3/g\u2032(1) is the coefficient of variation of the contact distribution. For a given transmissibility and mean number of contacts, the contact distribution that maximizes variance thus maximizes R0. The above equation is essentially the same as the expression for R0 given by c, and is similar to the expression for R0 used in deterministic network models  and mutation probabilities given by matrix U = [\u03bcij], we have aij = G\u2032(1)Ti\u03bcij = (g\u2032(1)(1 + c2) \u2212 1)Ti\u03bcij. We can thus express the mean matrix asM\u2014and hence its dominant eigenvalue\u2014on the mean and variance of the contact distribution, the transmissibility of each strain and the mutation scheme.In our multiple strain model, with contact distribution given by PGF 3.2.R0, and on the probability of emergence starting from one later-generation infective, 1 \u2212 q. Figure\u00a02a shows how R0 increases with transmissibility T: in all cases, this increase is linear, but at a different rate for each contact distribution depending on the variance in contacts. Figure\u00a02b plots the probability of emergence versus T for each contact distribution. This probability becomes non-zero at the point where R0 passes the critical value of one, which occurs at a different value of T for each distribution (as illustrated in figure\u00a02a). Thus, at low values of T, the disease can persist in some host contact structures but not others, despite the fact that they all have the same mean number of contacts. As T increases, however, the probability of emergence increases at different rates for different contact structures, such that the ordering is not preserved. This observation is highlighted in the inset, showing emergence probability over an extended range of T. In contrast, the ordering is consistent in figure\u00a02c, which plots the probability of emergence versus R0, comparable to Brauer's presentation Tm. Hence, criticality is independent of the precise details of a uni-directional mutation scheme. By contrast, a scheme that allows both forward and reverse mutation can affect criticality. R0  changes with the probability of reverse mutation (\u03bd) in the linear one-step reversible scheme; similar considerations apply to the hub-and-spoke one-step reversible scheme as well. A high rate of reverse mutation from a well-adapted to a poorly adapted strain can tip the branching process from supercritical to subcritical. Population R0 increases almost linearly with Tm, but is relatively insensitive to number of strains (m), earlier strain transmissibility (T1) and forward mutation probability (\u03bc). This is because population R0 is very close to amm = G\u2032(1)Tm(1 \u2212 \u03bd), but slightly influenced by contributions from small populations of other (poorly adapted) strains that persist asymptotically if the process escapes extinction.When there are multiple strains of pathogen, the pathways of mutation among them may also affect the threshold parameter. In the case of irreversible mutation, we can number strains such that 3.4.q1, we compute the extinction probability si < 1, \u221212 or 10\u221216 in the figures we present.We can compare not only the qualitative result of criticality, but also the quantitative probability of emergence across various supercritical branching processes. To obtain this probability, 1 \u2212 Tm is far from the critical threshold, and reverse mutation, even at a high rate equal to that of forward mutation, makes a negligible impact on the probability of emergence. This agrees with Sagitov and Serra's result that reverse mutation is negligible in a similar model with an arbitrary offspring distribution .ribution , and wit  varies. Although we have typically used the jackpot model, one might also consider other choices of transmissibilities. As one would expect, the higher the intermediate strain fitness, the higher the probability of emergence as observed by 3.5.et al.For any given mutation scheme, we can gain greater insight into the effect of mutation probabilities and number of strains by estimating the probability of emergence analytically. As other authors et al. have conet al. derived the following approximation for the probability of evolution to strain m (starting from strain 1) in the case of a Poisson-distributed number of infectious transmissions and one-step irreversible mutation  having Rm0, > 1; all other types i \u2260 m have Ri0, < 1.\u2014\u2003There is a single \u2018escape\u2019 type .\u2014\u2003All mutation probabilities Under these conditions, the above approximation has error of O(u2). However, the neglected error term increases as Ri0, \u2192 1, implying that the approximation will break down if strain fitness approaches the critical threshold. Ri0,, including the near-critical case, though only for a specific offspring distribution.More generally, y et al. , 2004. Sq1) scales as O(\u03bcm\u22121) + O(\u03c1), where \u03bc is the probability of one-step forward \u2018point mutation\u2019 and \u03c1 is the probability of jump-to-m \u2018recombination\u2019. m = 3 strains, illustrating the relative contributions of the different adaptive pathways. When \u03c1 \u226b \u03bc2, the probability of emergence is almost constant in \u03bc and scales approximately linearly in \u03c1. When \u03c1 \u226a \u03bc2, the probability of emergence is almost constant in \u03c1 and scales proportionally to \u03bc2.As an example, consider our \u2018mutation and recombination\u2019 scheme. Either by applying an Antia-like argument to this specific scenario or by substituting mutation probabilities into Serra and Haccou's result, we can show that the probability of emergence starting from a type 1 individual  or where the assumptions underlying these approximations are broken or pushed to their limits (e.g. relatively large mutation probabilities), as we will encounter in the next subsection.3.6.The same biological mechanisms  contribute to all mutation rates, not only those for beneficial mutations. Given this constraint, it is not immediately clear whether pathogens with the highest mutation rates will have the largest or smallest probability of emergence. We explore this question for two mutational schemes in which deleterious steps are possible: one-step reversible mutation and hub-and-spoke irreversible mutation.\u03bc, and from 2 to 1 with probability \u03bd, which we scale proportionally to \u03bc. Results demonstrate a non-monotonic relationship between mutation rate and probability of emergence , the poorly adapted strain 1 and the well-adapted strain 2. Mutation from 1 to 2 occurs with probability mergence . The proT1 for various mutation probabilities. When T1 is well below the critical threshold , emergence probability increases linearly with mutation probability, as we would predict from analytical approximations (\u00a73.5). Although this figure plots results for m = 10 strains, a virtually identical increase in emergence probability versus \u03bc at fixed small T1 was observed for m = 2, 3, 5 as well (results not shown). This indicates that regardless of how high the risk of deleterious steps, a pathogen that is guaranteed to go extinct unless it acquires a beneficial mutation (evolves to strain m) is always better off having a large mutation rate. Near the critical threshold, emergence probability shows a sharp jump; once strain 1 is sufficiently fit to escape extinction without further evolution, mutation probability makes only a small difference in results. Shortly after this point, a very high mutation probability becomes detrimental to the pathogen, even while strain 1 is not as well adapted as strain m. The risk of mutating to a poorly adapted strain now outweighs the potential benefit of mutating to a better adapted strain. Adding reverse mutation at a probability equal to that of forward mutation makes negligible difference to quantitative results (results not shown), as also observed with linear mutation schemes where Rm0, is not too close to one. We might suspect that increasing reverse mutation probability independently of forward mutation probability could provide a benefit to the pathogen if the transmissibility of deleterious strains is non-zero, such that mutation could \u2018rescue\u2019 the pathogen by a return to strain 1 (the hub). However, this does not appear to be the case within tested parameter ranges (results not shown): increasing reverse mutation probability decreases probability of emergence, though only marginally when Rm0, is well above one.With an irreversible hub-and-spoke mutation scheme, we assume that the initial strain is equally likely to mutate to any other, but only one mutational pathway is beneficial. As an extreme case, we set transmissibility of all deleterious strains to be zero. 4.We have linked phenomenological and contact network-based approaches to modelling disease spread as a branching process, clarifying how they may be viewed within a common framework. By explicitly considering the number of contacts along with a model of how transmission occurs to these contacts, a network-based model offers more detailed insights into factors contributing to pathogen fitness and probability of emergence. Such a model lends itself to comparison of the relative impact of various public health interventions, which may act by reducing either number of contacts or probability of transmission per contact .R0 = (g\u2032(1)(1 + c2) \u2212 1)T\u2014where g\u2032(1) is the mean of the contact distribution, c is its coefficient of variation and T is transmissibility\u2014highlights the contributions of both ecological factors (contact structure) and epidemiological factors (transmissibility) to pathogen fitness. That is, a pathogen's fitness depends on the particular host population in which it circulates, an observation that has been made more generally for pathogen fitness measures (M = (g\u2032(1)(1 + c2) \u2212 1)diagU is the mutation matrix. These expressions make it clear that for fixed transmissibilities, mean number of contacts and mutation scheme, maximizing pathogen fitness is equivalent to maximizing the variance of the host population's contact distribution; this agrees with the general observation that increasing heterogeneity among hosts increases R0 (Expressing the basic reproductive number as measures . Similareases R0 . Furthereases R0 .R0). This comparison more easily lends itself to questions about the sort of host population contact structure that is associated with greatest risk of disease emergence. In contrast, holding R0 fixed in comparisons requires that we simultaneously alter pathogen transmissibility when altering the contact distribution, thereby confounding these two factors.We have argued that the comparison of emergence probability across contact distributions should be made at given pathogen transmissibility, rather than at given pathogen fitness  Through comparison of contact distributions at given transmissibility, we found that heterogeneity can in fact increase the risk of emergence over a significant range.Although previous authors have often found that increased population heterogeneity increases the probability of extinction  by which the pathogen may possibly evolve . ProvideWe also considered more realistic situations where deleterious as well as beneficial evolutionary steps are available to the pathogen, either by reverse mutation or by including multiple pathways in strain space. Here, we constrained all types of mutations to occur with proportional probabilities, based on some overall mutation rate of the pathogen. We found that, when the pathogen was initially poorly adapted, increasing overall mutation rate was usually beneficial to an emerging pathogen (i.e. riskier to the host). On the other hand, as the pathogen's initial fitness increases beyond the critical threshold where survival without adaptation is possible, the risk of deleterious mutations comes to outweigh the benefit of potential mutation to a more fit strain, and a high mutation rate becomes a liability . Thus, o4.3.We have modelled the spread of an emerging pathogen following its introduction to a new host species. This model does not incorporate the dynamics of the introduction itself (or possibly multiple introductions), such as interspecific interactions  and the strain conversion process have been treated only phenomenologically in our present between-host model. We (and other authors) have implicitly assumed that any conversion of strains within an individual is instantaneous, with no possibility for co-infection. We have also ignored variation in the precise number of pathogen copies in the body. Realistically, once a mutation arises in a host there will be some dynamical process leading to fixation or loss over time, with coexistence of strains at least temporarily. Both R0 and conversion rates) supports a move to modelling at the within-host level.Furthermore, both the level of transmissibility (An important avenue for future work will thus be to develop an explicit model of within-host processes, and then to link the within- and between-host scales. In recent years, a number of authors have developed such \u2018nested models\u2019 (reviewed in"}
{"text": "Evolution involves both deterministic and random processes, both of which are known to contribute to directional evolutionary change. A number of studies have shown that when fitness is treated as a random variable, meaning that each individual has a distribution of possible fitness values, then both the mean and variance of individual fitness distributions contribute to directional evolution. Unfortunately the most general mathematical description of evolution that we have, the Price equation, is derived under the assumption that both fitness and offspring phenotype are fixed values that are known exactly. The Price equation is thus poorly equipped to study an important class of evolutionary processes.I present a general equation for directional evolutionary change that incorporates both deterministic and stochastic processes and applies to any evolving system. This is essentially a stochastic version of the Price equation, but it is derived independently and contains terms with no analog in Price's formulation. This equation shows that the effects of selection are actually amplified by random variation in fitness. It also generalizes the known tendency of populations to be pulled towards phenotypes with minimum variance in fitness, and shows that this is matched by a tendency to be pulled towards phenotypes with maximum positive asymmetry in fitness. This equation also contains a term, having no analog in the Price equation, that captures cases in which the fitness of parents has a direct effect on the phenotype of their offspring.Directional evolution is influenced by the entire distribution of individual fitness, not just the mean and variance. Though all moments of individuals' fitness distributions contribute to evolutionary change, the ways that they do so follow some general rules. These rules are invisible to the Price equation because it describes evolution retrospectively. An equally general prospective evolution equation compliments the Price equation and shows that the influence of stochastic processes on directional evolution is more diverse than has generally been recognized. Evolution involves both deterministic processes, such as selection, and random processes such as drift. When deterministic and stochastic processes are combined in the same model it is common to use the \"diffusion approximation\" \u2013 essentially assuming that populations are large (so that evolution can be approximated as a continuous process), that population size is relatively stable, and that selection is weak -4. The di.e. other moments) should do so as well. However, most of the models that have been studied have used methods  but also by the variances in possible fitness values associated with each strategy -10. If tcalculus ) which mThe most general (in the sense of making the fewest simplifying assumptions) mathematical description of evolution that we currently have, the Price equation , does noDespite this apparent limitation, the Price equation has been used extensively to study social evolution -16, the Below, I present a general equation for directional evolutionary change that treats fitness and offspring phenotype as random variables, rather than numbers, but imposes no restrictions on the distributions associated with these random variables. This is essentially a stochastic version of the Price equation, though it is derived independently and contains a term not found in Price's formulation. This theory accommodates all processes that influence directional evolution, both deterministic and stochastic. Using this result, I show that deterministic and stochastic processes interact in complex ways. One result is that stochastic variation in fitness amplifies the effects of selection in small or fluctuating populations. Furthermore, the role of fitness variation within an individual is more complex than has generally been recognized. The well known tendency for populations to be pulled towards phenotypes with minimum variance in fitness turns out to be one instance of a more general rule that, all else held equal, populations are pulled towards phenotypes with minimum symmetrical variation in fitness, as measured by all of the even moments of an individual's fitness distribution. This process can actually cause the variance in fitness to increase (so long as higher even moments decrease). There is also a tendency for populations to be pulled towards phenotypes with maximum positive asymmetry in fitness (as measured by the odd moments). Finally, this equation contains a term, capturing the direct effects of reproduction on offspring phenotype, that has no analog in the Price equation.w) measures the number of descendants that the individual has at some future time, potentially including the individual itself  is the nth central moment of x (this is just the delta method). We can now write For a random variable, w and w and The Taylor series expansion of Equation 10 does converge st mixed central moment of wk and 2 = 0, so If the actual number of descendants of different individuals are independent \u2013 meaning that the number of descendants of individual \u03d5, Substituting Equation 14 into cov. In such a case, we can write individual fitness as wi = si, where i, and si is the deviation from this expected fitness due to pure demographic stochasticity. In this case,Consider a case in which individuals with certain phenotypes are consistently influenced the same way by environmental variation across generations  = 2, var(w1) = 1.5, \u03bc3(w0) = 2, \u03bc3(w1) = 1.5, \u03bc4(w0) = 6, \u03bc4(w1) = 4.5.Equation 20 is derived using the fact that f0 and f1, the general rule is:Next, we need to specify the current frequencies of each phenotype and solve for the covariance terms. For the case of only two phenotypes, assigned values 0 and 1 and having frequencies n0 = n1 = N/2. For this case, we have \u03d5, \u03d5, var(w)) = -0.125, cov) = -0.125, cov) = -0.375, The dashed curves in the figure were derived by using the moments of the fitness distributions for each phenotype to approximate 1/H. We can thus iterate this process forward in time. If there are more than two phenotypes, then iteration is not possible unless we make further assumptions , that allow us to specify the entire distribution given only the mean.4(It is sometimes necessary to use moments higher than \u03bcN individuals, evenly divided between the two phenotypic values (so initially The monte-carlo simulations used asexual individuals with non overlapping generations. The value of \u0394SHR did the work and wrote the paper."}
{"text": "Tumorigenesis requires multiple genetic changes. Mutator mutations are mutations that increase genomic instability, and according to the mutator hypothesis, accelerate tumorigenesis by facilitating oncogenic mutations. Alternatively, repeated lineage selection and expansion without increased mutation frequency may explain observed cancer incidence. Mutator lineages also risk increased deleterious mutations, leading to extinction, thus providing another counterargument to the mutator hypothesis. Both selection and extinction involve changes in lineage fitness, which may be represented as \u201ctrajectories\u201d through a \u201cfitness landscape\u201d defined by genetics and environment.Here I systematically analyze the relative efficiency of tumorigenesis with and without mutator mutations by evaluating archetypal fitness trajectories using deterministic and stochastic mathematical models. I hypothesize that tumorigenic mechanisms occur clinically in proportion to their relative efficiency. This work quantifies the relative importance of mutator pathways as a function of experimentally measurable parameters, demonstrating that mutator pathways generally enhance efficiency of tumorigenesis. An optimal mutation rate for tumor evolution is derived, and shown to differ from that for species evolution.The models address the major counterarguments to the mutator hypothesis, confirming that mutator mechanisms are generally more efficient routes to tumorigenesis than non-mutator mechanisms. Mutator mutations are more likely to occur early, and to occur when more oncogenic mutations are required to create a tumor. Mutator mutations likely occur in a minority of premalignant lesions, but these mutator premalignant lesions are disproportionately likely to develop into malignant tumors. Tumor heterogeneity due to mutator mutations may contribute to therapeutic resistance, and the degree of heterogeneity of tumors may need to be considered when therapeutic strategies are devised. The model explains and predicts important biological observations in bacterial and mouse systems, as well as clinical observations. Tumorigenesis is a multistep process somatic mutator mutations occurring as a step in the evolution of somatic cells towards malignancy.The mutator hypothesis states that mutations leading to enhanced genomic instability (termed \u201cmutator mutations\u201d) drive cancer pathogenesis by accelerating the acquisition of oncogenic mutations. Originally formulated around DNA polymerases and repair enzymes On the contrary, it has been argued that mutator mutations (MM) are unnecessary for cancer development, and that the observed incidence rates of cancer may be explained by mutations occurring at the normal rate in conjunction with multiple rounds of lineage expansion and selection Efficiency is defined as the expected number of malignant lineages generated up to and including a reference timepoint by any particular tumorigenic mechanism. This shifts the issue from analyzing the waiting time to a single cancer cell, and fitting it to epidemiologic data, to the evaluation of the relative efficiencies of mutator and non-mutator pathways in cancer lineage production.A novel approach was recently suggested, based on the wider perspective that all potential mechanisms of tumorigenesis are in play, but those which produce malignant lineages most efficiently are most likely to contribute to clinical cancers In this framework, mutator mutations, lineage expansion, and selection are not mutually exclusive and could all simultaneously contribute to tumorigenesis. It is also noted that the conversion rate of normal cells to cancer cells (\u201ccancer lineage birth rate\u201d) likely far outstrips the number of clinically observed cancers, due to numerous malignant and premalignant lineages being eliminated by immune surveillance, failure to establish a blood supply, or competition from other premalignant lineages. Thus models which match the cancer lineage birth rate to clinical cancer incidence may have inherent limitations. It may be more relevant to evaluate the potential contribution of mutator mutations to the efficiency of tumorigenesis, as opposed to whether mutator mutations are necessary to explain a rate of cancer lineage birth rate equal to that of clinically observed cancer incidence. Furthermore, attempts to compare absolute theoretical cancer rates to absolute observed cancer rates are very sensitive to the underlying parameters and other assumptions, leading to variability in conclusions An analysis of the relative efficiency of tumorigenesis with and without a somatic mutator mutation, in the absence of lineage expansion (LE), demonstrated that mutator mutations enhance tumorigenic efficiency under many realistic scenarios, despite the need for an extra mutation step to acquire the mutator mutation itself Mutator lineages are also more likely to suffer deleterious mutations that reduce their fitness and potentially lead to extinction. This effect has been termed negative clonal selection (NCS) fitness landscape, or the multidimensional space representing cellular fitness, as a function of cellular genetic makeup within an environmental context. Pathways through this fitness landscape are termed fitness trajectories. Trajectories of special interest for tumorigenesis are those which begin with a normal cell and end with a transformed malignant cell.In order to account for the effects of selection and expansion of fitter lineages, as well as negative clonal selection, I systematically consider the This paper presents mathematical models which represent the general case of tumorigenesis across a variety of fitness trajectories, including multiple situations where the mutator lineage suffers reduced fitness (NCS), or achieves increased fitness leading to lineage expansion (LE). Four cases (\u201cfitness trajectories\u201d), which differ in the fitness of intermediate lineages in the tumorigenic process , are conincremental lineage expansion case, the lineages acquire a fixed increment in fitness with each successive oncogenic mutation, finally achieving their maximum fitness when they have acquired a full complement of oncogenic mutations. Given that most of the increased tumorigenic efficiency due to a mutator mutation can be captured by the case in which the mutator mutation is an initial step cooperative lineage expansion case with early mutator mutation, there is no increase in fitness until a subset of oncogenic mutations have been acquired, at which point the fitness increases rapidly and lineage expansion begins. Additional oncogenic mutations may then be required to achieve the fully malignant phenotype. The mutator mutation occurs early, e.g. at any point before the lineage expansion. In case 3, the cooperative lineage expansion case with late mutator mutation, the situation is analogous to case 2 except that the mutator mutation occurs after the onset of lineage expansion, during the period when additional oncogenic mutations are occurring towards reaching a fully malignant phenotype. Since cases 2 and 3 are alternate mutually exclusive subsets of the same fitness trajectory, their relative efficiencies (compared to non-mutator pathways) are additive. In case 4, the mutator and wild type lineages are subject to negative clonal selection may lead to reduction in fitness, depending on the genetic and environmental context. When a reduced fitness locus is mutated, the lineage is at risk for fitness reduction. Lineages with fitness reduction become extinct, thus potentially limiting the advantage conferred by a mutator mutation.In case 1, the While cases can be proposed that are mixtures of these four cases, it should be possible to infer their properties once these four archetypal fitness trajectories are analyzed. Thus, based on analysis of these pathways in combination  the minimum fold increase in mutation rate required from a mutator mutation before the mutator pathway has a relative tumorigenic efficiency greater than or equal to 1, termed \u03b150%.The results for the four cases below are presented in terms of two key model outputs: (1) relative tumorigenic efficiency of mutator vs. non-mutator pathways, Nrel is the ratio of malignant lineages produced by mutator and non-mutator pathways under the specified conditions. The fraction of clinical cancers arising by mutator pathways is given by Nrel/(1+Nrel), and mutator mechanisms predominate if Nrel>1. In non-lineage expansion models, in which progression to a malignant lineage is a rare event, Nrel can be expressed as a ratio of probabilities, PrelN50%, at which mutator pathways are expected to contribute to half of clinical cancers, and above which mutator pathways predominate. The fraction of total cancers caused by a mutator pathway with a given \u03b1 when compared to an alternative non-mutator pathway is given by \u03b1C/(\u03b1C+\u03b150%C), where C is the number of oncogenic mutations required for malignant transformation. Thus, a mutator mutation must confer a minimum level of genetic instability to be relevant. In evaluating the importance of mutator pathways in a particular model, we need to determine if \u03b150% is within a range commonly seen in known mutator mutations.\u03b1 is the multiplicative factor by which a mutator mutation increases the somatic mutation rate per cell generation, e.g. the magnitude of the genetic instability. In the lineage expansion (cases 1\u20133) and constant fitness 50% is at or below commonly observed values of \u03b1 (ca. 10\u2013500).Mutations in base selection and proofreading generally increase mutation rates 10\u2013100 fold optimal, which maximizes the importance of mutator pathways. This corresponds to an optimal mutation rate, kmut-optimal.For the negative clonal selection model (case 4), an additional key output is an optimal value of the fold increase in mutation rate, \u03b1R); 0\u2264Rp\u2264R, the component of R which is due to enhanced proliferation (the remainder would be due to decreased apoptosis); T, the time (in cell generations) to malignancy; NML, the number of genomic \u201cmutator loci\u201d, in nucleotides, mutation of which leads to genetic instability; and kmut, the mutation rate per nucleotide base per cell generation in wild type cells.The results depend on the properties of the tumor under consideration, which in turn define the inputs to the models. The key input parameters for all the models are: C, the number of oncogenic mutations required for transformation to the malignant phenotype; R\u22650, the natural logarithm of the relative fitness of a malignant cell compared to wild type , an additional input parameter is introduced: D, the number of oncogenic mutations required for an increase in fitness. In the negative clonal selection model (case 4), I introduce the input parameter NRFLN-D (Nreduced fitness loci net-dominant), which is an indicator of the vulnerability of the genome to mutations which may reduce cellular fitness In the Key input parameters and the ranges over which they have been varied in the calculations, as well as key model outputs, are summarized in ML is 100, a very conservative assumption ML\u200a=\u200a1000, \u03b150% would decrease by a factor of 101/C (relative to the same case with NML\u200a=\u200a100), further enhancing the potential role of mutator pathways.In the calculations, I assume NUsing the equations in R), and each successive oncogenic mutation leads to an incremental increase in fitness R/C , but for higher values of C, \u03b150% continues to be well within commonly observed ranges. For example, when three oncogenic mutations are required for cancer (C\u200a=\u200a3), the wild type mutation rate is low (kmut\u200a=\u200a10\u221211), and the relative fitness advantage eR of malignant cells relative to wild type is 2, a 770-fold increase in the mutation rate would be required for mutator pathways to be observed in 50% of the cancers  and to some extent at C\u200a=\u200a3 with a low wild type mutation rate /[(C+1)C] compared to the constant fitness case. However, \u03b150% would need to increase by a factor of only {RT (C\u22121)/[(C+1)C]}1/C to compensate for this. For example, with the relative fitness of malignant cells eR\u200a=\u200a2, the number of cell generations T\u200a=\u200a5000, and the number of required oncogenic mutations C\u200a=\u200a6, the relative tumorigenic efficiency Nrel 1\u22360 is reduced over 400-fold relative to the constant fitness case. But only a 2.7 fold increase in \u03b1 can restore the same relative importance of mutator pathways under these circumstances.When judged by relative efficiency Nrel 1\u22360 increases with increasing wild type mutation rate kmut and increasing fold-increase in mutation rate, \u03b1, similar to the constant fitness case The analytical model , but for higher values of C, \u03b150% continues to be well within commonly observed ranges. For example, when three oncogenic mutations are required for cancer (C\u200a=\u200a3), the relative fitness of malignant cells eR\u200a=\u200a2, and the wild type mutation rate is low (kmut\u200a=\u200a10\u221211), an 880-fold increase in the mutation rate would be required for mutator pathways to be observed in 50% of the cancers  and to some extent at C\u200a=\u200a3 with a low wild type mutation rate  shows that the relative contribution of mutator pathways is independent of number of cell generations T. Finally, the relative contribution of mutator pathways is shown by the analytic model (equations [13] and [16]) to be independent of the number of oncogenic mutations required for an increase in fitness, D, in contrast to the cooperative lineage expansion with ratio Nrel of malignant cell lineages produced by mutator and non-mutator pathways will be nearly equivalent to the probability ratio Prel previously derived for the constant fitness case rel (if the increased fitness includes more rapid proliferation). \u03b150% is calculated using equation [20], and Nrel by equation [21], in In this circumstance, D oncogenic mutations occur leading to a sudden cooperative increase in fitness. After this occurs, an additional C\u2013D oncogenic mutations must occur to complete the transformation to a malignant lineage. During this latter period, a somatic mutator mutation may occur . For bot50%. In the case of C\u2013D\u200a=\u200a3, for example, \u03b150% ranges from 12 to 252, depending on various parameter values . In the cooperative lineage expansion case with late mutator mutation, a greater fitness advantage increases the pool of cells which may acquire a late mutator mutation. For example, when the number of oncogenic mutations required for cancer C\u200a=\u200a6, the number of oncogenic mutations required for the cooperative fitness increase D\u200a=\u200a2, the number of cell generations to cancer T\u200a=\u200a5,000. and the wild type mutation rate kmut\u200a=\u200a10\u221211, \u03b150% is 29 when the relative fitness advantage eR of malignant cells relative to wild type is 1.2, 27 when the relative fitness advantage is 2, and 22 when the relative advantage is 7.4  and NRFLN-D (Nreduced fitness loci net-dominant), an indicator of the vulnerability of the genome to mutation rel  is calculated using equations [28\u201330] in The instantaneous risk of fitness reduction is the product of the mutation rate per nucleotide base per cell generation krel of mutator to non-mutator pathways does not continue to increase with greater fold increases \u03b1 in the mutation rate. Increased mutation rates speed the acquisition of oncogenic mutations, but at the same time increase the risk of fitness reduction and extinction. In this type of fitness landscape, the relative efficiency Nrel of mutator compared to non-mutator pathways increases with greater fold increases \u03b1 in mutation rate, until an optimum at which the growth of the malignant lineage begins to be limited by negative clonal selection. An approximate optimum for mutation rate kmut can be estimated for this circumstance from the theoretical treatment:In contrast to the other cases, the relative efficiency NNote that the treatment focuses on dominant reduced fitness mutations only. Recessive reduced fitness mutations, requiring mutation of both alleles, were found to be quantitatively insignificant mut optimal, varies from 2.1\u00d710\u221210 to 3.6\u00d710\u22126 per nucleotide base per cell generation. This optimum is generally higher than estimated mutation rates of wild type embryonic stem cells or somatic cells \u221211 to 10\u22129.Within the parameter ranges considered within this paper, the optimal mutation rate for tumor evolution, koptimal\u200a=\u200akmut optimal/kmut. In the presence of an anti-apoptotic mutation RFLN-D would be reduced to a very low value, further raising the optimal mutation rate and diminishing the potential effect of negative clonal selection. Thus, mutator pathways with \u03b1>\u03b1optimal would be more efficient if they occurred after an anti-apoptotic mutation.An approximately optimal value of the fold increase \u03b1 in mutation rate due to a mutator mutation is therefore given by \u03b1rel 1:0)>0] varies depending on the strength of negative clonal selection, as shown in mut\u200a=\u200a10\u221211, and a number of cell generations to cancer T\u200a=\u200a5,000. At maximal negative clonal selection, there must be at least 5 oncogenic mutations required for malignant transformation before mutator pathways are favored. Additional more detailed results are given in Supplementary The relative importance of mutator pathways increases with increasing number of required oncogenic mutations for malignant transformation, but in contrast to the other cases, the minimal number of oncogenic mutations at which mutator pathways are favored .In particular, malignant initiation events occur from lineages with C\u22121 oncogenic mutations, as they acquire their Cth oncogenic mutation: The total number of malignant initiation events by time T is simply the integral of this instantaneous initiation rate from 0 to T: In summary, the instantaneous initiation rate of lineages with one oncogenic mutation is given by a first order differential equation. Based on the instantaneous initiation rate of lineages with one oncogenic mutation, and their lineage expansion, one can derive an expression for the number of cells with one oncogenic mutation as a function of time. The instantaneous rate of initiation of lineages with two oncogenic mutations is proportional to the number of cells with one oncogenic mutation at any given time. In turn, based on the instantaneous initiation rate of lineages with two oncogenic mutations, and their lineage expansion, we derive an expression for the number of cells with two oncogenic mutations at any point in time. The instantaneous initiation rate of cells with 3 oncogenic mutations is then proportional to the number of cells with 2 oncogenic mutations, and so on.Expressions for the number of cells and lineage initiations were derived for several values of n (number of oncogenic mutations) and C , and based on the algebraic details, general expressions were hypothesized for all n and C, verified for the cases explicitly derived, and proven for all n and C by mathematical induction.0,C-mut(t), is given by the product of the initial number of cells N0, the number of mutator loci NML, and the mutation rate: For mutator pathways, the mutator mutation is assumed to occur first, based on previous work suggesting that mutator pathways are more efficient if the mutator mutation occurs early Once a lineage with a mutator mutation is formed, analysis of its progress parallels that of the non-mutator pathway, except for the increase in the mutation rate constant by the factor \u03b1.50%, the minimum fold increase in mutation rate at which mutator pathways account for 50% of observed cancers (derived by setting Nrel\u200a=\u200a1), is approximated by considering only the most rapidly growing exponential: The numbers of malignant cell initiations by both mutator and non-mutator pathways is the sum of exponentials representing lineages with 0 to C\u22121 oncogenic mutations. \u03b1The exact expression, considering all exponentials, is: A and B are proportional to the number of initiation events by time T for non-mutator and mutator pathways (with \u03b1\u200a=\u200a1), respectively. The approximate expression rel, the relative efficiency of mutator compared to non-mutator pathways, is given by the following expression when the mutator mutations increase the mutation rate by a factor of \u03b1: NIn this case, there is no change in fitness until D\u2264C oncogenic mutations have occurred. These first D steps, with or without a mutator mutation, can therefore be analyzed using the strategy previously outlined for the constant fitness case R per wild type cell generation (see equations [2\u20133] above). During this period, the fitness is also constant, although greater than it was prior to the D oncogenic mutations. The total number of cells with C\u22121 oncogenic mutations (by non-mutator or mutator pathways) at time T is given by the integral over t from 0 to T of the product of: the number of cells ND\u22121,C(t) or ND\u22121,C-mut(t) with D\u22121 oncogenic mutations  at time t; the instantaneous mutation rate per locus for conversion to cells with D oncogenic mutations ; the number of unmutated oncogenic loci at the time of conversion to cells with D oncogenic loci (NOL\u2212D+1), the exponential lineage expansion of cells with D oncogenic mutations from time t to time T (eR(T-t)); and the probability that any progeny of this expanded lineage would have acquired the final C\u2212D\u22121 oncogenic mutations in time T\u2212t  or PC-1|D,C-mut(T\u2212t)) for non-mutators and mutators, respectively, adjusted for the absence or presence of mutator mutations and the increased number of cell generations per unit time, if the fitness increase includes an increase in proliferation rate: Once D oncogenic mutations have occurred, the lineage has a natural log fitness advantage R, and expands by a factor of eOL-D remaining unmutated oncogenic loci, mut(T\u2212t) for non-mutators, \u03b1 kmut(T\u2212t) for mutators, adjusted by a factor of The probability of the expanded lineage acquiring C\u2212D\u22121 oncogenic mutations in time T-t is the product of the number of ways of selecting C\u2212D\u22121 oncogenic mutations from Nrel, the relative number of clinical cancers due to mutator compared to non-mutator pathways at time T or earlier, is given as before by the ratio of total number of malignant initiation events at or before time T for mutator divided by non-mutator pathways (see equation [4]). \u03b150% is again derived by setting Nrel\u200a=\u200a1.As in case 1, we use the number of cells with C\u22121 oncogenic mutations to calculate the instantaneous rate of malignant initiation events at any time, integrating that from 0 to T to obtain the number of malignant initiation events at or prior to T. N50%, in the limit of significant lineage expansion, and increasingly accurate as the fitness advantage R and the fold mutation rate increase \u03b1 get larger, is: rel is again given by An approximate expression for \u03b1In this case, there is no change in fitness and no mutator mutation until D\u2264C oncogenic mutations have occurred. These first D steps can therefore be analyzed using the strategy previously outlined for the non-mutator pathway in the constant fitness case R per wild type cell generation. During this time, an additional C\u2212D oncogenic mutations will also occur, with a mutator mutation occurring anywhere from the 1st to (C\u2212D)th step in the process. During this period, the fitness is also constant, although greater than it was prior to the D oncogenic mutations.Once D oncogenic mutations have occurred, the lineage has a natural log fitness advantage R, and expands by a factor of eC\u22121,C-mut(T), is given by the sum, over the possible steps k at which a mutator mutation can occur, of double integrals. These double integrals from t equals 0 to T are of the product of the number of cells with D\u22121 oncogenic mutations at time t, ND\u22121,C-mut(t); the instantaneous rate of conversion of these cells to cells with D oncogenic mutations, (NOL\u2212D+1) kmutdt; the lineage expansion from time t to T, eR(T\u2212t); and an internal integral representing the likelihood of subsequent acquisition of the remaining C\u2212D\u22121 oncogenic mutations and a mutator mutation in time T-t. This internal integral is over t' equals 0 to T-t, and the integrand is the product of the probability of having k\u22121 additional oncogenic mutations between time t and time t+t' before the mutator mutation occurs, Pk\u22121,t'; the instantaneous rate of occurrence of the mutator mutation (adjusted to account for the reduced cell generation time in the presence of a hyperproliferative mutation) at time t', eRp NML kmut dt'; and the probability that the remaining C\u2212D\u2212k oncogenic mutations will occur in the remaining T\u2212t\u2212t' cell generations, PC\u22121,k\u22121,t',t.The total number of cells with C\u22121 oncogenic mutations at time T by a mutator mechanism, NOL-D unmutated oncogenes, wild type cell generation by a factor In analogy with previous arguments, The number of cells with C\u22121 oncogenic mutations at time T by non-mutator pathways is the same as in case 2.rel, the relative number of clinical cancers due to mutator compared to non-mutator pathways at time T or earlier, is again given by the ratio of total number of malignant initiation events at or before time T for mutator divided by non-mutator pathways (see equation [4]). \u03b150% is again derived by setting Nrel\u200a=\u200a1.As in cases 1 and 2, we use the number of cells with C\u22121 oncogenic mutations to calculate the instantaneous rate of malignant initiation events at any time, integrating that from 0 to T to obtain the number of initiation events at or prior to T. N50%, in the limit of significant lineage expansion, and increasingly accurate as the fitness advantage R and the fold mutation rate increase \u03b1 get larger, is: rel is given by: An approximate expression for \u03b1rel. A late mutator (case 3) is more efficient (and therefore more likely) than an early mutator (case 2) if and only if: In the case of cooperative lineage expansion, we can determine whether an early or late mutator mutation is more efficient by comparing the respective values of NFor \u03b1\u226b1, We see that early mutator mutations (i.e. before the fitness increase) are much more likely in the cooperative case for larger values of D.RFLN-D. Lineages with reduced fitness become extinct. The loss of fitness is described by a first order differential equation, leading to exponential decay of surviving lineages , with exponent given by minus the product of the susceptibility constant \u03b2, the number 2 (given diploid cells), \u03b1 (for mutator lineages only) and the number of cell generations T. In turn, \u03b2 is the product of the mutation rate constant kmut and the net number of dominant RF lociIn this case, lineages are subject to negative clonal selection (NCS), or random dominant reduced fitness (RF) mutations that are deleterious with a certain probability proportional to NRFLN-DNAs this case does not involve lineage expansion, the model can be expressed in terms of probabilities rather than expectation values.cancer, 0, NCS, is the product of the probability of surviving negative clonal selection for T cell generations (equation [24a]) and the probability of having C oncogenic mutations at time T The probability of a malignant lineage initiation by a non-mutator pathway by time T, Pmut (bearing in mind equation [24c] for \u03b2), and setting this derivative equal to zero, leading to equation RFLN-D rather than the full genome length as the size of the target which can mutate to reduced fitness, and the factor of 2 to account for a diploid genome.The maximum probability of malignancy as a function of underlying mutation rate can be found by differentiating this expression with respect to the mutation rate kcancer, 1, NCS, is the integral from t equals 0 to T of the product of the instantaneous rate of occurrence of the mutator mutation at time t (NML kmut dt); the probability of surviving negative clonal selection until time t without a mutator mutation, P0, t, NCS; and the probability of acquiring C oncogenic mutations while surviving negative clonal selection in time T\u2013t after enhancement of mutation rate by an mutator mutation, PC-mut, 0, t, NCS: The mutator pathway probability of carcinogenesis is evaluated assuming the mutator mutation occurs first. Loss of lineages due to NCS is more rapid after a mutator mutation, but so is the acquisition of oncogenic mutations. The probability of malignant lineage initiation with a mutator mutation as step 1, P0, t, NCS, is given by [24a] and [24c] with T\u200a=\u200at. The probability of acquiring C oncogenic mutations while surviving negative clonal selection in time T\u2013t after enhancement of mutation rate by a mutator mutation, PC-mut, 0, t, NCS, is given by the product of: the probability of surviving negative clonal selection for T-t cell generations given a mutator mutation, e\u22122\u03b1\u03b2(T\u2212t); the number of ways to choose C oncogenes from a set of NOL oncogenes, mut(T\u2212t)]C:The probability of surviving negative clonal selection until time t, Prel or Prel) of a mutator pathway with an initial mutator mutation to that of a non-mutator pathway in the presence of negative clonal selection is the ratio of the malignant initiation probabilities for mutator vs. non-mutator pathways, and is given by:The relative efficiency or probability (NFor (\u03b1\u22121)\u03b2T\u226a1, an alternative expression for Z must be used to maintain adequate computational precision: To reproduce the derivations above, the following identities involving factorials are required. Proofs of these identities are available on request.Table S150%: incremental and cooperative lineage expansion cases (cases 1\u20133). \u03b150%, the minimum fold increase in mutation rate at which mutator pathways account for 50% of observed cancers; C, the number of oncogenic mutations required for commitment to cancer; D, the number of oncogenic mutations required for fitness increase in the cooperative lineage expansion model; eR, the relative fitness advantage per cell generation of malignant lineages compared to wild type; RP, the component of R due to enhanced proliferation rate; kmut, the wild type mutation rate per nucleotide per cell generation; T, the number of wild type cell generations at which the efficiency comparison is made (lineages with hyperproliferative mutations may have undergone more generations); LE, lineage expansion; MM, mutator mutation. For the cooperative lineage expansion case, \u03b150% is determined by the smaller of the two values (early and late mutator mutation). For the cooperative lineage expansion case with late mutator mutation, D and RP are required for the calculation, and we assume in these examples D\u200a=\u200a2 and RP\u22480.5 R. Calculated using equations [11], [12a\u2013h], ML, the number of \u201cmutator loci\u201d in nucleotides, mutation of which may lead to genetic instability, is 100. The fraction of cancers arising with an initial mutator mutation causing fold mutation increase \u03b1 in their pathogenesis is given by \u03b1C/(\u03b1C+\u03b150%C).\u03b1(0.11 MB DOC)Click here for additional data file.Table S250% for high fitness advantage R (R\u200a=\u200a2): incremental and cooperative lineage expansion cases (cases 1\u20133). \u03b150%, the minimum fold increase in mutation rate at which mutator pathways account for 50% of observed cancers; C, the number of oncogenic mutations required for commitment to cancer; D, the number of oncogenic mutations required for fitness increase in the cooperative lineage expansion model; eR, the relative fitness advantage per cell generation of malignant lineages compared to wild type; RP, the component of R due to enhanced proliferation rate; kmut, the wild type mutation rate per nucleotide per cell generation; T, the number of wild type cell generations at which the efficiency comparison is made (lineages with hyperproliferative mutations may have undergone more generations); LE, lineage expansion; MM, mutator mutation. For the cooperative lineage expansion case, \u03b150% is determined by the smaller of the two values (early and late mutator mutation). For the cooperative lineage expansion case with late mutator mutation, D and RP are required for the calculation, and we assume in these examples D\u200a=\u200a2 except for when C\u200a=\u200a2 (in which case we assume D\u200a=\u200a1) and RP\u200a=\u200a1.31 . Calculated using [11\u201312a\u2013h], ML, the number of \u201cmutator loci\u201d in nucleotides, mutation of which may lead to genetic instability, is 100. The fraction of cancers arising with an initial mutator mutation causing fold mutation increase \u03b1 in their pathogenesis is given by \u03b1C/(\u03b1C+\u03b150%C).\u03b1(0.10 MB DOC)Click here for additional data file.Table S3rel 1\u22360, NCS, Relative Efficiency of Mutator Pathways During Negative Clonal Selection (Case 4). Prel 1\u22360, NCS, the ratio of cancers arising with and without an initial mutator mutation in their pathogenesis in the presence of negative clonal selection (NCS); C, the number of oncogenic mutations required for commitment to cancer; NRFLN-D, the net number of dominant reduced fitness loci; \u03b1, the fold increase in mutation rate due to a mutator mutation; T, the number of wild type cell generations at which the efficiency comparison is made (lineages with hyperproliferative mutations may have undergone more generations). Calculated using equations ML, the number of \u201cmutator loci\u201d in nucleotides, mutation of which may lead to genetic instability, is 100, and kmut, the wild type mutation rate per base per cell generation, is 10\u221211. The fraction of cancers arising with an initial mutator mutation in their pathogenesis is given by Prel 1\u22360, NCS/.P(0.07 MB DOC)Click here for additional data file."}
{"text": "The yeast pheromone response pathway is a canonical three-step mitogen activated protein kinase (MAPK) cascade which requires a scaffold protein for proper signal transduction. Recent experimental studies into the role the scaffold plays in modulating the character of the transduced signal, show that the presence of the scaffold increases the biphasic nature of the signal response. This runs contrary to prior theoretical investigations into how scaffolds function. We describe a mathematical model of the yeast MAPK cascade specifically designed to capture the experimental conditions and results of these empirical studies. We demonstrate how the system can exhibit either graded or ultrasensitive (biphasic) response dynamics based on the binding kinetics of enzymes to the scaffold. At the basis of our theory is an analytical result that weak interactions make the response biphasic while tight interactions lead to a graded response. We then show via an analysis of the kinetic binding rate constants how the results of experimental manipulations, modeled as changes to certain of these binding constants, lead to predictions of pathway output consistent with experimental observations. We demonstrate how the results of these experimental manipulations are consistent within the framework of our theoretical treatment of this scaffold-dependent MAPK cascades, and how future efforts in this style of systems biology can be used to interpret the results of other signal transduction observations. The yeast pheromone response system is one of the first signal transduction systems to be identified and studied in detail In the yeast system, G protein activation leads to the activation of a MAPKKK, Ste11. Ste11 activates the MAPKK Ste7, which has two possible target MAPKs, Fus3 and Kss1 Scaffolds such as Ste5 have been a subject of extensive theoretical and empirical investigations, much of the work focusing on how the scaffold controls the output response of its pathway et alRecently, several studies have shown that the yeast Ste5 scaffold plays an important role in modulating the ultrasensitivity of the Fus3 response to pheromone. These reports have shown that the scaffold-dependent Fus3 response is ultrasensitive, whereas the scaffold-independent response of Kss1 is graded To summarize, past theoretical investigations into scaffold-free MAPK systems demonstrate a tendency towards a biphasic response, while systems involving a scaffold show in theory a strong and robust graded response, which is contrary to the recent experimental findings. To address this discrepancy between theory and experimental results, we devise a new model of the yeast pheromone response system. This model is tailored to several experimental results from the recent literature, and we demonstrate how the apparent conflict can be resolved by examining how the kinetic binding parameters influence the signal response output.This work is organized as follows. First, we revisit the classic MAPK cascade in the absence of scaffold and make a detailed investigation of its output signal characteristics. Then, we devise a simplified model of the yeast scaffold-MAPK system and study its dynamics and output response as functions of the kinetic binding parameters of MAPK enzymes to the scaffold. We show how these results are consistent with both prior theoretical studies and current experimental evidence. Finally, we offer several hypotheses, based on our results, which explain how experimental perturbations to this pathway reported in the literature lead to non-obvious changes to the biphasic nature of the transduced signal.We begin by seeking a better understanding of how the MAPK system transmits its signals in response to a stimulus. We thus start with a simple implementation of the classic MAPKKKThe system consists of three enzymes, each of which has one fully activated state and one or more inactive states . We assuThe parameters for this system may be found in We find that model (1) cannot be easily adapted to include the presence of the scaffold, as the number of intermediate steps and complexes in fully active scaffold formation process is quite sizable, even though many of those steps are never detected experimentally. A full system would include a combinatorial set of partially assembled scaffold complexes, and contain both fast-paced and slow reactions. Such complexity makes the description unnecessarily opaque. Instead, we will focus on a relatively simpler system in which we assume equilibrium levels of scaffold complexes have formed, and that the no-signal resting state for the system involves all three enzymes bound to the scaffold, although not in an orientation which is immediately permissive of processive phosphorylation A signalling event by pheromone through its receptor activates two separate processes, both of which are necessary for proper signal transduction see  [1]. FirIn a scaffold-free MAPK cascade described by system (1), the rate-limiting steps are the phosphorylation events. In the presence of a scaffold we hypothesize that the rate-limiting steps are the association events of proteins with the scaffold in an orientation which allows for processive phosphorylation, in accordance with other treatments of scaffold systems. Once the elements of the MAPK cascade are attached to the scaffold, the downstream activation can happen on a faster time-scale. The pertinent reactions are (i) signal-dependent scaffold complex binding to its partner in the plasma membrane, leading to a conformational change in the scaffold bringing the enzymes into alignment with one another; and, (ii) signal-dependent enzymatic activation of the first kinase on the scaffold. The forward and reverse rates for the first reaction are Inactivation of the scaffold is a two-step processes. We consider the kinase(s) upstream of the conformational shift are inactivated as a group, and the kinase(s) downstream of the shift are as well. A scaffold with inactive Ste11 but active Fus3 is still considered an active signaling complex. Dephosphorylation of Fus3 on a scaffold which is both properly aligned and has active Ste11 is disregarded, as we assume the Fus3 will immediately be rephosphorylated. However, once a Fus3-active scaffold detaches from its membrane target  it will remain an active signaler until Fus3 is dephosphorylated, independent of whether or not the upstream enzymes are active or not.Our model is graphically presented in Finally, we consider two control parameters which guide the flow of signal through the model. The first parameter, With the state variables given in Parameters values are listed in While our model takes as its conceptual basis the hypothesis of selective activation of the scaffold, We will use model (1) to study signal response sensitivity in the MAPK signaling pathway. The sensitivity is typically measured by fitting the output response of a signaling pathway as a function of the input signal strength  to a standard Hill function:We can analyze the behavior of model (1) by making several simplifying assumptions on the Michaelis-Menton (MM) activation terms. This simplification will allow us to calculate the steady state concentrations of the output product, The classic MM equation describes how the rate of formation of product Firstly, when Secondly, if we assume that The first assumption (weak interactions) can be easy to justify over the course of a reaction: if no new substrate is created, and if the binding constant has a much higher value than the initial substrate levels, then the binding constant will always be much greater than the substrate levels. On the other hand, the second assumption (tight interactions) is more drastic and can likely be violated during the reaction duration. Nonetheless, these two simplifications provide useful analytic bounds with which to describe the nature of the system output. We term the parameter regime which favors outputs of the nature of equation 5 to be the \u2018weak\u2019 regime, and that which favors equation 8 to be the \u2018tight\u2019 regime.To study signal-response sensitivity further, we simulated model (1) for many different randomly selected values of certain parameters. We pick coefficients As seen in the left panel of We next take a small sample of these parameter sets and determine the relationship between This result is very interesting in light of what is known about the yeast pheromone pathway. The MAPKK-MAPK interaction is reported to be quite tight We perform a similar numerical experiment, over a wide range of possible binding constant values, on the with-scaffold system (model 2). We randomly calculate these constants by drawing them from a uniform log distribution  spanning from As seen in et alThe numerical surveys of both the with-scaffold  and withet al, demonstrates a significantly different response profile than the yeast system An important distinction between the mammalian and yeast scaffold-MAPK systems is the requirement, in yeast, for membrane recruitment of the scaffold prior to full scaffold activation. Mammalian scaffolds, such as the MP1 protein, do not have such a requirement for proper activation With the results from the mammalian system in mind, we next explore what role the selective activation hypothesis plays in shaping the response curve. We modify the model by allowing scaffold realignment, the et al, which showed that loss of Fus3-Ste5 association converted the Fus3 response to a more Kss1-like response.This result is a strong theoretical indicator for the selective activation hypothesis. The one extra step involved in selective activation, the recruitment of the scaffold to the plasma membrane prior to formation of signal-competent scaffold, is critically involved in the formation of the ultrasensitive response signal. In light of this result, we can now begin to analyze and understand the both studies of Takahashi and Pryciak, in which signal is artificially induced at the Ste11 MAPKKK level while bypassing the receptor; and the studies of Hao The paper by Takahashi and Pryciak employs a Fus3-driven reporter gene to measure pathway activation We can use model (2) to model this experimental system and gain understanding of how membrane binding affects ultrasensitivity. By varying the complex-membrane association rate From these simulations, we observe that a scaffold with enhanced membrane binding ability signals with a graded response to input stimulus, whereas a diffusible scaffold signals with an ultrasensitive response. This mirrors the result from Takahashi and Pryciak, if we assume that the natural effect of tethering all the scaffold complexes to the plasma membrane is to increase scaffold-signal association rate, by forcing the two components into much closer long-term proximity.We have already observed that loss of the requirement for signal-induced membrane binding prior to full scaffold activation leads to a decrease in the ultrasensitive nature of the signal response. We now look to determine whether it is the scaffold alignment or initial enzymatic activation of Ste11 which is responsible for the majority of the ultrasensitive behavior. We perform a numerical experiment in which any scaffold, whether bound to the membrane or free in the cytoplasm, can undergo the enzymatic activation step. The relative level of cytosolic scaffold phosphorylation is controlled by the parameter Thus, in this system it is clear that the alignment of Ste11, Ste7 and Fus3 on the scaffold is the critical step needed to promote an ultrasensitive response.Next, we consider a second experiment by Takahashi and Pryciak et al, showed a strong tendency for a graded response We again note here that the results of this experiment contradict current theoretical understanding of how the MAPK pathway functions in the presence and absence of scaffolds. A pathway with a scaffold based on the mammalian MAPK system, as modeled by Levchenko In this system, there is no requirement for extracellular pheromone, so we assume the scaffold remains in its locked, non-permissive configuration (cis (from a scaffold's ste11 to its ste7 to its fus3), or in trans  by one scaffold acting as an enzyme transmitting the signal to another scaffold, which serves as the substrate.Our results suggest the following hypothesis. Scaffolds in the cytoplasm will assemble into their resting state, a noncompetent form; however, now they have dominantly active ste11 attached instead of wild-type, signal-dependent ste11. There are two possible modes of full activation: either the scaffolds transmit the signal in cis transmission is not responsible for the marked increase in Hill coefficient. Consider the scenario in which a misaligned scaffold can still transmit signal from Ste7 to Fus3 at some basal leakage rate. Then, based on the combinatorial nomenclature previously introduced, we can remove two of the three binary naming states  reducing the system to two dynamic variables, Using model 2 as a basis, we can show that trans. The difference in Hill coefficient is then a consequence of difference in binding constants of the scaffold-associated versus scaffold-free MAPK enzymes. We would predict that the steric constraints of the fully assembled\u2013but misaligned\u2013scaffold would exhibit very weak enzymatic associations between the components of the pathway, leading to very large binding constants and therefore a very high Hill coefficient . On the other hand, if pure localization is the key determinant in ultrasensitivity, then this joint system will revert to a graded signal response.To test the hypothesis that it is not just membrane localization, but selective activation at the membrane, which leads to ultrasensitivity, we propose this experiment. Consider the situation of co-expression of the tethered Ste5 scaffold with the dominantly active Ste11. If our hypothesis is correct, this system will still demonstrate a strong ultrasensitive character, as there is no external signal through the receptor to permit selective activation. We cannot predict the exact value of the Hill coefficient, as there will be new competing forces acting on the system  to generate both an ultrasensitive (Fus3) and graded (Kss1) response. However, in their model, the scaffold itself is not addressed, which we can now rectify. This system is still dependent upon addition of pheromone, and so we assume the enzyme-scaffold binding reactions occur as described in model (2) previously. Once pheromone is added, the final complex formed involves active Ste11 and active Ste7. The issue is how this system is now less ultrasensitive than the wild type, as it involves all the key steps identified earlier as components of the ultrasensitive nature of the signal. The results in ncreased . Taken tTo test the plausibility of this hypothesis, we modify our model to ablate the fus3 binding site. We assume instead that fus3 is present in the cytoplasm and interacts via standard Michaelis-Menton kinetics with activated, scaffold-associated ste7. We then simulate the model under two conditions: first, without modification with a slow In this work, we have formulated two complimentary models of the yeast pheromone response pathway, in the absence (model (1)) and the presence (model (2)) of the Ste5 scaffold. The first model is a revisit of the original MAPK model first discussed by Ferrel, while the second is a simplified system describing the influence of the Ste5 scaffold on the MAPK pathway. We show how in both cases a careful examination of the binding constants, which dictate how strongly the enzymes associate with one another, lead to results consistent with recent experimented observations. In particular, we describe how manipulation of the protein-protein binding constants can lead to a multitude of signal response profiles, trending from strongly graded to sharply ultrasensitive.Specifically we highlight the following:Selective scaffold activation modulates signal output. Based on the results of our models, the dual requirements of Ste5 binding to the plasma membrane and phosphorylative activation of Ste11 to jointly induce MAPK signaling is crucial for allowing the system to exhibit both graded and ultrasensitive response profiles. The extent to which the selective activation step is able to shift the signal response from the scaffold \u2018default\u2019 of graded response to a switch-like ultrasensitive profile is critically dependent upon the protein-protein interaction strengths of the components in the system. In particular, stronger association of the scaffold with its activator decreases the ultrasensitivity of the system, as does stronger association of the selectively activated component with the scaffold.Prediction of global system changes based on local changes. The experimental papers under consideration in the formulation of these models both involved biochemical manipulations of the pheromone response pathway, leading to changes in the signal response. We have shown, by coupling the results of our simulations with the results of these manipulations, how mathematical models can be used to predict wide-ranging effects caused by these small perturbations of the original system. We have presented several hypotheses concerning how a particular change in the system\u2013such as constitutive Ste11 activity or abrogation of Fus3-Ste5 interaction\u2013leads to global changes in the system so that the observed signal response might be formed.A common criticism of mathematical analysis of biological networks is that, for standard analytic techniques to be applied, the system must be simplified to such a great extent that is must lose various important, complex details, rendering the results of the analysis suspect at best. However, we have shown how even a fairly simple model, model (2), is capable of suggesting general hypotheses about the nature of systemwide interactions based on a single perturbation event. This lends credence to the idea of intelligent reductionism, a feature of systems biology. Rather than reduce a complex system into all its possible individual components and study each in isolation, a complex system can instead be broken into reasonably independent modules, and each module studied alone and with regulatory interactions with other modules. This approach has been successfully applied to the study of receptor tyrosine kinases"}
{"text": "The evolution of complex molecular traits such as disulphide bridges often requires multiple mutations. The intermediate steps in such evolutionary trajectories are likely to be selectively neutral or deleterious. Therefore, large populations and long times may be required to evolve such traits.We propose that errors in transcription and translation may allow selection for the intermediate mutations, if the final trait provides a large enough selective advantage. We test this hypothesis using a population based model of protein evolution.If an individual acquires one of two mutations needed for a novel trait, the second mutation can be introduced into the phenotype due to transcription and translation errors. If the novel trait is advantageous enough, the allele with only one mutation will spread through the population, even though the gene sequence does not yet code for the complettrait. Thus, errors allow protein sequences to \"look-ahead\" for a more direct path to a complex trait.This article was reviewed by Eugene Koonin, Subhajyoti De (nomimated by Madan Babu), and David Krakauer. E. coli cells appeared to acquire an excess of beneficial mutations. The idea that cells can somehow 'direct' evolution was thought provoking, and stimulated many investigations . Whiliews see ,7, the oiews see  have beephenotypic mutations) allow the selection of the intermediate mutations of a multiple-mutation requiring trait, and can thus speed up the evolution of complex traits.If mutations arise independently of their phenotypic consequences, then how can adaptations occur that require multiple amino acid mutations and for which the intermediate stages are either selectively neutral or disadvantageous? Large populations can climb multiple fitness peaks, even with disadvantageous intermediate alleles ,9. Altho-3 to 10-4 misreadings per codon k. Since the probability that allele 2 is generated k times follows a Poisson distribution with mean m, we find for the probability v that at least one of the mutations to allele 2 goes to fixationAssuming that  is u(s) . (In Appn(s\u03b2) and nloss(s\u03b2), setting m equal to either of these values. We assume that T is sufficiently large so that allele 1 has time to reach fixation within this interval (we assume T \u2273 2N). Then the probability u2 that allele 2 is generated and goes to fixation  isWe calculate this probability separately for u(s\u03b2), is multiplied by the probability v that at least one copy of allele 2 is generated and fixed. The second half corresponds to the case of loss of allele 1 from the population, where the probability of loss of allele 1, (1 - u(s\u03b2)), is multiplied by the probability of at least one mutation from allele 1 to allele 2 and subsequent fixation of allele 2. Taking into account allele 2 mutations during allele 1 loss is important especially for small s. Allele 1 is more likely to be lost than fixed for small s, but can occasionally drift for long times before being lost.The first half of the equation stems from the case when allele 1 eventually reaches fixation, where the probability that allele 1 becomes fixed, \u03b2 \u2192 0, i.e., in the absence of phenotypic mutations, we find with Eqs. (A2), (A27), and (A35)In the limit N \u226b 1, and neglect corrections of order 1 compared to N. Note that we cannot simplify (N + 1)/N to 1, because for small U, 1 - eNUu(s) -and (1 - eNU(T-N)u(s)-)/N are of the same order in N.) As we are interested in the effect of phenotypic mutations (\u03b2 > 0) compared to the case without phenotypic mutations (\u03b2 = 0), we define the increase in the probability of fixation from advantagous phenotypic mutations (the look-ahead effect) asu(s) -\u2192 1, in this limit, and thusWe can broaden the assumption of u(s\u03b2)/u(0) = Nu(s\u03b2).Apart from a correction for the case when allele 2 occurs while allele 1 is destined for extinction, Equation (5) is just the ratio of the probability of allele-1 fixation in the presence and absence of phenotypic mutations, s\u03b2, Eq. (5) simplifies to (Appendix A.6)To first order in N is on the order of 1/(s\u03b2).We can see from this equation that the look-ahead effect becomes important when Ns\u03b2 \u226b 1, only the first term contributes to the numerator in Eq. (5), and we obtain (Appendix A.7)For u2 and u2 by numerical simulation, for different values of s , (6) and (7). Figure s, the look-ahead effect can inflate the probability of fixation of allele 2 by several orders of magnitude. We also display the different analytic expressions for \u03be in Figure T \u2192 \u221e, works well for all values of s. The approximation (6), derived for small s\u03b2, captures correctly the magnitude of s at which the look-ahead effect starts to operate, i.e., s \u2273 1/(N\u03b2). Similarly, approximation (7), valid for Ns\u03b2 \u226b 1, approximates \u03be well for larger s.We confirmed our analytic results for the fixation probabilities \u03be for different population sizes. As expected from the condition s \u2273 1/(N\u03b2), the look-ahead effect will work with smaller selection coefficients s in larger populations. For large s, \u03be saturates at approximately N.Figure \u03b2  would be neutral. We refer to selection for the intermediate allele as the s\u03b2, Eq.(6), shows most clearly the relationship between the parameters. The look-ahead effect is proportional to N, s, and \u03b2, and sets in when N is on the order of 1/(s\u03b2). For large Ns\u03b2, the look-ahead effect saturates. The asymptotic value of \u03be is approximately N for NU \u226a 1.The approximation for small s, and saturates at a larger asymptotic value \u03be. Of course, even in the absence of the look-ahead effect, larger populations can more easily traverse multiple local fitness peaks  for alleles , giving the new number of alleles in a possibly larger population. Finally, the next population of N individuals is chosen by recursively sampling from the binomial distribution, representing random genetic drift. Allele 0 is first sampled with the mean = , and the  = N. Allele 1 is then sampled from the combined allele 1 and 2 individuals. The number of simulations where allele 2 becomes fixed is divided by the total number of simulations, giving an estimate of the fixation probability. The number of simulations for each parameter set was between 108 and 109.The population in each simulation is represented by three numbers, corresponding to the abundance of each of the three alleles. As described, the initial abundances are DJW, DV, COW, and EBB developed the original idea, DJW and COW performed the simulations and analysis, all authors contributed to the writing.Eugene V. Koonin, NCBI, NLM, NIH, Bethesda, MD 20894, United StatesThe idea of this paper is as brilliant as it is pretty obvious...in retrospect. A novel solution is offered to the old enigma of the evolution of complex features in proteins that require two or more mutations (emergence of a disulphide bond is a straightforward example). Whitehead et al. propose that selection for such traits could be facilitated by phenotypic mutations . Due to phenotypic mutations, rare variants of proteins will emerge that are \"pre-adapted\" to accommodate the second, beneficial mutation, yielding the complex, adaptive trait, even if transiently. Simply put, for the case of a disulfide bond, one cysteine appears as a result of a phenotypic mutation and the other one due to a genotypic mutation. The result will be that, for a while, the cell will have in its possession the protein molecule with a disulfide bond. Thus, \"pre-adaptation\" owing to phenotypic mutation would promote fixation of the second mutation which will be beneficial even without the first one \u2013 if the selective advantage of the complex trait is high enough . The actual fixation of the complex trait, then, requires only one (the first) mutation and is thus greatly facilitated. Mathematical modeling described in the paper shows that, if the selective advantage of the complex trait, i.e., the selection coefficient for the second mutation, is high enough, this look-ahead effect becomes realistic under the experimentally determined mistranslation rates. Obviously, the realization of the look-ahead effect will depend on a variety of factors including the overall translation fidelity, the local context of the codon involved, the stability of the protein etc. This allows a number of rather straightforward experimental tests of the model.From my perspective, this is a genuinely important work that introduces a new and potentially major mechanism of evolution and, in a sense, overturns the old adage of evolution having no foresight. It seems like, even if non-specifically and unwittingly, some foresight might be involved. At a more general conceptual level, this work is important in that it puts together, within a single conceptual framework, the evolutionary effects of genotypic and phenotypic mutations. There is much more to investigate here!I would like to mention a rather general biological implication. It seems obvious enough that, under conditions of stress , when translation fidelity drops, the look-ahead effect will be enhanced. Thus, this could be a general and crucial mechanism of adaptation during evolution.Eugene KooninAuthor response: We would like to thank Eugene Koonin for his enthusiastic and positive review.Subhajyoti De, MRC Laboratory of Molecular Biology Hills Road, Cambridge CB2 2QH, United KingdomI have read the revised manuscript, and have found that all points raised by the referees were fully addressed. The work is rigorous and very interesting, and I believe, will make a significant contribution in the field. I'll be happy to consider it for publication.Subhajyoti DeAuthor response: We would like to thank Subhajyoti De for feedback that improved the original manuscript, and the subsequent positive review.David Krakauer, Santa Fe Institute, United StatesIn this paper the authors demonstrate how phenotypic variation arising through errors in development (e.g. transcription and translation), can, when building on (amplifying) genetic variation, accelerate the fixation rate of neutral alleles. By assuming that neutral alleles are genetically closer to an optimum genotype than a mutation-free wild-type, this can also reduce the time required to reach the optimum. The result is illustrated through stochastic simulation and some limiting-case analytical approximations.This is an interesting paper that is technically rigorous, and correct in many of the conclusions that it reaches. The paper is now much improved as it now includes specific reference to the almost identical, Baldwin effect. As the authors correctly state, many of papers on the Baldwin effect emphasize learning, but a significant fraction explore the role of random ontogenetic variation on evolutionary dynamics, and a few, explicitly consider the adaptive value of errors in transcription and translation on the exploration of fitness landscapes. It is not yet clear how important the differences are between treating Baldwin effects in terms of individual ontogenetic programs versus population level dynamics. In both cases, the key insight is that random variation is capable of generating a more effective gradient for population dynamics.I think it worthwhile therefore to give a brief review of this mechanism and a little of its literature.A Synoptic Outline Of the Baldwin-Morgan-Osborn Effect1. The essential insight of Baldwin and several other 19th century biologists (listed above) was to understand that phenotypic plasticity can have a direct effect on genetic evolution. In some cases, this can give rise to the appearance of Lamarckian inheritance, as selection on plastic phenotypes derived from a single genotype, can lead to the fixation of polymorphic sequences generating these phenotypes without plasticity.2. The modern investigation of this effect is associated with the work of Hinton and Nowlan (1987) who showed that ontogenetic variability or plasticity, could lead to effective genetic optimization in neutral fitness landscapes.3. This has been followed by numerous papers exploring complex landscapes, diverse models of plasticity, including learning, homeostasis, diffusion, and combinatorial sampling. See Turney (1996) for a review with an emphasis on computational approaches.4. Ancel and Fontana (2000)  demonstrated for RNA secondary structure, the crucial requirement that phenotypic plasticity and genetic polymorphism should exhibit a particular correlational structure for the Baldwin effect to be effective.5. The most recent, and somewhat exhaustive analysis of the Baldwin effect has been conducted by Borenstein et al (2006) in fluctuating landscapes, exploring both directed and random phenotypic variation.6. Krakauer and Sasaki (2002) demonstrated a \"negative Baldwin effect\" whereby developmental errors could amplify mildly deleterious mutations in finite populations, thereby leading to their effective purging.Certainly the paper by Krakauer and Sasaki does not consider learning explicitly, but something much closer to the so called \"look ahead effect\" described by Whitehead et al, as it treats the ensemble of variant proteins generated by a single underlying sequence as a result of errors in transcription or translation. In both the Baldwin effect and the \"look ahead\" effect, genetically identical organisms generate phenotypically diverse populations. I think it an interesting subject for future work to establish the precise nature of any differences manifesting at the level of population dynamics, rather than at the incidental level, of mechanism.Author response: We appreciate this correction of a large hole in our background literature. We have cited relevant literature about the Baldwin effect, and discussed the main differences between the look-ahead effect and the Baldwin effect. While on the surface the look-ahead effect is very similar to the Baldwin effect, crucially the Baldwin effect is about individual learning, whereas the look-ahead effect is about errors that always produce different proteins from a single gene, at a given rate. Thus, in our model there is little difference between individuals with the same genotype, as no learning is involved, as opposed to the Baldwin effect, where, due to learning, two organisms with identical genotypes can have very different phenotypes. Therefore, we believe that it is important to distinguish clearly between the cases with and without learning, and to use different terminology to emphasize this distinction.I was somewhat confused by the remark that double mutations are neglected because they are very rare.Firstly, double mutations should be allowed within the binomial model presented by the authors. Secondly, the statement is empirically false for many haploid genomes. Bonhoeffer and Nowak (1997) showed that in large populations double mutants are likely to exist at fairly high abundance.Author response: We agree that for RNA-based viral genomes, which often have genomic mutation rates 1000 times greater than DNA-based organisms, double mutations occur frequently. Our model focused on DNA-based organisms, where double mutations are rare. If we wanted to apply our model to RNA viruses, we would have to include double mutations. However, the results from such a modification are obvious: If double mutations are frequent, the organism will happen upon the beneficial double mutation quickly and not require the look-ahead effect at all.The treatment of deleterious mutations remains a little confusing. Presumably developmental noise can both amplify existing deleterious effects  and contribute novel pathologies, orthogonal to those of the underlying transcript (e.g. gain of function mutations). This should be made an explicit, distributional property of the model rather than assuming a fixed background cost.Author response: The explanation of how we treat deleterious mutations was extremely brief in our original draft, and we have expanded and clarified the respective paragraph. We believe that a more explicit, complex treatment of deleterious effects would detract from the main message the model in this work was meant to convey. We have added to the discussion how phenotypic mutations can amplify deleterious genotypic mutations. A more complete treatment of deleterious phenotypic mutations will be a topic of future work.Here, we present the details of our analytic derivations.u(s) of a single allele with selection coefficient s is given byAccording to , the pros \u2273 1/N, this expression simplifies toFor Ns \u226b 1, this expression simplifies towhereas for s drifting to fixation or extinction, and ask how many mutations this allele generates until it is either fixed or lost. We will treat these two cases separately. Let nfix(s) be the expected number of mutations generated while the allele drifts to fixation, and let nloss(s) be the expected number of mutations generated while the allele drifts to extinction. We calculate these two quantities using diffusion theory, by integrating the sojourn times of the allele over all frequencies.We first consider a single allele with selective advantage s and starting at frequency p = 1/N, [This expansion follows directly from the definitions of Chi = nfix(s) + nT(s), where nT(s) is the total number of mutations generated once the first mutation has reached fixation. We haveWe calculate tfix(s) is the time to fixation of a mutation with selective advantage s. This time is given by the integral over all sojourn times,where withI2(a) = 2I(a), and thus we haveA partial fraction decomposition of the integrand reveals that Combining this result with Eqs. (A13) and (A30), we findn(s) = nfix(s) for T = tfix(s).Note that s \u2272 1/N, we findFor Ns \u226b 1, using Eqs. (A15) and (A19), we obtain the asymptotic expansionFor s\u03b2From Eq. (4), using Eqs. (A27), (A35), and (A2), we obtain to first order in NU(T - N)u(s) \u226a 1, we obtainIf further T \u2192 \u221e,and for Ns\u03b2 \u226b 1, only the first term contributes to Eq. (2), and we obtain from Eqs. (A36) and (A3)For Likewise, in this limit we can simplify Eq. (3) togivingT \u2192 \u221e, this expression simplifies toFurthermore, for NU \u226a 1, then \u03be \u2192 N in the limit s \u2192 \u221e.If"}
{"text": "Recent evidence suggests that mutation rates are fitness-dependent, broadening our view of the impacts of mutation on the genetic health of populations.  Many a research paper, textbook chapter, and grant proposal has begun with the phrase \u201cMutation is the ultimate source of genetic variation.\u201d Implicit in this phrase is the assumption that genetic variation is required for evolution. Without mutation, evolution would not be possible, and life itself could never have arisen in the first place. However, there is overwhelming evidence that the great majority of mutations with detectable effects are harmful . DeleterDeleterious mutations are known or thought to influence a wide variety of biological phenomena  for a comprehensive account), the most notable of which is sexual reproduction. To make a very long story short, there are several reasons that asexually reproducing taxa are expected to have a short-term evolutionary advantage over sexually reproducing taxa. Specifically, the fact that a gene is passed to all the offspring of an asexual individual and to only half the offspring of a sexually reproducing individual provides a 2-fold fitness advantage to a new mutation that confers asexual reproduction when it arises in a population of sexual organisms. This 2-fold difference in fitness is known as the \u201c2-fold cost of meiosis\u201d . Howeversine qua non of sex is genetic recombination, and deleterious mutations are more readily removed from a population in the presence of recombination than in its absence. A large body of theory predicts that, all else equal, the greater the mutation rate, the greater the probability that sexual reproduction will be favored. However, recent research by Aneil Agrawal [Understanding why and how sexual taxa have managed to consistently overcome the 2-fold cost has probably absorbed more intellectual energy than any other single problem in evolutionary biology. Many arguments have been advanced to explain the prevalence of sexual reproduction ,6, but t Agrawal ,11 callsEscherichia coli have higher mutation rates when starved . The bamstances .Importantly, the adaptive mutation scenario is only plausible in taxa that reproduce primarily asexually, because recombination breaks up the association between the alleles that influence mutation rate and alleles that influence fitness ,17. AnotPoor physiological condition may occur for two fundamental reasons, environment and/or genes. A simple example of the former is genetically identical plants raised under variable conditions of moisture: plants that are watered will be in better condition than those that are not. An example of the latter is harder to contrive, but it has been shown that under identical environmental conditions, there is genetic variation for fitness. Variation in fitness could be due either to variation in the number of deleterious alleles an individual carries in its genome or variation in the effects on fitness, or both. If individuals carrying more deleterious alleles tend to be in poor condition and if individuals in poor condition tend to have higher mutation rates, the existence of a positive-feedback process is suggested, which leads to an upwardly spiraling mutation rate and downwardly spiraling fitness.U), which is assumed to be constant, and is independent of the strength of selection [\u2212U and the genetic load is 1 \u2212 e\u2212U; this result holds for both sexual and asexual populations, in the absence of nonadditive effects [A useful way of quantifying population-genetic phenomena is in terms of the \u201cgenetic load,\u201d the reduction in fitness of a population of interest relative to a population composed solely of the most-fit genotype. Of particular interest is the genetic load at mutation-selection balance (MSB), the point at which the input of genetic variation from mutation is exactly balanced by the removal of deleterious mutant alleles by natural selection. A classic, perhaps surprising result is that the genetic load at equilibrium in an infinitely large population is determined solely by the genomic deleterious mutation rate , with individuals in the best-possible condition mutating at rate UMIN and those in the worst-possible condition (other than dead) at UMAX. The functional relationship between fitness and U is assumed monotonic but not necessarily linear, and the fitness effects of mutations are assumed multiplicative across loci.A functional relationship between fitness and mutation rate complicates the situation. The first theoretical treatment of fitness-dependent mutation rates was given by Agrawal  for the asex \u2248eUMIN\u2212, even though most genotypes in the population may have many more than zero mutations. Second, the equilibrium fitness of a sexual population depends critically on the nature of the functional relationship between mutation rate and fitness (sex will approach that expected at the maximum mutation rate (UMAX). Conversely, if the mutation rate is a concave upward (accelerating) function of genetic load, \u0174sex will be much closer to that expected at the minimum possible mutation rate (UMIN).Two important proximate results emerge from Agrawal's analysis. First, for an asexual population, mean fitness at equilibrium is a function of the mutation rate of the genotype with the fewest deleterious alleles . If the most-fit genotype has zero deleterious mutations, \u0174 fitness . If the This analysis points to a key difference in the equilibrium genetic load in sexual and asexual populations under constant and fitness-dependent mutation rates. If the mutation rate is constant, then sexual and asexual populations are expected to have the same genetic load at equilibrium. In contrast, if the mutation rate is fitness-dependent, the expected fitness in an asexual population at equilibrium will be that of the most-fit genotype, while in a sexual population, the equilibrium fitness will be less than that of the most-fit genotype, perhaps much less. Thus, surprisingly, the \u201ccost of sex\u201d may in fact be much greater than 2-fold.Chlamydomonas [Drosophila melanogaster that had been allowed to accumulate mutations for 160 generations increased 2.5-fold over the ancestral stock. That result is consistent with an increase in the mutation rate with genetic load (as claimed by the authors in the title of the article), but it is also consistent with an increase in the average deleterious effect of a new mutation with increasing genetic load, i.e., \u201csynergistic\u201d epistasis [Although condition-dependent, inducible increases in mutation rate are well-documented in microbes, including yeast  and the ydomonas , there iydomonas  showed tpistasis .PLoS Biology, Agrawal and Wang report a study designed to determine if the mutation rate is condition-dependent in D. melanogaster without the ambiguity associated with classical mutation accumulation studies [D. melanogaster, but DNA damage carried in sperm can be repaired post-fertilization in D. melanogaster embryos via maternal repair enzymes. Manipulating female condition could alter maternal repair processes. There are multiple maternal repair pathways, some of which are error prone and some of which are not. The error-prone pathways are believed to be less metabolically costly. Agrawal and Wang hypothesized that females in poor condition would preferentially use the less-costly error-prone mechanisms, whereas females in good condition would preferentially use the more-costly high-fidelity mechanisms. Condition was manipulated by diet; genetically identical females were fed high- or low-quality diets and fertilized with sperm that had been subject to mutagenesis. The relative frequency of recessive lethal alleles was 30% greater on paternally derived X chromosomes that had passed through low-condition females rather than high-condition females.In this issue of  studies . Their eDo these results prove that the mutation rate is condition-dependent? Not quite. It remains to be shown that the spontaneous mutation rate is condition-dependent. However, if the efficacy of DNA repair is in general condition-dependent, a lack of condition-dependence of the spontaneous mutation rate would require there be a compensatory reduction in the probability that DNA damage occurs. Nevertheless, this study provides the strongest evidence yet for condition-dependent mutation rate in a multicellular eukaryote.reduce the mutation load).Condition-  dependent mutation leads to some interesting possibilities for the long-term genetic health of populations, including our own species. For example, it has been argued that modern technology  has led to the relaxation of selection against mildly deleterious mutations in the developed world, leading to a build-up of genetic load ,25. If tA second interesting possibility is that condition-dependent mutation could, in effect, render temporary increases in mutagenesis due to environmental causes permanent. Many anthropogenic factors are known to be mutagenic, not only to humans but to many other organisms. If the mutation rate is condition-dependent, a short-term increase in the input of mutation due to (say) a mutagenic pollutant could lead to a long-term increase in the mutation rate, and thus in the genetic load. Similarly, genetic drift allows the fixation of slightly deleterious alleles in small populations. With condition-dependent mutation rate, a temporary bottleneck in population size that results in the increase in frequency of deleterious alleles could lead to an effectively permanent increase in the mutation rate."}
{"text": "Mutational fitness effects can be measured with relatively high accuracy in viruses due to their small genome size, which facilitates full-length sequencing and genetic manipulation. Previous work has shown that animal and plant RNA viruses are very sensitive to mutation. Here, we characterize mutational fitness effects in single-stranded (ss) DNA and ssRNA bacterial viruses. First, we performed a mutation-accumulation experiment in which we subjected three ssDNA  and three ssRNA phages  to plaque-to-plaque transfers and chemical mutagenesis. Genome sequencing and growth assays indicated that the average fitness effect of the accumulated mutations was similar in the two groups. Second, we used site-directed mutagenesis to obtain 45 clones of \u03a6X174 and 42 clones of Q\u03b2 carrying random single-nucleotide substitutions and assayed them for fitness. In \u03a6X174, 20% of such mutations were lethal, whereas viable ones reduced fitness by 13% on average. In Q\u03b2, these figures were 29% and 10%, respectively. It seems therefore that high mutational sensitivity is a general property of viruses with small genomes, including those infecting animals, plants, and bacteria. Mutational fitness effects are important for understanding processes of fitness decline, but also of neutral evolution and adaptation. As such, these findings can contribute to explain the evolution of ssDNA and ssRNA viruses. The fitness effects of mutations are the raw material for natural selection. It has been shown that point mutations typically have strongly deleterious effects in plant and animal RNA viruses, whereas cellular organisms are comparatively more robust. Here, we characterize the fitness effects of random mutations in DNA viruses and compare them with those found in RNA viruses, using six phage species of similar genome sizes. To achieve this goal, we introduced mutations by chemical and site-directed mutagenesis, identified the genetic changes by sequencing, and quantified their fitness effects using growth-rate assays. In all cases, mutations had a strong average impact on fitness. We conclude that mutational sensitivity is a general property of viruses with small genomes and discuss the evolutionary implications of these findings. Mutational fitness effects are important for understanding the genetic variability of populations, the relative roles of natural selection and drift, the origin of sex and recombination, or the ability to produce evolutionary innovations, among other processes Escherichia coli more than 90% of gene knock-outs are viable Some progress has been made in characterizing mutational fitness effects. For instance, in Viruses are a unique experimental system for characterizing mutational fitness effects because it is relatively easy to engineer sets of random single-nucleotide substitutions. This has been done previously for single-stranded (ss) RNA viruses, but not for ssDNA viruses. These two types of viruses have similar genome sizes and therefore might be expected to be equally sensitive to mutation. On the other hand, given the obvious ecological E. coli strain under identical environmental conditions Here, we compare mutational fitness effects in ssDNA and ssRNA viruses using six phages which can infect the same Preliminarily, we adapted each phage to our laboratory conditions by performing serial passages at high population sizes, until all lineages reached stable fitness values. For each phage, three independent mutation-accumulation lineages were initiated by picking single plaques at random from the adapted populations. Phage supernatants from plaques were subjected to chemical mutagenesis using nitrous acid and plated to isolate new plaques, a protocol that was continued until plaque sizes became drastically reduced. Under these conditions, selection is minimized and therefore, except for highly deleterious or lethal mutations, random genetic drift and mutational pressure are the only factors driving the fixation of mutations P\u200a=\u200a0.769) and there were no significant differences between the DNA and RNA groups (P\u200a=\u200a0.605). After sequencing the six ancestral and the 18 evolved genomes (GenBank accession numbers GQ153912-GQ153935) we found that DNA and RNA phages had accumulated 157\u00b13 and 146\u00b11 nucleotide substitutions in total, respectively (P\u200a=\u200a0.802). We calculated the per-mutation effect by dividing the fitness loss of each lineage by the number of mutations accumulated and again, this did not reveal significant differences between DNA and RNA phages (P\u200a=\u200a0.870). We therefore conclude that selection strength is similar in the two groups. F1 appeared to show the highest level of robustness, but differences between species were non-significant (P\u200a=\u200a0.088) and a Tukey's post-hoc test indicated that the six phages formed a single coherent group.The change in fitness was homogeneous among species (P\u200a=\u200a0.010). This might be due to their lower spontaneous mutation rates or to lower susceptibility to the mutagen. In both groups, the proportion of transitions was high, and this excess was significantly more marked in DNA phages . Since nitrous acid induces mainly transitions, this suggests that, in DNA phages, most substitutions were caused by the mutagen, whereas in RNA phages there was a greater contribution of spontaneous mutation. This could explain why more passages were required in the former group, although differences in susceptibility to the mutagen or in the proportion of transitions among spontaneous mutations cannot be discarded. Importantly, the higher transition/transversion ratio of DNA phages did not result in a significantly higher fraction of synonymous substitutions  and therefore is unlikely to have biased the above selection strength estimates.However, the number of passages required to reach similar fitness losses or mutation numbers was higher on average for DNA phages . All lethal mutations found in \u03a6X174 produced amino-acid changes, whereas in the case of Q\u03b2, one was synonymous (U2379A) and one intergenic (G1329A).We constructed 45 clones of \u03a6X174 and 42 clones of Q\u03b2 carrying single-nucleotide substitutions, choosing the target site and the substitution at random. First, we amplified the viral genomes by PCR using mutagenic primers, transfected ectively , i.e. a V(s)\u200a=\u200a0.162. For viable mutations only, V(vs)\u200a=\u200a0.047. In Q\u03b2, V(s)\u200a=\u200a0.181, V(vs)\u200a=\u200a0.018 . The above correction using controls implies that our inferences of the mean and variance should be free of experimental error or bias resulting from the presence of additional mutations or changes in the assay conditions, although it is still possible that the fitness values of some individual mutants were altered by the presence of additional mutations.We measured the growth rate of viruses carrying viable substitutions to obtain the distribution of mutational fitness effects. Growth rates were also determined for the above control samples and corrected means and variances were obtained by subtracting the mean and variance of the control group from the grand mean and variance of the mutants. In \u03a6X174, this yielded a mean selection strength of \u200a=\u200a0.018 . There ws-values, we performed non-linear regression to characterize the shape of the distribution. In \u03a6X174, an exponential model with an expected mean selection strength against deleterious mutations of R2\u200a=\u200a0.989) and was as accurate as a Gamma (R2\u200a=\u200a0.989) or a Beta (R2\u200a=\u200a0.985) model . Other statistical models were not considered here. In Q\u03b2, the data were better described by the Gamma distribution (R2\u200a=\u200a0.975) with an expected R2\u200a=\u200a0.973), whereas the exponential model was slightly worse . Finally, we must emphasize that mutants with positive values were not used for these inferences, which implies that they were assumed to be neutral or beneficial. Neglecting beneficial mutations, the fraction of deleterious to total mutations should thus be dp\u200a=\u200a (lp)/ds and the fraction of neutral to total mutations simply np\u200a=\u200a1\u2212lp\u2212dp , We analyzed \u03a6X174 using the same methodology and under the same environment as Q\u03b2 to make the comparison between ssDNA and ssRNA viruses. The two phages did not significantly differ in the fraction of lethal mutations or the average strength of selection, suggesting that ssDNA and ssRNA viruses are similarly sensitive to mutation. The strong parallel shown by viruses as different as \u03a6X174 and Q\u03b2 probably stems from the fact that both have small genomes with few and short non-coding regions, several multifunctional proteins, and little genetic redundancy The similar robustness shown by ssDNA and ssRNA viruses was also supported by the mutation-accumulation experiment, in which we examined three phage species of each group and found no significant differences in mean selection strength. Differences between species within the two groups were weak or absent, although F1 appeared to be the most robust phage. Related to this, it is worth noting that F1 has fewer overlapping genes. This might be related to the fact that inoviruses have filamentous capsids, which are structurally less constrained than icosahedral ones and can hence accommodate larger genomes It is possible to compare directly the mean selection strengths estimated by mutation-accumulation and site-directed mutagenesis, since the same viruses and the same environment were used. Because lethal mutations cannot be sampled during plaque-to-plaque passages we expected the former to be lower. However, even after excluding lethals, the The fitness effects of random mutations are relevant to evolution in many ways. For instance, they determine the fraction of nucleotide sites that evolves neutrally and thus the rate at which populations diverge through random genetic drift. Further, neutral and deleterious mutations can also determine the rate of adaptive evolution indirectly. The observation that mutations tend to be highly deleterious indicates that there are no efficient buffering mechanisms, which might result in large phenotypic variation and strong selection for beneficial mutations too The fitness effects of random mutations are not only relevant to neutral evolution and adaptation, but obviously, also to processes of fitness decline such as Muller's ratchet A conceptual dichotomy between DNA and RNA viruses has been established based on the observation that the latter evolve faster E. coli C strain IJ1862 Bacteriophages \u03a6X174, G4, F1, Q\u03b2, SP, and MS2 and the 5 particle forming units (pfu) were inoculated into 0.5 mL LB medium containing IJ1862 cells at their exponential growth phase. The appropriate cell density varied depending on the virus lytic activity and growth rate . Infected cultures were incubated in agitation (650 rpm) at 37\u00b0C in a Thermomixer 24-tube shaker (Eppendorf) and harvested during the late exponential growth phase of the virus (\u223c109 pfu/mL). Cells were removed by centrifugation and supernatants were aliquoted, stored at \u221270\u00b0C, and titrated using LB medium solidified with soft agar (0.7%). Initial and final titers were used to calculate growth rates and to adjust sampling times for the next passage accordingly. We performed 60\u201380 passages for each phage. In all cases, no significant changes in growth rate were observed during the last 30 passages, indicating that phages had reached a quasi-maximal fitness under these experimental conditions.Each phage was serially passaged at high population sizes in IJ1862 cells. For each transfer, \u223c10Three independent mutation-accumulation lines were seeded for each phage by picking random plaques from the high-fitness populations. Each plaque was resuspended in 50 \u00b5L LB and stored at \u221270\u00b0C. Lines were propagated plaque-to-plaque as previously described E. coli IJ1862 cells and from an overnight culture of E. coli K12 previously transformed with this cDNA, respectively. For PCR-based mutagenesis, full-length PCR amplicons were obtained from 500 pg of template using Phusion high-fidelity DNA polymerase  and a pair of adjacent, divergent, 5\u2032 phosphorylated primers, one of which carried the desired nucleotide substitution. PCR products were circularized using the Quick T4 ligase (New England Biolabs) and E. coli IJ1862 competent cells were transfected by the heat-shock method  in the presence of CaCl2 100 mM. The transfected cells were immediately plated onto LB plates using soft agar and individual plaques were picked after 5-9 hours of incubation at 37\u00b0C, resuspended in LB, and stored at \u221270\u00b0C. To verify that the desired mutation had been introduced and that no additional changes were present in the region flanking the target site, (RT)-PCR was performed directly from the resuspended plaque. Moloney murine leukemia virus reverse transcriptase (Fermentas) was used for cDNA synthesis and Phusion DNA polymerase for PCR. The products were column-purified and sequenced using sequence-specific primers.The High Pure Plasmid Isolation kit (Roche) was used to purify \u03a6X174 DNA and the pBRT7\u03b2 plasmid from partially lysed In cases where transfection yielded no plaques or phages recovered from plaques had not incorporated the mutation, the entire protocol was repeated and, after three consecutive failures, the mutation was classified as a candidate lethal. In all of these cases, the number of plaques obtained from transfection assays was abnormally low, suggesting that the mutation was lethal for the virus and that the few observed plaques came from the template wild-type DNA. To confirm lethality, we first sequenced the region of the PCR-based mutagenesis product flanking the target site to check that the mutation was present and that no additional changes had appeared in this region. Then, we designed new primers that did not carry the nucleotide substitution but were otherwise identical to those used for the mutagenesis reaction, and performed the PCR, circularization and transfection steps exactly as above. Lethality was assessed based on the comparison between the numbers of plaques recovered from mutagenesis versus control reactions .Similar control assays were carried out to estimate the contribution of non-desired mutations and other sources of error to the inferred distribution of mutational fitness effects. For each phage, we transfected three of the above control PCR-based mutagenesis products and picked 11\u201312 random plaques from each, yielding 36 and 33 plaques in total for \u03a6X174 and Q\u03b2, respectively. The relative fitness of each clone was determined as above and we obtained the mean and variance for each phage. These values were subtracted from the means and variances obtained for the real mutants. This allowed us to account for experimental error and, in particular, to control for any potential mutations present in the DNA templates, arising during PCR amplification, or during plaque growth.4 pfu were inoculated into 0.5 mL of LB medium containing IJ1862 cells at their exponential growth phase. Infected cultures were incubated in agitation (650 rpm) at 37\u00b0C in a Thermomixer 24-tube shaker and harvested when the wild-type reached an estimated titer of 108 pfu/mL. Cells were removed by centrifugation and the supernatants were aliquoted, stored at \u221270\u00b0C and titrated. The growth rate (r) was calculated as the increase in log-titer per hour. Relative fitness was obtained as W\u200a=\u200air/0r, where i and 0 refer to the mutant and wild-type, respectively , and the fitness effect (selection coefficient) was calculated as s\u200a=\u200aW\u22121. In all cases, mutant and wild-type samples were assayed in the same experimental block, and experimental blocks were done in triplicate.To measure growth rates, \u223c10Sequences were obtained using the Sanger method (Applied Biosystems). In general, we sequenced column-purified plaque-(RT)-PCRs directly. In cases where this yielded low-quality readings, we cloned the PCR fragments using the Zero Blunt TOPO PCR Cloning kit (Invitrogen) and sequenced the inserts with vector-based and internal primers, discarding mutations that were not present in at least 3/5 clones.v, the model can be written as ijk\u03bd\u200a=\u200a\u03bc+iG+ijS+ijkl, where \u03bc is the grand mean and l is the line (error term).For the mutation-accumulation experiment, we used a univariate linear model with two factors: Genetic material  and Phage species , whereas the line was the experimental replicate. The variables analyzed were the number of passages, the number of mutations fixed, and the expected fitness effect per mutation. For each variable ijkls\u200a=\u200a\u03bc+iS+ijA+ijkP+ijkl\u03b5, where \u03bc is the grand mean, \u03b5 is the error term.To test for differences in average fitness between the \u03a6X174 and Q\u03b2 mutant collections, we used a univariate linear model with three factors: Phage species , Assay type , and Plaque . Hence, each experimental determination can be expressed as V(ds) using the Levenberg-Marquardt algorithm implemented in SPSS v12.Non-linear regressions were performed to estimate Figure S1E. coli cells with these PCR producuts are shown below the brackets. (A) For \u03a6X174, all mutations except A402T were confirmed as lethal. (B) For Q\u03b2, all mutations except G780U, G1110A, and G2587U were confirmed.Confirmation of lethal mutations. Site-directed mutagenesis reactions and control reactions  were loaded in 0.8% agarose gels and stained with ethidium bromide. For each candidate mutation, the left and right lanes contain the mutagenesis and control reactions, respectively. Bands of the expected size (5.4 kb for \u03a6X174 and ca. 7.0 kb for Q\u03b2 insert plus the vector) were obtained in all cases . The number of plaques obtained after transfection of (0.43 MB PDF)Click here for additional data file.Table S1Genetic changes found in \u03a6X174, G4, F1, Q\u03b2, SP and MS2 mutation accumulation lines (three lines per phage). The final passage number, the relative fitness and its standard error (SEM), the nucleotide substitutions found (genomic position and substitution), the genes where they map, and the associated amino acid change (amino acid number within gene) are shown. Notice that some mutations fall at overlapping genes (the respective amino acid substitutions are indicated in these cases). Genomic positions in \u03a6X174 are numbered following the convention that nucleotide 1 corresponds to the Pst I cleavage site. Sequences covered nearly the whole genome (>98%) in all cases except for line SP.1 in which we failed to sequence genomic regions 1\u2013167, 653\u2013823, 2277\u20132633, and 2985\u20133186, and line MS2.3 in which we failed to sequence region 3048\u20133569.(0.03 MB PDF)Click here for additional data file.Table S2\u03a6X174 point mutants obtained by site-directed mutagenesis. For each mutant, the genomic position of the nucleotide substitution (relative to the Pst I cleavage site), the mutated gene, the amino acid change, and the relative fitness effect \u00b1 SEM are shown. Assays were done in triplicate and fitness values were corrected to account for the presence of additional mutations and other sources of experimental error see . Notice (0.02 MB PDF)Click here for additional data file.Table S3Q\u03b2 point mutants obtained by site-directed mutagenesis. For each mutant, the genomic position of the nucleotide substitution, the mutated gene, the amino acid change, and the relative fitness effect \u00b1 SEM are shown. Assays were done in triplicate and fitness values were corrected to account for the presence of additional mutations and other sources of experimental error see . Notice (0.02 MB PDF)Click here for additional data file."}
{"text": "In November 2007, the Society for Acupuncture Research (SAR) held an international symposium to mark the 10th anniversary of the 1997 NIH Consensus Development Conference on Acupuncture. The symposium presentations revealed the considerable maturation of the field of acupuncture research, yet two provocative paradoxes emerged. First, a number of well-designed clinical trials have reported that true acupuncture is superior to usual care, but does not significantly outperform sham acupuncture, findings apparently at odds with traditional theories regarding acupuncture point specificity. Second, although many studies using animal and human experimental models have reported physiological effects that vary as a function of needling parameters  the extent to which these parameters influence therapeutic outcomes in clinical trials is unclear. This White Paper, collaboratively written by the SAR Board of Directors, identifies gaps in knowledge underlying the paradoxes and proposes strategies for their resolution through translational research. We recommend that acupuncture treatments should be studied (1) \u201ctop down\u201d as multi-component \u201cwhole-system\u201d interventions and (2) \u201cbottom up\u201d as mechanistic studies that focus on understanding how individual treatment components interact and translate into clinical and physiological outcomes. Such a strategy, incorporating considerations of efficacy, effectiveness and qualitative measures, will strengthen the evidence base for such complex interventions as acupuncture. The 1997 National Institutes of Health (NIH) Consensus Development Conference on Acupuncture was a landmark event in the history of acupuncture research [In November 2007, the Society for Acupuncture Research (SAR) held an international symposium aimed at presenting and discussing the progress made in acupuncture research during the decade following the NIH Consensus Development Conference. The symposium presentations, as well as their published summaries , unequivThe emergent potential paradoxes that frame this paper are theA large number of well-designed clinical trials have reported that true acupuncture is superior to usual care, but does not significantly outperform sham acupuncture, findings apparently at odds with traditional theories regarding acupuncture point specificity and needling technique.While many studies in animal and human experimental models have reported physiological effects that vary as a function of needling parameters , the extent to which these parameters influence therapeutic outcomes in clinical trials is unclear.The goal of this White Paper is not to present a detailed review of the literature. Rather, this paper aims to identify gaps in current knowledge that underlie these paradoxes and to recommend research strategies that can help deconstruct and resolve them. We propose that resolution of the paradoxes lies in applying a bidirectional translational research approach, in which clinical trial design is informed by knowledge of mechanisms underlying acupuncture and by results from pragmatic trials of \u201creal world\u201d clinical practice. With this approach, findings from clinical trials will better inform the role of acupuncture in our evolving health care system.see ).The field of acupuncture research includes a number of terms that have created confusion and impeded progress, the word \u201cacupuncture\u201d notwithstanding. The term \u201cacupuncture\u201d has been used in the literature to convey not only the simple process of inserting a needle but also to describe a more complex intervention that includes a traditional exam, diagnosis, and the incorporation of additional modalities such as massage and moxibustion. \u201cSham acupuncture\u201d and \u201cacupuncture points\u201d are other examples of terms fraught with confusion in the literature  rather than needling or other components specific to acupuncture. If this is the case, the implication would be that the mechanisms underlying the therapeutic effects of acupuncture are essentially placebo-related. A central thrust of this White Paper is that there are other explanations that are just as plausible since a number of factors that could lead to Type II errors  remain poorly understood: (1) both verum and sham needling may have therapeutic effects, (2) acupuncture-specific nonneedle and/or needle components may be retained in sham treatments, and/or (3) the therapeutic effects of the provision of verum acupuncture treatments in the context of clinical trials may be suboptimal.A major factor potentially underlying Paradox 1 is the lack of consensus on which component(s) of acupuncture needling are therapeutically important. There has been little systematic investigation regarding the importance of needle placement and depth, type and intensity of stimulation, needle number, and so forth , as wellSham acupuncture treatments have consistently been shown to have greater therapeutic effects than conventional placebos \u201310. Placintention for a therapeutic outcome, traditionally described as yi. Some research has begun to experimentally investigate the therapeutic effects of healing intention as well as possible mechanisms associated with these effects [An additional factor that may contribute to the therapeutic effect of sham acupuncture treatments is the strong practitioner-patient interactions that commonly take place during interventions . One spe effects , 15. As In contrast to drug trials that evaluate well-defined and consistent pharmaceutical agents, acupuncture treatment protocols in clinical trials are rarely validated using objective, standardized approaches. Moreover, studies attempting to develop valid protocols based on consensus of multiple acupuncturists have revealed significant pluralism regarding what constitutes appropriate treatment . Unlike In summary, possible explanations for the generally small effect sizes observed in many sham-controlled trials are the following Verum and sham treatments are equivalent, because the therapeutic effects of both are due to nonspecific effects .Specific therapeutic effects of acupuncture treatments do exist but are not detected in sham-controlled trials becausesham acupuncture treatments are not inert , and/or verum acupuncture treatments are less than optimal.As with Paradox 1, a possible explanation for Paradox 2 is that the needle-related physiological effects observed in basic science experiments are unrelated to therapeutic effects. Another possible explanation is that at least some of the physiological effects of needling are therapeutic, but these effects are difficult to demonstrate in the context of a clinical trial.acute pain models  may have limited value for informing clinical trial design and practice-based treatment of chronic pain conditions given the emerging view of chronic pain as a qualitatively distinct neurological dysfunction from acute pain [As in most medical research, experiments in healthy humans and animal models have limitations in their applicability to clinical research. To date, the large majority of basic science research on acupuncture needling has been performed in healthy humans and animals; it is likely that physiological responses to acupuncture needling are different in pathological states . In partute pain . Furtherute pain , it is lor humans for functional concepts that are key to the traditional rationale underlying acupuncture treatments, such as acupuncture points, meridians, and \u201cqi\u201d.Mechanistic studies of acupuncture treatments and needling are also seriously hampered by three additional factors. First, similar to biomedical research, a significant proportion of acupuncture research evaluates conditions for which the pathophysiology is largely unknown, therapeutic outcomes are mostly subjective, and no good animal models or biomarkers exist . Second, there are no current animal models that investigate the mechanisms of acupuncture treatments within the explanatory framework of traditional East Asian medicine. Third, we do not understand what, if any, physiological equivalents exist in animals As discussed above, clinically relevant physiological effects  demonstrated in experimental models may be masked in clinical trials by the magnitude and variability of the multiple components of verum acupuncture treatment. In a sham-controlled clinical trial for chronic pain, an anti-inflammatory effect of verum needling may indeed be present, but the magnitude of this effect may be proportionally small compared with large and variable therapeutic effects produced by both verum and sham nonneedling components . A potentially important difference between basic science studies and clinical trials is that the former tend to use small numbers of needles, inserted at the same locations in every animal or human subject, while the latter, for the sake of reflecting clinical practice, often use 10 to 20 needles per session with at least some degree of practitioner-permitted choice of needling location. Thus, a small but specific therapeutic \u201csignal\u201d due to needling may be detectable in a physiological experiment but not readily detectable in clinical trials due to the additional \u201cnoise\u201d in the system. In summary, possible explanations for why specific effects of needling are clearly demonstrable in basic science studies, but not as evident in sham-controlled clinical trials are as follows: Some or all of the needle-related physiological effects observed in basic science experiments are unrelated to therapeutic effects.  Acupuncture needling does have therapeutic effects, but these effects are proportionately small compared with nonneedle and nonspecific effects in clinical trials.  Sham-controlled clinical trials are compromised methodologically because the influence of needling parameters for verum and sham needling are poorly understood, andobjective biomarkers relevant to the commonly treated clinical conditions are lacking.needling\u201d and \u201cacupuncture treatment\u201d  style of acupuncture, \u201cpattern differentiation\u201d is purported to be paramount for effective treatments and influences choice of needle location, needle stimulation techniques, and treatment frequency and duration. Consensus among acupuncturists regarding TCM diagnosis has ranged from good to poor in various interrater reliability studies \u201333 but wA particularly controversial question is the issue of \u201cpoint specificity\u201d with respect to clinical and/or physiological effects of acupuncture needling. It remains unclear whether effects observed at certain acupuncture points  are due to proximity of the needle to a major nerve, rather than its location at a specific acupuncture points. Few studies, for example, have included nonacupuncture point controls located along the same nerve as the tested acupuncture point. More studies including such controls therefore are urgently needed to advance our understanding of this important issue. A better understanding of potential differences in physiological responses elicited at acupuncture points and nonacupuncture points also is important for the future design of sham controls in clinical trials. Development of a modern scientific rationale for the traditional theories related to insertion of needles at specific locations is particularly challenging. Although it is possible that acupuncture points are truly unique anatomical structures, so far this has not been sufficiently and rigorously corroborated by research. In particular, a recent systematic review found little evidence in support of using skin conductance or impedance to identify acupuncture points . HoweverStudies are needed to identify the physiological effects associated with individual components of acupuncture, both needling and nonneedling. These include more in-depth and systematic investigations regarding the importance of such parameters as needle placement and depth , stimulaBiomarkers that relate to a given pathological condition and improve with treatment are needed to objectively evaluate treatment efficacy both in humans with these conditions and in animal models. If an understanding of disease pathophysiology is lacking, patients may be studied who have similar symptoms but who may have quite different disease mechanisms. Thus, the question \u201cis acupuncture effective for chronic low back pain?\u201d is likely not precise enough since low back pain is heterogeneous and may include subgroups with pathology that responds to treatment and subgroups that do not , 40. Morimmediate and delayed (hours to years) physiological responses to needling in humans be developed to facilitate assessments of effective needling parameters. This can be designed as a two-phase program in which the outcome measures are developed and correlated first in healthy volunteers, then repeated for assessment of clinical validity in patients. For example, quantification of changes in pulses (informed by Chinese and/or Japanese acupuncture theory) could not only qualify as an immediate response measure but would have an added advantage of beginning to develop biomarkers from within the acupuncture explanatory model. Further, suggestions as to why some parameters of needle stimulation appear to have physiological effects in basic science studies but are not associated with specific therapeutic effects in clinical trials (Paradox 2) may emerge from assessing immediate and/or delayed responses to needling  of healthy volunteers in comparison with medically diagnosed patients using the same specific needling parameters, for example, needle insertion depths, mode and amount of stimulation.An additional recommendation is that reliable biomarkers and clinical outcome measures of both and \u201cwhat to avoid\u201d. Most sham designs  are based mainly on the former consideration (aspects of treatment the patient sees) with insufficient attention to the latter. Thus, development of sham controls should be based on a systematic basic science evaluation of the components of acupuncture treatments  that the sham procedure should avoid. In order to gain clarity on the interpretation of these studies, we urge the scientific community to clearly state the hypothesis that is being tested and select a control procedure(s) that will most specifically test this hypothesis. For example, if a given physiological response in a basic science experiment is hypothesized to be due to electrical stimulation of acupuncture needles, an appropriate control would be needle insertion at the same location and depth but without electrical stimulation. If, on the other hand, the hypothesis is that there is an interaction between electrical stimulation, and needle location , then four experimental groups are needed . Thus, the frequent practice of comparing needle stimulation at acupuncture points to no stimulation at nonacupuncture points should be interpreted with caution as it risks confounding the two variables . Similarly, the common practice of comparing acupuncture points to nonacupuncture points in a different body region  risks confounding point type  and body region unless additional controls are included. Rational design of a sham needling procedure requires knowing \u201cwhat to mimic\u201d We thus encourage future clinical trials including both usual care (to test the effectiveness of acupuncture) as well as appropriate control group(s) designed to test the efficacy of specific treatment components . Such trials would be further strengthened by the inclusion of objective measurements of pathology relevant to the condition being treated. A summary of combined recommendations based on the paradoxes identified in this paper is shown in The experience gained by acupuncture researchers over the last ten years is invaluable both for the field of acupuncture itself and as an example for the study of other complex treatments that may wrestle (now or later) with similar issues. It is therefore vital to recognize the lack of clear difference between verum and sham acupuncture treatments in clinical trials, as well as the documented physiological effects of acupuncture needling in basic science studies. It is now the important responsibility of acupuncture researchers to face these results squarely and move the field forward. It is conceivable, as stated above, that the therapeutic effects of acupuncture treatments are all or largely based on nonspecific, placebo-related responses. If this is true, the implications for the investigation and practice of acupuncture are profound as this would certainly raise interesting questions as to the value of specific acupuncture point location and needling techniques. Before we consider such a perspective, however, it is important that the issues outlined in this White Paper are addressed.An opinion commonly voiced in the acupuncture community is that \u201cacupuncture cannot be studied using randomized controlled trials\u201d. We strongly disagree. Rather, we encourage clinicians and scientists to recognize that much patience and hard work will be needed before we fully understand how best to use the tools at our disposal, as well as develop new ones, to study this complex form of treatment. As has been shown in the field of ecology, complex phenomena are best tackled using a pluralistic strategy including both \u201ctop-down\u201d approaches that evaluate the behavior of a whole \u201cintact\u201d system, as well as \u201cbottom-up\u201d approaches that evaluate smaller-scale cause-and-effect links. Acupuncture treatments thus should be studied both from the \u201ctop down\u201d as multicomponent \u201cwhole-system\u201d interventions employing a pragmatic systems perspective, as well from the \u201cbottom up\u201d as mechanistic studies that focus on understanding individual treatment components and how the effects of these components interact and translate into clinical outcomes. Viewed through the lens of translational research , the schCreation of an acupuncture research agenda involves more than formulating lists of individualized goals for basic science and clinic research. In addition to providing a framework for addressing the paradoxes, the translational research approach will deepen our understanding of complex systems of care and refine our perspectives of human health and disease. Further, in calling for bidirectional translational research, SAR emphasizes the importance and value of building clinical relevance into the design of clinical as well as basic science research. The idea that research design should be framed in the context of ecological validity is an imperative, especially in light of the limited funds available for acupuncture research and its primary raison d'\u00eatre, which is improved patient care. A bidirectional translational research strategy, incorporating considerations of efficacy, effectiveness, and qualitative measures, will result in a broader and stronger basis of support for the study of complex treatments in general, including acupuncture and other CAM interventions."}
{"text": "Lethal mutagenesis is a promising new antiviral therapy that kills a virus by raising its mutation rate. One potential shortcoming of lethal mutagenesis is that viruses may resist the treatment by evolving genomes with increased robustness to mutations. Here, we investigate to what extent mutational robustness can inhibit extinction by lethal mutagenesis in viruses, using both simple toy models and more biophysically realistic models based on RNA secondary-structure folding. We show that although the evolution of greater robustness may be promoted by increasing the mutation rate of a viral population, such evolution is unlikely to greatly increase the mutation rate required for certain extinction. Using an analytic multi-type branching process model, we investigate whether the evolution of robustness can be relevant on the time scales on which extinction takes place. We find that the evolution of robustness matters only when initial viral population sizes are small and deleterious mutation rates are only slightly above the level at which extinction can occur. The stochastic calculations are in good agreement with simulations of self-replicating RNA sequences that have to fold into a specific secondary structure to reproduce. We conclude that the evolution of mutational robustness is in most cases unlikely to prevent the extinction of viruses by lethal mutagenesis. The high mutation rate of RNA viruses, such as HIV, allows them to rapidly evolve resistance to host defenses and antiviral drugs. A new approach to treating these viruses\u2014lethal mutagenesis\u2014turns the mutation rate of these viruses against them. It uses mutagens to increase the viruses' mutation rates so much that the accumulation of harmful mutations drives viral populations to extinction. Is there any way that a virus could adapt to a drug that increases its mutation rate? One way is that the virus could evolve so that mutations tend to be less harmful. In previous experimental work, there have been reports that virus populations can differ in robustness. Yet, the evolution of mutational robustness did not seem to inhibit extinction by lethal mutagenesis. In this work, we model viral populations under lethal mutagenesis in order to see when viruses might escape extinction by evolving robustness to mutations. We find that viruses can benefit from robustness only at relatively low mutation rates because the extent to which robustness increases fitness is rapidly drowned out by the extent to which higher mutation rates decrease fitness. The implication is that the evolution of mutational robustness is not a fundamental impediment to lethal mutagenesis therapy. Lethal mutagenesis is a proposed therapy for patients with viral infections. The general approach is to increase the deleterious viral mutation rate enough so that the viral population will go extinct Research on lethal mutagenesis and the question of how much mutational robustness can affect mutagenesis are of practical importance. In support of the promise of lethal mutagenesis as a treatment for many human and agricultural viruses, there are reports of the addition of a mutagen severely reducing or extinguishing populations of coxsackievirus B3 An important limitation to any pathogen treatment is the ability of the pathogen to develop resistance. Since lethal mutagenesis introduces deleterious mutations throughout the genome of viruses, it seems that there are only two types of effective resistance mechanisms. First, the virus could evolve a mechanism to reduce the number of mutations that the therapeutic mutagen introduces. Ref. Empirical studies of lethal mutagenesis appear to yield conflicting results. While Ref. The organization of this paper parallels our line of inquiry. First we ask, when will a population at equilibrium go extinct? We find with a deterministic model that an approximation for the critical mutation rate, i.e. the mutation rate beyond which the population goes extinct, is the log of reproductive capacity divided by the non-neutrality of the population at equilibrium. The implication is that small increase in the mutation rate can compensate for relatively large increases in neutrality. Next, we ask, how will elevating the mutation rate increase the rate at which populations move to areas of a neutral network with higher equilibrium neutrality? We find with a semi-deterministic model that the time it takes for a population undergoing mutagenesis to find the optimal area of the network grows exponentially with the size of the barrier to it. The implication is that we can usually disregard these shifts of the virus population, since the population will quickly shift to the optimal area if the barrier is small and the population will stay where it begins if the barrier is large. Finally, we ask, when will a population that is not at equilibrium go extinct? We show with a stochastic analytical model and simulations based on RNA-secondary structure networks both the critical mutation rate in these more complex models and the probability of stochastic extinction at mutation rates below the critical mutation rate. The implication is that the initial robustness of the population can be important in some cases, but not when the mutation rate exceeds the critical mutation rate.First, we consider the effects of mutational robustness in a deterministic model of lethal mutagenesis. In general, virus extinction is guaranteed if We can write In general, we can write the deleterious mutation rate as Throughout the remainder of this paper, we consider populations evolving on neutral networks. All sequences on the neutral network have the same reproductive capacity In the case where neutral sequences are distributed at equal density throughout the mutational network, Under the assumption that Of course, the critical mutation rate may be far above unity and the assumption that entropic barrier means that the probability to jump from one network to another with one mutational event is low.) In other words, there may be few possible paths in the network from one area to another. In this case, there is the risk that increasing mutation rates will increase the rate at which virus populations find rare paths to other areas of the neutral network in which it is possible to evolve greater neutrality. This process is comparable to that of demes drifting between equilibria (adaptive peaks) in the context of shifting-balance theory In general, a neutral network may be broken into separate areas of differing neutrality and separated by entropic barriers. . The bits are organized into The neutral-staircase landscape can be solved analytically, and the full derivation can be found in Ref. If we sum is large . This isThe prospect of the equilibrium neutrality increasing raises the question of how much increases in equilibrium neutrality may increase on rate  .When barriers are small, we can expect that the area of the neutral network with the greatest connection density can be found in a reasonable number of generations. In this case, the main question is whether the population can find areas with high connection density before it goes extinct under mutagenesis. In the following subsections, we will address this question using fully stochastic models.According to Equation (1), extinction is guaranteed if the mutation rate is so high that the equilibrium mean fitness of the population is less than 1. But lethal mutagenesis is not an equilibrium process. Therefore, we next explore how extinction occurs in a population out of equilibrium, using the mathematical framework of multi-type branching processes. Because this approach is a stochastic one, we calculate not only the mutation rate at which extinction is guaranteed but more generally the probability that extinction happens at any given mutation rate. Our main question here is how the extinction probability changes if the population resides initially in regions of the neutral network with particularly low or high connection density.The mathematical framework we use to calculate the extinction probability under lethal mutagenesis is that of multi-type branching processes. This framework has been used previously to calculate the fixation probability of a rapidly mutating virus on a neutral network Consider a population where all offspring are identical to their parents. A sequence produces a random number of offspring in the next generation. All these offspring sequences produce their own random number of offspring according to the same probability distribution. The number of progeny that a sequence has in two generations, then, is the sum of these random variables. The use of a probability generating function (p.g.f.) allows for convenient expression of these sums. We useWhen there is a finite number Extinction probabilities can easily be found numerically from Equation (12), but we next present two approximations to illuminate how extinction probabilities follow from offspring distributions.First, we need an explicit expression for the multivariate p.g.f.s in the fixed-point equation. If the number of offspring of type When extinction probabilities When extinction probabilities The previous subsection developed the general theory of stochastic extinction under lethal mutagenesis. We will now apply this theory to the special case of a neutral network of RNA sequences. To this end, we will first describe a model that links a sequence's location in a neutral network with the sequence's neutrality. This model yields the rates Consider how the probability-density function of the offspring distribution smaller the probability that offspring are neutral (see next paragraph). Instead of We define Our approach is inspired by Ref. Equations (19) and (20) say that, since there are only so many neutral sequences, if a sequence is in an area of the neutral network with a high connection density, then the connection density of neutral sequences must generally decline as we move away from it, and vice versa. This reasoning implies that re that  .We can use this framework to determine the Putting everything together, the probability that any one offspring of a parent of type The matrix tion of  . Since  by  and  . The relFirst, we present results based on the assumption that populations initially consist of a single sequence. This case is relevant to a scenario in which a patient is inoculated with a small dose of virus while on lethal mutagenesis therapy or a virus is establishing itself in a new tissue of a patient's body. With this assumption, we found that the probability of extinction declined with the initial sequence's neutrality, but also that the gradient in extinction probabilities rapidly leveled as the mutation rate increased . In agreNext we used the analytic calculations to study the effect of the size of the neutral network. When going from a smaller neutral network to a larger neutral network, the extinction threshold r values . Extinctpulation .Since lethal mutagenesis is intended to eliminate virus populations that have grown to high levels, we also considered the effect of the initial population size. We considered an initial population that was uniformly composed of sequences with a given initial robustness  changed . The gra was one .We verified our branching-process model by carrying out simulations with individual RNA sequences  prevailed in competition against a strain that was more fit in the absence of a mutagen when 5-FU doses were 20, 40, 60, and 80 While our models do show that the initial neutrality of a population can affect its probability of extinction, this relationship may be overshadowed in practice. For example, the models neglect the effect of defective interfering particles, which may contribute to extinction by lethal mutagenesis This work has provided quantitative support for the statement that the evolution of mutational robustness will have only a minor effect on lethal mutagenesis. In an extreme case, half of all non-beneficial mutations could evolve to become neutral. In this case, doubling the mutation rate will be sufficient to cause extinction . For lesWe evaluated the convergence times given by Equation (8), numerically derived We obtained the fixed point Sequences that folded into a target shape were considered neutral, and all others were considered inviable. The neutrality of a sequence was the fraction of neighbors at a Hamming distance of one that also had the target phenotype. The RNAfold function in the Vienna package ((((....)))).............................(((..........(((((.....))))))))........Here, positions that form base pairs are indicated with matching parentheses, and unpaired positions are indicated with dots. For the first target, which was used to generate the results in The extinction probability of a sequence was determined by simulation of a branching process on the RNA secondary-structure neutral network. Simulations began with a single neutral sequence. These sequences were selected from the sample of sequences used to estimate the size of the neutral network so as to get the full range of initial neutralities. At each iteration, each sequence in the population had a Poisson distributed number of offspring. Each letter of the sequence changed to any of the other three possible letters with a probability equal to the genomic mutation rate divided by the sequence length. Mutation rates ranged from zero to fifteen. Each sequence was tested to see if it folded into the target, and sequences that did not were removed. Simulation was continued until the population size reached zero or 10,000. Simulations were replicated 100 times for each of 500 initial sequences and the extinction probability was the number of simulations in which extinction occurred divided by the total number of simulations. A local polynomial fitting function  and (20) predict that as the parameter \u03b1 of a sequence increases, the epistasis parameter \u03b2 decreases. The panels are labeled with the sequence lengths of 40, 5000, and 10000. For each sequence length, the lines, from highest to lowest, are numerical solutions where log(0.02 MB EPS)Click here for additional data file.Figure S2p. Parameters: sequence length l\u200a=\u200a40, neutral-network size 29, reproductive capacity R\u200a=\u200a50, initial population size\u200a=\u200a1 sequence.Extinction probability as a function of initial neutrality and deleterious genomic mutation rate. Panel A displays results from simulations where sequence neutrality was determined by RNA folding. Panel B displays results from a branching process model derived from the correlation between sequence neutrality and epistasis. Only in a band of intermediate mutation rates does the extinction probability depend on initial neutrality 1-(0.26 MB EPS)Click here for additional data file.Dataset S1Raw data and computer code necessary to reproduce all results reported in this paper.(0.46 MB ZIP)Click here for additional data file.Text S1Supplementary text.(0.06 MB PDF)Click here for additional data file."}
{"text": "Despite the emerging experimental techniques for perturbing multiple genes and measuring their quantitative phenotypic effects, genetic interactions have remained extremely difficult to predict on a large scale. Using a recent high-resolution screen of genetic interactions in yeast as a case study, we investigated whether the extraction of pertinent information encoded in the quantitative phenotypic measurements could be improved by computational means. By taking advantage of the observation that most gene pairs in the genetic interaction screens have no significant interactions with each other, we developed a sequential approximation procedure which ranks the mutation pairs in order of evidence for a genetic interaction. The sequential approximations can efficiently remove background variation in the double-mutation screens and give increasingly accurate estimates of the single-mutant fitness measurements. Interestingly, these estimates not only provide predictions for genetic interactions which are consistent with those obtained using the measured fitness, but they can even significantly improve the accuracy with which one can distinguish functionally-related gene pairs from the non-interacting pairs. The computational approach, in general, enables an efficient exploration and classification of genetic interactions in other studies and systems as well. The systematic mapping of genetic interactions in biological systems has the potential to provide a better understanding of how genes function as networks to carry out and regulate cellular processes. In particular, recent advances in the experimental technologies which allow for the large-scale screening of the effects of combinatorial gene deletions are providing an exciting glimpse into the organization of complex genetic networks in terms of revealing novel interacting cellular components and compensatory pathways involved in many cell functions. Comprehensive maps of genetic interactions in model organisms, such as yeast, may also provide a valuable template for understanding the basic principles underlying the relationships between genotype and phenotype in other populations Saccharomyces cerevisiae, have already identified a number of synthetic lethal interactions, in which a combination of two individually non-lethal mutations results in lethality vs. non-interacting pairs), quantitative phenotypes, such as the relative growth rate of yeast colonies, have recently been explored systematically using high-throughput screening approaches like epistatic miniarray profiling (E-MAP) and genetic interaction mapping (GIM) Several large-scale studies, especially in yeast i and j can be defined by the deviation (ij\u03b5) of an observed double-mutant phenotype (ijP) from the expected neutral phenotype of an organism's fitness (ijE) under the hypothesis that it carries two non-interacting mutations (the null hypothesis). If the fitness ijP is evaluated in terms of the growth rate of double-mutant ijw, relative to the wild-type growth rate, and ijE is a function g of the relative single-mutant fitness values iw and jw, this definition can be formulated as:ij\u03b5| provides evidence for genetic interaction, while deviations close to zero indicate non-interacting gene pairs. Significant genetic interactions can further be classified into so-called synergistic interactions (ij\u03b5<0) and alleviating interactions (ij\u03b5>0). Synergistic interactions occur when a double-mutant has a more extreme effect on the fitness than would be expected from independent single mutants alone, and can therefore identify e.g. complementary pathways, with synthetic lethality being the extreme case (ijw\u200a=\u200a0). Alleviating interactions, in which the double-mutant phenotype is less severe than expected, can occur, for example, when the first mutation already impairs the function of a whole pathway and thereby masks the effect of the second mutation in the same pathway.Regardless of the experimental technology used, the screening strategies aim to quantify the extent to which a mutation in one gene modulates the phenotype (or fitness) associated with altering the second gene, either by explicitly measuring or analytically comparing the observed fitness of double-mutants to those of single-mutants. More formally, a genetic interaction between mutants g\u200a=\u200aiwjw provides a convenient null model in the sense that it yields a distribution with location close to zero and low dispersion over all of the measured deviations. The comparison was based on the principle that, as the vast majority of gene pairs should be non-interacting, the rare gene pairs sharing a specific function should be distinguishable from this background distribution as outlying cases with extreme absolute deviations. Accordingly, it was shown that the observed deviations based on the multiplicative null model were indeed most accurate at identifying functional relationships between the genes Recently, Mani et al. w is a 26-dimensional column vector and W is a 26\u00d726-dimensional symmetric matrix. Under the multiplicative null model, Eq. 1 takes the form:w with itself, and E is the n\u00d7n-matrix comprising the ij\u03b5 values as its elements for each gene pair i, j\u200a=\u200a1,2,\u2026,n. In the ideal case, when there are no measurement inaccuracies, the approximation problem of Eq. 2 could easily be solved using the well-established machinery of linear algebra. More precisely, using the spectral decomposition theorem, one can represent any symmetric real matrix as \u03bb is the largest eigenvalue of W and e is the corresponding eigenvector E equals zero. However, as the genetic interaction screens are bound to present with experimental variation, missing data, and hopefully also with significant genetic interactions, the estimation problem must in practice be solved by numerical means.As an initial study objective, we sought to assess the accuracy to which the single-mutant fitness vector W to provide a series of estimates for w as solutions of the weighted least squares problem In the present work, we developed a sequential matrix approximation procedure, which uses increasingly larger subsets of mutation pairs in ij\u03b5 and the ranked deviations p<10\u221211), whereas the functional enrichment was exceptionally low among the 50 pairs with the lowest p\u200a=\u200a0.998). These results show that the sequential matrix approximation procedure gives as its by-product a ranking of the mutation pairs that is in good agreement with their likelihood of sharing a specific function.Beyond the dynamic variability in the estimates of the single-mutant fitness values, the behavior of the approximation procedure with different subsets of mutation pairs revealed another interesting observation: the order in which the mutation pairs were added into the approximation process reflects on average the relative order of their actual measured deviations, even if the measurements of single-mutant fitness were not employed . In factp\u200a=\u200a0.003). The prediction capability was improved systematically at each specificity-level, demonstrating that the procedure could distinguish with high accuracy the functionally related gene pairs over the whole spectrum of the exceptional deviations (p\u200a=\u200a0.002). The prediction accuracy of the double-mutant fitness values alone was similar to that of a random classifier (AUC\u200a=\u200a0.506), demonstrating that the normalization by the measured or estimated single-mutant fitness values can, in any case, improve the prediction of the functional links. The estimates achieved using different subsets of mutation pairs can also lead to different degrees of prediction accuracy . Although the different measures of location and dispersion gave relatively similar results, the trimmed mean and interquartile range identified the distribution with least background bias and variability at k\u200a=\u200a136  with lth step of the procedure. Although the general formulation in Eq. 3 allows for an element of Cl) is used at step l. To select increasingly large subsets of non-missing and non-diagonal mutation pairs from the double-mutant fitness matrix W, we adapted the floating search method of Pudil et al. The subset of mutation pairs used at a particular step of the approximation process was encoded in the binary weight matrix l\u21900 and initialize the weight matrix C(0) (see the next subsection)Set \u0175l)Estimate While there are non-diagonal and non-missing entries with i, j) with select pair (\u0175l) with if there exists a pair (l\u2190l+1 and repeat step 3set tl)\u200a=\u200a2\u00d710\u22125 in the present work) or from a poor initialization of the weight matrix (see the next subsection for details), and it also made it possible to increase the size k of the subsets up to the maximal size K. K\u200a=\u200a323 mutation pairs in the dataset of St Onge et al. We modified the general subset search method by making the deletion of pairs an adaptive process over the evolution of the subset search. More specifically, the threshold Cl)\u200a=\u200a0 for the dataset under analysis. After calculating the initial residuals i) for each i, we set i, j) with the smallest residual C(0) non-zero; (ii) we made an additional setting of C(0); and finally (iii) we set pairs  with C(0) became non-zero. Every time a pair  was added in the initialization steps (i)\u2013(iii), we also set its transpose entry to one, that is, C(0) with 28 entries of ones.The approximation algorithm requires the weight matrix ijmeasured deviations \u03b5\u200a=\u200aijw\u2212iwjw were estimated in two different ways. The sequential approximation procedure gives as its by-product a surrogate for the deviations in the form of the residual errors of Eq. 4. More specifically, we defined the ranked deviationi, j) as its residual error l during which the pair was included into the approximation subset. In this way, even when there are multiple inclusions and deletions of a particular pair during the procedure, we obtained an unambiguous ranking of the pairs according to their The k, and by using the estimate of Eq. 3 in place of the measured single-mutant fitness vector w in the definition of the deviation in Eq. 1. For a mutation pair , this resulted in a sequence of estimated deviationsl at which the size of the approximation subset equals k. Note that since the residual errors in Eq. 4 are updated each time that a new pair is added, the estimated deviations can vary considerably as a function of k. These estimates are not generally congruent with the ranked deviations. The distributions of the estimated deviations with different subset sizes k are illustrated in The second set of estimates was obtained by stopping the sequential approximation procedure at a given subset size, S. cerevisiae. The detailed time course fitness measurements were performed in the presence and absence of the DNA-damaging agent methyl methaneusulfonate (MMS). The results of the present study were based on the growth measurements in the absence of MMS. The prediction of functionally related gene pairs was in fact more challenging in this case than in the data measured in the presence of MMS. The measured and estimated single-mutant fitness values and the double-deletion deviations in the dataset are shown in To evaluate the performance of the sequential matrix approximation procedure in practice, we applied it to a recent high-resolution genetic interaction study of St Onge et al. Functional links among the 26 genes were defined using the same approach as in many previous genetic interaction studies t is the number of top mutation pairs selected according to their residual errors, and M is the total number of the functionally-related links, then the probability of obtaining at least m functionally-related pairs when selecting pairs at random from the set of K mutation pairs can be calculated using the cumulative distribution function:Statistical enrichment of the specific functional links among a set of mutation pairs selected by the sequential approximation procedure was assessed using the standard hypergeometric test. Briefly, if M\u200a=\u200a35 functionally-linked pairs among the t\u200a=\u200a50 mutation pairs selected on the basis of either small (m\u200a=\u200a1) or large (m\u200a=\u200a22) residual errors is shown in M/K\u200a=\u200a35/323\u200a=\u200a0.108.The enrichment for the U-statistics to calculate an estimated covariance matrix and hence it can also take into account the correlated nature of the data. The ROC curves and the corresponding AUC values for the prediction of the 35 functional links in the dataset of St Onge et al. The predictive power of the measured and estimated deviations was assessed using the receiver operating characteristic (ROC) curves that characterize the relative trade-off between the true positive rate (sensitivity) and the false positive rate (1 - specificity). The overall predictive performance was summarized using the area under the ROC curve (AUC). The statistical significance of the difference in the AUC values between two genetic interaction measures was assessed using a custom written algorithm based on the method of DeLong et al. Figure S1Estimated vs. measured single-mutant fitness values. The comparison is shown both as histogram and scatter-plot. The two fitness values were highly correlated . The estimated values were calculated at the cut-off point k\u200a=\u200a317, in which the approximation procedure used all but the diagonal and missing entries of the double-mutant fitness matrix and also omitted those six pairs with the most extreme residual errors . This point can approximately be identified from the sharp increase in the trace of approximation error ((0.01 MB PDF)Click here for additional data file.Figure S2Scatter-plot between the measured and ranked deviations. The ranked deviations were highly correlated with the true measured deviations over all of the mutation pairs . The inset shows the five synthetic lethal mutation pairs. The dotted diagonal line corresponds to the one-to-one correspondence between the two deviations.(0.01 MB PDF)Click here for additional data file.Figure S3Distributions of the measured and estimated deviations. The non-scaled version of the (0.02 MB PDF)Click here for additional data file."}
{"text": "Poecilia formosa) is a small unisexual fish that has been suspected of being threatened by extinction from the stochastic accumulation of slightly deleterious mutations that is caused by Muller's ratchet in non-recombining populations. However, no detailed quantification of the extent of this threat is available.The Amazon molly  asexual species. Poecilia formosa, the Amazon molly.It is a general observation that asexual lineages do not last over very long periods of time, but the precise reasons for this are less clear . AncientQuantifications of the threat of extinction from Muller's ratchet are often not trivial theoretical work that requires either challenging mathematics  or complPoecilia formosa was the first unisexual vertebrate that was discovered . Howd models ,19. To sPoecilia mexicana or Poecilia latipinna as a mechanical trigger to start development . Wh. Whs )dominant mutations as follows. If and only if the ratchet can fix the first mutation at a site  then it will eventually also fix the second mutation . If a heterozygote cannot be fixed, there is no way the homozygote state can be fixed and the effective mutation rate used to compute the speed of the ratchet should be reduced accordingly to exclude such sites.We can treat recessive deleterious mutations, there are the two following extremes. If s is small (most mutations), then click times are hardly affected and almost no correction is necessary. If s is large enough to prevent fixation of heterozygotes, then again the effective mutation rate may be reduced, as fixation of the homozygous state is impossible. Only few intermediate s can become heterozygous but not homozygous, as they have to be close to the switch-like transition between 'can accumulate' and 'cannot accumulate' . This equation assumes that (i) recombination is free between sites, so that the ratchet only operates at homozygous sites, since selfing can restore the best class on a heterozygous site, (ii) Usdm, d is constant and independent from the number of mutations per individual, (iii) mutations at heterozygous sites are negligible, since selfing produces many more homozygotes, (iv) the standard model of Muller's ratchet without back mutations is valid [N0, can be computed by adding up all heterozygotes that are free from homozygous deleterious mutations and then scaling by the effective population sizewhere is valid ,104. TheUsing the well known result thatequations (4) \u2013 (6) yieldh = 0) this reduces to the haploid asexual case  that the degree of dominance of mutations with small selection coefficients does not significantly affect numerical values and hence the rate of the ratchet. Since the ratchet does not click for large s, one might as well neglect dominance altogether.For complete recessivity . To arrive at such an estimate we obtain corresponding diversity data from the control region of mtDNA, since it is linked to the rest of the genome by unisexual inheritance (see the Models section above for a justification of this approach). We use the DNA sequence diversity data from M\u00f6ller , where Ne is the effective population size of mitochondrial DNA in the presence of background selection, \u03bcbp is the mutation rate per site/generation and a = 1 + 1/2 + 1/3 + ... + 1/(n-1) for n sequences in the sample. Here we assume an mtDNA divergence rate of 3.6% \u00b1 0.46%/site/Myr as estimated in the species groups of the fish Centropomus [Ne between 153,000 and 198,000 that will have to be scaled downward, if actual mutation rates are higher, like in human pedigrees [For our simulations we need an estimate of the effective population size in the presence of background selection (nd 55 in ), who obterson's  equationtropomus  from divtropomus . This suedigrees . The nucedigrees ,116.Nt may be used to place upper bounds on Ne in most situations. Some mark and recapture experiments could only estimate that Nt > 10,000 in that local study area . One may speculate that about 10 million individuals of P. formosa may exist in total. If this is combined with the typical finding that vertebrates frequently have Ne-values that are about 10% of their census population sizes [Ne would be about a million. Given that a species as abundant as the fruitfly Drosophila melanogaster has effective population sizes of little more than a million [Ne < 106. On the other extreme it is highly unlikely that the Amazon molly would have an effective population size smaller than that of primates, suggesting Ne > 104.Comparative analyses suggest that the resulting values are reasonable. The total population size on sizes , then Ne million , suggestNe = 104 to 107 to demonstrate that population size has a rather small influence on the operation of Muller's ratchet in the presence of a distribution of mutational effects.We use deliberately wide upper and lower bounds of Tgen is between 4 month (min) and 2.5 years (max), so we assume 1 year as our middle value. The maximal reproductive capacity in the wild with a non-degraded genome Rmax is assumed to be 50 (min) 500 (mid) and 2000 (max) offspring per lifetime (see below). While Tgen scales extinction times linearly, Rmax has a much smaller effect. It is possible that our values for Rmax are too large since they are more based on the highest possible values conceivable under laboratory conditions than on any real life survival situation. Once such data are available, results from Figure Based on observations we estimate that generation time P. formosa comes from extensive aquarium cultures. In captivity the Amazon molly reaches maturity between 4 and 6 months, produces more offspring at larger body sizes and may live for up to 3 years under optimal laboratory conditions. Here we report that females caught in the wild contain a mean of 28 embryos  with the maximal number of embryos per period observed in the wild .Unfortunately there is very little information about life history from natural habitats. Most of the knowledge about  the lab ). Femalehis size ). We didPoecilia mexicana female and a Poecilia latipinna male [Tage for this event may be derived from comparing alleles in the Amazon molly with alleles in their corresponding parent species using a molecular clock. At the moment there are two datasets that are large enough to infer an evolutionary age that is different from zero. Comparisons of 1,377 bp in various nuclear genes (mostly introns) lead to a point estimate of Tage = 100,000 years assuming a divergence rate of 2%/Myr from standard mitochondrial clocks [Tage = 8,930 to Tage = 209,000 years due to the small number of substitutions observed [Ne above [P. formosa and P. mexicana and that this haplotype is at the center of P. formosa diversity ; lower and upper limits range from 63,000 to 104,000 years). This time will have to be scaled downward, if actual mutation rates are higher, as has been found in human pedigrees [Tage = 40 Kyr, 70 Kyr and 100 Kyr as estimates of the age of the Amazon molly in Figure The ancestor of the Amazon molly clone known to us was formed by a hybridisation event between a nna male ,12 and il clocks . This agobserved ,70. Thus. Since many of these mutations will be synonymous or affect only non-functional DNA, we have to scale this rate by the effective genome size [Drosophila melanogaster as a lower limit .-10/site/cell replication, respectively [P. formosa experience about 25 germ line cell divisions per adult generation (as female mice [Utot \u2248 0.4 potentially deleterious mutations/haploid genome/generation. Obviously, P. formosa is not a mouse and there is much room for improving the precision of such mutation rate estimates, even in mice. Recent high-precision measurements of the deleterious mutation rate in mutation accumulation experiments in Drosophila found Utot \u2248 0.6 deleterious mutations/haploid genome/generation [Utot \u2248 0.7.These estimates do not change much, even if we use a completely different way of obtaining the mutation rate. Observations in mice have lead to estimates of 1.8 \u00d7 10ectively . If an eale mice  that havneration . ScalingIt is encouraging to find such general agreement between these estimates that are based on very different approaches. This suggests that we can have some confidence in our estimates, even in the absence of direct observations in the Amazon molly. Since mutations are the inevitable consequence of DNA replication errors and the number of mitotic cell divisions per generation is a key determinant of mutation rates per generation ,155, we KA/KS, to infer that the fraction 1-KA/KS of all mutations must be more deleterious than about s = 1/Ne, since mutations with effects below that threshold accumulate as if they were neutral [Ne \u2248 10-6 [fsdm, could be perhaps around 30%, probably larger than 10%, but probably not much larger than 50%. We use these values as a point estimate with lower and upper bound. Thus Usdm probably shares the same order of magnitude with Utot. As this calculation has large errors, an array of other mutation rates is included as well when quantifying the ratchet. We assume that selection coefficients did not change much for most genes in the time since asexuality arose in the Amazon molly. This is compatible with the assumption that most genes in the Amazon molly are generic to similar fish species and thus well adapted, while only a small fraction is actually responsible for the specific adaptations of the Amazon molly.Recent progress has shown that the distribution of non-synonymous mutational effects is very leptokurtic and spans many orders of magnitude in many different species ,110-112. neutral ,156. If e \u2248 10-6 . Robust e \u2248 10-6 ,47. FromUtot \u2248 0.4 and Utot \u2248 0.9, while our best estimate of the fraction of mutations with critical slightly deleterious effects is expected to be between fsdm \u2248 10% and fsdm \u2248 50%. Combining these values makes us expect Usdm \u2248 0.2 slightly deleterious mutations with critical effects/haploid genome/generation with a lower limit of Usdm \u2248 0.04 and an upper limit of Usdm \u2248 0.45. These values may have to be doubled, depending on the genome model that is used for accounting for the effects of diploidy. To err on the side of caution and provide a better feeling for the effects of mutation rates we plot values from Usdm = 0.01 to Usdm = 1 in Figure As discussed above, our best estimate for the total mutation rate at potentially deleterious sites per haploid genome per generation is expected to be between LL conceived the study, wrote the software, managed the computation of simulation results, developed the theoretical aspects of the paper, estimated many parameters, analyzed the results and wrote the manuscript. DKL provided most details on the Amazon molly biology, contributed to parameter estimation and helped writing the manuscript. Both authors read and approved the final manuscript."}
{"text": "A novel method to infer the number and fitness effect of beneficial mutations reveals that the bulk of adaptive evolution is attributable to a few mutations with variable effects on fitness. Aspergillus nidulans evolving for approximately 800 generations at two population sizes using a novel maximum likelihood framework, the results of which were confirmed experimentally using sexual crosses. We find that adaptive walks do indeed tend to be short, and fitness increases become smaller as successive mutations fix. Moreover, we show that these patterns are associated with a decreasing supply of beneficial mutations as the population adapts. We also provide empirical distributions of fitness effects among mutations fixed at each step. Our results provide a first glimpse into the properties of multiple steps in an adaptive walk in asexual populations and lend empirical support to models of adaptation involving selection towards a single optimum phenotype. In practical terms, our results suggest that the bulk of adaptation is likely to be accomplished within the first few steps.The rarity of beneficial mutations has frustrated efforts to develop a quantitative theory of adaptation. Recent models of adaptive walks, the sequential substitution of beneficial mutations by selection, make two compelling predictions: adaptive walks should be short, and fitness increases should become exponentially smaller as successive mutations fix. We estimated the number and fitness effects of beneficial mutations in each of 118 replicate lineages of  adaptive walk, and used this to test predictions about the properties of adaptive walks in experimental populations of fungus. Our work shows that, in contrast to the gradualist view of adaptation dominant since the 1930s, adaptive walks tend to be fast and short, with beneficial mutations of large effect substituted first, followed by those of smaller effect.Adaptation is one of the least understood processes in biology because it relies on beneficial mutations, which are often too rare to study. We developed a method to infer the number and size of beneficial mutations substituted during adaptation, a process called an  Although recent work with microbial populations has shed light on the properties of a single bout of adaptation The rate and extent of adaptive evolution over long time periods depends ultimately on the sequential substitution of beneficial mutations by natural selection, a process termed an N) and mutation rate (\u03bc), is high, because clonal interference becomes increasingly important The dominant view championed since the 1930s by Fisher through his geometric model of adaptation has been that adaptive walks are driven by the substitution of many mutations with exceedingly small fitness effects With these caveats in mind, models of adaptation make three compelling predictions about the nature of the distributions of mutational effects over the course of an adaptive walk. First, the combined effects of genetic drift, which tends to eliminate small-effect mutations, and clonal interference transforms the typically L-shaped distribution of fitness effects available to selection into a distribution of fitness effects among fixed mutations that is unimodal with a substantially higher mean Aspergillus nidulans. This species forms spatially structured colonies on solid surfaces, making it relatively easy to detect beneficial mutations  of replicate lineages utations . We founAssumptions of the modelWe assume a strictly clonal population growing exponentially at rates that do not vary in time or depend on the frequencies of other clones. We assume that during propagation, clones are serially transferred to fresh medium so there is no density dependence. We assume that the minimum population size during each bottleneck is large enough to not affect the trajectories of allele frequencies between bottlenecks see .Derivation of the likelihood functionr\u200a=\u200a and times of origin t\u200a=\u200a of up to n clones  from data consisting of fitness estimates (observations) collected at k spaced time points during the adaptation of a single population. The likelihood of the data D\u200a=\u200a is thenE[ . ] is the expectation over all possible clones present at each time point in the population assayed and prob denotes the probability of observing a fitness iw at time point iT given a collection of n clones with associated fitness r1, r2,\u2026, nr. The model is very general and allows for clonal competition and could readily be extended to include a stochastic component for extreme bottlenecking or various forms of density dependence.In general, we wish to estimate a vector of Malthusian parameters Parameter estimation and hypothesis testingr and t  is used to decide which model (nM), and thus how many beneficial mutations, best fit the data in each population. Note that although the AIC criterion does not ensure that beneficial mutations in the final model have reached strict fixation (frequency\u200a=\u200a1), simulations evaluating the time to reach quasifixation (defined as 1\u22121/(e sN); see We maximize numerically this likelihood with respect to nd t see  and thert110\u200a=\u200a2.10, p\u200a=\u200a0.038). The variance in fitness among lineages follows a similar diminishing returns pattern but accumulates faster in the small bottleneck treatment , reaching comparable levels in both treatments by the end of the experiment . These results lend support to the idea that natural selection in populations of different size tends to lead to different effective fitness plateaus The fitness trajectories of all 118 lineages are shown in t-tests for all lineages between times t and t+1 using a false discovery rate criterion of \u03b1\u200a=\u200a0.3 to determine significance), as would be expected if mildly deleterious mutations had a higher probability of increasing in frequency following each transfer.Occasional fitness decreases occurred in both treatments although these were modest in size relative to the initial fitness increase at the first transfer and not sustained over multiple transfers. This variation is likely due to uncontrollable microenvironmental variation in environmental conditions arising during the selection experiment. Alternative explanations include frequency-dependent selection or drift causing a transient increase in the frequency of mildly deleterious mutations. Frequency-dependent selection is a priori unlikely because spatial structure, which facilitates frequency dependent selection by allowing persistent interactions among genotypes In previous work, the fungicide-sensitive strain from which the founder of our experiments was derived showed little evidence of adaptation to the same growth conditions used here, suggesting that the sensitive strain resides close to a fitness optimum n clones with fitness values r1, r2,\u2026, rn substituting in the population. Comparing the fit of the models to the data allowed us to estimate how many beneficial mutations arose in each lineage and the fitness effects associated with each new mutation. This procedure assumes an exponential model of population growth, and we provide experimental evidence to support this assumption in i+1 does not necessarily arise in the genetic background of clone i.Instead of relying on the fitting of fitness trajectories by step functions n\u200a=\u200a100,000 permutations comparing the absolute value of the difference in coefficients of variation, d, between two distributions: d\u200a=\u200a0.387, p<0.0001), the small bottleneck treatment appearing more L-shaped than in the large bottleneck treatment. This result suggests that weaker clonal competition arising from smaller population sizes leads to the fixation of more small-effect mutations and the fixation of mutations with a wider range of effects. Interestingly, the differences between treatments at steps 2 and 3 are not significant , implying that clonal competition becomes less important in shaping the distributions of fixed effects as the population adapts. Further support for this interpretation comes from the fact that significant differences were observed between the first and second steps in the large bottleneck treatment , but not between the second and third steps , nor between any of the steps in the small bottleneck treatment . Inspection of Z\u200a=\u200a3.15, p\u200a=\u200a0.0016, nlarge\u200a=\u200a58, nsmall\u200a=\u200a60), consistent with the higher probability of losing beneficial mutations by drift in smaller populations. Mutations restoring sensitivity to fludioxonil were rare in our experiment, being observed in just five of the 118 lineages. In two lineages from the small bottleneck treatment, sensitivity was restored by a single mutation. The remaining three fludioxonil-sensitive lineages  substituted multiple mutations that resulted in restored sensitivity. Removing these lineages from the analysis does not change our results. Thus, the short walks we observed are not merely due to the predominance of back mutations of the resistance mutation (fldA1). Moreover, we were able to detect significant genetic (GV) and genotype-by-environment interaction (GEV) variation among evolved lineages in both bottleneck treatments  when grown across a concentration gradient of fludioxonil  and a long right tail representing putatively accessible beneficial mutations. More striking is the relationship between mean fitness of the evolving population and the fraction of beneficial mutations available to selection, which is negative . Importantly, even in our small bottleneck treatment, N is unlikely to be so low as to prevent sampling of a large number of single-step mutations, as required by theory Interestingly, our conclusions are not much altered by the bottleneck size used between serial transfers. This result is attributable to the opposing effects of drift and clonal competition in small and large populations. In small populations, only mutations of moderate to large effect are likely to escape stochastic loss when rare, and those that do are nearly guaranteed to fix due to the absence of clonal competition. In large populations, more mutations of small effect will reach appreciable frequencies but are eventually lost due to clonal competition. The result is that the mean fitness effect of mutations that fix under different population sizes remains approximately the same at each step throughout the course of an adaptive walk. By contrast, population size does affect the mean fitness among lineages achieved by the end of the experiment, with small populations having lower fitness than large populations by the end of the experiment. This observation is consistent with our finding of fewer fixed mutations in small compared to large populations, which likely stems from changes in the mutation supply rate, As with most selection experiments, we have little direct information about the topography of the adaptive landscape; however, two results suggest that it may be fairly rugged. First, fitness tends to plateau at different levels in most lineages by about generation 500, and these fitness differences are maintained throughout the experiment The shortness of the adaptive walks is a striking feature of our results that stands in contrast to modestly longer walks of three to six steps documented in bacteria Second, it may be argued that short adaptive walks are expected a priori because we are studying compensatory evolution, that is, the recovery of fitness due to the presence of a costly resistance mutation Why did we observe shorter adaptive walks than previous experiments? One possibility is that our founding strain was already well adapted to the conditions of growth and so had only a few beneficial mutations available to it. This explanation is difficult to reconcile, however, with the observation of substantial genotype-by-environment interaction mentioned above, the apparently large fraction of beneficial mutations available to the ancestor  and a gaThat the relationship between the mean fitness increases with each successive step in an adaptive walk is negative suggests two important insights into the mechanics of adaptive evolution. First, the distribution of fitness effects among beneficial mutations prior to selection is expected to lie in the Weibull domain of attraction Our results have two important practical implications. First, if the environment changes regularly, populations may be expected to adapt perpetually, and it will be difficult to make strong predictions as to the outcome, for example, of long-term changes to the environment such as those expected to occur under many climate change scenarios. Second, compensatory evolution in response to losses in fitness due to mutations conferring resistance to fungicides, antibiotics, or other chemotherapies, is likely to be fast, requiring only a few mutational steps. More fundamentally, the picture emerging from these experiments is that adaptive evolution in mutation-limited populations can be readily understood from rather simple models that, because they are largely based on the statistical properties of extreme events, are likely to be robust to biological details.A. nidulans strain WG615  for the selection experiment, kindly provided by Fons Debets and Marijke Slakhorst at Wageningen University, the Netherlands. This strain is resistant to the fungicide fludioxonil (fldA1). Resistance confers a cost of around 46% relative to the sensitive strain from which it was derived when growing in the absence of fungicide yA1, veA1), which has the same genetic background as WG615. In the selection experiment, spores and mycelium were washed from the plate using 5 ml of saline-Tween (water containing NaCl 0.8% and Tween-80 0.05%), reserving 5 \u00b5l for transfer to fresh medium and storing 0.8 ml of the mixture mixed with 0.3 ml 80% glycerol at \u221280\u00b0C. Bottleneck sizes were adjusted by dilution. For all experiments, we used solid Complete Medium (CM) set at pH 5.8, consisting of NaNO3 6.0 g/l; KH2PO4 1.5 g/l; MgSO4.7H2O 0.5 g/l; NaCl 0.5 g/l; 0.1 ml of a saturated trace element solution containing FeSO4, ZnSO4, MnCl2, and CuSO4; tryptone 10 g/l; and yeast extract 5 g/l and (added after autoclaving) glucose 4.0 g/l. Cultures were incubated at 37\u00b0C.We used A. nidulans strain WG615 by placing 5 \u00b5l of a dense spore suspension  in the center of a Petri dish containing solid CM medium. We propagated these replicate lineages by serial transfer for a total of 800 generations. Every 5 d,  as the colony diameter  after 5 d of incubation, which is widely used as a measure of fitness and is highly correlated with other fitness measures such as total spore production and biomass r\u200a=\u200a0.81, t5\u200a=\u200a3.09, p\u200a=\u200a0.027). Selection coefficients were calculated as s\u200a=\u200a(iw\u2212wancestor)/wancestor. Each lineage was assayed in triplicate at multiple time points  in a single assay. Two lineages from the large bottleneck treatment were eliminated from the maximum likelihood analysis due to missing data. For 26 evolved strains that had a high relative fitness, we reassayed the fitness at 800 generations using at least seven replicates together with the fungicide-sensitive strain WG638. We estimated fitness of all lineages at the end of the selection experiment in CM supplemented with 0, 0.05, 0.2, or 0.4 ppm of fludioxonil in duplicate and used an analysis of covariance to test the main effect of lineage (genetic variance) and the interaction between lineage and fludioxonil concentration (genotype-by-environment interaction variance).We measured fitness of the fldA1, lysB5, veA1). We estimated the fitness of at least 50 progeny for each of 15 crosses and used the Castle-Wright estimator We performed sexual crosses 2 of mycelium including spores at three preassigned locations on the edge of the colony. Fitness was estimated for each spore sample, and the fraction of samples with fitness greater than that of the parent genotype was calculated for each time point. We assayed fitness in blocks and used strains WG615 and WG638 as reference strains to account for variation between blocks. Logistic regressions were performed in R version 2.6.1 using generalized linear models with binomial errors. We controlled for false positives, which would inflate our estimates of the fraction of beneficial mutations, using a false discovery rate analysis We initiated replicate populations of genotypes of interest by placing 5 \u00b5l of a dense spore suspension in the center of a Petri dish. After 5 d of colony expansion, we sampled approximately 3 mmFigure S1Fitness of 28 evolved lineages after 800 generations (grey bars), the ancestor that founded the selection experiment, and a strain that is fungicide sensitive but has the same genetic background. Error bars show 95% confidence intervals. The founding strain of the evolved lineages carries a fungicide resistance mutation that is costly under the growth condition used here  revealed that at least three strains have significantly higher fitness than the sensitive strain. (0.56 MB TIF)Click here for additional data file.Figure S2Relationship between the number of nuclei present in a mycelium (ln transformed number of colony forming units [CFU]) as a function of the size of the fungal colony (colony diameter in millimeters).(0.00 MB EPS)Click here for additional data file.Figure S3Mutations available to selection. Fraction of mutations sampled available to selection, scaled to the fraction available to the ancestor. Click here for additional data file.Figure S4Average time to quasifixation of beneficial mutations as a function of their selection coefficient and the size of the population see . Dots denote the average of simulations tracking the time to fixation or loss of 10,000 new mutations with selection coefficient s. Continuous lines are a diffusion-based approximation for the average time to quasifixation, assuming the strong selection limit .(0.02 MB EPS)Click here for additional data file.Table S1Comparison of number of loci contributing to adaptation estimated with Caste-Wright estimator and maximum likelihood. Number of segregating loci (en) in a cross between evolved genotypes and the non-evolved ancestor, using the Castle-Wright estimator (0.04 MB DOC)Click here for additional data file.Text S1Likelihood framework for estimating selection coefficients of beneficial mutations spreading in each population from fitness trajectory data.(0.06 MB DOC)Click here for additional data file.Text S2Experimental evidence for exponential population growth.(0.03 MB DOC)Click here for additional data file.Text S3Estimation of the number of segregating loci using the Castle-Wright estimator.(0.03 MB DOC)Click here for additional data file.Text S4Comparison between mycelial growth rate and competitive fitness.(0.03 MB DOC)Click here for additional data file."}
{"text": "The secondary structure of folded RNA sequences is a good model to map phenotype onto genotype, as represented by the RNA sequence. Computational studies of the evolution of ensembles of RNA molecules towards target secondary structures yield valuable clues to the mechanisms behind adaptation of complex populations. The relationship between the space of sequences and structures, the organization of RNA ensembles at mutation-selection equilibrium, the time of adaptation as a function of the population parameters, the presence of collective effects in quasispecies, or the optimal mutation rates to promote adaptation all are issues that can be explored within this framework.in silico evolution and adaptation. We calculate the distribution of the effects of mutations on fitness, the relative fractions of beneficial and deleterious mutations and the corresponding selection coefficients for populations evolving under different mutation rates. Three different situations are explored: the mutation-selection equilibrium (optimized population) in three different fitness landscapes, the dynamics during adaptation towards a goal structure (adapting population), and the behavior under periodic population bottlenecks (perturbed population).We investigate the effect of microscopic mutations on the phenotype of RNA molecules during their \u03bc at which evolution proceeds. In contrast, the selective value of mutations remains almost constant, independent of \u03bc, indicating that adaptation occurs through an increase in the amount of beneficial mutations, with little variations in the average effect they have on fitness. Statistical analyses of the distribution of fitness effects reveal that small effects, either beneficial or deleterious, are well described by a Pareto distribution. These results are robust under changes in the fitness landscape, remarkably when, in addition to selecting a target secondary structure, specific subsequences or low-energy folds are required. A population perturbed by bottlenecks behaves similarly to an adapting population, struggling to return to the optimized state. Whether it can survive in the long run or whether it goes extinct depends critically on the length of the time interval between bottlenecks.The ratio between the number of beneficial and deleterious mutations experienced by a population of RNA sequences increases with the value of the mutation rate  The fate of evolving populations is determined by a number of intrinsic properties of the ensemble and extrinsic mechanisms that interact in a highly non-trivial manner. The natural mutation rate of populations and their size, the effect that such mutations have on fitness and how these effects vary with the state of the population, or the environmental perturbations they have to cope with, all are relevant variables conditioning long-term survivability .Populations evolving in constant environments eventually reach a fitness plateau, often interpreted as an optimum in the fitness landscape. The time required to reach the optimum and the structure of the population at the mutation-selection equilibrium corresponding to that environment depend, in addition to the factors listed above, on the environment and on the initial state of the population. During adaptation, the effect of mutations is different from their effect at equilibrium, probably due, among others, to differences in the fitness of populations and to changes in the genomic context ,3. ThougThe relationship between the mutation rate of a population and the variability of the environment where it evolves has been a matter of interest for a long time -12. In sSimple, computational models of evolution explicitly separating genotype and phenotype might be of great aid in bridging the gap between phenomenological theories and experimental observations. The former are built on empirical observations which, as of yet, are far from complete. Actually, most experimental observations deal with a relatively small number of cases and model organisms, such that generalization is still a difficult enterprise . AnotherThe computational study of random ensembles of RNA sequences folding into their minimum free energy secondary structure (a proxy for the phenotype) has permitted one to assess the role played by compensatory mutations . More reGenomes within a population have to be highly correlated, since they share a phylogenetic history and have experienced similar selection pressures.a priori. They depend on the specific change that they produce in the secondary structure of the evolving molecules, in particular subsequences, or in the energy of the folded state. Our model also permits us to analyze the multiple mutations simultaneously present in a population, independently of whether they become fixed or not. Thus, mutations with highly deleterious effects can be pinpointed and those with a very small effect can be distinguished from neutral changes.The aim of this contribution is to investigate the internal organization of optimized, adapting and perturbed populations of RNA molecules evolving under different genomic mutation rates, with the goal of quantifying the effect of mutations on fitness and establishing links between the state of the population and the effect of mutations. In the case of optimized populations, different fitness landscapes are used to probe the generality of our results. The model we use does not presuppose any underlying distribution of mutational effects. Mutations arise according to the established mutation rate and their effects are not selected l = 50 nucleotides (nt). We begin with an ensemble of N random sequences. The ensemble evolves through discrete generations and the population size is kept constant. An exception is the case of perturbations through population bottlenecks, to be discussed in detail below. At every generation, all the sequences in the population are folded into their minimum free energy secondary structure with help of the Vienna package (see Methods). We define a target secondary structure S  that a molecule i in the population replicates is larger the closer to S it folds. This probability is defined asOur model system consists of a population of replicating RNA sequences, each of length di is the distance between the structure corresponding to sequence i and the target structure S. As a measure of structural distance, we use the base-pair distance (see Definitions). The constant l is a scale factor, since the maximum base pair distance between two molecules of length l is proportional to l. The overall normalization factor is S-landscape).where s \u2261 [p(di) - p(dj)]/p(dj) of a sequence i folding at distance di to the target with respect to a sequence j folding at distance dj = m + di is independent of di,With the above definition of replication probability, the selective advantage \u03b2 determines the relative advantage between different sequences in the population. In the limit \u03b2 \u2192 0, s \u2192 0 for any two sequences: the distinction between molecules folding closer or farther from S is lost and all have asymptotically the same probability to replicate. When \u03b2 \u2192 \u221e, only the sequence closest to the target is selected, the rest being eliminated in the next generation. The target structure S is found and fixed for most finite values of \u03b2, and only quantitative changes in the properties of the population are produced when it is varied. Increases in \u03b2 can be compensated by decreases in \u03bc, and vice versa. In this work, the value of this selection parameter will be fixed to \u03b2 = 2.This result implies that the selective advantage of a structure depends on the populational context, and not on its absolute distance to the target. Further, it reveals that the selection parameter \u03bc to be replaced by a nucleotide randomly chosen with equal probability among A, C, G, and U. In cases where the populations exceeds the maximum size N, we perform the usual Wright-Fisher sampling.When a molecule replicates, each nucleotide has a probability The Hamming distance between two RNA sequences or subsequences is given by the number of positions in which their nucleotides differ. Structural differences can be estimated by various means. In this work, we apply the base-pair distance, given by the number of base pairs that have to be opened and closed to transform one structure into the other .\u03c1 of structures in the population folding into the target structure. This quantity is zero when our simulation starts: note that the probability that a random sequence of length l folds into an arbitrary secondary structure is negligibly small, of the order of 10-15 in the present case [\u03c1 is attained at the statistically stationary state, once the target structure is fixed in the population. The process of fixation and the dependence of \u03c1 with the mutation rate \u03bc have been described in detail elsewhere [A relevant macroscopic quantity to characterize the degree of optimization of the population is the fraction ent case  (but seeent case ). The malsewhere .\u03bc is quantified by comparing the secondary structures of the mother i and daughter j sequences. We calculate the distances di and dj, as described, and the difference \u0394ij = di - dj. If \u0394ij = \u0394 > 0 (meaning that the mother sequence was farther from the target structure than its mutated daughter), the mutations increased fitness and count in the total of beneficial changes. If \u0394ij = \u0394 < 0, mutations have caused a negative change in fitness, and the daughter sequence is farther from the target than her mother was. When \u0394ij = 0, either mutations had no effect on fitness or no mutation has occurred.The change in fitness of an RNA molecule under replication with mutation rate i, j), as we will specify, to obtain a probability distribution \u03a0(\u0394) of the changes in fitness. That distribution eventually yields the fraction of deleterious changes p and the fraction of beneficial changes q:This procedure is repeated over a large number of mother-daughter pairs (n' = \u03a0(0)-regardless of whether no mutations occurred or because the ones occurring were neutral-, can be immediately obtained from p and q: n' = 1 - p - q.The fraction of replication events with no change in fitness, \u03bc, the probability that a sequence of length l acquires k mutations upon replication follows a Poisson distribution of average \u03bcl,For a mutation rate P(0) = e\u03bcl-, which for \u03bcl \u226a 1 reduces to P(0) = 1 - \u03bcl. The fraction of truly neutral mutations is thus n = n' - P(0). If one wishes to calculate the fraction of deleterious, beneficial or neutral mutations conditional on at least one mutation having taken place, it suffices to divide the values of p, q, and n above by the expected number of sequences with one or more mutations, 1 - P(0). The effect of single mutations can be estimated in this way only for sufficiently small values of the mutation rate \u03bc, since for relatively large values the probability of having two mutations in the same sequence becomes non-negligible. For example, for \u03bc = 10-3, the probability that a sequence of length l = 50 is hit by two or more mutations is P(k \u2265 2) = 1 - P(0) - P(1) \u2243 0.00121, thus one in a thousand molecules gets more than one mutation under replication. At sufficiently low mutation rates, the distribution of effects of mutations on fitness thus corresponds to the distribution of the effect of single mutations. As \u03bc increases, sequences at a distance of two and more mutations from the parental molecule can appear in a single replication event. This has implications in the mobility of the population in sequence space and in its evolutionary dynamics [The probability of incorporating no mutations is thus dynamics .\u03c3q and \u03c3p are calculated as the relative average change in fitness produced by all replication events affected by mutations with a beneficial or a deleterious effect on fitness, respectively:The average selection coefficients l corresponds to the maximum fitness change, such that 0 <  < 1. The fraction of deleterious and beneficial mutations and the corresponding average selection coefficients are calculated while keeping a fixed value of the genomic mutation rate \u03bc.where \u03bcc, the population is able to climb up towards the optimum of the phenotype space. After the transient, if mutation-selection equilibrium is reached, the population sits around the optimum, with a fraction \u03c1 of correctly folding sequences determined by \u03bc: the error rate at which a population evolves determines the degree of adaptation reached at the equilibrium. The mutation rate also determines the spread of the population in sequence and structure spaces. Populations perturbed through bottlenecks are forced to recover starting with a single sequence. If the time between bottlenecks is long enough, beneficial mutations can be found and fixed, recovery is possible, and the population is to be located near the optimum most of the time. However, for frequent perturbations it is likely that suboptimal sequences are repeatedly chosen. This favours the accumulation of deleterious mutations that separate the population steadily from the optimum: recovery is not possible, and eventual extinction might supervene.In the following we investigate the response of populations of RNA sequences evolving under the situations described. The three different cases explored are schematically represented in Figure S (plots (a), (c), and (e)) and the corresponding Hamming distance between all possible pairs of molecules in the population (plots (b), (d), and (f)). This quantitative picture agrees with the phenomenological description represented in Figure In the three cases described, and for a fixed value of the mutation rate, the diversity of each population in sequence and structure differs notably. Figure N = 1000 random RNA sequences subject to selection as a function of their distance to the target structure S, Eq. (1). In the statistically stationary state, the population has attained its maximum degree of optimization and is located as close to the optimum (represented by the target structure) as allowed by the operating mutation rate \u03bc. The fraction \u03c1 of sequences folding into S is a decreasing function of \u03bc for values of the mutation rate below the error threshold; \u03c1 becomes strictly zero for \u03bc > \u03bcc only for sequences of infinite length. In our case, where sequences are short, there is a non-zero probability that the target structure is found, even above the error threshold. However, it is rapidly lost due to the high mutation rate. Hence, though we sporadically see the appearance of S above threshold, it cannot be fixed in the population.We start the simulation as described, with \u03bc = 0.004, which yields a fraction \u03c1 \u2243 0.34 of correctly folded sequences. Larger values of \u03bc would yield broader distributions and smaller values of \u03bc, corresponding to larger values of \u03c1, would pack the population closer to the optimum. An important point is to notice that there is always place for improvement within the population for any \u03bc > 0. Suboptimal sequences (with di \u2265 1) might incorporate mutations (mostly compensatory once at the mutation-selection equilibrium) that diminish their distance to S and thus increase their fitness, while optimal sequences (with di = 0) might be affected by mutations that change their folded state, and thus count as deleterious. At equilibrium, the two processes balance so as to maintain the fraction \u03c1 constant. The situation is different regarding the genomic diversity of the population. Though at the first generations after fixation of the target structure all sequences are similar, in successive iterations the population explores the neutral network of genotypes. This permits the population to expand in sequence space, displaying broad variability in sequences while maintaining the optimized phenotype. Further, the topological properties of the neutral network allow a deep exploration of the space of configurations, a feature that has been shown to underly the extreme plasticity observed not only in RNA sequence evolution in silico [The distributions shown in Figure n silico , but alsn silico . The reo\u03bc. Beneficial mutations are very rare at low values of \u03bc, where the largest fractions \u03c1 of correctly folded sequences occur. The fraction of mutations with deleterious effects is slightly larger, though it still has low absolute value. As could have been expected, most replication events do not change fitness, particularly because mutations are absent. As \u03bc increases, mutations become more abundant, and changes of fitness upon replication increase in frequency. Simultaneously, the distribution becomes more symmetrical: the ratio between beneficial and deleterious mutations increases towards one. This analysis has been repeated for populations optimized at different values of \u03bc. Figure p, q, and the selection coefficients \u03c3p and \u03c3q as a function of the mutation rate \u03bc. We have indicated the approximate position of the error threshold \u03bcc. It is interesting to note that the effect of mutations on phenotype does not have any particular sensitivity to this important threshold. It only affects the probability of fixation of beneficial mutations, not their appearance, which increases monotonically with \u03bc. This is consistent with previous observations of structural stability, a kind of collective effect in RNA populations, which still maintain a distributed signal of the target secondary structure despite the fact that no sequence in the population folds into it [At the mutation-selection equilibrium, the effect of deleterious mutations is compensated by the incorporation of compensatory mutations , together with the replicative advantage of correctly folding sequences. Several examples of probability distributions \u03a0(\u0394) are shown in Figure  into it .\u03bc:Below the critical error threshold, the phenotypic mutation fractions seem to bear a simple functional relationship with q/p quantifies the number of compensatory mutations per deleterious mutation. Its dependence with \u03bc is displayed in the inset of Figure q/p \u2243 \u03bc\u03be, with \u03be = \u03b3 - \u03b1 = 0.87 \u00b1 0.03.with exponents \u03b1 \u2243 0.89 \u00b1 0.01 and \u03b3 \u2243 1.77 \u00b1 0.02, indicating that, as the degree of adaptation of a population reduces as a consequence of evolution at progressively increased mutation rates, compensatory mutations increase their frequency at a higher speed than do deleterious mutations. We expect the algebraic relationship between phenotypic and microscopic mutation rates to be generic, although the specific values of the exponents \u03b1 and \u03b3 will depend on the length of the sequences and on the particular secondary structure chosen. The ratio \u03bc, with \u03c3p taking about twice the value of \u03c3q. This relationship is dependent on sequence length and target structure.The average selection coefficients vary only slightly with N = 1000 random RNA sequences is the starting point, and we replicate and select sequences as described. Now, however, fitness changes are measured before the population has found the target structure, such that it is formed by a population of phylogenetically related, suboptimal structures. In practice, we measure the structure of the population and the effect of mutations during 50 generations after the initial random state. The average number of generations required to find and fix the target structure is well above this value.The study of adapting populations is performed in a way similar to that for optimized populations. An ensemble of d \u2243 9 to the target structure in Figure The internal structure of the population at generation 50 is illustrated through the representative example shown in Figure \u03bc. The overall shape of \u03a0(\u0394) is similar to that obtained at the mutation-selection equilibrium: a larger amount of deleterious mutations and a tendency of the distribution to become symmetrical as \u03bc grows.The distribution of changes in fitness along the first 50 generations of adaptation is shown in Figure \u03bc. In comparison to an optimized population, the fraction of deleterious mutations is slightly smaller than in the optimized case, while the fraction of mutations with a positive effect on fitness  is significantly larger. This fact is explained by considering that there are many more possibilities of improvement in the suboptimal states visited by an adapting population. This difference is bigger in the case of populations that evolve at low \u03bc than in populations evolving at high \u03bc. For \u03bc values above the error threshold, adapting and optimized populations behave similarly. The functional dependence of the phenotypic mutation fractions with \u03bc is of the same type as observed in an optimized population, Eq. (6). The exponents take different values in this case: using a least squares fit to the numerical data we obtain \u03b1 = 0.93 \u00b1 0.01 and \u03b3 = 1.06 \u00b1 0.01. Note the remarkable quantitative difference in the behaviour of the ratio q/p, represented in the inset. Though still increasing as a function of \u03bc, its variation with the mutation rate is much milder than in the case of optimized populations, as the corresponding exponent \u03be = 0.13 \u00b1 0.04 indicates. This result suggests that in the first stages of adaptation, populations evolving at different error rates are more similar than the same populations when equilibrium has been attained. In this case, however, substantial deviations observed in the q/p curve suggest that the power-law is not a good fit in the whole range of \u03bc. The selective value of mutations is, both for beneficial and deleterious changes, typically lower than in the optimized case. Adaptation takes place through an increase in the fraction of beneficial mutations. In natural systems, mutations with a large effect on fitness can mask beneficial mutations with a small effect, which are then not identified during evolution  replicate at a slower pace. Note that the relative advantage of sequences folding into structures at relative distance m is the same as for the case of constant populations, Eq. (2).This dynamical rule applies now to the case of a growing population and substitutes the definition of replicative ability given in Eq. (1), the latter applying to populations of constant size. If the sequence at the bottleneck folds into the target structure, then \u27e8g of generations elapsed between bottlenecks is large enough, the population will be able to recover and again attain mutation-selection equilibrium. As g decreases, the time for optimization becomes shorter and, for g small enough, the total population will not be capable of achieving the optimum. Still, survival might be possible. At too low values of g, however, the perturbation becomes too strong and the few sequences after the bottleneck might be unable to replicate, in which case the population goes extinct.If the number \u03bc is the main source of randomness in the system, permitting as well change and improvement. Population bottlenecks constitute an additional source of stochasticity and represent a stronger perturbation, since selecting a random sequence to found a new population implies choosing a suboptimal structure with a probability of at least 1 - \u03c1. In those cases, the population faces two important difficulties: first, it has to go again through the adapting transient, where the target structure has not yet been found; second, the reduction in the population size impairs the capability of the ensemble to generate beneficial variants.For optimized and adapting populations, the mutation rate Figure Q\u03b2 bacteriophage incorporated a lower amount of beneficial mutations than viruses isolated from a population previously displaced from mutation-selection equilibrium due to the application of successive bottlenecks [The dynamics of three populations under the action of bottlenecks with different periodicity is illustrated in Figure tlenecks .In the possible case that an optimal sequence is selected, it replicates fast and reaches high density in a small number of generations. In this case, almost all sequences in the first generations after the bottleneck fold into optimal structures, thus generating a superoptimal, unstable population that slowly relaxes towards the diversity characteristic of the mutation-selection equilibrium under the action of the mutation rate. Figure \u03bc. Low values of \u03bc correspond to a higher density \u03c1 of correctly folded sequences, thus favouring selection of an optimal sequence at the bottleneck. However, a sufficiently low value of \u03bc hinders the generation of diversity and with it the appearance of more optimal variants. Actually, when bottlenecks are frequent a good evolutionary strategy is to replicate under a not too low mutation rate, such that, in the case that a suboptimal phenotype is the founder of a new population, the time to recover mutation-selection diversity is minimized [The critical frequency of bottleneck application for the population to survive depends on other model parameters, especially on the mutation rate inimized . This reAn important question is whether our results are robust when different definitions of fitness for the RNA molecules are used. Up to now, we have only considered that, the closer a sequence folds to the target structure, the fitter it is. In this section, we introduce two additional definitions of fitness and discuss how they affect the dynamics and organization of optimized populations. As will be shown, the system is able to find a remarkably large number of genotypes compatible with the new selection pressures applied to the system, and the collective statistical properties of the population are only weakly affected.Q of length ls nucleotides is required at a certain position for a sequence to have maximal fitness, and that deviations from that composition are penalized. A sensible way to add this requirement is the use of a fitness function of the formMany molecules require a specific sequence of nucleotides for their function to be properly performed. Conserved subsequences often correspond to active sites which cannot be mutated, so variations in that subsequence are selected against. As an example, suppose that a target sequence i and the target sequence Q, ls is the length of the target sequence, and \u03b2H is a selection parameter that measures the relative strength of selection for target sequence Q versus selection for target structure S. The landscape defined by Eq. (8) is the S + Q-landscape. In the examples to be shown, we have chosen a target sequence GUAUCUUCAC, with ls = 10, placed at the 5' end of molecules in the population, and a selection parameter \u03b2H = 1. As previously, \u03b2 = 2.where Em for sequences of length l, and call Ei the energy of sequence i folded into its minimum free energy secondary structure. A suitable fitness function to favour low-energy configurations isA second important property of folded molecules is their thermodynamical stability. Usually, sequences folding into configurations of lower energy are at an advantage with respect to those of higher energy. Suppose that there is an absolute minimum energy i and the reference energy Em, and \u03b2E is a selection parameter that again measures the relative strength of selection for low energy versus selection for target structure S. Equation (9) defines the S + E-landscape. In the forthcoming examples, Em = -72 kcal/mol, which corresponds to the energy of a sequence of length l = 50 with 23 G=C pairs that contribute -3 kcal/mol each and where the positive contribution of the loop has been discarded. This value of Em is a lower bound to the energy of sequences of length l = 50. The selection parameter \u03b2E = 0.5, and \u03b2 = 2. Let us compare the mutation-selection equilibria of three populations evolving on each of the landscapes described under a mutation rate \u03bc = 0.003. In order to evaluate the degree of adaptation achieved for each of the traits under selection , three relevant quantities describing the macroscopic state of these populations are used: as before, the density of sequences correctly folding into the target sequence, the average energy \u27e8E\u27e9 = N-1\u2211i Ei of the population, and the fraction \u03c1H of sequences at distance Q. As previously performed, the simulation begins with an ensemble of N = 1000 random sequences. At each generation, the quantities id, Ei relevant in each landscape are evaluated. Substitution by a new generation of offspring sequences proceeds as described.where, analogous to previous definitions, S has \u03c1 \u2243 0.43, its average energy is \u27e8E\u27e9 \u2243 -12.3 kcal/mol and \u03c1H \u2243 0. That is, the required secondary structure has been successfully found but essentially none of the subsequences matches Q. The energy of the population is comparable to that of an ensemble of randomly chosen sequences folding into the target structure S. When selection for sequence is turned on in the S + Q landscape, there is a fraction \u03c1 \u2243 0.45 of sequences correctly folding into the target structure. However, this occurs simultaneously with a fraction \u03c1H \u2243 0.85 of molecules bearing the correct target sequence Q. The average energy in this case is comparable to that in the previous landscape, \u27e8E\u27e9 \u2243 -13.0 kcal/mol. Hence, the population has been completely displaced from its position in the space of sequences (compared to landscape S), allowing the optimization of a second trait (target sequence) while keeping other macroscopic values essentially unchanged. The third population, evolving on landscape S + E, is optimized at \u03c1 \u2243 0.36 with \u03c1H \u2243 0. However, its average energy has significantly decreased to \u27e8E\u27e9 \u2243 -28.4 kcal/mol. This again speaks for a major displacement in sequence space with respect to the two previous landscapes, favouring those sequences folding into S with minimum energy, and with a composition that does not match the target sequence Q.At the mutation-selection equilibrium, we observe the following. The population evolving in landscape S and the target sequence Q can be simultaneously (and partly independently) optimized since Q is compatible with folding into S. This is not a general situation, since demanding a target sequence too long or too biased in its composition might prevent the existence of solutions fulfilling both requirements. This is also the case for the selection of S together with the requirement of a low folding energy. If the parameter \u03b2E would take an exceedingly high value, structures of low energy (different from S) would dominate the population. This is probably the reason why \u03c1 takes a value slightly lower in landscape S + E than in the two former landscapes. While it is important to take these possibilities into account, our results indicate that, in this model, the simultaneous optimization of two traits is possible for a broad range of selection parameters \u03b2, \u03b2H, and \u03b2E.The target structure \u03bc the distribution of changes in \u03bcl, identical to Eq. (4). The landscape corresponding to selection for low energy shares characteristics with both. On the one hand, if mutations acquired through replication do not disrupt the folded state, changes in energy have to be small, since only the composition of the sequence is affected. However, if mutations lead to a different folded state, the minimum energy can suffer major changes, due to the appearance of a different distribution of structural motifs  having a major role in the folded energy.In addition to the different conceptual situations represented by the three landscapes introduced in this work, there are relevant geometrical differences among them. Selection for structure represents a rough landscape where changes in a single nucleotide can cause deep reorganizations in the folded configuration. On the contrary, selection for a specific sequence represents a smooth landscape where changes in the distance L) corresponding to landscapes S, S + Q, and S + E. The superindex L is generic and corresponds to a different variable \u0394L for each landscape. To make the three distributions quantitatively comparable, we have to use rescaled variables in the x-axes, analogous to x = \u0394/l, as introduced for landscape S. They are xQ = \u0394Q/l for landscape S + Q and xE = \u0394E/l for landscape S + E, withIn the next section, we study the distribution of fitness effects \u03a0 and of the corresponding cumulative probability function (CPF), R-squared values as shown. As a general trend, we observe that beneficial mutations typically have significantly smaller fitness effects than deleterious mutations - in agreement with the difference in the average selection coefficients calculated previously. This can be seen for instance in the \u03bb parameter of exponential fits, in the consistent changes in the shape parameter b for \u0393 and \u03b2 distributions, in the slope a of the Pareto distribution, or in the \u03c3 value of the Lognormal distribution.We have used five different probability density functions to fit the numerical, cumulative distributions of the effects of mutations on fitness. Table R-squared values corresponding to each fit as a measure of its goodness, we might conclude that most PDs represent the data reasonably well, though there is no function able to account for all numerical distributions. A closer inspection of the fits reveals systematic deviations from numerical results. Figure \u03b2 distributions) underestimate the number of small effects and overestimate the number of average-to-large effects. In the case of the \u0393 and \u03b2 distributions, the parameter a is in almost all cases close to 1 . This reveals that the fit to data is not improved by considering those two-parameter functions instead of the simpler, one parameter, exponential. The three functions are actually very similar. The Lognormal distribution slightly improves the fit in the region of small effects: It represents a best fit to beneficial distributions , though it systematically fails in the same regions as functions characterized by exponential decays.If we were only to consider None of the functions assayed are thus able to explain the shape of the distributions obtained in the whole domain of changes in fitness. One reason might be that our sequences are relatively short and thus fold into small secondary structures. We cannot discard that large effects on fitness could be affected by structural motifs of the particular target secondary structure studied in this work .R-squared coefficients are at least as good as the best fit in the whole range of fitness change, but now there are no systematic deviations from data. Two representative examples of Pareto distribution fits to numerical data are shown in the inserts of Figure The distribution of small effects on fitness, which considers mutations that slightly change the secondary structure of the parent sequence, should be much less affected by specific properties of the structure studied. We have thus conducted a systematic study of the distribution of small fitness effects, defined as those replication events affected by mutations where the change in the secondary structure was at most 22% with respect to the parent structure. This range is not too small: it can explain around 90% of the beneficial changes observed and around 70% of the deleterious changes (see Table Q(x \u2264 \u039b) for beneficial (B) and deleterious (D) changes in fitness and the three landscapes analysed. The visible similarity between the functional form in either case is supported by the quantitative analysis of the distribution of small effects, as summarized in Table Q(x \u2264 \u039b) for the landscapes and populations studied.Our analysis of optimized populations in different fitness landscapes agrees with the functional behaviour just described. Changes in the fitness landscape modify the distribution of effects on fitness, though only quantitatively. The three landscapes studied are compared in Figure S-landscape.In this section, we compare by means of explicit examples the overall effect of mutations, as obtained in our simulations with RNA sequences, with some measures performed in natural systems. Due to the important differences between populations, environments, and fitness definitions used in different works, the effect of mutations in those different systems should be compared with due care. Nevertheless, the values obtained might give clues about the degree of optimization of the population and about the likeliness that fitness is improved. In the following, the numerical results obtained in our simulations correspond to populations evolving on the \u03bc = 0.001. As described, there is a fraction P(0) = 0.951 of replication events without mutations and an amount 1 - P(0) - P(1) = 1.209 \u00d7 10-3 of daughter sequences incorporating two or more mutations. Hence, in most cases where mutations occur, the replicated sequence is hit by a single mutation (P(1) = 0.047). Let us consider only those cases, where a mutation has been incorporated. Our simulations indicate that, at equilibrium, 0.59% of such mutations have beneficial effects, while 46.34% diminish fitness. A large amount of mutations is neutral: 53.07%. Adapting populations suffer a similar amount of neutral mutations, 54.33% at the same mutation rate \u03bc, but they substantially differ in the amount of mutations with an effect on fitness. Deleterious mutations represent 38.51%, and beneficial mutations increase to 7.16%. It would be difficult to evaluate the effect of single mutations at higher rates of \u03bc. For example, if the average number of mutations per replication event is one per genome (\u03bc = 0.02 in our case), over 26% of the daughter genomes incorporate two or more mutations, and epistatic effects should not be discarded. This might entail a difficulty when translating the results of controlled experiments to natural situations involving fast-mutating replicators.Let us illustrate, with an example, the overall relative fractions of beneficial, deleterious, and neutral mutations affecting optimized and adapting populations. Take the mutation rate Empirical results concerning the effect of mutations on fitness have been obtained in different contexts and for different model systems. Single nucleotide substitutions in vesicular stomatitis virus yielded complex distributions of fitness effects, with the functional form of those corresponding to beneficial mutations (\u0393-distribution) differing from that of deleterious mutations , and both depending on how mutations were acquired . The oveWe have quantified the effect of mutations on fitness for populations of RNA sequences in different situations. Beneficial or compensatory mutations are systematically less probable than deleterious ones, the ratio between both types being strongly dependent on the degree of optimization of the population. Once the error threshold is crossed (at high error rates), optimized and adapted populations behave similarly. Selection coefficients are almost constant, regardless of the degree of adaptation of the population. They are only slightly lower in adapting populations than in optimized populations evolved at the same mutation rate, indicating that adaptation takes place through an increase in the fraction of beneficial mutations, preferably of those with small effect in fitness.Efforts to quantify the distribution of positive fitness effects rely on the interest to understand the dynamics of the adaptive process. Mutations of small effect are difficult to detect and quantify, so both theory and experiment have mainly addressed the tail of the distribution, where mutations with a large positive effect on fitness sit. Extreme value theory predicted an exponential shape for large effects . However\u03bc. In a first approximation, and in the absence of additional evidence, it would be advisable to maintain constant selection coefficients. As a consequence, at least in the system that we have studied, fitness equilibria are reached and maintained mainly through compensatory epistasis, and not through a variation in the average selective coefficients with fitness. This result is in good agreement with recent measurements of the same effects in populations of the bacteriophage \u03a6X174, where, moreover, the distributions of beneficial and deleterious fitness effects follow a single functional form [The results here presented could be used to design more realistic evolutionary models where an explicit representation of the microscopic mutation rate is not feasible. These suggest using phenotypic mutation fractions that increase algebraically with the genomic mutation rate nal form , as obtaS, S + Q, or S + E). One of our future objectives is to deepen the study of the universality of these and similar models, analyzing whether populations differing in their fitness values across environments, evolving towards different target structures, or incorporating a larger number of realistic phenotypic traits, still adapt through an increase in the amount of beneficial mutations with small effects on fitness, and whether the functional form of the distribution of fitness effects agrees with those obtained here.The restrictions imposed by the simultaneous selection of more than one phenotypic trait is an important subject worthy of additional analysis. Landscapes such as those introduced here could be a first step towards the quantification of constraints in evolution and adaptation of complex populations. Despite remarkable differences among the situations investigated, the distributions of effects on fitness are best explained by a unique functional form  irrespectively of the state of adaptation of the population (whether optimized or adapting) and of the fitness landscape on which the population evolves . For random number generation, we relied on the Mersenne Twister and Ziff's GFSR4 algorithms as provided by GNU Scientific Library (GSL), Version 1.7 . For secMS, EL, and SCM conceived and designed the research. MS performed the simulations. MS, EL, and SCM analyzed the data. EL and SCM performed the statistical analysis. SCM wrote the paper. All authors read and approved the final manuscript."}
{"text": "The ability for an evolving population to adapt to a novel environment is achieved through a balance of robustness and evolvability. Robustness is the invariance of phenotype in the face of perturbation and evolvability is the capacity to adapt in response to selection. Genetic robustness has been posited, depending on the underlying mechanism, to either decrease the efficacy of selection, or increase the possibility of future adaptation. However, the true effect of genetic robustness on evolvability in biological systems remains uncertain.Here we demonstrate that genetic robustness increases evolvability of thermotolerance in laboratory populations of the RNA virus \u03c66. We observed that populations founded by robust clones evolved greater resistance to heat shock, relative to populations founded by brittle (less-robust) clones. Thus, we provide empirical evidence for the idea that robustness can promote evolvability in this environment, and further suggest that evolvability can arise indirectly via selection for robustness, rather than through direct selective action.Our data imply that greater tolerance of mutational change is associated with virus adaptability in a new niche, a finding generally relevant to evolutionary biology, and informative for elucidating how viruses might evolve to emerge in new habitats and/or overcome novel therapies. Evolvability may be defined as the capacity to adapt in response to selection -3, or alUntil recently, it was controversial whether biological populations could evolve genetic robustness as posed by theory . HoweverPseudomonas syringae pathovar phaseolicola, under low versus high levels of virus co-infection , and gauging their relative ability to adapt to a novel environment. To do so, we tested whether populations founded by genetically-robust viruses were more evolvable than lineages founded by genetically-brittle viruses, in an environment that imposes strong directional selection. In particular, we explored whether robust virus lineages had the capacity to evolve faster than brittle populations, when viruses must adapt by evolving thermotolerance \u2013 resistance to the deleterious consequences of periodic heat shock.P. phaseolicola host bacteria. Bacteria-free lysates (~108 virus particles) of the wild type virus were subjected to thermal incubation at seven temperatures ranging between 35\u00b0C and 50\u00b0C, in repeated (3 <n < 5) assays. Results showed that temperatures increasingly higher than 40\u00b0C caused a progressively larger fraction of the virus population to become incapable of producing plaques on the host bacteria  of virus \u03c66 is adversely affected by virus exposure to temperatures above 45\u00b0C [10 W = log10 Wpost-bottleneck - log10 Wpre-bottleneck) was calculated for each lineage. The data revealed that variance in \u0394log10 W for the 12 lineages founded by high co-infection viruses (\u03c32 = 0.046) was over twice that of the low co-infection derived lineages . Robustness of one group relative to another can be defined as lesser variance in fitness changes brought on by mutation(s) added to the genome [10 W following mutation accumulation for lineages founded by high co-infection viruses (mean = -0.168) was over three-fold greater than that of the low co-infection derived lineages . This outcome is consistent with theory suggesting that increased mutational robustness should cause mutations to have reduced effects on mean fitness [Our prior mutation-accumulation experiment revealed that viruses which were evolved for 300 generations under low and high levels of co-infection could be generally categorized as robust and brittle, respectively . Using t \u03c32 = 0.06 was ovee genome . Thus, t fitness . AlthougS) following 5 min exposure to 45\u00b0C for all 24 strains, using repeated (n = 6) survival assays. Results  strains was 14.899 \u00b1 1.641 s.e.m, and that for the 12 high co-infection evolved (brittle) clones was 13.467 \u00b1 3.312 s.e.m. The average of these data across all 24 strains was 14.182 \u00b1 2.163 s.e.m., which did not differ according to robust versus brittle categorization . We concluded that the two groups of founding viruses were equivalent in average survival under heat shock at 45\u00b0C, regardless of their prior history of selection at low versus high co-infection.We then conducted preliminary assays to confirm that the two groups of strains  were equally poor at surviving the selective heat-shock environment. To do so, we measured the mean of percent survival (%P. phaseolicola) at low and high co-infection. Whereas the design of the original 300-generation experiment allowed pre-adsorption to cells to manipulate co-infection level  fitness assays. Consistent with earlier data for the entire 60-member collection [10 W at low MOI did not significantly differ according to prior adaptive history (low versus high co-infection) . We concluded that the intended thermal selection regime involving propagation in absence of co-infection would not bias in favor of viruses previously adapted to low co-infection conditions.We next conducted two preliminary experiments to determine whether the groups of viruses differed in fitness  fitness assays. Results showed that mean fitness did not significantly differ according to prior adaptive history (low versus high co-infection) . We concluded that the two groups of viruses were equivalent in fitness under low and high levels of co-infection (see further discussion below).For completeness, we also determined whether the two groups of founding viruses differed in performance at high co-infection, by estimating the mean logP. phaseolicola at 25\u00b0C. Thus, heat shock was imposed only every fifth generation of virus evolution. At the end of the experiment, we used replicated (n = 6) survival assays to measure mean %S at 45\u00b0C for each of the founding clones, and for each of their corresponding derived endpoint populations. Measurements of %S for a founding genotype and its evolved descendant population were conducted in parallel. The difference between the two mean values was then used to estimate \u0394%S, the change in average percent survival at 45\u00b0C following 50 generations of viral evolution with periodic heat shock.To test the formal hypothesis that evolvability correlates with robustness we used the 12 robust and 12 brittle clones as founders of independent lineages in an experimental evolution study. The 24 lineages were subjected to 50 generations (10 days) of viral evolution, involving daily exposure to 5 min incubation at 45\u00b0C, followed by five generations of growth on S for lineages founded by the robust strains was a significantly larger value than that for the populations initiated by brittle strains . Although the founding clones showed no difference in heat-shock survival prior to selection . Overall, these data provided strong support for the hypothesis that genetic robustness promotes evolvability of virus \u03c66 in a novel habitat where heat shock imposes directional selection.Results Figure  showed tS at 45\u00b0C with three-fold replication, for the founding strains and their descendant populations in parallel. Results showed that the two groups of evolved lineages in this control experiment did not statistically differ in \u0394%S at 45\u00b0C . We therefore concluded that change in resistance to heat shock was indeed an adaptive trait, and not simply a consequential response to the general conditions manipulated in our experiment to address the formal hypothesis.Aside from the imposition of periodic heat-shock, the selection experiment was designed to be highly similar to the general culture conditions that the founding clones previously experienced during their 300 generations of evolution under low versus high co-infection . We observed that the robust viruses tended to generate much higher frequencies of mutants, which suggested that robustness coincided with a higher mutation rate [P. atrofaciens and were then forced to obtain an additional mutation that allowed infection of P. tomato [Our earlier work hinted that elevated mutation rate may be a characteristic of the virus \u03c66 strains which we defined as relatively robust . In partion rate . Howeverion rate -33. Furt. tomato . The latP. phaseolicola host cell [n = 24, P = 0.355). Because variability in mutant frequency was seen among strains in these assays, we surmised that subtle differences in BHT-mutant frequency (as a proxy for mutation rate) might be associated with the observed differences in evolvability. Thus, we also examined the relationship between BHT-mutant frequency and mean adaptive change in our heat-shock experiment (\u0394%S) for the set of 24 clones. This analysis . In addition, correlation analyses conducted independently for the two groups were not significant either . These results strongly suggested that mutation rate differences among strains in our study did not influence their observed evolvability. Furthermore, these data for BHT mutant frequencies do not provide support for elevated mutation rate as the underlying mechanism governing the observed link between robustness and evolvability in virus \u03c66. However, although we cautioned against using host-range assays to discern mutation rate differences among robust and brittle clones [We identified a method for comparing mutant frequencies among robust and brittle strains of virus \u03c66 which should be less confounded by any of their underlying differences in genetic architecture in gene P3. The approach involved examining the frequency of mutants that are resistant to the chemical butylated hydroxytoluene (BHT). BHT is shown to be lethal to virus \u03c66 genotypes because it cleaves protein P3 from the virion, preventing attachment and subsequent entry of the virus into the ost cell . But oneost cell . Using ls Figure  showed ne clones , we note8 particles of a test clone to the challenge temperature (see Methods). These assayed lysates should be easily large enough to harbor rare spontaneous mutants; although the mutation rate and mutation spectrum for 45\u00b0C heat-shock resistance are unknown in \u03c66, we can expect between 10-4 and 10-6 particles should contain a point mutation for resistance to a selective challenge such as growth on a novel host [A somewhat related idea is that adaptation to the 45\u00b0C heat shock might require multiple mutational substitutions, and the robust clones evolved relatively faster because they happened to contain one or more of the needed mutations. We find this explanation unlikely, however. We observed that the initial survival of robust and brittle clones did not statistically differ at 45\u00b0C, using replicated survival assays which exposed lysates containing 10vel host ,35. ThusWe have yet to determine the exact molecular mechanism responsible for differences in genetic robustness among \u03c66 virus genotypes. However, data from other systems point to differences in protein stability as the responsible mechanism. In particular, it has been shown that proteins can be identical in shape, despite underlying sequence differences that impact protein sensitivity to the effects of mutations (i.e. robustness) . These rS at 45\u00b0C) in our selection experiment . Therefore, our data suggest that robust strains of \u03c66 are generally better at surviving moderate temperatures such as 42\u00b0C, but individual strain toleration of this temperature does not predict the adaptive success of its descendent lineage under 45\u00b0C selection. This analysis suggests that virus performance at moderate temperatures and evolvability under extreme temperature is not a trivial relationship, such as that resulting from a pre-existing mutation in a founding clone which places its descendent lineage closer to a selective optimum that is only two mutational steps away.We showed that the 12 robust and 12 brittle viruses used as founding genotypes in the current study were equally poor at surviving in the 45\u00b0C selective habitat  explanation for the transient nature of the trade-off suffered by high co-infection evolved viruses. The possibility exists that by generation 300 the high co-infection populations are no longer dominated by cheater viruses which present the trade-off; we discussed this possible outcome in a separate study . If the Genetic analyses should also prove useful for confirming the presumed role of latent genetic variation in our observed link between robustness and evolvability. Regardless of the molecular mechanism (e.g. protein stability) responsible for robustness in virus \u03c66, it is expected that a robust population should harbor more genetic diversity than its brittle counterpart. This assumption follows directly from the definition of robust genomes; i.e. they should be more easily capable of amassing neutral mutations because their robustness causes random mutations to be neutral in the current environment. All else being equal, we therefore might expect a larger number of molecular substitutions per genome in the generation-300 strains drawn from the low co-infection lineages, and a greater number of haplotypes per population in these treatment populations. Of course, the problem is that all else is not necessarily equal. Immediately above we discussed how cheaters might have been transiently successful in the high co-infection lineages, which suggests that virus-virus coevolution in these populations might have caused greater dynamic turnover of alleles and, hence, more extensive molecular evolution.Nevertheless, we acquired preliminary sequence data to examine the predicted larger number of substitutions/strain, and larger total number of haplotypes present (i.e. greater genetic diversity) in the generation-300 lineages evolved under low co-infection, which we defined as relatively robust. As part of a separate study , for all 24 strains we sequenced a 665 bp region on the large segment which is entirely protein-coding for the RNA-dependent RNA polymerase P2, a region that is thought to be highly conserved ,47. We aP0.05,4 = 0.007). We concluded that the \u03c66 populations evolved under low multiplicity showed greater genetic variability than the high MOI populations, on average, based on sequence data representing ~10% of the viral genome.As expected, very few changes were observed in the sequenced portion of the highly-conserved polymerase gene P2. Two synonymous substitutions were found in this protein-coding region of the large RNA segment: mutation N104N shared by clones L2.3 and L2.4, and mutation E255E shared by clones H1.1 and H1.7. Few changes were also observed in the coding region of the sequenced portion of gene P5 on the small segment. Here we observed two synonymous substitutions  and one non-synonymous change (A208E in clones H1.3 and H1.6). The sequenced non-coding region of gene P5 showed seven total mutations, all of which occurred in five clones drawn from populations in the low co-infection treatment: t2209c in L3.2, g2231a in L1.9, a2272g in L1.2, c2281t in L2.3 and L2.4, t2387c in L2.3 and L2.4. These five non-coding mutations explain the difference in the number of mutations observed in clones drawn from the two treatments . The data show that the low co-infection (robust) strains had an average of 1.08 substitutions/strain (13 mutations across 12 clones), compared to 0.58 substitutions/strain (7 mutations across 12 clones) for the high co-infection (brittle) strains. The sequence data also allowed us to estimate the inferred number of haplotypes per evolved population: L1, 3; L2, 3; L3, 3; H1, 2; H2, 1; H3, 1. The difference between robust and brittle populations in the inferred number of haplotypes was found to be statistically significant  was the host bacterium used in all experiments, and culture conditions are previously described [P. phaseolicola lawn for 24 hr; plaques were then collected and filtered  to remove bacteria. Virus lysates were stored at -20\u00b0C in 4:6 glycerol/LC (v/v).Virus \u03c66 is a lytic bacteriophage which contains a ~13 kb double-stranded RNA genome divided into three segments per particle . The curescribed ,55. Bact8 particles) in a PCR tube, followed by immediate sampling onto a host lawn to confirm the initial virus titer (Ni). The lysate was then placed in a preheated Eppendorf thermocycler for 5 min incubation, followed by sampling onto a host lawn to measure final titer (Nf). Percent survival (%S) equaled (Nf/Ni) * 100. Thus, survival at a test temperature was gauged through plaque-forming units (pfu), the phage particles that were viable for growth at 25\u00b0C.Temperature survival was assayed by placing 120 \u03bcl of a virus lysate  bacteria. This propagation scheme was repeated for 10 consecutive days. Because the overnight plaque growth corresponded to 5 viral generations [Virus lineage adaptation to heat shock occurred through experimental evolution involving periodic exposure to high temperature Figure . A lineaP. phaseolicola of a test genotype (or population) relative to a common competitor of \u03c66 bearing a genetic marker. In some assays the marked competitor was a host-range mutant containing a substitution in gene P3 of the medium RNA segment, allowing infection of P. pseudoalcaligenes bacteria. In other assays the competitor was an engineered mutant bearing the alpha subunit of the Escherichia coli beta-galactosidase  gene on the large RNA segment [P. phaseolicola lawn. Pre-adsorption to cells before plating was only allowed if the fitness assay examined impact of co-infection on relative fitness. After 24 hr incubation, the ~400 plaques were harvested and filtered to obtain a cell free lysate. When the common competitor was a host-range mutant, the ratios of competing genotypes in the starting mixture (R0) and in the harvested lysate (R1) were obtained by plating on mixed lawns of P. phaseolicola and P. pseudoalcaligenes (200:1 mixture), where ordinary and host-range genotypes form turbid and clear plaques, respectively. When the common competitor contained the \u03b2-gal gene insertion, ratios were tracked by plating on lawns of P. phaseolicola bacteria containing the beta subunit of the \u03b2-gal gene, allowing the marked competitor to produce blue plaques on agar containing X-gal (0.4% w/v). Fitness (W) was defined as the relative change in ratio of ordinary to marked virus, or W = R1/R0.Fitness assays consisted of paired-growth experiments , which c segment ; fitness10 viruses per mL) of a virus genotype was grown and titered on P. phaseolicola, and a sample of the lysate was then placed in 0.12 mM BHT for 5 min. Mutant frequency was calculated as the number of plaque-forming mutants per viruses in the inoculum following BHT exposure.We measured the frequency of spontaneous BHT-resistant mutants that occurred within virus populations grown in the absence of the chemical. A high-titer lysate .Genomic extraction was performed using QIAamp Viral RNA minikits (Qiagen). The dsRNA genome was reverse transcribed using random hexamer primers  and resultant cDNA was used as PCR template (primers available on request). PCR products were purified using the QIAquick PCR Purification kit (Qiagen) or ExoSAP-It . Sequencing was performed using the BigDye Terminator reaction v3.1 on an ABI 3100. Sequence reads were curated by eye using Sequencher software  and compared using ClustalX RCMcB, CBO and PET were involved in the conception, design and writing of this study. RCMcB conducted the experiments and analyses. All authors read and approved of the final manuscript."}
{"text": "E. coli requires the accumulation of multiple mutations, including those that alter target genes and genes regulating drug efflux. Previous studies have shown some drug-resistance mutations reduce bacterial fitness, leading to the selection of fitness-compensatory mutations. The impact of fluoroquinolone-resistance on bacterial fitness was analyzed in constructed isogenic strains carrying up to 5 resistance mutations. Some mutations significantly decreased bacterial fitness both in vitro and in vivo. We identified low-fitness triple-mutants where the acquisition of a fourth resistance mutation significantly increased fitness in vitro and in vivo while at the same time dramatically decreasing drug susceptibility. The largest effect occurred with the addition of a parC mutation (Topoisomerase IV) to a low-fitness strain carrying resistance mutations in gyrA (DNA Gyrase) and marR (drug efflux regulation). Increased fitness was accompanied by a significant change in the level of gyrA promoter activity as measured in an assay of DNA supercoiling. In selection and competition experiments made in the absence of drug, parC mutants that improved fitness and reduced susceptibility were selected. These data suggest that natural selection for improved growth in bacteria with low-level resistance to fluoroquinolones could in some cases select for further reductions in drug susceptibility. Thus, increased resistance to fluoroquinolones could be selected even in the absence of further exposure to the drug.Fluoroquinolones are antibacterial drugs that inhibit DNA Gyrase and Topoisomerase IV. These essential enzymes facilitate chromosome replication and RNA transcription by regulating chromosome supercoiling. High-level resistance to fluoroquinolones in  The increasing frequency of human pathogens resistant to important classes of antibiotics poses a serious and growing challenge for medicine and society. We need improved strategies to reduce the rate of resistance development, for established and novel drugs, based on knowledge of the factors that drive the increase in resistance. Resistance to fluoroquinolones in most bacteria develops via a series of sequential genetic changes affecting several different genes. These are selected and enriched in bacterial populations by exposure to the drug. Relevant factors driving this increase include overuse, and inappropriate use, of these drugs. In this paper we show that mutant bacteria with low-level resistance (not itself a problem to treat with standard drug doses) can evolve by natural selection (for improved growth rate) to acquire mutations that dramatically increase their level of drug resistance. This means that we may need to consider how to reduce inappropriate drug use that can enrich for bacteria with low-levels of resistance, because at that stage some of the mutant bacteria in the population may continue to evolve higher level resistance even in the absence of any further drug exposure. E. coli, but resistance is increasing with 28 of 29 countries in Europe reporting a significant rise between 2001\u20132007 E. coli require multiple genetic changes, including mutations altering topoisomerases and up-regulating drug efflux in vitro and in vivoE. coli increased from \u223c7\u201319% between 2001 and 2007 in the UK Fluoroquinolones are potent antibacterial drugs E. coli for decreased susceptibility to fluoroquinolones most lineages (16/18) suffered reduced growth competitiveness after only two or three selection steps Streptococcus pneumoniae carrying one or two resistance mutations in vivo in not clear. To examine the phenomenon we constructed isogenic strains carrying various combinations of five resistance mutations found commonly in fluoroquinolone-resistant clinical E. coli, and measured their drug-susceptibility and fitness. The relationships between the number of resistance mutations and bacterial fitness were complex and the addition of a resistance mutation was shown in some cases to improve bacterial fitness. These findings have implications for the evolution of fluoroquinolone resistance in the absence of antibiotic exposure.The fitness costs of drug resistance can be reduced by selection of low-cost mutations or by the accumulation of secondary fitness-compensating mutations that do not reduce resistance. During experimental evolution of clinical isolates of E. coli urinary tract infection isolate C1186 gyrA Ser83\u2192Leu, Asp87\u2192Asn; parC Ser80\u2192Ile), and up-regulating drug efflux . These 5 mutations are typical of highly resistant clinical isolates gyrA, parC) or constructed by \u03bb-red recombineering  and then separately introduced into MG1655 by P1 transduction. The gyrA and parC mutations were transduced by selection for a linked genetic marker , and all strains with 4 or 5 mutations had MIC's above the 1 \u00b5g/ml breakpoint that defines clinical resistance in Europe MIC for ciprofloxacin was measured for each strain . The margyrA S83L, gyrA D87N, and parC S80I) were statistically neutral whereas others (\u0394marR and \u0394acrR) caused a significant reduction in fitness . Strains carrying three resistance mutations had fitness values that ranged from \u223c1 down to as low as 0.60, with 8/9 strains suffering a fitness deficit of \u22655% per generation. Interestingly, the addition of a fourth resistance mutation to these strains restricted the minimum fitness value to 0.66 per generation, higher than that measured with three mutations  has high level resistance (MIC 32) with no significant reduction in fitness relative to the wild-type  in two of the three cases, LM695>LM707 and LM882\u2192LM707 , and the average fitness of all strains carrying marR is 0.76 (SD 0.08). MarR protein regulates, directly and indirectly, transcription of many genes mar is most likely because of disruption of gene regulation. This suggests a possible mechanism of fitness compensation associated with resistance mutations in topoisomerase genes. Thus, some topoisomerase mutations that alter chromosomal supercoiling levels may partially restore expression of growth-limiting gene(s) regulated by mar, reversing the fitness deficit. This hypothesis predicts that the improvement in fitness measured in LM695\u2192LM707 should be associated with a change in global supercoiling level. This was tested by electrophoresis of pUC18 and pUC19 plasmids purified from MG1655, LM695 and LM707 in chloroquine-agarose gels topA and pgyrA) whose expression is sensitive to, respectively, increased or decreased supercoiling levels topA / pgyrA) of reporter gene expression from these two promoters, defined as the quotient of supercoiling (Qsc), is a measure of the relative level of negative supercoiling in isogenic strains gyrA promoter construct in LM707 relative to LM695. Thus the introduction of the parC S80I mutation to make strain LM707 caused a significant increase in the Qsc, coincident with the improvement in fitness. Although we cannot explain the absence of an effect in the chloroquine-agarose assay, the reporter gene assay suggests that some changes in chromosomal supercoiling associated with the acquisition of topoisomerase mutations may provide a mechanism linking bacterial fitness and decreased susceptibility to fluoroquinolones. However, an alternative explanation is that the combination of mutations in LM707 reduces expression of gyrA by a mechanism that does not change global supercoiling levels, and that it is a consequence of the reduced expression of gyrA that causes the increase in growth fitness. Additional experiments will be required to distinguish between these models.The  strains . The marparC S80I mutation into LM695 increased its MIC for ciprofloxacin and its competitive growth fitness versus the wild-type. This predicted that the resulting constructed strain, LM707, should outcompete LM695 in a head-to-head competition, and also raised the following questions: (i) could a mutant with an increased MIC for ciprofloxacin and a higher growth fitness be selected spontaneously from LM695 in the absence of drug; and (ii) would such a mutant be exclusively associated with of the acquisition of the parC S80I mutation. These predictions and questions were addressed experimentally. First, in head-to-head growth competition experiments in LB medium with no drug, LM707 (four resistance mutations) outcompeted LM695 (three resistance mutations), gaining \u223c8% per generation, in good agreement with the relative differences in growth fitness of each strain versus the wild-type  in a volume of 200 \u00b5L. An aliquot of 5\u00d7106 cfu from each lineage was tested, after the completion of 2, 3, and 4 cycles of growth, for the presence of resistant mutants on solid medium . Growth of LM695 is completely inhibited on 3 \u00b5g/mL ciprofloxacin and the spontaneous mutation rate to resistance on this media, measured in fluctuation tests, is 2\u00d710\u22128. Accordingly, we expected that virtually none of the 96 independent lineages would contain a resistant mutant at the initiation of the experiment, but that a small number of resistant mutants would arise in each lineage during each growth cycle. If these mutants out-competed the parental LM695 they would be expected to increase relative to LM695, and thus have a greater probability of being transferred to the next growth cycle. The number of lineages from which resistant mutants were obtained was found to increase with successive growth cycles: from 2/96 (cycle 2)\u21928/96 (cycle 3)\u219215/96 (cycle 4). Because the spontaneous mutation rate to resistance (2\u00d710\u22128) if much lower than the number of cells being tested from each lineage (5\u00d7106) it is very unlikely that these mutants arose on the selective media. Instead, the most reasonable conclusion is that the resistant mutants arose spontaneously and randomly during the growth of lineages in the absence of drug and were enriched because they out-competed the parent population. Three random drug-resistant mutants were chosen from independent lineages and tested by DNA sequencing for the presence of mutations in gyrA and B, in parC and E, and in marR and acrR. In each case the mutations originally present in LM695 were confirmed and in addition each of the mutants was found to have acquired a new mutation in parC . The MIC CIP of each of the mutants had increased from 0.75 to >32 \u00b5g/mL, and the exponential doubling time in rich medium had increased significantly, by \u223c10% per generation  could also be generated by a variety of spontaneous mutations, and that the growth advantage phenotype could be enriched by growth selection in the absence of drug.In the sections above it was shown that the transfer of the ild-type . Second,neration . Thus, tE. coli strains was constructed and used to measure the relationship between the accumulation of fluoroquinolone resistance mutations, drug susceptibility, and growth fitness. The question was whether the accumulation of up to five resistance mutations, commonly found in resistant clinical isolates, would progressively reduce bacterial fitness. Most of the published data on the relationship between drug resistance and bacterial fitness would predict two possibilities: (i) that these mutations would cause little or no fitness cost, explaining their high frequency among resistant isolates; or (ii) that their accumulation would cause a progressive decrease in bacterial fitness, and require additional fitness-compensating mutations to restore fitness A set of 28 isogenic The main conclusions from the data set were the following:marR mutation had the largest negative effect on fitness.Some resistance mutations, individually or when incorporated in an already mutant strain, reduced drug susceptibility without significantly reducing fitness. Other mutations were individually or in combination associated with significant fitness costs. This suggests that selection for low-cost mutations, and/or compensating mutations could be selective forces determining the frequency of particular mutations individually or in combination. The and an increase in relative fitness . Thus, the combination of target mutations studied here is typical of resistant clinical isolates. As measured by organic solvent tolerance, drug efflux was phenotypically up-regulated in 15/30 of these resistant isolates, associated in most cases with mutations in acrR and/or marORThe particular How fluoroquinolone resistance evolves in nature will depend on the genotype being selected and on the selective environment E. coli K12 MG1655 wild-type was the starting strain for all constructions (C1186 is a multiply mutant fluoroquinolone resistant UTI isolate previously described ructions . Liquid www.clsi.org).MIC was determined by Etest  on Mueller-Hinton agar plates incubated for 16 to 18 h at 37\u00b0C with quality control reference strains gyrA and parC were selected sequentially in E. coli MG1655 in LB with ciprofloxacin at 2\u20138-fold MIC and mutations were identified by DNA sequencing. Individual mutations were always moved into a clean genetic background (MG1655) after initial selection. Deletion-replacement mutations in marR, acrR, yfaH, metC and araB were made by \u03bb-red recombineering + Nad+ derivative of DY329) using PCR amplified linear DNA from pCP16 with an FRT-bounded TcR cassette Spontaneous resistance mutations in marR and acrR was selected directly on LA+Tc. When transducing the gyrA and parC mutations selection was made for the linked markers, yfaH<>Frt::TcR::Frt and metC<>Frt::TcR::Frt, each \u223c10 kb from gyrA and parC, respectively. Note that we are using the symbol <> to indicate a replacement generated by \u03bb red homologous recombination technology. After transduction the TcR marker was removed by Flp-catalyzed excision expressed following transformation with pCP20 Isogenic derivatives of MG1655 were constructed by phage P1-mediated transduction. Transduction of the deletion-replacement mutations in araB<>FRT) was used as the standard wild type strain in growth competitions against which each of the constructed mutant strains was competed. This strain was tested against its parent MG1655 showing that the \u0394araB mutation was neutral (relative growth rate 1.002\u00b10.005). To support statistical analysis each competition was tested in at least 5 independent experiments  was made the number of times indicated in ara+) and white (\u0394araB) colonies were scored. The change in the ratio of mutant/wild-type was used to estimate the selection coefficient per generation of each of the constructed strains according to the formula: S\u200a=\u200aln2 (mutant/wild-type) / generation S+1. Note that the fitness defects could be due to defects at any stage of the growth cycle .LM347  at 10-min intervals, using a BioscreenC machine .Exponential doubling times were calculated by measuring the increase in optical density at 600 nm  is 2\u00d710topA-luc, and, pgyrA-luc, The relative supercoiling degree was determined using a published assay. The quotient of supercoiling, Qsc Table S1Oligonucleotides used to amplify linear PCR fragments for \u03bb-red recombineering(0.03 MB DOC)Click here for additional data file.Text S1The ascending urinary tract infection model in mice.(0.03 MB DOC)Click here for additional data file.Text S2Supercoiling assay.(0.03 MB DOC)Click here for additional data file."}
{"text": "The role of mutation rate in optimizing key features of evolutionary dynamics has recently been investigated in various computational models. Here, we address the related question of how maximum mutation size affects the formation of species in a simple computational evolutionary model. We find that the number of species is maximized for intermediate values of a mutation size parameter \u03bc; the result is observed for evolving organisms on a randomly changing landscape as well as in a version of the model where negative feedback exists between the local population size and the fitness provided by the landscape. The same result is observed for various distributions of mutation values within the limits set by \u03bc. When organisms with various values of \u03bc compete against each other, those with intermediate \u03bc values are found to survive. The surviving values of \u03bc from these competition simulations, however, do not necessarily coincide with the values that maximize the number of species. These results suggest that various complex factors are involved in determining optimal mutation parameters for any population, and may also suggest approaches for building a computational bridge between the (micro) dynamics of mutations at the level of individual organisms and (macro) evolutionary dynamics at the species level. H. Sturtevant posed this question from above are perhaps harder to untangle. A source of variation is obviously necessary for the process of natural selection. On the other hand, too high a mutation rate has obvious negative consequences optimum amount of variability?The origins of limits on mutation rates Drake's studies of microbial genetics showed similar mutation rates across a wide range of genome sizes current organism on the basis of that organism's descendants' increased ability to radiate into new ecological niches. In other words, the fact that a higher mutation rate will help later generations does not explain how it can be selected for in the current generation, for which it does not have a clear advantage. As a result, mutations in genes that control mutation rate may often hitchhike along with other mutations that confer immediate selective advantage The question of mutation rate optimization is compounded by the problem of causality, to the extent that an increased mutation rate cannot be selected for in a whether an intermediate mutation rate can optimize fitness. In the present paper, we investigate a closely related problem, the optimization of the number of species as a function of maximum mutation size. Since we pose similar questions and take a similar approach to those of the three papers just cited \u2013 though in the context of speciation rather than of individual fitness \u2013 we briefly review each of those works.Several recent computational studies have addressed the problem of optimum mutation rate. Three of these studies, Bedau and Packard novelty (adaptability) and evolutionary memory (adaptedness). They explored the optimization of fitness as a function of mutation rate in a model of reproducing agents on a toroidal lattice; the agents consumed energy from a \u201ccontinually augmented external source\u201d, and reproduced when they had accumulated sufficient resources to split into two organisms. The organisms were characterized by their strategies of interacting with the environment and gathering resources; their fitness, defined as the amount of food gathered, was found to be optimized for an intermediate mutation rate, where the mutation rate represented the rate at which strategy elements were selected from a pool of possible behaviors. Bedau and Packard also performed simulations in which agents with different mutation rates competed against each other; they found that those with a specific intermediate mutation rate were the most successful. Using measures of diversity, they also demonstrated that \u201cthe mutation rate governs a transition between two qualitatively different phases of evolutionary dynamics\u201d, namely a more ordered state characterized by long periods of stasis for low mutation rates, and highly disordered dynamics where \u201cthe gene pool tends to be a continually changing plethora\u201d of strategies at high mutation rates Bedau and Packard different evolvability characteristics can be optimal under different circumstances. This result was already suggested by the bacteriophage and Drosophila studies cited above; it appears again in another computational evolutionary model, that of Clune et al. Earl and Deem is optimal, it may not be reachable via a natural selection algorithm operating on a highly rugged landscape. The authors interpret this in terms of the short term vs. long term cost of mutation rates: a high mutation rate would provide the benefit of rare large mutations that could \u201ccarry the organism over a valley to the next fitness peak\u201d, but would also exact a high energetic cost due to the occurrence of mutations that are \u201cnot quite large enough\u201d, costing the organism dearly, but leaving it stranded in the valley it was trying to escape. Clune et al. contrast their results with studies suggesting that high mutation rates can be optimal in all circumstances, commenting that \u201cit seems unlikely that stably high mutation rates, such as those for RNA viruses, are maintained primarily because of the rapid adaptive capacity they bestow, as has sometimes been argued\u201d Clune et al. The three studies summarized above address the optimization of a measure of individual organismal fitness. However, individual fitness is not the only quantity that can be optimized by natural selection. Darwin himself explored the idea that diversity itself may be selected for, and that phyla that are better at radiating may also be better at flourishing. This can be envisioned as an optimal filling of morphospace or, in more nineteenth-century terms, as a \u201cBenthamite optimization calculus\u201d variability, heritability and overpopulation, in the simplest possible manner. Organisms exist in a two-dimensional morphospace, where each axis represents a hypothetical phenotype. At each time step, a new generation of organisms is produced via an assortative mating algorithm. The number of new organisms depends on an underlying fitness landscape; the locations of new organisms in the morphospace are determined by the locations of their parent organisms, as well as by the mutation size. We investigate the clustering of organisms, where clusters are defined as reproductively isolated groups and serve as an analogue of species, as a function of maximum mutation size.The motivation for the design of the present model, implemented in MATLAB, was to incorporate the three fundamental aspects of Darwinian evolution, Simulated organisms exist on a landscape in a two-dimensional morphospace, with the x- and y-coordinates corresponding, respectively, to a given organism's two traits. This is illustrated in The model uses assortative mating, whereby, in each generation, every organism picks the nearest other organism in the landscape and mates with it to produce new organisms for the next generation. The choice of an assortative mating scheme is motivated at once by its simplicity and its realism. Recalling that the organisms exist in a morphospace rather than a physical space, it should be immediately apparent that the simplest realistic mating scheme is one in which phenotypically similar organisms mate with each other rather than with more phenotypically distant organisms.Assortative mating schemes have been extensively used in various studies, such as investigations of the mechanisms of sympatric cx1,cy1) and  are the morphospace coordinates of the two parent organisms, the coordinates  of a next-generation organism are given byuniform distribution between 0 and 1, and where \u03bc represents the maximum possible mutation size.If  of a new organism are calculated as follows:normal distribution with zero mean, and b\u200a=\u200a0.1581. Again, \u03bc represents the maximum mutation size. Thus, for both versions of the model, the coordinates of each new organism are randomly chosen to lie within a range defined by the coordinates of the parents, but with the boundaries of the range extended by the parameter \u03bc. Note that \u03bc can be easily related to a mutation rate M by integrating the rate over (generation) time. Thus,M is constant, then the maximum mutation size \u03bc will be directly proportional to the mutation rate M.In another version of the experiment, the coordinates  shifting gradually or (2) being altered by feedback from the local density of organisms.n+1 position, leaving a \u201chole\u201d at the first column of the matrix, which is replaced by 12 new, randomly-generated values, after which interpolation is performed again to generate a 45\u00d745 landscape. These operations have the effect of shifting the landscape gradually to the right. The parameter \u03bb was set at 2 throughout the simulations shown here.For the randomly shifting landscape, every \u03bb generations, the last column is deleted from the 12\u00d712 random matrix underlying the fitness landscape. The other n columns are shifted to the For landscapes modulated by feedback, in every generation, the fitness value at each location in the landscape grid was decreased by an amount proportional to the number of organisms living in the region. For the models implemented here, the proportionality was set at 0.0071. These reactive changes in the landscape symbolize the depletion by over-use of the available resources in a given ecological niche. The total summation of fitness values available across the entire landscape was conserved in each generation; this was done by adding back the entire subtracted quantity after dividing it equally amongst each of the 144 elements of the fitness landscape matrix (before interpolation). Through this method, areas in the morphospace which were unaffected by the subtraction (areas whose resources were not depleted) become increasingly advantageous for reproduction.In order to introduce further randomness into the model, a fraction \u03c1 of the new organisms are randomly eliminated before the start of the new generation, where, for each generation, \u03c1 is a random number selected from a uniform distribution between 0 and 0.70. The effects of overpopulation are implemented by setting a distance limit within which only one organism can exist. In all the implementations of the model shown here, the overpopulation limit is set at 0.25.In order to investigate the interactions between organisms with different mutation parameters, the model was modified so that each organism was assigned not only coordinates in the morphospace, but also a distinct value of \u03bc . The mutation parameter \u03bc for each individual in the initial population was selected randomly from a uniform distribution between 0 and 1. Note that the choice of a maximum value of \u03bc\u200a=\u200a1 is motivated solely by the range of \u03bc values used in other simulations shown here; there is no a priori limit for the value of \u03bc.The simulation was performed identically to the other simulations , except that each new organism takes the \u03bc value of one of its parents.biological species, in which species are defined as reproductively isolated groups, i.e., groups with the ability to interbreed, developed by Dobzhansky, Mayr and others in the early days of the modern synthesis Clusters, the analog of species in our model, are determined by who mates with whom. The development of this algorithm was motivated by the concept of As implemented here, the clustering algorithm is carried out as follows. For a given organism in a given generation, a search is performed to find all the organisms that it, as well as its nearest neighbor (its mate) and its second-nearest neighbor, have mated with. Then a similar search is performed for each of the organisms found during this first search. This iterative search continues until a closed set \u2013 a cluster \u2013 is obtained, where all organisms within the set have mated, in that generation, only within the set. This algorithm assigns each organism to one, and only one, cluster, and arrives at a unique solution for each generation.inspired by, rather than being an explicit implementation of, the biological species concept. Several points need to be mentioned in this regard.It should be clarified that the definition of clusters implemented here is based on \u201cwho does mate with whom\u201d rather than \u201cwho can mate with whom\u201d, and thus we have described it as being can be interpreted as defining \u201cwho can mate with whom\u201d. Consider one individual in the cluster, and its mate. A third organism which mates with the mate is also included in the cluster, and therefore the first individual chosen to seed the cluster could presumably mate with this third individual, under an expanded version of our assortative mating criterion. To this extent, we do indeed implement a criterion of \u201cwho could mate with whom\u201d.First, let us consider the extent to which our cluster definition top-down definition of clusters . Such a top-down definition would undermine the crux of the approach taken here, which is to capture fundamental dynamical features which emerge naturally from a model satisfying certain basic criteria of evolving systems.A second point to be emphasized is that a more explicit implementation of the rigorous definition of biological species would necessitate a After all the parent organisms in a given generation have produced a new generation of organisms and some of the new organisms have been culled, the parent organisms vanish and a new generation begins, with the previous offspring now playing the role of parents. In the implementation of the model used here, the initial generation consisted of 300 individuals randomly placed within the landscape; during subsequent generations the population fluctuated between several hundred and nearly ten thousand organisms.Five experiments were performed: (1) uniformly distributed mutations with shifting fitness landscape, (2) uniformly distributed mutations with feedback-modulated landscape, (3) normally distributed mutations with shifting fitness landscape, (4) normally distributed mutations with feedback-modulated landscape, and (5) competition between organisms with different maximum mutation parameters (\u03bc) on a shifting fitness landscape with uniformly distributed mutation values within the limits set by \u03bc.Experiments (1)\u2013(4) were run for a range of values of \u03bc, with all other parameters held constant. For each experiment and at each value of \u03bc, the simulation was allowed to run for 1000 generations; in some cases, the population became extinct before 1000 generations were reached. Over the course of each simulation, various parameters were recorded at each generation, such as the total population size, the number of clusters, the mean distance between individuals in a cluster, etc. For each experiment, five runs were performed at each value of \u03bc.The results of a typical simulation after 1000 generations are shown in In some cases, simulations with identical parameters exhibited a high degree of historical contingency, as illustrated in In In order to obtain a measure of the diversity within species/clusters, we calculated the mean Euclidean distance in the morphospace between individuals in a cluster. This value was averaged over all clusters and all generations to give the within-cluster diversity at a given \u03bc. This diversity measure is shown, averaged over five realizations of the model at each value of \u03bc, in the time series of the population size and the time series of the number of clusters. At values of \u03bc for which the population size and number of clusters sharply increase, however, the correlation coefficient drops, and also shows an increase in variability from one simulation run to another, indicated by the increased standard deviation. For some runs, there is a positive correlation between population and clusters; for others, a negative correlation. As \u03bc reaches values corresponding to the population plateau, a consistent anticorrelation is observed between the two quantities.does not preclude others from doing the same. A traced organism has the original labeled organism as one of its ancestors, but this does not imply that this was its only ancestor. The fact that the final fraction of traced organisms increases with \u03bc is likely a result of the increased mixing of the population as \u03bc increases.In order to investigate the spread of an organism's descendents through the population, a random organism of the initial generation was labeled, and its descendents traced through subsequent generations. The maximum ratio of labeled organisms to total organisms is illustrated in different values of \u03bc dominate in different runs of the simulation, and the surviving \u03bc  that gives a maximal number of species for the shifting landscape model with uniformly distributed mutations  will increase the width of the normal distribution of mutation sizes, shifting the plots to the left.The model studied here is remarkably robust across various modifications, including changes in how the landscape varies  and in the distribution of mutation sizes . However, some differences do occur between the various implementations of the model. For example, the cases where mutation sizes are normally distributed  and 6 exA second difference among the different versions of the model concerns the sharpness in the drop of the correlation between population size and number of clusters. For the models without feedback  and 5d torder parameters characterizing the system. Not only do these parameters exhibit sharp changes in their mean values as a function of \u03bc, but they also exhibit large fluctuations  during the transition, another characteristic of phase transitions. It is possible that further investigation of this and related models from such a statistical physics perspective may be of critical importance in understanding the role of mutation rate and mutation size in modulating speciation. Moreover, such models may eventually begin to touch upon a fundamental problem of evolutionary biology: the bridge between micro  and macro  levels. The phase transition behavior observed here is particularly tantalizing in this regard, since a key characteristic of phase transitions is the co-existence of multiple scales of behavior. Further, in the model presented here, properties at the individual level (such as the parameter \u03bc) affect the global dynamics of the entire population, both by modulating the formation of clusters, and modulating the average properties of these clusters. It should be emphasized that this relation between scales is fundamentally in the spirit of statistical physics, where microscopic dynamics determine global, macroscopic behavior.For all versions of the model, sharp changes are observed in various quantities characterizing the system as \u03bc is varied. These sharp changes are strongly reminiscent of phase transitions, with population size, number of clusters and population-cluster correlation serving as If an intermediate mutation parameter maximizes the number of clusters, does this mean that this value of \u03bc is optimal? The competition experiment illustrated in It is striking that multiple stable populations with different values of \u03bc have not been found to coexist in this model. However, the simulations did not allow for the reemergence of extinct mutation values, and thus perhaps the eventual dominance of a single value of \u03bc may be inevitable, especially given the system's tendency in the limit of a large number of generations to exhibit a fully mixed population where all organisms share at least one common ancestor (as shown in"}
{"text": "It is commonly thought that large asexual populations evolve more rapidly than smaller ones, due to their increased rate of beneficial mutations. Less clear is how population size influences the level of fitness an asexual population can attain. Here, we simulate the evolution of bacteria in repeated serial passage experiments to explore how features such as fitness landscape ruggedness, the size of the mutational target under selection, and the mutation supply rate, interact to affect the evolution of microbial populations of different sizes.We find that if the fitness landscape has many local peaks, there can be a trade-off between the rate of adaptation and the potential to reach high fitness peaks. This result derives from the fact that whereas large populations evolve mostly deterministically and often become trapped on local fitness peaks, smaller populations can follow more stochastic evolutionary paths and thus locate higher fitness peaks. We also find that the target size of adaptation and the mutation rate interact with population size to influence the trade-off between rate of adaptation and final fitness.Our study suggests that the optimal population size for adaptation depends on the details of the environment and on the importance of either the ability to evolve rapidly or to reach high fitness levels. Understanding the factors that influence the evolution of microbial populations not only provides fundamental insights into evolutionary processes -4, but iIt is widely believed that the higher supply of beneficial mutations allows large asexual populations to adapt more rapidly to new environments compared to small populations -13. HoweOn the other hand, a small population will generate only a subset of all possible 1-step beneficial mutations, with few mutations that confer large fitness effects ,15,16. BN0 identical clones. The bacteria go through D rounds of division, and each bacterium produces offspring depending on fitness, f, as 2f. After D divisions, serial transfer, modeled as multinomial sampling, reduces the population size back to N0 which initiates another round of exponential growth. This procedure is iterated until the desired number of generations is reached. Because bacterial death is ignored, the only way a given clone can be eliminated is if it is not sampled during serial transfer [We simulate the evolution of bacteria as they undergo repeated cycles of growth and serial dilution ,14. At ttransfer ,18.L mutants that can be reached by a single mutation. The ancestral clone is assigned a fitness value of 1 and the fitness values for the L mutants are 1 + si, with values for si drawn from an exponential distribution p(s) = \u03b1e-\u03b1s [L 1-step mutants is generated with a probability \u03bc. Whenever a new mutant is generated, it obtains its own neighborhood of L 1-step mutants with fitness values of 1 + si , the 1-step neighborhood of a newly created clone with fitness 1 + si can generate mutants that have higher or lower fitness, depending on the randomly chosen values of si for these mutants. This generates a potentially rugged fitness landscape with multiple peaks. L can be interpreted as the mutational target size of selection, or more generally the number of possible beneficial mutations from a given starting genotype.Every clone is assigned a 1-step neighborhood of  = \u03b1e-\u03b1s -23. ThisL-mutant neighborhood is chosen, we can tune the fitness landscape from one that is completely smooth to one that is completely rugged. For the smooth landscape, each newly created mutant is assigned a mutant neighborhood that is identical to that of the ancestral strain. In other words, the mutation does not alter the fitness effects of any subsequent mutations that might be obtained. Under these conditions there is a single fitness peak. At the other extreme, for a completely rugged landscape, every new mutant is assigned an entirely new L-mutant neighborhood with values for the fitness effect of each new mutation re-sampled from p(s). This means that a new mutation changes the fitness effects of all other possible mutations. In this scenario there is no correlation between the fitness effects of the L-mutants from a parent clone and those available to its mutant offspring. By changing the fraction, r, of the L sites that are replaced, we can tune the amount of ruggedness of the landscape from smooth (r = 0) to completely rugged (r = 1). By considering a broad range of values for r and L, we can explore a range of scenarios in order to identify conditions where changes in parameters lead to qualitative changes in adaptation. Figure r = 0.6.By adjusting the rules for how the fixed fitness landscape, rather than to differences arising from the fact that different lineages experience distinct ecological or genetic conditions [While the ability to tune between smooth and rugged fitness landscapes is analogous to that of previously studied NK-models and their variants -31 therenditions ,33. The local fitness peak, where, if asexual, they can become trapped. In contrast, a smaller population size allows for more stochastic trajectories on the fitness landscape, and this can occasionally lead to higher fitness peaks. The transition from more stochastic to more deterministic trajectories occurs as the mutation supply rate, S, becomes so large that a population is able to completely sample all possible 1-step beneficial mutations, i.e. if S \u2248 L [S = Ne\u03bc. For the three initial population sizes we consider here, N0 = 102, 104 and 106, an effective population size given by Ne \u2248 DN0 [\u03bc = 10-6, the mutation supply rates are Ss = 0.001, Sm = 0.1 and Sl = 10 for the small, medium and large populations respectively. We initially choose the size of the 1-step neighborhood to be L = 50, which means Sl \u2248 L, Sm <L and Ss \u00ab L. Thus we expect the large population to evolve mostly deterministically, while the medium population is expected to evolve somewhat slower, but with the potential of reaching higher fitness peaks. Because the small populations have Ss \u00ab 1, they are expected to operate in the strong selection weak mutation limit, where evolution will be slow because it is limited by the infrequent creation of beneficial mutations [On a smooth fitness landscape, all populations will eventually reach the sole fitness peak, with the larger populations doing so more rapidly. However, this can change during adaptation on a rugged landscape, as explained above. Here, large populations are expected to evolve almost deterministically. This allows them to quickly reach the highest if S \u2248 L . The mututations ,36.r = 1). An example of the results for a single landscape is shown in Figures For each of the three population sizes, we simulated 100 evolutionary trajectories for 50 different rugged fitness landscapes , populations of intermediate size retain their ability to sample the fitness landscape more broadly and to reach higher fitness peaks than large populations. However, as the landscape becomes smoother (r = 0.1), this advantage disappears. This is expected, since for a completely smooth landscape (r = 0), there is only a single globally optimum peak which would be reached by all populations eventually, simply more rapidly by the larger ones. The small populations are less affected by the change in ruggedness because they are still far away from any peak on which they could become stuck. This is confirmed by the mean final ranks  peak, compared with the L = 50 situation (see Rank and CV in Table L = 5 with L = 50). For small L, the small populations are less disadvantaged in terms of adaptive \"speed\" and are able to more frequently, although still quite rarely overall, reach higher fitness peaks than larger populations .For instance for L = 500) scenario, evolution for the large population becomes markedly more stochastic (see CV in Table L = 50 with L = 500). This supports the intuitive understanding that if more beneficial mutations are accessible, the population size that optimizes the trade-off between the speed of adaptation and the magnitude of the adaptive response shifts towards larger populations. Indeed, in the limit of L \u2192 \u221e, every clone has access to all possible other mutants, in essence reducing the system to a smooth landscape on which the large populations are always favored [For the large ( favored ,34.S, and the mutant neighborhood, L, are relevant for determining whether adaptation tends to be dominated by stochastic or deterministic change. The mutation supply rate is the product of population size and mutation rate. It is known that population size and mutation rate can have differential effects on the evolutionary dynamics [Above, we explained how the relation of the mutation supply rate, dynamics ,40,41. FWe therefore examined how changing mutation rate and population size, while keeping mutation supply rate fixed, influenced the results of our simulations. As Figure It is generally accepted that large populations will tend to evolve more rapidly than smaller ones. This is caused by two related factors. First large populations have an increased supply of beneficial mutations each generation, which decreases the waiting time for new advantageous mutations. Second, large populations have increased access to mutations that confer large benefits. These factors imply that larger populations gain an advantage by taking larger adaptive steps during population evolution. However, as we have shown in a previous study , sometimWe further showed that for a rather smooth fitness landscape, there is no advantage in following more stochastic adaptive trajectories; however, even an intermediate amount of ruggedness can be sufficient to occasionally favor more stochastically evolving populations of smaller size. Experimental studies suggest that at least some amount of ruggedness is present in natural situations ,44-47. WLastly, we found some evidence that for a fixed mutation supply rate, small populations evolved more rapidly and more stochastically, which allowed them to reach higher fitness compared to larger populations in a majority of simulations. We suggest that this can be attributed to clonal interference acting in larger populations, which limits the amount of within population variation and can retard the rate of adaptation . To keepAlthough medium and small populations can exceed the fitness of larger populations, we note that this outcome does not occur in all, or even most, simulations. More important, the degree to which this result is realized is highly dependent upon underlying landscape architecture. For example, as is most clearly evident in Figure N0 = 100.As with any model, we have made several simplifications. For instance we excluded death of bacteria and only allowed the loss of novel mutants to occur through stochastic loss during sampling via serial dilution. The inclusion of stochastic drift  would liA second simplification is our exclusive focus on asexual populations. A number of studies have shown that the incorporation of recombination can help to overcome clonal interference or can help populations to more easily escape from local fitness peaks -53, thouWe used our simulation to study populations that ranged in size over 4 orders of magnitude. In this range, we found that our large populations exhibited clonal interference and very rarely escaped from local fitness peaks. However, a number of recent studies suggest that if the population size is large enough, the impact of clonal interference might be reduced ,55-57. AIn summary, we have shown that for asexual populations evolving on rugged fitness landscapes, there can be a trade-off between speed of adaptation and the attainable fitness, which strongly depends on the underlying fitness landscape. This suggests that the optimal population size likely depends on both the details of the fitness landscape and the relative importance of speed versus final fitness.AH and DR conceived of the study. AH wrote and analyzed the simulations. AH and DR wrote the manuscript. Both authors read and approved the final manuscript."}
{"text": "The goal of this paper is to develop a mathematical model that analyzes the selective advantage of the SOS response in unicellular organisms. To this end, this paper develops a quasispecies model that incorporates the SOS response. We consider a unicellular, asexually replicating population of organisms, whose genomes consist of a single, double-stranded DNA molecule, i.e. one chromosome. We assume that repair of post-replication mismatched base-pairs occurs with probability  Genetic repair is an essential component of cellular genomes. Without mechanisms for repairing damaged and mutated DNA, genomes could not achieve sufficient information content to code for the variety and complexity of modern terrestrial life Genetic repair mechanisms fall into two main categories: Those that correct base mis-pairings during the replication cycle of a cell, and those that repair mutated and damaged DNA during the growth (G) phase of the cellular life cycle Two important examples of the first class of repair mechanisms are DNA proofreading and mismatch repair (MMR). DNA proofreading is a repair mechanism that is built into the DNA replicases themselves. During daughter strand synthesis, an erroneously matched base is excised, and a second attempt at a base pairing is made Two important examples of the second class of repair mechanisms are Nucleotide Excision Repair (NER) and the SOS response The SOS response is a genomic repair mechanism that only activates when there is extensive damage to the cellular genome. When DNA damage is sufficiently extensive, the cell stops growing, and the SOS repair pathways attempt to restore complementarity to the genome In recent work with quasispecies models of evolutionary dynamics, quasispecies models This paper continues the theme of incorporating various details characteristic of cellular genomes by developing a quasispecies model that takes into consideration the SOS repair mechanism. The model is highly simplified, and therefore only a first step in developing proper evolutionary dynamics equations with SOS repair. Nevertheless, because our model is analytically tractable, we believe it is a useful and important initial approach to mathematically modeling the evolutionary aspects of the SOS repair pathway. A proper modeling of the SOS response is an essential component of developing a quantitative theory of mutation-propagation in cellular organisms, which is important for understanding phenomena such as the emergence of antibiotic drug resistance in bacteria, and cancer in multicellular organisms We consider a unicellular population of asexually replicating organisms, whose genomes consist of a single DNA molecule, i.e. one chromosome. The genome may then be denoted by fitness landscape. It should be emphasized that fitness in this model only refers to replication rate. In particular, this model does not consider cell death.We let The semiconservative replication of the DNA genomes happens in three stages:Strand separation, whereby each strand of the chromosome separates to act as a template for daughter strand synthesis.Daughter strand synthesis. We assume a genome and base-independent mismatch probability Lesion repair, where any post-replication mismatches are removed. Here, there is no longer the parent-daughter strand discrimination that was available during daughter strand synthesis, so in contrast to DNA proofreading and mismatch repair, lesion repair has a In our simplified model, the SOS response is triggered if a given genome has at least  by  see .In this paper, we will consider the behavior of the model in the limit of infinite sequence length. If The assumption of infinite sequence length is a common assumption in quasispecies theory, because it is the mathematical formalization of the long genome-length regime that makes the neglect of backmutations exact. While finite genome length effects need to be considered in dynamic fitness landscapes, where adaptation to specific genomes is important Finally, we assume that the fitness landscape is defined by a master genome The justification for this choice of fitness landscape is as follows: If a genome has a fixed mutation, then neither DNA strand corresponds to either of the master strands However, in the case of a mismatch where one of the bases is the same as the corresponding base in one of the master strands, the information contained in the master genome is still preserved in one of the strands, so that the organism is assumed to remain viable if the total number of such mismatches does not exceed some cutoff value For convenience, We can develop the infinite sequence length equations for our model, assuming an initially prepared clonal population consisting entirely of the wild-type (mutation-free) genome The Hamming distance between two sequences Now, in the limit of infinite sequence length, it may be shown that, with probability As a result, we can define a strand ordering A given genome Note that the fitness landscape depends only on By the symmetry of the fitness landscape, and by the symmetry of the initial population distribution, we can group all genomes of identical To develop the dynamical equations for both the We first consider the case where this genome is not undergoing the SOS response. Then, due to the semiconservative nature of DNA replication, this genome is being destroyed at a rate given by If the The probability of a given set of mutations corresponding to The degeneracy is given by If We also obtain an overall transition probability of It is important to note from the Furthermore, note that strands Then for the genomes Then for the population number A similar expression is obtained for the population number It should also be noted that, by the symmetry of the fitness landscape, we have that Taking into consideration the contribution to Note that we do not write down the dynamical equations for The factor of It should be noted that this factor is missing in the contribution to The factor of To obtain the steady-state behavior of our model, we begin by introducing some definitions that will allow us to simplify the calculations.where we set We can add these equations to obtain,For the purposes of computing the mean fitness at steady-state, we can simplify the system of equations somewhat by defining To obtain the steady-state behavior of this system of equations, we begin by first solving for the steady-state of the population undergoing SOS repair.For For This expression has the form of the recursion relation, If we define Using a similar argument, we obtain,For the steady-state value of Plugging our expressions for From these equations we may derive the equality,Beyond the error catastrophe, the mutation rate is sufficiently high that the selective advantage for remaining localized about the The error catastrophe occurs at the mutation rate for which the two expressions for the mean equilibrium fitness become equal. As with previous quasispecies models, the error catastrophe here also corresponds to a localization to delocalization transition over sequence space We now proceed to consider the behavior of the steady-state mean fitness for a number of limiting cases, in order to better understand our model. We consider the following cases: (1) Case 1:Case 2:Case 3:Taking If we assume that Therefore, for large Now, because In the regime where Now, because Therefore, in any case, we can maximize the SOS response will confer a maximum selective advantage if it is activated when and only when the genome has sustained sufficient genetic damage so that it will be unviable without SOS repair. However, it should be emphasized that this only holds when We reach the conclusion that, when the fitness penalty for having a non-viable genome is sufficiently great, We developed stochastic simulations of a unicellular population capable of undergoing the SOS response, in order to numerically test the analytical predictions of our model. We consider a constant population of genomes that is cycled over every time step. During each cycle, every genome is allowed to replicate with a probability We assume that the population initially consists of a clonal population of wild-type (mutation-free) genomes. The fitness of a given genome If a genome replicates during a cycle, then it is removed from the population, and the two daughters are added to the population of genomes. To maintain a constant population size, another, randomly chosen genome is removed from the population as well. Because this approach is simply the stochastic implementation of the quasispecies dynamics of the system, it converges to the infinite population, continuous time result as the population size gets larger and the time steps get smaller.If a daughter genome is produced that has at least The stochastic simulation is allowed to run for a sufficient number of time steps so that the mean fitness of the population does not change significantly, at which point the system is assumed to be at steady-state.This paper developed a quasispecies approach for describing the evolutionary dynamics of a unicellular population that incorporated a simplified model of the SOS response. The model was a generalization of the single-fitness-peak landscape that is often used in quasispecies theory to study various problems in evolutionary dynamics. The model was shown to be analytically solvable, and it was found that the solution led to a maximal selective advantage to the SOS response in a manner that is broadly consistent with the behavior of actual organisms. Specifically, we showed that the SOS response should only be activated in a cell with a sufficiently damaged genome that it will be unviable if the SOS response is not activated. In such a situation, the cell has \u201cnothing to lose,\u201d meaning that it is better to attempt to repair the genome and risk introducing deleterious mutations, than it is to leave a highly damaged genome alone.For future research, it will be important to consider more realistic models that will allow for quantitative models that can be used in collaboration with experiment. Because the SOS response is a genetic repair pathway that works in conjuction with other cellular repair pathways, a proper understanding of the SOS response is important for developing a coherent theory of mutation-propagation that will be useful for understanding the emergence of antibiotic drug resistance in bacteria, and cancer in multicellular organisms"}
{"text": "An essential, conserved characteristic of proneural genes is their ability to restrict their own activity to single or a small number of progenitor cells from their initially broad domains of expression. This is achieved through a process called lateral inhibition. A regulatory element, the sensory organ precursor enhancer (SOPE), is required for this process. First identified in Drosophila, the SOPE contains discrete binding sites for four regulatory factors. The SOPE of the Drosophila asense gene is situated in the 5' UTR.An increasing number of publications demonstrate conservation of function of cis-regulatory elements without sequence similarity. In invertebrates such functional conservation has only been shown for closely related species. Here we demonstrate the existence of an ancient arthropod regulatory element that functions during the selection of neural precursors. The activity of genes of the asense-like genes in species belonging to all four arthropod groups . The SOPEs of the spider Cupiennius salei and the insect Tribolium castaneum are shown to be functional in transgenic Drosophila. This would place the origin of this regulatory sequence as far back as the last common ancestor of the Arthropoda, that is, in the Cambrian, 550 million years ago.Through a manual comparison of consensus binding site sequences we have been able to identify a SOPE in UTR sequences of The SOPE is not detectable by inter-specific sequence comparison, raising the possibility that other ancient regulatory modules in invertebrates might have escaped detection. They are initially expressed during development in groups/domains of equivalent neuroectodermal cells. An essential, conserved characteristic of proneural genes is their ability to restrict their own activity to single or a small number of progenitor cells within these domains  Gal4 , UAS-sc (FlyBase ). UAS-co1 aseflies were crossed to hsp70Gal4 > UAS Dm-ase, hsp70Gal4 > UAS Tc-ase and hsp70Gal4 > UAS CsASH2 flies, respectively, and allowed to lay eggs in culture bottles for 3 days. Heat shocks were preformed between 16 hours and 8 hours before puparium formation. Heat shock expression was driven by three 1-hour heat shocks at 37\u00b0C, separated by 2 hour intervals at 25\u00b0C. Wings were mounted in glycerol and analyzed under a compound microscope (Leica).Gal4 lines that activated the constructs in the expression domains of toll-8, patched, scabrous and ac. Flies of the appropriate genotype were selected, mounted and the bristles were counted under the dissecting microscope (Leica). Statistical analysis was performed using Microsoft Excel.Three independent lines were generated for each UAS construct (ORF only and ORF+SOPE) and crossed to four different D. pulex (Daphnia_pulex 2006-09 JGI) and S. maritima  were searched using tblastn with the ASH and Ase proteins of D. melanogaster, T. castaneum and C. salei as queries. Hits with relevant homology to the bHLH domain were further characterized. Three genes were identified in the D. pulex genome: an ASH homologue (JGI_V11_254034), an ase homologue (JGI_V11_254038) and a truncated copy of ase (JGI_V11_232740). In the S. maritima genome only one ASH homologue was identified; a second gene analyzed was too divergent in the basic region of the bHLH domain to be classified as an ASH gene.The sequenced genomes of ASH and ase genes from the insects D. melanogaster and T. castaneum, the crustaceans T. longicaudatus and D. pulex, the chelicerate C. salei and the myriapods G. marginata and S. maritima. Amino acid sequences have been aligned with ClustalW2 [Phylogenetic trees were constructed using all lustalW2 , manualllustalW2 . The reslustalW2 .AC: achaete; AS-C: Achaete-Scute complex; ASE: asense; ASH: achaete-scute homologue; ATO: atonal; BHLH: basic helix-loop-helix; BP: base pair; NF: nuclear factor; SC: scute; ORF: open reading frame; SOP: sensory organ precursor; SOPE: sensory organ precursor enhancer; UTR: untranslated region.The authors declare that they have no competing interests.Conceived and designed the experiments: SA, PS, AS. Performed the experiments and analyzed the data: SA, BN, AS. Wrote the manuscript: AS, PS. Contributed to writing and editing: BN.asense rescue experiment. Comparison of the number of stout bristles exhibiting differentiation defects in 1 aseflies (first column) and 1 aseflies carrying hsp70Gal4 > UAS Dm-sc, hsp70Gal4 > UAS Dm-ase, hsp70Gal4 > UAS Tc-ase and hsp70-Gal > UAS-CsASH2 transgenesClick here for fileAlignment of the SOP enhancer elements. Nucleotide sequence alignments of the individual transcription factor binding sites of Drosophila, Tribolium, Daphnia, Strigamia and Cupiennius.Click here for fileSequence logos of the SOPE boxes. Graphic representation of the aligned sequences of the SOPE binding sites of Drosophila melanogaster, Tribolium castaneum, Daphnia pulex, Strigamia maritima and Cupiennius salei.Click here for fileMisexpression experiment. Comparison of the number of ectopic bristles in flies carrying the UAS-ase/ASH2 ORF only and ORF+SOPE constructs.Click here for filesenseless 5' UTRSOP enhancer in the . Arrangement of the SOPE boxes in the 5' UTR of the senseless transcript.Click here for filease-like UTRsSecondary structure of the . Graphic representation of the secondary structure of the UTRs of the Drosophila melanogaster, Tribolium castaneum and Daphnia pulex asense genes and the UTRs of Strigamia maritima ASH and Cupiennius salei CsASH2.Click here for file"}
{"text": "Systemic Epstein-Barr-virus-positive T cell lymphoproliferative disease of childhood is an extremely rare disorder, characterized by clonal proliferation of Epstein-Barr-virus-infected T cells with an activated cytotoxic phenotype. The disease is more frequent in Asia and South America, with only few cases reported in Western countries. A prompt diagnosis, though often difficult, is a necessity due to the very aggressive clinical course of the disease.+, Epstein-Barr-encoded-RNA-positive T cell phenotype with clonal rearrangement of the T cell receptor genes, the final diagnosis being systemic Epstein-Barr-virus-positive T cell lymphoproliferative disease. On reviewing the literature we found only 14 similar cases, all presenting with very aggressive clinical courses and requiring extensive phenotyping and molecular techniques for final diagnosis.We report the clinicopathological features of fulminant T cell lymphoproliferative disease that arose in the setting of acute primary Epstein-Barr virus infection. Our patient, a 23-year-old man, presented to our facility with persisting fever, hepatosplenomegaly and severe pancytopenia. On bone marrow biopsy, an abundant lymphoid infiltrate was observed. Immunophenotypic and molecular studies revealed that the atypical lymphoid cells displayed a CD8Though extremely rare, this disease can occur in Europe, and a comprehensive diagnostic approach is thus recommended in all case of Epstein-Barr-virus-positive lymphoproliferative disorders. Unfortunately, at present no specific treatment is available; however, prompt administration of anti- Epstein-Barr virus treatment and rapid attempts to control the hemophagocytic syndrome are indicated. Howeval blood . UnfortuIn addition, EBV is implicated in the pathogenesis of different types of lymphoproliferative diseases (LPD), which are related to diverse immune alterations or peculiar clinical backgrounds . Typical+ LPD, and is currently classified as systemic EBV+ T cell LPD of childhood in the World Health Organization classification of tumors of hematopoietic and lymphoid tissues [In this regard, fulminant mononucleosis has recently been demonstrated to be a monoclonal CD8 tissues . This en tissues ; the dis tissues .+, CD3+, CD8+, CD56-, and TIA+ [+. Neoplastic cells have monoclonally rearranged T cell receptor (TCR) genes, and consistent Epstein-Barr encoded RNA (EBER) positivity at in situ hybridization (ISH). Differential diagnosis mainly concerns reactive conditions as well as aggressive natural killer (NK) cell leukemia.At morphology, neoplastic T cells are usually small and lack significant cytological atypia . HoweverHere, we report the clinicopathological features of fulminant T-LPD that arose in the setting of acute primary EBV infection in our patient, characterized by a monoclonal proliferation of EBV-infected T cells.9 cells/L, white blood cells 2.2 \u00d7 109 cells/L, neutrophils 410 \u00d7 109 cells/L, lymphocytes 1.570 \u00d7 109 cells/L), increased LDH, signs of disseminated intra-vascular coagulopathy (CID), and anti-EBV IgM positivity, while a chest X-ray showed diffuse pulmonary infiltrates. No prior immunological abnormalities were recorded.A 23-year-old Caucasian man was hospitalized for persisting fever resistant to conventional therapies. On physical examination, our patient presented with marked hepatosplenomegaly and abnormal sounds at thoracic auscultation. Laboratory findings consisted of severe pancytopenia  Figure . Immunohescribed ,10.Based on the above findings, a final diagnosis of systemic EBV+ T cell LPD of childhood was made. Our patient was initially treated with two sequential doses of VP16 with moderate improvement of his clinical and laboratory data. In particular, the fever transiently improved, hepatosplenomegaly was reduced, and coagulation parameters were partially corrected; however, severe peripheral blood cytopenia persisted. Soon after, the patient developed a fever recrudescence in association with pulmonary fungal infection.-, CD3+, CD4-, CD8+, EBER- small lymphocytes and absence of the previously observed CD8+ large cells. VP16 was then replaced with cyclosporine, obtaining a white blood cell count increase and a further decline of splenomegaly, but with no improvement in thrombocytopenia. A third bone marrow biopsy showed an increased cellularity with reappearance of numerous CD8+ lymphocytes and evident hemophagocytosis.A second bone marrow biopsy was performed, revealing (hypo)aplasia with a minimum percentage of CD79aOur patient then developed rectal hemorrhages only treatable with surgery, which turned out to be sustained by microvascular thrombosis on histological examination. Finally, after a short period of relative good health, our patient had a relapse of rectal bleeding and died soon after with cerebral manifestations.Systemic EBV+ T cell LPD of childhood is a rare disorder characterized by an aggressive disease course and dismal prognosis . As deatOn reviewing the literature, we found only 14 cases reported in Western countries ,11-14, s+ phenotype, two a CD4+ phenotype and one showed double positivity for CD8 and CD4; in one case phenotype was not interpretable and in three cases it was not reported. TCR\u03b1 presented with clonal rearrangements in nine out of 10 patients and EBV genome was clonal in all but one case. In contrast, among patients with T cell LPD after CAEBV infection two were CD4+, one was CD45RO+ and one presented with an admixture of CD8+ and CD4+ lymphocytes; three cases presented with monoclonal patterns with regard to rearrangement of both TCR\u03b1 genes and EBV genome. In the remaining case, only the EBV positivity was assessed.Our review of Western cases showed that four of those 14 patients developed a T cell LPD after CAEBV infection ,11, and TCR\u03b1 rearrangement in systemic EBV+ T cell LPDs, and also sCD3/CD8 negativity, CD56 positivity and germline TCR\u03b1 patterns in aggressive NK cell leukemia cases.In all described cases, an accurate diagnostic investigation including clinical, morphological, immunohistochemical, and molecular analyses was necessary in order to formulate a correct diagnosis. In particular, the differential diagnosis with aggressive NK cell leukemia was based on surface sCD3 and CD8 positivity, CD56 negativity, and evidence of + LPDs. In fact, though, at present, specific therapies are not available, the correct description of rare disorders is essential for improving current knowledge and possibly future therapeutic approaches.In conclusion, our case report underlines the importance of a comprehensive diagnostic approach in the management of atypical EBVWritten informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.VT performed research, analyzed data and wrote the manuscript; CA performed research and analyzed data; ES and FB analyzed data; SC and GM were responsible for patient care and provided clinical information; AG, CM, SR, and MTS analyzed data; SAP and PPP performed research, analyzed data and wrote the manuscript. All authors read and approved the final manuscript. VT and CA contributed equally to this work; SAP and PPP contributed equally to this work."}
{"text": "Meta-analyses play an important role in synthesizing evidence from diverse studies and datasets that address similar questions. A major obstacle for meta-analyses arises from biases in reporting. In particular, it is speculated that findings which do not achieve formal statistical significance are less likely reported than statistically significant findings. Moreover, the patterns of bias can be complex and may also depend on the timing of the research results and their relationship with previously published work. In this paper, we present an approach that is specifically designed to analyze large-scale datasets on published results. Such datasets are currently emerging in diverse research fields, particularly in molecular medicine. We use our approach to investigate a dataset on Alzheimer's disease (AD) that covers 1167 results from case-control studies on 102 genetic markers. We observe that initial studies on a genetic marker tend to be substantially more biased than subsequent replications. The chances for initial, statistically non-significant results to be published are estimated to be about 44%  relative to statistically significant results, while statistically non-significant replications have almost the same chance to be published as statistically significant replications . Early replications tend to be biased against initial findings, an observation previously termed Proteus phenomenon: The chances for non-significant studies going in the same direction as the initial result are estimated to be lower than the chances for non-significant studies opposing the initial result . Such dynamic patters in bias are difficult to capture by conventional methods, where typically simple publication bias is assumed to operate. Our approach captures and corrects for complex dynamic patterns of bias, and thereby helps generating conclusions from published results that are more robust against the presence of different coexisting types of selective reporting. In many research fields, meta-analyses play an increasingly important role for synthesizing evidence from studies that have been published in the past. Since not all studies and analyses that have been undertaken are eventually published in the scientific literature, meta-analyses typically rely on incomplete samples of study outcomes. These samples might be biased, because the result of a study or of a specific analysis might influence its chances to become reported. Studies with results that achieve formal statistical significance might, for example, have increased chances to become published Statistical methods have been developed to detect and correct for selective reporting biases Here, we present an approach to quantify selective reporting from large-scale datasets of published results. Such datasets are currently emerging in a number of research fields, including molecular medicine. Our approach extends previous work by Hedges and others We use our approach to investigate data from the AlzGene database Despite different association strengths, there is little reason to believe that fundamental differences exist between markers in how selective reporting bias is acting. Thus, even though selective reporting bias cannot be studied properly on a single marker, because most markers are only covered by a handful of studies each, the combined data are likely highly informative for quantitative models of selective reporting. Our approach exploits this and allows us to investigate whether initial publications are more biased than subsequent replications, and whether early replications are biased against initial results.1,X \u2026, nX are assumed to come from a normal distribution i \u223c NX with a known within-study variance i2\u03c3, an unknown between-study variance 2\u03c3, and an unknown mean effect size \u0394. The relative chance for a result to be published is assumed to be depend on i\u200a=\u200aXi/\u03c3iZ, because the value of iZ typically determines the p-value associated with a study result. It is described by the weight functionWe build our approach on the selection model proposed by Hedges i, \u03c3i)\u03c9w. The function changes when i/\u03c3iX crosses one of the interval boundaries 1,z\u200a=\u200a z. While the parameters 1,w\u200a=\u200a w are estimated from the data, the interval boundaries 1,z\u200a=\u200a z take fixed, pre-defined values. The discontinuities in the weight function at these pre-defined boundaries may be justified by the salience of p-values such as 0.05 in the current practice of interpreting study outcomes The weight function Applying the weight function to the probability density for the unbiased study results yields a weighted probability density given byi, \u03c3i)\u03c9w denote the parameters of the weight function (eq. 1), and the terms ijB are given bywhere 1,X\u200a=\u200a X is given byThe log likelihood for the data \u0394 and 2\u03c3 in parallel with the parameters of the weight function, 1,w\u200a=\u200a w.This function and first and second derivatives as given in 1,w\u200a=\u200a w describing selective reporting bias. We now assume that there are m datasets (1),X\u200a=\u200a )X with (i) \u200a=\u200a (X1(i),X \u2026 n(i)(i))X. These datasets may differ in the underlying mean (m)\u0394 and between-study variance (m)2\u03c3. However, all the datasets are assumed to be subject to the same selective reporting bias, i.e. the parameters 1,w\u200a=\u200a w are the same for all datasets. For the analysis of the AlzGene dataset, this means that we assume while the effect sizes and between-study variance might be different for different markers, selective reporting affects all markers in the same way. We therefore can simultaneously estimate  (1),\u0394\u200a=\u200a )\u0394, (1),\u03c3\u200a=\u200a )\u03c3, and 1,w\u200a=\u200a w by maximizing the log likelihoodThe above approach simultaneously quantifies the parameters underlying the distribution of the unbiased data, and the parameters of the weight function i(k)X is associated with a categorical variable i(k)K, different weight functions can be used for the different categories. Instead of using a single weight function as in eq. 5 and 6, weight function (j)\u03c9 is used if i(k)K falls into category j. Using this approach we can, for example, use different categories for the initial studies, immediate follow-ups, and subsequent studies. This allows testing whether initial studies on a marker are more biased towards significant outcomes than late studies, or whether immediate replications tend to be biased against the result of the initial study. The use of multiple weight functions for quantifying the Proteus phenomenon is illustrated in Moreover, if each study result Although estimating the parameters for these extensions requires somewhat more effort in handling the data, the likelihood function and its derivatives remain analogous to the ones given by Hedges The unrestricted maximum likelihood approach used in our algorithm may not always converge. In our analysis, convergence problems arise for 22 of the 124 markers. To improve convergence, the between-study variance could be determined by non-iterative methods iX and the corresponding standard errors i\u03c3 are estimated using a logistic regression. It should be noted that the p-values associated with observations in our model via the value of i\u200a=\u200aXi/\u03c3iZ are not necessarily identical to the ones given in the actual publications, where often additional factors such as age, gender or specific diagnosis (such as early-onset AD) might be included, and different statistical and genetic models might be used.The AlzGene database In the analysis presented here, we included results regardless of whether allele frequencies are in Hardy-Weinberg equilibrium. We included only markers with results from at least 4 independent publications, as judged by PubMed identifiers. In total, 124 markers fulfill this criterion. 22 markers are excluded because of convergence issues described above.Categories. For modeling the Proteus phenomenon, we assigned one of three categories to each case-control study: initial findings, early replications, and late replications. Any studies on a specific marker that were published in the same year as the first case-control study on this marker fall into the first category . Studies published within the next two years fall into the second category (early replications). All subsequently published studies fall into the third category (late replications). If a marker appears, for example, in publications from 2000, 2000, 2002, 2004, and 2005, the findings would fall into categories 1, 1, 2, 3 and 3, respectively. In an additional analysis we use PubMed IDs for an alternative categorization. Compared to using the publication year, PubMed IDs allow sorting papers at a finer temporal resolution. However, because particularly for publications from the 90's there is no strict relation between PubMed ID and date of publication, a PubMed ID based ranking is much less robust that a year-wise ranking. Results from the additional analysis are shown in the To estimate selective reporting bias we use fixed-effect models and random-effects models see . We use i, \u03c3i) \u200a=\u200a1\u03c9. The weight function is set to one for the outer intervals, i.e. i, \u03c3i) \u200a=\u200a1\u03c9. The weight function for this model, and for all following models, is set to one for the outer intervals, i.e. i, \u03c3i) \u200a=\u200a1\u03c9w, describing bias for all studies with Z-values that fall in the mid interval.We use a single weight function to model the data, i.e. do not use different categories for initial studies, early replications and late replications. The boundaries are chosen at (I)w and (S)w, and allows detecting whether initial studies are more biased then subsequent ones.This model is similar to Model 1, but uses two different weight functions. One weight function describes bias for the initial study, while the other weight function describes bias for all subsequent studies . The resz\u200a=\u200a . The model has four parameters: (I)w for initial studies that fall into the interval between \u22121.64 and 1.64, (E)1w for early replication studies with z-values between \u22121.64 and 0, (E)2w for early replication studies with z-values between 0 and 1.64, and (S)w for all subsequent studies that fall into the interval between \u22121.64 and 1.64. This model allows investigating whether early replication studies differ from later ones, and is designed to match the model for the Proteus phenomenon described in the next paragraph.In this model, a third category is added to separately model early and late replication studies. The additional weight function is described by two additional parameters and uses boundaries at \u22121.64 and 1.64 and has the opposite sign of the initial study, the weight function is set to (E)1w. If it falls into the interval between \u22121.64 and 1.64 and has the same sign, weight (E)2w is used. If several studies on a marker are categorized as initial studies, we use the sign of the most extreme result. The Proteus model allows us to investigate whether subsequent studies that contradict the initial one are more likely to be published than studies that are in agreement with the initial one but do not show a strong effect. It has the same number of parameters as Model 3. A comparison between the two models is used to determine whether explicitly taking into account the sign of the initial study for modeling bias in early replications improves the model.As in Model 3, we use three different weight functions, describing initial studies, early replications and late replications. The weight functions and parameters are analogous to the ones used in Model 3. However, the weight function for early replications is assumed to further depend on the outcome of the first study see . If the \u22121.64 and 1.64 are relatively constant. The weights start increasing as |z| increases over a value of 1.64. This is what one would qualitatively expect for selective reporting bias. Results that do not reach formal statistical significance are less likely published, irrespective of where exactly the result falls. The estimated weights in the 10 intervals covering the region between \u22121.64 and 1.64 fall roughly around \u22120.5 which corresponds to about 60% of non-significant results in any of these intervals being reported. For a fixed effects model, where the between-study variances are assumed to be zero  seem to be underrepresented when compared to results with a two-sided p-value smaller than 0.01 (|z| >2.58), and with two-sided p-values between 0.05 and 0.1 (1.64<|z|<1.96). This observation is somewhat surprising for classical publication bias where the p\u200a=\u200a0.05 value is considered to be a salient threshold for publication, but is in line with previous observations The high-resolution model reveals an interesting shape of the weight function for results with z-values outside the interval between z\u200a=\u200a\u22121.64 and z\u200a=\u200a1.64, we used these boundaries in the subsequent models (Model 1\u20133 and Proteus model). Estimates for the simple random-effects one-parameter model (Model 1) are shown in \u22120.33 for the random effects model. This corresponds to a probability of reporting of about 72% (95% CI from 58% to 90%) relative to studies that fall into the outer intervals. This estimate is similar to the corresponding estimates in the high-resolution model, but note that we now use different outer intervals . The error in the estimate of the bias is much lower, because in the one-parameter model each interval contains data from several intervals of the high-resolution model (Because the change of the weight function in the high-resolution model is highest at the boundaries of on model .(I) \u200a=\u200a\u22120.81w, suggesting that initial studies with standardized effects in the 1.64 to \u22121.64 interval have a chance of only 44% (95% CI from 32% to 63%) of being reported, relative to initial studies with results outside that interval. This bias is considerably stronger than the estimates for subsequent studies ((S) \u200a=\u200a\u22120.17log w, respectively, corresponding to a probability of 84% compared with studies outside the interval; 95% CI from 66% to 107%). Thus, initial studies tend to be much more biased than subsequent ones. The potential origin and consequences of this effect are discussed further below. All estimates and their standard errors are summarized in The results for the models designed to investigate the initial study bias and the Proteus phenomenon are summarized in (E)1w and (E)2w, and yields a smaller change in the log likelihood score than the Proteus model. Based on the Akaike Information Criterion (AIC), the Proteus model would be favored for describing the given AD dataset. The single most important parameter in these models is the one to distinguish bias in initial publications from bias in subsequent ones, which indicates that the initial study bias is a very robust phenomenon.The results for the Proteus model show that studies that confirm the direction of the effect of an initial study but do not achieve formal statistical significance face more bias than studies with non-significant results that oppose the initial result in terms of the direction of the effect Click here for additional data file.Supporting Information S2Uncorrected and corrected estimates for the association strengths and between-study variances for the markers included in our analysis.(CSV)Click here for additional data file."}
{"text": "Arca subcrenata is documented in the literature of marine Traditional Chinese Medicine. Polypeptide fraction from A. subcrenata, coded as P2, was demonstrated to possess significant antitumor activity in our previous study. However, the underlying mechanism remains undefined. The present study was carried out to investigate the underlying antitumor mechanism of P2 in human cervical cancer HeLa cells by MTT, FCM, LSCM, and western blot assays. The results revealed that P2 significantly induced apoptosis of HeLa cells in a concentration- and time-dependent manner. High level of ROS was provoked by P2, which was in turn responsible for induction of apoptosis through activation of intrinsic mitochondrial pathway and JNK1/2, p38 MAPK pathways, as well as inhibition of ERK1/2 pathway, as evidenced by the abrogation of P2's effect on HeLa cells preincubated with the ROS scavenger NAC. P2 also was observed to display significant effect on G2/M phase arrest by downregulating the expression of cyclin B1/cdc2 complex and upregulating the expression of p21. These findings demonstrate that P2 induces apoptosis and G2/M phase arrest in HeLa cells through ROS-mediated MAPKs pathways, suggesting that P2 would be worth investigating as a promising agent within the scope of marine drugs for treatment of cervical cancer. Cervical cancer, the second most common type of tumor in females surpassed only by breast cancer , becomesMarine organisms are known for their capability of producing large amount of bioactive materials, with properties such as antitumor, anti-inflammatory, and antioxidant activities mainly due to the marine environment being characterized with high salinity, high pressure, low temperature, low light intensity, and an oligotrophic condition. Cytarabine, a nucleoside firstly obtained from sponge in the deep sea, was approved by FDA in 1969 and is regarded as one of the main drugs used to treat hematologic malignancies , 5. Rece\u03baB pathway [ROS, a series of natural metabolites of intracellular oxygen, plays a predominant role in cellular process and involves multiple signaling pathways as a second messenger , such as pathway , and PI3 pathway . A moder pathway  by induc pathway . ROS wou pathway . Activat pathway , 19. The pathway . Numbers pathway \u201322. Evid pathway . Low lev pathway . Numbers pathway , 23, 24.Arca subcrenata Lischke, a member from Arcidae family, is classified as a marine invertebrate species [ A. subcrenata [ A. subcrenata can be adopted in the treatment of a number of diseases such as inflammation, tumor, anemia, and dyspepsia in China [ A. subcrenata is mainly composed of polysaccharide complexes and proteins [ A. subcrenata was reported to possess the bioactivity of improving the immunological function such as promotion of the thymus/spleen index of normal mice as well as immunosuppressed mice in vivo tests [ A. subcrenata could inhibit the proliferation of several tumor cell lines in vitro [ A. subcrenata had antioxidant activity against DPPH radicals and hydrogen peroxide [ species . It is wbcrenata . The bodin China . A. subcproteins , 26. Recvo tests . Our prein vitro  and the peroxide . A. subcrenata, displayed significant antitumor activities in both in vitro and in vivo tests [More recently, we had confirmed that P2, a polypeptide fraction fromvo tests . However3--2,5-diphenyltetrazolium bromide (MTT), N-acetylcysteine (NAC), Z-Val-Ala-Asp(OMe)-fluoromethylketone (Z-VAD-FMK), and 2\u2032,7\u2032-dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Sigma . RPMI-1640 medium and fetal bovine serum (FBS) were purchased from Gibco . Primary antibodies against cleaved caspases 3, 7, and 9, PARP, p-cdc2, p21, cyclin B1, ERK1/2, p-ERK1/2, p38, p-p38, JNK1/2, p-JNK1/2, and anti-rabbit or anti-mouse IgG horseradish peroxidase- (HRP-) linked secondary antibodies were purchased from Cell Signaling Technology . Primary antibodies against Bax, Bcl-2, and cytochrome c were purchased from Abcam . Primary antibody against GAPDH was from Hangzhou Xianzhi Biotech. Co., Ltd. . Enhanced chemiluminescence (ECL) detection kit for western blot was obtained from Nanjing KeyGEN Biotech. Co., Ltd. . A. subcrenata was purchased from Huangsha seafood market, Guangzhou, China, and identified by Professor Rongmin Yu . The visceral mass of A. subcrenata was washed with distilled water for 3 times and homogenized with phosphate-buffered saline solution . The supernatant was collected after centrifugation and to which ammonium sulfate was added to precipitate the protein components. The crude polypeptides were further purified by ion-exchange chromatography eluted with different concentrations of sodium chloride solution at 4\u00b0C and the effluent fractions were detected using a protein detector at 280\u2009nm. P2 was one of the effluent fractions with the highest content of protein. The collected fraction of P2 was concentrated, dialyzed against distilled water at 4\u00b0C, and lyophilized.The material of\u03bcg/mL streptomycin, and 100\u2009U/mL penicillin in a cell incubator with 5% CO2 at 37\u00b0C.Human cervical cancer HeLa cells were purchased from the American Type Culture Collection  and cultured in RPMI-1640 medium containing 10% FBS, 100\u20094 cells/mL and incubated with P2 at various concentrations for setting time. The cells were pretreated by inhibitors for 1\u2009h before treatment. Thereafter, MTT solution (5\u2009mg/mL) was added and incubated with cells for another 4\u2009h. The 96-well plates were added with 200\u2009\u03bcL/well of DMSO to dissolve the MTT-formazan product and the data were spectrophotometrically estimated at 570\u2009nm. The cell growth inhibitory rate was determined according to the absorption.MTT assay was used to detect the viability of HeLa cells according to the literature with some necessary variations . The celApoptosis measurement was carried out mainly according to the literature  with som4 cells/mL and incubated with 0, 4, 12, and 36\u2009\u03bcg/mL of P2 for 48\u2009h. After the treatment with P2, the attached cells were washed twice with PBS and fixed with 4% formaldehyde for 10\u2009min. The fixing solution was then removed and the cells were washed twice with PBS before staining with DAPI solution . After staining for 15\u2009min in the dark, cells were washed again and observed under LSCM . Ten fields were randomly selected to observe and images were taken [Cells were seeded at a density of 4 \u00d7 10re taken .5 cells/mL onto 6-well plates and incubated for 48\u2009h with different concentrations of P2. After being treated with P2, cells were harvested and washed twice with cold PBS. Then cells were fixed in 70% alcohol for 12\u2009h at 4\u00b0C. The pellet was washed and resuspended again in PBS containing PI (50\u2009mg/mL), Triton X-100 , and RNase A (100\u2009mg/mL). After incubation for 30\u2009min, the cell cycle distribution in each treatment was detected using a flow cytometer. Data were analyzed by WinMDI 2.9 software.Cell cycle distribution analysis was performed according to the literature  with som5/well) were seeded in a 6-well plate for 24\u2009h. After being treated with different concentrations of P2  for 24\u2009h, cells were harvested and washed twice with PBS and then incubated with JC-1 at 37\u00b0C for 30\u2009min in the dark. Loss of MMP in HeLa cells was detected by measuring a change in fluorescence intensity using a flow cytometer, with excitation at 484\u2009nm and emission at 501\u2009nm [MMP of HeLa cells in each treatment was measured by FCM using JC-1 staining. Briefly, cells (4 \u00d7 105/well) for 24\u2009h. After incubation, cells were treated with different concentrations of P2  for setting time. Then, the cells in each treatment were detached using trypsin-EDTA and washed twice with PBS. Cells were incubated with 10\u2009\u03bcM of DCFH-DA for 30\u2009min in the dark. ROS level in each treatment was measured using a flow cytometer, with excitation at 485\u2009nm and emission at 525\u2009nm [DCFH-DA, a fluorescent probe of ROS, was used to measure the generation of intracellular ROS in P2-treated HeLa cells. Cells were seeded in 6-well plates (4 \u00d7 10t 525\u2009nm .\u03bcg) of protein of each sample were separated on SDS-PAGE and further transferred to a methanol preactivated-polyvinylidene difluoride (PVDF) membrane . The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 90\u2009min at room temperature and incubated with primary antibodies at 1\u2009:\u2009400 to 1000 dilutions with 5% bovine serum albumin (BSA) in TBST overnight at 4\u00b0C. The blots were washed 3 times with TBST and incubated with HRP-conjugated secondary antibodies (1\u2009:\u20092000) for 1\u2009h at room temperature. Antibody binding was detected using ECL detection kit.The procedure of western blot was mainly performed according to the literature . HeLa cet-test and P < 0.05 was considered statistically significant. All results are representation of at least three independent tests and data are expressed as mean \u00b1 SD.Data were analyzed with Student's +/PI\u2212) and late apoptosis (Annexin V+/PI+) [\u03bcg/mL of P2 for 12, 24, and 48\u2009h, both the percentages of early apoptotic cells and late apoptotic cells increased in a concentration- and time-dependent manner  . After b Figures . These r\u03bcg/mL of P2. The result showed that Z-VAD-FMK significantly reduced the inhibitory effect of P2 on HeLa cells, indicating that P2's antiproliferative effect was caspase-dependent  [Cell growth and cell division are known to be controlled by several genetically defined cell-cycle checkpoints to ensure the correct progression of cell cycle through different stages . Cell cyn (PCNA) , 37. In n (PCNA) . ROS wasn (PCNA) , 53 and n (PCNA) , 54. In n (PCNA) . Our stuThe current study, for the first time, demonstrates that P2 exerts antitumor effect via induction of apoptosis and cell cycle arrest at G2/M phase. The underlying mechanism is mainly related to ROS-mediated activation of JNK1/2 and p-38 MAPK pathways, inactivation of ERK1/2 pathway, and subsequent trigger of intrinsic mitochondrial apoptotic cell death in a caspase-dependent manner, including the activation of caspases 7, 9, and 3, and shifting the Bcl-2 family proteins. These results exhibit the exciting potential for development of antitumor agents from marine organisms through apoptosis induction and cell cycle arrest by ROS overproduction and targeting MAPKs pathways."}
{"text": "Streptococcus pneumoniae is the most common cause of pediatric and frequent cause of adult empyema. We investigated whether S. pneumoniae can proliferate within human pleural fluid and if growth is affected by the cellular content of the fluid and/or characteristics of pneumococcal surface proteins. Invasive S. pneumoniae isolates (n = 24) and reference strain recovered from human blood or empyema were inoculated (1.5\u00d7106CFU/mL) into sterile human malignant pleural fluid samples (n = 11). All S. pneumoniae (n = 25) strains proliferated rapidly, increasing by a median of 3009 (IQR 1063\u20139846) from baseline at 24hrs in all pleural effusions tested. Proliferation was greater than in commercial pneumococcal culture media and concentrations were maintained for 48hrs without autolysis. A similar magnitude of proliferation was observed in pleural fluid before and after removal of its cellular content, p = 0.728. S. pneumoniae (D39 strain) wild-type, and derivatives (n = 12), each with mutation(s) in a different gene required for full virulence were inoculated into human pleural fluid (n = 8). S. pneumoniae with pneumococcal surface antigen A (\u0394psaA) mutation failed to grow (2207-fold lower than wild-type), p<0.001, however growth was restored with manganese supplementation. Growth of other common respiratory pathogens (n = 14) across pleural fluid samples (n = 7) was variable and inconsistent, with some strains failing to grow. We establish for the first time that pleural fluid is a potent growth medium for S. pneumoniae and proliferation is dependent on the PsaA surface protein and manganese.Empyema is defined by the presence of bacteria and/or pus in pleural effusions. However, the biology of bacteria within human pleural fluid has not been studied.  Streptococcus pneumoniae the most common bacterial cause. As many as 20\u201340% of patients with pneumonia develop a simple parapneumonic pleural effusion [Pneumonia affects 450 million patients worldwide each year,  with Streffusion .S. pneumoniae accounts for the vast majority of pediatric empyema and is among the most frequent causative organisms of empyema in adults [Empyema represents the most severe end of the pleural infection spectrum. It is defined by the presence of bacteria, or pus, in the pleural fluid. n adults \u20136. The in adults \u20139.S. pneumoniae seems to have a particular affinity for causing pleural infection, but the reasons why are not fully understood. During pleural infection, the pleural fluid is exudative and contains a diverse milieu of biologically active molecules. Despite the clinical importance of empyema, no studies have investigated the biological interactions between bacteria and pleural fluids. It is unknown, for example, if bacteria are merely shed from the pleural tissues into the fluid and if the fluid influences bacterial growth. On one hand, exudative fluids are rich in nutrients that can potentially enhance microbial growth; on the other hand, many proteins and enzymes  in the fluid can serve to defend against bacterial invasion. These questions are difficult to study in empyema fluid as the presence of bacteria can profoundly alter the pleural fluid composition. In clinical settings, patients are often given systemic antibiotics before the fluid is sampled, taking away the opportunity to study the effects of bacterial growth.Compared to most other pathogens that cause pneumonia, S. pneumoniae strains. We hypothesized that pleural fluid can serve as a growth medium for common empyema bacteria such as S. pneumoniae. We further investigated the role of common pneumococcal surface proteins, in particular nutrient transporters, on pneumococcal growth and survival within human pleural fluid.Pleural effusion is not unique to pleural infection but is the common end-product of a variety of other non-infective pleural diseases/inflammation that leads to vascular hyper-permeability and plasma leak into the pleural cavity. In this paper, we studied whether the pleural fluid of patients with other non-infective pleural etiologies could support survival and growth of Pleural fluid samples were collected from patients attending Sir Charles Gairdner Hospital (SCGH) that required pleural fluid drainage for clinical indications, with approval (SCGH Human Research Ethics Committee 2012\u2013156).g for 10 minutes.Pleural fluid samples were collected using aseptic techniques from patients with pleural effusions of non-infective etiologies. All samples were subjected to routine bacterial culture for which the fluid was injected into and transported in blood culture bottles and processed in an accredited hospital laboratory . Pleural samples confirmed to be culture-negative were used in experiments. Patients who received antibiotics within 72 hours were excluded. Pleural fluid biochemistry  was measured as previously described . SupernaPleural fluids (n = 53) were collected from 45 patients; effusions obtained from the same individual (n = 8) were collected on separate occasions at least 7 days apart. Most samples were malignant pleural effusions  and 10.6% (n = 5) were transudates; . SamplesS. pneumoniae (n = 24) from patients with invasive pneumococcal infection were obtained from PathWest . Serotypes of the isolates include type 1, 11A and 19F from pleural fluid and 1, 6B, 6C, 8 (n = 3), 10A, 11A, 12F, 19A (n = 8), 21, 22F (n = 2) and 35B from blood cultures. In addition, a S. pneumoniae reference strain of capsular serotype 3  was used , Staphylococcus aureus ATCC\u00ae9144, Escherichia coli ATCC\u00ae105365 and ATCC\u00ae25922, Moraxella catarrhalis NCTC\u00ae3622, Pseudomonas aeruginosa ATCC\u00ae25668 and clinical isolates (n = 5) capable of causing invasive disease including methicillin-resistant S. aureus, P. aeruginosa (n = 2), Klebsiella pneumoniae and Klebsiella oxytoca (PathWest) were used for comparison growth studies with S. pneumoniae  including eumoniae .S. pneumoniae, a capsule type-2 isolate, and its derivatives (n = 11) with mutations in genes required for full virulence have previously been described [Wild-type D39 ives n =  with mutescribed , and thoS. pneumoniae were prepared as previously described [To standardize counts between experiments, frozen inocula of escribed . Inocula6 CFU/mL of bacteria and incubated for 24hrs at 37\u00b0C and 5% CO2 unless specified. Baseline inoculum and final concentrations (CFU/mL) were verified by manual counting log serial dilutions from blood agar.Pleural fluid samples  were warmed to 37\u00b0C then inoculated with approximately 1.5\u00d710S. pneumoniae isolates (n = 25). Six pairs were inoculated within 6 hours from collection and all within 24 hours (range 1 to 18 hours). Growth was compared in parallel to Todd-Hewitt Broth  containing 17% FCS , [S. pneumoniae clinical isolate (n = 2) controls, at the same starting inoculum of 1.5\u00d7106 CFU/mL, for comparison purposes. The CFU/mL of bacteria failing to grow at 24 hours was subsequently measured at 4 and 8 hours in pleural fluid (n = 3).Pleural fluid characteristics and bacteria used in each experiment are presented in ia, WA), , Dulbecc\u0394psaA in parallel to wild type in pleural fluids (n = 16) with and without supplementation of 3\u03bcM manganese chloride tetrahydrate  were performed. The concentration was determined following concentration dependent supplementation experiments ranging from 0.003 to 30\u03bcM of manganese.D39 wild and mutant pneumococci (n = 12) were tested in pleural effusions (n = 8). Further experiments with Statistical analyses were performed using SigmaPlot 12.5 . Results are presented as mean (SD) or median (IQR) based on normality of data. The Wilcoxon Signed-Rank test and paired t-test (for non-parametric and parametric data respectively) were used to compare baseline to 24 hour pneumococcal concentrations and Student\u2019s t-test to compare growth between pleural fluid and other medium at various time-points over 48 hours. The Mann-Whitney Rank Sum test was used to compare differences between samples containing cells and correspondent supernatant and the Pearson Correlation was used to determine any relationship between spun and unspun samples. Significance was defined as p<0.05.6 CFU/mL S. pneumoniae (n = 3), with a median increase of 6875 (IQR 3494\u201315744) fold at 24 hours compared to baseline. Volumes ranging from 1 to 10mL in both tubes and multi-well plates demonstrated similar growth, however volumes less than 1mL had inconsistent or poor growth (data not shown). Inoculum concentrations in the range of 1.5 x 103 to 1.5x 106 CFU/mL demonstrated similar bacterial growth at 24 hours; an initial concentration of 106 CFU/mL was used across experiments. Samples were incubated for 24 hours following inoculation as both S. pneumoniae isolates and D39 mutant strains were found to plateau at this time point (data not shown). No difference in CFU/mL of pneumococci was observed in fluids stored for up to 96 hours at 2\u20138\u00b0C following collection.Significant growth was observed when 3mL volumes of pleural fluid were inoculated with 1.5 \u00d7 10S. pneumoniae proliferated rapidly in all (n = 11) effusion samples tested (5 CFU/mL to 1.3 \u00d7 109 CFU/mL), p<0.001 after 24 hours. S. pneumoniae growth was consistent with no difference in CFU/mL when data was analyzed as growth per S. pneumoniae isolate, growth in each pleural fluid sample or growth per serotype (p = 0.776).All 25 strains of s tested  by a medS. pneumoniae growth, with a median fold increase at 24 hours of 2857 . Growth of S. pneumoniae in pleural fluid with cells correlated closely to growth in pleural fluid without cells present  THB with fetal calf serum (17%), a supplemented culture medium commonly used to cultivate pneumococci, 2) DMEM, a glucose rich cell culture medium and 3) 0.85% saline as a control. Rapid proliferation of S. pneumoniae was observed in THB with FCS, DMEM and pleural fluid within 8 hours following inoculation , p<0.001. Furthermore, inconsistent growth between strains of the same bacterial pathogen was evident, including within the viridans streptococci group (n = 3) which are a frequent cause of adult empyema. The bacterial pathogens which failed to grow at 24 hours in pleural fluid did not demonstrate increased growth at earlier time points of 4 or 8 hours (data not shown).We investigated the growth of other bacterial pathogens (n = 14) across pleural fluid samples (n = 7). Growth measured at 24 hours was variable across other bacterial pathogens  with a mS. pneumoniae growth in pleural fluid, growth experiments were repeated using the D39 wild-type strain and eleven mutant strains  in eight pleural fluid samples  whereas the D39 wild strain had increased 1214-fold . A decrease in \u0394psaA concentrations from baseline was apparent at 4 hours and further decreased at 8 and 24 hours. Importantly, manganese supplementation of pleural fluid by the addition of 3uM of manganese prior to inoculation restored \u0394psaA proliferation in the pleural fluid, increasing the median fold-change in CFU from 0.11 (0.07\u2013501) to 1546 (509\u20134775) at 24 hours, p <0.001 (\u0394pit and \u0394adcAII/I mutants respectively had no impact on growth compared to the D39 wild type control (data not shown).To identify factors required for  samples . These m in vivo , 20, 22.p <0.001 . 3\u03bcM manS. pneumoniae is a common cause of both pediatric and adult empyema, yet the reasons why this pathogen can cause pleural infections have only been partially investigated. This is the first study to investigate the interactions of bacteria, namely S. pneumoniae, and human pleural fluid ex vivo. We found that pleural fluid provides a rich medium to support the growth of all relevant S. pneumoniae strains tested and that pneumococci appear to be much better adapted to grow in pleural fluid than most other common respiratory pathogens. More importantly, growth of S. pneumoniae in pleural fluid was dependent on PsaA, a manganese transporter making this a potential future therapeutic target. Mutations of other key virulence factors of S. pneumoniae did not affect the growth of pneumococci in human pleural fluid ex vivo.S. pneumoniae from infected pneumonia tissues invades the pleural cavity within 4 hours of infection via transcytosis across the mesothelial cells [We have previously shown in our mouse model that al cells . Little S. pneumoniae consistently proliferated across eleven human exudative pleural fluid samples tested with a median increase of approximately 3000-fold from baseline at 24 hours. In contrast, the median growth of other bacterial pathogens in pleural fluid was approximately 10-fold, and interestingly the growth was variable with some bacterial isolates demonstrating similar levels of growth to S. pneumoniae, whilst other isolates consistently failed to grow.To investigate the capacity of pleural fluid to support bacterial growth we used human pleural effusions, most with malignant etiology. Parapneumonic effusions and malignant pleural effusions are both exudative in nature and have indistinguishable biochemical compositions with elevated protein and lactate dehydrogenase and reduced glucose and pH. The effusions used for this study display the same biochemical features of non-infective simple parapneumonic effusions. Our results show that all 25 isolates of S. pneumoniae was similar to optimal laboratory media at 8 hours demonstrating that pleural fluid represents an excellent growth medium for S. pneumoniae. Furthermore, pneumococci naturally undergo autolysis after reaching stationary phase during growth which was observed in broth at 18 hours, yet bacterial CFU/mL density was maintained for 48 hours after growth in human pleural fluid. This persistent growth is likely to promote significant neutrophil influx and may be a reason why pneumococci are a frequent cause of empyema. This failure of autolysis would ensure S. pneumoniae strains are capable of maintaining high level infection even in a closed environment such as a pleural effusion. The reasons why autolysis did not occur after growth in pleural fluid are not known. The cellular content of the pleural fluid did not influence S. pneumoniae proliferation, indicating it is the pleural fluid itself that provides the medium for S. pneumoniae growth. The ability to grow in pleural fluid did not vary between individual S. pneumoniae isolates or serotypes investigated.When tested in parallel, the growth of S. pneumoniae have been identified as essential for virulence and growth of the bacterium. We investigated eleven of these virulence factors for their role during growth in pleural fluid, using mutants that are known to have reduced growth in blood  [ [psaA mutant. PsaA is a lipoprotein required for manganese transport and resistance to oxidative stress [S. pneumoniae and growth in laboratory medium that has been depleted of manganese. Providing biochemical complementation of the \u0394psaA mutation by supplementing pleural fluid with 3uM manganese was sufficient to restore growth in pleural fluid, confirming that it is the low concentration of manganese that causes failure of growth of the \u0394psaA strain [S. pneumoniae in pleural fluid yet pneumococci are capable of overcoming this using the PsaA transporter. Potential PsaA inhibitors that have recently been identified open opportunities to control S. pneumoniae proliferation in the pleural space [S. pneumoniae with mutations affecting transporters required for iron or zinc uptake was not affected, suggesting that manganese results are specific for growth within pleural fluid.Multiple cell surface proteins that function as adhesins, in complement resistance, autolytic enzymes and nutrient transporters in d \u0394livH) , 24\u201327 o\u0394adcAII) , 28, 29.e stress , 30, 31.al space . InteresS. pneumoniae in vitro. Next, due to the nature of the study, experiments were performed in ex vivo pleural fluid and therefore host defenses, including neutrophils that may modify the bacterial proliferation were not accounted for. However, as a proof of principle study our aim was to investigate the direct effect of pleural fluid on S. pneumoniae proliferation. Furthermore, our data showed no difference in proliferation with or without cellular content of the pleural fluid. Finally, we studied 25 invasive strains of S. pneumoniae that had caused empyema or septicemia. Future studies that include other serotypes may be needed, though the very consistent nature of the results indicates that the majority of S. pneumoniae isolates are likely to grow in pleural fluid.The study has a number of potential limitations. We were unable to use pleural fluid of infected etiology in the present study for reasons stated in the introduction. However, malignant pleural effusions have a close resemblance in biochemistry to parapneumonic effusions and were therefore used for the majority of our experiments. Although the cellular content of these effusions may differ, we found that the cellular content of the fluid did not influence the growth/proliferation of S. pneumoniae and may be one reason why pneumococci are a common bacterial cause of empyema. The high density and persistent growth of S. pneumoniae in pleural fluid highlights the importance of draining infected effusions. Pneumococcal growth was independent of the cellular content of pleural fluid but dependent on the PsaA surface protein, making PsaA a potential future therapeutic target. The variable growth of other bacterial pathogens in pleural fluid may account for the lower incidence of otherwise common respiratory pathogens in empyema and warrants further exploration in order to understand pathogenesis of pleural infection.Pleural fluid is a potent growth medium for S1 Table(DOC)Click here for additional data file."}
{"text": "The burden of pleural diseases has substantially increased in the past decade because of a rise in the incidence of pleural space infections and pleural malignancies in a patient population that is older and more immunocompromised and has more comorbidities. This complexity increasingly requires minimally invasive diagnostic options and tailored management. Implications for patients are such that the limitations of current diagnostic methods need to be addressed by multidisciplinary teams of investigators from the fields of imaging, biology, and engineering. Ignored for a long time as an epiphenomenon at the crossroad of many unrelated medical problems, pleural diseases are finally getting the attention they deserve and have spurred a vibrant and exciting field of research. Interestingly, surgical removal of the pleura, or chemical fusion of the visceral and parietal membranes for therapeutic purposes (pleurodesis), seems to have little physiologic effect on lung function or work of breathing in long-term human studies and this raises the question of why such a complex structure was selected for in the first place5.The term pleura refers to the membrane separating the lungs from the chest wall, which is constituted by two leaflets attached to the outer surface of the lung  and the inside of the chest wall , joining at the hilum of the lungs, hence forming a genuine sac surrounding both lungs. The small amount of pleural fluid separating visceral from parietal pleural membranes serves as a lubricant, optimizing respiratory mechanics by facilitating synchronous chest wall and lung movements during respiratory efforts6. Approximately 150,000 of these effusions are caused by malignancy\u2014lung and breast cancer are by far the most common offenders\u2014generally due to parietal pleural involvement in advanced disease7. Other common causes of pleural effusions include congestive heart failure, pleural space infections, pulmonary embolism, and manifestations of connective tissue diseases such as lupus or rheumatoid arthritis. It should be intuitively obvious, given the profound implications of such diagnoses for patients, that timely and accurate diagnosis of unexplained pleural effusions is of considerable importance. Pleural fluid aspiration followed by application of the time-honored Light\u2019s criteria remains a remarkably useful step in the diagnostic algorithm of pleural effusions, although an expanding panel of diagnostic tools in the past decade has slowly transformed our approach to pleural diseases, the increasing complexity of which has motivated the development of multidisciplinary pleural subspecialty programs at many institutions in recent years12. In this brief report, we will attempt to summarize what we believe are the most significant and promising recent advances in the diagnosis of pleural diseases and highlight current research efforts in the field.Notwithstanding this lack of clear physiologic function, pleural disorders represent one of the most common reasons for consultation of lung specialists, and an estimated 1.5 million new pleural effusions identified every year in the US alone are caused by more than 60 distinct disease processes16. In fact, the evidence for improved outcomes and reduced complication rates with ultrasound for thoracic procedures is so overwhelming that its use is now universally considered the standard of care18. Bedside ultrasound examination of the pleura can easily confirm the presence of pleural fluid when conventional chest x-ray findings are equivocal. In addition, it allows an estimation of its volume and complexity and occasionally identifies pleural nodules or masses suggestive of pleural malignancy19. While most physicians use ultrasound merely to mark the site for needle insertion before setting up for the procedure, real-time ultrasound using a sterile sleeve allows sampling of previously unreachable small loculated effusions and occasionally allows parietal pleural biopsies. Furthermore, recent evidence suggests that pre-procedural identification of the intercostal artery, the course of which can be unpredictable, is easily achieved with a high-frequency probe and could help avoid rare but potentially fatal bleeding complications22. Ultrasound examination immediately after the procedure can indirectly suggest the occurrence of pneumothorax by the disappearance of the typical \u201csliding\u201d sign, representing the sliding of visceral and parietal pleura over each other. Rare but potentially serious bleeding complications from injury of the intercostal artery or one of its collateral vessels may also be identified in the form of rapid re-accumulation of echogenic pleural fluid24.Thoracic ultrasound, widely available in the form of increasingly sophisticated and affordable portable and handheld units, has unquestionably changed the paradigm in bedside medical evaluation. Basic ultrasound education now complements traditional physical examination training at many medical schools, residency, and fellowship programs. Point-of-care ultrasound for diagnosis and management of thoracic diseases in particular is recommended and endorsed by most relevant scientific societies, and there are established training and certification standards26. An intriguing recent report suggests that ultrasound may allow the identification of unexpandable lung before thoracentesis. In some cases, such as when chronic inflammatory effusions have resulted in the creation of a thick peel around the lung, fluid drainage is not accompanied by lung re-expansion. The vacuum generated can expose patients to complications including pain, pneumothorax, and re-expansion pulmonary edema. Transmission of the heartbeat results in different lung movement and deformation changes that can be identified by using specific ultrasound modes such as the M-mode and speckle tracking27. In this small study, ultrasound in fact performed better than pleural manometry, long regarded as the gold standard for the diagnosis of unexpandable lung and the utility of which is being assessed in an ongoing randomized clinical trial .Interestingly, there has been relatively little research on pleural ultrasound as a predictive tool. Two small studies suggest that a highly organized pleural space with loculations and septations during pleural space infection predicts poorer outcomes, but these studies are small and unadjusted for possible confounders28. However, in the absence of such specific findings, computed tomography appears neither sensitive nor specific enough to obviate the need for invasive diagnostic interventions30. Positron emission tomography is often used to assess the probability of malignant pleural effusion but with modest sensitivity and specificity, which in a recent meta-analysis were estimated to be 81% and 74%, respectively31. Recently, magnetic resonance imaging has been proposed as a potentially superior imaging modality for pleural effusions in general and malignant pleural mesothelioma in particular but remains largely underutilized compared with computed tomography34. Radiomic approaches to thoracic diseases represent an early and exciting field in imaging science, and very preliminary work in quantitative imaging for the diagnosis of malignant pleural effusion\u2014and staging and diagnosis of mesothelioma in particular\u2014is generating considerable interest36.There have been comparatively less recent data on other imaging modalities. Whereas loculations (sequestrations of fluid in non-dependent areas) are well identified on computed tomography, the degree of organization within the effusion is better appreciated on ultrasound. In addition, the presence of any of the four following radiologic signs is highly predictive of malignancy: parietal pleural thickening of more than 1 cm, thickened mediastinal pleura, circumferential thickening, and the presence of pleural nodules11. Briefly, at the risk of oversimplifying complex processes, transudative effusions typically result from local alterations of the Starling rules that govern capillary microcirculation in the parietal pleura with increased hydrostatic  or decreased oncotic  pressures, whereas exudative effusions suggest a disease process involving the parietal pleura itself, whether from malignancy, infection, or inflammatory diseases. However, these processes are complex and overlaps are frequent in terms of both pathophysiology and pleural fluid analysis. For example, an estimated 25% of effusions due to congestive heart failure may classify as exudates (\u201cpseudo-exudates\u201d), particularly if the patient is on diuretics37, and conversely a small percentage of documented malignant pleural effusions may classify as transudative effusions. While a high index of suspicion should lead to pleural biopsies in the latter scenario, recent data suggest that the pleural fluid\u2013to\u2013serum albumin gradient could help clarify the etiology of heart failure\u2013related pseudo-exudative pleural effusions38. Pleural and serum brain natriuretic peptide and amino-terminal pro-brain natriuretic peptide levels are also useful to establish heart failure as the cause of the effusion39. Pleural space infections, potentially life-threatening conditions, are usually characterized by typical pleural fluid biochemistry and hence are more straightforward from a diagnostic standpoint, and an interesting recent observation is that the yield of pleural fluid cultures can be increased by 20% by simply inoculating blood culture bottles with pleural fluid at the bedside rather than sending the fluid directly to the laboratory40.The \u201cdiagnostic separation of transudates and exudates\u201d using a combination of pleural fluid protein and lactate dehydrogenase levels was published in 1972  and to this day remains the single most important step in determining the etiology of a pleural effusion41, although some reports suggest much lower estimates42. Others focusing on modern immunohistochemistry techniques seem much more optimistic, particularly for mesothelioma, a primary pleural malignancy which traditionally requires pleural biopsies for definitive diagnosis44. In addition, pleural fluid has been shown to be a biospecimen suitable for the majority of molecular analyses required for targeted therapy45. High-throughput techniques and modern molecular techniques promise to identify biomarkers expressed by cancer cells or their environment that could ultimately transform our diagnostic approach. Novel proposed techniques include single-cell mechanophenotyping which evaluates the deformability of pleural cells46, circulating tumor cells and cell-free tumor DNA48, and metabolic-based assays to identify non-leukocyte metabolically active tumor cells49. These recent developments, though exciting, remain preliminary and are still closer to the bench than they are to the clinic. The search for optimal biomarkers has been hampered by a lack of standardized methodology and failure to externally validate promising results, as in the case of fibulin-3, a biomarker once anticipated to transform our approach to malignant mesothelioma53. Large ongoing research projects are attempting to identify reliable alternative candidates.The diagnostic utility of pleural fluid analysis for malignant pleural effusions has been the object of several recent studies. The sensitivity of cytology for malignancy is estimated around 60%, and there is a 15% increase with a second procedure54. A large multicenter observational prospective study to validate this score is ongoing. Similarly, the LENT  score is a clinical prediction model that provides estimates of survival for patients with malignant pleural effusions, which could prove very useful in subject selection in clinical trials and ultimately individualized medical or surgical management of pleural effusions55.An unprecedented number of well-designed randomized controlled studies published in the last decade by a growing international pleural research network have clarified and sometimes transformed patient management for malignant pleural effusions and pleural space infections in particular. However, one major obstacle faced by researchers and clinicians has been the lack of clinical prediction models allowing appropriate patient selection and stratification. The RAPID  score was derived and validated from two large datasets and proposes to risk-stratify patients with pleural space infections, and ultimately its purpose is to individualize management, which currently is subject to local preferences and expertise rather than patient characteristics56. An example from a Bayesian approach to diagnosis might be helpful to illustrate the limitations of this approach. Given a sensitivity for cytology of 60% and a specificity of 100%, a patient with a 50% pre-test probability of pleural malignancy would have, after a negative cytology, a remaining 27% post-test probability of disease. After a second thoracentesis, this number decreases to only approximately 18% (a generous estimate as the same test is used twice). Given the profound implications of a diagnosis of malignant pleural effusion, it should be intuitively obvious that moving forward with additional, ideally minimally invasive, diagnostic tests would be desirable.Approximately 25% of exudative pleural effusions remain without identifiable causes after pleural fluid analysis and cytology. Parietal pleural biopsies are the recommended next step in the diagnostic assessment but often are not pursued as they sometimes require thoracic surgery which, though less invasive since the advent of video-assisted thoracoscopic surgery, remains a major operation requiring general anesthesia, double-lumen endotracheal intubation, large-bore chest tube placement, and a hospital stay. Hence, clinicians often default to observation after an unfruitful pleural fluid analysis61. While pleuroscopy is slowly gaining traction in the US, centers offering the procedure have exponentially increased over the past decade in Europe and other parts of the world and presumably this is due to more favorable regulatory environments and sometimes more selective access to thoracic surgery. The development of dedicated interventional pulmonology training programs in the US, with accreditation standards that include pleuroscopy training endorsed by all relevant medical societies, may facilitate a more widespread adoption of this safe and effective diagnostic modality15. Even less invasive interventions, such as \u201cmini-thoracoscopy\u201d, using increasingly smaller instruments, have been proposed62.Reclaiming a procedure initially introduced by Hans Christian Jacobeus, a Swedish Professor of Internal Medicine at the Karolinska Institute in Stockholm from 1916 to 1937, interventional pulmonologists and pleural specialists have popularized minimally invasive, so-called \u201clocal anesthetic\u201d pleuroscopy, which allows pleural exploration and biopsies in awake patients and often is performed on an outpatient basis63.When focal pleural lesions can be identified by computed tomography or ultrasound, percutaneous image-guided biopsies performed by interventional radiologists or pulmonary specialists are a useful and minimally invasive approach to diagnosis which in recent studies has had a yield similar to that of thoracoscopy but without the option to offer definitive treatment in the same setting The rising burden of pleural disease in an increasingly complex patient population demands a more tailored approach to diagnosis and management than ever before. Thoracic ultrasound, the application of new bioassays in addition to foundational biochemical analysis of pleural fluid, the development of models for prognosis and prediction of treatment response, and the resurgence of medical thoracoscopy-pleuroscopy comprise recent advances in pleural disease, and there is a great need for further basic, translational, and clinical research in this field."}
{"text": "The consultation is open until 28 November 2016.On 17 October, the European Centre for Disease Prevention and Control (ECDC) launched a public consultation on the The aim of the consultation is to harvest input for the ECDC expert opinion which should provide European Union/European Economic Area Member States with scientific opinion and expert opinion to support the decision-making process on the possible introduction and monitoring of routine vaccination against rotavirus-induced gastroenteritis in infants.here on how to submit contributions.Please read more"}
{"text": "Regiella insecticola on the life table parameters of Sitobion avenae. The results showed that R. insecticola can decrease the intrinsic rate of increase (r), the finite rate of increase (\u03bb) and birth rate and can increase the mean generation times (T) of S. avenae clones, suggesting that R. insecticola may decelerate the normal development of the hosts. No significant differences of these parameters were observed between the examined Sitobion avenae clones, and the symbiont treatment by genotype interaction affected only the net reproduction rate R0, pre-adult duration and total longevity but not the other parameters. Additionally, a population projection showed that R. insecticola decelerated the growth of the S. avenae clones. The evocable effects of R. insecticola on the S. avenae clones may have significant ramifications for the control of S. avenae populations under field/natural conditions.Many insects harbor heritable endosymbionts, whether obligatory or facultative, and the role of facultative endosymbionts in shaping the phenotype of these species has become increasingly important. However, little is known about whether micro-injected endosymbionts can have any effects on aphid clones, which was measured using various ecological parameters. We examined the effects between symbiotic treatments and the vital life history traits generated by  Buchnera aphidicola, which belongs to the class \u03b3-proteobacterium. B. aphidicola synthesizes essential amino acids and other nutrients that are ingested in very small quantities from restricted diets, such as plant phloem, for their host aphids23Regiella insecticola, Hamiltonella defensa and Serratia symbiotica belong to the class \u03b3-proteobacterium, and Rickettsia and Spiroplasma belong to the class \u03b1-proteobacterium56789101112Many insect species harbor endosymbionts, which are indispensable to their survival and common reproduction. Because the hosts and their endosymbionts share a common fate, endosymbionts can often profoundly affect the ecology and evolution of their hosts. Among them, the endosymbionts in aphids are ideal targets for studying their role in ecological fitnessAcyrthosiphon pisum (Harris), including resistance to heat shock . However, a key component of this knowledge is the life table. The life table analysis is the most reliable method for evaluating the survival and reproduction of a populationr) represents the reproductive potential of a population in relation to its capacity to increaseR0) has been used to summarize the mean number of offspring that an individual can produce during its lifetime, with the mean generation time (T) defined as the period that a population needs to increase its size R0-foldBuchnera, on which aphid performance and fecundity dependsR. insecticola on hosts, it is necessary to obtain S. avenae clones with R. insecticola inoculated via micro-injection, which would have the same genetic background as symbiont-free aphids. Additionally, different aphid species or different genetic backgrounds within one species in the field can harbor various endosymbionts, and it is not clear whether the genotypes of the hosts could impact the ecological effects of endosymbionts. Therefore, the study of the interactive effects between micro-injected endosymbionts and the genotypes of the hosts and the ecological effects of endosymbionts on the aphid population is warranted.Secondary endosymbionts do not present in all aphid individuals, suggesting they are not generally required for the survival and reproduction of aphids. However, endosymbionts can significantly affect host aphid ecological fitness and behavior. In the past few decades, secondary endosymbionts have been found to confer benefits on the pea aphid R. insecticola on different S. avenae clones based on the life table data. The results that we present here might provide new insight for the future systematic study of endosymbionts and its ramifications in suppressing S. avenae populations.The objectives of the study are to investigate the ecological effects of R. insecticola significantly impacted the population parameters . Clone nested in \u2018R. insecticola\u2019 had no impact on r (P\u2009=\u20090.091), T (P\u2009=\u20090.236) and \u03bb (P\u2009=\u20090.090), but significantly impacted R0(P\u2009=\u20090.001).A two-way nested analysis of variance was used to analyze the effect of endosymbionts on the population parameters in the different genotypes of aphid host . R. inseF\u2009=\u200927.418, P\u2009<\u20090.001 and F\u2009=\u200928.216, P\u2009<\u20090.001 for r and \u03bb, respectively) than the controls Linyi-NA (Linyi clone) and Neixiang-NA (Neixiang clone). There were no differences between the treatments  . However, the mean generation times (T) of treatments Linyi-1, Linyi-2, Neixiang-1 and Neixiang-2 were significantly longer  than that of the controls Linyi-NA and Neixiang-NA, and no differences were found between the treatments  (P\u2009=\u20090.119).The population parameters of these original aphid clones (controls) were compared to those corresponding to infected clones (treatments) . Body flR. insecticola clearly affected the birth rate (P\u2009<\u20090.001), pre-adult duration (P\u2009<\u20090.001) and total longevity (P\u2009=\u20090.001). Clone nested in \u2018R. insecticola\u2019 had no impact on birth rate (P\u2009=\u20090.091) but had a significant impact on pre-adult duration and total longevity .A two-way nested analysis of variance was used to analyze the effect of endosymbionts on fitness traits in the different genotypes of the aphid host . R. inseR. insecticola had a remarkable effect on birth rate, and birth rates in the treatments Linyi-1, Linyi-2, Neixiang-1 and Neixiang-2 were significantly lower  than that in the controls Linyi-NA and Neixiang-NA. No differences in birth rate were found between the treatments  (P\u2009=\u20090.163). The pre-adult duration of the treatments Linyi-1, Linyi-2, Neixiang-1 and Neixiang-2  were clearly longer than that of the controls Linyi-NA (7.03\u2009\u00b1\u20090.126) and Neixiang-NA (6.62\u2009\u00b1\u20090.950). In addition, the total longevity of the treatments Linyi-1, Linyi-2, Neixiang-1 and Neixiang-2  were clearly higher than that of the control Linyi-NA (22.29\u2009\u00b1\u20090.756).Fitness traits  of the controls clones were compared to those corresponding to infected clones (treatments) . R. insest instar nymphs, are shown in th, 15th, 16th and 16th day, respectively, and the corresponding controls Linyi-NA and Neixiang-NA both occurred on the 14th day. This showed that the outbreak times of the treatments were delayed compared to those of the controls, and it suggested that R. insecticola might postpone the timing of the adult populations in the studied S. avenae clones.The simulated population growth and stage structure of the adult stages for 30 days, beginning with an initial population of 10 pairs of 1A. pisum, Aphis craccivora and Myzus persicae) have been extensively studied in recent decades56789183132333435H. defensa can protect A. pisum from its natural enemies11618et al.H. defensa has little effect on the resistance to the natural enemies in S. avenae, indicating that secondary endosymbionts might, to some extent, have different effects on related species. Therefore, it is necessary to study the effects of the secondary symbionts on different aphid clones using the life table.The diverse infections and functions of secondary endosymbionts of aphids , though the interaction did affect the net reproductive rate (R0), pre-adult duration and total longevity. For a comprehensive understanding of the treatment by genotype interaction, more studies are needed.A linear two-way nested analysis of variance (ANOVA) showed that the treatment by genotype interaction did not affect aphid fitness , the total developmental time of nymphs (DT5), and 10 d fecundity. However, their research did not focus on the population parameters. In life table research, the population parameters represent the intrinsic rate of increase r, the net reproductive rate R0, the mean generation times T and the finite rate of increase \u03bb. In our study, the main life table parameters researched were the population parameters.Understanding the entire life history plays a significant role in evaluating the population parameters that are influenced by secondary endosymbionts, and the key component of this analysis is the life table, which can provide a comprehensive description of the development, survival, and fecundity of a populationr) of the treatments was significantly lower than that of the controls. The intrinsic rate of increase is a key demographic parameter that has been used to summarize the reproductive potential of an animal population in relation to its capacity to increaseR. insecticola on the potential growth of S. avenae clones is intriguing. This notion was confirmed by the mean generation times (T) of the treatments, which were shorter than that of the controls. Oliver et al.H. defensa shortened the mean generation time (T) of A. pisum. Although R. insecticola might decelerate the potential growth of S. avenae populations, the finite rates of increase (\u03bb) were all over 1 between the treatments and the controls, indicating that the studied S. avenae population increased continuously. The net reproductive rates (R0\u2009>\u20091) between the treatments and the controls also confirmed this observation. However, the finite rates of increase (\u03bb) of the treatments with the secondary symbionts were remarkably less than that of the controls, suggesting that the daily increase of S. avenae clones in the treatment groups was significantly lower than that of the controls. This was confirmed by the birth rates of the treatment groups, which were significantly lower than that of the controls. In conclusion, R. insecticola may reduce the potential growth of S. avenae clones, and this is consistent with the findings of Wang et al.The intrinsic rate of increase , finite rates of increase (\u03bb), mean generation time (T) and birth rate demonstrated that R. insecticola infection had a negative effect on the natural development of S. avenae clones, and the impact levels differed among different genetic backgrounds. The difference between genotypes might be due to variations in host nutrients. For example, the effect of nitrogen content and protein quality might affect the fecundity of herbivores383940S. symbiotica, H. defense and Rickettsia, need to be examined individually, and (3) more donor and recipient clones or species could be used, as only 2 aphid clones were used as donors and 2 aphid clones were recipients in the current study. Such efforts would provide us with a comprehensive perspective that would aid our understanding of the complex symbiotic relationships between aphids and bacterial endosymbionts.The intrinsic rates of increase , pre-adult duration and total longevity. In addition, the significant reduction of fitness traits  and the clear increase of the mean generation times (T) indicated that R. insecticola might have a negative effect on the potential population growth of S. avenae.In conclusion, the findings of this study indicate that the interaction effects between R. insecticola were collected in Neixiang , Henan Province and in Linyi , Shandong Province in May 2014. Collected clones were genotyped at four microsatellite loci  as described in the R. insecticola donor clones were collected in April 2014 in two quadrats, at a distance of 300\u2009m from each other, in Yangpingguan , Shaanxi. They were named Yangpingguan 1 and Yangpingguan 2. Each clone was derived from a single parthenogenetic S. avenae female, and the endosymbionts of the four clones were detected. The primers are shown in the Triticum aestivum L.) seedlings (variety \u20181376\u2019) that were changed at ten-day intervalsAphid clones (considered genotypes in this paper) used in this study were established from individuals collected from winter wheat plants on private farm land, with the permission of the owners. Clones without R. insecticola into either 2nd or 3rd instar S. avenae individuals. To do this, the Yangpingguan clones 1 and 2 (from Shaanxi) that hosted R. insecticola were chosen as the donor clones. Body fluids from the Yangpingguan clones 1 and 2 were transferred into the Linyi (from Shandong) and the Neixiang clones (from Henan), respectively, resulting in the creation of four new clones . Therefore, the Linyi-1 and Linyi-2 clones and the Neixiang-1 and Neixiang-2 clones have the same genetic background as the Linyi (Shandong) and the Neixiang clones (Shandong), respectively. The surviving aphids  after the injection were transferred to fresh wheat plants (one aphid per plant). Twelve hours later, the dead aphids were discarded, and the remaining surviving aphids were reared till they became adults and produce nymphs. Next, one single offspring individual from each clone that tested \u201cpositive\u201d was subjected to PCR using PCR primers described in Sandstr\u00f6m et al.Taq DNA polymerase mix, and the following cycle parameters: 94\u2009\u00b0C for 7\u2009min and 35 cycles of 94\u2009\u00b0C for 30\u2009s, 57\u2009\u00b0C for 45\u2009s and 72\u2009\u00b0C for 30\u2009s. The resulting PCR products were separated in a 1% agarose gel and visualized under UV after gel staining with ethidium bromide for the presence of R. insecticola and were then placed on T. aestivum and allowed to reproduce for several days. At this time, adults were removed and 5 individuals were tested again for the presence of R. insecticola. One positive clone was then used to conduct the following study. To ensure that R. insecticola densities in S. avenae produced clones approached equilibrium, the original data of the life table were collected after a minimum of 10 generationsThe micro-injection technique advocated by Chen & PurcellS. avenae clones (unpublished data). Experiments were performed using three groups for each individual clone, with a total of 30 aphids in each group. Newly emerged 1st instar nymphs were individually transferred within 24\u2009h to a 9-cm Petri dish containing a fresh wheat leaf blade to start the experiment. The collection of life table data was initiated approximately 24\u2009h later. Each aphid was observed daily at the same time until all individuals died. All offspring were removed daily, and the wheat leaves were replaced every 2 days. Molting and mortality events, as well as the number of produced nymphs, were recorded.Linyi-1 and Linyi-2 clones were compared with the Linyi clone control as they all shared a common genetic background. This clone was named Linyi-NA for convenience. Similarly, Neixiang-1 and Neixiang-2 clones were compared with the Neixiang clone control, and the control was named Neixiang-NA for convenience. A preliminary experiment showed that the control clone (no injection) and the negative control (injection of water) showed no difference in the growth and development of the studied r) was estimated using an interactive bisection method and the following Euler\u2013Lotka equation  was calculated as follows  to analyze the effects of \u2018R. insecticola\u2019 and clone  nested in \u2018R. insecticola\u2019. We used Tukey\u2019s procedureP\u2009<\u20090.05. Mean values (\u00b1standard errors of the mean) were calculated and used in the above analyses. If needed, data were transformed to meet the required assumptions of normality and homoscedasticity. Graphs were generated using SigmaPlot 12.5.A 2\u2009\u00d7\u20093 factorial randomized in complete blocks with 30 replications was deployed to test How to cite this article: Luo, C. et al. Ecological impact of a secondary bacterial symbiont on the clones of Sitobion avenae (Fabricius) (Hemiptera: Aphididae). Sci. Rep.7, 40754; doi: 10.1038/srep40754 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."}
{"text": "N-acetylcysteine, and the recovery of cell viability was observed. The increase in ROS production in response to 6-shogaol was associated with cell death. Similarly, glucose uptake decreased with incremental concentrations of 6-shogaol, and an increase in the expression of mTOR-p and Akt-p proteins was observed; PTEN was active in all the treatments with 6-shogaol. Thus, the results suggest that cells activate uncontrolled signaling pathways, such as phosphoinositide 3-kinase (PI3K)/Akt/mTOR, among other alternative mechanisms of metabolic modulation and of survival in order to counteract the pro-oxidant effect of 6-shogaol and the decrease in glucose uptake. Interestingly, a differential response was observed when non-cancerous cells were treated with 6-shogaol. Ginger is a plant that is native to southern China. In the last decade and research on the components of ginger has significantly increased; of these components, 6-shogaol exhibits the greatest potential antitumor capacity. However, the molecular mechanism through which 6-shogaol exerts its effects has not yet been elucidated. In this study, the effect of 6-shogaol on tumor cells that were derived from human fibrosarcoma (HT1080) was evaluated. Cell viability was determined by a -2,5-diphenyltetrazolium bromide) MTT assay testing different concentrations of 6-shogaol (2.5\u2013150 \u03bcM). Subsequently, the effect of 6-shogaol on reactive oxygen species (ROS) production, glucose uptake, and protein expression of the signaling pathway phosphatase and tensin homolog/ protein kinase B /mammalian target of rapamycin (PTEN/Akt/mTOR) was measured. 6-Shogaol reduced the viability of the tumor cells and caused an increase in ROS production, which was attenuated with the addition of  Zingiber officinale Roscoe, Zingiberaceae) has been used for thousands of years as a spice, and it has been considered to be an important ingredient in traditional Chinese medicine for the treatment of certain diseases, such as diabetes, cardiovascular diseases, rheumatism, and cancer [Ginger  of 6-shogaol in HT1080 cells was 52.8 \u03bcM. Low concentrations of 6-shogaol exhibited effects on the cell viability of the HT1080 tumor model. From 30 \u03bcM, a clear decrease in cell viability was observed, even below 80%. No cells survived at 150 \u03bcM 6-shogaol. Fibroblasts that were derived from human periodontal ligament (HPdLF) exhibited greater resistance to treatment with 6-shogaol as compared to the HT1080 cells (p = 0.0058). HPdLF decreased its viability from 70 \u03bcM 6-shogaol (6-Shogaol (2.5\u2013150 \u03bcM) decreases HT1080 cell viability in a dose-dependent manner. The half maximal inhibitory concentration (IC-shogaol A.p = 0.0011) than fibroblasts that were derived from the periodontal ligament . The fibroblasts did not show alterations following treatment with 6-shogaol. Importantly, it can be concluded that, relative to ROS production in response to treatment with 6-shogaol, a different effect is observed between the tumor and non-tumor cells (p = 0.001 HT1080) (p = 0.0029 HPdLF). This allowed us to conclude that the increase in ROS production was due to the effect of 6-shogaol.To evaluate cell dynamics in response to treatment with 6-shogaol, concentrations of 30\u201370 \u03bcM were used, which resulted in a significant decrease in cell viability when applied in the tumor model. An increase in dose-dependent ROS production was observed in HT1080 cells, which was significant at 50 \u03bcM 6-shogaol (or cells C. When tp = 0.0091). Therefore, we concluded that cell death generated by 6-shogaol is associated with an increase in ROS production.Finally, the cell viability of the tumor model was evaluated under treatment with 6-shogaol (30\u201370 \u03bcM) and joint treatment with NAC (5 mM). Cells only treated with 6-shogaol showed decreased viability at all concentrations D, whereap = 0.0114), which can be explained by the high metabolic activity and high energy demand of tumoral cells for the maintenance of their proliferation [The assay results showed that basal glucose uptake by the tumoral cell model was greater than for normal fibroblasts , however, from 60 \u03bcM 6-shogaol, a progressive decrease in glucose uptake was observed with increasing 6-shogaol concentration , there was a decrease in the expression of these proteins A. It was observed that 6-shogaol (10 and 50 \u03bcM) induced phosphorylation of the mTOR protein, whereas PTEN was not observed to be phosphorylated at any of its three activation sites; this suggests that PTEN could be in its active (dephosphorylated) form B.p = 0.0091)  C.The differential effects of 6-shogaol on normal and tumor cell lines have already been described, however the cause of this compound having this differential effect is still unknown. It is thought that the effect is related to the increased production of ROS induced by 6-shogaol, which acts as a pro-oxidant; it is likely that this effect is more effectively neutralized by non-tumor cells through more efficient endogenous antioxidant mechanisms. In addition, it can be considered that tumor cells already have a high amount of intracellular ROS, which can make them more sensitive to this type of insult, which compromises their viability.It should be considered that 6-shogaol is not the only volatile compound with biological activities against cancer that is present in ginger. It is also known that 6-gingerol has similar antitumoral properties, but to a lesser degree .The gingerols are present at higher concentrations in fresh root. The main isoform of gingerol has a double bond in carbon 6, and it is called 6-gingerol A. 6-GingIt has been shown that 6-gingerol inhibits the progression of skin cancer induced by phorbol ester in addition to inhibiting metastasis in breast cancer (MDA-MB-231), induced apoptosis in prostate cancer (LNCaP), and it could inhibit tumor progression in melanomas and kidney cancer in murine models; there are few reports in relation to other types of cancer .Shogaols B are anaSimilarly, it has been reported that 6-shogaol has greater anti-inflammatory capacity than 6-gingerol, which inhibits the production of prostaglandin E2 (PGE2) as well as the expression of proinflammatory cytokines, such as interleukin 1\u03b2, tumor necrosis factor (TNF-\u03b1), p38, and nuclear factor kappa \u03b2 (NF-kB). In addition, it inhibits the release of nitric oxide and the expression of the enzyme nitric oxide synthase in mouse microglia cells (BV-2) treated with lipopolysaccharide (LPS), primary cultures of glia, and in vivo models of neuroinflammation . There a50 of 29.6 and 22.1 \u03bcM, respectively, when treated with 6-shogaol for 48 h [50 between the tumor lines might be due to the histological differences of each of the cell lines, which affects their differential sensitivity to the compound. The cytotoxic effects of 6-shogaol have been described in several tumor lines and using different concentrations, such as in the case of HT-29 cells of colorectal cancer, whose viability was reduced by 50% when treated with 60 \u03bcM and they were completely inviable upon treatment with 80 \u03bcM . Other tfor 48 h . The difp = 0.0001) coincide with reports that were described in human gingival fibroblasts (HGF-1), where a 10% decrease in cell viability was observed when treated with upwards of 80 \u03bcM 6-shogaol [In the present work, the significant differences in viability that were observed between non-tumor cells (HPdLF) and the HT1080 line in response to 6-shogaol treatment . However, the mechanism of 6-shogaol uptaken by cells is unknown .Studies of the SAR (structure\u2013activity relationship) have shown that the free hydroxyl group of 6-gingerol contributes to its antitumor activity against breast cancer, and that the length of the aliphatic side chain increases this activity, since this chain loses activity when its length is reduced, as shown in the tumor models . HoweverIn tumor cells, the presence of complex alterations, such as disruption of some metabolic pathways and the permanent activation of signaling pathways involved in survival, as well as the increase in nutrient consumption, can lead not only to a higher production of ROS, but also to a decrease in the ability to neutralize them effectively; therefore, the tumor cell exhibits a higher production of ROS. To counteract the effect of increased ROS on viability, recent studies have indicated that tumor cells develop alternative mechanisms of activation of antioxidant pathways, which are not sufficient when cells undergo additional pro-oxidant stimuli (6-shogaol). It is well known that increased ROS production in tumor cells induces the uncontrolled activation of the PI3K signaling pathway (PI3K/Akt/mTOR), in comparison with the response of normal cells. Consequently, the RAS pathway is activated, which leads to metabolic modulation and survival. Additionally, the resulting hyperactivation of HIF-\u03b1 is also mediated by factors, such as VEGF, modular angiogenesis, and increased proliferation and cell migration, among others .Similar results to those that were observed in the present work have also been described in other tumor models, such as mouse kidney epithelial cells (TMCK-1) and mesangial cells (MC) . These aSimilarly, Akimoto et al. 2015 demonstrated that the pro-oxidant capacity of 6-shogaol and its effect on the reduction of cell viability can be maintained with the treatment of 10 mM NAC in a cellular model of PANC-1 (pancreatic tumor cells) . It has The effect of 6-shogaol on the increase of glucose uptake in non-tumor cell models was described for the first time in adipocytes (3T3-L1) and myotubules (C2C12), demonstrating that this compound generated a greater uptake in comparison with 6-gingerol and zingerone . This efp = 0.0114); this can be explained by its high metabolic activity and its high energy demands for the maintenance of its proliferation rate (10). Additionally, the effect is characteristic of the so-called Warburg effect, which, with its modifications, has been described in recent literature reports as aerobic lactate genesis, characterized by a metabolic reprogramming that leads to an increase in glucose uptake, higher production of lactate, and increased translocation of GLUT-1 to the cell membrane [The basal glucose uptake that was determined in both cell models showed that the tumor model captures more glucose than the non-tumor model  exhibited a significant reduction in glucose uptake. It is already known that glucose competes with ascorbic acid for GLUT-1. However, more studies are needed that characterize GLUT transporter activity in this type of cell .Otherwise, the canonical concept of the role of Akt as a protein that promotes proliferation, survival, and cell growth is based on downstream Akt inducing the activation of proteins, such as mTOR, which promotes cell growth in addition to the inactivation of several proapoptotic factors such as BAD, procaspase-9, and transcription factor FKHR (forkhead). Additionally, the activation of Akt promotes the increase of transcription factors that increase the expression of anti-apoptotic genes, such as CREB, as well as direct phosphorylation of NF-kB and HIF-1\u03b1.Another mechanism through which Akt promotes survival is through the inactivation of the p53 tumor suppressor gene . TherefoIt has recently been described that the activation of Akt might be related to an increase in glucose in the medium, a process that induces ROS production and is characteristic of hyperglycemia mechanisms, where, if glucose is unable to enter the cell, either by decreasing the translocation of GLUT-1 or by any other mechanism, its extracellular accumulation will allow for the activation of Akt and the induction of premature senescence .Premature cellular senescence is an irreversible state of cell growth arrest that is characterized by a set of physicochemical changes and cellular functions . CurrentWhile considering the results that were obtained and the information outlined in previous paragraphs, it is very likely that 6-shogaol induces premature senescence through increased ROS production, and that this is related to the activation of Akt; similarly, the recently described mechanisms suggest that the resulting decrease in glucose uptake makes the cells more sensitive to the induction of ROS-mediated apoptosis, even if the order in which they are triggered is not yet clear. Undoubtedly, research in this field includes a large number of possibilities that should be explored.2. 6-Shogaol was obtained from Sigma-Aldrich  [The fibrosarcoma cell line (HT1080) was obtained from American Type Culture Collection (ATCC-CCL-12), and the human periodontal-derived ligament fibroblast (HPdLF) line from Lonza Ref. CC-7049 . Both cells lines were maintained in complete DMEM Dulbecco\u2019s modified Eagle medium that was supplemented with 10% fetal bovine serum  and a 1% antibiotic\u2013antimycotic cocktail, and at 37 \u00b0C and 5% COMO, USA) .2. Upon conclusion of the treatment, the medium containing 6-shogaol was replaced with 100 \u00b5L of fresh DMEM and then incubated with 10 \u00b5L of MTT reagent for 2 h at 37 \u00b0C, followed by 10 \u00b5L detergent reagent at room temperature overnight. Viability was measured at 570 nm while using a FLUOstar microplate reader . Cell viability was assessed while using a MTT assay . Briefly2\u2212 [2. The cells were then incubated with 1.5 \u00b5L of DHE dye for 30 min. at 37 \u00b0C and 5% CO2. Mean intensity fluorescence was measured at 535/610 nm using a FLUOstar microplate reader . Hydrogen peroxide was used as a positive control. N-acetylcysteine was used as negative control in co-treatment with 6-shogaol to measure the recovery from death induced by ROS.A dihydroethidium (DHE) probe was used to quantify the levels of superoxide anion O2\u2212 . Cells wN-amino)-2-deoxyglucose) probe was used to quantify glucose uptake. The cells were treated as previously described. Briefly, the cells were trypsinized, centrifugated, and resuspended in 200 \u00b5L of staining buffer (1\u00d7 PBS and 50 \u00b5M 2-NBDG). After 30 min. of incubation, cells were washed twice with 200 \u00b5L of 1\u00d7 PBS. Mean intensity fluorescence of glucose uptake was measured with a green laser (430 nm) while using a Flow Cytometer Guava easyCyte  [A 2-NBDG (2-(Germany) .2, 1% Triton X-100, and protease and phosphatase inhibitor cocktail). Equal concentrations of total cell lysate were resolved by 10% SDS-PAGE and then transferred to a polyvinylidene fluoride membrane (PVDF). Nonspecific binding sites were blocked with BSA (10%), followed by an incubation with primary antibodies for anti-mTOR, anti-phospho-mTOR-S2448, anti-PTEN, anti-phospho-PTEN-S380/T382/383, anti-Akt, and anti-phospho-Akt-Ser473. Protein complexes were detected with peroxidase-conjugated secondary antibodies and enhanced with chemiluminescence reagent  [A total of 150,000 cells were harvested in lysis buffer , 10 mM MgCl Canada) . The fil2. After the treatments were complete, cells were fixed with 1.6% formaldehyde for 10 min. Subsequently, cells were permeabilized with methanol for 1 h. The treatments were replaced with two changes of 600 \u00b5L staining medium (1\u00d7 PBS and 2% BSA) and then incubated with a different antibody. The following human antibodies were from Cell Signaling: anti-mTOR (#2972), anti-phospho-mTOR-S2448 (#2971), anti-PTEN (#9552), anti-phospho-PTEN-S380/T382/383 (#9554), anti-Akt (#9272), anti-phospho-Akt-Ser473 (#9271), anti-GAPDH (14C10), and rabbit mAB (#2118) . Anti-\u03b2-actin was from ABCAM reference (#ab8227)  and was used as the loading control. The secondary antibodies used were either anti-mouse Alexa 488 (1:400) and anti-rabbit Alexa 488 (1:400). Mean intensity fluorescence was measured with a green laser (430 nm) while using Flow Cytometer Guava easyCyte . Incubations with only secondary antibody were used as the negative controls [A total of 50,000 cells/well were seeded in a 24-well plate. The cells were treated for 24 h with 10\u201350 \u00b5M 6-shogaol concentrations at 37 \u00b0C and 5% COp < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). The results were subject to statistical analysis while using GraphPad Prism 7  [Data are presented as the mean \u00b1 SD of at least three independent experiments and they were analyzed by two-way ANOVA followed by a post hoc Dunnett\u2019s test. Data were classified at different levels of significance corresponding to CA, USA) .6-Shogaol showed different effects when used to treat either HT1080 tumor cells or fibroblasts. The decrease in HT1080 viability was associated with the increase in ROS that was caused by treatment with 6-shogaol, unlike fibroblasts, which showed no significant changes. The increase in ROS was attenuated by NAC. It is suggested that in the tumor model (HT1080), 6-shogaol induces premature senescence through increased ROS production, the activation of Akt/mTOR, and the decrease in glucose uptake. Taken together, these effects can make the cells more sensitive to the induction of apoptosis by mechanisms that have not yet been completely described. In future, it will be necessary to evaluate other proteins of the signaling pathway (PI3K/Akt/mTOR) to check the modulation efficacy of pathway activation in inducing apoptosis."}
{"text": "As the aging population increases, large changes have occurred among household structures in Japan. Half of older adults lived with their children\u2019s families in 1980. Now around 60 percent of them live by themselves: 27% in single households and 31% as older couples alone. Older adults in single households are said to be at higher risk of social isolation. A Japanese white paper reported that they had scanty of social interactions compared to other types of households. This study examines differences in the social relationships and health statuses among household types by gender and explores the risk factors of social isolation. Nationally representative 2012 Japanese Social Survey data were used for analyses; a subsample comprised participants aged 60 to 74 years. A series of ANCOVAs were conducted. The distribution of the gender and household types were single male 105 (10.0%), married male 387 (36.8%), single female 180 (17.1%), and married female 381 (36.2%); the main effects were being female and married. An interaction effect between them  was observed. Neighborhood relationships were better among females and married participants. Married participants were more active in community meetings, social participation, and volunteering. However, no difference was observed in social network size. Thus, network size alone was not related to social isolation, but being active in social relationships and the quality of relationships influenced social isolation and well-being. Being married and female may facilitate higher quality relationships and may lead to activity and buffer social isolation."}
{"text": "The captions for"}
{"text": "Many therapeutically active molecules are non-soluble in aqueous systems, chemically and biologically fragile or present severe side effects. Lipid-based nanoparticle (LBNP) systems represent one of the most promising colloidal carriers for bioactive organic molecules. Their current application in oncology has revolutionized cancer treatment by improving the antitumor activity of several chemotherapeutic agents. LBNPs advantages include high temporal and thermal stability, high loading capacity, ease of preparation, low production costs, and large-scale industrial production since they can be prepared from natural sources. Moreover, the association of chemotherapeutic agents with lipid nanoparticles reduces active therapeutic dose and toxicity, decreases drug resistance and increases drug levels in tumor tissue by decreasing them in healthy tissue. LBNPs have been extensively assayed in in vitro cancer therapy but also in vivo, with promising results in some clinical trials. This review summarizes the types of LBNPs that have been developed in recent years and the main results when applied in cancer treatment, including essential assays in patients. Cancer nanotechnology as a way of anticancer drug delivery has been developed as a promising cancer treatment . NanoparLipid-based nanoparticles (LBNPs) such as liposomes, solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) have received great attention in drug discovery and cancer treatment. These nanoparticles can transport hydrophobic and hydrophilic molecules, display very low or no toxicity, and increase the time of drug action by means of a prolonged half-life and a controlled release of the drug . Lipid n\u00ae, a liposome formulation encapsulating DOX, was one of the first anticancer drug nanosystems commercially used.Many antitumor agents, such as cisplatin, irinotecan (IRI), paclitaxel (PTX), doxorubicin (DOX) oxaliplatin, daunorubicin, cytarabine or vincristine, have been studied in nanoformulations, and some of them have been analyzed in clinical trials and/or are commercially available for clinical use in patients . In factThis review highlights the main contributions to LBNPs that have been developed in recent years and applied in cancer treatment, including essential assays in patients.Liposomes are the most studied delivery systems due to the biocompatibility and biodegradability that they present. The main components of these nanoparticles are phospholipids, which are organized in a bilayer structure due to their amphipathic properties. In presence of water, they form vesicles, improving the solubility and stability of anticancer drugs once they are loaded into their structure. They are capable of encapsulating either hydrophobic or hydrophilic drugs . In addiIt is well known that there are two general ways to achieve nanocarrier vectorization. One of them is passive targeting, which occurs when liposomes only enter the tumor cell by molecular movement through the cellular membrane. The other is active targeting, which involves structurally modified liposomes holding antibodies that recognize tumor cells . A third3O4 cores are increasingly being used to functionalize different types of nanoparticles , which can be degraded by sonication, leading to drug release. On the other hand, liposomes modified with PEG and anacardic acid have been developed, and these were employed to encapsulate docetaxel, improving the stability of this anticancer drug [The synthesis and development of new liposomes have been extensively studied in recent years. In fact, Fearticles . In 2014lesterol . Moreovelesterol , such ascer drug .Yan et al.  develope0.75Sr0.25MnO3 and iron oxide to combine self-controlled hyperthermia and chemotherapy. PTX was encapsulated in this type of hybrid liposome. In vitro and in vivo biological studies demonstrated the biocompatibility and therapeutic efficacy of this new magnetic liposome [Gogoi et al.  exploredliposome . In 2018liposome  designedliposome . In anotliposome . Studiesliposome . A notewliposome , who encSLNs represent a relatively new colloidal drug delivery system, composed of physiological lipids that remain in a solid state at both room and body temperature. These particles are in the size range of 50\u20131000 nm. The solid lipid used forms a matrix material for drug encapsulation and include mono-, di- or triglycerides, fatty acids and complex glyceride mixtures. This matrix is stabilized by a mixture of surfactants or polymers. SLNs have significant advantages, such as site-specific targeting, physical stability over a long period, possibility of controlled release of both lipophilic and hydrophilic drugs, protection of labile drugs, low cost, ease of preparation and nontoxic. Furthermore, in reference to toxicity, SLNs have exceptionally low toxicity effects against human granulocytes. All these outstanding advantages make them an important candidate for drug delivery systems . In contGiven their unique properties, SLNs have been extensively studied for drug delivery of active anticancer compounds, incorporating them into the SLN formulation in order to improve the oral bioavailability of drugs, protect labile anticancer drugs, and also to reduce side effects by decreasing the dosage by effective targeting to the site of action. The main contributions reported in the last year show that SLNs can be used for encapsulated anticancer drugs. For example, in 2018, Pindiprolu et al.  developeNLCs represent a second generation of lipid-based nanocarriers, developed from SLN, which comprise a combination of solid and liquid lipids. This system was developed in order to overcome the limitations of SLNs; hence, NLCs have higher drug loading capacity, and could also avoid drug expulsion during storage by avoiding lipid crystallization due to the presence of liquid lipids in the NLC formulation. While SLNs are composed of solid lipids, NLCs are a mixture of solid and liquid lipids, such as glyceryl tricaprylate, ethyl oleate, isopropyl myristate and glyceryl dioleate. The mean particle sizes are highly similar to SLNs, generally in the range of 10\u20131000 nm, and are affected by the nature of the containing lipids and the manufacturing process . The maiAmong the contributions reported in recent years, a first relevant example is presented by fluvastatin, which, when combined with lipoic acid and ellagic acid in a NLC, could be used as a candidate for prostate cancer therapy due to the effects of the combination with respect to cell death in comparison to free drugs . AnotherNanoparticles constitute nano-sized carriers (5\u2013200 nm)  whose vaActive targeting is one of the most promising applications for NPs, since they make it possible to take advantage of the specific characteristics and specific profile of each tumor niche to direct treatments to it by functionalizing the NPs with antibodies, tumor-specific antigens (TSA), microRNAs or siRNA, which, together with the properties of certain nanomaterials used in their synthesis, such as pH and/or temperature dependent degradation, response to light, magnetic or ultrasound stimuli, make it possible to release its load specifically at the tumor site, decreasing systemic toxicity enormously with respect to traditional treatment ,46,47.Currently, NPs have acquired a dual profile, being able to help in the diagnosis of disease in addition to granting targeted and specific therapy of disease (teragnosis) . In addi\u00ae or Abraxane\u00ae) have already been approved for BreC treatment [Lipid-Based NPs (LBNPs) constitute a broad and diverse group of nanoparticles that are particularly relevant in BreC treatment. However, despite their variety, liposomes are so extensively used due to their high degree of biocompatibility and their ability to encapsulate a wide range of cargos. LBNPs are currently being used in several trials, and some of them  is the fifth most common cancer and the third most common cause of cancer-related death worldwide ,42. Only5\u03b21 . Additiong cells . In adding cells . Preliming cells  showed ang cells  designedng cells . Jiang eng cells  designedng cells , which eng cells , compris188Re)-liposome in combination with radiotherapy to evaluate its effectiveness in BE-3  tumor-bearing mice. This system determined an increased tumor growth inhibition, with no alterations of WBC, hemogram profile or weight loss. Another interesting approach, developed by Feng et al. [On the other hand, for esophageal cancer (EC), the seventh most frequent cancer worldwide ,42, someg et al. , was theMDR-P-gp overexpression) to IRI by reducing the IC50 of the free drug 6 and 9.5 times at 72 and 96 h of exposure, respectively.Colorectal cancer represents a serious health problem due to its high mortality (it is the second most common cause of cancer-related death) and the continuous increase in its incidence in recent years ,42. LBNPThere are no feasible screening tests for early detection of pancreatic cancer (PaC). Therefore, PaC is often detected in advanced stages, when surgery cannot be applied. However, the failure of pancreatic cancer treatment is not only caused by the advanced state of the tumor , but also by the difficulty the drugs have in penetrating deeply, given the nature of the tumor stroma. Nanotechnology offers some therapeutic strategies to improve the prognosis of these patients. Chirio et al.  prepared\u00ae, benefitting its accumulation and permanence in plasma [Liver cancer (LivC) therapies are often limited by poor drug physicochemical properties. In fact, chemotherapy and targeted drugs such as sorafenib induce a minimal impact in patient survival. In addition, radiotherapy is generally ineffective. Combining drugs with some nanoplatforms has been proposed as a strategy to increase overall treatment efficacy and patient survival. In this context, 5-FU-loaded cubosomes and PTX-loaded NLCs have been used in LivC treatment. On the one hand, 5-FU-loaded cubosomes prevent the drug from its rapid enzymatic degradation, increasing its accumulation in the liver. On the other hand, PTX-loaded NLCs improve the effect of the commercial formulation Intaxeln plasma ,76. In an plasma . Furthern plasma  and Qu en plasma  developen plasma  designedn plasma  or CUR-gn plasma  have shon plasma .6-(4-bromothenyl)guanine (O6BTG) derivative/N9-modified liposomes capable of inhibiting the expression of the MGMT gene in SMA-497 and ZH-161 cells. Furthermore, the intravenous application of O6BTG derivative liposomes and TMZ in a GBM intracranial model using SMA-497 cells resulted in a reduction of tumor growth and in an increase in survival rates in comparison with other treatments. In conclusion, the results of some clinical trials reinforce the potential application of these new therapeutic strategies in GBM. In fact, Myocet\u00ae, a non-PEG-DOX liposome, was used in pediatric patients with high-grade glioma  after TMZ-based chemotherapy  [Glioblastoma multiforme (GBM) is the most aggressive and frequent type of malignant brain tumor (50% of glioma cases), with a very low 5-year survival rate (10%) . The curl trial) . With thl trial) .Lung cancer (LuC) is the most common cancer and the primary cause of cancer-related death worldwide in both men and women ,42. Desp\u00a9 with cyclophosphamide (CM) or vinorelbine (MV) in patients with metastatic BreC has also been developed [Breast cancer, the primary cause of cancer-related death in women ,42, is ceveloped .On the other hand, niosomes have become one of the most interesting LBNPs in BreC treatment, which has led to the development of various systems to facilitate the vehiculization of gingerol , tamoxifOther LBNPs used in BreC research include SLNs. Yu et al.  proposedCurrently, NEs, liposomes and solid-lipid NPs (SLNs) are the main LBNPs being tested as new therapeutic strategies in prostate cancer (PrC). Recently, Ahmad et al.  developep53, MALT1, KRAS, HGAL, ERBB2, PIK3CA, AKT) [Drug resistance (DR) is an essential aspect that limits the effectiveness and application of cancer treatment . A distiCA, AKT) ,129. In CA, AKT)  synthesiFinally, the epithelial-mesenchymal transition (EMT), by which epithelial cells lose typical markers of differentiation (mainly E-cadherin) and begin to express markers of mesenchymal cell differentiation (N-cadherin), may be relevant in cancer resistance. This transition has been linked to processes such as cancer metastasis. Therefore, it is suspected that EMT could induce cancer stem cell (CSC) characteristics by increasing MDR phenomena ,131. In \u00ae, Abraxane\u00ae, nal-IRI and Myocet\u00ae, have been approved in clinic for the treatment of cancer. This makes LBNPs a very promising group for the treatment of cancer in the future.LBNPs constitute a diverse and extensive group that has been used in the treatment of numerous pathologies, mainly in cancer. Liposomes are the most widely used LBNPs, due to their great biocompatibility and versatility; however, recently, SLNs and NLCs have been gaining great attention. Even so, research is not focused solely on these NPs, and there are many publications focused on new strategies using LBNPs to treat different types of cancers. Some of these have even taken the leap to the next level and have begun their path in clinical trials, and what is more, some examples, such as Doxil"}
{"text": "The attitudes of care staff toward the sexuality of residents with dementia they care for is assumed to influence the residents\u2019 expression of their sexuality in the way they want. This paper examines the effect of organizational factors, person-centered care, and the culture of the organization on the attitudes of care staff toward the sexuality of residents with dementia in residential care facilities (RCF) .N\u2009=\u2009187) participated. Using a survey, we gathered information on demographics and care-staff careers, attitudes toward resident sexuality, the culture of the organization, person-centered care, and knowledge of resident sexuality. Ordinary least square (OLS) hierarchical analyses were performed to analyze results.Care staff in different functions at six RCF organizations (Care staff attitudes were found to be positively affected by person-centered care, and marginally positively affected by a supportive culture in the organization, Moreover, knowledge of resident sexuality positively affected care staff \u2018attitudes toward resident sexuality, and the presence of policy regarding resident sexuality affected them negatively .Despite different study limitations, these results give a first insight in a broad perspective on care staff attitudes toward resident sexuality. In addition to improving knowledge of the care staff, enhancing person-centered care and a supportive culture in the organization will improve care-staff attitudes toward resident sexuality. Positive intimate and sexual experiences are found to influence health and quality of life (QoL) positively in the elderly \u20133. HowevA small body of research exists on caregiver attitudes toward the sexuality of residents with dementia, based on the assumption that a more open or positive attitude positively influences the expression of sexuality by residents . AlthougSeveral factors at the level of the individual caregiver and their careers were reported to influence their attitudes toward resident sexuality. First, age was an influence. Older employees had more positive attitudes toward sexuality of residents with dementia than their younger colleagues . This diIn addition to these individual factors and career characteristics (e.g. participation in a training program), previous literature has emphasized the importance of factors at the level of the organization , 13, 14.In addition to the organizational culture, in recent years the concept of person-centered care has generally gained popularity in dementia care. In this concept, care is meant to be holistic and empowering, and should aim at increasing resident QoL , 22. ThiThe aim of this study is to examine in a broad perspective the attitudes of care staff toward the sexuality of residents with dementia, by examining the possible influence of organizational factors on care staff attitudes. In this study, organizational factors are operationalized as person-centered care and the culture of the organization. Next to the organizational factors, the role of individual factors, knowledge of resident sexuality, and some characteristics of care staff career (e.g. years of tenure and current function) over the possible effect on the main target variable \u201cattitude\u201d are included.The data for this study were collected at psychogeriatric care units of RCFs located in the south of the Netherlands. The RCFs participated in an academic collaborative network with the aim of connecting research and practice to elderly care. The participating RCFs varied in size between 590 and 1000 residents. An admission into a RCF becomes inevitable when the cognitive and physical impairments of patients with dementia rise to severity levels that make care at home with help of a private and formal care network impossible. In the Netherlands, this highly intensive care is provided in psychogeriatric care units. These are protected living environments, where approximately six to ten persons with moderate to severe dementia reside in a closed unit.Employees with both direct and indirect contact with residents of such psychogeriatric care units were recruited. Direct caregivers are employees with a vocational level of education, who work together in a care team that belongs to a specific psychogeriatric unit and so provide direct daily care to residents with dementia. However, Dutch psychogeriatric care is organized in a multidisciplinary way. This means that several professionals with different expertise and tasks, and mostly higher levels of education, are indirectly involved in the care of residents . Although RCF care staff other than direct caregivers are less involved in daily care, they are mostly responsible for policy, guidelines, and treatment or care advice for the direct caregivers.Initially, a convenience sample of 191 employees participated in the study. One minor (<\u200918\u2009years of age) was excluded because of considerations concerning the ethical review. Two respondents were excluded because they barely filled in the questionnaire (<\u200910% of the questionnaire). To maximize the sample size in analyses, we included all cases for which 80% of the items on the dependent variable, attitude toward resident sexuality, were completed. This resulted in a final sample size of 187.n\u2009=\u2009179, 95.7%). Mean age was 40.8\u2009years (range 18\u201364), and they had an average of 16\u2009years of tenure within care (range 1\u201343). Most of the participants completed an average vocational education and worked as direct caregivers.The participant characteristics are shown in Table\u00a0The Tilburg University psychological ethics committee granted ethical approval (Reg. No. EC 2015.60), and approval of the executive and ethical boards of the participating organizations was also obtained.Hard-copy surveys were distributed after scheduled (team) meetings. A convenience sample was gathered through the selection of the meetings, in which all employers were represented. Approximately ten members of care staff attended these meetings and care staff with different functions were present during most meetings. Participation was voluntary and participants could withdraw from the study at any time. The participants received an information letter, an informed-consent form, and the survey for immediate completion. The informed consent form and anonymous questionnaires were stored separately. One author (TR) or a student assistant was present during data collection to answer all questions. The author and assistant aligned their answers in frequent discussions about the data-collection process.The survey questionnaire was divided into five sections; one includes the main outcome measure , and four include the covariates. To estimate reliability of the measures based on the sample, we used Cronbach\u2019s alpha test for internal consistency. Values between .7 and .9 were considered acceptable .The Dutch version of the aging sexual knowledge and attitudes scale (AKSAS) , 25 was The attitude subsection consists of 25 items, rated in a seven-point Likert scale from 1  to 7 , it included such items as \u201cAged people have little interest in sexuality.\u201d Ten items were reversed after completion of data collection. An example of such an item is \u201cMasturbation is an acceptable sexual activity for older males.\u201d Total scores were calculated ranging from 25 (most positive attitude) to 175 (least positive attitude) for analysis, as used before .For this subsection, the developers of the scale found high internal consistency and reliability (Cronbach\u2019s \u03b1\u2009=\u2009.88) and sufficient content validity , based on the judgment of ten experts . ReliabiPerson-centeredness is defined as a holistic view of residents, and in this maintaining personhood, despite increasing cognitive and physical impairments . The perThe FOCUS Questionnaire  was usedThe four cultural orientations are measured from a descriptive and an evaluative perspective. The complete questionnaire consists of 54 items that are distributed over eight different variables (four orientations measured from two perspectives). The descriptive perspective measures directly observable behavior and consists of 25 items rated in a six-point scale . An example of an item is \u201cHow often is constructive criticism accepted?\u201d The evaluative part measures the perception of employees regarding typical characteristics of the organization and consists of 29 items, rated on a six-point scale . An example of an item is \u201cHow typical is mutual understanding?\u201dReliability  was assessed for the eight different variables separately . The intThe subsection of the Dutch version of the ASKAS  was alsoGender , age (in years), level of education ), tenure within care (in years), and current function  were assessed. Second, participation in training in handling resident sexuality  was assessed. Finally, care organization (labeled as A through F) and the reported presence of policy concerning resident sexuality (yes/no) were included.Before data collection, a power analyses was performed to find an appropriate sample size, using the program G*power . To reacT-tests were used for two categories: policy regarding sexuality within an organization (yes/no). Analysis of variances (ANOVA) was used for more than two categories: level of education, employer, function, and sexual education. Differences among continuous independent variables  were assessed using linear regression analysis.Descriptive analyses were performed to assess participant characteristics. Means and standard deviations were assessed for continuous measures, percentages for categorical measures. More descriptive statistics were estimated to assess preliminary differences in attitudes regarding residents\u2019 sexuality between groups of employees. p values. The .1 of p level is considered marginally significant, owing to the large number of variables in the model.Hierarchical OLS regression analyses were conducted to examine the effects on and the addition to the variance explained in the dependent variable: attitude toward resident sexuality. Knowledge of resident sexuality was entered in Model 1. Because of the expected interrelation between knowledge and attitude, the control variables age, level of education, and years of tenure were included in Model 2 (gender was excluded because of the overrepresentation of women). Employment , policy regarding resident sexuality, current function, and training were included in Model 3. Finally, in the last model, our main independent variables of interest, person-centered care and the eight variables measuring organizational culture, were entered (Model 4). Dummy variables were constructed for four variables with categorical measures: level of education , employer (Organization B is the reference category), function (caregivers is reference category), and training (none is reference category). Values of .1, .05, and .01 on \u03b1 level were used to test for significance of the M) and standard deviations (SD)) have demonstrated that differences in attitude toward resident sexuality were found between groups of employees based on levels of education F \u2009=\u200911.36, p\u2009<\u2009.01. Employees with high levels of education reported more positive attitudes than other employees; function F \u2009=\u200910.88, p\u2009<\u2009.01. Therapists reported more positive attitudes than direct caregivers and other employees. Finally, attitudes were significantly influenced by the presence or absence of policy t (165)\u2009=\u2009\u2212\u20093.79, p\u2009<\u2009.01. Employees, who reported that policy regarding resident sexuality was not present, were found to have a more positive attitude than employees who did report that policy was present in their RCF organization. Descriptive results from analyses between care staff of different employers cannot be reported owing to inequality in group sizes. No single effects were found for age, years of tenure, and if employees received training concerning resident sexuality .Descriptive analyses  24.39, p\u2009<\u2009.01), and this effect remains in all subsequent models (yet changes slightly in magnitude). More knowledge of resident sexuality goes together with a positive attitude toward resident sexuality. Model 2 shows that care staff with a \u2018high vocational level\u2019  are found to have more positive attitudes toward resident sexuality than the \u2018average vocational level\u2019, the reference category. At first, the highest educated caregivers were also found to enhance more negative attitudes but this result diminishes as more variables are included. We found no effects of age on caregiver attitudes and years of tenure. In Model 3, significantly more negative attitudes were found in care staff of Employers B and F than Employer D, the reference category. This effect, however, diminished in Model 4, in which the culture of the organization and person-centered-care are included in the model. The presence of policy regarding resident sexuality  was significantly and positively associated with the attitudes of care staff in Models 3 and 4.Complete results for all models of the effects on care staff attitudes toward resident sexuality are presented in Table\u00a0F \u2009=\u20092.94, p\u2009<\u2009.01. In this final model, person-centered care and the eight variables on the culture of the organization explained an additional 5% of the variance in attitudes. Person-centered care was found to significantly predict differences in attitudes . Employees reporting to provide more person-centered care, report more positive attitudes with regard to resident sexuality. Of the six included variables measuring organizational culture, only the descriptive measurement of the support orientation was marginally significant in predicting differences in attitudes . Employees reporting more supportive behaviors, policies, and procedures reported more positive attitudes toward resident sexuality.In the final model (Model 4) 44% of the variance in attitudes toward resident sexuality was explained by all of the variables together, Person-centered care was found to have a significant effect on the attitudes of care staff towards the sexuality of residents with dementia. Employees of care organizations, who feel they provide more person-centered care, have more positive attitudes toward the sexuality of residents. The person-centered care paradigm advocates that residents are viewed in a holistic and empowering way , 22. TheRegarding the culture of the organization, only the descriptive part of the support orientation, including observable supportive behavior, procedures, and policy in the organization, marginally affected care staff attitudes toward resident sexuality. Care staff that report their care organization to be observably more supportive had more positive attitudes toward the sexuality of residents with dementia. Our results are in line with the assumption made by Roach (2004), who noted the influence of the culture of an organization on care staff attitudes.Results also demonstrated that the presence of policy had a significant impact on attitudes of care staff. When reporting the absence of a policy considering resident sexuality in their organization, they had a more positive attitude toward resident sexuality. This result is in contrast with previous research, in which the presence of policy was perceived as having a positive influence on care staff attitudes , 20. ThiAlso contrary to previous research , our samFinally, we found that knowledge of the sexuality of residents influenced care staff attitudes. This variable was included as a control variable in this study, although the results confirm previous research  that theThis study is characterized by several strengths and limitations. A first strength is that this study is, to our knowledge, the first attempt to assess the effect of organizational factors on care staff attitudes toward sexuality of residents with dementia. Although assumptions were made in previous research, an actual assessment has not yet been undertaken , 14, 20.A second strength lies in the data collection. The completion of the questionnaires was planned after scheduled (team) meetings. Remaining participant questions could be answered before, during, and after completion. Most questions were related to textual and lingual ambiguities; none were related to completing the knowledge question. During some team meetings, discussion arose on the topic of resident sexuality. The topic raised thoughts and concerns among care staff, as they potentially reevaluated their knowledge, attitude, and skills in responding to the sexual needs of residents with dementia.This study also has limitations. First, although validated instruments are used, person-centered care and organizational culture are considered difficult to operationalize. For example, the definitions of both constructs are subject to different definitions.Second, the way data were collected yielded a limitation, because care staff might have felt peer pressure to participate. Although both the author and trained student assistant emphasized that participation was voluntary and withdrawal was possible at any time, the care staff still chose to participate.Third, we encountered limitations with the questionnaires. First, the way measures are granted in the knowledge section of the ASKAS questionnaire prompted a discussion among the authors. An incorrect answer was granted a \u201chigher\u201d score than the \u201cI don\u2019t know\u201d option, which seems counterintuitive. It means that respondents who admit to not knowing the answer are perceived as having less knowledge of resident sexuality than respondents who gave an incorrect answer. However, this questionnaire has been well studied and validated , and wasA fourth limitation lies in the study\u2019s rather small scale. Only one region in the Netherlands was selected, which is a limitation with regard to generalization. Moreover, in this region of the Netherlands, most residents are Christian (Catholic or Protestant), which is why we did not include religion as an individual factor .Finally, the gender distribution was a limitation, because women were overrepresented in the sample. A gender effect on the attitudes could not be explored owing to this distribution. However, this distribution is representative of the actual situation in clinical practice, and this was also the experience of researchers who conducted previous studies , 10.Next to known benefits , 22, theHowever, neither implication is straightforward in its practical implementation. Both imply a profound change on the organizational level , such asFurthermore, greater knowledge of resident sexuality was specifically found to benefit care staff attitudes toward resident sexuality. The way to improve this knowledge needs more detailed consideration, because participation in education in resident sexuality did not significantly influence these attitudes in our study. In previous research, greater knowledge was found as a result of a training program .To further close the lacuna in research, the assumed influence of the attitude toward resident sexuality on the actual behavior of care staff needs further investigation. The behavior of care staff was found to be important, because they influence the possibility of residents and their possible partners expressing sexuality as they want. Spouses of residents mentioned, in qualitative research, that the behavior of care staff (both direct and indirect care staff) was important to their experiences of intimacy and sexuality . HoweverFinally, replication of this study, in another cultural setting, could add detail to the image that is presented here, especially as future generations of the elderly enter RCFs, and will probably demand more facilitation with regard to their intimate and sexual needs . MoreoveTo establish a broader understanding of the attitudes of the care staff toward the sexuality of residents with dementia, it is important to know more about the organizational factors (person-centered care and the culture of the organization) that might influence factors these attitudes, next to factors on an individual level . The aim of this study was to determine the effect of these two organizational factors.Person-centered care was found to influence the attitudes and a supportive culture of the care organization was found to marginally influence care staff attitudes toward the sexuality of residents with dementia that they care for positively.Moreover, knowledge of the sexuality of residents influenced care staff attitudes positively and the known presence of policy regarding resident sexuality influenced the attitudes negatively, which contradicted results from previous research."}
{"text": "ACR) in first morning void (FMV) urine samples collected over three days is the recommended method for measuring and monitoring albuminuria in adults in the clinical setting. Such a guideline is not available for toddlers and young children. We tested several urine collection strategies for albuminuria measurement in toddlers in a prospective observational study.Urinary albumin:creatinine ratio (UAC) and UACR were compared using only the first measurement and using all three measurements per time point. In addition, these were compared with published CV of adults.Both a FMV and a random daytime urine sample were collected on three consecutive days at week 0, 4, and 8 in toddlers aged 12\u201348 months. Intra-individual coefficients of variation (CV) of urinary albumin . CV of UAC was similar to values published for adults. However, UACR CV was considerably higher in toddlers.A total of 80 toddlers  were included. Intra-individual CV of FMV samples appeared lower than with random samples. The intra-individual CV in UThese data show that\u2014in analogy with adult data\u2014multiple first morning void urine samples should be preferred to single or random urine samples for establishing and monitoring albuminuria in toddlers. Further studies are needed to investigate why creatinine correction for differences in urine dilution is less effective in children. Measurement of urine albumin is important, both in clinical practice and in epidemiological studies and clinical trials. To accurately quantify albuminuria in the adult population, a 24-hour urine collection is considered the gold standard. This method is rather cumbersome and has thus been replaced in clinical practice by alternatives such as collection of a first morning void (FMV) or a random daytime urine sample. To correct for urine concentration in these samples, albumin concentration is divided by creatinine concentration, where the latter is assumed to be excreted at a constant rate over 24 hours.ACR, represented by the coefficient of variation (CV), is lower in FMV than in random samples. Furthermore, FMV performs at least as good as a 24-hour urine collection, so that a first morning void urine sample constitutes a good alternative to a 24-hour urine collection.[To validate these alternative albuminuria measurements against the 24-hour urine albuminuria measurement, several studies have been performed. These stHowever, there is still no consensus on how to optimally measure and monitor albuminuria over time in toddlers. Because a 24-hour urine collection is even more cumbersome in young children than in adults and is not feasible for regular controls, it is very important to have reliable alternatives that can be easily implemented in clinical practice.Therefore, in this study we have compared various urine collection techniques to measure and monitor albuminuria in healthy toddlers. The results are compared with previously reported intra-individual CV\u2019s of albuminuria and creatinine measurements in adults.In this observational prospective cohort study, healthy toddlers (12\u201348 months of age) were recruited from May 2015 until October 2016 via well-baby clinics and children\u2019s daycare centers in Groningen, Drenthe and Friesland (three Northern provinces in the Netherlands). Children with previously diagnosed kidney disease were excluded from the study. Both parents or the legal guardian of the child signed for informed consent. The study was approved by the medical ethical committee of the University Medical Center of Groningen (UMCG) (METc reference number 2014\u2013512) and was conducted according the principles of the declaration of Helsinki.Data was collected on three consecutive days at three different time points: 0, 4 and 8 weeks. Data consisted of three first morning voids (FMV), three random daytime samples and a questionnaire at each time point . All wer\u00ae device, which is a validated tool for urine collection in both children who are continent for urine and children who are not.[AC) was measured with the Roche Modular P using the immunoturbimetric assay and expressed in mg/L. Urinary creatinine concentration (UCR) was measured with the Roche Modular P using creatinase to sarcosine oxidase based colorimetric method and expressed in g/L. To correct UAC for urine dilution, urinary albumin:creatinine ratio (UACR) was assessed. Urine samples were tested for leukocytes with urinary dipsticks (Combur-test\u00ae strips), in order to exclude an intercurrent urinary tract infection.Urine was collected with the PeeSpot are not. It consi are not.,5 Tubes Before starting the study, we calculated that a study population of 100 subjects would provide 80% power in detecting a CV between 14% and 24% (difference of 5% relative to 19%), assuming a SD of the within subject CV of 20%. However, in an interim analysis halfway the study, we discovered that the SD of the within subject CV was considerably lower (17.5%). Therefore, we decided to include 80 participants.AC and UACR are represented by geometric mean and sample standard deviation. Geometric mean was used as albuminuria has a non-parametric distribution. In order to assess the intra-individual variability of albuminuria, individual standard deviation and individual geometric mean were assessed to calculate coefficients of variation (CV) of albuminuria per individual with the following formula: AC \u2265 20 mg/L or UACR \u2265 30 mg/g).[Descriptive characteristics of the study population were reported as mean \u00b1 standard deviation. U30 mg/g). DifferenOf the 95 children of whom informed consent was signed, a total of 80 toddlers (mean age 26.6 months (SD 10.8), 53% male) completed follow-up and were included in this analysis. The remaining 15 children were lost to follow-up. The main characteristics of the study population are shown in AC and UACR) than random sample collections, although the differences did not always reach statistical significance. In addition, AC and UACR) compared to a single collection per time point. The lowest median intra-individual CV was observed when UAC was measured in FMV on three consecutive days (38.3%). When comparing the CV\u2019s of albumin concentration with albumin:creatinine ratio, UACR consistently showed a higher CV. This was the case both with a single urine sample per time point or three urine samples per time point and with either FMV or random samples.AC \u2265 20mg/l or UACR \u2265 30mg/mg) as well as using the geometric mean of the three consecutive samples (geometric mean of 3 consecutive samples within time point UAC \u2265 20mg/l or UACR \u2265 30mg/mg). It is clear from the results in AC) is very low (around 2%) using the adult guideline, and is higher (around 5%) using the geometric mean. Interestingly, creatinine correction gives very high prevalences (17.5 to 33.8%) when using both the adult guideline and the geometric mean.In clinical practice, urine albumin excretion is classified in normo-, micro, and macroalbuminuria, or recenAC and UCR in this population were similar. However, UACR CV in our cohort was much higher.We compared the intra-individual CV found in this study with results from a previous study in adults. In this In this study we compared for the first time various urine collection strategies to optimally measure and monitor albuminuria in toddlers. As expected, albuminuria CV\u2019s are lower when measured in first morning void compared to random samples, and when done with 3 consecutive urine collections versus one collection. This supports implementation of multiple first morning void urine samples for establishing and monitoring albuminuria in toddlers, conform to adult guidelines.ACR was used as compared to UAC. This could be due to a larger variability of urine creatinine in children. However, we did not observe a difference in urinary creatinine CV\u2019s between adults and toddlers. We therefore do not have a clear explanation why creatinine correction does not work in toddlers, other than that urine creatinine measurement itself is flawed in children.However, we also identified differences with the adult population. In adults, urine albumin correction for urine creatinine  helps to correct for differences in urine dilution and reduces variability in the measurements in first morning void or random urine samples. In adults this indeed leads to a more precise assessment of albuminuria and a smaller intra-individual variability in albuminuria. Surprisingly, in our study we observed higher CV\u2019s when UACR \u2265 30mg/mg) also increases substantially as compared to the prevalence defined on the basis of UAC (\u2265 20mg/l). The most likely explanation for this is the fact that children have a lower muscle mass, which accounts for an overall lower urinary creatinine excretion and therefore higher physiologic values of UACR. Indeed, multiple studies reported that UACR is higher in younger children.[AC and UACR in FMV samples and compare these results with 24-hour urine albumin excretion to establish optimal age-dependent reference values for UACR. In addition, the urine creatinine measurement itself in children should be validated and compared with adults to find an explanation why UACR correction does not work in children. At this stage, we recommend to interpret UACR in young children with extreme caution.Another difference we encountered with the adult population is that when using urine albumin correction for creatinine in children, the prevalence of microalbuminuria Click here for additional data file."}
{"text": "Herein, we present a scalable approach for the synthesis of a hydrogen-bonded organic\u2013inorganic framework via coordination-driven supramolecular chemistry, for efficient remediation of trace heavy metal ions from water. In particular, using copper as our model ion of interest and inspired by nature\u2019s use of histidine residues within the active sites of various copper binding proteins, we design a framework featuring pendant imidazole rings and copper-chelating salicylaldoxime, known as zinc imidazole salicylaldoxime supramolecule. This material is water-stable and exhibits unprecedented adsorption kinetics, up to 50 times faster than state-of-the-art materials for selective copper ion capture from water. Furthermore, selective copper removal is achieved using this material in a pH range that was proven ineffective with previously reported metal\u2013organic frameworks. Molecular dynamics simulations show that this supramolecule can reversibly breathe water through lattice expansion and contraction, and that water is initially transported into the lattice through hopping between hydrogen-bond sites. Heavy metals and metalloids pose major threats to health and environmental ecosystems, thus systems for low-cost remediation are needed. Here the authors report the scalable design of a hydrogen-bonded organic\u2013inorganic framework for selective removal of trace heavy metal ions from water. Despite intensive efforts, access to safe drinking water in particular is still at risk, due in part to unacceptable levels of toxic heavy metals found in various water streams and food webs. Heavy metals and metalloids, released from geochemical cycles and anthropogenic activities, pose major threats to environmental ecosystems and can cause severe adverse health effects in humans, plants, and microorganisms. While some of these metals are essential for biological functions, they can also escape control mechanisms\u2014such as transport, compartmentalization, and binding to designated cell constituents\u2014via coordination chemistry and redox pathways, leading to misregulation of trafficking machinery and ion homeostasis3. Undesirable binding of heavy metals to DNA and nuclear proteins often leads to cell malfunction, oxidative deterioration of biological macromolecules, and eventually toxicity3. Effective solutions for efficient remediation of trace heavy metal ions from water with low costs are key pieces of the puzzle to promote environmental and human well-being.Rapid population growth, industrialization, and climate variability are just a few of the factors that have contributed to pushing the global ecological balance near a threshold where immediate attention is required to ensure a future of sustainability. Addressing the inextricably linked water-energy-food security nexus is critical to promote societal stability and human prosperity4. A copper imbalance can impact heart function and lipid metabolism as well as causing inflammation and resistance to chemotherapeutic drugs5. The U.S. Environmental Protection Agency has established a maximum level of copper in drinking water at 1.3\u2009mg\u2009L\u221216. However, copper levels reported in drinking water in Europe, Canada, and the USA can range from \u22640.005 to >30\u2009mg\u2009L\u221216. Historically, there have been several outbreaks of copper poisoning from ingestion of drinks that were contaminated with copper salts7. Given that millions of tons of copper are produced annually worldwide and it is ubiquitous across industries8, maintaining low levels of copper contaminants in water streams is a present concern. Therefore, seeking highly efficient approaches for copper removal from water is important. Beyond human health, selective separations involving copper is a relevant area of research for which traditional metallurgical solutions are limited. For example, in electrolytic nickel refining, selective removal of copper from nickel electrolysis anolytes has long been a problem plaguing the global metallurgical industry, and requires more effective solutions9.Copper, one of the most widely used heavy metals, is toxic to humans in large quantities, despite its essential role in our physiology10. Indeed, isolating and disposing of only toxic contaminants from a myriad of species coexisting in water streams\u2014some of which are valuable catalyst materials\u2014appears to be a costly bottleneck for the water industry. Several methods have been used to remove heavy metal ions from wastewater, including evaporation, chemical precipitation, coagulation-flocculation, photocatalysis, and membrane separation, each with advantages and disadvantages11. Adsorption has been shown to be an effective and economic approach for heavy metal ion removal, as it offers flexibility in operation, a low volume of harmful secondary products, reversibility, and is capable of producing high-quality effluent with low energy and maintenance costs10. The efficacy of adsorption methods is limited largely by the ability to design an adsorbent with the necessary physical or chemical functionality for the contaminant of choice.Challenges inherent to heavy metal ion remediation from water include incomplete ion removal, high costs, and the generation of toxic, indistinguishable ion mixtures in sludge waste streams13, polymer-based adsorbents15, layered metal sulfides17, porous aromatic frameworks4, and metal\u2013organic frameworks20. The thioether-functionalized porous aromatic framework PAF-1-SMe was shown to be highly selective for the detection of copper ions in biological and aqueous samples4, but it was exclusively investigated as a diagnostic tool for the detection of Wilson\u2019s disease. ZIF-8 was also recently reported to exhibit an extremely high Cu(II) adsorption capacity (up to ~800\u2009mg\u2009g\u22121), establishing it among the state-of-the-art adsorbents for copper ion capture18. In general, however, most materials investigated to date for copper ion capture often suffer from one or more drawbacks, such as low selectivity, low capacity, or limited function ), and their syntheses have not been designed for affordable copper removal at scale, often using costly organic solvents or expensive preparations20. The design of selective adsorbents for efficient removal of ions with fast kinetics and high adsorption capacities, using simple and versatile synthetic routes for economic production at scale, is thus critical.A promising ion adsorbent should exhibit high adsorption kinetics, large adsorption capacities even at low contaminant levels, selectivity for the target ion, stability in the water environment, scalability, regenerability, and affordability. Several materials have been shown to be effective for copper ion adsorption, such as carbon-based adsorbents25. Such promising results motivated us to explore and exploit crystal frameworks built predominantly on the propagation of hydrogen-bonding networks ubiquitously found in sophisticated functions performed by nature26 for selective ion removal. Herein, we present, zinc imidazole salicylaldoxime supramolecule (ZIOS), a HOIF that is capable of selective copper capture without the need for postsynthetic modification and exhibits excellent separation performance relative to ZIF-8. We couple aqueous coordination chemistry and supramolecular assembly to achieve a simple and scalable synthesis of ZIOS, which is composed of trinuclear units of zinc(II), 2-methylimidazole, and salicylaldoxime. Our material exhibits unprecedentedly fast adsorption kinetics as well as high adsorption capacities and selective performance at low pH values, rendering it a promising candidate for the adsorptive removal of copper from acid mine drainage-polluted water27. In addition, ZIOS crystals are highly stable in water, exhibiting no changes in crystallinity or structure after exposure to water for 52 days. Interestingly, this stability results from the ability of the material to reversibly breathe water through lattice expansion and contraction. We envision that such supramolecular structures may inform the further development of functional crystalline structures that can be translated to macroscopic platforms for selective removal of toxic metals from water.Hydrogen-bonded organic\u2013inorganic frameworks (HOIF) are an emerging class of adsorbents that have recently been demonstrated for selective separations due to their flexibility and the highly reversible nature of their guest uptake3)2, 2-methylimidazole (Hmim), and the copper chelator salicylaldoxime  in water around 50\u2009\u00b0C results in the formation of a zinc imidazole salicylaldoxime supramolecule, hereafter referred to as ZIOS. The material forms rapidly as small crystals in relatively high yield (76%) following the combination of separate mixtures of H2salox/Zn(NO3)3 and H2salox/mim. Synchrotron single-crystal X-ray diffraction (SC-XRD) data 4(mim)2 4(CH3C3H2N2)2) trinuclear units, wherein H2salox and 2-methylimidazole are deprotonated and bonded directly to Zn2+ nodes through tetrahedral and pentahedral coordination by oxygen and nitrogen, respectively. Intermolecular hydrogen bonds between the hydroxyimino oxygen of H2salox and the pyrrolic nitrogen of 2-methylimidazole lead to the formation of a two-dimensional supramolecular network. The ZIOS structure is essentially nonporous, in contrast to MOFs and ZIFs28, with a Brunauer\u2013Emmett\u2013Teller (BET) surface area of ~12\u201314\u2009m2\u2009g\u22121 confirmed by both BET measurements at different activation conditions, e.g. 100 and 200\u2009\u00b0C and grand canonical Monte Carlo simulations . Even still, we find that the Cu2+ uptake capacity of ZIOS is 40% higher than that of a sample of ZIF-8 prepared in our hands, and the supramolecular structure exhibits unprecedented rapid adsorption kinetics (via infra). This surprising performance suggests the role of a chemisorption-based uptake mechanism, based on the coordination of Cu2+ by the aldoxime and histidine-inspired imidazole pendant groups present in the host structure. Powder X-ray diffraction characterization of ZIOS revealed that the bulk, as-synthesized material is highly crystalline  weight loss at ~50\u2009\u00b0C, likely due to traces of uncoordinated water, followed by a two-step decomposition beginning at 250\u2009\u00b0C, as a result of ligand degradation. Differential scanning calorimetry (DSC) analysis of ZIOS  at the room temperature, and within the first 30\u2009min both samples underwent a color change, from white to pale blue or green, respectively  images of ZIOS revealed that the material partially retains its rod-like structure following 24\u2009h of exposure to Cu2+  to observe their adsorption kinetics, isotherms, distribution coefficient, and selective performance in complex aqueous environments containing copper in the presence of several competing ions. Details of these experiments are listed in the Supporting Information. Samples of ZIOS and ZIF-8 were initially exposed to an aqueous CuClely Fig.\u00a0. This chu2+ Fig.\u00a0, and eneu2+ Fig.\u00a0. In otheu2+ Fig.\u00a0. Crystalake Fig.\u00a0.2 solution are shown in Fig.\u00a02+ solution results in a significant drop in ion concentration to a steady state concentration <1.5\u2009ppm in ~30\u2009min  and is significantly larger than rates reported for other state-of-the-art copper capture adsorbents (Supplementary Table\u00a0t) as a function of time shown in Fig.\u00a030. Notably, the fast adsorption kinetics exhibited by ZIOS are accompanied by a substantial adsorption capacity: at equilibrium, the amount of adsorbed copper ions (qe) was found to be 162.9\u2009\u00b1\u20099.4\u2009mg\u2009g\u22121. For ZIF-8, a smaller equilibrium adsorption capacity of 116.4\u2009\u00b1\u20095.7\u2009mg\u2009g\u22121 was determined from our measurements . We note this capacity for ZIF-8 is much lower than the uptake of ~800\u2009mg\u2009g\u22121 reported previously (reported surface area\u2009=\u20091340\u2009m2\u2009g\u22121)18, although the authors in ref. 18 determined the material uptake after only 30\u2009min of exposure to a copper ion solution, and our data indicate that equilibrium does not occur until ~75\u2009min. As a comparison at this 30\u2009min mark, the uptake of our sample of ZIF-8 was 151.7\u2009\u00b1\u20094.3\u2009mg\u2009g\u22121. It is worth to note that the ratio of mass concentration (in mg\u2009L\u22121) of Cu: ZIF-8 used in our study is ~0.17:1, whereas that reported in Zhang and coworkers\u2019 work is 1.26:1. We also note that adsorption capacity can be sensitive to slight differences in material preparation and activation conditions.Adsorption kinetics data for ZIF-8 and ZIOS in the presence of 895\u2009ppm CuClmin Fig.\u00a0, close tn\u22121 Fig.\u00a0. This raqsat of ~208\u2009mg\u2009g\u22121 4, our material is advantageous in that it can be prepared through a much simpler synthetic route. In addition, copper uptake kinetics in ZIOS are ~30 times faster than in PAF-1-SMe4. Using the Langmuir constant, KL, obtained from single-site Langmuir model16, and Blanchard\u2019s32 original proposed equation (see the Supporting Information), we determined that the Gibbs free energy of copper ion adsorption by ZIOS is \u221227.23\u2009kJ\u2009mol\u22121, indicating that uptake is also thermodynamically favorable.We further equilibrated ~2.5\u2009mg ZIOS with solutions featuring a wide range of copper concentrations . Each solution was then analyzed by inductively coupled plasma-optical emission spectrometry (ICP-OES) to determine the quantity of copper adsorbed, and the resulting data are presented in the adsorption isotherm in Fig.\u00a02+, Na+, Ca2+, Fe2+, Fe3+, Mn2+, and Ni2+ ions, prepared from chloride and nitrate salts of these cations . Notably, even in the presence of these other ions, ZIOS adsorbed ~98.0 % of the copper(II) in solution, nearly double the amount adsorbed by ZIF-8 (52.3%), and uptake of Na+, Ca2+, Mn2+ was less than 10% in ZIOS and ZIF-8. However, we found that ZIOS also adsorbs substantial Fe2+/Fe3+ and Ni2+, namely ~93.9% and ~92.2%, respectively, compared with only ~56.1%, and ~8.3% for ZIF-8, respectively 33. Indeed, when adsorption tests were carried out using metal ion solutions prepared with pH\u2009=\u20092.45, copper uptake by ZIOS remained high (~97.5%) while the material adsorbed negligible iron (~14.3%) and nickel (~2.9%); in contrast, ZIF-8 adsorbed more iron (~30.0%) than copper (~14.6%)  and 4.1 (pH\u2009=\u20094) were reported for the commercial resin Purolite S98428 and an N-octyl-2-aminomethylpyridine-functionalized chelating resin34 under equimolar concentrations of Ni2+ and Cu2+. We further evaluated the copper selectivity of ZIOS in an ion mixture having Zn2+ as one of the contaminants and found this behavior almost unchanged  Fig.\u00a0. SignifiKd, mL\u2009g\u22121), or the ratio of copper in the adsorbed versus the solution phase  in addition to copper(II) (see below). All peak positions were referenced to the C 1s peak of adventitious carbon at 284.5\u2009eV. Initial deconvolution of the Cu 2p spectra for both materials  and a Cu+ complex and/or a heteronuclear Cu2+Zn2+ complex (for ZIOS-Cu), both supported by macrocyclic ligands36. For ZIF-8-Cu, pairs of Cu 2p1/2,3/2 doublets (at 952.4/932.6\u2009eV and 954.8/934.8\u2009eV) and the charge transfer shake-up satellites (at 962.8 and 941.8\u2013944.8\u2009eV) were observed, indicative of Cu+ and Cu2+, respectively. For ZIOS-Cu, the Cu 2p spectrum exhibits peaks at 952.78 and 932.98\u2009eV characteristic of Cu+ , in addition to weaker features at 954.48 and 934.78\u2009eV that can be ascribed to Cu2+ . The copper(I) peaks could indicate the presence of a five-coordinate Cu+ diamagnetic adduct or a four-coordinate Cu+ complex with nitrogen macrocyclic ligands, while the copper(II) peaks are indicative of a heteronuclear Cu2+Zn2+ complex37.In order to better understand the mechanisms leading to copper ion uptake in ZIOS and ZIF-8, we utilized X-ray photoelectron spectroscopy (XPS), synchrotron radiation near-edge X-ray absorption fine structure (NEXAFS) spectroscopy, and extended X-ray absorption fine structure (EXAFS) spectroscopy to characterize the materials before and after copper ion adsorption. XPS analysis of ZIOS-Cu and ZIF-8-Cu was carried out in the binding energy regions of the Cu 22+ in ZIOS results in a downshifting of the O 1s binding energies of free salicylaldoxime from 531.68 and 533.08\u2009eV to 531.28 and 532.48\u2009eV (HO\u2013N and HO\u2013C (phenol), respectively). Similar values of 531.28 and 532.38\u2009eV (HO\u2013N and HO\u2013C (phenol), respectively) are observed following copper adsorption in ZIOS. In general, coordination to a metal ion results in a shifting of binding energies to lower values as a result of the electron donating/accepting ability of the metal ions38. For ZIOS, the 0.1 eV-downshift of the binding energy of the phenolic oxygen O 1s peak, along with the enhanced intensity of this peak after copper adsorption, show that this site plays an active role in scavenging copper ions. Moreover, the N 1s binding energy of ZIOS exhibits a downshift from 398.88 to 398.78\u2009eV at the pyridinic nitrogen following copper adsorption and, interestingly, a significant increase in intensity of the peak at ~400.18\u2009eV for C=N(\u2013O) and/or a pyrrolic nitrogen /I(C\u2013N) from ~0.65 for ZIOS to ~1.54 for ZIOS-Cu indicates significant translational/rotational movement of C=N(\u2013O) and/or pyrrolic nitrogen bonding from the bulk to the surface sites at which copper ions are coordinated. It remains unclear what imidazole nitrogen participates in copper coordination. Given the tautomeric property of the ionizable imidazole ring, both are possible if the Zn-imidazole bond was cleaved at the pyridinic nitrogen by copper ions. Note that the pH value of the stock copper chloride solution is ~5.5.In ZIOS, it is possible that copper chelates with salicylaldoxime by partially transmetalating with zinc nodes originally present in ZIOS and/or with 2-methylimidazole by coordinating with the free pyrrolic nitrogen. As seen in Fig.\u00a0s\u21921\u03c0* transition. Both features are lower in energy than the corresponding 1s\u21921\u03c0* transition of the pyridinic nitrogen in the imidazole ring of the ZIF-8 structure (~400.5\u2009eV). As the imino group typically shows a transition at 398\u2013399\u2009eV40, the peak at 399\u2009eV for ZIOS and ZIOS-Cu likely stems from the N 1s\u21921\u03c0* transition of the oximate nitrogen (C=N\u2013O) or the imidazolate pyridinic nitrogen41. After copper(II) ion adsorption in ZIOS, this peak for the 1s\u21921\u03c0* transition intensifies significantly compared with the broad peaks at 403.2, 406.3, and 410.5\u2009eV, which correspond to the ionization of aromatic N\u2013H and transitions from N 1s\u2192\u03c3*N\u2013H and \u03c3*C\u2013N, respectively42. This intensity increase suggests that the oxime hydroxyimino nitrogen and/or the imidazolate pyridinic nitrogen play a critical role in interacting with copper ions during adsorption. Given the close atomic radii of Zn (134\u2009pm) and Cu (128\u2009pm), along with the higher stability of complexes formed by copper(II) than zinc(II)43, there could be a transmetalation event where Cu2+ partially ion-exchanges with some of the Zn2+ nodes in ZIOS. During this event, the zinc\u2013oxime bonds may be partially cleaved, leaving oxime ligands poised to scavenge copper ions. On the other hand, the unbound Lewis basic pyrrolic nitrogen atoms of the imidazole side chains in ZIOS should have a high affinity for Lewis acidic Cu2+ ions. These sites could actively coordinate with copper to form a CuZn-based heteropolynuclear complex, reactivity that could potentially mimic that found in Cu\u2013Zn superoxide dismutase, an antioxidant enzyme found in almost all cells26 of the human body.The roles of oxime and imidazole in coordinating with copper ions in ZIOS were further elucidated with NEXAFS spectroscopy. At the N K-edge, both ZIOS and ZIOS-Cu exhibit a sharp peak around 399\u2009eV corresponding to a 12+ adsorption does not significantly change the local environment around the Zn2+ node in the ZIOS structure. This result is indicative of negligible adsorption of Cu2+ at sites already occupied by Zn2+ and preferential coordination at oxime and/or imidazole ligands. In contrast, a stark difference in the Zn K-edge data for ZIF-8 and ZIF-8-Cu  and ~8985 and 8987\u2009eV, respectively , which confirm the presence of Cu2+ in the samples46. However, a more detailed examination reveals some modest differences related to the peak intensities and sharpness between the two samples. The Cu L-edge NEXAFS profiles of the two samples , CuCl2 is prone to fully dissociate in the aqueous solution, rendering the inner hydration shell of copper with water molecules and nitrate anions. ZIF-8-Cu shows another predominant peak at ~2.85\u2009\u00c5 which is within range of reported Cu\u2013C bonds45. Furthermore, there is no evidence of Cu\u2013N bonding in ZIF-8-Cu, supporting the fact that the parent ZIF-8 structure featuring metal\u2013imidazole bonding is completely transformed following copper uptake. On the other hand, peaks at ~2.01 and 2.41\u2009\u00c5 for ZIOS-Cu (averaged from different scans) align well with reported Cu\u2013Nequatorial and Cu\u2013Oaxial distances54, respectively, in a pentacoordinated complex with a square pyramidal geometry, suggesting some degree of chemisorption. Other peaks observed in the range of 3.2\u20133.9\u2009\u00c5 for ZIOS-Cu correspond to Cu\u2013C bonds47. From these analyses, we infer that copper predominantly chemisorbs in ZIOS by coordinating with the oxime and/or imidazole ligands, while copper ions in ZIF-8-Cu appear to be weakly bound through physisorptive interactions and also perhaps weakly coordinated within the structure. The extent to which oxime and imidazole moieties coordinate with copper in ZIOS-Cu remains undetermined.At the Cu K-edge Fig.\u00a0, both ZI-Cu Fig.\u00a0, inset w-Cu Fig.\u00a0. The fir2 solution at pH 5.5. These results indicate that a large amount of Zn2+ still remains in ZIF-8-Cu, in spite of the structural collapse observed via SEM and PXRD, indicating that ion exchange is not the main mechanism governing copper ion uptake in this material. Rather, our data indicate that copper adsorption in ZIF-8 leads to collapse of the ZIF-8 parent structure and the formation of a new phase that has retained the majority of the zinc ions as well as incorporated physisorbed and also possibly some chemically bound copper ions. In contrast, for ZIOS, a fraction of the Zn2+ ions appear to be replaced with copper ions, leading to a structure featuring Cu2+/Cu+ coordination pockets near the coordinated Zn2+ ions in ZIOS.From ICP-OES analysis of acid-digested solid samples, we were able to estimate that our samples of ZIF-8, ZIF-8-Cu, ZIOS, and ZIOS-Cu contain ~23.4, 19.9, 18.0, and 10.1\u2009wt% Zn, respectively. Moreover, the ratios of Cu:Zn (by mass) in ZIF-8-Cu and ZIOS-Cu are 0.9:1 and 1.7:1, respectively. In these tests, ZIF-8-Cu and ZIOS-Cu were analyzed after soaking ZIF-8 and ZIOS for 24\u2009h in a ~895\u2009ppm CuCl2+ nodes of the trinuclear units, chelating with salicylaldoxime ligands and leading to a partial transformation of the crystalline structure. Adsorption evidently occurs quickly enough such that ZIOS does not have enough time to further interact with water and collapse. This behavior of ZIOS aligns well with the guest-binding property of metallohosts, structures that contain one or more transition metal atoms56. This property is regulated by metal coordination or redox reactions leading to site-selective transmetalation and ultimately to the construction of heteropolynuclear supramolecular structures. The transmetalation is evident by the amount of zinc ion transferred from ZIOS into the supernatant during copper adsorption quantified by ICP-OES -containing aqueous environment, the framework expands to some extent by gradually inviting water molecules to \u201cassimilate\u201d themselves into the framework through the propagation of a hydrogen-bonding network. This process may then open up channels that can accept copper ions. In addition to spontaneously coordinating with imidazole pendants at Lewis basic pyrrolic nitrogen sites, copper ions may partially exchange with the Znx, Ly, and Lz  simulations were performed using the LAMMPS package with standard three-dimensional periodic-boundary conditions. Further details are listed in the Experimental. Our results show that immersion of ZIOS unit cells in water leads to an expansion of the crystal lattice and the formation of nanochannels that can accommodate water molecules through the propagation of a hydrogen-bonding network. Water incorporation occurs quite rapidly\u2014a snapshot at 15\u2009ns Fig.\u00a0 shows a  Lz Fig.\u00a0. When wa Lz Fig.\u00a0, indicat Lz Fig.\u00a0. A slighg(r) , and a moderately high copper ion adsorption capacity . Furthermore, selective copper removal is possible with ZIOS at pH <3, a value inaccessible with previously reported metal\u2013organic frameworks for copper adsorption. Unlike a majority of metal\u2013organic frameworks, our hydrogen-bonded framework is also stable to water exposure for several weeks. MD simulations show that this framework reversibly breathes in the presence of water through lattice expansion and contraction. Increasing the concentration of water in the unit cell further results in a transition from hopping to diffusion-based transport. Future efforts will investigate the regenerability of our material through copper adsorption\u2013desorption cycling. More broadly, this study is a valuable starting point to guide further development of novel organic\u2013inorganic hybrids that can be effectively leveraged to address at-scale removal of toxic metals from water.\u22121) were used in the synthesis of ZIF-8/ZIOS and ion solution preparations, respectively.Zinc(II) nitrate hexahydrate (\u226598%), 2-methylimidazole (>99%), salicylaldoxime, copper(II) chloride dehydrate, calcium(II) nitrate tetrahydrate, iron(II) chloride tetrahydrate, iron(III) chloride, nickel(II) nitrate hexahydrate, manganese(II) chloride, and sodium chloride were purchased from Sigma-Aldrich and used as received. Anhydrous methanol  and Milli-Q water 2\u20276H2O  and 2-methylimidazole  were separately dissolved in methanol (20\u2009mL) and then mixed together under ambient conditions with vigorous stirring for 4\u2009h. Solid ZIF-8 was then separated from the suspension by centrifuging for 5\u2009min at 8000\u2009rpm and removal of the reaction solvent. The resulting solid was then rinsed with methanol (20\u2009mL), centrifuged, and again separated from the solvent. This rinsing process was repeated twice more, and the final product was dried in a vacuum oven at the room temperature for 18\u2009h.ZIF-8 crystals were prepared following the procedure reported by Song and coworkers3)2\u20276H2O , 2-methylimidazole  and salicylaldoxime  for 2\u2009h at 53\u201357\u2009\u00b0C and atmospheric pressure. ZIOS was then separated from the reaction suspension by centrifuging for 5\u2009min at 8000\u2009rpm, rinsed with deionized water (25\u2009mL), centrifuged, and reisolated; this rinsing was repeated two times. The resulting solid was finally rinsed once with methanol (20\u2009mL) and isolated via centrifuge under the same conditions. The final product was then isolated by decanting the methanol and dried in a vacuum oven at the room temperature for 18\u2009h. Single-crystal X-ray diffraction confirmed that the structure of ZIOS contains Zn3(L1)4(L2)2 trimers, where L1 is salicylaldoxime and L2 is 2-methylimidazole. (Supplementary Tables\u00a01\u20133) and XPS confirmed elemental composition  after degassing samples at 100\u2009\u00b0C for 20\u2009h. Chemical and electronic states of elemental compositions of MOFs before and after Cu2+ adsorption were evaluated with XPS collected with a monochromated Al K\u03b1 source. All peak positions were referenced to the C 1s peak at 284.5\u2009eV. Synchrotron soft- and hard- X-ray absorption  were conducted to understand the local environment surrounding Zn and Cu metal nodes to provide insights into adsorption mechanisms. ICP-OES test was used to quantify the total trace amount of ions in the supernatant after a given time of adsorption.Further details are reported in the supporting information. Briefly, after synthesized, ZIF-8 and ZIOS were grinded with a mortar and pestle before characterization and adsorption tests. The crystal structure of ZIOS was elucidated with synchrotron SC-XRD collected on beamline 12.2.1 at the Advanced Light Source using a Bruker D8 diffractometer with a PHOTONII CPAD detector, with \u03bb\u2009=\u20090.7288\u2009\u00c5, equipped with an Oxford Cryosystems Cryostream 800 plus, at 100\u2009K. Representative morphology of MOFs before and after Cukd as well as the Gibbs free energy of copper ion adsorption on ZIOS. Furthermore, these MOFs were challenged with a selectivity test in which solutions having several cations and anions and adjusted pH were used. Details of experiments and calculations are listed in the supporting information.ZIF-8  and ZIOS were evaluated with different adsorption tests from which experimental data were fitted accordingly to extract adsorption performance and behavior of MOFs. In essence, adsorption kinetics test was conducted where results were fitted with a pseudo-second-order model to determine the rate of adsorption and the amount of copper ion adsorbed on MOFs at equilibrium. Adsorption isotherm was then obtained by challenging ZIOS with solutions having copper concentration values covered a wide range from 0.45 to 450\u2009ppm for 24\u2009h. Results were then fitted with single-site and dual-site Langmuir models. Results from this study was also used to determine the distribution (or partition) coefficient U were used in the MD simulation of this system.All-atom AMBER force fields for potential energy 63. The force field parameters of atomistic oxidized salicylaldoxime and methylimidazole were developed in previous work65 and summarized in Supplementary Tables\u00a066 was employed for water molecules, which depicts well the dynamic and structural properties of liquid water. The VdW interaction parameters between unlike atoms were obtained by the Lorentz\u2013Berthelot combining rule. The nonbonded interactions separated by exactly three consecutive bonds (1\u20134 interactions) were reduced by related scaling factors68, which were optimized as 0.50 for VdW interactions and 0.83 for electrostatic interactions, respectively. Electronegativity equalization method69 was used to calculate the atomic charges by using Multiwfn70. The schematic molecular structures and partial charges of the components for the ZIOS unit and SPC/E water molecule are presented in Supplementary Fig.\u00a0In Eq. , the firSupplementary InformationPeer Review File"}
{"text": "In-bed cycling is a novel modality for the initiation of early mobilization in the intensive care unit. No study has investigated its use in the critically ill, off-track post cardiac surgery population. Before conducting an effectiveness trial, feasibility data are needed. The aim of this study was to determine the feasibility of in-bed cycling in a population of off-track cardiac surgery patients.We conducted a prospective feasibility study in a 16-bed adult cardiac surgery intensive care unit in Ontario, Canada. Previously ambulatory adults (\u2265 18\u2009years) who were mechanically ventilated for \u2265 72\u2009h were enrolled within 3 to 7\u2009days post cardiac surgery. Twenty minutes of in-bed cycling was delivered by ICU physiotherapists 5\u2009days/week. The primary outcome, feasibility, was the percent of patient-cycling sessions that occurred when cycling was appropriate. The secondary outcome was cycling safety, measured as cycling discontinuation due to predetermined adverse events.We screened 2074 patients, 29 met eligibility criteria, and 23 (92%) consented. Patients were male (78.26%) with a median [IQR] age of 76 [11] years, underwent isolated coronary bypass (39.1%), and had a median EuroScore II of 5.4 [7.8]. The mean (SD) time post-surgery to start of cycling was 5.9 (1.4) days. Patients were cycled on 80.5% (136/169) of eligible days, with limited physiotherapy staffing accounting for 48.5% of the missed patient-cycling sessions. During 136 sessions of cycling, 3 adverse events occurred in 3 individual patients. The incidence of an adverse event was 2.2 per 100 patient-cycling sessions .In-bed cycling with critically ill cardiac surgery patients is feasible with adequate physiotherapy staffing and appears to be safe. Future studies are needed to determine the effectiveness of this intervention in a larger sample.NCT02976415). Registered November 29, 2016.This trial was registered with Clinicaltrials.gov (The online version contains supplementary material available at 10.1186/s40814-020-00760-5. What uncertainties existed regarding the feasibility?What are the key feasibility findings?It is feasible to enroll critically ill, off-track patients post cardiac surgery although stringent inclusion criteria limited our sample size.What are the implications of the feasibility findings for the design of the main study?Future studies involving critically ill, off-track patients post cardiac surgery should consider broader, less strict inclusion criteria, including patients who are extubated, those who failed extubation, and those who were readmitted to the ICU from the rehabilitation ward.It was not known whether it was feasible to enroll a population of critically ill, off-track patients post cardiac surgery, how easy it would be to identify potential patients and gain consent, and whether they would tolerate this rehabilitation modality.The number of cardiac surgeries has decreased significantly over the past decade, precipitated primarily by improvements in the use of percutaneous coronary interventions (PCI). As a result of increasing PCI use, patients who qualify for cardiac surgery are older with more co-morbidities . The devEarly mobilization of patients receiving intensive care unit (ICU)-level care may reduce the iatrogenic effects of critical care , 4. EarlThe benefits of in-bed cycling in critically ill patients \u20138 are coBetween August 28, 2017, and March 29, 2019, we conducted a single-center pilot, prospective, feasibility study in a 16-bed adult cardiac surgery ICU in Hamilton, Ontario, Canada. Our study was approved by the Hamilton Integrated Research Ethics Board (project number 1999) and was registered with Clinicaltrials.gov (NCT02976415). Inclusion criteria were (1) cardiac surgery patients \u2265 18\u2009years old, (2) ICU stay for > 3 but < 7\u2009days , (3) mecCycling was performed using the RT300 Supine Cycle, a portable in-bed device which provides passive cycling, active cycling, or a combination of both with the patient in a supine or semi-recumbent position . ThroughCritical care physiotherapists delivered 20\u2009min of in-bed cycling, Monday to Friday, to patients as part of their post-operative care. We chose 20\u2009min for consistency with previous cycling research that included patients\u2019 post-cardiac surgery , 9 and mWe recorded the route of oxygen delivery, mechanical ventilation settings, presence of dialysis ), agitation, and delirium as assessed by the Richmond Agitation-Sedation Scale (RASS) , ConfusiThe primary outcome was feasibility. The feasibility outcomes were 1) the ability to implement in-bed cycling into daily physiotherapy practice at least 80% of the time that patients had no cycling exemptions  sustained hypertension for \u2265 2\u2009min ), (2) sustained hypotension for \u2265 2\u2009min ), (3) cardiorespiratory arrest, (4) oxygen desaturation less than 88% for \u2265 2\u2009min, (5) removal of any lines or tubes, (6) cardiac arrhythmias , (7) saphenous vein graft incision site dehiscence confirmed by the ICU intensivist, and (8) sustained increased agitation for \u2265 2\u2009min (RASS score > + 2) .Other secondary outcomes included consent rate, hospital all-cause mortality, ICU length of stay, 28-day mortality, EuroScore II, New York Heart Association Functional Classification scores, handgrip strength, Functional Status Score for the Intensive Care Unit (FSS-ICU), 2-min walk test (2MWT), Clinical Frailty Scale, and the number of cycling sessions per patient.As no previous safety and feasibility study has been conducted in the cardiac surgery population, we based our enrollment rates on a previous safety and feasibility study of in-bed cycling in a similar-sized medical-surgical ICU . The likDescriptive statistics were used to analyze participant demographics  and baseline data . The feasibility outcome was the percent of cycling sessions that occurred compared to the number of eligible cycling opportunities. The safety outcome was the percentage of cycling sessions terminated prematurely due to the development of one of the 8 a priori adverse events. We calculated means and standard deviations (SD) and medians and interquartile ranges [IQR] for normally and non-normally distributed data respectfully. Incidence of adverse events was reported as number of events per 100 patient-cycling sessions with 95% confidence intervals (CI) . AnalysiProgression criteria are defined as criteria that inform the decision to progress from a pilot study to a larger definitive trial . MeetingWe screened 2074 patients admitted to the ICU over the 19-month period from August 28, 2017, to March 29, 2019  time from ICU admission post cardiac surgery to the initiation of cycling was 5.9 (1.4) days. Overall, our patients cycled a median [IQR] of 4  sessions which yielded 136 patient-cycling sessions and completed a mean (SD) of 113.7 (90.7) min of cycling per patient. Of the 136 patient-cycling sessions, 104 (76.5%) occurred while participants were mechanically ventilated via an endotracheal tube, 17 (12.5%) with a pulmonary artery catheter, and 14 (10.3%) during dialysis (8/136 CRRT (5.9%), 6/136 hemodialysis (4.4%)). Participants cycled during receipt of inotropes/vasopressors , benzodiazepines , and propofol . The median [IQR] RASS score pre-cycling was \u2212 1 [1.75]. The 28-day mortality rate was 34.8%.For patients enrolled in the study, 150 routine physiotherapy sessions were also conducted. These sessions occurred separately from cycling sessions. The most common routine interventions were airway clearance techniques, including manual percussions, vibrations, and tracheal suctioning , followed by passive range of motion exercises  and sitting at the edge of the bed .Outcome measure results are available in Additional File There were 169 eligible cycling days with cycling being delivered on 136 of those days (80.5%). Of the 33\u2009days that cycling was not delivered, 16 (48.5%) were due to physiotherapy staffing shortages  and 6 (18.2%) occurred by participant discharge from ICU without advanced notification, preventing rescheduling of the cycling sessions . Two patients developed hypotension while cycling. One patient became agitated with a RASS of greater than + 2. All 3 events lead to cycling termination with no further medical management required. Patients were able to cycle on subsequent days with no further events. The EuroScore II scores for these three patients were 1.58 (developed hypotension), 1.84 (developed agitation), and 3.71 (developed hypotension) respectively. These EuroScore II scores indicated low risk of post-operative mortality. We examined the saphenous vein graft incision sites before, during, and after cycling. No wound dehiscence was noted during our study period as determined by the intensivist on staff.During routine physiotherapy, 5 adverse events occurred in 5 individual patients : 3 oxygen desaturations to < 88% for > 2\u2009min despite attempts to improve oxygenation and 2 developments of cardiac arrhythmias  from previous normal sinus rhythm. No further medical management beyond terminating the physiotherapy intervention was required.In the 136 patient-cycling sessions, participants cycled a total of 164.6 kilometers (km) with the median [IQR] distance cycled per participant of 5.07 [7.28] km and per session was 1.23 [0] km. The proportion of active versus passive cycling during a single patient-cycling session varied based on each patient\u2019s status and available effort given at the time of cycling. Active cycling was noted in 45/136 patient-cycling sessions (33.1%). The minimum and maximum distances cycled during an individual session ranged from 0.49 to 2.04\u2009km. The maximum total cumulative distance cycled by a single patient was 19.51\u2009km. The majority  of the patient-cycling sessions were completed to the full protocol with the median [IQR] length of each patient-cycling session being 20.0 [1.3] min. The shortest cycling session was 7.9\u2009min. The median [IQR] time to set up and complete cycling was 34 [5.7] min.For our enrollment feasibility, we enrolled 92% of our qualifying patients, which exceeded our target by 42%. Similarly, for our cycling feasibility, we were able to offer cycling on 80.5% of eligible cycling days, 10.5% above our target. This would correspond with the green traffic light color, as per Avery et al. .Our study builds on the body of knowledge investigating the safety and feasibility of in-bed cycling with critically ill patients , 11, 20 We obtained a high consent rate of 92% considering the acuity of our population. We screened 2074 patients; however, due to our narrow inclusion criteria, only 29 patients qualified. While our inclusion criteria allowed us to include patients with high acuity, it limited the enrollment of other patients who may have benefited from early in-bed cycling, such as those who required reintubation within 48\u2009h of extubation, extubated patients who required prolonged hemodynamic support, and patients readmitted to ICU from the rehabilitation ward. While we exceeded our progression criteria with respect to recruitment of eligible patients, in consideration of future interventional trials in this population, the combination of our strict inclusion criteria and a high post-operative mortality rate would have limited the ability to assess the effectiveness of in-bed cycling on patients\u2019 post-operative strength and functional abilities as well as their long-term functional outcomes. Future effectiveness trials of in-bed cycling in this population should consider broader inclusion criteria to prevent the exclusion of patients who may benefit from the early initiation of this modality, to ensure enrollment targets are met, and to increase the likelihood of assessing long-term functional outcomes.Feasibility was dependent upon adequate physiotherapy staffing. \u201cAdequate\u201d staffing for critical care physiotherapists is not defined in the literature. However, staffing limitations have been identified as a common structural barrier that can have a significant impact on patient care \u201326. In aAll patients were cycled on the day of their study enrollment with the mean (SD) time to initiation of cycling from ICU admission of 5.9 (1.4) days. This is more conservative than previous cycling literature , 9. TimeMore than half our study patients (56.5%) had New York Heart Association Functional Classification scores greater than 3, signifying moderate to severe symptoms of congestive heart failure prior to surgery, and 78.2% of our sample scored between class I and II on the Canadian Cardiovascular Society Scores, indicating that symptoms of angina were present only during strenuous or moderate exertion. Over 80% of our sample were identified as either \u201cWell\u201d or \u201cManaging Well\u201d on the Clinical Frailty Scale , 29. DesIn comparison, patients identified as \u201con-track\u201d post cardiac surgery tend to be younger in age, male, have intact left ventricle ejection fraction, no diagnosis of diabetes, and no previous history of either congestive heart failure or cardiac surgery . The majOur protocol of 20\u2009min of in-bed cycling was consistent with previous cycling literature in which patients post cardiac surgery were enrolled , 9. EijsAdmission to ICU is not benign. The strength and functional impairments acquired during extended stays may remain well beyond hospital discharge. Physiotherapy may mitigate the effects of prolonged critical care stays , 31, 32.There are several presumed mechanisms on how muscles may respond to physical stress during the course of a critical illness, including the ability of mechanical signals to induce protein synthesis . If muscIn this study, we demonstrated that in-bed cycling was potentially feasible and safe to implement with off-track critically ill patients post cardiac surgery. Future studies can build upon this safety and feasibility evaluation and previous effectiveness studies to determine how best to challenge critically ill patients post cardiac surgery in an effort to moderate the effects of prolonged ICU stays.This study is novel in that it is the first study to investigate the feasibility of in-bed cycling with a population of solely acutely critically ill, off-track cardiac surgery patients. We obtained a consent rate of 92% across our 19-month enrollment period. While our patients had high median pre-operative EuroScore II results, our findings suggest that in-bed cycling can be implemented with some of the most critically ill patients in the cardiac ICU.Our study had limitations. We conducted a single-center study; thus, our results may not represent the broader critically ill cardiac surgery population. We did not meet our target due to our strict inclusion criteria. Future studies should incorporate broader inclusion criteria and target subgroups of the critically ill post cardiac surgery population missed by our strict criteria.In-bed cycling can be safely implemented in a population of off-track, critically ill patients post cardiac surgery and can be feasibly conducted by critical care physiotherapists with adequate staffing. The results of this single-center feasibility study can be the basis for future studies evaluating the effectiveness of this intervention in this high-risk group. The low adverse event rate can provide confidence to broaden the inclusion criteria to include and enroll patients with or without mechanical ventilation who continue to require critical care interventions in future research.Additional file 1. Outcome Measure Results."}
{"text": "Patients with hemianopic field defects (HFD) might benefit from reading text in vertical orientation if they place the text in the seeing hemifield along the vertical midline.We assigned 21 patients with HFD randomly to either vertical or horizontal reading training. They trained reading single lines of texts from a computer screen at home for 2\u2009\u00d7\u200930 min/day, 5\u00a0days/week, for 4\u00a0weeks. The main outcome variable was reading speed (RS) during reading standardized paragraphs of printed text (IReST) aloud. RS was assessed before training (T1), directly after training (T2) and 4\u00a0weeks later (T3). Quality of life (QoL) was assessed by Impact of Visual Impairment (IVI) questionnaire.Vertical training improved RS in the vertical direction significantly. Only patients with right HFD benefited. Horizontal training improved RS in horizontal diection significantly, but much more in patients with left than in those with right HFD. Both effects remained stable at T3. RS during training at the computer improved highly significantly and correlated strongly with RS of printed text (Pearson r=\u2009>\u20090.9). QoL: Vertical training showed a statistically significant improvement in the complete IVI-score, patients with right HFD in the emotional IVI-score.The improvements of RS were specific for the training. The stable effect indicates that the patients can apply the newly learned strategies to everyday life. The side of the HFD plays an essential role: Left-HFD patients benefitted from horizontal training, right-HFD patients from vertical training. However, the vertical RS did not reach the level of horizontal RS.The study was registered in the German Clinical Trials register (DRKS-ID: DRKS00018843). If data were normally distributed, parametric confirmatory statistical analyses were applied . In all other cases, we used non-parametric methods. Descriptive data were presented as medians with their interquartile range (IQR). We also used the Wilcoxon signed-rank test, Friedman test, and the Mann-Whitney U test for independent samples. With 3 test points and a significant Friedman test result, we did not perform a Bonferroni correction because it is not indicated (\u201cclosed testing procedure\u201d ).https://libguides.library.kent.edu/SPSS/Explore).The required level of significance \u03b1 was set to 0.05 (two-sided) in all statistical tests. Unless otherwise stated, given a sample size of 21, the Shapiro-Wilk normality test and graphical Q-Q plots were used to determine the shapes of distributions. All calculations were performed with listwise exclusion  and side of the HFD (left vs. right).Median individual changes (delta RS between T1 and T2) of the subgroups were calculated and the individual changes of each patient are presented as scatter-plots.The statistical values are shown in Table IReading speed (RS) during reading printed text (IReST)Reading in vertical orientationn\u00a0=\u200911). The median vertical RS improved by 14 wpm from T1  to T2 (104 wpm) and remained stable at T3 (104 wpm)  was only 6.3 wpm (10%). Only 3/11 patients improved by at least 10 wpm , there was no significant change: T1: 70 wpm; T2: 77 wpm; T3: 73 wpm. The patients with right HFD (n\u00a0=\u20096) yielded a statistically significant and clinically relevant improvement by 11 wpm from T1 (94 wpm) to T2  (delta RS T1-T2) shows a wide overlap between right (red dots) and left HFD (blue dots). Only 3 patients  improved by more than 10 wpm , there was statistically significant improvement of RS  from T1 to T2, which remained stable at T3.For the total cohort  did not show any statistically significant changes during horizontal reading.Training group V (n\u00a0=\u200910) showed a statistically significant increase of RS of 14.9 wpm from T1 (112.50 wpm) to T2 (127.40 wpm), of 12 wpm from T1 to T3 (124.50 wpm), and no change from T2 to T3, i.e. the training effect remained stable.Training group H  (delta RS T1-T2) was 11 wpm for group V and 12.5 wpm for group H.The individual change of RS during the training period showed a wide overlap between the groups without a clear advantage for the horizontal training group :HFD Fig.\u00a0:Fig. 5Ren\u00a0=\u200921) into groups according to the side of the HFD (independent of the training group), patients with left HFD (n\u00a0=\u200911) showed a statistically significant increase of median RS during horizontal reading from T1 (113.0 wpm) to T2 (129.8 wpm), see Fig. Separating all patients  was 15.1 wpm (22.2%) in left HFD and 5.6 wpm [8.8%] in right HFD (delta RS T1-T3).The individual change RS correlated highly with cRS (RS during the training) (see below). No correlations were found between RS at T1 and the change of RS (from T1 to T2), regarding age, disease duration, and size of macular sparing.n\u00a0=\u200911) showed a statistically significant improvement of the reading speed at the computer during the training (cRS) between the median of the first 3\u00a0days (72.93 wpm) and the median of the last 3\u00a0days (103.35 wpm) of the vertical reading training (see Table Group V ( 3\u00a0days 7.93 wpm an\u00a0=\u200910) improved cRS during the horizontal reading training, but this change was not statistically significant: the mean of the first 3\u00a0days of training was 126.72 wpm; the mean of the last 3\u00a0days of training was 148.70 wpm. (see Table Group H (III.Training intensitycRS (during the home training) correlated highly with RS (at testing in the clinic with the printed IReST charts) see Fig.\u00a0.III.Train\u00a0=\u200921) was 1236.45\u00a0min  (103%). As the actual average training time per day for each patient was 62\u00a0min, this means that there was good compliance. There was neither a statistically significant correlation between training intensity and change of RS (T1-T2), nor between training intensity and change of cRS during the training at home .IV.QuestionnairesQuality of Life (IVI)The study design requested that each patient should train for 4\u00a0weeks, 5\u00a0days per week, 2\u2009\u00d7\u200930\u00a0min per day (1200\u00a0min\u2009=\u200920\u00a0h). The actually measured median cumulative training time of all patients , we found a statistically significant improvement in the functional IVI-score from T1 (1.45) to T2 (1.05), which remained stable at T3 (1.05). The IVI reading-score showed an improvement from T1 (1.5) to T2 (1.0) and from T1 to T3 (1.00), which was statistically marginally significant. The complete IVI-score showed statistically significant improvement from T1 (1.25) to T2 (1.00) and from T1 to T3 (1.11) indicating that the effect remained stable after training. An improvement of the complete IVI-score was found in 11/12 patients who improved their RS by more than 10 wpm, but was found by only 3 patients who had not improved their RS. This indicates that a successful training was considered beneficial in everyday life by the patients.When all patients were analyzed together  to T2 (1.00), group H did not show any significant improvement.When the total cohort was separated by side of HFD, in left HFD, no significant improvement was observed. In right HFD, the improvement was significant in the emotional IVI-score from T1  to T3  . The reading IVI score tended to improve: T1: 1.50 (IQR 1.00 to 2.00); T2: 1.0 (IQR 0.50 to 1.50); T3: 1.00 (IQR 0.50 to 1.00), p\u00a0=\u20090.07, Friedman\u2019s test. Post-hoc tests were not applied, as Friedman\u2019s test did not show a significant improvement.b)Specific questions regarding reading performancec)Feedback regarding the training programSome patients reported spontaneously that reading was \u201efun again\u201c, that they were reading \u201cmuch more than before\u201d, and that they were reading \u201cmore comfortably\u201d. The questionnaire showed that the amount of reading in daily life increased in 6 patients, the strain decreased in 5 patients. This was assessed at T1, T2 and T3.As not all patients answered all the questions, we can only report the absolute numbers. The questions were asked after the training at T2.How demanding was the training for the patients: Five patients described the training as slightly demanding , 7 patients reported the training to be demanding , 4 found it exhausting . This effort decreased during the training period in 9  and remained equal in 10  patients.d)Cognitive StatusQuestion regarding the handling of the program: It was easy for 9 and needed help in another 5 patients. Four patients reported difficulties. Reading in daily life was experienced as easier in 5, equal in 13 patients and harder in none.The MoCA score was not statistically different, neither between the reading training groups nor between the groups divided by the side of the HFD, and did not change during the training period , experienced a statistically significant improvement of their median vertical RS. This effect remained stable at T3, and they improved their RS not only during the training at the computer, but also during reading the printed IReST. This means that the patients applied their improved reading performance to everyday life. The finding that only patients with right HFD benefited regarding vertical RS is in accordance with the finding by de Jong et al. . This isHorizontal training (group H) led to statistically significant improvement of RS in horizontal orientation. The effect remained stable at T3. Despite the improvement during vertical training, the vertical RS at T2 (104 wpm) did not reach the level of horizontal RS . It should be taken into account that most people have practiced horizontal reading throughout their lives, whereas vertical reading is unfamiliar and challenging, e.g. because of the perceptually unusual word shapes and the necessity to perform reading saccades in a vertical direction. In an unpublished pilot study, normally sighted subjects found that vertical reading was quite demanding and tiring. However, it should be kept in mind that normally sighted people will not have any functional advantage using a vertically orientated text, whereas HFD-patients might have this advantage by shifting the line of text into their seeing hemifield. Our results allow hypothesizing that these patients might benefit only after much longer training of reading in the vertical direction.In fact, the difference between horizontal and vertical reading performance disappears, if readers are used to read in both directions, as in Chinese and Japanese . FurtherIn our total cohort of patients (V\u2009+\u2009H), we found a statistically significant increase of RS from T1 to T2 for horizontal reading. This effect remained stable at T3. Regarding the side of the HFD, patients with left HFD showed stronger improvements from T1 to T2 than those with right HFD from T1 to T3 during horizontal reading, but not in vertical reading. Patients with right HFD have the problem of having to perform saccades into the blind hemifield, but can at least improve by reading more frequently. The patients with left HFD have the advantage of getting through the line rather easily during horizontal reading and might need some more (unspecific) training of the return sweep to the next line.Even though we found statistically significant effects in the groups and subgroups, we should consider that the changes of RS were individually very different and did not show very clear effects. One limitation of the current study is the small sample size, which makes it difficult to form subgroups. The recruitment of the patients was very difficult, which is shown in Fig. We consider a change of 10 wpm in these patients as clinically relevant. The value is based on our long-standing clinical experience and has been used in previous studies e.g. , 35). In. In35]).There are two RCTs on reading training in patients with HFD that used different approaches. Spitzyna et al., (2006)  applied The strong correlation between reading speed during home training at the computer (cRS) with reading speed of printed paragraphs (RS) in the clinic shows that the method of measuring reading speed by software allows an assessment of the training results at home. The advantage is that the training effect can be determined by software without an additional visit in the clinic. This could save additional effort for the patient and lowers the costs for the health insurance, which would allow monitoring the status of patients with reduced mobility by \u201ctelemedicine\u201d. The strong correlation between RS during reading printed text and cRS during the home training at the computer shows that the measurement of reading speed in both conditions is reliable and shows that the data is robust. This also indicates that the patients actually read the texts during the training at home without a supervising person.Not all of the objective improvements were reflected in the subjective reports by the patients. The fact that QoL improved in group V and in right HFD is in accordance with the finding that patients with right HFD and vertical training benefited most.The cognitive status (MoCA) of our patients did not change. There was neither an increase (by more intensive reading) nor a decrease  of the cognitive status of our patients.In summary, we found significant improvements of reading speed in two groups using reading training in horizontal or vertical orientation. Training in vertical text orientation improved RS in patients with a right hemianopic field defect, but does not reach the speed attained by reading text presented in horizontal orientation.In patients with a left HFD, the vertical training did not offer an advantage, because their main problem is finding the beginning of the new line, which they can train more easily by horizontal reading."}
{"text": "New tools to calibrate different types of scattering experiments suitable for static and moving detectors are presented. pyFAI suite for processing scattering experiments acquired with area detectors are presented. These include a new graphical user interface for calibrating the detector position in a scattering experiment performed with a fixed large area detector, as well as a library to be used in Jupyter notebooks for calibrating the motion of a detector on a goniometer arm (or any other moving table) to perform diffraction experiments.New calibration tools in the  The calibration issue is currently worked around by providing users with the proper geometry description or even the reduced data; however, this prevents re-processing at home institutes and also re-interpretation of data, which is needed as a part of the open-data initiative (Wilkinson etc.). Using a fixed-position setup combined with the speed of modern detectors (up to a few kHz), it is easy to follow chemical reactions or other physical processes in situ. Even with fixed geometries, determining the detector position is still an issue for the majority of users. Hence, a new graphical user interface has been developed for the Python fast azimuthal integration library pyFAI  area detectors mounted on moveable goniometer arms for powder diffraction . Detectors at synchrotrons are often mounted on goniometer or translation tables to provide the degrees of freedom (hereafter DoFs) needed to align the beamline, although those DoFs are rarely used for data acquisition. One counter-example is reported by Gao  al. 2016, where tq-range in one single frame  to acquire powder diffraction or PDF data on a larger q-range with no additional cost or investment.Pair distribution function (PDF) analysis typically requires very large area detectors and higher energies to be able to cover the needed high pyFAI library  as a function of motor positions is described.The 2.pyFAI is a Python /\u03bb. pyFAI also provides additional tools to calibrate the detector position, i.e. to determine its location in space by means of Debye\u2013Scherrer conical rings resulting from the intersection of the diffracted X-ray beam with the detector surface. The observed rings are deformed into ellipses when the detector is planar but slightly inclined. The azimuthal integration is performed in two steps. The first is a pixel-wise transformation corresponding to the image correction,Iraw is the raw signal of the detector, Idark is the dark current image , F is the factor accounting for the flat-field correction, \u03a9 is the solid angle subtended by a given pixel, P is the polarization correction term and A represents the detector\u2019s apparent efficiency due to the incidence angle of the photon on the detector . Iraw may be normalized by the incoming flux I0, which is independent of the pixel position. The numerator of equation (1)r). The intensity averaged over all pixels located at a given radial value is then simply the ratio of the two histograms which bin corresponds to the radius of interest [equation (2)]ci is used to describe pixel splitting and corresponds to the fraction of the pixel area falling into the specific histogram bin (0 \u2264 ci \u2264 1). The multiple pixel-splitting schemes available in pyFAI, called full-splitting, bounding-box-splitting and no-splitting, have been described by Ashiotis et al. , which used equation (4)The integration scheme presented in equation (2)3.pyFAI integration scheme of multiple images taken at various positions was first reported by Kieffer & Wright . Hence, integrated intensities in multi-geometry mode are orders of magnitude larger than usual, the scale factor being the solid angle of one pixel at the PONI, i.e. dist2/(pixel1 \u00d7 pixel2). Errors are propagated in an similar way to equation (3)The ight 2013. The pro4.4.1.calibrant. The rings are extracted automatically from the image (by subtracting a smoothed version of the image) and control points are placed at the local maxima on the rings. The geometry of the experiment is obtained by refining geometric parameters using a least-square fitting of the 2\u03b8 values calculated for the different control points.The calibration of a detector position is performed using several Debye\u2013Scherrer rings collected from a reference powder called the FIT2D . The geometry used in pyFAI is based on the definition of the PONI  on the detector plane (or the plane z = d3 = 0 when the detector is non-planar and z varies from pixel to pixel). This geometry is inspired by SPD . Some geometry conversion tools are provided by pyFAI and documented by Detlefs & Kieffer  from the sample position to the PONI. (ii) poni1 and poni2: space coordinates of the PONI (in metres) within the detector plane (z = d3 = 0) along the slow- and fast-reading dimension of the detector image . (iii) rot1, rot2 and rot3: the rotation angles (expressed in radians) of the detector placed at the proper distance from the sample, with respect to the three axes of the laboratory reference system. The detector is first rotated around the vertical axis (rot1), then around the horizontal axis (rot2) and finally around the incoming beam direction (rot3). In a more mathematical way, this givesR1 and R2 are left-handed, while R3 is right-handed, which is a legacy from previous versions of pyFAI.The geometrical parameters to be refined are the following: (i) The strength of this geometry parameterization is that it describes any detector position in space. The drawback is that some parameters are correlated or not optimized:dist-wavelength: reducing the wavelength is nearly equivalent to increasing the distance unless the diffraction angle is large (2\u03b8 > 30\u00b0). It is advisable to fix one of these two variables unless the data quality are good enough and the scattering range is very large. This is an intrinsic limitation of the single-image approach.(i) rot1-poni2 and rot2-poni1: as small rotations can be mistaken for larger translations. Fixing one of the two decreases the uncertainty of the other by several orders of magnitude. The user interface offers a \u2018SAXS constrains\u2019 button to lock the detector normal to the direct beam.(ii) rot3: cannot be refined from Debye\u2013Scherrer cones as they are, by definition, invariant by rotation around the incoming beam. The rot3 parameter can be assessed using an anisotropic contribution such as the polarization effect or some textures in the sample. This rot3 parameter is used to describe the azimuthal rotation of the experimental setup or of the sample, for example, when calculating pole figures.(iii) azimuthal integrator object, ready to perform the azimuthal regrouping of images coming from the detector.Those calibration parameters containing the geometry of the experimental setup are saved in text files with a .poni extension, also containing the detector definition and the wavelength. Such a file can subsequently be loaded into an 4.2.pyFAI since the origin of the project  design was developed on top of (i) Experimental settings. This first tab lets the user select the wavelength (or the energy), the reference material for calibration  and choose the type of detector, either from a list of 50 provided, or by defining a new detector Fig.\u00a02.(ii) Mask drawing. This tab allows us to mask-out unwanted regions/pixels from the image, either based on their intensity (thresholding) or simply by drawing polygons on the image. Different masks can be saved, retrieved and assembled Fig.\u00a03.(iii) Peak picking. Individual peaks and arcs of rings can be segmented out  and assigned  to different rings, each associated with a single reflection of the calibrant Fig. 4.(iv) Geometry fitting. At this stage, the detector position and wavelength are fitted against peak positions and ring numbers. Any of the parameters can be fixed or let free for refinement, possibly with boundaries. The  positions of the beam centre and the PONI, and the expected positions of rings are overlaid onto the diffraction image to visually assess the quality of the fit. The sample, detector and direct beam can also be visualized in 3D Fig. 5.i.e. powder diffraction profile and caked-image) to further validate the modelling of the experimental setup  Integration. This tab displays the 1D and 2D integrated patterns .The calibration of the detector position on a fixed goniometer position can be performed with 5.1.pyFAI accepts an arbitrary number of motors attached to the goniometer/translation stage. Let motors be a list of n motors moved during the acquisition: .Here, the difficulty is not in the calibration, but rather in the variety of goniometers and translation stages which may be controlled by one or multiple motors. The goniometer description implemented in Calibration of the goniometer involves defining a model and optimizing it:m static parameters for the model which are used to describe the position of the detector, m being the number of DoFs of the model: params = .(i) Choose a list of transformation functionsmotors) and goniometer parameters (params) into the six PONI parameters used to initialize an azimuthal integrator in pyFAI:(ii) Define the six (iii) Optimize the parameter set of the goniometer (params) so that, for any position of the motors, the detector position is expressed as a PONI-parameters set.exp position of the peaks over all control points. Those 2\u03b8exp are compared with the theoretically expected 2\u03b8theo values calculated from the calibrant d-spacing and the wavelength,scipy.optimize.minimize function from SciPy , but should be prevented from executing any arbitrary code (security). Moreover, those functions should be made persistable on disk (i.e. saved) and their restoration should be possible without having to re-perform the calibration, nor creating vulnerabilities.The six PONI parameters returned are then used to calculate the experimentally observed 2\u03b8NumExpr library , with a list of motor names (pos_names), parameter names (param_names), as well as a set of six formulae, one for each of the six PONI parameters.In the following example, the definition of those functions is\u00a0simply the creation of an object from the 5.2.q or longer distances for better Bragg peak separation.At the ESRF, the protein crystallography beamlines use large area pixel detectors  placed on a translation table which allows collection of the data at the optimal distance: shorter sample-to-detector distances to explore the region for high-MX-calibrate tool from the pyFAI suite is available for the calibration of many images taken at various distances. This can also be interpreted in terms of the goniometer setup , where the sample-to-detector distance is modelled as a simple linear function of the table position:The pyFAI: poni1, poni2, rot1 and rot2. rot3 is forced to zero while the distance is defined as a linear function of the motor position.In the example above, a six-parameter model was chosen for the goniometer geometry, most parameters matching exactly those of Jupyter notebook  from the metadata written in the image headers, thus a set of control points is available prior to data processing. The translation table is calibrated using all control points and this geometry transformation object. As a result, all images have been integrated azimuthally. Fig. 72 calibrated using this methodology. The blue curve corresponds to this initial model refined; the two peaks look \u2018doubled\u2019, indicating a poor modelling of the\u00a0geometry.The content of this tutorial is available as a dist, poni1 and poni2) have been re-fitted and all data integrated with the new model, one obtains the orange curve in Fig. 7To address this poor modelling, another transformation function is defined, with a few additional DoFs on the PONI position . Once three parameters  for powder diffraction measurements. The initial idea was to use a small region of interest (ROI) in the centre of an area detector, and check whether it was possible to rebuild a powder diffraction pattern when moving the detector at various goniometer angles. This ROI has to be of limited size in the dimension orthogonal to the ring and as extended as possible in the tangential direction, with the limit of the curvature of the ring . We considered an ROI 10\u2005pixels wide within a Pilatus 100k (487\u2005pixels \u00d7 195\u2005pixels) which means using only 2% of the total width of the detector (different lines being used for different bins). By using the full detector area, the signal/noise ratio could be improved by a factor of seven [(487/10)1/2] if the movement of the detector on the goniometer is perfectly predictable and reproducible.Probably the main application of this work is to place a small area detector on a goniometer 2\u03b8 arm in replacement of a punctual detector (geometry transformation is defined with rot2 (in radians) as a linear transformation of the motor position (pos is the default motor name), here measured in degrees by the goniometer. The scale parameter, scale = \u03c0/180, is used to convert angular units. The other parameters dist, poni1, poni2 and rot1 are directly mapped to pyFAI\u2019s parameters. As done previously, rot3 is kept fixed at zero:When the detector arm is moving in the vertical plane , a simple Jupyter notebook , most have only one ring and only a few images display two rings. Even if those images are technically suitable for static detector calibration, they still present a challenge due to the large diffraction angles (2\u03b8 > 30\u00b0), the low curvature of the rings and the difficulty in assigning the picked peaks with the proper calibrant ring number.This example is also available in a rot1 = 0) to guarantee some consistency between the calibrations.Four images have been manually calibrated, fixing the vertical rotation axis of the detector  over all images, but Fig. 10rot1. A new model was tested where both the rot1 and rot2 values are scaled with the motor position. After refinement, the cost function dropped by a factor two and the low-angle peaks became sharp  is a factor of ten worse than the highest achieved resolution with secondary monochromator on insertion devices . The average peak resolution full width at half-maximum FWHM = 0.02\u00b0 obtained on LaBet al., 2003et al., 1998rot3 = \u03c0/2.A third example of goniometer calibration is available in the work by Kieffer & Blanc 2017. It corrThis generic method has even been extended to strip detectors like the Mythen detector manufactured by Dectris as shown in the tutorial on the calibration of the array of nine Mythen detectors , this shrinkage of pixel sizes naturally leads to higher quality powder diffraction patterns. Unfortunately, to keep the covered 2\u03b8 range constant, one would need to multiply the number of pixels by the square of the pixel size reduction factor, and the associated infrastructure for read-out and data transfer accordingly. Moving the detector offers a flexibility which removes this limitation but makes the experiment slightly slower \u2013 though still suitable for 7.pyFAI has been developed to ease the calibration of an experimental setup with static detectors, especially for novice users. The concept of calibration of the detector position has been extended to fit the detector position as a function of the motion of a goniometer. Once a few fixed positions of the goniometer have been calibrated, a model can be optimized and the detector position can be extrapolated at any goniometer configuration. By acquiring multiple images at various positions, these images can be integrated together to produce a high-q powder diffraction pattern of quality equivalent to that acquired with a much larger detector, opening up new opportunities for insitu experiments and PDF measurements on most synchrotron diffraction beamlines.The new graphical user interface of"}
{"text": "Tumours with high expression of B7-H3 showed larger spatial differences of CD8+ T cells (86.4/mm2 in tumour centre vs. 414.9/mm2 in invasive front) when compared to B7-H3-low group  (p < 0.001). This study provides insight into the expression pattern of B7-H3 in GC of Western origin. In GCs with higher level of B7-H3 expression, CD8+ T cells were spatially suppressed in the tumour centre suggesting that B7-H3 might be involved in tumour escape mechanisms from the immune response.CD8+ T cells are the main effector cells of anti-cancer immune response that can be regulated by various costimulatory and coinhibitory molecules, including members of the B7 family. B7 homolog 3 (B7-H3) appears as a promising marker for immunotherapy; however, its significance in gastric cancer (GC) is unclear yet. We evaluated the spatial distribution of CD8+ T cells in relation to the expression of B7-H3 by double immunohistochemical staining. The level of B7-H3 intensity was scored manually (0\u20133) and dichotomized into B7-H3-low and B7-H3-high groups. The distribution and density of CD8+ T cells was analysed using whole slide digital imaging. B7-H3 was expressed mainly in the stromal compartment of GC ( The tumour immune microenvironment (TIME) can be categorized into three classes : (a) theB7 homolog 3 protein  is one of the immune checkpoint molecules of the B7 family. Although the exact receptor of B7-H3 has not been discovered yet , this mon = 48; diffuse, n = 27; mixed, n = 6; unclassified, n = 15) were included in the study. Among 73 B7-H3 positive GCs (76%), B7-H3 expression was found within the tumour stroma (n = 60) or both in stromal cells and neoplastic cells (n = 13). After assessing the intensity of B7-H3 expression, 55 cases showed negative or low staining, and 41 cases showed moderate or strong staining .The median overall survival (OS) for the entire cohort was 17.5 months, and the median cause-specific survival (CSS) was 18.4 months. No significant differences were found in OS and CSS between B7-H3-low and B7-H3-high groups (2) differed significantly between both tumour compartments  than in the invasive front (IF) (258.1 cells/mm2). Tumours with higher B7-H3 expression in the stroma showed greater spatial differences of CD8+ T cells (86.4/mm2 in TC and 414.9/mm2 in IF) when compared to the B7-H3-low group  (The median density of CD8+ T cells (cells/mm< 0.001) . Addition = 415) was significantly higher than that in normal gastric tissue  .Data from The Cancer Genome Atlas Stomach Adenocarcinoma (TCGA STAD) cohort were retrieved for quantitative validation analysis using TCGA Xena computational tool (UC Santa Cruz) . The B7-Studies have demonstrated that the analysis of tumour infiltrating lymphocytes (TIL) alone cannot explain the complex crosstalk between tumour cells and their TIME. Here, we have presented a potential relationship between B7-H3, an immune checkpoint molecule, and CD8+ T cells, the main effector cells involved in tumour surveillance. The major findings of this study are the following: (1) B7-H3 is expressed mainly within the tumour stroma; (2) B7-H3 expression correlates significantly with tumour location, Lauren phenotype, and pT category; (3) median density of CD8+ T cells differs significantly between TC and IF; (4) in cases with higher stromal B7-H3 expression, restricted CD8+ cell infiltration in the TC was observed. Taken together, our results suggest a potential B7-H3 role in the immunosuppressive TIME of GC. However, while there is no specific human receptor for B7-H3 described yet, the exact mechanism of this interaction remains unclear.Most studies of TILs (including CD8+ T cells) and B7-H3 in GC are carried out on Asian cohorts. According to their meta-analyses, higher intratumour CD8 expression is associated with an improved overall survival ,12. Distn = 24), Gullo et al. demonstrated that T cells are enriched at the invasive margin of GCs, as well as in EBV+ cases [n = 18) showed T cell exclusion and possessed CD8+ cells predominantly at the invasive margin [By analysing GCs with lymphoid stroma , B7-H3 expression in stromal cells was described. By examining the co-expression of B7-H3 with CD31 and alpha-SMA, they detected stromal B7-H3 expression in 62.7% of GC cases, mainly in cancer-associated fibroblasts, which were the major component of the tumour stroma [The association between B7-H3 positivity and CD8 expression has been reported. Guo et al. examined the expression of PD-L1, B7-H3 and B7-H4 during different stages of gastric carcinogenesis. In their set of 50 GCs, 78% cases were B7-H3 positive, and a negative correlation between B7-H3 in the tumour and the density of CD8-expressing cells was reported . Here, wIn conclusion, the results indicate that B7-H3 within GC stroma could also play a role in regulating cell-mediated immune responses against cancer in patients of Western descent. Thus, it could serve as a great target of interest for different novel cancer immunotherapeutic strategies, especially in immunologically \u201ccold\u201d tumours.n = 48; diffuse, n = 27; mixed, n = 6; unclassified, n = 15) were included in the study. Specimens were routinely fixed in formalin and embedded in paraffin.A total of 98 gastric adenocarcinoma cases were selected by stratified sampling out of a consecutive series of GC patients treated with primary total or partial gastrectomy from the archive of the Institute of Pathology, University Hospital Schleswig-Holstein, Kiel, Germany. The following clinicopathological characteristics were collected: gender, age at diagnosis, tumour location, tumour phenotype by Lauren, pTNM stage, histological grade (G), UICC stage, as well as survival data. All patient-related data were pseudonymized. Two cases were excluded from the cohort as there was no clear invasive margin evaluable in the slides. Finally, a total of 96 cases . In the first step, antigen retrieval was performed using BOND Epitope Retrieval Solution 2  for 20 min, followed by incubation with anti-human B7-H3 . Detection and visualization were done with the Bond Polymer Refine Detection kit . In the second staining step, antigen retrieval was performed again using BOND Epitope Retrieval Solution 2 , followed by the second antibody incubation with anti-human CD8\u03b1 . Finally, detection and visualization were done with the Bond Polymer Refine Red Detection .B7-H3 expression was scored manually by two pathologists (D.U. and C.R.) for staining intensity and localization. Four representative cases were selected for the adjustment of staining intensity  and used as reference standard for the evaluation of the entire cohort. Inconsistent results were resolved by consensus review. For statistical purposes, cases were divided into B7-H3-low (score 0/1) and B7-H3-high (score 2/3) groups as proposed by a previous study .2; cell simulation with 2 \u00b5m of maximum growth as the optimal threshold for cell border generation; cell classification by double staining immunohistochemistry markers with red chromogen threshold of 0.32 and brown chromogen threshold of 3.0, respectively. These settings were used for all the images and carried out by a single author, who was blinded to patient outcomes. To avoid the false results due to different artefacts , marking of tumour compartments was done manually by using the viewer and painting program VMP. The tumour centre (TC) was defined as intratumoural area comprising cancer cells and desmoplastic tumour stroma with no direct connection to the peritumoural non-tumourous tissue. The invasive front (IF) was defined as a narrow band-like area at the tumour\u2013host interface with a width of up to 250 \u00b5m between the invasive margin of GC and adjacent non-tumourous tissue.Digital images of tissue sections were obtained using a Leica SCN400 microscopic whole-slide scanner  at 40\u00d7 nominal magnification, which corresponds to a resolution of 0.25 \u00b5m per pixel. To detect CD8+ T cells, digital image analysis with Definiens Tissue Studio  was performed. The following detection settings were used: 20\u00d7 nominal magnification for digital analysis; nucleus detection with haematoxylin threshold of 0.15 and typical nucleus size of 25 \u00b5mp < 0.05 was assumed.Data were analysed using SPSS v25.0.0.2 . Differences in clinicopathological variables between B7-H3 staining intensity groups were determined using Kendall\u2019s tau test for ordinal variables and Fischer\u2019s exact test for non-ordinal variables. Survival data were analysed by the Kaplan\u2013Meier method and compared using log-rank test. Differences in CD8+ T cell densities between B7-H3 groups were analysed using Wilcoxon Signed Ranks test and Mann\u2013Whitney U test. A significance level of http://xena.ucsc.edu (accessed on 14 February 2021)) [http://timer.cistrome.org (accessed on 14 February 2021)) [Data from TCGA STAD cohort were retrieved for quantitative validation analysis using TCGA Xena computational tool ) . The effy 2021)) ."}
{"text": "Type 1 Diabetes (T1D) is a T cell-mediated autoimmune disorder caused by the destruction of insulin-secreting cells. B7-H3 (CD276) plays a vital role in T cell response. However, B7-H3 expression and its clinical significance in T1D remain unclear. The aim of this study was to investigate the correlations between the expression of B7-H3 and clinical parameters in T1D patients. The possible role of B7-H3 gene variants with T1D was also discussed.Four B7-H3 single nucleotide polymorphisms (SNPs) were genotyped in 121 T1D patients and 120 healthy controls by polymerase chain reaction (PCR) direct sequencing. Expression of membrane B7-H3 (mB7-H3) in peripheral blood lymphocytes was determined by flow cytometry. Levels of soluble B7-H3 (sB7-H3) in serum were analyzed by enzyme linked immunosorbent assay (ELISA).P\u2009<\u20090.05). The concentration of sB7-H3 in serum increased in T1D patients (P\u2009<\u20090.0001). We also observed that B7-H3-T-A-C-T was associated with the decreased release of sB7-H3 but not the membrane form.The B7-H3 haplotype T-A-C-T was less frequently observed in T1D patients compared to the controls . B7-H3 expression on monocytes showed significant upregulation in T1D patients and was positively correlated with several clinical features including ALT, fast C-peptide 120\u2009min, HbAlc, IFN-\u03b3, IL-6 and TNF-\u03b1 (B7-H3 may act as a potential biomarker related to the pathogenesis of T1D. The B7-H3-T-A-C-T polymorphism variant is associated with the low risk of T1D as well as less release of sB7-H3. Type 1 diabetes (T1D) is a chronic autoimmune disease that leads to the selective loss of insulin-producing \u03b2-cells by activating the T cells \u20136. B7-H3+ and CD8+ T cell proliferation, cytotoxic T lymphocytes (CTLs) induction and interferon-gamma (IFN-\u03b3) secretion in vitro  range; R is the correlation coefficient between alleles.Additional file 5: Fig. S4. Expression of mB7-H3 in HC and T1D patients. Bars show the mean and SD of mB7-H3 mean fluorescence intensity (MFI)."}
{"text": "NPM1 mutated AMLs, which expressed B7-H3. These different B7 immune profiles suggest that specific immunotherapies are required to target the distinct immune evasion strategies of this genetically heterogeneous disease.Acute myeloid leukemia (AML) is generally considered a poorly immunogenic malignancy, displaying a \u201cnon-inflamed\u201d leukemia microenvironment (LME), leading to T cell tolerance. However, the immune landscape of AML is much more heterogeneous. Since B7 expression is regarded as a consequence of an interferon-mediated \u201cinflammatory\u201d phenotype, we have investigated by flow cytometry the B7 checkpoint ligands B7.1, B7.2, programmed death ligand 1 (PD-L1), PD-L2, ICOS-L, B7-H3, and B7-H4 on the AML blasts of 30 newly diagnosed patients and their corresponding receptors  on bone marrow (BM) T cell maturation populations. We could thus evidence B7-negative and B7-positive leukemias either with an isolated expression or part of eight different checkpoint ligand \u201csignatures\u201d that always included an inhibitory B7 molecule. B7-positive AMLs encompassed intermediate and adverse European Leukemia Net (ELN) risk cases and displayed mainly central memory CD4+ T cells with high ICOS levels and effector CD8+ T cells with high PD-1 expression. B7-negative cases were rather classified as AML with recurrent genetic anomalies and displayed predominantly naive T cells, with the exception of  The last decade has witnessed dramatic advances in the field of cancer immunotherapy. Immune checkpoint blockade (ICB) is reshaping the treatment paradigm of solid tumors  and hemaSimultaneously, gene expression profiling (GEP) of tumor immune microenvironments is revolutionizing our understanding of cancer-immune interactions. Several recurrent pan-cancer immune profiles have been identified and could serve as biomarkers for predicting clinical responses to immunotherapy or for tailoring personalized treatment strategies , 8. BrieThe development of immunotherapy in acute myeloid leukemia (AML) has been hindered so far not only by its remarkable genetic, antigenic, and clonal heterogeneity \u201313 and tNoteworthy, the LME of some AML cases displays evidence of a prior antileukemic immune response, restrained by immune escape mechanisms such as cytotoxic T lymphocyte associated protein-4 (CTLA-4)/B7.1/B7.2 and programmed death 1 (PD-1)/PD-1L signaling or Treg expansion . B7 moleThe expression of IFN-\u03b3-responsive genes has been correlated with primary refractory AML and is able to predict responses to ICB or flotetuzumab (CD3/CD123 DART\u00ae), showing that immune signatures within the LME can serve as reliable biomarkers to predict responses to immunotherapy , 29.Previous research was rather focused on finding prognostic relevance of isolated B7 molecule expression in AML , 30\u201332, Well-beyond investigating the expression of isolated molecules, our study aims to simultaneously evaluate the B7 checkpoint ligand phenotype of AML blasts  and the expression of immune checkpoint receptors (ICRs)  on helper and cytotoxic T cell maturation populations and to correlate these data to standard prognostic factors. We advanced the notion of \u201cB7 checkpoint ligand signatures\u201d to systematize the co-signaling output of AML blasts toward T cells. Since the expression of the immune escape PD-1/PD-L1 axis is correlated with an IFN-rich \u201cinflamed\u201d LME , it willde novo, non-promyelocytic AML between 2016 and 2019 at the Ia\u015fi Regional Oncology Institute, Romania, were included in this study. This study has been approved by the institutional ethics committee. BM and peripheral blood (PB) samples were collected at diagnosis. AML diagnosis was established according to the WHO diagnostic criteria  on erythrocyte-lysed fresh BM and PB samples. This study comprised four phases:  confirmaData acquisition was performed on a BD FACS ARIA III cytometer, and data were interpreted using the FACS DIVA v6.1.3 software. An identical investigation protocol was applied for all healthy subjects. The MoAbs used in this study are detailed in AML blasts gating was performed on CD45+/CD34+/CD117+/HLA-DR+ events. Subsequently, the expression level of each B7 molecule was assessed.T cell gating was performed on CD3+/CD4+ and CD3+/CD8+ events. The following T cell maturation subsets were defined based on their differential expression of CD28, CD27, and CD45RA: naive (N), central memory (CM), intermediate effector memory (iEM), late effector memory (late EM) , 39. Tren, total number of patients, the significance p-value, the statistical test used. The chi square, Fisher's exact test, Mann\u2013Whitney test, and Student's t-test were used to analyze the associations between different variables. The Pearson correlation coefficient was calculated to investigate the relationships between numerical variables. The two-way ANOVA test was used to analyze the differences among multiple variables. A p < 0.05 was considered as statistically significant.Statistical analyses were performed using the IBM\u00ae SPSS Statistics 21.0 Software. Each figure contains the relevant statistical information: the FLT-3-ITD mutations, and three patients had NPM1 mutated status. FLT-3-ITD and NPM-1 mutations did not coexist in our study group.Patient data are summarized in p = 0.028, B7\u2013 vs. HD: p = 0.739), B7.2  and ICOS-L . Out of the B7-positive cases, 10 expressed B7 molecule signatures and eight had isolated B7 expression.Within the healthy donor (HD) group, we could evidence the isolated expression of two molecules, PD-L1 and B7.2, in two cases , while 1n = 15), followed by PD-L1 , PD-L2 , ICOS-L , B7-H3 , and B7-H4 . B7.1 was expressed at extremely low levels and was thus considered negative. B7.2 was equally expressed isolated or co-expressed, while all the other B7 ligands were mainly co-expressed on AML blasts as B7 signatures.B7.2 was the most frequently expressed molecule  had intermediate or adverse ELN risk AML-Not Otherwise Specified (NOS). Out of these, complex karyotype AML-NOS expressed either B7.2 isolated or co-expressed B7.2, ICOS-L, and PD-L1, while normal karyotype AML-NOS also expressed PD-L2, B7-H3, and B7-H4 alongside B7.2, ICOS-L, and PD-L1 . The twopression .NPM1 mutated AML was the only B7-positive subtype and was correlated with B7-H3 expression (p = 0.02) and significantly higher levels of B7-H3 when compared to the AML-NOS cases (p = 0.019). AML with t  was B7 negative and expressed significantly lower percentages of B7.2 (p = 0.036) when compared to the AML-NOS cases . Out of this group, OS cases .p = 0.017, chi square test) and worse overall survival  (data not shown).Further details regarding the expression of each B7 checkpoint ligand relative to age, WHO AML type, and ELN risk are provided in We have identified eight different B7 checkpoint ligand signatures in 10 patients : co-exprp = 0.0002), B7.2\u2013ICOS-L (p = 0.019), PD-L1\u2013ICOS-L (p = 0.0009), PD-L1\u2013B7-H4 (p = 0.0051), PD-L2\u2013B7-H4 (p < 0.0001). B7.2 expression was rather associated with ICOS-L and PD-L1 in the B7.2/PD-L1/ICOS-L (three cases) and B7.2/PD-L1/ICOS-L/B7-H4 signatures (one case). Moreover, PD-L2 was correlated to B7-H4 expression (p < 0.0001). However, B7.2 expression was not correlated to PD-L2, B7-H3, and B7-H4. Finally, B7-H3 and B7-H4 expression was mutually exclusive  and lower naive (p < 0.0001) and CM (p < 0.0001) cells than CD4+ cells  and were rarely polarized as naive or effector cells. CD8+ cells displayed significantly higher late EM frequencies (4+ cells .p < 0.0001), while CD8+ expressed higher PD-1 levels  and iEM CD8+  T cells . By contrast, on CD8+ cells, the CM and iEM subpopulations expressed the highest PD-1 levels, and late EM cells displayed the lowest levels  and lower CD4+ T cells ; and (p = 0.016) and late EM (p = 0.022) T cells. Similarly, naive CD8+ T cells were poorly represented in B7-positive AMLs ; and  higher pive AMLs .p < 0.0001, iEM: p = 0.009, lateEM: p < 0.0001) and PD-1  levels on the effector CD4+, but not CD8+ cells  and was mostly characterized by the presence of molecules with known or most probably inhibitory role in cancer and AML in particular  , 44. B7.NPM1 mutated AML. ICOS-L was the only co-stimulatory ligand that was identified in 40% of B7 signatures. However, its facilitating role is probably either outbalanced by the co-expression of inhibitory ligands PD-L1 or B7.2 or, according to literature data, is detrimental in itself for successful antileukemic immunity by inducing Treg expansion  (data not shown).In complex karyotype AML-NOS, the B7 phenotype was rather restricted to isolated B7.2 expression or the co-expression of B7.2, PD-L1, and ICOS-L, which was also the most frequent B7 signature. On the other hand, normal karyotype AML-NOS cases expressed more diverse B7 ligands, including PD-L2 and B7-H4, which were statistically correlated, but also B7-H3. Interestingly, B7-H3 and B7-H4 expression was mutually exclusive, and B7-H3 expression was correlated with xpansion , PD-1 exxpansion , 48. Brixpansion , we alsoThe B7-negative patient group was smaller (40%) and encompassed most of the AML cases with recurrent genetic anomalies, most notably with t and t.The expression of B7 ligands on AML blasts has been further mirrored by polarization differences in T cell populations and ICR expression. When the B7+ and the B7\u2013 cases were analyzed separately, it turned out that the B7+ patient cases had significantly higher cytotoxic and lower T helper cell percentages, while this ratio was reversed within the B7\u2013 group. When analyzing the maturation subsets, we were able to show that most of the CD4+ T cells fall in the CM category, unlike the CD8+ T cells where the effector subsets outnumber the central memory ones. Furthermore, B7+ patients had a significantly lower number of naive CD4+ and CD8+ T cells than the B7- patients . The simOn an overall analysis of our AML patients, setting aside the maturation polarization, the ICOS expression was predominant on CD4+ T cells, while PD-1 was higher on CD8+ cells. When compared to B7 negatives, B7-positive patients expressed higher levels of both ICOS and PD-1 on CD4+ T cells, unlike the CD8+ cells that expressed similar levels of ICOS and PD-1 regardless of the B7 positivity or negativity. Despite the significant predominance of CD8+ T cells in B7+ AML patients, it so seems that B7 checkpoint ligands are rather impacting the immunoediting of CD4+ BM T cells.Analyzing further the ICOS and PD-1 expression on maturative subsets, we have noted a progressive increase in PD-1 expression from the naive toward the CD4+ effector compartment, while ICOS expression was highest on lateEM cells. By contrast, PD-1 expression on CD8+ cells was highest on CM, but not effector cells that displayed higher ICOS levels. Since CD4+ T cells promote the CD8+ T cell antitumor activity and prevent their exhaustion , we can When extending the investigation of ICOS and PD-1 expression on maturative subsets in B7-positive and B7-negative patients, we could stress further that the highest ICOS expression was present on late EM CD4+ T cells, followed by CM and iEM CD4+ T cells, and it always surpassed the ICOS expression on CD8+ T cells. Even though the level of expression was constantly higher on the cells of B7-positive patients, no statistically significant differences vs. negative ones or healthy donors emerged. PD-1 was instead always expressed at higher levels on CD8+ T cells as compared to CD4+ T cells, with the highest level reached by the CM CD8+ T cells, followed by iEM and late EM CD8+ T cells. However, the differences between B7+ and B7\u2013 patients and healthy donors were again not significant, even though iEM and late EM CD8+ T cells constantly displayed higher levels of PD1. As ICOS and PD1 binding mediate activating, respectively, inhibitory signals, these results might suggest that, irrespectively of B7 expression on AML blasts, CD8+ T cells are more prone to PD-1-mediated apoptosis, while their CD4+ counterparts are more susceptible to activation, but also to activation-induced cell death. Furthermore, the presence or absence of the B7 molecules on the AML blasts seems to have a minimal impact in influencing the levels of expression of ICOS and PD-1 on the T cell surfaces.NPM1 mutated AML, that displayed features holding indirect evidence of an \u201cinflammatory\u201d microenvironment, including the expression of mainly inhibitory B7 ligands, correlated with higher percentages of CD4 effector cells, less CD4+ and CD8+ naive T cells, as well as higher ICOS and PD-1 expression. NPM1 mutated AML also presented effector differentiation of CD8+ PD-1+ T cells, which is likely a feature of immune exhaustion. The rather inflammatory polarity of the immune microenvironment in NPM1 mutated AML is further supported by research that revealed that NPM1 mutation generates immunogenic peptides  but also in AML harboring the t9,11), an entity with rather low immunogenicity, as shown by previous research , 54. Thu,11p21;q.TP53 mutation and adverse karyotypes. In our similarly sized group of newly diagnosed AML patients, we were able to confirm this association between the expression of B7 ligands, including PD-L1 and the adverse-risk ELN patient subgroup.It is most clear that further research is needed to improve the characterization of the T cell composition and immune checkpoint landscape of the BM microenvironment. Recently, a comprehensive study conducted by Williams et al.  addresseWilliams et al.  did not Our study has obvious limitations, including the deliberate simplification of the leukemia immune biology, which depends on many other factors, such as functional T helper polarization , NK cells, macrophages or myeloid derived suppressor cells, and a relatively small patient group made of exclusively newly diagnosed AML cases however reflecting the heterogeneous AML patient population and confirming several conclusions emerging from the study of Williams et al. .Our data provide a more in-depth insight on the immune biology of AML. The B7 expression and antileukemic immunity in general are continuously regulated by a plethora of factors, including tumor-independent ones . Hence, All in all, our results reinforce the concept that this genetically heterogeneous disease has distinct and versatile patterns of antitumor immune response that depend on factors beyond the intrinsic genetic traits of the tumor cells. This finding is particularly relevant since new AML drugs are being rapidly developed and immune profiles emerge as a powerful biomarker in guiding and personalizing the new immunotherapy approaches.Gene expression profiling of the leukemia immune microenvironment is laborious, time-consuming, and not widely available. Hence, alternative markers for a faster and less expensive evaluation of the AML immune landscape are needed. In this paper, we have demonstrated that B7 checkpoint receptor and ligand profiling by flow cytometry is able to generate relevant data.In our study group, in more than half of the cases, the AML blasts expressed B7 molecule signatures or isolated B7 ligands. Regardless of the B7 combination, a co-inhibitory B7 molecule, such as B7.2, PD-L1, or PD-L2, was always present. B7 molecule expression was more frequent in intermediate and adverse ELN risk AML (78.2%) when compared to favorable risk cases (28.5%). Finally, B7 positivity was correlated with primary refractory AML and reduced overall survival.B7-positive AMLs displayed a predominance of effector BM CD4+ T cells with significantly higher levels of ICOS and PD-1. B7-negative AMLs were characterized by a significant predominance of naive CD4+ and CD8+ T cells and lower CD4+ T cell ICOS and PD-1 levels.As B7 ligands and their counterpart T cell receptors are regarded as indirect indicators of an IFN-driven \u201cinflammatory\u201d microenvironment, we can thus hypothesize that the B7 immune profiling of AML blasts and T cells could serve as a useful biomarker to rapidly discriminate between AMLs with \u201cinflamed\u201d vs. \u201cnon-inflamed\u201d microenvironment. Rather than defining an AML subtype in itself, the B7 phenotype more likely offers a momentary perspective of the versatile interaction between leukemia and immune cells that could predict resistance to standard chemotherapy and could guide personalized immunotherapy.We have undoubtedly entered a new therapeutic era in AML, where new and effective drugs are being rapidly designed, but the progress of immunotherapy clinical trials has a tendency to outpace our understanding of leukemia immune biology. The immune profiling of the tumor microenvironment will likely have a major impact on future clinical trial design, drug development, and integration of personalized immunotherapy in current therapeutic strategies.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by Ethics Committee, Grigore T. Popa University of Medicine and Pharmacy, Iasi, Romania. The patients/participants provided their written informed consent to participate in this study.IA, MZ, and PC are responsible for the study design. IA, AD, and CD were involved in the clinical management of the patients. MZ, II, and AS performed the immunophenotypic, molecular, and cytogenetic analyses. MP performed the statistical analysis and contributed to the graphical illustrations. IA and PC wrote the manuscript. All the authors were involved in the revision of the manuscript.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."}
{"text": "Cannabis sativa L.) is currently one of the most controversial and promising crops. This study compared nine wild hemp (C. sativa spp. spontanea V.) accessions with 13 registered cultivars, eight breeding lines, and one cannabidiol (CBD) hemp strain belonging to C. sativa L. The first three groups had similar main essential oil (EO) constituents, but in different concentrations; the CBD hemp had a different EO profile. The concentration of the four major constituents in the industrial hemp lines and wild hemp accessions varied as follows: \u03b2-caryophyllene 11\u201322% and 15.4\u201329.6%; \u03b1-humulene 4.4\u20137.6% and 5.3\u201311.9%; caryophyllene oxide 8.6\u201313.7% and 0.2\u201331.2%; and humulene epoxide 2, 2.3\u20135.6% and 1.2\u20139.5%, respectively. The concentration of CBD in the EO of wild hemp varied from 6.9 to 52.4% of the total oil while CBD in the EO of the registered cultivars varied from 7.1 to 25%; CBD in the EO of the breeding lines and in the CBD strain varied from 6.4 to 25% and 7.4 to 8.8%, respectively. The concentrations of \u03b49-tetrahydrocannabinol (THC) in the EO of the three groups of hemp were significantly different, with the highest concentration being 3.5%. The EO of wild hemp had greater antimicrobial activity compared with the EO of registered cultivars. This is the first report to show that significant amounts of CBD could be accumulated in the EO of wild and registered cultivars of hemp following hydro-distillation. The amount of CBD in the EO can be greater than that in the EO of the USA strain used for commercial production of CBD. Furthermore, this is among the first reports that show greater antimicrobial activity of the EO of wild hemp vs. the EO of registered cultivars. The results suggest that wild hemp may offer an excellent opportunity for future breeding and the selection of cultivars with a desirable composition of the EO and possibly CBD-rich EO production.Hemp ( Cannabis sativa L.) is a new-old crop, one of the most controversial and promising crops due to its multiple utilizations, and contains a wide array of biologically active substances synthesized and accumulated in different plant parts [C. sativa spp. spontanea Vavilov, also known as spontaneous), which is native to both Central and Eastern Europe as well as parts of Asia, and is found as a weed in agricultural fields.Hemp  can be extracted using various extraction methods, the simplest and most natural using either steam or hydro-distillation, as is the case with many other EO crops. Hydro-distilled or steam distilled hemp EO are generally preferred by consumers and can be incorporated into a number of certified organic products; the market for organic food and non-food products reached US$55 billion in 2019 in the USA alone . That acHemp terpenes in the EO contribute to the aroma of various cannabis genotypes, and so far, around 140 different terpenes have been reported in this plant ,7,8. Cur2, or solvent extraction) [The hemp EO profile depends on genotype, growth conditions, and extraction method  . Of the raction) . These Eraction) ,10. Monoraction) . Overallraction) . Hydro-dS. aureus, H. pylori, Candida, and Malassezia spp., enzymes, and cancer cell lines [+ and G- bacteria and yeast, and the effect depended on the cultivar and seeding date [Hemp EO  has shown biological activity against several targets of pharmaceutical interests ll lines . The EOsing date .C. sativa spp. spontanea) sampled from agricultural fields in northeastern Serbia with 13 EU registered cultivars, eight breeding lines, and one CBD hemp strain (belonging to C. sativa) with respect to their EO profile and antimicrobial activity.This study addresses a knowledge gap and current industry interest towards hemp EO with different origins and profile. The hypothesis was that wild hemp would have a different EO content, composition, and antimicrobial activity compared with the EOs of registered industrial hemp cultivars, new hemp breeding lines, and a hemp strain (unregistered cultivar) that is currently used for the commercial production of CBD. The objective of this study was to compare nine wild hemp accessions  were randomly selected among the nine locations  to be used as two replications to represent wild hemp in the statistical analyses. As indicated in the Materials and Methods section, one-way ANOVA was completed to determine the significance of differences between the mean constituents obtained from nine hemp cultivars from Northeast Serbia. The constituents were: \u03b1-pinene, \u03b2-pinene, isocaryophyllene (\u03b3-caryophyllene), \u03b2-caryophyllene, \u03b1-(E)-bergamotene, (Z)-\u03b2-farnesene, caryophyllene oxide, humulene epoxide 2, selina-6-en-4-ol, caryophylla-4(12),8(13)-dien-5\u03b1-ol, caryophylla-4(12),8(13)-dien-5\u03b2-ol, 14-hydroxy-(Z)-caryophyllene, \u03b2-bisabolol, \u03b1-bisabolol, CBD, and \u03b49-tetrahydrocannabinol.Overall, the EO profile of the wild hemp was different from that of one of the registered cultivars and the new breeding lines . The EO The concentration range of \u03b1-pinene (bicyclic monoterpene) in the EO was from non-detected (n.d.) in three accessions of wild hemp to 2.5% in wild hemp Slavka; from n.d. to 8.4% in the registered cultivars; and 0.15 to 2.95% in the new breeding lines . HoweverThe concentration range of \u03b3-caryophyllene in the EO was 0.2 to 1.23% in wild, 0.6 to 1.4% in the registered cultivars, and 0.57 to 1.6% in the EO of the breeding lines . \u03b3-CaryoC. sativa EO caryophyllane- and humulane-type sesquiterpenes that include sesquiterpenes (E)-\u03b2-caryophyllene, (Z)-\u03b2-caryophyllene, caryophyllene oxide, and the ring-opened isomer \u03b1-humulene (\u03b1-caryophyllene) [The concentration of \u03b2-caryophyllene was 15 to 30% in the EO of wild hemp, 22 to 55% in the registered cultivars, and 11 to 22% in the EO of the breeding lines . Overallhyllene) . This EOhyllene) .The concentration of \u03b1-(E)-bergamotene (bicyclic sesquiterpene) ranged from 0.75 to 1.9% in the wild hemp, 0.36 to 4.4% in the registered cultivars, and 0.5 to 2.8% in the breeding lines . OverallThe concentration of caryophyllene oxide (bicyclic sesquiterpenoid) was 0.24 to 31% of the total EO in wild hemp, 3.9 to 6.8% in registered cultivars, and 8.9 to 17% in the EO of the breeding lines . The conThe concentration of humulene epoxide 2 (bicyclic sesquiterpenoid) was 1.3 to 3.2% in wild hemp, 0.4 to 2.3% in registered hemp cultivars, and 2.3 to 5.6% in the EO of the breeding lines . OverallSelina-6-en-4-ol (bicyclic sesquiterpenoid) was 0.23 to 1.6% in the EO of wild hemp, n.d. to 1.8% in the EO of the registered cultivars, and 1.1 to 2.8% in the EO of the breeding lines . Its conThe concentration of caryophylla-4(12),8(13)-dien-5\u03b2-ol (bicyclic sesquiterpenoid) was 1.1 to 5.4% in the EO of wild hemp; from n.d. to 2.3% in the EO of the registered hemp cultivars; and 2.3 to 6.6% in the EO of the breeding lines . Its conThe concentration of \u03b2-bisabolol (monocyclic sesquiterpenoid) was 0.9 to 4.5% in the EO of wild hemp; from n.d. to 1.8% in the EO of the registered cultivars; and 2.9 to 4.0% in the EOs of the breeding lines ; Table 2The concentration of \u03b1-bisabolol (monocyclic sesquiterpenoid) was 0.4 to 2.9% in the EO of wild hemp, n.d to 6.9% in the registered cultivars, and 0.5 to 3.5% in the EO of the breeding lines . \u03b1-BisabThe major EO constituents of the USA hemp strain that was grown in close vicinity had a different chemical profile, with major constituents of myrcene (9.2 to 12%), \u03b2-caryophyllene (6.5 to 7.5%), limonene (3.8 to 4.2%), \u03b2-(E)-ocimene (5.3 to 5.6%), and \u03b1-bisabolol (3.9 to 4.4%). Some previous reports identified a different number of EO constituents in hemp EO  ; 84 EO cC. sativa [Previous research has suggested that metabolic fingerprinting can be used for chemotaxonomic purposes in . sativa . However. sativa ,18 have In this study, the concentration of CBD in the EO of wild hemp varied from 6.9 to 52.4% of the total oil, the CBD in the EO of the registered cultivars was from 7.1 to 25.4%, while the CBD concentration in the oil of the breeding lines was from 6.4 to 25.4% . HoweverOverall, the concentration of THC in the EO was significantly different between the three groups of hemp . The THCOverall, the content of monoterpenes fluctuated from n.d. to 8.0% in the EO of wild hemp, 0.3 to 13% in the EO of the registered cultivars, and 0.2 to 10% of the EO of the breeding lines . MonoterSesquiterpenes were the largest group of chemical constituents. The content of sesquiterpenes was 41 to 79% of the EO of wild hemp, 65 to 89% of the EO of the registered cultivars, and 70 to 89% of the EO of the breeding lines . Sesquit9-THC %), however, they were selected to have <0.3% total THC in dried biomass in order to be compliant with the current rules and regulations that are evolving [Cannabinoids also comprised the second largest group of chemical constituents in the wild hemp. The concentration of cannabinoids was 7 to 56% of the EO of wild hemp, 4.6 to 28% of the EO of the registered cultivars, and 6.4 to 26% of the EO of breeding lines . Cannabievolving .In a study of spontaneous hemp, Nagy et al.  reportedThe pharmacological power of hemp is based on the content of \u03949-tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA) . Other m-) bacteria and gentamicin for Gram-positive (G+) bacteria, and fluconazole for yeast. From G- we used SE, Salmonella enterica subsp. enterica; PA, Pseudomonas aeruginosa; and YE, Yersinia enterocolitica. From G+, we used SA, Staphylococcus subsp. aureus; EF, Enterococcus faecalis; and SP, Streptococcus pneumoniae. From yeast, CA, Candida albicans; CK, Candida krusei; and CT, Candida tropicalis were used. The method has been described previously [We used antibiotics as a positive control: cefoxitin for Gram-negative  activity to the antibiotics gentamycin, cetofoxin, and fluconazole.Staphylococcus subsp. aureus (SA), followed by the EO of wild hemp Saykaj . The EOs of wild hemp Buro and cv. Dioica were the most potent against Streptococcus pneumoniae (SP) (Streptococcus pneumoniae compared with that of other EOs (The antibiotic gentamycin was used as a positive control in the data presented in p Saykaj A. Similaiae (SP) B. The EOther EOs C.Pseudomonas aeruginosa compared with that of the EOs of the other hemps , the bioactivity of EO of wild hemp Perez was not significantly different, while the bioactivity of the EOs of the other hemp EOs was lower than that of Saykaj (Candida krusei (CK), while the bioactivity of the EO of Sequieni was not different from the above  (The antibiotic fluconazole was the positive control for the data presented in f Saykaj A. The EOhe above B. The EOlis (CT) C.S. aureus, H. pylori, Candida and Malassezia spp., enzymes, and cancer cell lines [Previously, hemp EO has shown biological activity against several targets of pharmaceutical interest: ll lines . In anotll lines .Overall, the findings in this study are consistent with the ones in a recent report . This stCannabis sativa L.) seeds were obtained from the Institute for Field and Vegetable Crops in Novi Sad, Serbia. Field experiments were set up at the Alternative Crops and Organic Production Department in Backi Petrovac, Serbia (N502138 E395689) using 13 different cultivars of industrial hemp  was applied as a broadcast treatment at 300 kg/ha before deep plowing. An additional 50 kg/ha of nitrogen was applied in the spring before sowing. Weed control was conducted using burnout with glyphosate prior to seeding. Mechanized weed control was performed twice during the first four weeks of vegetation stage though the use of sweep-type cultivators. Hemp closes the canopy at 5-6 weeks after emergence and hence, suppresses weeds very well after that. The trial of the breeding lines was at the same field, approximately 60 m from the main trial, and was subjected to the same agricultural conditions. Plants in both trials were cultivated the same way including seeding time, seeding depth, fertilization, and weed control.Fresh biomass samples  were obtained on 27 June, 2019 at the beginning of flowering of the male plants. Hemp tissue samples were generated by cutting the top 1.5 feet (46 cm) from the top of female plants . Fresh weight was measured, then the samples were hung in a shady area (tobacco dryer) until constant air-dried weight and then extracted.The EO extraction was conducted via hydro-distillation of hemp air-dried material using 4-L hydro-distillation Clevenger-type units as described previously for another plant material . The samm/z range of 40\u2013400. The injector and detector temperature (FID) was set at 220 \u00b0C and 280 \u00b0C, respectively.Hemp volatile compounds were analyzed by GC-FID and GC-MS as described previously , with th8 to C40 under the same conditions described above.All constituents present in the EO samples were identified by comparing their linear retention indices (LRI) and MS fragmentation patterns with those from the National Institute of Standards and Technology (NIST\u203208) and Adams mass spectra library. The estimated LRI were determined using a mixture of a homologous series of aliphatic hydrocarbons from CPseudomonas aeroginosa (CCM 1959), Salmonella enterica subsp. enterica (CCM 3807), Yersinia enterocolitica (CCM 5671); Gram-positive bacteria Enterococcus faecalis (CCM 4224), Staphylococcus aureus subs. aureus (CCM 4223), Streptococcus pneumoniae (CCM 4501); and yeasts Candida albicans (CCM 8186), C. krusei (CCM 8271), and C. tropicalis (CCM 8223) . The bacteria cultures were incubated in Mueller Hinton broth  at 37 \u00b0C, but yeast cultures were in Sabouraud broth  at 25 \u00b0C overnight.The microorganisms used for antimicrobial activity testing included Gram-negative bacteria 6 CFU/mL bacterial suspension after incubation was spread on the Mueller Hinton Agar . Filter paper discs (6 mm in diameter) were infused with 15 \u00b5l of the EO, tested, and placed on the inoculated MHA. MHA was kept at 4 \u00b0C for 2 h and then at 37 \u00b0C for 24 h. For yeasts, 100 \u00b5L of the yeast suspension was spread on Sabouraud agar  and agars were cultivated at 25 \u00b0C for 24 h. After the incubation period, the diameter of inhibition zones was measured (mm). Growth inhibition was compared with the standard drugs. The standard drugs cefoxitin for G\u2212 bacteria, gentamycin for G+ bacteria and fluconazole for yeasts were used as positive controls. Tests were performed in three separate experiments, and the means were calculated.For the agar disc diffusion method, a 100 \u00b5L of 10One-way analysis of variance (ANOVA) with two replications was completed to determine the significance of differences among the mean oil content and constituents obtained from nine cultivars. The constituents were: \u03b1-pinene, \u03b2-pinene, isocaryophyllene (\u03b3-caryophyllene), \u03b2-caryophyllene, \u03b1-(E)-bergamotene, (Z)-\u03b2-farnesene, caryophyllene oxide, humulene epoxide 2, selina-6-en-4-ol, caryophylla-4(12),8(13)-dien-5\u03b1-ol, caryophylla-4(12),8(13)-dien-5\u03b2-ol, 14-hydroxy-(Z)-caryophyllene, \u03b2-bisabolol, \u03b1-bisabolol, CBD, and \u03b49-tetrahydrocannabinol (Dronabinol). This completely randomized design has two replications.The differences among the mean antimicrobial activities  obtained from the seven registered cultivars  and seven wild accessions  were also compared using one-way ANOVA with three replications.p-value < 0.05) or marginally significant  on most of the constituents, further multiple means comparison was completed using the lsmeans statement of Proc Mixed  at 5% level of significance and letter groupings were generated. The overall mean was calculated for the constituents where cultivar effect was not significant. For the antimicrobial activities, cultivar effect was highly significant  on all nine activities, and considering the large number (14) of cultivars and accessions compared, multiple means comparison was done using Tukey\u2019s multiple range test, which controls the Type II experiment-wise error rate.The analyses were completed using the mixed procedure of SAS . Since tFor each response variable, the validity of model assumptions was verified by examining the residuals as described in Montgomery .Cannabis sativa var. spontanea Vavilov, (a synonym of Cannabis sativa L.), considered native to Central and Eastern Europe and parts of Asia. However, the taxonomy of hemp is still debatable and there is no consensus among taxonomists as to whether it is a single species with several subspecies or multiple species.The results from this study demonstrated that the essential oil (EO) of wild hemp from Serbia is different in its chemical profile and bioactivity from the EO of registered industrial hemp cultivars, the breeding lines, and the hemp strain grown for CBD production in North America. Wild hemp EOs were also somewhat different from the EO profile of wild and spontaneous hemps collected in Austria and Hungary, as reported in the literature. However, although having been named differently, the wild hemp in this study, the wild hemp collected in Austria, and the spontaneous hemp from Hungary might have common origins; the collection sites were approximately in the same region (with a dimeter of approximately 500 km) although collected in three different countries. These wild/spontaneous hemps have been exposed to significant environmental and agricultural (pesticide) pressure over the last few decades. These populations may all belong to The concentration of the four major constituents in the industrial hemp lines and wild hemp varied as follows: \u03b2-caryophyllene 11 to 22% and 15.4 to 29.6%; \u03b1-humulene (\u03b1-caryophyllene) 4.4 to 7.6% and 5.3 to 11.9%; caryophyllene oxide, 8.6 to 13.7% and 0.2 to 31.2%; humulene epoxide 2, 2.3 to 5.6% and 1.2 to 9.5%, respectively. The major EO constituents in the USA hemp strain that was grown in the vicinity of the field trials had different chemical profiles, with the major constituents myrcene (9.2 to 12%), \u03b2-caryophyllene (6.5 to 7.5%), limonene (3.8 to 4.2%), \u03b2-(E)-ocimene (5.3 to 5.6%), and \u03b1-bisabolol (3.9 to 4.4%).Overall, the EO of wild hemp has shown greater antimicrobial activity against Staphylococcus susp. aureus, Enterococcus faecalis, and G\u2212Yersinia enterocolitica, Salmonella enterica subsp. enterica, and yeasts Candida albicans and Candida tropicalis compared with the EO of registered cultivars.This is the first report to show that a significant amount of CBD can be accumulated in the EO of wild and registered cultivars of hemp following hydro-distillation. One of the wild hemp EOs showed a very high concentration of CBD.The EO of the wild hemp had a significantly higher concentration of THC relative to the one from the registered EU cultivars of industrial hemp breeding lines. In addition, it was interesting to see that wild hemp had a higher concentration of CBD in the EO relative to the EO from a strain grown for commercial production of CBD in the USA Therefore, wild hemp collected in Serbia could be used for the development of varieties (registered cultivars) with specific desirable chemical composition, and may also provide excellent material for the selection and breeding of hemp cultivars with high CBD for the commercial production of CBD and other high-value chemicals."}
{"text": "Aging is often accompanied by exacerbated activation of cell death-related signaling pathways and decreased energy metabolism. We hypothesized that transcranial near-infrared laser may increase intracellular signaling pathways beneficial to aging brains, such as those that regulate brain cell proliferation, apoptosis, and energy metabolism. To test this hypothesis, we investigated the expression and activation of intracellular signaling proteins in the cerebral cortex and hippocampus of aged rats (20 months old) treated with the transcranial near-infrared laser for 58 consecutive days. As compared to sham controls, transcranial laser treatment increased intracellular signaling proteins related to cell proliferation and cell survival, such as signal transducer and activator of transcription 3 (STAT3), extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), p70 ribosomal protein S6 kinase (p70S6K) and protein kinase B (PKB), also known as Akt that is linked to glucose metabolism. In addition, ERK is linked to memory, while ERK and JNK signaling pathways regulate glucose metabolism. Specifically, the laser treatment caused the activation of STAT3, ERK, and JNK signaling proteins in the cerebral cortex. In the hippocampus, the laser treatment increased the expression of p70S6K and STAT3 and the activation of Akt. Taken together, the data support the hypothesis that transcranial laser photobiomodulation improves intracellular signaling pathways linked to cell survival, memory, and glucose metabolism in the brain of aged rats. In the world, the number of people aged 60 and over has been growing faster than any age group Desa, . However2 per week for 5 weeks improved the cognitive function and EEG rhythms of older adults with memory complaint. Using D-galactose-induced aging mice, Salehpour and collaborators  and all efforts were made to minimize animal suffering in accordance with the proposals of the International Ethical Guideline for Biomedical Research  were used in this study. The colony room was maintained at 21 \u00b1 2\u00b0C with a 12 h light/ dark schedule (light: 7 am until 7 pm), and food and water were provided n = 6) and control (n = 5). One week before the treatment protocol, the animals were adapted to manual handling (with no anesthesia). This procedure was adopted to minimize discomfort during treatment. After this, the animals from the laser group were manually immobilized and received treatment with a laser diode of 810 nm wavelength and 100 mW power for 30 s (3 Joules of energy/point) by approximation at each of the 5 irradiation points of application . The daily laser treatment total was 15 Joules of energy, 150 s of irradiation, and fluency of 535.7 Joules/cm2. Also, no difference in scalp temperature measured in the animals was noted with a non-contact thermometer during the treatment protocol, corroborating with the results of Wang and collaborators : 810Operating mode: CWAverage radiant power (W): 0.1Aperture diameter (cm): 0.62): 3.571Irradiance at aperture (W/cmBeam shape: Circular2): 0.028Beam spot size (cm2): 3.571Irradiance at target (W/cmExposure duration/point (s): 302) per session: 535.7Radiant exposure  per head: 15g for 10 min at 4\u00b0C and supernatants were transferred to a new tube.Twenty-four hours after the final laser/placebo session (59th day of the experiment), the rats were euthanized by decapitation and their cerebral cortex  and hippocampus (Ammon\u2019s horn and dentate gyrus) were immediately collected and frozen. The tissue samples were homogenized in ice-cold RIPA lysis buffer  with freshly added protease  and phosphatase  inhibitors. Homogenates were centrifuged at 10,000\u00d7 \u00ae MAP kits magnetic bead panel assay  following the manufacturer\u2019s specifications. This multiplex immunoassay allows the simultaneous quantification of the expression and activation of cortical and hippocampal proteins: Akt, p70S6K, STAT3, STAT5, ERK, JNK, and p38. The plates were read on a LuminexTM MagpixTM instrument and results were analyzed with the Milliplex Analyst 5.1 Software using a Logistic 5P Weighted regression formula to calculate sample concentrations from the standard curves.The expression and activation  of intracellular proteins in the brain samples were quantified using the MilliplexP-value was lower than 0.05. All plots were acquired using the Graph Pad Prism (6.0). Data are presented as individual points to show dispersion and as mean and standard error of the mean (\u00b1SEM).Statistical procedures were conducted using the Mann-Whitney U-Test for independent group comparisons of nonparametric data. The Z-score was used to remove outlier values (\u2212/+2 SD). In the cortex, we removed one outlier of p-ERK/ ERK from the control group and one outlier of p-p38/p38 from the laser group. In the hippocampus, we removed one outlier of STAT3 and STAT5 from the control group and one outlier of p-ERK/ERK from the laser group. All analyses were performed using the Statistical Package for the Social Science . A statistical difference was considered significant when the two-tailed The expression and activation of intracellular signaling proteins  were investigated in the cerebral cortex and hippocampus of rats from the laser and control groups. The cortical and hippocampal expression and activation are presented in , 2. The U = 8.000; p = 0.201), p70S6K , STAT3 , STAT5 , ERK , JNK  and p38 . With regard to the activation of signaling proteins , no significant difference was observed between the groups studied in the cortical activation of Akt , p70S6K , STAT5  and p38 . However, PBM significantly increased the cortical activation of STAT3 , ERK  and JNK , STAT5 , ERK , JNK  and p38 . However, PBM significantly increased the hippocampal expression of p70S6K  and STAT3 , STAT3 U = 12.000; p = 0.917), STAT5  ERK , JNK  and p38 . However, PBM significantly increased the hippocampal activation of Akt , while our protocol consisted of 58 consecutive days of laser treatment with 810 nm wavelength and fluence of 535.7 J/cm2. Therefore, our laser treatment was 10 times greater. PBM is well known to show hormetic dose-responses, with opposite effects at lower and higher doses . These results suggest a neuroprotective effect of PBM since these proteins are linked to cell survival, apoptosis, and protein synthesis .Conceived and designed the experiments: FC, FM, and SG. Performed the experiments: FC and FM. Analyzed the data: FC and SG. Contributed reagents, materials, and analysis tools: FC and SG. Wrote the manuscript: FC, RL-M, FG-L, and SG. Approved the final version of the manuscript: FC, FM, RL-M, FG-L, and SG.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": "Little is known about how cancer cells adapt their mechanical properties in complex 3D microenvironments. Here we generated different types of tumor spheroids within compliant or stiff hydrogels. We then quantitatively mapped the mechanical properties of these spheroids in situ using Brillouin microscopy. Maps acquired for tumor spheroids grown within stiffer hydrogels showed elevated Brillouin shifts, hence spheroids became \u201cstiffer\u201d compared to the ones cultured within compliant gels. The spheroid\u2019s mechanical properties were modulated by various microenvironment properties including matrix stiffness and degradability and the resultant compressive stress but also depending on whether single cells or cell aggregates were analyzed. Moreover, spheroids generated from a panel of invasive breast, prostate and pancreatic cancer cell lines within degradable stiff hydrogels became stiffer and at the same time, less invasive compared to those in compliant hydrogels. Taken together, our findings contribute to a better understanding of the interplay between cancer cells and their microenvironment, which is relevant to better understand cancer progression.Altered biophysical properties of cancer cells and of their microenvironment contribute to cancer progression. While the relationship between microenvironmental stiffness and cancer cell mechanical properties and responses has been previously studied using two-dimensional (2D) systems, much less is known about it in a physiologically more relevant 3D context and in particular for multicellular systems. To investigate the influence of microenvironment stiffness on tumor spheroid mechanics, we first generated MCF-7 tumor spheroids within matrix metalloproteinase (MMP)-degradable 3D polyethylene glycol (PEG)-heparin hydrogels, where spheroids showed reduced growth in stiffer hydrogels. We then quantitatively mapped the mechanical properties of tumor spheroids in situ using Brillouin microscopy. Maps acquired for tumor spheroids grown within stiff hydrogels showed elevated Brillouin frequency shifts  with increasing hydrogel stiffness. Maps furthermore revealed spatial variations of the mechanical properties across the spheroids\u2019 cross-sections. When hydrogel degradability was blocked, comparable Brillouin frequency shifts of the MCF-7 spheroids were found in both compliant and stiff hydrogels, along with similar levels of growth-induced compressive stress. Under low compressive stress, single cells or free multicellular aggregates showed consistently lower Brillouin frequency shifts compared to spheroids growing within hydrogels. Thus, the spheroids\u2019 mechanical properties were modulated by matrix stiffness and degradability as well as multicellularity, and also to the associated level of compressive stress felt by tumor spheroids. Spheroids generated from a panel of invasive breast, prostate and pancreatic cancer cell lines within degradable stiff hydrogels, showed higher Brillouin frequency shifts and less cell invasion compared to those in compliant hydrogels. Taken together, our findings contribute to a better understanding of the interplay between cancer cells and microenvironment mechanics and degradability, which is relevant to better understand cancer progression. Tumor cells reside within a complex microenvironment that regulates essential cellular functions and also plays an important role in tumor progression and metastasis ,2. The uSince increased ECM stiffness has been correlated with tumor aggressiveness ,12,13, iMulticellular 3D cell culture models such as tumor spheroids recapitulate relevant features of epithelial tumors, including more realistic cell-cell contacts, proliferation rates, diffusion gradients and drug responses ,26,27. WFor this reason, we set out here to investigate the mechanical properties of tumor spheroids in situ using Brillouin microscopy (BM) . It is aWe report here that different types of tumor spheroids growing in stiff degradable hydrogels are characterized by a larger Brillouin frequency shift, hence increased elastic moduli, as compared to spheroids in compliant degradable hydrogels. This indicates that the spheroids adapted their mechanical properties to their microenvironment. Systematically comparing different culture systems where differences in compressive stress occur, e.g., after blocking hydrogel degradation, probing single cells or free spheroids, revealed a positive relationship of increasing Brillouin frequency shifts and compressive stress. Spheroids formed from invasive breast, prostate and pancreatic cancer cells formed in stiffer degradable hydrogels, showed higher Brillouin shifts and were less invasive compared to those in compliant degradable hydrogels. Thus, we find that the spheroids\u2019 mechanical phenotype and cell invasiveness are modulated by matrix stiffness, matrix degradability, multicellularity and concomitant changes in compressive stress levels. Together, our results bring new insights into the mechanical interactions between tumor cells and their microenvironment, which is relevant to better understand cancer progression.\u00ae was used. All cell culture reagents were from Thermofisher unless otherwise stated.MCF-7, MDA-MB-231 and PC3 cells were obtained from the \u201cDeutsche Stammsammlung f\u00fcr Mikroorganismen und Zellkulturen\u201d (DSMZ). PANC1 cells were kindly provided. MCF-7 cells were maintained in RPMI 1640  supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1 mM sodium pyruvate, MEM non-essential amino acids, 10 \u00b5g/mL human insulin  and penicillin (100 U/mL) -streptomycin (100 \u00b5g/mL). MDA-MB-231, PANC1 and PC-3 cells were cultured in DMEM/F-12, DMEM-GlutaMAX and RPMI 1640 respectively, supplemented with 10% fetal bovine serum (FBS) and penicillin (100 U/mL) -streptomycin (100 \u00b5g/mL). Before starting 3D cultures, cells were grown in standard T25 culture flasks and sub-passaged 2\u20133 per week. For cell detachment, TrypLE\u00ae, resuspended in cell culture medium, centrifuged down, and resuspended in PBS. Then they were mixed with the freshly prepared heparin-maleimide (MW~15000) solution and RGD peptide , at a final density of 5.6 \u00d7 105 cells/mL and a final heparin-maleimide concentration of 1.5 mM. For a molar ratio, PEG/heparin-maleimide of gamma = 1.5, PEG precursors (MW~16000) were reconstituted with PBS at a concentration of 2.25 mM and placed into an ultrasonic bath (Merck) for 20 s (medium intensity). Lower stiffness gels were prepared by diluting the stock solution of PEG precursors accordingly (1:2 for gamma = 0.75). To prepare PEG-heparin hydrogel droplets, heparin-cell suspension was mixed in a chilled microtube with the same amount of PEG solution using a low binding pipette tip. Then, a 25 \u00b5L drop of the PEG-heparin-cell mix was pipetted onto hydrophobic, Sigmacote\u00ae (Sigma Aldrich)-treated glass slides (VWR). Gel polymerization started immediately, and stable hydrogels were obtained within 1\u20132 min. Hydrogels were gently detached from the glass surface after 4 min using a razor blade and transferred into a 24 multi-well plate supplemented with 1 mL cell culture medium. Cell culture medium was exchanged every other day.PEG and heparin-maleimide precursors were synthesized as previously described . Cells wAfter their preparation, hydrogels were immersed in PBS and stored at 4 \u00b0C until mechanical characterization on the following day. After equilibrating them at RT for 1 h, gels were mounted using CellTak  onto glass object slides . A Nanowizard I or IV  was used to probe the gel stiffness. Cantilevers  that had been modified with polystyrene beads of 10 \u03bcm diameter  using epoxy glue , were calibrated using the thermal noise method implemented in the AFM software . Hydrogels were probed at RT in PBS using a speed of 5 \u03bcm/sec and a relative force setpoint ranging from 2.5 to 4.0 nN in order to obtain comparable indentation depths (0.5\u20131 \u00b5m). Force distance curves were processed using the JPK data processing software . Indentation parts of the force curves (approximately 1.5 \u03bcm indentation depth) were fitted using the Hertz/Sneddon model for a spherical indenter, assuming a Poisson ratio of 0.5 ,43.formaldehyde/PBS for 45 min, followed by a 30 min permeabilization step in 0.2% Triton-X100. Spheroids were stained for 2\u20134 h with 5 \u00b5g/mL DAPI and 0.2 \u00b5g/mL Phalloidin-TRITC in 2% BSA/PBS. Then hydrogels were immersed at least for 1 h in PBS. Spheroids were imaged with a LSM700 confocal microscope using a 40\u00d7 objective . Quantitative analysis of cell morphology was done using FIJI. Briefly, a threshold was applied to the fluorescent images , which were then transformed into binary pictures, and shape factor (defined as 4 \u00d7 \u03c0 \u00d7 area/(perimeter)2) and area were calculated.On day 14, cultures were fixed with 4% Brillouin maps were acquired using a custom-built confocal Brillouin microscope employing a two-stage VIPA spectrometer as previously described in ,45. The p < 0.05). As indicated in the figure legends, for pairwise comparisons a Mann\u2013Whitney (non-parametric) test was used since most datasets were not normally distributed. In the case of more than two groups, a Kruskal\u2013Wallis (non-parametric) with multiple comparisons (Tukey) was chosen. To test for normality, a D\u2019Agostino\u2013Pearson omnibus normality test was performed.GraphPad Prism was used to plot data and to perform statistical tests. A two-tailed significance level of 5% was considered statistically significant  spheroids by embedding single cells into PEG-heparin hydrogels and letting them grow to multicellular aggregates A. HydrogAfter seeing a difference in spheroid growth and compaction in response to hydrogel stiffness, we set out to investigate how matrix stiffness would affect the mechanical phenotype of the spheroids in situ. We employed BM A to quanIn order to investigate whether the seen changes in Brillouin frequency shift were a response of compressive stress acting on the tumor spheroids during growth, we next set out to manipulate the levels of compressive stress on growing tumor spheroids. Firstly, we tested how microenvironment degradability would affect the spheroid\u2019s mechanical properties since our bead pressure sensors had indicated that matrix degradability had a large effect on the growth-induced compressive stress H. Therefp = 0.001), with a trend of increasing Brillouin frequency shifts with hydrogel stiffness. Moreover, unconfined spheroids were generated by MCF-7 cell aggregation in low-adhesive U-well plates (p = 0.73 compared to non-degradable compliant). BM on unconfined spheroids , PANC1 (pancreatic cancer cell line) and PC3 (prostate cancer cell line) in compliant and stiff non-degradable gels A\u2013C, all We then set out to explore how these invasive cancer cell lines behaved in a microenvironment that would permit cell invasion. Therefore, we seeded MDA-MB-231, PANC1 and PC3 cells in compliant and stiff degradable hydrogels to generate spheroids. In the compliant degradable hydrogels, MDA-MB-231 cells formed no spheroids as they dispersed through the gel A, which In the presented study, we have investigated the influence of microenvironment stiffness on tumor spheroid growth, morphology, invasion and mechanical properties using a platform combining engineered tumor microenvironments and BM, an emerging tool in the biomechanics field. To investigate spheroid mechanics in 3D microenvironments of defined stiffness, we have chosen in our study a biohybrid PEG-heparin hydrogel system. Our 3D engineered model incorporates different aspects of mechanical cell interactions, such as growth-induced compressive stress, cell-cell, as well as cell-matrix interactions (by including RGD peptides). Importantly the hydrogel\u2019s mechanical properties can be tuned independently of ligand density. Young\u2019s moduli of the predominantly elastic hydrogels, as characterized by AFM, ranged between 1 and 20 kPa and were therefore within the range of elastic moduli reported for breast tumor specimens . When weWhile these results were obtained on hydrogels, also various experiments have been conducted in the past to reveal biological properties that influence the Brillouin frequency shift measured for cells. For instance, drugs that interfere with F-actin polymerization were combined with BM, which resulted in decreased Brillouin frequency shifts in different cell types ,51. AddiWhen we compared the spheroids\u2019 mechanical properties within the BM maps, we found increased Brillouin frequency shifts for spheroids in stiff degradable gels. Plotting Brillouin frequency shift of spheroids versus Brillouin frequency shift of hydrogels, a direct relation was seen , which sUnder invasive conditions, i.e., invasive cell lines in compliant degradable gels, we have found lower overall Brillouin frequency shifts, which might also indicate decreased cell stiffness of invasive cells or result from a looser structure. However, also for PC-3 cells, where a similar cell density was seen in compliant and stiff degradable hydrogels, a lower Brillouin frequency shift was detected for invasive spheroids in compliant hydrogels. Since we suspected that the Brillouin frequency shift for the invading single cells might have been biased by averaging with the surrounding hydrogel within the scattering volume, we decided to look at the maximum values within each map. In compliant degradable gels, we observed lower Brillouin frequency shifts for invading cells compared to no non-invading cells in stiff hydrogels, which fits the picture of invasive cells having decreased stiffness values . In contThe finding that so little invasion occurs in stiff degradable hydrogels appears at first sight contradictory to previous work showing greater invasion in stiffer microenvironments . HoweverAs mentioned before, an important aspect of stiff tumor microenvironments is compressive stress . This coTaken together, our study brings new insights into the mechanical interplay between tumor spheroids and their complex 3D microenvironment. We have shown that the level of compressive stress varies in our system with matrix stiffness and degradability and modulates growth, morphology, invasion and mechanics of tumor spheroids. Increased elastic moduli of spheroids were associated with lower invasion into the matrix, which suggests that the cancer cells\u2019 adaption of their mechanical properties in response to their microenvironments might also play a functional role in cancer progression. Since all these aspects can be modified with our experimental setup, we have a powerful model at hand to next investigate molecular mechanisms regulating the seen cellular responses. Ultimately, more insights into these mechanisms may provide a basis for identifying new targets that could potentially be used to interfere with tumor progression."}
{"text": "Bioaugmentation aims to use the capacities of specific bacterial strains inoculated into sites to enhance pollutant biodegradation. Bioaugmentation results have been mixed, which has been attributed to poor inoculant growth and survival in the field, and, consequently, moderate catalytic performance. However, our understanding of biodegradation activity mostly comes from experiments conducted under laboratory conditions, and the processes occurring during adaptation and invasion of inoculants into complex environmental microbiomes remain poorly known. The main aim of this work was thus to study the specific and different cellular reactions of an inoculant for bioaugmentation during adaptation, growth and survival in natural clean and contaminated non-sterile soils, in order to better understand factors limiting bioaugmentation.Pseudomonas veronii 1YdBTEX2. The strain proliferated in all but one soil types in presence and in absence of exogenously added toluene. RNAseq and differential genome-wide gene expression analysis illustrated both a range of common soil responses such as increased nutrient scavenging and recycling, expression of defense mechanisms, as well as environment-specific reactions, notably osmoprotection and metal homeostasis. The core metabolism of P. veronii remained remarkably constant during exponential growth irrespective of the environment, with slight changes in cofactor regeneration pathways, possibly needed for balancing defense reactions.As inoculant we focused on the monoaromatic compound-degrading bacterium P. veronii displayed a versatile global program, enabling it to adapt to a variety of soil environments in the presence and even in absence of its target pollutant toluene. Our results thus challenge the widely perceived dogma of poor survival and growth of exogenous inoculants in complex microbial ecosystems such as soil and provide a further basis to developing successful bioaugmentation strategies.The online version contains supplementary material available at 10.1186/s40793-021-00378-x. The recovery and restoration of soils polluted by organic compounds may be enhanced by introducing specific biodegrader bacteria \u20134. This Sphingomonas wittichii RW1 express differently during transition and growth in (polluted) sand as compared to liquid growth with the same carbon substrate [S. wittichii with those of Pseudomonas veronii strain 1YdBTEX2, isolated from sites contaminated with aromatic compounds [S. wittichii RW1, inoculation into sandy soil provoked a major reorganization in global gene expression of P. veronii 1YdBTEX2, implicating one-third of all genes, but with very few pathways and biological processes in common between the two [Previously, we suggested that more attention should be given to the combination of factors representing the actual expected conditions at polluted sites , and theubstrate . A complubstrate . This suubstrate . In ordeompounds , during ompounds . Similar the two .P. veronii 1YdBTEX2 gene expression changes during adaptation to a soil environment we focus here on its growth and establishment. The questions we aimed to address here were whether P. veronii would be capable to grow and survive in natural non-sterile soils, and whether this would be dependent on the presence of contaminated material or specific added carbon substrate (toluene). We further aimed to understand whether P. veronii would react differently in response to soil type and whether its cellular reactions could be revealed from global gene expression changes. In our experimental design, we inoculated P. veronii and measured population growth in three different natural non-sterile soils, in comparison to regular liquid culture and inert silica matrix, in the presence or absence of externally added toluene or in historically contaminated material with (polycyclic) aromatic hydrocarbons. Community RNA was extracted in exponential phase of P. veronii growth and in stationary phase, which was reverse-transcribed, ribosomal RNA depleted, and sequenced (RNA-seq). Adaptation and cellular reactions of P. veronii under different conditions were interpreted from a combination of global tools, including gene ontology (GO) [P. veronii 1YdBTEX2 (iPsvr) [To complement previous studies on ogy (GO)  and clusogy (GO) , a previ (iPsvr) , as wellP. veronii is a toluene, benzene and m- and p-xylene degrading bacterium originating from contaminated soil [P. veronii colony-forming units (CFUs) in soil, we used a derivative with a mini-Tn5 insertion constitutively expressing the green fluorescent protein (GFP) from the Pcirc promoter of the ICEclc element [P. veronii and the tagged strain , and was grown for 48\u2009h at 30\u2009\u00b0C. P. veronii colonies were restreaked on 21C-type minimal medium (MM) [v/v) in MM with 10\u2009mM succinate and again incubated as before. For soil growth experiments, cells were recovered from exponentially growing cultures on succinate (at culture turbidity OD600\u00a0=\u20090.4) by centrifugation (4000\u00d7g for 5\u2009min at room temperature), resuspended in MM without further carbon source and then transferred to the soil microcosms (see below). Growth of P. veronii was determined by CFU counting of appropriate dilutions on MM plates with toluene (gas phase). P. veronii colonies were differentiated from any background soil bacterial colonies growing on MM plates with toluene by their green fluorescence, and counted under a digital dual band microscope (Dino-Lite model AM4115T-GRFBY) using the 485\u2009nm blue LED excitation and 510\u2009nm emission filter.ted soil . To more element . We founium (MM)  agar plaP. veronii. These consisted of (i) a sandy soil sampled in a lake Geneva beach near in St. Sulpice \u2013 named Sand, (ii) a silty soil sampled from the bank of the local stream \u2018Sorge\u2019 on the university campus \u2013 named Silt, and (iii) a clay soil sampled in a forest area on campus near to Lake Geneva \u2013 named Clay. Quantities of ~\u20095\u2009kg were spread on aluminium foil and air-dried on the laboratory bench. Sand and Silt were dried for 7 days (further water losses were not evident with longer drying periods), whereas Clay was dried for 15\u2009days. At the moment of microcosm inoculations, the Sand, Silt, and Clay had gravimetric water content (GWC) of 0.14\u2009\u00b1\u20090.01%, 0.20\u2009\u00b1\u20090.01%, and 2.22\u2009\u00b1\u20090.06%, respectively. pH-H2O of the materials was 7.14\u2009\u00b1\u20090.02 (Sand), 8.57\u2009\u00b1\u20090.2 (Silt) and 7.78\u2009\u00b1\u20090.02 (Clay). Total organic matter content amounted to 0.028 (Sand), 0.13% (Silt) and 4.0% (Clay). Total cell counts on washed material and stained by SYBR Green I were quantified according to Weinbauer et al. [Three natural soils were used for microcosm growth studies with r et al. . The eff. According to previous characterization [10 \u2013 C40; 9500\u2009mg\u2009kg\u2212\u20091) and polycyclic aromatic hydrocarbons (2300\u2009mg\u2009kg\u2212\u20091). In addition, minor concentrations of benzene (3.3\u2009mg\u2009kg\u2212\u20091) and methylated monoaromatic compounds were found (22\u2009mg\u2009kg\u2212\u20091). The original material consisted mainly of gravel (1\u20135\u2009cm) covered with a layer of tar-containing mud. In order to obtain more easily handleable material, it was mixed 65/35 (w/w) in small portions with air-dried Silt and sieved through 3\u2013mm diameter to remove the gravel. In the main text we refer to this mixed and sieved material as Jonction. At the moment of inoculation, Jonction had a GWC of 6.55\u2009\u00b1\u20090.15%.The polluted material (Jonction) originated from a former gasification work in Geneva and was obtained through collaboration with Biotech S.A., Geneva (CH)rization , the matP. veronii miniTn5::gfp population growth in soil was assessed in microcosms artificially contaminated with toluene. Four replicate microcosms were prepared, each consisting of 95\u2009g of dried Sand, Silt or Clay inside 500\u2013ml glass Schott bottles and closed with Teflon-lined screw caps. As control for porosity effects, we used autoclaved quartz at 5% GWC ; hereafter referred to as artificial porous matrix or APM.The kinetics of P. veronii to obtain 2.5\u2009\u00d7\u2009104\u2009CFU\u2009g\u2212\u20091 material at the start. Microcosm flasks were mixed on a horizontal roller mixer  at 80\u2009rpm for 30\u2009min, with manual shaking every 5\u2009min to detach the soil from the walls. After mixing, the tip containing the toluene was placed back inside and the bottles were incubated upright at 24\u201326\u2009\u00b0C in the dark for 60\u2013120\u2009h with regular sampling (see below).Toluene was dosed through the vapor phase from a sealed 1\u2013ml micropipette tip, placed inside the glass bottle and containing 0.2\u2009ml of pure toluene. Tubes with toluene were removed before inoculation with 5\u2009ml suspension of washed preculture of P. veronii miniTn5::gfp in Jonction was followed in (triplicate) microcosms of 10\u2009g in 50\u2009ml polypropylene screw-cap tubes  containing either 100% Jonction (see above), 100% Silt, or mixtures of 75% (g\u2009g\u2212\u20091), 50, and 25% Jonction with Silt. Microcosms were again inoculated with washed P. veronii preculture (0.5\u2009ml) to achieve 2.5\u2009\u00d7\u2009104\u2009CFU\u2009g\u2212\u20091 at the start. Microcosms were homogenized and incubated as above, either without any further addition of carbon, or amended with a sealed 1\u2009ml micropipette tip containing 20\u2009\u03bcl pure toluene (as described above).Growth of P. veronii on toluene in liquid suspended culture was measured from four replicate 100\u2013mL screw-cap conical flasks containing 15\u2009ml MM and starting cell concentrations of ~\u20091\u2009\u00d7\u2009107\u2009CFU\u2009ml\u2212\u20091. As we found in preliminary experiments that P. veronii growth on toluene in liquid culture was less consistent with gas-phase dosing, we deployed an inert oil-toluene mixture instead. For this, we mixed 1:25 (v/v) of toluene:tetradecane  and added 0.5\u2009ml per 15\u2009ml liquid culture. Flasks were incubated at 30\u2009\u00b0C and at 180\u2009rpm on an orbital shaker.Growth of P. veronii CFU as described above. Identities of P. veronii miniTn5::gfp colonies were verified by their GFP fluorescence.Soil and liquid microcosms were sampled directly after inoculation (1\u2009h) and then four times per day at approximately 6\u2009h intervals . Samples of 5\u2009g (or 2.5\u2009g for Jonction) were retrieved from the microcosms with a spatula and transferred to clean 50\u2009ml polypropylene tubes. 10\u2009ml of sterile saline solution (0.9% NaCl) was added to each tube and cells were extracted by vortexing for 1\u2009min. Larger soil particles were allowed to sediment for a few seconds, after which the supernatant was transferred to a fresh 50\u2009ml tube and serially diluted in sterile saline. For liquid cultures, a 1\u2013mL aliquot was taken directly from the flask and serially diluted. Serial dilutions were drop-plated (10\u2009\u00d7\u20095\u2009\u03bcl drops of each dilution) on MM agar, which was incubated with toluene vapor to quantify the number of max) of P. veronii were calculated from the slope of the mean log10 CFU g\u2212\u20091 over time across quadruplicate assays . We further refer to the maximum population size (max pop size) as the highest mean CFU g\u2212\u20091 or ml\u2212\u20091 observed during the entire experiment for each condition, averaged from four (soils) or three (Jonction) replicates at the same sampling time point. Growth rates were compared among treatments by ANOVA, followed by post-hoc Tukey testing.Growth rates , during estimated exponential (EXPO) or in stationary (STAT) phase.For genome-wide expression analysis we used 7\u2009ml\u2212\u20091P. veronii cells into soil microcosms with 95\u2009g material  in 500\u2009ml capped-glass Schott bottles, with toluene dosage through the gas phase. Cells were prepared from exponentially growing liquid cultures as described above, but resuspended in MM with 0.5\u2009mM succinate to avoid starvation [For the transition phase we inoculated 5\u2009ml (2.5\u2009ml for Jonction) of a suspension of ~\u200910arvation . We usedP. veronii during exponential growth and stationary phase was sampled from similar inoculated microcosms , but in this case with a low starting cell density of ~\u2009104P. veronii CFU g\u2212\u20091 or ml\u2212\u20091 to achieve sufficient growth in the soils. Cells were precultured as before, but washed and resuspended in MM without succinate. Microcosms were dosed with toluene as before (four replicates each), and sampled (10\u2009g) at approximate exponential and stationary phase for P. veronii based on initial growth experiments as in Fig. P. veronii was inoculated into 100\u2009ml screw-cap conical flasks containing 25\u2009ml of MM amended with 0.5\u2009ml of a 1:19 mixture of toluene:tetradecane to obtain a starting OD600 of 0.16 (four replicates). Flasks were incubated at 30\u2009\u00b0C and 180\u2009rpm on an orbital shaker. Cells were sampled after 4\u2009h  and 24\u2009h , and harvested by centrifugation at 3500\u00d7g, for 6\u2009min at 30\u2009\u00b0C. Pellets were snap-frozen in liquid nitrogen and stored at \u2212\u200980\u2009\u00b0C until RNA extraction.In order to improve yields for RNA extraction and limit humic acid interference, we first washed cells from solid samples of 10\u2009g by adding 20\u2009ml of 4\u2009\u00b0C-cold sterile saline solution (0.9% NaCl) to the tube (50\u2009ml volume) and vortexing for 2\u2009min. Suspensions were allowed to sediment for 1\u2009min, after which the cleared liquid was transferred into a clean 50\u2009ml tube, which was centrifuged for 4\u2009min at 4000\u00d7g at 4\u2009\u00b0C to collect the cells. After centrifugation, the supernatant was immediately discarded by inversion, the excess of liquid from the walls was quickly removed with a clean paper towel, and the cell pellet was snap-frozen in liquid nitrogen and stored at \u2212\u200980\u2009\u00b0C until RNA isolation. Estimated cell recoveries from the washing procedure are reported in Supplementary Figure RNA was extracted from frozen cell samples using the RNA PowerSoil Total RNA Isolation Kit (MoBio Laboratories). Cells in thawed samples were disrupted in a bead-beating protocol as recommended by the manufacturer (MoBio Laboratories). RNA was purified following MoBio procedures and the final RNA pellet was resuspended in a volume of 20\u2009\u03bcl of RNase-free water. Contaminating DNA was removed by two consecutive treatments with TURBO DNase (Ambion), followed by purification using an RNeasy MinElute Cleanup kit .P. veronii genome. RNA samples were then depleted from ribosomal RNAs by using the Ribo-Zero rRNA Removal Kit Bacteria protocol . Subsequently, the RNA was reverse-transcribed, indexed and amplified by PCR using the ScriptSeq\u2122 v2 Bacteria kit and ScriptSeq\u2122 Index PCR primers set 1 (Epicentre). The resulting directional RNAseq libraries were sequenced using single 100-nt read chemistry on an Illumina HiSeq 2500 platform  at the Lausanne Genomic Technologies Facility.RNA quantity (reported in Supplementary Table\u00a0P. veronii genome sequence as reference (European Nucleotide Archive under bioproject number PRJEB11417). A summary of the total number of mapped reads per condition is listed in Supplementary Table\u00a0P. veronii transition response (1\u2009h) in liquid suspension and in Sand were taken from previous work [Raw reads were quality-filtered, mapped, sorted and indexed with Bowtie2  and Samtous work .p-value of <\u20090.01 and a log2 fold-change >\u20092, and were subsequently interpreted by using Gene Ontology (GO) analysis [P. veronii genes were inferred using the program BLAST2GO [Genes were called significantly differentially expressed between two conditions at a false-discovery rate (FDR) of <\u20090.05, analysis . GO termBLAST2GO . The samBLAST2GO  with nodP. veronii 1YdBTEX2 metabolic capacities was extracted from its reconstructed genome-scale metabolic model (iPsvr), which accounts for 1234 metabolic genes [2 normalized expression attributed to that reaction. Those reactions whose mean expression differed by two or more standard deviations in EXPO phase among cells in liquid, Silt or Jonction, were highlighted in different color. Maps were exported to Adobe Illustrator (vs 2020). The mean normalized expression of all genes attributed to KEGG reactions under all conditions was further visualized as a heatmap with rows  clustered in Euclidian distance .A survey of ic genes  and all ic genes . Identific genes  with linThe raw unmapped RNA-seq reads related to this study have been deposited in the NCBI Short Read Archive under Bioproject accession number PRJNA682712.P. veronii in different soils, we inoculated microcosms dosed with toluene in comparison to liquid suspended medium or to artificial porous medium  and two natural soils (Sand and Silt) were slightly higher and statistically different from those measured in liquid or in APM . Population size variations in Clay suggested alternating growth and decline, perhaps as a result of cycles of predation (given that the soils were not sterilized). Predation may have been more pronounced in the Clay microcosms because of their slightly higher starting water content (Methods), in which protozoan may have remained alive for longer than in air-dried Sand or Silt [In contrast to the other microcosms, the  or Silt \u201338. Grow1.28\u2009\u00d7\u200910\u2009CFU\u2009g\u2212\u20091P. veronii could grow in field-collected polluted soil. Since contaminated material from Jonction contains monoaromatic compounds (see above) [P. veronii might be able to grow in absence of externally added toluene. Contrary to our expectations, however, P. veronii miniTn5::gfp developed poorer in microcosms with a higher degree of Jonction material but without added toluene  within 24\u2009h, in 100% Jonction it only reached 2.7\u2009\u00d7\u2009105\u2009CFU\u2009g\u2212\u20091. Mixing Jonction material with Silt overproportionally reduced the final attained P. veronii population size  Silt, but that the strain is inhibited by components or factors originating from Jonction. The poorer growth in microcosms mixed with Jonction material further suggested that P. veronii found little available aromatic substrates , in order to determine to what extent e above) , we hypoene Fig. . WhereasP. veronii grew very rapidly in all Jonction microcosms to which external toluene was supplied through the gas phase , exponential growth and stationary phase , both in presence of toluene  and metabolism  of putrescine , genes from the nar operon for nitrate respiration (narGJKLY), a gene encoding oxygen-independent coproporphyrinogen-III oxidase (hemN), and genes coding for the cbb3 cytochrome C oxidase isoforms, ccoN1, ccoO and ccoG2 . These effects may thus be solely a response to change in porosity from the growth matrix itself.P. veronii in different soil types  caused a further common core of 68 genes (60 higher and 8 lower) to change expression  involved in malonate transport and metabolism, and of bkdA1A2B, involved in the metabolism of branched-chain amino acids, were higher expressed  and the narGIJKY gene cluster for nitrate respiration were also significantly induced in P. veronii in soils after 1\u2009h  operon, which is responsible for molybdopterin biosynthesis  were also higher expressed in cells in Jonction, suggesting further induction of anoxic respiration pathways.Among the genes induced specifically in Jonction and not in clean soils  and for the phosphate starvation-inducible protein PsiF. The GO enrichment analysis of transcriptomic response in Jonction was coherent with these observations , elongation factors (EF), fusA (EF-G), efp (EF-P), tsf (EF-Ts), and tuf (EF-Tu); (ii) DNA replication; (iii) ATP synthesis; (iv) sulfate transport; (v) TCA cycle, and (vi) amino acid biosynthesis.As expected, both GO analysis and individual gene annotations confirmed most of the commonly higher expressed genes in exponentially growing cells to be related to growth and energy production Table\u00a0 and 23. In contrast, signatures of commonly lower expressed genes were globally less clear. Around 50% of those encoded hypothetical proteins (Table meta-cleavage metabolism (dmp gene cluster) and urea transport  showed P. veronii during stationary phase conditions and different growth environments. In comparison to 1\u2009h, cells sampled in stationary phase conditions , glycogen (glg), and polyhydroxyalkanoates (phaG), the latter being induced up to 55-fold in APM and 200-fold in Silt , putrescine (spuD), spermidine (spuE), nitrate (nasA), and sulfate (PVE_r1g3919). Genes associated with resistance mechanisms were higher expressed, for example, organic hydroperoxide resistance protein (ohr), and mercury resistance (merACTP). Interestingly, several signaling genes such as cheA, cheB2, cheW, and cheY, PVE_r1g893, and PVE_r1g2555 were also higher expressed in APM and Silt compared to LQ  in controlled conditions of growth under sublethal solute and matric stress that mimicked water stress in soils, in order to identify common strategies [P. veronii transcriptomic response observed here in APM and, previously, under imposed matric and solute stresses [P. veronii with toluene in APM was worse than in liquid or natural soils, suggesting that porous conditions caused differences in the regulation of toluene metabolism in P. veronii.Previous studies had indicated the massive difference in gene expression of criptome . Furtherrategies . By inclrategies , 49, 50.rategies . On the stresses , was difP. veronii, and to understand if there are soil factors that would determine its adaptation and survival. The soils we worked with here have different microbiota background levels, and some showed growth on toluene, which may have invoked substrate competition on the introduced P. veronii cells. Unfortunately, we do not understand the nature of signs for competition in the P. veronii transcriptome. Soils further varied in pH, organic carbon content, structures and textures, leading to expected differences in e.g., substrate and water availability, matric stress, oxygen fluxes [P. veronii and induction of cytochrome c oxidases cbb3, which have been observed as low oxygen environmental responses of P. aeruginosa [Pseudomonas stutzeri A15, and Pseudomonas fluorescens YT01 [P. veronii to adapt to soil microniches with low oxygen concentrations, as previous studies in controlled porous matrices have shown [P. veronii to adapt to living in soil.One of the aims of this work was to study the effect of soil type on physiological responses and growth of n fluxes , or protn fluxes . Indeed,ruginosa , Pseudomens YT01 , 54. Nitve shown . PossiblP. veronii cells to induce an osmoprotective response (putrescine and potassium uptake), to induce genes for exopolysaccharide production, and to regulate genes for copper homeostasis, indicating the importance of these processes for the initial soil adaptation. Of particular interest is the strong upregulation of systems involved in putrescine uptake and metabolism. The roles of putrescine (and other polyamines) are manifold, having been described both as signals for and during carbon or nitrogen starvation, regulation and oxidative stress defense [P. veronii, whose reaction is in line with the three classical physiological responses to osmotic stress: polyamine transport and accumulation of glutamate and potassium [Regardless of the soil type and even in contaminated material such as Jonction, transition caused  defense , and rec defense . The acc defense . Our datotassium . Particuotassium , 58.P. veronii population developed in all non-sterile environments, more extensively when toluene was added as specific growth substrate, but also in its absence. This is in contrast to the much cited incapacity of inoculants to grow and be metabolically active in natural non-sterile materials, which may lead to bioaugmentation failures [P. veronii would not be able to adapt and proliferate, but unfortunately, we could not really exploit this at transcriptomic level. Growth in Jonction material without added toluene was clearly not favorable for P. veronii, but the transcriptomic response from 1\u2009h exposure in Jonction with toluene (in which it could grow) did not particularly show signs of poor adaptation. Except for increased induction of stress defense systems and changes in expression of genes for respiratory activity, there were no particular signs of physiological breakdown in cells in Jonction. The only other environment, which did not lead to strong population development of P. veronii on toluene was Clay. We think that the most likely causes for population loss here were predation and, possibly, competition of native soil microbiota for toluene. Unfortunately, we did not manage to isolate sufficient RNA for transcriptome analysis from P. veronii during exponential growth in Clay. However, during the 1-h transition in Clay we observed a decrease of expression of genes for flagellar assembly and motility, which was not present in Silt or Sand. Flagella allow bacteria to explore their environment, to search for nutrients and to escape from predators or adverse conditions, but activation of flagellar genes have also been implicated in the solvent stress tolerance response, where the flagellar export apparatus is used to export other proteins unrelated to flagellar assembly [S. wittichii RW1 in sand [S. wittichii RW1, Artrobacter chlorophenolicus A6 and P. veronii reduce expression of flagella synthesis under solute and matric stress [P. putida KT2440 exposed to water stress on ceramic plates did not show significant difference in flagellar gene expression [P. veronii during growth in Silt with toluene upregulated flagellar gene synthesis  is necessarily a \u2018narrative\u2019 of understanding how cells adapt and grow, concluded from the conglomerate of global  analysis as well as that of individual gene annotations. However, specific cellular reactions are clearly different between typical liquid cultures and in soil. Therefore, it seems crucial to us to study strain behaviour under the conditions of the expected complex environments , and not in standard liquid culture. In the future, this knowledge may help to better predict the success of inoculants.Additional file 1: Supplementary Figure 1. Comparison of growth rates on toluene of P. veronii wild-type and the P. veronii gfp-tagged variant. Supplementary Figure 2. Background soil microbiota counts. Supplementary Figure 3. Cell washing recoveries of wild-type P. veronii cells inoculated to soils. Supplementary Figure 4. Background growth of P. veronii miniTn5::gfp on soil organic carbon or soil microbiota on toluene.Additional file 2: Table S1. Summary of total mapped reads of inoculated microcosms exposed to toluene. Table S2. Subset of genes whose expression levels responded to inoculation into glass beads (APM) versus liquid (FDR\u2009<\u20090.05). Table S3. Subset differentially expressed genes of P. veronii 1YdBTEX2 in all soils vs controls (Liquid&APM). Table S4. Relevant KEGG reactions with outlier expression in exponential phase conditions. Table S5. Biochemical term enrichment in the exponential phase outlier reactions compared to all metabolic reactions. Table S6. Commonly differentially expressed genes in Liquid, glass beads (APM) and Silt in stationary phase. Table S7. Subset of commonly differentially expressed genes in glass beads (APM) and Silt in stationary phase.Additional file 3: DATA\u00a01. Complete list of differentially expressed genes in APM-1H versus liquid-1H (FDR\u2009<\u20090.05). DATA\u00a02. Enriched GO terms among the significantly differentially downregulated genes in cells growing on glass beads (APM) versus liquid media after 1\u2009h inoculation. DATA\u00a03. Complete list of differentially expressed genes in Sand-1H versus APM-1H (FDR\u2009<\u20090.05). DATA\u00a04. Complete list of differentially expressed genes in Silt -1H versus APM-1H (FDR\u2009<\u20090.05). DATA\u00a05. Complete list of differentially expressed genes in Clay-1H versus APM-1H (FDR\u2009<\u20090.05). DATA\u00a06. Enriched GO terms among the significantly differentially upregulated genes in Sand versus APM after 1\u2009h contact. DATA\u00a07. Enriched GO terms among the significantly differentially expressed upregulated genes in Silt versus glass beads (APM) after 1\u2009h contact. DATA\u00a08. Enriched GO terms among the significantly differentially expressed downregulated genes in Silt versus glass beads (APM) after 1\u2009h contact. DATA\u00a09. Enriched GO terms among the significantly differentially expressed upregulated genes in Clay versus glass beads (APM) after 1\u2009h contact. DATA\u00a09. Enriched GO terms among the significantly differentially expressed downregulated genes in Clay versus glass beads (APM) after 1\u2009h contact. DATA\u00a011. Differentially expressed genes in all natural soils-1H vs controls-1\u2009h (ANOVA. FDR\u2009<\u20090.05). DATA\u00a012. Complete list of differentially expressed genes in Jonction-1H vs APM-1H (FDR\u2009<\u20090.05). DATA\u00a013. Commonly differentially expressed genes in soils and Jonction soils after 1\u2009h contact versus control conditions -1H (ANOVA. FDR\u2009<\u20090.05). DATA\u00a014. Unique differentially expressed genes in Jonction-1H vs APM-1H. DATA\u00a015. Enriched GO terms among the significantly differentially expressed upregulated genes in Jonction versus glass beads (APM) after 1\u2009h contact. DATA\u00a016. Enriched GO terms among the significantly differentially expressed downregulated genes in Jonction versus glass beads (APM) after 1\u2009h contact. DATA\u00a017. Comonly differentially expressed genes in Pseudomonas veronii cells growing exponentialy in Liquid, Sand and Silt vs same material -1H (FDR\u2009<\u20090.05). DATA\u00a018. Enriched GO terms among the significantly differentially expressed upregulated genes in LIQUID-EXPO vs LIQUID-1\u2009h. DATA\u00a019. Enriched GO terms among the significantly differentially expressed downregulated genes in LIQUID-EXPO vs LIQUID-1\u2009h. DATA\u00a020. Enriched GO terms among the significantly differentially expressed upregulated genes in SILT-EXPO vs SILT-1\u2009h. DATA\u00a021. Enriched GO terms among the significantly differentially expressed downregulated genes in SILT-EXPO vs SILT-1\u2009h. DATA\u00a022. Enriched GO terms among the significantly differentially expressed upregulated genes in JONCTION-EXPO vs JONCTION-1\u2009h. DATA\u00a023. Enriched GO terms among the significantly differentially expressed downregulated genes in JONCTION-EXPO vs JONCTION-1\u2009h. DATA\u00a024. Complete list of differentially expressed genes in SILT-EXPO vs SILT-1H (FDR\u2009<\u20090.05). DATA\u00a025. Complete list of differentially expressed genes in Jonction-EXPO vs JN-1H (FDR\u2009<\u20090.05). DATA\u00a026. Commonly differentially express genes in APM and Silt in stationary phase (FDR\u2009<\u20090.05). DATA27. Unique differentially express genes Silt in stationary phase (FDR\u2009<\u20090.05). DATA\u00a028. Unique differentially express genes in APM-STAT vs APM-1H (FDR\u2009<\u20090.05). DATA\u00a029. Enriched GO terms among the significantly differentially expressed upregulated genes in SILT-STAT vs SILT-1\u2009h. DATA\u00a030. Enriched GO terms among the significantly differentially expressed downregulated genes in SILT-STAT vs SILT-1\u2009h. DATA\u00a031. Enriched GO terms among the significantly differentially expressed upregulated genes in AS-STAT vs AS-1\u2009h . DATA\u00a032. Enriched GO terms among the significantly differentially expressed downregulated genes in AS-STAT vs AS-1\u2009h. DATA. Enriched GO terms among the significantly differentially expressed upegulated genes in LIQUID-STAT vs LIQUID-1\u2009h. DATA. Enriched GO terms among the significantly differentially expressed downregulated genes in LIQUID-STAT vs LIQUID-1\u2009h."}
{"text": "Dental fear is a prevalent problem that can lead to poor dental health. The Kleinknecht\u2019s Dental Fear Survey (DFS) is one of the used scales to assess dental fear. The present study aims to evaluate the psychometric properties of the Lebanese Arabic version of the DFS (DFS-A) and to determine the optimal cut-off to identify dental fear as well as the correlates of dental fear in a group of Lebanese adults dental patients.A cross-sectional study was conducted among a group of 442 dental patients (18\u201365\u00a0years) recruited at 29 dental clinics from March to June 2019. Patients completed a questionnaire including questions about demographic characteristics, previous bad dental experience, trauma\u2019s experience period, the sensation of nausea during dental treatment, the DFS-A scale, the Lebanese Arabic version of the Modified Dental Anxiety Scale (MDAS-A), and a general question about dental fear..DFS-A revealed evidence of adequate psychometric properties. DFS-A scale demonstrated high internal consistency . Test\u2013retest reliability assessment demonstrated strong reproducibility of the DFS-A scale score (ICC\u2009=\u20090.92 with 95% CI (0.83\u20130.96), p value\u2009<\u20090.0001 (N\u2009=\u200930). Confirmatory factor analysis revealed a three-factor structure of the DFS-A reflecting fear associated with specific dental stimuli and procedures, patterns of dental avoidance and anticipatory anxiety, and physiologic arousal during dental treatment. A significant correlation was found between DFS-A and the MDAS-A indicating a good convergent validity. The optimal cut-off point to identify patients with and without dental fear is 41. Considering this cut-off score, the prevalence of dental fear in our sample was reported at 33.8%. Multivariable analysis showed that having previous scary and painful dental experiences, a sensation of nausea during treatment, and having dental anxiety were identified as predictors of dental fear.The adapted Arabic version of the DFS (DFS-A) is a valid tool to evaluate dental fear among Lebanese adult patients Dental fear is a prevalent condition that affects all populations worldwide . It is dTo diagnose dental fear in clinical settings, it is important to have a valid and reliable diagnostic tool. In response to this need, researchers have developed various specific instruments to evaluate dental fear. One of the most used tool is the Kleinknecht\u2019s Dental Fear Survey (DFS) . DFS wasDFS was initially established in the English language and authors have revealed good psychometric properties supporting its validity and reliability among adults in the USA , 5. Due Cross cultural adaptation through testing Arabic translations of the measuring system in different countries can assign more information about its validity and reliability. So far, no studies have assessed the feasibility of the DFS in the cultural context of the Lebanese population. Thus, the current study aims to test the properties of the DFS Lebanese Arabic version and to identify the optimal cut-off to detect dental fear. In addition we sought to evaluate the factors associated with dental fear in a group of Lebanese adult dental patients.A cross-sectional study was conducted over 4\u00a0months extending from March until June 2019 in different dental clinics from all Lebanese districts. Patients aged between 18 and 65\u00a0years who were able to read the Arabic language were included in our study. Pregnant women as well as patients with malignant diseases and mental disabilities were excluded. In addition, patients with missing information about DFS-A were excluded from the analysis (9 of 451 eligible patients). Thus, a total sample of 442 adult patients was collected. The study was approved by the Neuroscience Research committee at the medical Lebanese University (Reference number 12/2/2019). The guideline for Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) was followed when reporting this study.Information about the demographic characteristics , previous bad dental experience, trauma\u2019s experience period, perception of a periodontal problem, the sensation of nausea during dental treatment, the Arabic version of the Dental Fear Scale (DFS-A), the Arabic version of the Modified Dental Anxiety Scale (MDAS-A) were collected from the participants. To determine the test\u2013retest reliability of the DFS-A scale, 30 patients were recruited to complete the DFS-A scale twice within 2\u00a0weeks. The methodology used in this study is described elsewhere. The questionnaires were distributed to 500 patients, 442 were completed which corresponded to an overall effective response rate of 88.4%.This scale consists of 20 items divided into three components: Avoidance of dental treatment, physiologic reactions to the dental treatment, and fear aroused by different dental procedures . Each quThe translation and Cross-cultural adaptation of the DFS-A, into the Arabic language, was performed according to the five steps proposed by Beaton et al. , after oThe MDAS consists of 5 questions to measure the degree of dental anxiety in 5 situations: preparing for a dental visit, waiting in the dentist\u2019s office for treatment, sitting in the dental chair for drilling, getting ready in the dental chair for scaling, and preparing for local anesthetic injection. Evaluations range from \u201cnon-anxious\u201d  to \u201cextrData entry and analyses were performed using the statistical software SPSS version 21.0. Descriptive statistics were reported using means and standard deviations (SD) for continuous variables and frequency with percentages for categorical variables. Floor and ceiling effects were evaluated for the DFS-A total score. These effects were considered to be present if they exceeded 15\u201320% of patients receiving the minimum or maximum scores . The Cro2) and degrees of freedom (df), Root Mean Square Error of Approximation (RMSEA), and Comparative Fit Index (CFI).The total group was randomly divided into two groups using the randomization function on SPSS 22.0. In the first random-half sub-sample (n\u2009=\u2009221), exploratory factor analysis was conducted using the principal components analysis with Varimax rotation. Sampling adequacy was assessed by the Kaiser\u2013Meyer\u2013Olkin (KMO) measure and Bartlett\u2019s test of sphericity. The number of factors retained in the scale was determined based on Eigenvalues greater than 1, and visual inspection of the scree plot. Confirmatory factor analysis (CFA) was performed in the second random-half sub-sample (n\u2009=\u2009221) using the Amos software version 22.0. The goodness-of-fit of the models were evaluated using Chi-square  analysis. The best cut-off point was identified by calculating the Youden index (Youden index\u2009=\u2009sensitivity\u2009+\u2009specificity\u2009\u2212\u20091). The score at which the Youden index is maximal was considered the best cut-off point. The Pearson chi-square (\u03c72) test was used for statistical bivariate analysis. Multivariable logistic regression analyses were performed to identify associated factors of dental fear. Adjusted odds ratio and their 95% confidence intervals were reported. The final logistic regression model was reached after ensuring the adequacy of our data using the Hosmer and Lemeshow test. All statistical tests were two-sided, and the significance level was set at 0.05.Convergent validity using Spearman correlation was assessed to evaluate whether the total DFS-A scale was associated with MDAS-A. The discriminant validity of the scale was also assessed by comparing the means of the anxiety group and non-anxiety group using an independent-samples Table The internal consistency of the DFS-A was assessed using Cronbach\u2019s alpha coefficients and composite reliability (CR) coefficients. The DFS-A scale showed high internal consistency with a Cronbach's alpha\u2009=\u20090.93 and a composite reliability of 0.94. The Corrected\u2013item to total correlation coefficients varied from 0.45 to 0.82. Removing an item from the construct did not significantly affect the Cronbach's alpha which ranged between 0.93 and 0.94 . The scree plot of the Eigen values revealed a three-factor structure of the DFS-A scale: the first factor was related to fear of specific stimuli/procedures (item 14\u201320) and accounted for 25.56% of the scale\u2019s total variance, the second factor was associated with patterns of dental avoidance and anticipatory anxiety and accounted for 22.65% of the scale\u2019s total variance , and the third factor concerned physiological arousal during dental treatment and accounted for 16.22% of the scale\u2019s total variance (items 3\u20137). The factor loadings for each item are presented in Table The exploratory factor analysis of the DFS-A scale showed a KMO measure of 0.92 indicating adequate sampling adequacy and a highly significant Bartlett\u2019s Test of sphericity  using the Mann\u2013Whitney test. A statistically significant mean rank difference was found between the two groups with higher scores for fearful compared to the non-fearful group  indicating that DFS-A has good discriminant validity. The receiver operating curve (ROC) analysis was also conducted to evaluate the diagnostic validity of the DFS-A scale. The area under the curve (AUC) was 0.9  indicating a good diagnostic accuracy of the DFS-A Fig.\u00a0. A cut-oOf the total, 33.8% of the participants have reported having dental fear. Overall, previous scary and painful dental experience, a sensation of nausea during dental treatment, and dental anxiety were identified as predictors of dental fear Table .Table 5F.The present study was conducted to evaluate the psychometric properties of the Lebanese Arabic version of the DFS and to identify the factors associated with dental fear. Results showed evidence of good validity and reliability of the scale that can be used to assess dental fear in Arab-speaking individuals. Having previous scary and painful dental experiences, a sensation of nausea during dental treatment, and having dental anxiety were identified as predictors of dental fearThe results showed an adequate level of internal consistency with a Cronbach\u2019s alpha of 0.93 for DFS-A, indicating the homogeneity of the scale items. The internal consistency of the translated versions of the DFS was 0.95 for the Brazilian version , ranged Test\u2013retest reliability of the DFS-A, as assessed by the ICC (0.92) was high reflecting stability over time. This is also concordant with the results of the Oliviera study where the ICC results were 0.882 (95% CI 0.793\u20130.930) for avoidance, 0.874 (95% CI 0.810\u20130.917) for physiological arousal, and 0.897 (95% CI 0.829\u20130.937) for fears of specific stimuli/situations . This vaThe exploratory factor analysis revealed three factor structure of the DFS-A three constructs underlying dental fear namely \u201cfear of specific stimuli/procedures\u201d, \u201cpatterns of dental avoidance and anticipatory anxiety\u201d and \u201cphysiological arousal during dental treatment\u201d together explaining about 64% of the variance. Studies that inspected the factor structure of the DFS have shown inconclusive results, with some showing best fit for a three-factor structure , 15, 27,The discriminant validity of the DFS-A scale was confirmed using the ROC curve. The cut-off score of\u2009>\u200941 was identified as the best score to differentiate between patients with and without dental fear with a sensitivity of 90% and a specificity of 76%. The Brazilian DFS reported a different cut-off of 53 that met a sensitivity of 88.9% and a specificity of 92.5% . This coHaving previous scary and painful dental experiences, reporting having a sensation of nausea during dental treatment, and having dental anxiety were found to be risk factors for having dental fear. The previous scary and bad dental experiences can not be erased from the memories of a young brain, so attention should be made when treating young patients, since this will influence their behavior in all the upcoming dental appointments. Usually pedodontic dental practitioners are trained to deal with young dental patients and especially difficult patients by prescribing some antihistaminic drugs that alleviate the patient, using nitrous oxide, benzodiazepines and sometimes treating the patient under general anesthesia. Furthermore, nauseated patients have been proved to suffer from dental fear. This could be related to the difficulty in accepting some dental treatments, in the worst cases, all dental treatments, taking intra-oral radiographs and sometimes brushing their teeth. This disturbance while treating their teeth could lead to dental fear and consequently avoiding dental treatment which will worsen their dental health. Anti-emetic drugs can be prescribed to alleviate their nausea, one hour before the appointment. Finally, being anxious about dental treatment can lead to being afraid of dental treatment. In the literature, these two expressions were used interchangeably  althoughGiven that dental fear is an international problem that may lead to avoiding dental treatment, it is important to have a valid and reliable scale to identify patients with dental fear. The DFS-A scale could act as a screening tool to identify these patients thus appropriate strategies could be applied to alleviate their fears. Several methodological limitations can impact the results of this study such as the possibility of selection bias due to the convenience sampling strategy that was applied to recruit patients. It is worth noting that this translated Arabic-language form may not be suitable for other Arabic-speaking communities. The linguistic characteristics of other Arabic-speaking societies may impose some adjustments to the scale.This study was the first to explore the psychometric properties of the Arabic version of the DFS in Lebanon. Results revealed that the DFS-A has good validity and reliability. Therefore, it is considered a useful screening way for assessing dental fear among Lebanese adult patients in clinical settings. For this population, having previous bad scary and painful dental experience, reporting having a sensation of nausea during dental treatment, and diagnosed as having dental anxiety are risk factors for having dental fear."}
{"text": "Extracellular matrix (ECM) synthesis and metabolism abnormalities may influence the pelvic supporting system and lead to the occurrence and development of pelvic organ prolapse (POP). Genetic polymorphisms of such related genes have been increasingly studied. This study aims to explore the association between the single-nucleotide polymorphisms (SNPs) of genes encoding ECM processing enzymes , ECM degrading enzymes  and their tissue inhibitors of metalloproteinase (TIMPs), and POP.We conducted an association study including 48 women with POP at stages III and IV and 48 women without prolapse in Chinese groups. SNPs were identified using the target region sequencing technique. We performed Fisher\u2019s exact tests to assess the association between SNPs and POP in the unadjusted model and logistic regression analysis in the adjusted model, adjusting for delivery and pregnancy.TIMP2 SNP rs2277698 , ADAMTS13 SNP rs149586801 , and ADAMTS1 SNPs rs370850 and rs422803 , rs402007, rs428785, rs434857, and rs445784 , and POP in the adjusted model.There was a significant association between TIMP2, ADAMTS13, and ADAMTS1 might be candidate genes for POP. Our results provide preliminarily new evidence for future investigation of these genes in the pathophysiology of POP.The online version contains supplementary material available at 10.1007/s00192-021-04917-5. Pelvic organ prolapse (POP) is a common pelvic disorder among older women. It is characterized as the downward bulging of uterus, bladder, rectum, etc., urinary and fecal inconvenience, and sexual dysfunction, which negatively affect women\u2019s quality of life and social activities . The preCOL1A1) [COL3A1) [LAMC1) [MMPs) [The etiology of POP is complicated. Race/ethnicity, advancing age, obesity, higher parity, and menopause are known risk factors for POP. In addition, it is well demonstrated that POP has the characteristics of familial aggregation, as the risk of POP in women increases if their mothers or sisters have suffered from the disease, indicating a genetic contribution to POP . PreviouCOL1A1) , 5, coll[COL3A1) , laminin [LAMC1) , and mat) [MMPs) , which aMMP1, 3, and 9 genes, which could alter the expression of these genes and increase the risk of POP [MMP9 SNPs  and found rs17576 to be associated with POP. Wu et al. [MMP9 SNPs in an association study and found two SNPs, rs3918253 and rs3918256, associated with prolapse. Wang et al. [MMP10 SNP rs17435959 was associated with POP in a Chinese group of 91 cases and 172 controls. Besides these limited studies, other MMP members have not been studied in POP.MMPs are a family of multiple catabolic proteases involved in the degradation of collagen fibers and other components of ECM. Several previous studies have identified the existence of polymorphisms in the promoter regions of k of POP \u201311. Chenk of POP  detectedu et al.  further g et al.  demonstrTIMP2, which has been considered to be involved in connective tissue disorders and vascular diseases.The bioactivity of MMPs is regulated by tissue inhibitors of metalloproteinase (TIMPs), which are also crucial factors of ECM remodeling. Allen-Brady et al.  carried ADAMTSs, known as a disintegrin and metalloproteinase with thrombospondin motif, are a family of proteinases involved in procollagen processing. They can cleave the N-terminal of the peptide chains of procollagen molecules, which are the precursors of mature collagen . Thus, AMMP1, 3, 9, and 10, SNPs from other MMP members, TIMPs, and ADAMTSs would be associated with POP. Using a target region sequencing technique, we (1) confirmed the previous findings on MMPs and (2) investigated the susceptible loci of some of the other MMP, ADAMTS, and TIMP family genes for POP. We believe that these results might offer novel insights into the molecular mechanisms of POP development in Chinese women.As the fine balance between the synthesis and degradation of ECM components is essential to the integrity of the pelvic floor supportive structures, and given the previous limited findings on some of the MMP studies, and also given the fact that there has been no research on the association between the single-nucleotide polymorphisms (SNPs) of TIMPs/ADAMTSs and risks of POP, we have carried out a case-control association study in a group of Chinese women. We supposed that besides This was part of a case-control association study in which we recruited women from Peking Union Medical College Hospital (PUMCH) in Beijing from October 2016 to May 2017. Cases were POP patients diagnosed at Pelvic Organ Prolapse Quantification (POP-Q) stages III and IV from the Department of Gynecology and Obstetrics, and control women were diagnosed with no prolapse and have not suffered from prolapse surgery from the Physical Examination Center. All the participants signed the informed consents, and the study was approved by the Ethics Committee of PUMCH.We excluded women with the well-known connective tissue diseases, including Marfan syndromes, Ehlers-Danlos syndromes, rheumatoid arthritis or scleroderma, and women with neurological diseases including sclerosis or stroke. As the genetic variants vary between different race/ethnic groups, all the samples were limited to Chinese ancestry. Finally, we recruited 48 cases and 48 controls subjected to the next genotyping. Sociodemographic data and physical information were collected as summarized in our previous study .Genome DNA was extracted from the peripheral venous whole-blood samples using a Puregene Blood Kit . The target region sequencing approach was used to achieve a comprehensive assessment of the specific genes; 1\u20132\u00a0\u03bcg DNA of each blood sample was sequenced by Agilent Liquid Capture System  at Novogene  based on the Illumina HiSeq 4000 platform to provide a more than 200\u00d7 deep of sequencing.http://broadinstitute.github.io/picard) were used to sort the files and mark the duplicated reads. Samtools mpileup and bcftools were used to call variants, insertions, and deletions. The SNP quality control included SNPs with read depth\u2009>\u20094, mapping quality\u2009>\u200930, and the variant quality\u2009>\u200920. Variants with a minor allele frequency (MAF)\u2009>\u20095% remained. SNPs were assessed by PolyPhen-2, SIFT, MutationTaster, and CADD software, respectively, for the prediction of their functional effects. ANNOVAR was applied to provide the annotations of the position, type, and conservative prediction of the altered alleles and other information.The valid sequencing data landed in a BAM file by Burrows-Wheeler Aligner (BWA) software based on the reference genome (UCSC hg19). Samtools and Picard . The linkage disequilibrium (LD) graphs indicating r2 between SNPs were drawn by Haploview .Odds ratio (OR) and 95% confidence interval (CI) were shown for each of the variants with OR\u2009<\u20091 indicating a protective effect and OR\u2009>\u20091 indicating a risk effect . P\u2009<\u20090.0P\u2009=\u20090.663) and body mass index (BMI)  between case and control groups. However, the case group had approximately \u2265 1 delivery  and pregnancy  in contrast to the control group. In the case group, 41 women (85.42%) were diagnosed at POP-Q stage III and 7 women (14.58%) were at stage IV as previously described [We have described sample characteristics in our previous study. Briefly, we matched age  and degradation  in Chinese women. These genes are listed in Table P values < 0.1 in the unadjusted model and 16 SNPs with P values < 0.1 in the adjusted model that were considered to be significant or suggestively significant  after the adjustment, which was an intronic SNP. For other MMP members, we have identified two new MMP13 SNPs, rs3758853 and rs78356340, which were intronic SNPs, which showed a suggestively significant association with POP in the adjusted model   and a suggestively significant association with POP in the unadjusted model . TIMP3 SNP rs9862 showed a suggestively significant association with POP in the adjusted model . rs2277698 and rs9862 were synonymous SNPs that did not result in amino acid changes. TIMP4 SNP rs10433537 showed a suggestively significant association with POP in both the unadjusted and adjusted model , which was an intronic SNP . There was also a suggestively significant association between rs1055432 and POP in the adjusted model . rs1055432 was a synonymous SNP that did not lead to the amino acid alteration  in the adjusted model, which was an intronic SNP . They also showed a trend toward significance with POP in the unadjusted model . rs402007, rs428785, rs434857, and rs445784 were respectively significantly associated with POP in the adjusted model . rs402007 was in the 5\u2019UTR region of the ADAMTS1 gene. rs428785 was a missense SNP. rs434857 and rs445784 were synonymous SNPs that did not lead to amino acid changes. There was also a trend toward significance for the SNP rs436525 and POP in the adjusted model . rs436525 was also a synonymous SNPs without amino acid changes (Table ADAMTS1 gene were described. rs436525 was in good LD with six other SNPs (r2\u2009>\u20090.8), and rs370850, rs422803, rs402007, rs428785, rs434857, and rs445784 were in perfect LD between each other (r2\u2009=\u20091)  Fig. . The misMMP9 and 10 polymorphisms and further analyzed the association of some other MMP genes, TIMPs, and some ADAMTS genes with risks of POP for the first time. Besides MMP9 and 10, our study provided novel evidence that MMP13, TIMP2, 3, 4, and ADAMTS1, 13, 14 might be possible candidate genes for POP.POP is a common female pelvic disorder due to the decline of the pelvic floor supportive tissues. The imbalance of synthesis and degradation of collagen and other ECM components plays a role in the pathogenesis of POP. Such regulation of ECM stability mainly relied on MMPs, which can cleave collagen, elastin, and proteoglycans, etc., and their inhibitors TIMPs . ADAMTSsMMPs act on ECM breakdown in multiple physiological and pathological processes, including embryonic development, tissue remodeling, wound repair, angiogenesis, inflammatory processes, and tumor progression . AccordiMMP1, 3, and 9 in our target region sequencing approach, we did not verify our data in relation to the previous polymorphisms in the promoter regions of MMP1, 3, and 9. However, we compared our results to the previous MMP9 and 10 studies [MMP9 SNPs, rs3918242, rs17576, and rs2250889, and found that rs17576 G allele was a risk site for POP. The authors recruited 92 POP patients at POP-Q stage \u2265 II and 152 control women at stage 0\u2013I of Taiwanese women. Wu et al. [MMP9 SNPs and found rs3918253 and rs3918256 associated with POP in non-Hispanic white women in 239 cases with stages III and IV and 197 controls with stages 0\u2013I. In our study, we did not find the previously reported MMP9 SNPs rs3918253 and rs3918256 by Wu et al. [MMP9 SNPs rs3918242, rs2250889, rs3918278, rs2274755, rs17577, rs2236416, and rs3787268 by Wu et al. and Chen et al., we confirmed that these SNPs also showed no association with POP in our study. Besides validating the previous study, we additionally identified a MMP9 SNP rs3918254 that had a suggestively significant association with POP, which was an intronic SNP. Wang et al. [MMP10 SNP rs17435959 genotype G/C was distributed differentially between 91 women with POP at stage \u2265 II and 172 control women at stage 0-I of Chinese ancestry. However, we did not find a significant association in the MMP10 gene in our study. The inconsistency of these studies may partially be attributed to the differences in the recruitment criteria and the characteristics of the women, for example, the race, age, BMI, parity, etc., which were key influencing factors for POP, or in the detection methods.The role of MMPs in the development of POP has been primarily explored in their expression changes as well as their susceptible loci for POP. Previous analysis demonstrated that women with POP showed higher expression levels of MMP1 and MMP8 compared with the control women . The exp studies \u201314. Chen studies  conducteu et al.  additionu et al.  in signiu et al.  reportedu et al.  and our MMP9 and MMP10, we have sequenced multiple MMP genes and aimed to find the association between these genes and POP. However, we did not observe any positive associations among MMP1, 2, 3, 8, 10, and POP, respectively. Additionally, we have found two new MMP13 SNPs, rs3758853 and rs78356340, which showed borderline significant associations with POP and may have a risk effect for POP.Studies on other MMP polymorphisms have not been conducted in POP. In this study, besides TIMP1, 2, 3, and 4 genes for the first time. We found a significant association between a TIMP2 SNP rs2277698 and POP. Our data suggested that it may show a protective effect for POP development. We also reported a TIMP3 SNP rs9862 and a TIMP4 SNP rs10433537 that had a borderline association with POP, which should be further investigated in future research.There were few studies focusing on TIMP family genes and POP. TIMP2 expression level in the POP group was lower than that in the control group . TIMP3 aADAMTS1 SNPs rs370850, rs422803, rs402007, rs428785, rs434857, and rs445784 and POP, which may be risk factors related to POP, and a significant association between ADAMTS13 SNP rs149586801 and POP, which showed potential protective effects for this disease. We also found three additional SNPs, ADAMTS1 SNP rs436525, ADAMTS13 SNP rs1055432, and ADAMTS14 SNP rs4747097, which have suggestively significant associations with POP. Additionally, ADAMTS1 SNP rs428785 is a missense SNP and can lead to a substitution of alanine to proline, which might exhibit a benign and tolerant effect predicted by PolyPhen-2 and SIFT. Therefore, ADAMTS1, ADAMTS13, and ADAMTS14 could be novel candidate genes for POP, and this needs further validation.The ADAMTS protease family plays crucial roles in ECM remodeling and tissue morphogenesis as well as in inflammation and other physiological and pathological processes . This faMMP9 and 10, but also analyzed the association between MMP1, 2, 3, 8, and 13 and POP for the first time. Additionally, the polymorphisms of TIMP and some ADAMTS family genes have been first studied in POP, suggesting new susceptible SNPs. Third, we focused on unrelated Chinese women from mainland China in this candidate gene association study, choosing extreme symptoms of severe POP with POP-Q stages III and IV, tying to improve the detection of the potential variants.Our study had several strengths. As previous studies were few and focused on certain limited SNPs of a gene, we sequenced several MMP, TIMP, and ADAMTS family genes by target region sequencing technique to assess the entire genes with their coding regions. Second, we not only validated the data of the previously identified SNPs in MMP, TIMP, and ADAMTS polymorphisms for future investigation of the involvement of these candidate genes in the etiology of POP. The genetic contributions to POP remain poorly understood. Additional work needs to be done to provide further validation of POP predisposition variants in a variety of different populations to establish the role of these genes in the pathogenesis of prolapse [However, the main limitation of our study was the small sample size. Since our nationwide epidemiological survey in mainland China found that the prevalence of POP at stages III and IV was 2.04% , this maprolapse .MMP9 and 13, TIMP2, 3, and 4 and ADAMTS1, 13, and 14 genes in Chinese groups, preliminarily revealing the underlying mechanisms for the pathophysiology of this common disease.MMP, TIMP and ADAMTS family genes are crucial for ECM synthesis, modification, and metabolism. In this study, we provide initial evidence that the genetic variants in these genes may have an association with POP. We identified several susceptible SNPs in the Table S1All of the variants with a minor allele frequency of 5% (XLS 66 kb)"}
{"text": "NCDN gene encoding Neurochondrin (NCDN), NM_014284.3:c.1206G\u2009>\u2009A, p.(Trp402*). The variant was associated with a\u2009~\u200931% reduction in full-length protein levels in the patient\u2019s brain, suggesting that this mutation leads to NCDN haploinsufficiency. We examined the effects of NCDN haploinsufficiency on FUS and found that depleting primary cortical neurons of NCDN causes a reduction in the total number of FUS-positive cytoplasmic granules. Moreover, we found that these granules were significantly larger and more highly enriched with FUS. We then examined the effects of a loss of FUS function on NCDN in neurons and found that depleting cells of FUS leads to a decrease in NCDN protein and mRNA levels. Our study identifies the NCDN protein as a likely contributor of FTLD-FET pathophysiology. Moreover, we provide evidence for a negative feedback loop of toxicity between NCDN and FUS, where loss of NCDN alters FUS cytoplasmic dynamics, which in turn has an impact on NCDN expression.Frontotemporal dementia (FTD) is a heterogeneous clinical disorder characterized by progressive abnormalities in behavior, executive functions, personality, language and/or motricity. A neuropathological subtype of FTD, frontotemporal lobar degeneration (FTLD)-FET, is characterized by protein aggregates consisting of the RNA-binding protein fused in sarcoma (FUS). The cause of FTLD-FET is not well understood and there is a lack of genetic evidence to aid in the investigation of mechanisms of the disease. The goal of this study was to identify genetic variants contributing to FTLD-FET and to investigate their effects on FUS pathology. We performed whole-exome sequencing on a 50-year-old FTLD patient with ubiquitin and FUS-positive neuronal inclusions and unaffected parents, and identified a de novo postzygotic nonsense variant in the The online version contains supplementary material available at 10.1186/s40478-022-01314-x. Frontotemporal dementia (FTD) is a common form of dementia characterized by a progressive neuronal loss, primarily across the frontal and temporal lobes, leading to changes in executive functions, personality, abnormal behaviors and language impairments , 54. FTDFUS mutations have been identified in FTLD-FET patients or even in cases of ALS-FTLD .FTD shares clinical, genetic and pathogenic features with ALS , 52. ALS\u201310% ALS , 44, 82.\u201310% ALS , 44, 82,\u201310% ALS , 51, 66.nowledge , 80. In NCDN was not associated with any Mendelian disorder. Missense variants were however reported in patients with a neurodevelopmental phenotype with epilepsy. One family exhibited a missense homozygous variant, with unaffected heterozygous parents, while 3 unrelated patients showed missense de novo variants with a more severe phenotype [NCDN function. In addition, although there are no truncating NCDN variant predicted to trigger NMD in variant databases or deletions in controls, a few patients with a developmental disorder were reported with deletions encompassing NCDN and multiple other genes. However, it is unclear (i) whether NCDN plays a role in the developmental phenotype of the latter patients and (ii) whether the same patients as well as recently described patients with missense variants will eventually develop FTLD-FET. Here, the nonsense mutation was detected as a mosaic. We hypothesize that such an NMD-triggering variant, if carried in the germ line, might be lethal during in-utero development, which could explain absence from control databases and absence of association with developmental disorders.Until very recently, henotype . FunctioIn a non-Mendelian manner, NCDN has been implicated in several neurological disorders including epilepsy, depression, schizophrenia and spinal muscular atrophy (SMA) \u201385, 87. NCDN translation and whether NCDN:FUS:SMN are part of a converging pathway involved in maintaining synaptic homeostasis.NCDN is also linked with SMA, a degenerative muscular disease caused by a reduction in the survival motor neuron (SMN) full-length protein , 76. ThemGluR5\u2212/\u2212 neurons [A previous study showed that NCDN interacts with a subset of group 1 mGluRs, including mGluR5, where NCDN has been shown to promote mGluR5 cell surface expression and activation of mGluR5 through a direct interaction with this receptor . mGluR5  neurons . In resp neurons . Under c neurons . In our  neurons , 68, 77, neurons . NCDN variant in a FTLD-FET patient that results in haploinsufficiency of NCDN. We conclude that NCDN has an important function in regulating FUS granule dynamics, and that these changes cause a misregulation of NCDN expression. Based on the common biological functions of both NCDN and FUS, as well as their link with FTLD-FUS and ALS-FUS, there is an intriguing possibility that these proteins are part of a common regulatory pathway for the maintenance of synaptic homeostasis. Taken together, our findings suggest that disruption of this pathway would lead to neuronal defects and neurodegeneration.In summary, we have identified a rare de novoAdditional File 1: Supplementary tables and figures:Table S1. Exome sequencing data or DNA samples from multiple international cases with FTLD-FET. Fig. S1. Validation of secondary antibody specificity for immunocytochemistry studies. Fig. S2. Immunocytochemistry validation of NCDN knock-down in neurons. Fig. S3. Knock-down of FUS in N2a affects NCDN protein and mRNA levels. Fig. S4. Model for NCDN haploinsufficiency and FTD-FET. Supplementary References. Citations for Table S1."}
{"text": "Musculoskeletal ailments affect millions of people around the world and place a high burden on healthcare. Traditional treatment modalities are limited and do not address underlying pathologies. Mesenchymal stem cells (MSCs) have emerged as an exciting therapeutic alternative and Wharton\u2019s jelly-derived mesenchymal stem cells (WJSCs) are some of these. This review reports the clinical and functional outcomes of the applications of WJSCs in orthopedic surgery. A systematic review was conducted utilizing the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines. The studies that used culture-expanded, mesenchymal stem or stromal cells, MSCs and/or connective tissues procured from Wharton\u2019s jelly (WJ), from January 2010 to October 2021, were included. Conventional non-operative therapies and placebos were used as comparisons. Six studies that directly discussed WJSCs use in an animal model or the basic scientific testing using an injury model were identified. Five publications studied cartilage injury, three studied degenerative disc disease, one was related to osteoarthritis, and one was related to osteochondral defects. The results of these studies suggested the benefits of WJSCs in the management of these orthopedic pathologies. To adequately assess the safety and efficacy of WJSCs in orthopedic surgery, further randomized controlled clinical studies are necessary. Orthopedic musculoskeletal ailments involve inflammatory and/or degenerative conditions in muscles, tendons, ligaments, nerves, and bones. These conditions are estimated to affect one in four people in developed countries, thus representing a significant burden on healthcare . TraditiThe field of regenerative medicine has undergone a tremendous growth as of late, especially the field of orthopedic surgery . MesenchBMC and adipose-derived stem cells (ADSCs) are clinically available and have a long history of being used with robust clinical data, in comparison to other sources . HoweverWJ is an allogenic tissue comprised of connective tissue located within the umbilical cord. Wharton\u2019s jelly resists torsional and compressive stresses during fetal development levied upon the umbilical vessels. The primitive mesenchymal stem cells reside within the UC-derived WJ . These pWharton\u2019s jelly is easily accessible and available in comparison to autogenic tissues. The UC, and the Wharton\u2019s jelly within it, is an after-birth tissue, and is normally discarded after every birth, presenting ample opportunity for harvest . The easGiven the possible advantages of Wharton\u2019s jelly, the present systematic review evaluated the quality of the published evidence related to the safety and efficacy of WJSCs for orthopedic regenerative applications. The primary goal of this review is to document the clinical and functional outcomes of WJSCs for orthopedic, regenerative medicine applications. The secondary goal of this review is to identify the methodological characteristics associated with the application outcomes.Of the 20 publications describing the use of Wharton\u2019s jelly in regenerative medicine application for orthopedic surgery, only seven directly discussed human WJSCs are undergoing human or animal model testing, or basic scientific testing using an injury model. Five of the publications studied cartilage injury: three related to degenerative disc disease, one related to osteoporotic vertebral compression fractures, one related to osteoarthritis, and one related to osteochondral defect. Han et al. analyzed the effect of Wharton\u2019s jelly cells on degenerative nucleus pulposus cells isolated from a degenerative intervertebral disc. Wharton\u2019s jelly cells were co-cultured in vitro with nucleus pulposus cells for seven days with and without direct cell-to-cell contact. Gene expression was quantified using a polymerase chain reaction (PCR) analysis. Compared to a Wharton\u2019s jelly cell control and a degenerative nucleus pulposus cell control, the expression of type II collagen, aggrecan, and SOX-9 were significantly elevated for Wharton\u2019s jelly and the nucleus pulposus co-culture. The gene expression was at its highest with direct cell-to-cell contact using a ratio of 75:25 Wharton\u2019s jelly cells to nucleus pulposus cells. The polymerase chain reaction gene expression of the co-cultured Wharton\u2019s jelly cells and degenerative nucleus pulposus cells differed from each individual control. Human Wharton\u2019s jelly cells could be induced to differentiate toward nucleus pulposus-like cells when co-cultured with degenerative nucleus pulposus cells .Cheng et al. analyzed the effects of a single injection of Wharton\u2019s jelly-derived cells on acute spinal cord injury in a rat model. At day 28, the L3 transected rats that received the Wharton\u2019s jelly injection exhibited a statistically significant increase in motor function versus the rats that did not receive Wharton\u2019s jelly. The use of transmission electron microscopy in the Wharton\u2019s jelly group demonstrated considerably increased neurofilaments and microtubules at the injury site compared to the rats that did not receive Wharton\u2019s jelly. Additionally, the authors demonstrated an increase in the neural differentiation factor (NGF) expression and a decrease in the inflammatory marker interleukin-1\u03b2. Wharton\u2019s jelly cells injected after spinal cord injury in a rat model produced better functional clinical results .6 Wharton\u2019s jelly cells labeled with a viral vector were injected into L6-7. L5-6 was injected with saline, L4-5 served as the injured control, and L3-4 served as the uninjured control. Throughout the experiment, the disc height index and relative gray index were measured via a radiograph and magnetic resonance imaging (MRI), respectively. At 24 weeks, the intervertebral disc injected with Wharton\u2019s jelly cells exhibited a statistically significant slower progression of disc height loss than the injured control and saline injected control. The intervertebral disc injected with Wharton\u2019s jelly cells also exhibited a statistically significant and higher relative gray index than the injured control and saline injected control. At 20 weeks after injection, the discs were removed. Immunohistochemistry confirmed the presence of Wharton\u2019s jelly cells at 20 weeks [Yan Zhang et al. studied the effects of Wharton\u2019s jelly on degenerated nucleus pulposus in a canine model. The degeneration of L4-5, L5-6, and L6-7 were induced. Intervertebral discs were exposed through an anterolateral approach, and 14.5 \u00b1 2.7 mg of nucleus pulposus was aspirated from each disc. Four weeks after the procedure, 10Shim et al. presented the results of a randomized, open-label, phase I/IIa study examining the safety and effectiveness of managing osteoporotic vertebral compression fractures with WJSCs and teriparatide. Twenty subjects were followed for 12 months. All subjects received a daily subcutaneous injection of 20 mg teriparatide and 20 mg oral bazedoxifene daily for 6 months. The subjects in the experimental group underwent an injection of WJSCs intramedullarily on day 0 and intraveniously on day 7. Three subjects from the control group dropped out because of an adverse reaction to teriparatide. Four subjects in the experimental group experienced an adverse event. Three patients chose to drop out of the study: one secondary to a urinary tract infection shortly after WJSC injection, another secondary to a pulmonary embolus discovered 30 days after WJSC injection on chest CT, and the third secondary to a diagnosis of pancreatic cancer discovered on CT. The clinical outcome scores exhibited statistically significant improvements in VAS, ODI, and SF-36 after 12 months versus the baseline. The pain score in the VAS, as well as the ODI and SF-36 scores, for the experimental group were statistically significant when compared to the control group at 12 months. Bone turnover markers measured did not demonstrate a statistical significance between the control and experimental groups. Bone mineral density improved significantly for both the control and experimental groups, but there was no statistically significant difference between the two groups. CT analysis demonstrated an improved microarchitecture for the experimental group compared to the control group at 12 months .Shalaby et al. examined the effect of Wharton\u2019s jelly cells added to a nerve conduit on the functional recovery of a 10 mm sciatic nerve deficit. At 12 weeks, the Functional Recovery Index was \u22125.2 \u00b1 2.1 in the uninjured control group, \u221255.3 \u00b1 12.3 in the injured control group, \u221223.8 \u00b1 5.6 in the injured group treated with nerve conduit alone, and \u22129.8 \u00b1 2.5 in the injured group treated with nerve conduit and Wharton\u2019s jelly cells. There was a greater significant improvement in the Wharton\u2019s jelly group. For the pin prick-functional analysis, there was a statistically significant improvement in the treated groups, but no significance for the nerve conduit group and the group treated with Wharton\u2019s jelly. Histologic analysis of the surgically treated nerve exhibited more normally appearing nerve fibers and axons with thin a myelin sheath than nerve conduit and control groups. The real-time PCR showed a significant increase in innetrin-1, ninjurin, the glial cell-line-derived neurotrophic factor (GDNF), the brain-derived neurotrophic factor (BDNF), the vascular endothelin growth factor (VEGF), and angiopoitin-1 gene expression versus the other three groups .Sofia et al. conducted a basic science study observing the matrix metalloproteinase-13 (MMP-13) gene expression of synoviocytes isolated prior to a total knee arthroplasty versus those cells combined with Wharton\u2019s jelly cells. This study analyzed the gene expression of two pro-inflammatory markers: MMP-13 and RELA. The addition of Wharton\u2019s jelly to synoviocytes isolated from human knees with grade IV osteoarthritis reduced the expression of MMP-13 and RELA. The findings were statistically significant compared to the synoviocyte control .Zhang and colleagues seeded Wharton\u2019s jelly cells to an acellular cartilage extracellular matrix scaffold. The seeded scaffold was then tested against a microfracture for the restoration of a 6.5 mm diameter, femoral condyle osteochondral defect in a caprine model. At nine months, the Wharton\u2019s jelly group demonstrated more abundant glycosaminoglycans and type II collagen with highly organized fibers compared to that of the microfracture group. The modulus of elasticity was 2.9 \u00b1 9 MPa for the Wharton\u2019s jelly group compared to 2.2 \u00b1 5 MPa for the microfracture group. An MRI analysis of the treated osteochondral defect demonstrated an appearance that was similar to the native articular cartilage than the microfracture group. Of note, two knees of goats from the microfracture group were deemed to have a meniscus tear at the time of euthanasia .The Wharton\u2019s jelly extracellular matrix is partly comprised of glycosaminoglycans and collagen, similar to cartilage ,30,31. TThe present study evaluated the quality of published evidence regarding the safety and efficacy of WJSCs for orthopedic regenerative medicine applications. To date, there is only one scientific publication in the literature using culture-expanded WJSCs for orthopedic applications in clinical practice. Shim et al. presented the results from a randomized, open-label, phase I/IIa study examining the safety and effectiveness of managing osteoporotic vertebral compression fractures with WJSCs and teriparatide. The study reported a statistically significant improvement in the pain and functional scores for the experimental group compared to the control. The bone mineral density and bone turnover markers did not significantly differ from the control and experimental subjects. Of note, that study included a large percentage of participants who did not complete the 12-month trial. More clinical research must be completed to determine the safety and efficacy of WJSCs in a human compression fracture model.7 WJSCs for moderate hip, knee or glenohumeral osteoarthritis. One hundred subjects will receive an injection of WJSCs every three months for one year. The subjects will be followed throughout the study and 24 months post-administration of last injection, studying clinical responses, inflammatory markers, and magnetic resonance imaging. The clinical trial is still in the recruitment phase [There is another current phase I/II clinical trial at the Medical University of Warsaw evaluating the efficacy of an intra-articular injection of 10nt phase .Aging negatively affects stem cells. This makes stem cells cultured from the umbilical cord or placenta advantageous over stem cells cultured from adipose tissue, bone marrow, or other autogenic adult cell sources. Birth-derived products have shown potential for use in the orthopedic sector. Multiple companies now have a flowable placental allograft formulation that is under consideration for approval by the US FDA as Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/P\u2019s) for eventual clinical use. While there is growing interest from companies, consumers, and healthcare providers, a lack of insurance reimbursement and limited safety and efficacy studies limit the development and use of these products. Additionally, to our knowledge the current commercial products on the market do not offer living cells .Similar to placental tissue, Wharton\u2019s jelly is obtained after birth. This alleviates the controversial aspects of harvesting embryonic cells. Unique to WJSCs, the process used to extract WJSCs can be performed without the use of digestive enzymes, cryoprotectants, or the in vitro expansion of cells . All revIn the study by Yan Zhang et al., human Wharton\u2019s jelly-derived cells were injected into the intervertebral disc in a canine model. Immunohistochemistry confirmed the presence of Wharton\u2019s jelly cells at 20 weeks . SimilarThe methodology of this systematic review followed the protocol that was previously published and registered on the international prospective register of systematic reviews (PROSPERO), registration number CRD42020182487 . The varThe literature describing the use of WJSCs for musculoskeletal regenerative medicine is limited. However, the safety profile and the effectiveness in managing musculoskeletal ailments described in this review are encouraging. The benefits of WJSCs for cartilage restoration seem to be the most promising, given the similarities between chondrocytes and Wharton\u2019s jelly cells and the cellular matrix of cartilage and Wharton\u2019s jelly. Further well-designed and appropriately powered, prospective, non-randomized and randomized studies evaluating the safety and efficacy of WJSCs in a human model are warranted to justify their clinical use."}
{"text": "Patients\u2019 spontaneous speech can act as a biomarker for identifying pathological entities, such as mental illness. Despite this potential, audio recording patients\u2019 spontaneous speech is not part of clinical workflows, and health care organizations often do not have dedicated policies regarding the audio recording of clinical encounters. No previous studies have investigated the best practical approach for integrating audio recording of patient-clinician encounters into clinical workflows, particularly in the home health care (HHC) setting.This study aimed to evaluate the functionality and usability of several audio-recording devices for the audio recording of patient-nurse verbal communications in the HHC settings and elicit HHC stakeholder (patients and nurses) perspectives about the facilitators of and barriers to integrating audio recordings into clinical workflows.This study was conducted at a large urban HHC agency located in New York, United States. We evaluated the usability and functionality of 7 audio-recording devices in a laboratory (controlled) setting. A total of 3 devices\u2014Saramonic Blink500, Sony ICD-TX6, and Black Vox 365\u2014were further evaluated in a clinical setting (patients\u2019 homes) by HHC nurses who completed the System Usability Scale questionnaire and participated in a short, structured interview to elicit feedback about each device. We also evaluated the accuracy of the automatic transcription of audio-recorded encounters for the 3 devices using the Amazon Web Service Transcribe. Word error rate was used to measure the accuracy of automated speech transcription. To understand the facilitators of and barriers to integrating audio recording of encounters into clinical workflows, we conducted semistructured interviews with 3 HHC nurses and 10 HHC patients. Thematic analysis was used to analyze the transcribed interviews.Saramonic Blink500 received the best overall evaluation score. The System Usability Scale score and word error rate for Saramonic Blink500 were 65% and 26%, respectively, and nurses found it easier to approach patients using this device than with the other 2 devices. Overall, patients found the process of audio recording to be satisfactory and convenient, with minimal impact on their communication with nurses. Although, in general, nurses also found the process easy to learn and satisfactory, they suggested that the audio recording of HHC encounters can affect their communication patterns. In addition, nurses were not aware of the potential to use audio-recorded encounters to improve health care services. Nurses also indicated that they would need to involve their managers to determine how audio recordings could be integrated into their clinical workflows and for any ongoing use of audio recordings during patient care management.This study established the feasibility of audio recording HHC patient-nurse encounters. Training HHC nurses about the importance of the audio-recording process and the support of clinical managers are essential factors for successful implementation. Patients\u2019 spoken language provides a window into a wide range of pathological entities, including pulmonary hypertension , respiraDespite the promising findings of these studies, the audio recording of patients\u2019 spontaneous speech is not a part of routine clinical workflows. Most studies on patients\u2019 speech were cross-sectional and conducted in laboratory (controlled) settings, where patients were instructed to follow specific diagnostic and screening tests  without having any interaction with clinicians -8. TheseHealth care stakeholders\u2019 perspectives toward audio recording patient-clinician verbal communication have been discussed in previous studies. A recent study that investigated policies for audio recording patient-clinician encounters in the 49 largest health care systems in the United States found that despite physicians\u2019 willingness to audio record patient-clinician encounters, none of the health care systems had a dedicated policy or guidance for integrating audio recordings of patient-clinician encounters into clinical workflow  exploredThere is a growing consensus on the usability of home health care (HHC) technology as a significant factor affecting the use of technology in the HHC settings. HHC is a health care setting where services are provided by skilled practitioners (often registered nurses) to patients in their homes . HHC patAlthough some studies have reported on audio recording of clinical encounters mainly for patients\u2019 personal use  ,18, few To address the gaps in the literature, this study aimed to (1) evaluate the functionality and usability of several audio-recording devices for audio recording patient-nurse verbal communication in the HHC setting and (2) elicit the perspectives of HHC stakeholders (patients and nurses) about the facilitators of and barriers to integrating audio recording into clinical workflows.This descriptive feasibility study was conducted at the largest not-for-profit home health agency in the United States. The agency has approximately 10,600 staff, including 1470 nurses and >6500 home health aides. In 2019, the agency served >106,000 unique patients across >1.08 million clinical visits. A summary of patients\u2019 demographic information in 2019 showed that most of the Visiting Nurse Service of New York (VNSNY) patients were aged >65 years, predominantly women , and almost half were African American or Hispanic . This study was approved by the Visiting Nurse Service of New York Institutional Review Board (reference #E20-003).In the first phase of the study, we created a list of criteria for selecting the audio-recording devices. The criteria included the portability of the device, wearable features, functionality of the device (memory size and battery life), and voice activation features. We reviewed the features of >50 audio-recording devices from different web-based sellers, such as Amazon, BestBuy, and SpyCenter. We selected 7 devices that met the criteria for the audio-recording device for the study. We evaluated the 7 devices for audio recording HHC patient-nurse verbal communication. All the devices were portable, with relatively simple operation, and could be used easily for recording verbal communication without disrupting the participants\u2019 movements during communication. The devices used were Black Vox 365, SOTA Surveillance-USR500, INSTAMIC PRO, Sony ICD-TX6, Mini Wristband Voice Activated Recorder, Apple Watch, and Saramonic Blink500 Pro B2. Transcription Accuracy section for evaluation metrics of automated voice activation features). The SOTA Surveillance-USR500, INSTAMIC PRO, Mini Wristband Voice Activated Recorder, and Apple Watch were excluded from further evaluation in the clinical settings because of their low battery life; low memory capacity; no indicators for recording (on or off), battery, and memory status; and low accuracy of automatic transcription of recorded communications. A total of 3 recorders, Black Vox 365 (further referred to as Vox), Sony ICD-TX6 (further referred to as Sony), and Saramonic Blink500 Pro B2 Pro (further referred to as Saramonic), were included for further exploration in HHC because of the relatively high accuracy of automatic transcription, easy operation, good battery life, and memory capacity.In the first phase of evaluation, 2 study investigators (MZ and SV) used the 7 devices to audio record verbal communications between themselves in a controlled environment, which resembled the verbal communication between patients and nurses in HHC settings. The investigators evaluated the devices using 8 criteria: memory size; battery life; indicators for memory size, battery life, or recording status; automated voice activation feature; ease of device attachment to clothing; and accuracy of automatic transcription of recorded verbal communications as an indicator of voice quality , which can facilitate the process of separating the patient\u2019s voice from the nurse\u2019s voice for analysis purposes. By contrast, Sony and Vox embedded microphones with single-channel features. In contrast with Vox, Sony and Saramonic could be easily attached to the patients\u2019 and nurses\u2019 clothes and included indicators for recording status (on or off). Vox had a voice activation feature that was not available in Saramonic and Sony.Next, we collaborated with 2 HHC nurses who used the 3 selected devices to audio record their verbal communication with patients during HHC encounters. With HHC organizational support of the study and after institutional review board approval, we approached nurses through email and advertising at the VNSNY site. Interested nurses provided written consent. The consent form included information about the aim of the study and its potential risks and benefits. A research assistant (RA) trained the nurses to use the audio-recording devices.Several different strategies were used to engage patient participants. The first strategy involved nurses providing flyers to patients with a brief description of the study. Nurses provided the RA with the name and contact information of patients who expressed interest. The second strategy was that after a waiver of authorization was granted, the RA reviewed the nurse\u2019s schedule in the EHR to identify patients on the nurse\u2019s caseload. The RA contacted the potential patient participants by phone and described the study\u2019s aim, potential risks, and benefits. For those who provided verbal consent, the RA mailed the consent form to the patients for their reference. Both nurse and patient participants received a gift card as a token of appreciation.The participating nurses audio recorded their routine HHC encounters with consented patients. Audio-recorded encounters were uploaded to a secure server. Nurses also completed the System Usability Scale (SUS) questionnaires  for eachThe quality of audio-recorded communications affects the transcription accuracy of a specific automatic speech recognition system such as the Amazon Web Service General Transcribe System (AWS-GTS) . For exaWe evaluated 2 components of transcription quality, word error rate (WER) and speaker identification accuracy, using the steps described in Step 1For each device , we randomly selected 3 audio-recorded home health care encounters.Step 2One of the investigators transcribed the audio files manually and assigned each utterance  to an appropriate speaker (patient or nurse). Manual transcription was reviewed by a second investigator to ensure the quality of the transcription.Step 3All audio files were transcribed using the Amazon Web Service General Transcribe System application programming interface. The application programming interface returns the transcriptions as JSON files, including the start and end times of each transcribed word and the assigned speaker  to each word. The transcribed words were joined to form an utterance using the type of speaker and converted to a Microsoft Excel sheet. We define an \u201cutterance\u201d as a continuous block of the uninterrupted speech of a single speaker.Step 4The quality of automatic transcriptions at the utterance level was compared with manual transcription and measured using the word error rate (WER). WER is a common metric for measuring the performance of speech recognition systems. It is computed based on the number of substitutions, insertions, and deletions that occur in a sequence of recognized words using a speech recognition system. The WER score starts from 0 (indicating no error in transcription) and can reach any score >1 depending on the length (number of words) in the utterance or document. For comparison, the average WER for human transcriptions is 0.04 (4%). Our earlier preliminary study for measuring the quality of transcription of an open-source automatic speech recognition system, Wav2Vec, on a subset of audio-recorded patient-nurse encounters provided a WER of 0.98 (98%). Wav2Vec is an unsupervised pretraining for speech recognition that learns representations of raw audio and was developed by the Facebook Company .Second, we used the speaker identification feature of AWS-GTS to measure the accuracy of the automatic transcription of audio-recorded patient-nurse verbal communication by the devices. We expected that multiple-channel audio-recording devices  would provide higher accuracy for speaker identification than that provided by single-channel devices . To measure the accuracy of speaker identification, we used the following steps:Step 1: All manually transcribed audio files with assigned speakers at the utterance level (patient or nurse) were tokenized into words, and each word was linked to the assigned speaker.Step 2: The words of each manually transcribed utterance were mapped to the corresponding utterances and words provided by AWS-GTS.Step 3: We computed the percentage of words with accurate speaker identification with references to the total number of transcribed words in each audio file.To understand the facilitators and barriers to the integration of audio recording of patient-nurse encounters into the clinical workflow, we conducted semistructured interviews with patients and nurses. The questions for nurses mainly covered their experience, concerns , potential benefits of recording (for both patients and clinicians), and their overall attitude toward the integration of audio-recording processing into HHC clinical workflows. The questions for patients covered their motivation to participate in the study, their concerns, and their attitudes toward audio recording their conversations with nurses. All the research questions were reviewed and discussed by the research team, 2 nurses with expertise in HHC services, a patient representative , and 2 health informaticians. The study team ensured that the topics of the semistructured interviews would lead to the discovery of major facilitators and barriers to the development of a practical approach for audio recording patient-nurse encounters in a clinical setting. The questions for the semistructured interviews are presented in A total of 5 nurses audio recorded their communication with patients. However, during the study period, 40% (2/5) of nurses left the participating agency and were therefore not available to participate in the interviews. The nurses participated in audio-recorded encounters with 45 patients in this ongoing study. After securing additional consent, we conducted interviews with the remaining 60% (3/5) of nurses.To reduce the likelihood of selection bias in creating a sample of HHC patients for the interviews, we used a stratified sampling technique. Using this technique, we stratified the pool of patients who participated in the audio recording of patient-nurse verbal communication (45 patients) based on the study nurse who recorded the encounter. Next, from each group of patients, the RA randomly contacted 2 patients for interviews. If the patient agreed to participate, the RA consented the patient for the study. This process was repeated until data saturation was achieved by interviewing a sample of 22% (10/45) of HHC patients.To address the patient\u2019s potential concern about privacy and confidentiality of the information collected during the interviews and ensure that the patient expressed their unbiased perspectives, our RA consented the patients by explaining our commitment and strategies to protect the patients\u2019 privacy and confidentiality. In addition, patients were informed that they could withdraw from the study  without any consequences and had the freedom not to answer any questions.In addition, our RA, who conducted the interviews, was well trained for effective communication  with VNSNY\u2019s patient population and had extensive experience in interviewing HHC patients for different qualitative studies. Gift cards were offered to both nurses and patients for participating in the qualitative interviews.The team created an initial codebook to summarize the open-ended interviews. Next, we used a thematic analysis approach for the systematic coding of the interviews. Thematic analysis is a qualitative descriptive approach for identifying, analyzing, and reporting themes within data -25. The Sony had the highest SUS score compared with Vox and Saramonic. Although the Saramonic device had a slightly lower SUS score than the SUS score of the Sony device, nurses found Saramonic easier in terms of approaching HHC patients for permission to audio record the verbal communication because of the appearance of the device and flexibility in attaching the microphone to the patient\u2019s clothes. In addition, the quality of audio-recorded communication using Saramonic was higher than that of the other 2 devices when measured using the WER of automatic transcription and accuracy of speaker identification provided by AWS-GTS. As expected, the desirable usability feature of Vox was automatic voice-activated recording. However, this feature might compromise the patient\u2019s or nurse\u2019s privacy if the nurse forgets to pick up the device from the patient\u2019s home or if the nurse forgets to turn off the device, which in turn would start recording unrelated conversations. This feature was not available for Sony or Saramonic. Overall, we found that Saramonic is the most appropriate device for recording patient-nurse encounters with the highest SUS score and accuracy of speaker identification, as shown in We investigated the perspectives of HHC stakeholders (patients and nurses) toward integrating the audio-recording of patient-nurse verbal communications into the clinical workflow. This study is the first to develop a practical approach for audio recording HHC patient-nurse verbal communication and evaluate the feasibility of integrating this approach into the clinical workflow. We showed that the type of recording device can have a differential effect on the accuracy of downstream tasks built on audio-recorded patient-nurse encounters, such as automatic transcription and provided speaker identification. In addition, our results suggest that HHC stakeholders\u2019 attitudes toward the recording process are a crucial factor affecting the successful integration of the audio recording of patient-nurse verbal communications into the clinical workflow.Selecting an appropriate recording device is of great importance to designing and implementing an effective approach for audio recording clinical encounters in HHC settings. Some devices are lightweight, can be easily attached to the participants\u2019 clothes, and have a very simple operation mechanism with one on or off button. However, these devices often have functionality issues, such as short battery life or limited memory size, which reduce their usability by increasing the number of clinicians or administrative staff. The INSTAMIC PRO device is an example of such a device with high ease of use but low functionality because of its small memory capacity. In contrast, some devices are easy to use with good functionality; however, because of their size or weight, they cannot be easily attached to a patient\u2019s or clinician\u2019s clothes. Vox is an example of a device that needs to be set on a flat surface  during the process of recording. Although this solution is practical for recording patient-clinician encounters where no movement is required during communication, it is not an appropriate solution in HHC settings where there is a constant required movement of the patient or nurse for physical examination or treatment. The patient\u2019s or nurse\u2019s movement will change their position with reference to the device location and, therefore, would affect the quality of audio-recorded communication, in turn affecting downstream tasks built on the audio data .In addition, the quality of the audio-recorded communication by devices substantially affects the quality of automatic transcription and speaker identification provided by an automatic speech recognition system. This is particularly important when the goal of the study is to develop an automatic analytic pipeline for processing and modeling patient-nurse verbal communication to develop a risk identification or diagnostic algorithm . Regarding the possibility of background noise in HHC settings, which comes from different sources such as television, air conditioner, or a caregiver\u2019s speaking, it is important to set the microphone as close to the patient\u2019s and nurse\u2019s mouths to reduce the possibility of background noise captured by the device\u2019s microphone. An option for reducing background noise is to use a unidirectional (cardioid) overhead microphone; however, as this study was conducted during the COVID-19 pandemic and the study participants were recognized as being at high risk for COVID-19, we avoided any devices that touched the patient\u2019s face. Unidirectional microphones pick up audio  from only the front compared with omnidirectional microphones that pick up audio from all directions. Another reason that convinced us not to use overhead microphones was the risk of patient discomfort during the recording procedure when the microphone touches the patient\u2019s face.Another important feature of audio-recording devices is the number of channels with the ability to separate audio tracks for each individual participating in the communication. Devices with this type of feature usually have multiple microphones that are used by several individual speakers. The voice captured by each microphone is transmitted to an individual channel. This feature is important for downstream tasks, especially the differentiation of the patient\u2019s voice from the nurse\u2019s voice. Among all the devices evaluated in this study, the Saramonic device was the only device that included this feature; consequently, it had a higher score for speaker identification when it was measured using AWS-GTS. Overall, because of the better quality of automatic transcription and speaker identification (measured using AWS-GTS) of verbal communications recorded by this device and the high score for usability, we selected this device for audio recording further encounters beyond the pilot assessment.Understanding HHC patients\u2019 and nurses\u2019 perspectives toward audio recording is a key determinant of the successful integration of the audio recording of patient-nurse verbal communication into the HHC clinical workflow. Overall, patients found the process of audio recording to be satisfactory and convenient, with minimal impact on their communication with nurses. Patients expressed that they were able to freely share their concerns and health care issues with nurses, and they even forgot about the presence of audio devices soon after the visit started. This expression implies that the Hawthorne effect on patients\u2019 communication patterns with nurses was minimal. It also shows the practicality of the recording procedure designed for this study.The Hawthorne effect refers to a study participant\u2019s reactivity, in which the participant changes an aspect of their behavior in response to their awareness of being observed by the study\u2019s investigator . At the formal rather than personal. Therefore, we may conclude that the presence of a recording device can introduce a Hawthorne effect on nurses\u2019 communication with patients. Nurses expressed concern about sharing audio-recorded encounters with supervisors for evaluation purposes. Owing to this concern, we experienced difficulties in recruiting nurses to audio record their communication with patients; however, after educating nurses about protecting their privacy and confidentiality, 5 nurses agreed to participate in the study. Nurses were not very optimistic about the usability of integrating audio recording in clinical workflows as they perceived limited benefits in audio recording the HHC encounters. This is contrary to the findings of numerous studies [Similar to patients, nurses found the audio-recording procedure satisfactory and easy to learn. However, in contrast to patients, nurses expressed that audio recording of HHC encounters can affect their practice and communication patterns in the HHC settings. Some nurses suggested that their communication with patients might become more  studies -8 showinThe findings of this study should be considered in light of several limitations. First, although the audio-recording devices selected for this study included a wide range of useful and convenient features for audio recording HHC patient-nurse encounters, they may not represent all features of existing devices in the market. For example, future studies may investigate the usability and functionality of Amazon Alexa (a device that was developed by the Amazon company) for audio recording patient-nurse encounters, the possibility of connecting to a secure server for storing the audio-recorded data, and the potential risks to patient privacy and confidentiality. Second, we quantified the quality of audio-recorded communication by measuring the accuracy of the automated transcription provided by AWS-GTS. Although the quality of audio-recorded data is correlated with the accuracy of automated transcription, it may not provide comprehensive insights into the quality of audio recorded by a device. Future studies may investigate other measures such as the sensitivity of the device for filtering background noise in noisy clinical settings to better evaluate the quality of the audio-recorded voice by the device. Third, as this study was conducted during the COVID-19 pandemic, recruitment and retention of HHC nurses were challenging. This was mostly because of nurses\u2019 heavy workload, and precautions needed to be taken to reduce the risk of COVID-19 transmission in the HHC setting. In addition, because of the COVID-19 pandemic, it was challenging for the research team to reach out to all nurses with a potential interest in participating in this study. Overall, we were able to recruit 5 nurses for the study of audio recording patient-nurse verbal communication conducted at VNSNY. Of the 5 nurses, 2 (40%) nurses left the VNSNY during the study. Hence, unfortunately, we could not reach the 2 nurses to solicit their perspectives. Although a sample size of 3 nurses may not provide sufficient data to achieve data saturation, the 3 nurses who participated in the interviews had extensive experience in HHC services, HHC workflow, and working with a racially diverse patient population in VNSNY. Therefore, they were able to provide a valuable evaluation of the facilitators and barriers to the pipeline designed for audio recording and its integration into the HHC workflow. Aggregation of the 3 nurses\u2019 perspectives sheds light on the facilitators and barriers to a large extent and was very informative for HHC managers and policy makers. For example, the manager of the VNSNY research center informed us that they were willing to address some of the barriers  to encourage more HHC nurses to participate in similar studies in the future. Currently, we are actively recruiting more nurses for this study, which will enable us to provide a better picture of nurses\u2019 opinions toward this process in our future report. Fourth, the racially diverse sample size of patients  who participated in this study was achieved through an iterative process and data saturation of thematic analysis of interview findings, which represents the patient population at the study agency to a large extent. However, the findings regarding patients\u2019 opinions may not provide a deep insight into the attitudes of ethnic minority patients or those with complex clinical conditions. Finally, this study was conducted at one agency, the largest HHC organization in the United States. However, there might be differences in the types of health care services, communication, and practice patterns across HHC. Therefore, the overall findings of this study may not be representative of all HHC settings.To develop an effective practical approach for integrating audio recording of patient-nurse verbal communication in HHC settings, it is essential to select an audio-recording device with high functionality and usability. Training nurses and clinical managers on the importance of audio-recorded verbal communication can encourage them to support the process of integration.In addition, training can reduce the potential concerns of nurses about protecting their privacy and confidentiality during the recording process."}
{"text": "The simulation results revealed that 59% of the catchment does not contain or benefit from flood-mitigating land cover features. Adding 0.6 km2 (4% of the catchment) of green roofs alongside major stormwater flow paths resulted in a nearly three-fold decrease (11%) in the unmitigated flooding area. These results suggest that green roofs could help manage stormwater and mitigate flooding in the densely built areas of the catchment. Using ecosystem service assessment tools, like Nature Braid, can inform the design of more regenerative and resilient urban green infrastructure networks that help mitigate climate change impacts on urban residents.Many cities are vulnerable to flooding due to their high proportion of impervious surfaces and lack of vegetated land cover. This vulnerability will often be exacerbated by changing rainfall and storm patterns due to climate change. Using the principles of urban biomimicry, this study aims to show an ecosystem service-based approach to designing an urban green infrastructure network for stormwater management in densely built areas that more closely emulates natural hydrology processes. Nature Braid (next-generation LUCI) is an ecosystem services assessment tool that was used to simulate flood mitigation ecosystem services in a 13.7 km As population growth and urbanisation drive the expansion of urban environments ,2, the pHabitat provides the foundational abiotic and biotic resources, such as soil and plants, that support flood mitigation ecosystem services, such as rainfall absorption, evapotranspiration, and disturbance prevention . TypicalSpatial multi-criteria analyses, such as the ones conducted by Venter et al.  and LangPredicting and managing flooding risk will be critical to the well-being and resilience of urban residents as climate change progresses. Despite a large field of literature on the benefits of green infrastructures, like green roofs, there remains a reliance on grey stormwater management infrastructure to manage flooding in urban environments. Better design and simulation tools that help optimize the design of urban green infrastructure networks and quantify their benefits could help increase the uptake and implementation of green infrastructures by local governments and landowners, thereby helping cities emulate ecosystem processes and functions more effectively. Using Wellington, New Zealand, as a case study, this research aims to show how an ecosystem service assessment tool, Nature Braid, can be used to guide the design of an urban green roof network to increase flood mitigation ecosystem services across a city. Nature Braid is one of the ecosystem service tools most suitable to Oceanic sites  and is c2 of green space per capita in central Wellington, which is higher than the minimum 9 m2 recommended by the World Health Organization [Located in the South Pacific on the North Island of Aotearoa New Zealand, Wellington is a temperate coastal city with a population of approximately 203,000 . Wellingnization . Howevernization .2)  , particuNature Braid, the next generation of the Land Utilisation and Capability Indicator (LUCI), is a GIS-based framework and accompanying embedded tools that simulate the impacts of land cover and management on different ecosystem services and allows the user to compare where synergies and trade-offs occur . Nature Nature Braid requires a set of input data, including digital elevation, land cover, soil, and climate data . The WelThe outputs generated from pre-processing were then used in the flood mitigation ecosystem service tool. This tool uses soil permeability and land cover to generate maps of features that help mitigate flooding and high stream flow and where overland stormwater flow accumulates . Feature2 (4% of the catchment) of green roof area was added to the base land cover file . Nature Braid is able to represent overland and subsurface flow based on topography, land cover, and soil, but does not yet include subsurface flow due to grey infrastructures, such as sewers and stormwater drains. However, its output remains useful  as it identifies where stormwater flows and flooding most need to be managed, whether that be through green, grey, or hybrid infrastructures. The average water flow paths highlight areas where overland flows are most significant and where more mitigation is needed to slow their flow. These flow paths were used to guide the design of a green roof network that could help manage stormwater in the catchment . The buiver file . The lan2 or 4% of mitigating features area (green roofs) resulted in a nearly three-fold decrease (1.49 km2 or 11%) in the non-mitigated flood area  in non-mitigated flood areas in the catchment, demonstrating the positive impacts they can have for flood mitigation beyond their individual sites. While these are promising results, there is still room to improve the design of the green roof network. As highlighted by area A in The results of the Nature Braid simulation demonstrate that the proposed green roof network can aid in stormwater management in a densely built urban catchment. Though previous research has evidenced the stormwater management benefits of green roofs on a single building ,21,22, tWhile the Nature Braid tool can estimate the supply and spatial extent of ecosystem services in a given area, some important limitations must be mentioned. Gaps or inaccuracies in the base or input data can result in potential errors while running the tools or in the outputs produced. The LiDAR information for buildings was unavailable, and though a workaround was achieved using building footprints and heights, the DEM input could be improved by filling these gaps. This will be particularly important for refining the building selection for green roof networks, as roof shape and slope will determine their suitability for green roofs. Many other factors will also impact buildings\u2019 suitability for green roofs, such as structural capacity, building code restrictions, and costs. These are beyond the scope of this analysis but could be done in future research to enhance the feasibility of the proposed green roof network. The information to supplement or amend gaps in the soil data was limited. The majority of the catchment was classified as the soil type \u2018town\u2019 with no differentiation between the soil classification of densely built areas and significant green spaces . More data on the soil types and permeabilities of the green spaces in the study area, as well as determining an appropriate soil classification for green roofs, would help improve the accuracy of the Nature Braid results and other similar biophysical and geospatial analyses. Though the primary focus of this study was to determine the amount and location of green roofs needed to reduce flooding in the study area, green roof type  impacts how they manage stormwater ,54. The Though this is the first study using the Nature Braid tool to design and simulate an urban green roof network, similar stormwater management benefits of widespread urban green roofs have been found using other modelling and simulation tools. Mora-Meli\u00e0 et al.  used theThis paper focused on the flood mitigation ecosystem service tool of Nature Braid; however, there are several other ecosystem service tools  that could be used in future research to develop an urban green infrastructure design that optimises the outputs for multiple ecosystem services, responds to additional climate change impacts, or addresses other societal challenges. The applications of Nature Braid and other ecosystem system service assessment tools are relevant to many disciplines. As has been demonstrated in this paper, they can help urban designers and planners map risk areas and design urban green/grey infrastructures to mitigate them. The tools can also help policymakers identify and draft suitable regulations and incentives to support urban-scale biomimicry projects. However, the successful implementation of these projects will depend upon knowledge from other fields. Architects and engineers can help determine the suitability and capacity of buildings and existing grey infrastructure to support green infrastructures, like green roofs. Local ecologists will also need to be consulted regarding the target species and habitats that green infrastructures should emulate, conserve, restore, or regenerate. Interdisciplinary collaborations are essential to creating buildings and urban green infrastructures that contribute to the regeneration of urban ecosystem services and help cities adapt to climate change.As cities expand and increase in density, and climate change drives more frequent and severe rainfall events, the need for urban ecosystem services increases in quantity and urgency. This research found that large-scale implementation of green roofs (4% of the catchment area) resulted in a three-fold decrease (11% of the catchment area) in the unmitigated flood risk area. Urban biomimicry strategies and tools, like Nature Braid, can help planners, designers, policymakers, and stakeholders create more resilient urban green infrastructure networks that mimic natural hydrological processes and increase flood mitigation. Robust urban green infrastructure networks that increase flood mitigation and other ecosystem services, like habitat provision, air purification, and evaporative cooling, will be critical to the health, well-being, and resilience of urban residents as climate change progresses."}
{"text": "From the perspective of social relationships, this study extends the understanding of employee voice by examining voice outcomes, especially a voicer\u2019s influence in their work team. In particular, we explore how two different social relationships, LMX and peer relationship, separately and jointly affect the \u2018voice-influence\u2019 relationship. Drawing on social network theory, we propose that higher LMX and central positions in peer networks  strengthen the positive impact of voice on individual influence. From a sample of 128 employees from three firms in South Korea, we found that two types of voice (promotive and prohibitive) are positively related with individual influence. This study also found that LMX strengthened the positive effect of promotive voice on a voicer\u2019s influence. Moreover, LMX and peer relationship jointly affect the voice-influence relationship as follows: (1) a voicer with a high LMX-high centrality (in the peer network) is most influential within their team, (2) as for a low LMX-high centrality member, speaking up rather decreases individual influence. These results suggest that voice outcome is not unilateral. Rather, whose voice it is and where a voicer stands may matter more. We discussed the theoretical and practical implications of these findings in employee voice research. Employee voice is a key driver of group or organizational success and constructive change ,2,3. GivTo enable employees to speak up, we need to understand the social outcomes of voice behavior and the mechanism through which it is produced. Several studies have emphasized that voice increases the status, power, and personal influence of a voicer ,8. ConseFurthermore, employee voice is a social phenomenon. It has a target or audience to speak to. Even voice conveying the same content can be recognized or evaluated differently by the supervisor and colleagues in the workplace ,7,10,11.In light of this stream of research, we examine the moderating role of leader\u2013member exchange (LMX) and peer relationships in the process of determining the voice outcome. Drawing on social network theory, we especially focus on the separate and joint effects of LMX and peer relationships. Although the main effects of LMX have been well addressed ,16 in voMoreover, this study goes further to examine the combined effects of these two social relationships. In a real work team, a member\u2019s relationships with the formal leader and with other members (excluding the leader) are inextricably interwoven . SeveralIn conclusion, our research question aims to better understand under what conditions employee voice enhances voicers\u2019 influences in the workplace. Regardless of voice content, voice outcomes may be beneficial or harmful to voice-performers, along with the social context in which they are located. We advance the theory of voice outcomes by examining the possible combination of LMX and informal peer relationships, which in turn delineates the boundary conditions of harvesting voice outcomes for individuals. Thus, we attempt to answer the question of whose voice will be heard, appreciated, or devalued within a team, and understand the factors amplifying or mitigating the impact of voice.Employee voice, referring to the discretionary communication of improvement-oriented suggestions, ideas, options, or concerns, is a critical component in enhancing organizational or work-unit functioning ,22,23. TFurther, the nonconforming nature of voice behavior can illuminate a voicer\u2019s competence. Nonconformity  directs the focus of the group to the contributions made by the voicer . From thHypothesis\u00a01. (H1).Voice behavior is positively related to individual influence within a team.Voice behavior is target-sensitive, implying that peers\u2019 perspectives of voice and that of the supervisor play a critical role when individuals decide whether to speak up or not . TherefoFirst, the relationship with the leader can impact the voice-influence relationship. LMX is the quality of relationships between a supervisor and his or her subordinates . BecauseIn addition, employees with high LMX not only receive valuable resources from the leader but also inflated performance ratings because of leniency bias resulting from the leader\u2019s intention to reciprocate ,40. GiveHypothesis\u00a02.\u00a0(H2).LMX moderates the relationship between voice behavior and individual influence within a team, such that the positive relationship is stronger when a voicer has higher LMX.Peer relationships have gained attention as most organizations now pursue a more horizontal structure . With thHypothesis\u00a03.\u00a0(H3).Peer relationships moderate the relationship between voice behavior and individual influence within a team, such that the positive relationship is stronger when a voicer has high-quality peer relationships .In this study, we maintain that the two aforementioned social relationships, LMX and peer relationships, have their own interaction effects on individual influence with voice behavior in a team. However, previous social exchange research has suggested that the relationship between the leader and coworkers is inextricably interwoven within a work team ,49. In tSpecifically, an employee\u2019s voice may be more strongly associated with their influence when it is combined with both high LMX and quality peer relationships. Employees with high LMX and high centrality in friendship networks can receive specific supports and opportunities necessary for effective job performance through unique relationships with the supervisor  and haveNext, although prior research has suggested that employees with high LMX could be evaluated positively by supervisors ,54, voicHypothesis\u00a04.\u00a0(H4).LMX and peer relationships jointly moderate the relationship between voice behavior and individual influence.Hypothesis\u00a04a.\u00a0(H4a).With high-quality peer relationships , the relationship between voice behavior and individual influence is stronger for a voicer with high LMX than for a voicer with low LMX.Hypothesis\u00a04b.\u00a0(H4b).With low-quality peer relationships , the relationship between voice behavior and individual influence is weaker for a voicer with high LMX than for a voicer with low LMX.The overall research model is illustrated in To test our hypotheses, we collected data from 3 organizations in South Korea. These organizations were an IT company, a manufacturing and retail company, and a public enterprise. Before distributing the survey, we asked HR managers for permission for data collection. We explained this survey\u2019s objective and interviewed a sample of company HR managers. We gained a team roster from organizations with supportive attitudes to the survey. We allocated a serial code to the respondents to recognize their answers. Of the 179 employees in total, 166 replied, representing a response rate of 92.7%. We collected data by questionnaire survey. The survey items and scales of this research were translated into Korean and received precise validity checks using back-translation by a bilingual Korean\u2013American translator independent of our research team . Our surDemographic characteristics of the respondents consisted of male (70%) and female (30%). The age distribution was as follows: 20s (23.4%), 30s (47.7%), 40s (24.2%), and 50s (4.7%). The average organization tenure was 87 months (s.d.= 84.9). The distribution of rank showed that the majority were junior managers (49.2%), followed by staff (25%), senior managers (17.2%), and directors (8.6%). The average team size was 6.82 (s.d: 2.29), with the smallest team comprising 4 members and the largest team containing 10 members. The table with demographic characteristics is given in All team members, including formal leader, assessed each member\u2019s influence. We provided the team roster to all team members (respondents). The question was \u201cFor each person in your team, on the scale from 1 (none) to 5 (very much), please indicate how much influence the person you checked has in the everyday activities of this team\u201d . BecauseWe measured promotive and prohibitive voice behavior rated by the formal leader. To date, most of the previous researchers have focused heavily on both promotion and prevention aspects of voice behavior e.g., ,3,13]. E,13. E3,1We measured the individual perception of LMX using the seven-item LMX-5 Scale . Items aPeer relationship was assessed by team members, excluding the formal leader. We measured indegree centrality in friendship networks among team members using the roster method . Team meWe controlled for organizational characteristics using firm dummy because the three organizations are different in structure and management style. We also controlled for team size because it influences team processes and outcomes. Demographic characteristics were also included in control variables: age, gender, organizational tenure (in months), and rank.p < 0.01) and prohibitive voice  were positively related with individual influence. Also, significant positive correlations were between social relationships and individual influence . b = 0.319, p < 0.001; b = 0.211, p < 0.01). Thus, H1 was supported.We tested the hypotheses using hierarchical regression analysis. b = 0.168, p < 0.05), but the interaction with prohibitive voice was not supported . The interaction effect of promotive voice and LMX on individual influence was plotted in b = 0.420, p < 0.001) and low . Also, as shown in b = \u22120.464, n.s.; b = \u22120.302, n.s.). Thus, H3 was not supported.Next, H2 and H3 proposed the moderating effects of LMX and peer relationship. Following Aiken and West (1991), interaction terms were mean-centered in order to reduce multi-collinearity. As shown in 2 = 0.739; prohibitive voice, f2 = 0.592). As shown in model 5 of b = 0.889, p < 0.05) and prohibitive voice .H4 was about the three-way interaction of LMX, peer relationship, and voice. To perform a configurational analysis, we entered the three-way interaction term in the final step . Conversely, b = \u22121.039, p < 0.05; prohibitive voice: b = \u22121.215, p < 0.05). As for members with high LMX and low centrality, their voices were unrelated to individual influence . Also, employee voice had a marginally significant relationship with individual influence with low LMX and low centrality . Following Dawson and Richter (2014), we ran the slope difference test )This study makes several theoretical contributions. First, this study can answer the recent calls for investigating the social side of voice consequences. Most voice research has focused on the antecedents of voice, such as psychological safety , disposiSecond, our study contributes to the development of the voice literature from a social relationship perspective. Recent research suggests that not only LMX but also relationships among coworkers impact workplace outcomes ,50. HencMost importantly, our results pave the way for studying, not only the interactions of the relationships with the leader and peers, but also the optimal balance of LMX and peer relationships from the social network perspective. Although previous social exchange research has argued that LMX and TMX are interwoven in a work team , it has In a practical sense, our study suggests that managers can consider voice behavior as an important way of increasing members\u2019 influence in the work team. Along with the recent research emphasizing the voice consequences ,8,71, weMore importantly, this study found that voice and influence relationships are affected by various social relationships. Extant studies have claimed that leaders and coworkers provide benefits for employees who speak up, but sometimes leaders and peers may ignore or reject their colleague\u2019s voice ,10,68. AIn addition, our results imply that LMX is a more critical factor than centrality in friendship networks for employees to gain influence through voice behaviors. Although peer relationships are considered important in the workgroup, their salience may be weaker than that of the vertical relationship between leaders and subordinates. Taken together, this study\u2019s results indicate that managers should consider not only high-quality peer relationships but also sound supervisor-subordinate relationships.Despite its numerous contributions, this study also has several limitations. First, to reduce the common method bias, this study gathered data from two distinct sources: member and leader . HoweverSecond, we tested our research model using data collected from the firms with collectivistic cultures. The unexpected results of this study may be attributable to specific cultural characteristics. Yum (1988) revealed that the main difference between East Asians and North Americans is their emphasis on communication in relation to social relationships. As our data are from Korean companies which emphasize harmonious social relationships, preserving others\u2019 honor based on relational norms is of great importance, rendering straightforward voice behavior less appropriate ,74. ThisAs business environments continue to emphasize flexibility and interdependency in workgroups, the importance of voice behavior cannot be overemphasized. This study investigated the consequences of voice behavior by focusing on voice as a social phenomenon. Overall, from a social relationship perspective on voice behavior, this study revealed the effect of voice behavior on employees\u2019 influence in a team. We also shed new light on the importance of the social relationships within a team\u2014both LMX and peer relationships\u2014by demonstrating how different social relationships separately and jointly affect the voice-influence relationship. This study can be a meaningful step toward discovering voice outcomes from the perspective of social relationships. As employees\u2019 proactivity and change-oriented behavior are required more than ever, managing social relationships within a work team will be a critical task for managers to foster employees\u2019 continuous voice behavior in organizations. This study suggests that designing a work team with high-quality LMX and peer relationships may be an effective means of accomplishing goals. Further studies are needed for a deeper understanding of the consequences of voice behavior."}
{"text": "Humans perceive the external world by integrating information from different modalities, obtained through the sensory organs. However, the aforementioned mechanism is still unclear and has been a subject of widespread interest in the fields of psychology and brain science. A model using two reservoir computing systems, i.e., a type of recurrent neural network trained to mimic each other's output, can detect stimulus patterns that repeatedly appear in a time series signal. We applied this model for identifying specific patterns that co-occur between information from different modalities. The model was self-organized by specific fluctuation patterns that co-occurred between different modalities, and could detect each fluctuation pattern. Additionally, similarly to the case where perception is influenced by synchronous/asynchronous presentation of multimodal stimuli, the model failed to work correctly for signals that did not co-occur with corresponding fluctuation patterns. Recent experimental studies have suggested that direct interaction between different sensory systems is important for multisensory integration, in addition to top-down control from higher brain regions such as the association cortex. Because several patterns of interaction between sensory modules can be incorporated into the employed model, we were able to compare the performance between them; the original version of the employed model incorporated such an interaction as the teaching signals for learning. The performance of the original and alternative models was evaluated, and the original model was found to perform the best. Thus, we demonstrated that feedback of the outputs of appropriately learned sensory modules performed the best when compared to the other examined patterns of interaction. The proposed model incorporated information encoded by the dynamic state of the neural population and the interactions between different sensory modules, both of which were based on recent experimental observations; this allowed us to study the influence of the temporal relationship and frequency of occurrence of multisensory signals on sensory integration, as well as the nature of interaction between different sensory signals. The brain uses numerous sensory inputs to recognize the external world and acquire knowledge. In addition, handling multiple pieces of information in an integrated manner increases the reliability of perceiving the external world and knowledge acquisition. Researchers have investigated this phenomenon, termed sensory integration or multisensory integration, in the fields of psychology, medicine, neuroscience, and artificial intelligence.1. The co-occurrence and combination of specific visual and auditory stimuli exert a significant impact on perception3. In neuroscience, most experimental studies employ the experimental design requiring perceptual decision-making to distinguish between multimodal and unimodal sensory effects5. The models based on Bayesian decision theory succeed in explaining the mechanism underlying multisensory integration, especially the mechanism of perceptual enhancement for combinations of multiple sensory stimuli. The models based on the drift\u2013diffusion theory could explain reaction time in perceptual decision-making of multisensory stimuli7. These models are based on the hierarchical structure, namely feedforward structure, in which sensory information is processed in the corresponding unimodal sensory system  and then transferred to association areas (via a single pathway) for integration with other types of sensory information. According to evidence that the view of the feedforward system is not necessarily consistent with the results of perturbation studies, in which a relevant brain region is inactivated, systems for multimodal sensory integration consist of multiple pathways and are more multifaceted than the feedforward view8. Such interaction between sensory modalities and integration of sensory information through these pathways before it reaches the association cortex would allow for faster perception than the conventional feedforward processing. In addition, because responses of single neurons during multisensory integration are heterogenous, it is also suggested that the contribution of population-level dynamics to the mechanism should be considered8. From the perspective of computer science, one made attempts to model a visual system based on the convolutional neural network (CNN)9 and the mechanism for sound source localization10. Regarding audio-visual integration, the model with self-supervised learning, CNN, and Bayesian theory proposed the mechanism that utilizes correspondence and temporal coincidence between features, each of which is extracted from visual or auditory stimuli24.In psychology, the \"McGurk effect\" describes the perception of a third phoneme when people listen to one phoneme being vocalized while simultaneously seeing another phoneme being vocalized in a video25. In the early stage of multimodal integration, sensory organs or low-level brain regions, such as the retina/primary visual cortex for the visual system or cochlea/primary auditory cortex for the auditory system, need to extract useful information from such a continuous signal and to encode extracted information as a dynamical state of neural activity. Most of the studies concerning multisensory integration have focused on how to utilize acquired information, i.e., the features obtained after processing the continuous signals, but not on how to extract information from continuous signals27. In addition, the encoded sensory information is assumed to be represented by steady firing rates of a neural population , not by a dynamical state of them. Indeed, the time-varying activity of a neural population has been observed in various brain regions and showed its relevance to brain functions28. Perception of multisensory stimuli depends on the timings of stimuli such as stimuli presented with a certain delay3. This dependence of multisensory integration on the timings could be achieved only with the model based on the dynamical state. Furthermore, because the process is carried out in an unsupervised manner, it is likely that frequent simultaneous presentation of signals from different sensory modes should serve as the cue. Although how multimodal information is constructed from continuous signals has been studied22 from the perspective of machine learning, most of the studies utilize a steady state of the neural population, rather than a dynamical state, as the encoding relies on an artificially layered structure that is not seen in the brain24.Sensory information comprises an uninterrupted time series, and sensory organs transmit it as a continuous signal to downstream areas. Sensory neurons during such a sensory stimulus presentation exhibit dynamic firing patterns, but not steady firing intensities29. The model is based on reservoir computing (RC) because RC is a recurrent network model that handles time series data31. While most models for chunking focus on the transition probability between elements that make up a chunk35, the model utilizes trajectories of states of the neural network responding to sequential inputs. Because sensory signals are not always comprised of separable elements such as letters in a string, the model is more likely to provide a plausible mechanism. Furthermore, because the neuronal network in sensory organs, such as the retina for the visual system, rarely shows experience-dependent plasticity, RC in which recurrent connections are fixed is structurally closer to the system engaging in sensory processing than other types of recurrent neural networks in which all connections are supposed to be modified for learning. Similarly, a reservoir computing model based on predictive coding (PCRC) was proposed36. The PCRC model was constructed such that the sensory information of each modality was supposed to be encoded as predicted information, namely, the encoding neural activity is supposed to reproduce the input. Therefore, in the model, each sensory input is essentially unaffected by the other types of sensory signals, and interaction between different sensory modalities was not considered. On the other hand, the proposed model enables us to consider the effect of direct interactions between sensory modules, which has been suggested by several studies39. In this study, we intended to apply the previously proposed model29 to realize information integration between different modalities through the segmentation of specific stimuli co-occurrence in repeatedly presented time series data and to encode it as a dynamical state.In this study, we aimed to propose a potent model for understanding the mechanism of multisensory integration in the early stage implemented by the brain based on the previously proposed model for chunking29. In the brain, there are different systems, each of which is responsible for the processing of a specific sensory signal, such as signals from the visual system or the auditory system. Therefore, in the model used in this study, two RC networks, each of which is later referred to as an RC module, interacted with each other by feeding their output to the other module for learning. As described below, the model received different modal inputs (images and text tones) to be integrated, each of which is processed by an RC module that corresponds to a sensory system. Each RC module consisted of rate-based neurons in a recurrent network and linear readout units in the output layer. An RC module received the time-varying inputs of a modality, which differed among the modules . The teaching signals are defined by the following equations.We demonstrated the mechanism by which the model learned multiple chunks. The input was made by alternating input sequence and a chunk that was randomly chosen with the same probability among the three. Fig.\u00a0A, left. We trained the model by using input sequences in which three chunks appeared randomly and consecutively with equal probability Fig.\u00a0A, right.Next, we investigated the mechanism by which activities of reservoir neurons encoded chunks. We trained the network on sequences containing three chunks and random sequences Fig.\u00a0A left. EFollowing learning, the norm of ler Fig.\u00a0A1 and A2N. During each chunk presentation, the activity of the RC internal nodes drew a specific trajectory in the PC1 to PC3 space  of reservoir neurons and the strength (g) of recurrent connections among reservoir neurons  and the integrated output from the model. The missing input signal replaced one of the signals in a chunk pair with another signal or a random sequence, with a probability m  was by the interaction of feedback from the outputs of appropriately learned sensory modules with alignment between sensory modules, suggesting that anatomical connectivity facilitates such an interaction between different sensory systems.i-th readout in either RC module, the teaching signal is supposed to reinforce the i-th readout in the partner RC module by adjusting reservoir-readout connections. Thus, the input will be encoded by the i-th readouts of both RC modules.\u00a0Considering that the brain possesses sensory module such as visual and auditory cortices and the modules directly interact each other, the interaction between RC modules might be the key for understanding the brain mechanism of multisensory integration. The earliest region where different sensory information converges is the superior colliculus (SC)46. Input from the association cortex assists the SC neurons in learning how to respond to multisensory inputs. In the proposed model, neurons in the association layer correspond to SC neurons, and connections between readout and association neurons should be regulated by the association cortex. However, the proposed model could not incorporate such learning because the association neurons are simplified and the higher layer was not included. In the computational model of multisensory integration based on such anatomical connectivity48, the unisensory modules that send sensory signals to SC neurons are designed to compete with each other in a winner-take-all fashion during the learning phase; this corresponds with the teaching signals employed in the proposed model.Given that an input activates the 36. In the model with a similar structure to the present model, each sensory signal is encoded by the corresponding sensory RC such that the error between a sensory input and the RC output is minimized. Then, a multisensory signal is encoded by combining visual and auditory sensory information processed by the sensory RCs at the upper layer. Because the PCRC model uses predictive coding for encoding50, each sensory RC is trained such that it can generate the output that is the same as the given sensory input. Therefore, a sensory signal of a modality is essentially processed within the corresponding sensory module, while the proposed models interact with the other sensory module.  If direct interaction between sensory modalities is to be considered, it may be necessary to redefine the essential mechanism; for example, what an RC module predicts is the input of a single modality or integrated inputs of related sensory modalities. On the other hand, the interaction between modalities is incorporated in the proposed model, although it is designed as the teaching signal. Furthermore, the model can be transformed as shown in Fig.\u00a0The PCRC model has been proposed as a model to process time-varying sensory signals and encode them as the dynamical states of neural activity51. Perception by multimodal integration decreased as the delay became longer. The percentage of correct perception drops to 50% when the delay increases to 200\u2013300 ms3. Although the accuracy shown in Fig.\u00a03. Furthermore, such perception highly depends on stimulus types , suggesting that a higher cortical area may be involved in the process. This property would be difficult to reproduce within the present model because the top-down control that would arise from such a higher structure is not represented in the present model.The co-occurrence\u00a0of stimuli of different modes would be attributable to an identical cause, particularly in the case of frequent presentation. The proposed model was capable of integrating simultaneously presented paired stimuli, and its performance deteriorates with decreased probability of co-occurrence or an increased delay of the presentation of one of the paired stimuli Fig.\u00a0. Concern3 and \u201cventriloquism effect\u201d52; the \u201cMcGurk effect\u201d is a phenomenon in which an unfamiliar combination of sensory information, each of which is familiar, leads to the new perception, and \u201cventriloquism effect\u201d is the auditory illusion that sound is perceived as coming from the different location, but not from the location where the sound is actually generated owing to visual stimuli. Both are major issues in multisensory integration because the dependence of perception on temporal aspects of the stimuli would imply that the integration process depends on dynamic shifts in properties of neural activity; the chunking mechanism based on neural population dynamics may also be involved in the illusional perception. The \u201cMcGurk effect\u201d can be resolved by treating the multimodal information as a static representation of a joint probability distribution representing combined stimuli of different modalities54; however, it is impossible to treat the \u201cventriloquism effect\u201d by such representation because it does not incorporate the time course of sensory signals. In particular, it is reported that the \u201cventriloquism effect\u201d is affected by the temporal difference between visual and auditory stimuli and the symmetrical/asymmetrical effect between visual and auditory stimuli exits52. The present model would enable us to consider the effect of the temporal difference or how to interact between the modules. In the ventriloquism effect, when there is a mismatch between the location of auditory and visual stimuli, a calibration occurs in which the perception of the location of auditory stimulus is drawn to the location of visual presentation. Traditionally, it has been assumed that repeated experience of such mismatch stimuli is required for this calibration to be achieved, but one study found it to occur after only the immediately preceding trial55. Extensions of this model that can encode stimulus location may allow us to test whether such short-term calibration can occur via a bottom-up mechanism, such as memory retention by recurrent dynamics. Therefore, the proposed model may contribute to a unified understanding of how temporal characteristics affect multisensory integration efforts beyond the McGurk and ventriloquism effects.The probability of co-occurrence of paired stimuli and the temporal delay between these stimuli displayed a strong association with the \u201cMcGurk effect\u201d2. In our model, the interaction between the different modal modules (RC1 and RC2) was direct. The interaction via a higher layer may be possible because perception requires top-down control from higher-order brain areas, such as the association cortices, whereas direct connections between primary sensory areas allow those areas to interact with each other. In the ventriloquism effect, the influence of visual information on auditory information could be stronger than vice-versa, although either is influenced by the accuracy of modal information.57. It is important to determine whether the above-mentioned asymmetric result is caused by the characteristics of bottom-up processing , by an asymmetric interaction between different sensory modules, or by asymmetric top-down control61.In the proposed model, neurons in the higher association layer were modelled to encode the results of multimodal integration by responding to specific co-occurrences of paired stimuli. This layer should be modelled to integrate multimodal information from the lower layers in a self-organizing manner, which would enable each neuron in this layer to display its response to specific combinations of multisensory information among numerous combinations. This modified version could be tested as a possible underlying mechanism of the \u201cMcGurk effect.\u201d For example, the model could be validated by evidence showing that the degree of the \u201cMcGurk effect\u201d depends on the temporal order and intervals of auditory and visual stimuli62, each of which has three layers: the input, reservoir, and output. The reservoir layer is a recurrent neural network that consists of N rate-based neurons. A neuron in the reservoir layer obeys the following dynamics:i(t) is the activity of the neuron in the input layer, g is the parameter that scales connections between neurons in the reservoir layer and controls the complexity of the network behavior; the reservoir network shows chaotic spontaneous activity for g\u2009>\u20091. W, Win, and Wback are connections within reservoir neurons, those from the input to the reservoir layer, and those from the reservoir to output layers, respectively. r(t) and Wout are the output of the reservoir neurons, and connection matrix from the reservoir neurons to output neurons that is also called the readout weight matrix, respectively. Each output neuron receives inputs from S reservoir neurons by the weights Wout. The output neurons projected back to all reservoir neurons. Learning was conducted by modifying the readout weight matrix Wout based on the FORCE algorithm that minimizes the error between the output and the teaching signal40. The elements of W are generated by a Gaussian distribution of N) where p is the probability of connections between reservoir neurons. The elements of Win were determined such that each row had only one non-zero element generated by a normal distribution N. Elements of Wback were drawn from a uniform distribution . The initial values of Wout are generated by a Gaussian distribution N. Table 63.In this study, we adopted a collaborative RC model that was originally developed to extract meaningful segments from information streams. The model consisted of two recurrent networks called echo state networks (ESNs)mentclass2pt{minimT\u2009=\u200915,000\u00a0ms.In the learning rule, we normalized the outputs such that the mean and the standard deviation of outputs were satisfied with 0 and unity, respectively:i-th normalized output. The threshold linear function [x]+ returns 0 for x\u2009\u2264\u20090 and x for x\u2009>\u20090; therefore, the output value was positive. The numerator inside the function tanh takes a positive value if the i-th readout showed the largest response because the second term of the numerator represents an average except the i-th readout. Therefore, when the i-th readout of an RC module responded to a chunk the most, the teacher signal reinforces the i-th readout of the other RC module. Each output neuron in an RC module received the teacher signal defined by Eq.\u00a0 for the final performance comparison of the model. We set the constants to \u03b4\u2009=\u20090.5.From the symmetry of the teacher signals for RC1 and RC2, i-the readout in the RC1 is responsible for the chunk \u201capple\u201d, while the j-th readout in the RC2 may respond to the chunk \u201capple\u201d. The neurons in the association layer must organize the outputs of RC1 and RC2 so that each neuron in the layer represents one of the learned chunks. For this purpose, we introduced the learning rule into connections from the readouts of RC1 and RC2. Let j-th readout of RCx to the i-th neuron in the association layer. The output of oi(t) and the dynamics N is the number of readouts or neurons in the association layer (N\u2009=\u20093). The first and second term of the right-hand side represents the Hebbian rule and the normalization, respectively.In the alternative models presented in Fig.\u00a0o(t) be the output to be analyzed. Convolution of o(t) with the window function w(t): Let C represents the total number of stimulus presentation epochs, including random sequences . cij represents the element in the i-th row and the j-th column. Therefore, the numerator is the sum of diagonal elements, indicating the sum of correct responses for all presentations. Considering learning was unsupervised, the stimulus selectivity was not explicitly imposed on each unit. Therefore, we determined the assignment of chunk selectivity for each unit such that the accuracy could be maximized.According to the obtained confusion matrix, the accuracy rate was calculated as follows:To define the response selectivity of reservoir neurons, we sorted them based on their mean activation phases:t when an input X is presented for the i-th trial is denoted by t averaged over trials, which is denoted as the center point at t for input X, is obtained as And nX is the number of trials for input X.A state of an RC module in low-dimensional space spanning from PC1 to PC3 at t is defined as Thus, the distance between the center points of input X and Y, t\u2009=\u20090. Therefore, we decided to characterize it by cutting out the 100 steps before and after t\u2009=\u20090 and integrating the time points.Therefore, we defined the degree of separation between trajectories of input X and Y as The smaller the value of 44. For example, in the effective dimension, if n principal components equally share the total variance and the remaining N principal components have zero variance, then Effective dimension is a measure of the effective number of principal components describing a trajectoryThe effective dimension is given bySupplementary Information."}
{"text": "We conclude that AE-AE is an effective and safe technique for the treatment of acquired stage Ib and II cholesteatoma present in the attic and tympanic cavities.This study aims to test the effectiveness and safety of exteriorization surgery comprising atticotomy and obliteration of the additus ad antrum, also referred to as attic exposition\u2013antrum exclusion (AE-AE) surgery. This surgery combines otoendoscopy with surgical microscopy for the treatment of acquired pars flaccida cholesteatoma in stages Ib and II  present in the attic and the tympanic cavity. We reviewed a historical cohort of 65 patients. Of the total, 21 were treated with canal wall-up tympanomastoidectomy (CWU). Patients in whom the AE-AE technique was performed had residual and recurrence rates of 0% and 9.1%, respectively, compared with 28.6% and 9.5%, respectively, for those treated with CWU. In the AE-AE procedure, surgery is performed in one stage compared with the two stages in CWU, to address the risk of residual cholesteatoma. Auditory thresholds were higher in the CWU group compared with the AE-AE group in the pre-surgery (53 \u00b1 16 vs. 44 \u00b1 15 dB;  Middle-ear cholesteatoma is characterized by the existence of epithelial tissue with aggressive and destructive behavior in the tympanic and mastoid cavities . AlthougAE-AE is technically and conceptually characterized by the complete exposure of the attic by reaming the upper wall of the external auditory canal , which allows the exclusion of the antrum and mastoid via closure of the additus through cartilaginous grafts. Using the classification of the IOOG, the categorization of this technique corresponds to an atticotomy with partial obliteration of the additus . The attFinally, we find that the use of otoendoscopy in this technique reduces recurrence due to persistent cholesteatoma, mainly in the epitympanum and mastoid antrum. The introduction of an endoscope is also significant in ossicular reconstruction.The objective of this study is to estimate the effectiveness and safety of AE-AE with endoscopic support when used to treat cholesteatoma occupying the attic and tympanic cavity in stages Ib and II according to the classification of the Japan Otological Society  Table 1.Using a historical cohort design, 65 patients undergoing their first surgeries between 2001 and 2020 with a diagnosis of stage Ib or II acquired attic and tympanic cholesteatoma were included [This research was approved by the Ethics Committee (2020/86). All patients were operated on with either (a) the AE-AE technique with the use of an otoendoscope or (b) the canal wall-up tympanomastoidectomy technique with only a microscope. The stage of cholesteatoma was established from the operative findings recorded in the clinical history as well as by reviewing the extent described in the surgical protocol and confirming the histopathological diagnosis of the pathological anatomy.All patients underwent preoperative computed axial tomography and surgery with intraoperative facial nerve monitoring according to a previously published protocol . AudiomeWe determined the difference between the AC and BC PTAs (ABG) pre- and post-surgery and determined whether the values were less than 20 dB, which was used as the criterion for a good post-surgical hearing outcome. When necessary, patients underwent an ossiculoplasty procedure in the same surgical act as cholesteatoma exeresis, using either pinna or tragus cartilage over the stapes or total or partial titanium prosthesis. Treatment of the tympanic membrane was performed with temporalis muscle fascia or tragus perichondrium.The inclusion criteria were as follows: attic cholesteatoma of stage Ib or II affecting only the attic or both the attic and the tympanic cavity; first intervention on the pathological ear; and patients undergoing AE-AE with the use of a microscope and an otoendoscope or undergoing CWU with only the use of a microscope. The exclusion criteria were the absence of cholesteatoma during surgery, cholesteatoma not acquired from pars flaccida, and prior surgery in the affected ear. Age, sex, affected ear, residual disease, and time of recurrence (if any) were also recorded in the clinical history.Five surgeons with more than 10 years of experience in otologic surgery operated on the patients using the CWU technique. These surgeons were not exclusively dedicated to the treatment of ear pathologies but were specialists in otolaryngology and general cervical pathology. The series of patients who underwent CWU were operated on between 2001 and 2013, a period prior to the creation of super-specialty units in our hospital. The otology unit currently has 2 surgeons who have performed all surgeries with microscopes and endoscopes since 2013. We performed AE-AE hybrid surgeries using standard microsurgical and otoendoscopy instruments.The following is a description of the surgical steps, which are based on the IOOG categorization . Retroauricular approach (A4);Tympanomeatal flap is lifted, and the middle ear is explored;Excision of middle-ear cholesteatoma and exploration of ossicular chain and incudostapedial articulation;Mastoidectomy with canal wall preserved and posterior tympanotomy M1b with additional combination of M1b and M2a (atticotomy);No external ear canal reconstruction (Ex);No obliteration (Ox);No bone removal from external ear canal wall (Ax);Tympano-ossicular reconstruction if necessary .CWU reconstruction technique:All cases were scheduled for second-look (S2p) revision between 1 and 2 years. Surgeons did not use otoendoscopes or diffusion MRI to assess residual disease.AE-AE hybrid exteriorization technique is shown in Following the steps previously described by other authors ,7,8, we 9.Retroauricular approach (A4);10.Tympanomeatal flap is lifted, and middle ear is explored;11.Excision of middle-ear cholesteatoma and exploration of ossicular chain and incudostapedial articulation using endoscope and microscope;12.Atticotomy (M2a). Superior wall of EAC is drilled with two hands and microscopic control, exposing entire tegmen tympani. Once attic is completely exposed , cholest13.No external ear canal reconstruction (Ex);14.Partial obliteration of additus (O1) using cartilage. Once cholesteatoma is removed, we proceed to closure of additus and, therefore, antroexclusion using several pieces of cartilage. Here, again, the use of an endoscope allows for perfect antroexclusion.15.No bone removal from external ear canal wall (Ax);16.Tympano-ossicular reconstruction, if necessary , using endoscope and microscope.17.Superior meatoplasty. To favor marsupialization of attic and self-cleaning, we perform an upper meatoplasty by incision at 12 o\u2019clock in upper wall of EAC and removal of cartilaginous tissues. Place tamponade with gauze edge soaked in antibiotic and corticosteroid creams and remove after 10 days.Using endoscopes in different stages allowed us to access areas of the middle ear for which visualization was very difficult, allowing cholesteatoma elimination and epithelium cleaning, mainly in the epitympanum and tympanic sinus in addition to the hypotympanum, protympanum, and supratubaric fossa.This study is observational in nature, involving a comparison of the surgical performances of otologists (2 surgeons) and general ENT specialists (5 surgeons) during different periods of time. Although the patients were not randomized, the fact that the recurrence rates are similar to those reported in other studies leads us to conclude that the surgeon is not a factor influencing the recurrence rate. We do not know if the results would change (better outcomes) if the otologists had performed the CWU procedures, although we consider super-specialization to be important in ENT surgeries.Categorical variables are expressed as frequencies and percentages. Quantitative variables are expressed as means and standard deviations. The proportions between surgical groups for sex, affected ear, stage of cholesteatoma, facial nerve dehiscence, reconstruction, recurrence, and PTA were compared using chi-squared tests or Fisher\u2019s exact tests, when appropriate. Comparisons between surgical groups for age, time to recurrence, and audiometry were carried out using Mann\u2013Whitney tests. The determination of the pre\u2013post-surgery period within each surgical group was carried out with Friedman or Wilcoxon tests, as appropriate. p-values below 0.05 were considered to indicate statistically significant differences. Statistical analysis was performed using SPSS  and LogXact-4 for Windows .All p = 0.005.Sixty-five patients were included in the analysis. They were divided into two groups according to whether the surgical technique used was AE-AE with endoscopes or CWU. The distribution and demographic variables are shown in p = 0.04.Regardless of the type of surgery, the residual disease and recurrence rates were higher in patients with stage II cholesteatoma  than in those with stage Ib cholesteatoma , with The residual disease, but not recurrence, rate was lower for patients in whom the AE-AE technique was performed compared with those treated using CWU see : 28.6% aIn the CWU group, 6 (28.6%) patients had an affected sinus tympani due to previous surgery compared with 8 (18.2%) patients in the AE-AE group. In both groups, no recurrence was found for previously affected sinus tympanicum ears. All cases of recurrence in the CWU group were due to failure in attic reconstruction, whereas failure in the AE-AE group was due to an error in the obliteration of the additus ad antrum. n = 26 (59.1%)) and in the CWU group (n = 11 (52.4%)). No difference was found in surgery time between the stage Ib group (158 \u00b1 58 min) and the stage II group (181 \u00b1 51 min), with p = 0.17. No differences were observed in the neurophysiological dehiscence of the facial tympanic portions.The most frequent reconstruction technique for the chain was cartilage over stapes, both in the AE-AE group (p = 0.039) but not at one or more years post-surgery  or in pre\u2013post-surgery comparisons, either within the AE-AE technique (p = 0.89) or within the CWU technique (p = 0.96). Overall, no differences in PTA were found between both groups (p = 0.49) or when comparing the hearing levels of partial reconstruction with cartilage over stapes  greater than 20 Db post-surgery in four patients from the CWU group and one patient from the AE-AE group. All of these patients had previous high-frequency affectations prior to surgery. The main objective of surgical treatment for cholesteatoma is removal with a minimum rate of recurrence. Canal wall-up and canal wall-down techniques have classically been performed depending on multiple factors. For stage Ib or II cholesteatoma of the attic and tympanic cavity, we modified an AE-AE technique performed in the 1980s ,6,7,8 inWe provide evidence here for lower residual and recurrence rates in acquired stage Ib and II cholesteatoma of the attic and tympanic cavity when using the AE-AE 0\u20139.1%) technique in combination with otoendoscopy in comparison with the CWU technique 28.6\u20139.5%). When we contrast our results with those of other authors, we can see that the CWU technique has residual and recurrence rates between 17 and 61% .1% techn reported.6\u20139.5%. The impression that the residual rate in the CWU group is high is due to a number of factors. Mainly, exposure of the attic, antrum, facial recess, and sinus timpani is more limited following the CWU procedure, which may lead to difficulty in the complete removal of all involved air cell tracts and in the elimination of cholesteatoma during the initial procedure . Due to Second, the epitympanum and mastoid cavity are ultimately relined with nitrogen-absorbing cuboidal mucosal epithelium after the CWU procedure, and the presence of this mucosa can result in the development of continued mucosal inflammation and recurrence . We conclude that there are several factors contributing to the decrease in the recurrence rate for the AE-AE procedure. As described in other articles ,8,11, a In contrast to other authors , we moniSimilarly to other authors ,16,17, wAs with other research , the attFurthermore, in other research , the surIn the group of patients treated with AE-AE using otoendoscopes, the most frequent treatment of the chain was partial reconstruction with cartilage on the stapes superstructure. We preferred autologous material to other types of materials, as we find, in line with other authors, that it provides functional benefits and allows us to reinforce the tympanic membrane, achieving acceptable functional results with post-surgical thresholds of 41 dB and a residual ABG of less than 20 dB, consistent with other studies .In 18.2% of the patients, we performed total reconstruction with titanium prostheses, obtaining thresholds of 46 dB and a residual ABG of less than 20 dB in 28.6% of the patients. We found no statistically significant differences between partial reconstruction with cartilage over stapes and total reconstruction with titanium prosthesis when studying ABG, which is consistent with the literature [There are reports  of high-The AE-AE procedure utilizing an endoscope is a safe technique that significantly reduces the rates of cholesteatoma persistence and recurrence and shows good auditive results. It allows patients to have excellent quality of life, normalized bathing, and a reduction in surgical revisions when used in the treatment of attic acquired cholesteatoma. There is a need for controlled clinical trials to demonstrate the benefits of the AE-AE method compared with other techniques."}
{"text": "A COVID-19 vaccine booster dose is effective and safe for older adults. This study investigated facilitators and barriers to take up a COVID-19 vaccine booster dose among older adults in Hong Kong. Participants were Chinese-speaking community-dwelling adults aged \u226565 years. Telephone numbers were randomly selected from up-to-date telephone directories. A total of 395 participants completed the telephone interview. Logistic regression models were fitted. Among the participants, 31.6% received a COVID-19 vaccine booster dose. After adjustment for significant background characteristics, positive attitudes toward the booster dose, perceiving significant others would support them to receive the booster dose, and less uncertainty regarding the choice of the booster dose was associated with higher uptake of a COVID-19 vaccine booster dose. Concerns about poorer responses to the booster dose due to older age and the presence of chronic conditions were negatively associated with the dependent variable. In addition, the belief that governmental promotional materials could address their concern and were helpful for them to make decisions was associated with a higher COVID-19 vaccine booster dose uptake. Improving booster dose health promotion materials, modifying perceptions, involving significant others and reducing uncertainty are potentially useful strategies to improve COVID-19 vaccine booster dose uptake among older adults. The cru = 8787) . The maj = 8787) .COVID-19 vaccination is effective and safe for preventing severe consequences and deaths caused by COVID-19 among older adults . HoweverPeople may be hesitant to receive a COVID-19 vaccine booster dose. At least eleven studies investigated willingness to receive a COVID-19 booster dose ,29,30,31Across countries, history of seasonal influenza vaccination, perceived higher risk and severe consequences of COVID-19, belief in vaccine efficacy, and trust in information obtained from social media and significant others were found to be facilitators to receive primary COVID-19 vaccine series among older adults ,43,44,45This study investigated facilitators and barriers to receive a COVID-19 vaccine booster dose among a random sample of community-dwelling persons aged 65 years or above in Hong Kong. We hypothesized that older people who had supportive perceptions of the booster dose, with less uncertainty when choosing a booster dose, and a higher level of satisfaction with governmental booster dose promotion materials would have higher uptake of COVID-19 vaccine booster dose.n = 56,827) on 2 March 2022. By the end of this study (13 April 2022), daily confirmed cases dropped to 1043. We present the COVID-19 situation in Hong Kong during the study period in A random telephone survey was conducted among community-dwelling Chinese-speaking individuals aged \u226565 years in Hong Kong from 14 February to 13 April 2022. During the study period, Hong Kong was experiencing the fifth wave of COVID-19 outbreak caused by the Omicron BA.2. The number of daily confirmed COVID-19 cases was 1619 on 14 February 2022, and this peaked (The inclusion criteria were: (1) community-dwelling Chinese-speaking individuals aged \u226565 years, and (2) having a Hong Kong ID card. We excluded those who were not able to communicate effectively with the study interviewers.Our target sample size was 400. We assumed the uptake rate of the COVID-19 vaccine booster dose to be 50%. Assuming the uptake rate in the reference group (without a facilitating condition) to be 10\u201340%, the sample size could detect the smallest odds ratio of 1.76 between people with and without a facilitating condition . According to the Hong Kong census data in 2021, 18.2% of Hong Kong residents were 65 years or above . AssuminThe research team input all household telephone numbers listed in the most up-to-date telephone directories about 350,000) into an Excel file and randomly selected 4000 household telephone numbers by using the function of \u201cselect random cells\u201d. Trained interviewers conducted the telephone calls during 6\u201310 p.m. on weekdays and 2\u20139 p.m. on Saturdays to avoid under-sampling of working individuals. If the initial call was not answered, four more follow-up calls were made during different time slots before we considered the household to be non-valid (one without an eligible participant). In case there was more than one person in the household who was aged \u2265 65 years, the interviewers invited the one whose last birthday was the closest to the interview date to complete the survey. This was to avoid data contamination and the introduction of extra confounding factors. After screening the eligibility, interviewers briefed prospective eligible participants about the study, and made guarantees of anonymity, the right to quit at any time, and that refusal would have no consequences. Since there was no face-to-face contact and the study was anonymous, we obtained verbal informed consent from the participants. The interviewers signed a form pledging that the participants had been fully informed about the study. The same procedures have been used in previous random telephone surveys targeting local older adults ,49,50. T,000 intoWe performed in-depth interviews to understand older adults\u2019 perspectives of a COVID-19 vaccine booster dose. Five community-dwelling Chinese-speaking individuals aged \u2265 65 years were recruited through purposive sampling . Prior to the interview, fieldworkers explained the purposes and nature of the interviews. With verbal informed consent, interviews were conducted through telephone and audio recorded with informants\u2019 consent. The interviews lasted for about one hour. We transcribed interviews and kept a code book to record special data and transform the data into categories to identify main themes. Three out of five informants intended to receive a COVID-19 vaccine booster dose in the future. We identified three themes related to facilitators: (1) concern that the protection conferred by the primary vaccine series would decline over time, (2) belief that a booster dose could reduce the risk of having Omicron BA.2 and the risk of death, and (3) suggestions from the government, doctor, and family members to receive a COVID-19 vaccine booster dose. Four other themes were related to barriers. They were: (1) not knowing which type of vaccine was better for them as a booster dose, (2) belief that older people would respond less well to a booster dose (less effective and more side effects), (3) concern about potential interactions between chronic diseases and a booster dose, and (4) concern about the duration of protection of a booster dose. Based on the qualitative findings, a panel of researchers in public health, behavioral health, and vaccination behaviors developed the questionnaire. The questionnaire was tested among 10 older adults to assess clarity and readability. All older adults participating in the pilot study believed that the questions were understandable and the length was acceptable. These older adults did not participate in the actual survey. The panel finalized the questionnaire based on the pilot testing results.Participants reported sociodemographic characteristics, the presence of chronic conditions, history of COVID-19, and history of seasonal influenza vaccination and pneumococcal vaccination.Participants reported the number of doses and types of COVID-19 vaccines received as primary vaccine series and the booster dose. Participants who had received a booster dose were asked for some details, such as the severity of side effects of the booster dose and whether such side effects were milder or more severe than the primary vaccine series.We adapted the 3-item scale measuring positive attitudes toward a COVID-19 vaccine booster dose validated in the Chinese population . One iteWe used the questions validated in Hong Kong older adults to assess satisfaction with COVID-19 vaccine booster dose health promotional materials produced by the Hong Kong government . The quep < 0.05 considered as statistically significant.The frequency distribution of all variables was presented. Mean and standard deviation (SD) of the items and scales representing perceptions related to the COVID-19 vaccine booster dose were also presented. Cronbach\u2019s alphas were calculated by using reliability tests. We used principal component analysis with varimax rotation to perform exploratory factor analysis. The dependent variable was self-reported uptake of a COVID-19 vaccine booster dose. Univariate logistic regression models first assessed the significance of associations between background characteristics and the dependent variable. We then fitted a single logistic regression model involving all significant background characteristics and one independent variable of interest. A summary multiple logistic regression model was fitted considering all significant variables in univariate analysis. Crude odds ratios (OR), adjusted odds ratios (AOR), and their 95% confidence interval (CI) were obtained. In addition, correlations between perceptions and satisfaction with the health promotional materials were investigated. SPSS 26.0  was used for data analysis, with About half of the participants were 65\u201369 years (49.9%) and female (60.3%). The majority of them were married or cohabited with a partner (75.4%), did not receive tertiary education (89.9%), without a full-time or part-time work (85.8%), and had a monthly household income below HKD 20,000 (USD 2580) (73.9%). Over 60% of them had at least one chronic condition (60.8%). The most prevalent chronic condition was hypertension (47.6%), followed by diabetes mellitus (19.2%), chronic cardiovascular diseases (10.9%), and chronic lung diseases (10.9%). Among the participants, 10.6% reported a history of COVID-19, and 44.3% reported having an acquaintance with a history of COVID-19. At the survey time, 63.8% had received a seasonal influenza vaccination in their lifetime, and 42.5% received it both in the current and previous seasonal influenza seasons. In addition, 27.6% received pneumococcal vaccination in their lifetime .n = 125) received a COVID-19 vaccine booster dose. More participants chose Comirnaty  rather than CoronaVac  as their booster dose. Twelve participants mixed COVID-19 vaccine booster doses; eleven switched from CoronaVac to Comirnaty, and one switched from ZAD1222 to Comirnaty when receiving the booster dose. Over half of them reported no side effects of the COVID-19 vaccine booster dose, and 88.8% reported such side effects were about the same or even milder than those of their primary series , could effectively protect them from COVID-19 variants of concern (60.0%), and could prevent severe consequences of COVID-19 (82.5%). Relatively few participants were concerned that older age would lead to a lower level of protection (10.9%) and a higher level of side effects of the COVID-19 vaccine booster dose (14.2%). About 70% of them were sure about which type of the COVID-19 booster dose was suitable for them and which type they should choose. About 90% of the participants believed that governmental COVID-19 vaccine booster dose promotional materials were easy to understand (87.8%), 42.3% believed such materials could address their concerns related to the booster dose, and 48.6% perceived such materials as helpful for them when making the decision to receive the booster dose .In univariate analysis, higher education level and history of pneumococcal vaccination was associated with higher uptake of a COVID-19 vaccine booster dose . After aUncertainty Scale remained significantly associated with the dependent variable in the multiple logistic regression model . However, education level, history of pneumococcal vaccination in the lifetime, positive or negative attitudes, perceived subjective norm, or satisfaction with the health promotional materials became statistically non-significant in the multiple logistic regression model .Positive attitudes, perceived subjective norm, perceived behavioral control, and satisfaction with the health promotional materials were associated with less uncertainty, while negative attitudes were associated with higher uncertainty. Positive attitudes, perceived subjective norm, and perceived behavioral control were also associated with higher satisfaction with the health promotional materials, while negative attitudes were associated with lower satisfaction .This is one of the first studies examining facilitators and barriers of COVID-19 vaccine booster dose uptake among older adults in China. During the study period, 91.9% of participants received at least one dose of the COVID-19 vaccine, and 31.6% received a booster dose. Perceived threat of the ongoing outbreak of COVID-19 might motivate many older adults to complete their primary vaccine series and to receive the booster dose, as the proportion of taking up at least one dose of COVID-19 vaccine in this group was much higher than that observed between November 2021 and January 2022 (about 60%) . The uptHigher education level was positively associated with COVID-19 vaccine booster dose uptake among our participants. Such a finding was similar to those of previous studies, as higher education level was associated with a higher completion rate of primary COVID-19 vaccine series among older adults in Hong Kong  and highThis study provided some practical implications to increase coverage of the COVID-19 vaccine booster dose among older adults in Hong Kong. First, it is necessary to improve the existing COVID-19 vaccine booster dose promotional materials in Hong Kong. The information on these materials was friendly to older people, as about 90% of the participants found it easy to understand. However, about half of the participants thought such materials could not address their greatest concern related to the COVID-19 vaccine booster dose. A similar proportion of participants believed such materials were not helpful for them when it came to deciding to receive the booster dose. Since the satisfaction with the health promotional materials was a facilitator to receive the booster dose, it was important to improve the contents. Our studies revealed some concerns specific to older adults in Hong Kong. About 10\u201320% of the older adults in our study had concerns about interactions between old age, chronic conditions, and COVID-19 vaccine booster dose, such as whether older age and the presence of chronic conditions would result in poorer responses to COVID-19 vaccine booster dose . Local evidence showed that the protection against severe/fatal diseases and death conferred by the Comirnaty and CoronaVac was similar in older adults and their younger counterparts . MoreoveAlthough this study was based on a random and population-based sample, it had several limitations. First, compared to census data , people Community-dwelling older adults aged 65 years or above in Hong Kong reported a lower uptake of COVID-19 vaccine booster dose than their younger counterparts. Existing COVID-19 booster dose health promotional materials might not adequately address older adults\u2019 main concerns. Such materials could be improved by addressing concerns about how older age and the presence of chronic conditions would affect responses to the COVID-19 vaccine booster dose. Strengthening positive attitudes, involving significant others of older adults, and reducing uncertainty about the choice of the booster dose might be useful strategies to increase the uptake of COVID-19 vaccine booster dose in this age group."}
{"text": "The reluctance of individuals to obtain solid vaccine-induced immunity represents a fundamental challenge to containing the spread of SARS-CoV-2, including its highly mutated variants. We aimed to assess vaccination acceptance and associated factors for the COVID-19 vaccine booster dose among elderly people (\u226560 years old) in China, providing a theoretical and practical reference for universal vaccination policy.Via a backward stepwise method, multivariable logistic regression models were established to assess factors associated with booster dose acceptance. Two-sided P < 0.05 was considered statistically significant.A national anonymous survey was conducted in mainland China from May 25 to June 8, 2022, using a stratified random sampling method. Individuals 60 years of age and above were the target population. A chi-squared test and Cochran-Armitage test for trend were used to compare and examine vaccine acceptance rates by characteristics. Of 3,321 eligible participants, 82.8% (95% CI: 81.5\u201384.1%) were willing to receive COVID-19 vaccine booster shots. Concerns about contraindications (38.3%), vaccine safety (32.0%), and limited movement (28.0%) were the main reasons for vaccine hesitancy. Nearly one-third still believed that the booster dose was unnecessary after receiving the initial vaccination. Older adults with a low level of perceived barriers , a high level of perceived benefit , and higher cues to action  were more likely to accept the booster dose. Other major factors affecting the booster dose acceptance rate were occupation, time spent on social media, vaccination history, and a high knowledge score for COVID-19 and vaccines. In addition, for those over 70 years of age, rising awareness of susceptibility could be a better gateway for improving their willingness to get vaccinated.A total of 82.8% of recruited older adults were willing to receive the booster dose. Acceptance behaviors were closely related to occupation, time spent on social media, vaccination history, knowledge factors, perception of barriers, and benefit, as well as action cues. Targeted public health measures are a priority for improving the vaccination coverage of valid immunity among the elderly population, not only to prevent infection and poor prognosis caused by emerging variants but also to reduce the huge disease and economic burden caused by the long-term sequelae after SARS-CoV-2 infection. Withter dose . Moreoveter dose .The reluctance of people to obtain solid vaccine-induced immunity represents a fundamental challenge to containing the spread of SARS-CoV-2 , 13. AccThe entire medical community has invested enormous efforts in developing and delivering COVID-19 vaccines, and vaccination campaigns among adults are in full swing, such as in China. The Chinese government has continued to encourage older people to get vaccinated, especially for booster shots , 34. Comvia an online platform called Wen Juan Xing . Covering at least 2.6 million registered members in China, we could easily reach an authentic, diverse, and representative sample  Chinese citizens; (2) \u226560 years old; (3) people who agreed to participate in this survey; and (4) completed the survey between May 25, 2022 and June 8, 2022. To recruit enough representative respondents, we randomly sampled potentially eligible subjects in 31 provinces according to the proportion of adults aged 60 and above reported in the Seventh National Census  among older people . The reliability of this questionnaire  was confirmed by Cronbach's alpha coefficient by different dimensions . Each question was followed by a reminder that all answers were supposed to reflect the reality and true thoughts of those older adults.The questionnaire was constructed and revised by a panel of experts, including one public health expert and two epidemiologists specializing in infectious diseases . We perfThe primary outcome was the acceptance rate of the COVID-19 vaccine booster, defined as the proportion of participants who answered \u201cyes\u201d when asked whether they were willing to receive the COVID-19 vaccine booster dose if available. If a respondent had any concerns or reluctance, the questionnaire would automatically jump to the specific reasons for hesitation.Sociodemographic characteristics and health status of the sample population were investigated, including region, age, sex, marital status, education level, occupation, income, history of chronic disease, and COVID-19 vaccination. The amount of time people spent checking the news about COVID-19 or vaccines on social media each day and people's satisfaction with the government's response to COVID-19 were also obtained.For knowledge factors, we set six and four questions to determine the knowledge level of the elderly on COVID-19 and COVID-19 vaccines, respectively. Sources of SARS-CoV-2 infection, common symptoms, transmission routes, high-risk groups, self-protection measures, and herd susceptibility were the investigational scope of the former aspect, with a total of 19 scores. Scores of \u201c0\u20136,\u201d \u201c7\u201313,\u201d and \u201c14\u201319\u201d represented a \u201clow,\u201d \u201cmoderate,\u201d and \u201chigh\u201d degree, respectively. For the knowledge on COVID-19 vaccines component, we mainly focused on price, time for booster vaccination, immunity induced by vaccines, and adverse reactions after vaccination. Each correct choice received 1 point, and a total of four scores were assigned to this part. Then, all respondents were divided into three groups  from low to high.As one of the most common theories reflecting individual behavior change, the HBM is an organic combination of motivation theory, cognitive theory, and expectancy-value theory, which has been widely used to explain public attitudes toward vaccines and predict people's vaccination behavior \u201321. ConsP < 0.05 was considered statistically significant.We performed descriptive analyses to summarize the characteristics of the recruited population by frequencies and percentages. The independent Chi-squared test and Cochran-Armitage test for trend were used to compare the differences between groups stratified by the abovementioned characteristics. To identify factors that influence vaccination willingness, multivariable logistic regression analyses were performed in different groups of participants, including three age groups  and those who had not yet received the booster dose. All independent variables were added to our multivariable models, and adjusted odds ratios (aORs) and 95% CIs were calculated via a backward stepwise method (P < 0.2). The Hosmer and Lemeshow test was used to assess the goodness of model fitting. All statistical analyses in this study were conducted using SPSS 26.0  and R 4.1.3 , and two-sided Ptrend < 0.05). Significant differences in vaccine acceptance were not found between groups stratified by region, sex, occupation, and economic condition .A total of 3,321 respondents were eventually included in our analyses , and 1,0P < 0.05) (P > 0.05). Elderly people who believed in the safety and effectiveness of COVID-19 booster shots showed higher acceptance rates of 83.20% (81.90\u201384.50%) and 83.80% (82.40\u201385.10%), respectively. Similar results were exhibited for the dimension of the perceived benefit of the COVID-19 booster shot. Based on the analysis of five dimensions of the HBM, the higher the perception of susceptibility, benefit, and cues to action, the higher the vaccination acceptance rate among the elderly .Concerns about the susceptibility of themselves and family members to SARS-CoV-2 infection indicated a higher acceptance rate of the booster dose among the older population ( < 0.05) . On the For all 3,321 participants, our multivariable logistic regression indicated that the main factors related to COVID-19 vaccine booster dose acceptance were occupation, time spent on social media, and higher knowledge scores on COVID-19 and vaccines . VaccinaAmong the 1,554 participants who did not receive booster shots, 65.3% showed a willingness to receive the booster vaccination. A higher level of perceived benefit  and cues to action  indicated a higher willingness to receive a booster dose among those elderly people. Additionally, occupation, time spent on social media, and vaccination history could also affect the attitudes of elderly people toward booster vaccination .Among the 571 (17.2%) participants who were reluctant or refused to receive the booster dose, nearly one-third were still unaware of the importance of booster shots, believing that it was unnecessary to receive a booster dose as they had already received one or two doses of the initial vaccination . A totalAs a high-risk population with poor prognosis after infection, older adults are the key population for follow-up booster vaccination. We found that, of the 3,321 participants recruited, 82.8%  were willing to receive COVID-19 vaccine booster shots. Concerns about contraindications, vaccine safety, and limited movement were the main reasons for vaccine hesitancy. Factors related to vaccine acceptance rate were occupation, time spent on social media, vaccination history, knowledge factors, and perception of barriers and benefit, as well as action cues. For the over 70s, increasing their awareness of susceptibility could be a better gateway for improving vaccination acceptance. Given the current global epidemic situation and the prevalence of highly infectious variants, it is likely that the subsequent second and third booster shots or vaccines against specific variants will soon be administered globally . Older aBased on our study, 82.8% of 3,321 participants recruited were willing to receive COVID-19 vaccine booster shots. As the high-risk group for severe illness and death after SARS-CoV-2 infection, 87.6 million people (33.2%) aged 60 years and above in China have yet to receive booster shots as of August 10, 2022 . To dateP > 0.05). Multiple studies have shown that higher perceived severity is negatively associated with vaccine hesitancy (P < 0.05), which highlighted the importance and advantages of families and healthcare workers encouraging vaccination for eligible older persons. Similar results can also be verified . The patients/participants provided their written informed consent to participate in this study.Conceptualization: CQ, MD, and WY. Methodology and analysis, visualization, and writing\u2014original draft preparation: CQ. Review and editing: LT, WY, MD, QL, and ML. Supervision: JL. All authors have read and agreed to the published version of the manuscript."}
{"text": "Objective: The COVID-19 pandemic is still continuing throughout the world, with newer genetic variants regularly appearing from different parts of the world. Considering the waning of immunity against COVID-19 infection even with two doses of the COVID-19 vaccine, regulatory authorities have authorised booster COVID-19 vaccination in many countries, especially for vulnerable populations, including healthcare workers. The current study analysed factors predicting the third (booster) dose of COVID-19 vaccine intention, including the health belief model (HBM), among the healthcare workers in Saudi Arabia. Methods: The current study was a cross-sectional online survey performed from 1st October 2021 to 30th November 2021, using a questionnaire prepared in GoogleTM form among healthcare workers in Saudi Arabia. The questionnaire asked demographic factors, COVID-19 experience of participants, subjective assessment of health, intention of COVID-19 booster dose vaccination, preferences for local/foreign-made vaccines, and health belief of the study population related to COVID-19 infection and COVID-19 booster dose. Results: This study received 2059 complete responses. The study population reported mixed health belief with respect to the susceptibility of COVID-19 infection, and higher health belief perception regarding the severity. The perceptions of the study participants regarding the benefits of COVID-19 booster dose were positive. There were few barriers to COVID-19 booster dose expressed by study participants. A total of 1464 (71.1%) study participants reported positive intent for receiving a COVID-19 booster dose. The study showed significant association between definite intention to receive a booster dose and nationality (p = 0.001), marital status (p = 0.017), gender (p < 0.001), education level (p = 0.001), monthly income (p < 0.001), and co-morbid medical illness (p = 0.045). The perception of the COVID-19 booster vaccine as a good idea to minimise worries about getting COVID-19 , and perceptions that receiving the third (booster) dose reduces the risk of COVID-19 infection and associated complications , of the perceived benefit construct of HBM, predicted significantly higher definite intention to receive a booster dose. The concern with the safety of the vaccine  under the perceived barriers construct of HBM predicted as significantly higher no definite intention to receive a booster dose. Conclusions: The results of the present study can guide policy makers in their efforts to promote booster doses of COVID-19 vaccination among the healthcare workers in Saudi Arabia. Since the World Health Organization (WHO) declared the novel coronavirus COVID-19) as a pandemic on 11 March 2020, its spread continues throughout the world, with newer genetic variants regularly appearing from different parts of the world 9 as a pa. UnfortuSince the beginning of the pandemic, KSA had put very active steps in controlling the pandemic. Soon after the release of the results of the Phase III clinical trial with the Pfizer-BioNTech COVID-19 vaccine, it was approved by the Saudi authorities for use . COVID-1Vaccine hesitancy for a COVID-19 vaccine booster is a public health challenge. Morbidity and mortality associated with the spread of the Omicron variant can be reduced by an increased acceptance of the COVID-19 booster dose strategy, especially among the high-risk population including HCWs. However, emerging evidence suggests that vaccine hesitancy for a COVID-19 vaccine booster dose is present among a considerable proportion of the fully vaccinated general public . A recenTM forms. This study was conducted from 1 October 2021 to 30 November 2021. The Saudi government announced the availability of the COVID-19 booster dose for high-risk individuals including healthcare workers on 20 October 2021, and the COVID-19 booster dose became available for everyone above the age of 18 on 7 November 2021. The institutional ethical committee has approved this study with approval number UJ-REC-027. The STROBE guidelines for cross-sectional studies were followed while reporting this study\u2019s findings [The present study was an online survey using the snowball technique to recruit participants using GoogleThe questionnaire was prepared in English along with its Arabic translation. The translation of the questionnaire was performed according to the standard procedure involving independent bilingual experts. The questionnaire was pilot tested among 20 participants before the beginning of the study. The questionnaire\u2019s link was shared with the author\u2019s social media contacts in the health sector . The intention to receive a COVID-19 booster dose was rated using a four-point scale . The vaccination preference was also rated using a four-point scale (completely confident to completely non-confident).The fourth part of the questionnaire was related to the health belief model (HBM) in the context of the COVID-19 illness and booster vaccination [t-test. Binary logistic regression using the vaccination intention as the dependent variable was utilised to analyse the HBM construct predictors of COVID-19 booster dose uptake. The model fit of logistic regression analysis was tested using the Hosmer\u2013Lemeshow goodness-of-fit test. The forward LR method of regression was used to obtain odds ratios (OR) and 95% confidence intervals (95% CI). The statistical significance level was kept at p < 0.05.The Statistical Package for Social Sciences  software was employed in data analysis. The characteristics of the study population were explored using descriptive statistics. The statistical significance among study variables was analysed using chi-square tests as well as independent sample This study received 2059 complete responses. Health beliefs about the susceptibility for COVID-19 infection were mixed for the study participants. Of all participants, 64.2% disagreed with the statement regarding the greater chance of getting infected with COVID-19, and only 40.2% reported their worries regarding the higher chance of getting COVID-19. However, 74.7% of survey participants still thought that acquiring COVID-19 is a possibility for them right now. The study participants had relatively higher health belief perceptions regarding the severity of the COVID-19 infection. Of all participants, 84.2% agreed that the COVID-19-related complications are serious, and 57% still admitted their fear of getting COVID-19 infection. However, only 28.3% agreed that getting COVID-19 will make them severely sick. The benefits of COVID-19 booster dose vaccination were viewed positively by the trial participants. Of the participants, 54.7% agreed with the statement that the booster COVID-19 vaccination is a good step to reduce their fear of catching COVID-19, and 69.3% agreed that receiving the booster COVID-19 vaccine decreases their chances of catching COVID-19 infection and its complications. The study participants expressed fewer perceived barriers to COVID-19 booster dose vaccination. A total of 46.1% were concerned with the possible side effects, 43.1% were concerned with the efficacy of the COVID-19 booster dose vaccination, 37.9% expressed concerns regarding the safety of the COVID-19 booster dose vaccination, and 34.2% expressed concern regarding faulty/fake COVID-19 booster dose vaccination. Regarding cues to action, 75.4% of the study population reported that they would take the booster dose of COVID-19 vaccine only if they were given adequate information about the vaccine, and 62% reported that they would only take it provided the booster dose was taken by many healthcare workers in Saudi Arabia. p = 0.001), with more people choosing locally manufactured vaccines. There was also a statistically significant association between vaccine preference and confidence in the foreign-made vaccines , with a higher number of respondents who reported confidence in foreign-made vaccines preferring foreign-made vaccines. Males , non-Saudi nationals , education level masters and above , monthly income more than SAR 15,000 , and those with family history of COVID-19 infection  expressed significant preference for foreign-made vaccines. The findings are summarised in A majority of the study participants (64.5%) expressed their confidence in the domestically made COVID-19 vaccine. However, a higher proportion (80.6%) reported their confidence in the foreign-made/imported vaccines. Of the participants, 47.6% expressed a preference for foreign-made COVID-19 vaccines, whereas only 14.2% expressed a preference for domestically made vaccine. There was a statistically significant link between vaccination preference and confidence in local vaccines  reported their intention for the COVID-19 booster dose uptake: 39.8% responded definitely yes and 31.3% responded probably yes. On the other hand, 17.4% of the respondents expressed that they were probably not intending to receive the COVID-19 booster dose uptake, and 11.5% also responded with definitely no intention for COVID-19 booster dose uptake. p < 0.001). There was also significant association between definite intention to receive a COVID-19 booster dose and nationality , marital status , education level , monthly income , and co-morbid medical illness . The findings are summarised in p < 0.001). There was a significant association between the constructs in the HBM model and COVID-19 vaccine (booster) intention. The findings are summarised in In the chi-square test, there was a significantly higher definite intention for COVID-19 booster dose uptake among males  dose of COVID-19 vaccine as a good idea to decrease fears regarding getting COVID-19  and perceptions that receiving the booster dose reduces the risk of getting COVID-19 or its complications , under the perceived benefit construct, were the strongest predictors of a definite intention to receive a COVID-19 booster dose. Being concerned with safety  under the perceived barriers construct was the strongest significant correlate of having no definite intention to take the COVID-19 vaccination. The findings are summarised in This study found that overall, 71.1% of the study population reported their intention for COVID-19 vaccination booster dose uptake, and a definite intention was reported by 39.8%. The results of the present study are in agreement with similar published studies. A cross-sectional survey among HCWs from Czechia reported that 71.3% reported positive intention for a booster dose of COVID-19 vaccination uptake . AnotherIn addition, factors influencing the intention to receive a third (booster) dose of COVID-19 immunisation and the role of the HBM in predicting vaccination intention among KSA healthcare workers (HCW) were investigated in this study. The study was started before the Saudi government approved the COVID-19 booster dose vaccination for HCW. The findings of the present study showed that HBM constructs of perceived benefit and perceived barriers significantly predicted COVID-19 booster dose vaccination uptake. However, the other HBM constructs  did not significantly influence the definite intention for the third (booster) dose of COVID-19 vaccination among HCWs in the KSA. To the best of our understanding, no published studies are available exploring COVID-19 booster uptake intention using HBM. However, previous studies that analysed COVID-19 vaccination intention using HBM also reported similar results. Similar to our study results, a Malaysian study reported that the HBM constructs  significantly predicted a definite intention for COVID-19 vaccination uptake . A recenThe study results showed that perceptions regarding the susceptibility to COVID-19 and worries regarding the likelihood of getting COVID-19 infection were less among the majority of the study population. One of the reasons for the lower level of perceived susceptibility among HCWs could be that the majority of them were fully vaccinated and the rate of COVID-19 infection in KSA at the time of the study was significantly less. However, lower perceived susceptibility for COVID-19 infection might lead to less compliance with preventive measures against COVID-19 infection . There iThis study\u2019s results also showed that males, older participants, higher educational achievers, and respondents belonging to the high-income category had more acceptance for the COVID-19 booster dose vaccination, which is consistent with the published literature. Pal et al. explored attitudes towards the COVID-19 booster dose vaccine among US HCWs and found that younger age and lower level of educational attainment were associated with higher vaccine hesitancy . AnotherThere are numerous limitations to this study that must be considered when evaluating its findings. The present study utilised an online survey methodology to collect data, and such study designs are prone to selection bias. The HCWs were inquired regarding the behavioural intention for COVID-19 booster dose vaccination uptake in this study, which may not reflect actual behaviour. Various confounding factors which might influence vaccination hesitancy such as co-morbid psychiatric symptoms were not included in this study. A number of factors that could predict hesitancy toward receiving vaccines, such as adverse events after previous vaccine administration, being pregnant, and hesitancy toward other types of vaccines, were not included in this study. Finally, the questionnaire did not investigate COVID-19 vaccine anamneses.This study found that 71.1% of the study population reported their intention for COVID-19 booster dose vaccination uptake. Moreover, we also found that many HBM constructs predict vaccination intention for COVID-19 booster dose among HCWs in the KSA, which can be utilised to promote COVID-19 booster vaccination among HCWs."}
{"text": "Health authorities have struggled to increase vaccination uptake since the COVID-19 vaccines became available. However, there have been increasing concerns about declining immunity after the initial COVID-19 vaccination with the emergence of new variants. Booster doses were implemented as a complementary policy to increase protection against COVID-19. Egyptian hemodialysis (HD) patients have shown\u00a0a high rate of hesitancy to COVID-19 primary vaccination, yet their willingness to receive booster doses is unknown. This study aimed to assess COVID-19 vaccine booster hesitancy and its associated factors in Egyptian HD patients.A face-to-face interview was conducted with closed-ended questionnaires distributed to healthcare workers in seven Egyptian HD centers, mainly located in three Egyptian governorates, between the 7th of \u00a0March and\u00a0the 7th of April 2022.n\u2009=\u2009341) were willing to take the booster dose. The main reason for booster hesitancy was the opinion that a booster dose is unnecessary . Booster vaccine\u00a0hesitancy was associated with female gender, younger age, being single, Alexandria and urban residency, the use of a tunneled dialysis catheter, not being fully vaccinated against COVID-19. Odds of booster hesitancy were higher among participants who did not receive full COVID-19 vaccination and among\u00a0those who were not planning to take the influenza vaccine .Among 691 chronic HD patients, 49.3% (COVID-19 booster-dose hesitancy among HD patients in Egypt represents a major concern, is associated with\u00a0vaccine hesitancy with respect to other vaccines and emphasizes the need to develop effective strategies to increase vaccine uptake.The online version contains supplementary material available at 10.1007/s40620-023-01586-z. On the 11th March, 2020, coronavirus disease 2019 (COVID-19), the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was declared a pandemic by The World Health Organization (WHO) . It is cPatients on maintenance hemodialysis (HD) have a substantial risk of developing COVID-19. Such patients on HD typically have several medical comorbidities, including diabetes mellitus, obesity, and compromised immune system, all of which increase their risk of developing devastating COVID-19\u2013related complications. One study found that the estimated 90-day mortality among COVID-19-infected HD patients exceeds 20% in the United States . There iNevertheless, several studies showed that a considerable proportion of patients on dialysis were reluctant to take the COVID-19 vaccines \u201315. Vari, Alexandria, and Fayoum. HD patients with dementia and those with language barriers that would impair basic comprehension of the questions were excluded. After obtaining informed consent from eligible patients, a healthcare worker belonging to each HD unit explained the aim of the questionnaire to each patient and fulfilled the related questions. The study was conducted between the 7th of both\u00a0March and April 2022.This cross-sectional study recruited 691\u00a0chronic HD patients aged 18\u00a0years or above from seven different HD centers distributed mainly in three Egyptian governorates; DakahliaG*Power software was used for power analysis to enable sample size calculation in order to detect hypothesized effect , 18. UsiThe survey consisted of 27 questions with three major subheadings: demographic data (7 items), COVID-19 vaccine (10 items), and health of the responder and family (10 items). These subheadings followed the conceptual framework recommended by the Strategic Advisory Group of Experts on Immunization (SAGE) Working Group on Vaccine Hesitancy , 20, 21.The study protocol was reviewed and approved by the Mansoura University institutional research board with approval number: R.22.03.1639.P value\u2009<\u20090.05 was considered significant.The collected data were coded, processed and analyzed using Statistical Package for the Social Sciences (SPSS) program for Windows. Qualitative data were represented as frequencies and relative percentages. Chi-Square test was used to test the association between categorical variables. Binary logistic regression was utilized to test the association between independent and dependent variables. Subsequently, multivariate logistic regression was performed to compute the adjusted odds ratio (AOR) and 95% confidence interval (95% CI) for predictors of booster hesitancy. Among 692 chronic HD patients assessed for eligibility, 691 participants completed the survey , television , treating physician , and social media . Few patients  reported they had no information about COVID-19 vaccines. Of the studied patients, 486 (70.3%) were fully vaccinated against COVID-19. The most commonly received vaccine was Sinopharm , while Janssen / Johnson & Johnson was the least received vaccine . Table Participants of this study received information regarding COVID-19 vaccines mainly from dialysis staff , followed by safety uncertainties  and the side effects experienced after the previous doses . Unwillingness to receive the booster dose of the COVID-19 vaccine was significantly higher among females, those aged 18\u201330\u00a0years versus age above 50\u00a0years, single individuals, Alexandria and urban residents, dialysis via tunneled catheter, those who are not fully vaccinated, and vaccinated patients with moderate to severe side effects  were willing to take the booster dose, and the remaining 350 (50.7%) were booster-dose hesitant. The primary reason for hesitancy was the opinion that a booster dose is unnecessary  had no preference with regard to a specific vaccine. However, most participants who completed their initial regimen with AstraZeneca and BioNTech-Pfizer wished to receive a potential booster dose provided by the same manufacturer . In the case of Sinopharm, 46.4% (n\u2009=\u200952) of surveyed individuals were interested in receiving it as a booster dose, while in the case of Sinovac, 36.2% (n\u2009=\u200921) of subjects. The only patient who initially received Janssen / Johnson & Johnson vaccine and was willing to receive a booster dose preferred to receive the same vaccine.In general, 29.6% (101) of patients willing to receive a COVID-19 vaccine booster dose could not decide at the moment of the survey which vaccine to take, while 20.2% . The model correctly classified 71.8% of participants with 61.1% sensitivity and 82.7% specificity, and it explained 38.4% of the variance in booster hesitancy (Nagelkerke R2\u2009=\u20090.384). Of the 10 predictor variables, only not being fully vaccinated and not planning a new flu shot were the two statistically significant independent predictors of the likelihood of booster hesitancy. Participants who did not receive full COVID-19 vaccination and those who were not planning to take the influenza vaccine had 10.8- and 4.2-times higher odds of exhibiting booster hesitancy, respectively.Table Convincing the Egyptian population to receive the COVID-19 vaccine has already been a challenging process. Despite good public knowledge and awareness about COVID-19 severity and vaccine safety , hesitanThe present study shows male predominance (58.9%) among our surveyed HD patients. This is in agreement with the latest published statistics from the Egyptian Renal Data System (ERDS) which shows a 58.7% male predominance in the Egyptian HD population . The majApproximately 57% of surveyed individuals lived in multigenerational housing, which represents an independent risk factor for COVID-19 infection . While dOnly about one-quarter of the participants in the present study reportedly became infected with SARS-CoV-2. This could be partly explained by the fact that some patients might have had asymptomatic COVID-19 infection as reported in previous studies \u201335. AnotVaccination practice, hesitancy, and acceptance are influenced by the source of information , especiaAs the Sinopharm vaccine against COVID-19 was the first vaccine to be approved in Egypt, it was the most commonly received one by participants of this survey with nearly one-third of surveyed individuals receiving the full vaccination schedule (2 doses of Sinopharm vaccine). Patients reported many reasons for not receiving the vaccine at all or not completing the full schedule. Reasons reported by the patients were classified into 5 main categories; their confidence (which represented 50%), complacency, convenience or constraints, risk calculation and collective responsibility for protection of others through herd immunity .The current study highlights an ongoing dilemma encountered since the beginning of the COVID-19 pandemic, it reflects frustration and indecisiveness regarding COVID-19 vaccines and the booster dose. About half of the participants (49.3%) exhibited their willingness to receive the booster dose of the COVID-19 vaccine. The majority of booster-hesitant individuals (44.9%) considered the booster dose unnecessary, while surprisingly only a minority were booster-hesitant owing to the side effects experienced after previous COVID-19 vaccine doses. This is in agreement with the Algerians  but contAlthough results obtained from surveys in developed countries were encouraging, with rates of willingness to receive vaccine boosters varying from 61.8 to 95.5% in the USA, Poland, Czech Republic, Germany, Japan, China, and Denmark , 41\u201347, Indeed, these dissimilarities among countries regarding COVID-19 vaccine hesitancy could be ascribed to differences in sociodemographic and anamnestic characteristics, human behavior, sources of information about vaccines, control of rumors and misinformation, trust in the health care systems, available vaccines, uncertainties about vaccine side effects, and belief in vaccine benefits. Surprisingly, the results of this study indicate that not only differences in the rate of booster hesitancy are evident among countries but also within the same country. Residents of Alexandria were more likely to be booster hesitant than their counterparts in other governorates included in this survey, a finding that could be attributed to the high frequency of patients without full primary vaccination against COVID-19 in Alexandria (\u223c47%) compared with Dakahlia and Fayoum . Similarly, urban residents constituted more than two-thirds of the not fully vaccinated participants.The present study showed that various social and demographic factors are significantly associated with vaccine booster hesitancy. Regarding gender, females were significantly more booster-dose hesitant than males . This finding is in accordance with findings from other studies about initial vaccination hesitancy , 51. ThiRegarding the age of the participants in the present study, older participants were more likely to accept vaccine boosters than younger ones. This finding seems to match other studies that were carried out addressing the initial COVID-19 vaccination not only in the dialysis population , 54 but Concerning marital status, single individuals who had never married were more likely to be booster-dose hesitant than married, divorced, or widowed individuals . This is in agreement with a study carried out in Saudi Arabia which demonstrated that married individuals were more likely to accept vaccination .Educational level has been a matter of debate as regards its correlation with vaccine hesitancy, with most studies reporting an inverse relationship between education and vaccine hesitancy , 48, 51,A striking finding in multivariate analysis in the current study is that participants who were already fully vaccinated against COVID-19 were nearly eleven times more likely to accept booster doses. This suggests that acceptance of primary vaccination is strongly associated with willingness to accept a booster dose once recommended. This finding is similar to that found in adult Americans .Concordance between the answers on the influenza vaccine and the COVID-19 vaccine booster dose deserves further discussion: although, as expected, not having received the influenza vaccine was associated with a significantly higher likelihood of COVID-19 vaccine booster-dose hesitancy , the prevalence of influenza vaccine hesitancy was greater compared with COVID-19 vaccine booster-dose hesitancy (\u223c75% versus \u223c50%). Interestingly, those who were not planning or who had not yet\u00a0decided whether to receive the influenza vaccine were more likely to be COVID-19\u00a0booster-hesitant in the multivariate analysis. This could be attributed to a lower perceived severity of seasonal influenza than COVID-19 by patients on dialysis  or lack The current study demonstrates that the participants\u2019 preferences for a specific COVID-19 vaccine to be used as a booster dose did not necessarily match the previously administered vaccine. Surprisingly, nearly half of the participants willing to receive a COVID-19 booster dose did not specify any particular vaccine preference. They\u00a0did not choose a specific vaccination or make a decision at the time of the survey. This finding is similar to that found in the Polish population, of whom \u223c55% did not know which vaccine to receive as a booster dose or showed no preference for a specific vaccine. Although the highest level of vaccine agreement in the present study was seen for AstraZeneca, a low level of agreement was observed in surveyed participants in Poland and the authors attributed this finding to safety concerns and lower degree of trust in this vaccine perceived by the Polish population . On the This study provides significant insight into factors associated with COVID-19 vaccine booster hesitancy among Egyptian HD patients. To our knowledge, this is the first study to explore this issue. A notable strength of the study is that it is multicenter and the sample is representative of HD patients in three Egyptian Governorates. The questionnaire was completed by professional health care workers through face-to-face interviews, not web-based as in the case of most published literature, eliminating the idea of the presence of missing questions or data. Nevertheless, the study has several limitations. First, it is a cross-sectional study taking a snapshot of HD patients' willingness to take the booster vaccine, while in reality, individual attitudes are dynamic and evolving, and the intention to vaccinate is generally context-dependent. Second, as is the case for all cross-sectional studies, causality cannot be inferred from this design.Healthcare authorities in Egypt should be aware of the magnitude of the vaccine hesitancy problem among hemodialysis patients and act accordingly to resolve such a problem. The results of the current study could be used to identify solutions to the vaccine hesitancy problem. Focusing on the younger generations, female gender, unmarried, and unvaccinated patients to raise their awareness and fight back against false information is mandatory to decrease vaccine hesitancy. Since healthcare workers played a critical role in the delivery of information to the participants of the present study, they should raise awareness regarding the general attitude toward vaccination focusing on the importance of vaccination and the safety and efficacy of the available vaccines. Another effective strategy for reducing booster hesitancy, particularly for in-center HD patients, is the provision of COVID-19 vaccination as a component of routine in-center care which significantly reduced the odds of vaccine hesitancy in South Africa .COVID-19 booster-dose hesitancy is highly prevalent among hemodialysis patients in Egypt. Health authorities should invest in health promotion to disseminate the right medical information to improve vaccine uptake.Supplementary file1 (DOCX 24 KB)Below is the link to the electronic supplementary material."}
{"text": "The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is evolving,the newly emerged Omicron variant being the dominant strain worldwide, and this has raised concerns about vaccine efficacy. The purposes of this survey were to examine the extent to which healthcare workers (HCWs) intend to receive a second booster dose of the COVID-19 vaccine and the factors that influence their willingness to accept it.The study was conducted among HCWs who were randomly selected from four public hospitals in the Campania region, Southern Italy.A total of 496 HCWs answered the questionnaire (a response rate of 61.2%). Among the respondents, 20.8% indicated a score of 10, using a 10-point Likert-type scale, regarding the usefulness of a second COVID-19 vaccine booster dose. Physicians, HCWs who believed that COVID-19 was a severe disease, and those who have acquired information about the second booster dose from scientific journals were more likely to have this positive attitude. Slightly more than half of HCWs self-reported willingness to receive a second booster dose. Respondents who believe that HCWs are at higher risk of being infected by SARS-CoV-2, those who have a higher belief that COVID-19 is a severe disease, and those who have a higher belief that a second booster dose is useful were more willing to receive a second booster dose. The main reasons for those who had a positive intention were to protect their family members and patients, whereas, the main reasons for not getting vaccinated or for uncertainty were that the dose does not offer protection against the emerging variants and the fear of its side effects. HCWs of younger age, physicians, those who have a higher belief that a second booster dose is useful, and those who were willing to receive a second booster dose were more likely to recommend the booster dose to their patients.This study's findings highlight the necessity for designing and implementing educational interventions for improving second booster dose uptake and beliefs among HCWs and their capacity to recommend the vaccine to the patients. The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has generated more than half a billion confirmed cases and almost 6.5 million deaths around the world , 4, and From this point of view, it is, therefore, extremely important and crucial to understand and assess HCWs' willingness to have the second booster dose of the COVID-19 vaccine; no literature is available on this topic. Therefore, the purposes of this present survey were to examine the extent to which a large sample of HCWs in Italy intends to receive a second booster dose of the COVID-19 vaccine and the factors that influence their willingness for accepting it.The study was carried out from 12 July to 9 September 2022. The source population included all 4,000 HCWs who worked in different wards in four randomly selected public hospitals, one teaching and three nonteaching, located in the Campania region, Southern Italy. The sample for the present study included 496 HCWs who had been selected by a simple random sampling technique. A sample size of 384 HCWs was estimated assuming that 50% of the study population would intend to receive a second booster of the COVID-19 vaccine, 95% confidence interval, and a margin of error of 5%.Initially, the research team asked for permission from the health director of each hospital to conduct the study. After the approval, the team identified in each ward an HCW to distribute the questionnaire to the HCWs who were randomly selected from the list of those present at that moment in each ward and to collect the filled questionnaires within an envelope to maintain anonymity and to return the envelope. The questionnaire contained a brief introduction about the objectives, procedure, confidentiality, and anonymity of the survey, that the participation was voluntary, that the information provided will be used only for research purposes, and that the participant was able to withdraw at any moment. HCWs gave their informed consent to participate by handing in the questionnaire. The participants received no incentive to complete the questionnaire.All data were collected through a self-administered questionnaire adopted and modified from previously published studies of the research group \u201320. The Ethical approval of the study protocol and questionnaire was received from the Ethics Committee of the Teaching Hospital of the University of Campania \u201cLuigi Vanvitelli.\u201dt-test, to evaluate predictors of the different outcomes of interest. Only those variables with a p < 0.25 in the univariate analysis were entered into three multivariate logistic regression models to assess associations between the main dependent variables and the several independent variables. Then, multivariate logistic regression analysis with backward elimination of any variable that did not contribute to the model on the grounds of the Likelihood Ratio test (cut-off at p = 0.05) was performed. Variables whose exclusion altered the coefficient of the remaining variables were kept in the model. Backward stepwise selection has been used with a threshold of p = 0.2 and p = 0.4, respectively, for the entry or removal of the variables from the final models. Odds ratios (OR) and their corresponding 95% confidence intervals (CI) were calculated in the models. Three outcomes of interest have been identified: a) belief that a second booster dose of the COVID-19 vaccine was useful  (Model 1); b) willingness to receive a second booster dose of the COVID-19 vaccine  (Model 2); c) recommendation of a second booster dose of the COVID-19 vaccine to the patients  (Model 3). The following potential determinants were included in all models: gender ; age, in years (continuous); marital status ; physicians ; length of practice, in years ; having worked in a COVID-19 ward ; having underlying at least one chronic medical condition ; having been tested positive for COVID-19 ; at least one family member/colleague/friend who had been tested positive for COVID-19 ; perceived risk of getting infected with SARS-CoV-2 during the working activity ; belief that COVID-19 is a serious disease ; belief that HCWs are at a higher risk of being infected by SARS-CoV-2 ; scientific journals as source of information about the second booster dose of the COVID-19 vaccine ; and needing additional information regarding the second booster dose of the COVID-19 vaccine . Moreover, the variables belief that the second booster dose of the COVID-19 vaccine was useful  and belief that the second booster dose of the COVID-19 vaccine was effective  were included in Models 2 and 3; and the variable willingness to receive the second booster dose of the COVID-19 vaccine  was included in Model 3. For all analyses, two-tailed tests were used and statistical significance was determined with a p-value equal to or less than 0.05.All statistical analyses were conducted using the software STATA version 15.1. Descriptive statistics were used with frequency, mean, and standard deviation to describe the principal characteristics of the participants, as well as behavior and attitude toward having a second COVID-19 booster dose. Multiple logistic regression models were built using the strategy suggested by Hosmer et al. . Each vaA total of 496 HCWs, out of the 810 selected, answered the questionnaire with a response rate of 61.2%. The main sociodemographic, general, and professional characteristics of the respondents are summarized in The results regarding the attitudes, measured on a 10-point Likert-type scale, showed that the mean scores of the respondent's belief that COVID-19 was a severe disease and whether they feel at risk of being infected by SARS-CoV-2 during the working activity were 7.4 and 6.8, respectively, with 19.6% believing themselves to be at an elevated risk . The mean scores regarding the usefulness and efficacy of a second booster dose of the COVID-19 vaccine were 6.7 and 6, respectively, with only 20.8% and 16.4% of participants who had indicated a score of 10. Among those respondents who had not had the second booster dose of the COVID-19 vaccine, 52.6% self-reported a willingness to receive it, and 25.1% and 22.3% indicated that they had \u201cno intention\u201d or showed \u201cuncertainty.\u201d The main self-reported reasons for those who had a positive intention were to protect their family members (49.6%) and their patients (42.9%) and the fear of acquiring the infection (37.6%). The main reasons for not getting vaccinated or for uncertainty, however, were that the dose does not offer protection against the emerging variants (54.6%) and the fear of its side effects (27%). Three variables were found to be associated with the HCWs' willingness to receive a second booster dose in the multivariate logistic regression analysis. Respondents who believed that HCWs are at higher risk of being infected by SARS-CoV-2 , those who have a higher belief that COVID-19 was a severe disease , and those who have a higher belief that a second booster dose is useful  were more willing to receive a second booster dose of the COVID-19 vaccine . The internet (51.8%), mass media (48.6%), scientific meetings (48.2%), and scientific journals (41.5%) were indicated as primary sources for this information, followed by social networks (26.7%). More than one-third of the respondents expressed an interest in acquiring additional information about the second booster dose (36.3%).To our knowledge, this is the largest survey of HCWs' willingness to have a second booster dose of the COVID-19 vaccine and the factors associated with this decision conducted in Italy. The major findings can be summarized in the following five points. First, slightly more than 50% of the sample would accept a second booster dose of the COVID-19 vaccine. Second, the main reasons behind the willingness to have a second booster dose were to protect family members and patients. Third, the main reasons for the intention to not receive or uncertainty toward the second booster dose were the belief that it does not offer protection against the emerging variants and the fear of side effects. Fourth, scientific meetings and journals were among the primary sources of information on the second booster dose. Fifth, several determinants have been observed to be significantly associated with the different outcomes of interest.Overall, the present survey revealed that only 52.6% of respondents self-reported a willingness to receive a second booster dose. Though it is only mandatory for HCWs to have the first COVID-19 booster dose, it was nonetheless a striking and unexpected finding that very few (48.1%) eligible HCWs had received a second booster dose. The prevalence of this willingness was lower than the values observed among HCWs in Saudi Arabia (55.3%) , CzechiaThis study highlighted that the protection of their family members and patients and the fear of acquiring the infection were the most frequent reasons for the willingness to receive a second booster dose of the COVID-19 vaccine. These findings are consistent with other recent similar research studies \u201338. A poThe results of the multivariate logistic regression analysis showed that several factors were significant predictors of attitude, vaccine willingness, and vaccine recommendation. Of the several sociodemographic and professional characteristics, only age and professional role were associated with the outcomes of interest. Indeed, physicians indicated a higher score regarding the usefulness of a second booster dose of the COVID-19 vaccine, and as, those younger, they were more likely to recommend this booster dose to the patients and more willing to receive it. Moreover, three variables related to the respondents' attitudes have had a significant impact. HCWs who believed that COVID-19 was a serious disease and who believed that they are at higher risk of being infected by SARS-CoV-2 were more likely to believe that the second booster dose is useful and more willing to receive the booster dose, and HCWs who believed that the second booster dose is useful and who were willing to receive it were more likely to recommend the booster dose to the patients. Therefore, it is extremely important that the HCWs should be aware of the vaccine's effectiveness in preventing SARS-CoV-2 infection and to improve their attitudes as an effective way to enhance HCWs' willingness to be vaccinated with a second booster dose of the COVID-19 vaccine or to recommend it. Some of these associations have been observed in a previous investigation .This present survey showed that almost all HCWs had received information related to a second booster dose of the COVID-19 vaccine, with scientific meetings and journals being two of the most trusted sources. It is important to highlight that scientific journals have a significant effect on the higher belief regarding the usefulness of a second booster dose. This finding confirms that these sources are an important factor in the vaccination process and decision. Indeed, this association is in accordance with previous studies that showed that scientific journals played a significant role in determining a higher level of knowledge, a more positive attitude, an increase in the willingness to receive a vaccine, and a higher vaccination coverage among those who have acquired information from these sources , 18. MorThe results from the present survey should also be considered with some potential methodological limitations. First, as in all cross-sectional studies, no causal relationships between the independent variables and the different outcomes of interest can be established. Second, the survey was administered to HCWs in a single geographic area, and therefore, the findings may not necessarily apply to other areas of Italy. Third, a self-reporting questionnaire may have introduced social desirability bias and the surveyed HCWs may tend to have more positive attitudes that lead to an overestimation of their intention to have a second booster dose of the COVID-19 vaccine. However, an anonymous questionnaire has been used to reduce this bias. Despite these limitations, this study was the first to assess the willingness to have a second booster dose among HCWs in Italy, and it thus provides an important picture with important implications for health policymakers.In conclusion, this survey reveals a low willingness to receive a second booster dose, the facilitators and barriers influencing this willingness, and the factors associated with this choice. The findings have important implications and highlight the necessity for designing and implementing targeted education interventions for improving the second booster dose of the COVID-19 vaccine uptake among HCWs and their capacity to recommend the vaccine to the patients. In the future, investigations are expected to quantify the coverage level in HCWs and to evaluate whether they can promote this vaccination with special attention toward more vulnerable people.Walter Longanella , Mario Massimo Mensorio , Federica Cantore .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 Teaching Hospital of the University of Campania Luigi Vanvitelli. The patients/participants provided their written informed consent to participate in this study.GDP, GMdG, LF, and AN participated in the conception and design of the study and contributed to the data collection, data analysis, and interpretation. IFA the principal investigator, designed the study, was responsible for the statistical analysis and interpretation, drafted and wrote the article. All authors have read and approved the final version of the article and agree to be accountable for all aspects of the work."}
{"text": "Backgroundand objectives: Although several vaccines have been produced and administered around the world, new SARS-CoV-2 worsened the COVID-19 infection risk and impacted the initial vaccine dosage effectiveness. Based on studies indicating that the third and fourth COVID-19 vaccine doses significantly reduced COVID-19 transmission, Saudi Arabia has been administering COVID-19 booster vaccine doses to its citizens. The purpose of this study was to evaluate the uptake of the COVID-19 vaccine booster in relation to the socio-demographic characteristics and other associated factors among the Saudi population. Materials and Methods: This study was an online analytical cross-sectional study using a self-administered questionnaire. Pearson Chi-square test and multiple logistic regression analyses were used to determine factors associated with the uptake of COVID-19 booster dose vaccines. Results: A total of 2332 responded to our study. Overall, 527 (22.6%) participants had received a booster dose. An age of 55 and above , Eastern region , history of influenza vaccination at annual intervals , the first dose of Moderna vaccine , and cancer  were independent factors most associated with a higher uptake of the COVID-19 vaccine booster dose. In contrast, the second dose of Moderna vaccine , AstraZeneca vaccine , strong symptoms from side effects after the second dose of the COVID-19 vaccine  were independent factors most associated with a lower uptake of the COVID-19 vaccine booster dose. Conclusions: Our findings indicate low COVID-19 vaccine booster uptake. This necessitates the need for strategies to address discouraging factors of the COVID-19 vaccine booster dose uptake and engage the Saudi population to raise awareness about the importance of the booster dose. In December 2019, cases of atypical pneumonia due to an unknown cause were reported to the World Health Organization (WHO) from Wuhan, China . SeveralImmunization experts have carefully considered whether COVID-19 booster doses for specific susceptible groups or everyone are necessary to bolster immunity in order to combat the newly emerging strains of COVID-19 and there is uncertainty about the duration of protection offered by the vaccines . As publA study at Ajman University showed that the average knowledge score was 44.6% for the third COVID-19 booster dose, but the attitude score was 70.2% . In thisWe conducted a cross-sectional study from December 2021 to March 2022. All adults aged 18 years and older who were vaccinated against COVID-19 and had taken the vaccine booster dose were eligible for this study. We used an anonymous and self-administered questionnaire designed following the examples of previous similar studies assessing COVID-19 vaccine acceptance and uptake ,14,15,16The uptake of the COVID-19 vaccine booster dose was our study outcome variable. Participants were asked if they had received the vaccine booster dose and had to answer YES or NO.The age variable was categorized into: 18\u201325, 26\u201335, 36\u201345, 46\u201354, and 55 and above years. Gender was binary: male or female, as was marital status: married or single. Residence was divided in five regions: Northern, Southern, Western, Eastern, and Central. Education level was divided into four levels: below secondary school, secondary school, bachelor\u2019s, and postgraduate degrees. Employment status, healthcare work employment type, obesity status, and smoking were dichotomous, and responses were YES or NO. Monthly income in Saudi Riyal (SAR) was grouped into 3 categories: less than SAR 5000, SAR 5000\u201310,000, and more than SAR 10,000.Participants were asked about vaccination history. Answering a question about influenza vaccine, they had to choose from \u201cnever got before\u201d,\u201d in irregular intervals\u201d, or \u201cannual intervals\u201d. Answering a question about the first or second dose of COVID-19 vaccine, they had to choose from \u201cPfizer\u201d, \u201cModerna\u201d, \u201cOxford AstraZeneca\u201d, or \u201cother\u201d. The respondents were asked if they contracted COVID-19 infection and had to answer YES or NO. Regarding when they got COVID-19 in relation to vaccination, they had to choose from \u201cbefore the first dose\u201d, \u201cafter the first dose\u201d, or \u201cafter the second dose\u201d.Vaccine side effect symptoms or COVID-19 symptoms experienced were grouped into: no symptoms, mild symptoms , moderate symptoms (symptoms needing treatment taken at home), strong symptoms , and critical (ICU admission). Then, participants were asked to list the symptoms experienced.On a three-point Likert scale , participants gave answers to statements about their trust in vaccines, healthcare professionals, public health and government institutions, different reasons for them to get COVID-19 vaccines, preventive recommendations, and keeping up with new COVID-19 information.Participants were asked about chronic diseases, and they had to mention which chronic disease they had if they responded \u201cYes\u201d. Finally, to know about the sources of COVID-19 information, participants were asked to list their most common sources of information about COVID-19 and its vaccines.p value less than 0.05 was statistically significant. A descriptive analysis based on the frequency and percent distribution was conducted for all variables. Crosstabulation was used to compare the intention to take up COVID-19 vaccine booster dose among variables and groups. Relationships between variables were tested using Pearson Chi-square test and an exact probability test for small frequency distributions. Subsequently, all variables with a statistically significant association with the dependent variable (received booster dose) were included in the multiple predictive models based on the backward stepwise method with a p-value of <0.05 as an entry criterion and a p-value of >0.05 as an exclusion criterion. Multiple logistic regression analyses were used to estimate adjusted odds ratios (aOR) and their 95% confidence intervals (95% CI) for the association of getting the COVID-19 booster dose with the predictors.Data were extracted, revised, coded, and entered into IBM SPSS version 22  statistical software. All statistical analysis was done using two-tailed tests, and a The Research Ethics Committee at Security Forces Hospital Program in Holy Capital (HAP-02-K-052) reviewed and approved this research project being conducted. The approval number is ECM#0460-190122.A total of 2332 people participated in this study. Of all the participants, the majority (49.1%) were aged 18\u201325 years, were not obese (88.0%), and 51% were males. Most (61.4%) were not married, had a bachelor\u2019s degree (70.8%), and were not employed (57.7%). Most employed participants (14.5%) worked outside the healthcare sector (85.5%). Of all the participants, the majority (57.6%) earned less than SAR 5000 monthly (57.6%), lived in the Central region (39.0%), and were nonsmokers (81.9%) .p < 0.01) got vaccinated more with the booster dose (47.5%) than those aged 18\u201325 years (13.9%) despite the latter being the most predominant age group. More males (26.5%) significantly received the booster dose (p < 0.01) than females (18.5%). Being married was associated with almost double take-ups of the booster dose (30.1%) (p < 0.01) than being single (17.5%). The booster dose was received significantly more by participants in the Eastern region (33.6%) (p < 0.01). A higher education level was associated with more booster doses, with those with postgraduate degrees (41.3%) (p < 0.01) receiving significantly more booster doses. The COVID-19 vaccine booster dose was received significantly more by healthcare workers (p < 0.01) and employed participants (p < 0.01) in general . Taking up the booster dose significantly increased with the increase in monthly income (p < 0.01). Those who earned more than SAR 10,000 were more than twice as likely to receive the booster dose than those who earned less than SAR 5000 (34.8% vs. 15.6%). Obese participants and smokers were also significantly more likely to take up the booster dose (p < 0.01).Overall, 527 (22.6%) participants had received the booster dose. Older age was associated with a higher uptake of the COVID-19 booster vaccine, which increased with age. Those aged 55 years and more significantly (p < 0.01). Participants who took influenza on an annual basis had a higher booster dose uptake rate (38.6%). The types of the vaccine on the first dose (p < 0.01) and the second dose (p = 0.002) were significantly associated with the uptake of the booster dose. Participants who took the first or second doses of the Moderna COVID-19 vaccines had the lowest booster dose uptake rates . However, 71.8% and 77.4% received Pfizer vaccines as the first and second doses, respectively. Experiencing side effects on the second dose (p < 0.01), being infected with COVID-19 (p = 0.006), COVID-19 symptom severity (p < 0.01), having COVID-19 after receiving both doses (p < 0.01), and types of symptoms experienced (p = 0.028) were significantly associated with lower COVID-19 booster dose uptake. Participants who reported experiencing adverse effects following their second doses, and those who faced critical symptoms (admitted to ICU) were the least vaccinated with booster doses . However, the difference was not statistically significant for the first dose receivers (p = 0.34).Regarding COVID-19 and infection vaccination history, participants with a history of previous influenza vaccines were significantly more likely to take up the COVID-19 vaccine booster dose  . In termp < 0.01). However, practicing preventive measures and following up with COVID-19 news did not substantially impact the booster uptake (p > 0.05) , WHO (85.3%), healthcare practitioners (82.1%), the effectiveness of all vaccines (85.7%), COVID-19 vaccines (84.3%), and were not satisfied with the previous COVID-19 vaccine doses taken (84.2%). Overall, loss of trust in healthcare and public health professionals and institutions was significantly associated with less booster uptake ( > 0.05) .Having comorbidities (n = 501) was significantly associated with booster uptake (p < 0.01), with the most uptake (53.8%) in cancer patients, while the least was in patients with immune deficiency diseases .p < 0.05)  .Participants aged 55 and above were three times more likely to get a booster dose than those aged 26\u201335 years . Living in other regions other than the East and West were not significantly associated with the booster dose uptake. However, living in the East was almost two times more associated with the booster dose uptake . Influenza vaccination at annual intervals was two times more associated with more vaccine booster doses  than irregular influenza vaccination. Although the first dose of the Moderna vaccine was associated with more booster dose uptake , the second dose of the Moderna vaccine was associated with about 21% less booster dose uptake , while the second dose of AstraZeneca vaccine was associated with 30% less booster dose uptake . On the other hand, participants who did not lose or have faith in COVID-19 were also more likely to get booster doses . Participants who had cancer were two times more likely to get booster vaccine doses than those without cancer .Even if it is expected that the worse their side effect symptoms are, the less people are less likely to take up the booster dose, participants who experienced critical symptoms were 19% less likely to get a booster dose , while those with strong and moderate symptoms were 39%  and 29%  less likely to get a booster dose, respectively. Other variables analyzed were not statistically significant predictors.p = 0.000). However, those who followed scientifically developed resources, such as articles, health practitioners, the WHO, and the Ministry of Health, had the highest uptake of booster doses. In contrast, those who got information from social media, celebrities, influencers, friends, and family had the lowest COVID-19 vaccine booster uptake , and uptake of the COVID-19 booster dose increased as the education levels increased. This can be explained by the fact that most high-income earners are leaders in different sectors who had to be exemplary to other workers by getting vaccinated earlier, which made them qualify for a booster dose earlier too. That is also in line with education\u2019s impact on the take up of the COVID-19 booster dose. Highly educated people usually occupy high-income job positions and are more likely to be better informed on vaccines as their sources of information are more likely to be genuine, such as scientific papers and healthcare and public health professionals\u2019 contacts. Some studies have reported similar findings as ours, and in addition, it has been reported that higher education institutions were more likely to impose vaccine mandates . This miMore participants who had obesity and who were smokers received booster doses than others. COVID-19 was found to be more severe in individuals with chronic conditions and obesity, and the COVID-19 vaccine booster dose is recommended more in people with comorbidities. Obesity and smoking are known risk factors for most chronic diseases, and most participants who were smokers might have other chronic diseases. Therefore, these conditions might have encouraged them to receive the booster doses .Our findings also indicated that the majority of the participants received Pfizer vaccines, followed by Oxford AstraZeneca. The efficacy of these two vaccines is believed to be the same with similar side effects. In Saudi Arabia, Pfizer vaccines were distributed more than any other vaccine type. Generally, the Pfizer and AstraZeneca vaccine side effects were mostly mild. We found a decrease in side effects as the severity increased for Pfizer and AstraZeneca vaccines, with more severe side effects reported after the second dose. Side effects for Moderna vaccines were mostly the same throughout. Previous studies have reported reduced side effects for the Pfizer vaccine compared to Moderna vaccines . On the A history of taking annual influenza vaccine was associated with a high uptake of COVID-19 booster vaccines. This might be because participants who take regular influenza vaccines are well informed about vaccines and perceive them as safe since they are used to them. In a study carried out in the UAE, they found similar influenza vaccine uptake as a factor for acceptance of COVID-19 vaccines .We found that participants who had COVID-19 infection after receiving a double dose of COVID-19 and who reported critical symptoms were the least vaccinated with booster doses. Several studies have shown that COVID-19 vaccines reduce the likelihood of infection and COVID-19 severity ,16,25. BThis study showed that adverse effect symptoms significantly influence the booster uptake, with shortness of breath being associated with the highest booster uptake rate, followed by anosmia, myalgia, and fatigue. These findings are similar to what was found in a study conducted by Meo et al. , who fouOverall, loss of trust in healthcare and public health professionals and institutions was significantly associated with less booster uptake. This might be caused by the frequent changes in the scientific information that comes out as science uncovers more about COVID-19, which can lead to mistrust in science, scientists, scientific institutions, and healthcare professionals and institutions. Most participants who did not receive a booster dose were not satisfied with the previous COVID-19 vaccine doses taken and lost trust in the Saudi Ministry of Health, WHO, healthcare practitioners, and the effectiveness of all vaccines. This can be explained by the possibility of reinfection after being fully vaccinated, which is contrary to what they thought before taking vaccines when they thought that it was completely protective against COVID-19. Strategies to approach them, more education programs focusing on different reasons why vaccines are not 100% protective, such as the new variants, and the effectiveness percentage of each vaccine, as well as education on the importance of a booster dose and COVID-19 vaccination in general, are needed. Our findings are also supported by other previous studies indicating that mistrust of science and health institutions could impede preventive measures and vaccination ,28. ThisComorbidities, such as diabetes, hypertension, cancer, respiratory disease, asthma, and renal and immunodeficiency diseases, were positively correlated with receiving booster doses. Cancer was associated with the highest booster vaccine uptake, followed by diabetes. People with chronic diseases have been encouraged to get vaccinated to lower their increased risk of mortality and morbidity from COVID-19, and it was found that people with higher perceived risks demonstrated lower vaccination hesitancy . This miVaccinated people have been gradually increasing as time goes on. In Saudi Arabia, data indicate that less than 5% of people were fully vaccinated in July 2021 . HoweverMultiple logistic regression analysis showed that age, residence region, being employed, previous influenza vaccination, type of COVID-19 vaccine received, side effect strong symptoms, faith in COVID-19 vaccine and having cancer remained significant independent predictors of COVID-19 vaccine booster dose uptake. This confirms that these factors had strong associations with the uptake of COVID-19 booster vaccine doses. These results are similar to comparative results we discussed above. However, logistic regression showed that participants who regularly tool influenza vaccine were two times more likely to take up COVID-19 booster doses than the ones who tool it irregularly. This is expected since people with a history of irregularities are more likely to keep up with COVID-19 vaccination schedules. Living in the Eastern region was two times more associated with the booster uptake than living in the Western region. The first cases of COVID-19 in Saudi Arabia were discovered in the East, which might have led to more efforts to combat the pandemic and more awareness among the residents and subsequently more and earlier uptake of vaccines . The earWe found that participants who followed scientifically developed resources such as scientific articles, health practitioners, the WHO, and the Ministry of Health had a higher uptake of booster doses compared to those who got information from social media, celebrities, influencers, friends, and family. This indicates that trusted sources of COVID-19 information are scientific articles, health practitioners, the WHO, and the Ministry of Health. Previous studies have indicated that social media and other unofficial sources of information were associated with more conspiracy theories about COVID-19 and vaccines, which could explain the lower uptake rates among participants who use them as their sources of information ,31. ZhanThe quantitative study design used limited the exploration of some aspects in detail, rather than in a structured format. A qualitative study design alongside the quantitative one would generate more information about COVID-19 vaccine booster doses. The convenience sampling method and online nature of this study cause under- or over-representation of the Saudi population and are prone to bias. Therefore, extensive experimental or longitudinal studies would mitigate these limitations. Since more people are becoming eligible for a vaccination with the COVID-19 vaccine booster dose every day, the results presented in this study might not accurately represent the current situation. We recommend further studies to remain up to date.This study showed that older age, male gender, being married, higher education levels, high income, previous influenza vaccine, having comorbidities, consulting the Ministry of Health, the WHO, healthcare professions, and scientific articles as sources of information were associated with a high uptake of the COVID-19 booster dose. In contrast, being vaccinated with Moderna COVID-19 vaccines, experiencing side effects on the second dose and critical symptoms, infection with COVID-19 after full vaccination, and loss of trust in healthcare, public health professionals and institution, and using social media, celebrities, influencers, friends, and family as sources of information were associated with a lower uptake of the COVID-19 booster vaccines. We recommend healthcare organizations, the government, and other authorities design strategies that augment the public\u2019s knowledge, awareness, perception, and debunk myths about the COVID-19 vaccine booster dose. Extensive longitudinal and offline studies with larger samples are also recommended to explore, in detail, the booster dose uptake."}
{"text": "A second COVID-19 vaccine booster dose is effective and safe for older adults. This study investigated hesitancy to take up a second COVID-19 vaccine booster dose and its determinants among older adults in Hong Kong. Participants were Chinese-speaking community-dwelling adults aged 65 years or above. Telephone numbers were randomly selected from up-to-date telephone directories. A total of 370 participants completed the telephone survey. Logistic regression models were fitted for data analysis. Among the participants, half (52.4%) were hesitant to receive the second COVID-19 vaccine booster dose. After adjustment for significant background characteristics, perceived benefits , cues to action , and perceived self-efficacy  of receiving the second booster dose were associated with lower vaccine hesitancy. Perceived barriers  and vaccine fatigue (tired of receiving repeated COVID-19 vaccination)  were associated with higher vaccine hesitancy. Level of hesitancy to receive the second booster dose was high among older adults in Hong Kong. Health authorities should address vaccine fatigue and modify perceptions related to the second booster dose. Globally, the ongoing pandemic of coronavirus disease 2019 (COVID-19) is still a severe public health issue. As of 12 December 2022, there have been 646 million confirmed cases and 6.6 million deaths caused by COVID-19 worldwide . The majCOVID-19 vaccination is one of the most cost-effective measures to control the pandemic. It is proven to be effective and safe in preventing deaths and other severe consequences caused by COVID-19 among older adults ,5,6. HowA second booster dose could help increase protection levels, especially for individuals in high-risk groups, such as older adults. Numerous studies demonstrated that a second booster dose was effective in reducing the risk of COVID-19-related outcomes , such as polymerase chain reaction-confirmed infection, symptomatic infection, severe illness, hospitalization, emergency department and urgent care counters and deaths ,16,17,18Based on the vaccine effectiveness and safety of receiving the second COVID-19 vaccine booster dose, on 18 August 2022, the World Health Organization (WHO) issued a practice statement and recommended countries consider a second COVID-19 vaccine booster dose in the populations at risk, including older people . Health Despite the promising evidence of COVID-19 vaccine effectiveness and safety, vaccine hesitancy is still a significant threat to the rollout of COVID-19 vaccine booster doses. Previous studies showed that older adults were less willing to receive the primary COVID-19 vaccination series and first booster dose than the younger groups in China ,30,31,32Vaccine fatigue refers to people\u2019s inaction toward vaccine instructions due to perceived burden and burnout . The neeCOVID-19 vaccinations and booster doses are hot topics on different media. Exposure to information related to COVID-19 vaccination influenced people\u2019s decision to take up such vaccines. The general population in China with a higher frequency of exposure to positive information supporting COVID-19 vaccination were more likely to complete the primary vaccination series . It is pIt is essential to understand vaccine hesitancy to receive the second COVID-19 booster dose and its associated factors among older adults in order to design effective health promotion strategies. However, there is a dearth of studies investigating the hesitancy to receive the second booster dose among older adults. To address the knowledge gaps, this study investigated the vaccine hesitancy to receive the second COVID-19 vaccine booster dose among older adults in Hong Kong, China. In addition, we examined the factors associated with the vaccine hesitancy to receive the second booster dose.We conducted a random telephone survey among community-dwelling Chinese-speaking adults aged 65 years or above in Hong Kong between 11 May 2022 and 11 July 2022. The number of local daily-confirmed COVID-19 cases was 254 on 11 May 2022 and slowly increased to 2558 on 11 July 2022. The COVID-19 situation in Hong Kong during the study period is illustrated in The inclusion criteria of the participants were: (1) community-dwelling Chinese-speaking individuals aged 65 years or above, and (2) having a Hong Kong ID card. Those who were not able to communicate effectively with the study interviewers were excluded.Simple random sampling was used. All the household telephone numbers  listed on the up-to-date telephone directories in Hong Kong were inputted into an Excel sheet. Using the random selection function in the software, about 4000 telephone numbers were randomly selected. Experienced interviewers conducted the telephone interview during 6:00\u201310:00 p.m. on weekdays and 2:00\u20139:00 p.m. on Saturdays to prevent under-sampling of individuals who worked on weekdays. Each number was called up to 5 times at different timeslots. Households were regarded as \u201cnon-valid\u201d  if no one answered the call after five times of calling. To avoid clustering effects, if there was more than one individual in a household aged 65 or more, the one with a birthday closest to the survey date was invited to join the study. Interviewers screened the eligibility of prospective participants and provided a study briefing to participants. Verbal informed consent was obtained from all participants. Prior to the interview, the interviewers used a checklist to confirm that the participant was fully informed about the study. There were six parts on the checklist: (1) questions to confirm eligibility, (2) scrips about study information, (3) the interviewers confirming they had introduced the research purpose, research process, main content of the survey, time required for completing the survey, rights of the participant, that non-participation would not affect the use of any services, and confidentiality of the research data, (4) the interviewers confirming that the participant fully understood the above contents, (5) the interviewers confirming that the participant verbally expressed his/her willingness to participate in the study, and (6) signature of the interviewers. No incentive was offered for study participation. The whole survey took around 20 min to complete. The same data collection method was used in a number of published studies ,40,41,42A panel of researchers in public health, behavioral health, and vaccination behaviors was formed to design the questionnaire used in this study. The questionnaire was pilot tested among 10 older adults to assess clarity and readability. All the older adults participating in the pilot study indicated that the items of the questionnaire were easy to understand and the length of the questionnaire was acceptable. These older adults did not participate in the actual survey. The panel finalized the questionnaire based on their comments.Information on socioeconomic characteristics was collected, such as gender, age, educational level, relationship status, employment status, household income level, whether living alone, and whether they were receiving Comprehensive Social Security Assistance (CSSA). Participants also reported their current health condition and history of seasonal influenza vaccination, pneumococcal vaccination, and COVID-19 infection.Information on the vaccination status of the participants, including the number of vaccine doses, the timing and type(s) of vaccine received and any side effects after vaccination, was collected. Vaccine hesitancy was measured using the same definition as in published studies ,44. It wRegarding perceptions about COVID-19, we added one new item \u201cHow high is your chance of having close contact with people having COVID-19?\u201d to a validated item measuring perceived susceptibility to COVID-19 ,38, and One single item was constructed to measure vaccine fatigue . The response categories for the above items were 1 = disagree, 2 = neutral, 3 = agree.Validated items were adapted to measure the frequency of exposure to the following contents on TV, radio, newspaper, and Internet: (1) people infected with COVID-19 after receiving three doses of COVID-19 vaccines, and (2) people recovered from COVID-19 without seeking medical consultation . We usedThe targeted sample size of this study was 360. We assumed 50% of the elderly population intended to receive a second COVID-19 vaccine booster dose. With the assumption of the prevalence of behavioral intention in the reference group (without a facilitating condition) to be 10\u201340%, this targeted sample size could be able to detect the smallest odds of 1.76 between individuals with or without a facilitating condition . The same sample size planning approach was used in published studies .p < 0.05 was considered statistically significant.We followed the statistical methods used in a number of published studies ,41,46. TAbout half of the participants were 65\u201369 years (49.2%) and female (60.8%). The majority of them were married or cohabited with a partner (74.6%), did not receive tertiary education (89.2%), were without a full-time or part-time job (86.2%), and had a monthly household income below HKD 20,000 (USD 2580) (73.8%). Over half had at least one chronic condition (60.3%). The most prevalent chronic condition was hypertension (46.8%), followed by diabetes mellitus (18.9%) and chronic cardiovascular diseases (10.8%). Among the participants, 25.4% reported a history of COVID-19 infection. At the survey time, 66.2% had received a seasonal influenza vaccination and 28.6% had received pneumococcal vaccination in their lifetime .Among the participants, 3.5% (n = 13) received a second COVID-19 vaccine booster dose. More participants chose Comirnaty  rather than CoronaVac  as their second booster dose. The majority reported no side effects (46.2%), and the side effects were very mild or mild (46.2%) with the second booster dose. The prevalence of hesitancy to receive the second booster dose was 52.4% .For perceived susceptibility, 3.8% and 24.0% of the participants perceived the chance of COVID-19 infection and having another wave of COVID-19 outbreak in Hong Kong was high/very high, respectively. For perceived severity, 21.1%, 10.5%, and 33.0% perceived the chance of having severe illness, financial difficulties caused by COVID-19, and transmitting COVID-19 to family members was high/very high, respectively .Regarding perceptions related to the second COVID-19 vaccine booster dose, the majority had positive attitudes toward the second COVID-19 vaccine booster dose, such as the belief that a second booster dose was highly effective in preventing severe consequences of COVID-19 (73.8%), could maintain their antibody level and strengthen the protection against COVID-19 (59.2%), and was highly effective in protecting them from the Omicron variant (47.8%). About 34.1% and 20.0% were concerned that the presence of chronic diseases would decrease the protection of the second booster dose and that the duration of protection offered by the second booster dose was shorter among people with older age, respectively. About a quarter (27.0%) reported that their family doctors would suggest them to receive the second booster dose and less than half (43.8%) reported that their family members would suggest them to receive the second booster dose. The majority (86.8%) were confident to receive the second booster dose. However, over half (54.3%) reported that they were tired of receiving repeated COVID-19 vaccination (vaccine fatigue) .Females had a higher level of hesitancy to receive the second COVID-19 vaccine booster dose than males  . After aKey findings of the study:Half of the participants (52.4%) were hesitant to receive the second COVID-19 vaccine booster dose.Perceived benefits, cues to action, and perceived self-efficacy of receiving the second booster dose were associated with lower vaccine hesitancy.Perceived barriers and vaccine fatigue (tired of receiving repeated COVID-19 vaccination) were associated with higher vaccine hesitancy.To our knowledge, this is one of the first studies to examine the hesitancy to receive the second COVID-19 vaccine booster dose and its associated factors among older adults in China. Factors at the individual level were determinants of hesitancy to receive the second booster dose. The findings provided a knowledge basis to develop tailored behavioral interventions to reduce vaccine hesitancy to receive the second booster dose among older adults.In this study, the socioeconomic and educational status of the study populations were similar to the general population aged 65 years or above reported by the Hong Kong government ,48,49. HThe findings provided some empirical insights for developing interventions to decrease vaccine hesitancy of receiving the second COVID-19 vaccine booster dose. The majority of participants perceived some benefits of receiving the second COVID-19 booster dose, notably that it was highly effective in preventing severe consequences of COVID-19 (73.8%) and it could maintain their antibody level and strengthen the protection against COVID-19 (59.2%). Those who scored higher in perceived benefit were less hesitant to receive the second booster dose. Future health promotion campaigns should strengthen such beliefs among older adults. About one-third of participants (34.1%) had concerns about the presence of chronic diseases that would decrease the protection of the second booster dose. One-fifth of the participants (20.0%) had concerns about the shorter duration of protection offered by the second booster dose among older adults. It is important to address such concerns among older adults as they were significantly associated with higher hesitancy to receive the second booster dose. Health promotions should clearly convey a message to the older adults that receiving the second booster dose could protect people with chronic diseases and there is no significant difference between the duration of protection for older adults and younger adults. Perceived cue to action and self-efficacy were both facilitators. Future programs should encourage significant others, such as family doctors and members, to give reminders to receive the second booster dose as a strong cue to action. To increase self-efficacy, creating a promotional video including a role model  demonstrating the specific procedures to take up the second booster dose and facilitating them to form an action plan are potentially useful strategies. This study had several limitations. First, selection bias existed due to non-response. The refusals might have different characteristics compared to the participants and we were not able to know how non-response would affect the results. However, our response rate was comparable to random telephone surveys on vaccination behaviors among community-dwelling older adults of previous studies ,41,42. SCommunity-dwelling older adults aged 65 years or above in Hong Kong reported a high level of vaccine hesitancy to receive the second COVID-19 vaccine booster dose. Health authorities should address vaccine fatigue and concerns about the interaction between the presence of chronic diseases and the second booster dose. Strengthening perceived benefits, involving significant others of older adults, and increasing perceived self-efficacy to receive the second booster dose might also be useful strategies in this age group."}
{"text": "Participants who reported reliance on information about the COVID-19 vaccination from the Ministry of Health websites were more willing to accept booster doses . The leading causes behind booster dose rejection were the beliefs that booster doses have no benefit (48.35%) and have severe side effects (25.6%). Determinants of booster dose acceptance were age (odds ratio (OR) = 1.02, 95% confidence interval (CI): 1.01\u20131.03, p = 0.002), information provided by the Ministry of Health , perceived susceptibility to COVID-19 infection , perceived severity of COVID-19 , and perceived risk of side effects . Booster dose acceptance in EMR is relatively high. Interventions based on HBM may provide useful directions for policymakers to enhance the population\u2019s acceptance of booster vaccination.Coronavirus disease (COVID-19) booster doses decrease infection transmission and disease severity. This study aimed to assess the acceptance of COVID-19 vaccine booster doses in low, middle, and high-income countries of the East Mediterranean Region (EMR) and its determinants using the health belief model (HBM). In addition, we aimed to identify the causes of booster dose rejection and the main source of information about vaccination. Using the snowball and convince sampling technique, a bilingual, self-administered, anonymous questionnaire was used to collect the data from 14 EMR countries through different social media platforms. Logistic regression analysis was used to estimate the key determinants that predict vaccination acceptance among respondents. Overall, 2327 participants responded to the questionnaire. In total, 1468 received compulsory doses of vaccination. Of them, 739 (50.3%) received booster doses and 387 (26.4%) were willing to get the COVID-19 vaccine booster doses. Vaccine booster dose acceptance rates in low, middle, and high-income countries were 73.4%, 67.9%, and 83.0%, respectively ( Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome (SARS-CoV-2), is still sweeping the globe. The pattern of infection and mortality due to COVID-19 differed significantly across countries . As of 10) by 53% , whereas NPIs and vaccination reduced transmission by 35% and 38%, respectively. So, both measures should be implemented simultaneously.COVID-19 control is based on the strength of non-pharmaceutical interventions (NPIs) and the pace of infection development or decay, the speed with which the vaccine can be rolled out, vaccine targeting and uptake, and vaccine characteristics . AlthougGlobally, a total of 12,613,484,608 vaccine doses have been delivered as of September 12, 2022 , in an aRecent clinical investigations have shown that the frequencies of confirmed COVID-19 and severe illness dramatically decreased with the third and fourth doses of inactivated or mRNA vaccine . Other rThe East Mediterranean region (EMR) consists of 22 countries , Tunisia, Syrian Arab Republic, Sudan, Somalia, Qatar, Pakistan, Oman, Occupied Palestine Territory, Morocco, Libya, Lebanon, Kuwait, Jordan, Iraq, Iran, Egypt, Djibouti, Bahrain, and Afghanistan). These countries are of different income levels and health system capacities . This gaStudies about the acceptance of COVID-19 vaccine booster doses in the EMR are scarce. A study in Jordan reported that about 50% of the population have concerns about the side effects of vaccination, which might prevent them from receiving booster shots of vaccines, and 45.3% thought that receiving a third dose of the vaccine would exacerbate the side effects . Higher In this study, we aimed to explore the acceptance of COVID-19 vaccine booster doses in low, middle, and high-income countries of the EMR and its determinants by using the Health Belief Model (HBM) constructions. The Health Belief Model is one of the most popular models for analyzing individuals\u2019 behavior towards vaccination against COVID-19 . The modA cross-sectional method was adopted to collect data from 14 EMR countries from March to June 2022. Based on the World Bank classification, the countries chosen were either low-income , middle-income , or high-income countries . An anonymous online survey was distributed by a team of researchers via social media platform and messaging platforms. A convenience and snowball sampling techniques were used to recure the participants who must meet the following eligibility criteria for participation in this study: aged 18 years or older, had received two doses of the COVID-19 vaccine (or a single dose of the Johnson & Johnson vaccine), had a mobile phone or computer, educated to self-complete the survey, and residents in the EMR during the COVID-19 pandemic.Epi-info 7 was used to estimate the minimum size of the required sample. With a 5% margin of error, 95% confidence level, 50% response rate, and a previously estimated rate of 60.0% for COVID-19 vaccine acceptance, a sample size of 642 (from the EMR) was considered sufficient. However, the sample size was doubled to compensate for stratification during the analysis of data and enhance the power of the study findings.A questionnaire of two domains was created to collect the data. The first domain collected (i) sociodemographic and health condition data  and (ii) attitude towards the booster dose .The second domain (HBM questionnaire) included 11 items that were used to assess the perceptions of COVID-19 infection and booster vaccination. The HBM consists of the following 6 domains: (i) Perceived susceptibility to COVID-19 infection: it consists of two items and refers to one\u2019s beliefs about the chances of worsening his/her health condition. (ii) Perceived severity of COVID-19 which refers to one\u2019s feeling about the seriousness of his/her health condition, ability to worsen, or failure to treat the illness. This item also consists of two items. Moreover, the perceived severity domain includes one\u2019s evaluation of medical and clinical outcomes such as fatality, infirmity, and discomfort, as well as potential consequences on her/his daily social activities such as the effects of their medical condition on work productivity, family activities, and social interactions and relations. According to the HBM, perceived threats were defined as the combination of perceived susceptibility and perceived severity. Thus, the first and second domains can be applied under one domain labeled \u201cperceived threats\u2019\u2019. (iii) Perceived benefit of COVID-19 booster indicates that the individual\u2019s beliefs about the perceived benefits of the various available actions for reducing the disease threat are influenced by his/her perception of the threat, regardless of whether or not that perception ended with a behavior change. This domain consists of three items, (iv) a perceived barrier to receiving COVID-19 booster doses, which indicates the impending negative consequences and aspects of a particular health action that may considerably act as obstacles to engagement in recommended health behaviors. This domain consists of three items. As per the HBM, perceived susceptibility and perceived benefits could only be potentiated by other independent factors, particularly by clues used to prompt actions, namely \u201ccues to action\u201d, such as physical condition, or by environmental factors, such as media. (v) self-efficacy has been elaborated on by the HBM as an essential factor that may encourage certain behavior. Moreover, it is defined as \u201cthe conviction that one can successfully execute the behavior required to produce the outcomes\u201d . The responses to the questionnaire\u2019s items were aggregated as follows: high (high/very high), neutral, and low (low/very low). All domains of the HBM questionnaire had an acceptable level of validity as Cronbach\u2019s Alpha value was \u22650.6, except the perceived barrier was 0.59 . Of the The questionnaire was administered in Arabic  and English  to eliminate the difficulties of language as previously suggested . The facAn electronic form of the questionnaire was designed using Google Forms and distributed via different social media (Facebook and Twitter) and messaging platforms (WhatsApp and emails) from March to June 2022. Before actual data collection, the research team tested the feasibility and accessibility of the online tool in a pilot study. Each collaborator was asked to submit at least two responses to determine the time required for completing the survey and the feasibility of the study. A total of 2327 individuals responded to the questionnaire. About 806 (34.6%) were excluded as they either did not receive the vaccination at all or did not complete the compulsory schedule of COVID-19 vaccination. Another 53 (2.3%) responses were omitted as the responders were not living in EMR countries or the responses were unsatisfactory. Finally, 1468 were included in the analysis; 375 (25.5%) refused to get the booster, 739 (50.3%) received booster doses, and 387 (26.4%) were willing to get the booster doses. In The Ethics Committee of the High Institute of Public Health, Alexandria University, Egypt approved the study. Information about the study\u2019s purpose, anonymity, confidentiality, voluntary participation, and privacy statements was provided on the survey cover page to all participants who were prompted \u201cto agree\u201d or \u201cnot to agree\u201d to participate in the study. Those who clicked \u201cI agree to participate\u201d were able to access the questionnaire. Data were protected and saved in a password-accessible computer available only to the principal investigator.p-value < 0.05 was considered statistically significant.The data were managed and analyzed using the R 4.2.1 software . Numerical variables were presented using mean \u00b1 standard deviation (SD), whereas nominal and categorical variables were expressed as a percentage (%). A Chi-Square test was used to assess the association between the nonnumerical variables, and the responses were categorized into Yes or No based on receiving COVID-19 booster doses. An independent t-test was performed to compare the difference between the means of two independent groups. Cronbach\u2019s alpha test was deployed to assess the internal consistency of the domains and their items. A binary logistic regression analysis was conducted to estimate the significant predictors\u2019 odds ratios, and a confidence interval of 95%  was reported. The dependent variable was the actual or virtual acceptance of COVID-19 booster vaccination, which was defined based on the following questions: \u201cHave you received the booster dose of COVID-19 vaccine\u201d or \u201cWill you get the booster dose of COVID-19 vaccine\u201d (Yes/No)\u201d. A The mean age of the respondents was 36.53 \u00b1 13.45, ranging from 18\u201388 years; females represented 62.7% (920); more than half of them were married 55.2%, (811); nearly one half 48.0% (705) had a university degree; about one-third 35.8% (526) were working in the medical field; 16.1% (237) had chronic diseases; and those who had COVID-19 infection before accounted for 51.1% (750); those who had relatives that had COVID-19 infection represented 86.4% (1268); and those with relatives who were immunocompromised and living in the vicinity of the participant made up 14.3% (210) of respondents .p < 0.001 There was a significant difference in booster dose acceptance among the included EMR countries, being the highest in low-income countries at 73.4% (141/192), and lowest in middle-income countries at 17.0% (480/707) at p = 0.001. The mean age of respondents who accepted vaccination was significantly older than those who accepted vaccination . The mean BMI of respondents who accepted booster doses tended to be higher than those who rejected (26.2 \u00b1 5.5 vs. 25.6 \u00b1 5.8). However, this difference was insignificant (p = 0.075). Being married was significantly associated with booster dose acceptance, as 77.7% of those who were married versus 69.6% of those who were single accepted booster dose, with p = 0.003. About 71.1% (533/750) of respondents -19 previously infected with COVID vs. 78.0 (560/718) of non-previously infected respondents accepted booster doses (p = 0.003). About 75.5% (959/1258) of those who accepted vaccination had no immunocompromised person living in the same context compared to 68.1% (143/210) of participants who had immunocompromised relatives (p = 0.028). Neither occupation, chronic diseases, nor having a relative who was infected with COVID-19 were associated with booster dose acceptance , ceptance .The leading causes behind booster dose rejection were the beliefs that the booster vaccine has no benefit 48.35% (181/375) and that it has severe side effects 25.6% (96/375). About 15.5% (58/375) refused to answer this question. p < 0.001) , the Center for Diseases Prevention and Control (CDC) website 53.6% (787/1468), followed by the Ministry of Health (MOH) website 41.3% (905/1468). However, 79.3% (718/905) of those who did follow the information provided by the MOH website accepted booster doses compared to 66.6% (375/863) of those who did not follow this source of information; this difference was statistically significant (< 0.001) .p < 0.05) except for question 13 (cues to action) as shown in All domains of the HBM  were statistically significant across vaccinated and unvaccinated groups (p = 0.002), information source as Ministry of Health and population  were significant determinants of COVID-19 booster dose acceptance. Regarding the HBM, perceived susceptibility to COVID-19 infection , perceived severity of COVID-19 , and perceived risk of side effects  were the significant predictors of COVID-19 booster dose acceptance.The present study demonstrated a high COVID-19 vaccination booster acceptance rate (74.46%) among the population in the EMR. This figure is substantially higher than a recently published study by Abuzaid et al., . They reIn the current study, vaccine acceptance was significantly higher in high-income countries compared to low- and middle-income countries. Indeed, high-income countries received more doses from manufacturers in order to vaccinate a larger proportion of their population. On the other hand, Arce et al.,  reportedIn our univariate analysis, being female, younger age, being single, having a university degree or being postgraduate, and previous COVID-19 infection were significantly associated with booster dose acceptance. However, in multiple regression, age and the information provided by the Ministry of Health were the only significant determinants of vaccine acceptance. On the other hand, Lai et al.,  reportedWe used the HBM to predict booster dose vaccine acceptance in the current study, and the questionnaire has a satisfactory level of internal consistency among the studied sample. In line of previous studies, our study findings showed that the HBM aspects served as a valuable framework for this study\u2019s assessment of participants\u2019 attitudes concerning a booster dose of the COVID-19 vaccination. In the univariate analysis, except for question 13, all items of all constructs of the HBM were significantly associated with booster dose acceptance. In contrast, in the multivariable analysis, perceived susceptibility, perceived severity, and perceived barriers were significant predictors of booster dose acceptance. For example, Wang et al.,  found thOur study aimed to look into the individuals who were hesitant to receive a COVID-19 vaccine booster to better understand the contributing factors. Interestingly, we found that most of them thought that booster doses had no benefit, and about one-fourth refused vaccination as they witnessed severe side effects among vaccinated individuals. A prior study by Al-Qerem et al., , similarIn the same vein, Lin et al.,  found thInfodemic have a negative impact on the COVID-19 pandemic in terms of the increased frequency of self-treatment,  non-compFinally, although the COVID-19 pandemic has been successfully controlled in many countries, SARS-CoV-2 still seriously threatens the world\u2019s health . Indeed,There are several limitations to this study. First, due to the intrinsic disadvantages of cross-sectional online surveys, a sampling bias may exist to limit the representativeness of the results . Second,A third dose of the COVID-19 vaccination was acceptable to 74.47% of the EMR inhabitants in this regional cross-sectional study. Acceptance behaviors, increased age and the HBM construct were highly associated. However, there may be some reluctance due to public skepticism regarding the safety and efficacy of current vaccines in practical settings. Our findings can therefore assist policymakers in creating more precise and scientific roll-out plans for the third COVID-19 vaccine, which is crucial when a further outbreak is still conceivable."}
{"text": "Females, individuals with normal body mass index, history of COVID-19 infection, and influenza-unvaccinated individuals were significantly associated with declining the booster dose. Higher fear levels were observed in females, rural citizens, and chronic and immunosuppressed patients. Our results suggest that vaccine hesitancy and fear in several highlighted groups continue to be challenges for healthcare providers, necessitating public health intervention, prioritizing the need for targeted awareness campaigns, and facilitating the spread of evidence-based scientific communication.COVID-19 vaccines are crucial to control the pandemic and avoid COVID-19 severe infections. The rapid evolution of COVID-19 variants such as B.1.1.529 is alarming, especially with the gradual decrease in serum antibody levels in vaccinated individuals. Middle Eastern countries were less likely to accept the initial doses of vaccines. This study was directed to determine COVID-19 vaccine booster acceptance and its associated factors in the general population in the MENA region to attain public herd immunity. We conducted an online survey in five countries  in November and December 2021. The questionnaire included self-reported information about the vaccine type, side effects, fear level, and several demographic factors. Kruskal\u2013Wallis ANOVA was used to associate the fear level with the type of COVID-19 vaccine. Logistic regression was performed to confirm the results and reported as odds ratios (ORs) and 95% confidence intervals. The final analysis included 3041 fully vaccinated participants. Overall, 60.2% of the respondents reported willingness to receive the COVID-19 booster dose, while 20.4% were hesitant. Safety uncertainties and opinions that the booster dose is not necessary were the primary reasons for refusing the booster dose. The willingness to receive the booster dose was in a triangular relationship with the side effects of first and second doses and the fear ( The coronavirus disease 2019 (COVID-19) outbreak has prompted remarkable research responses in various fields ,4,5. TheSix COVID-19 vaccines were approved in the Middle East and North Africa (MENA) region: mRNA-1273 , given as two doses with at least 28 days between doses ; Ad26.COThe clinical studies supported by legitimate reports and observations showed great effectiveness toward SARS-CoV-2 infection ,13. HoweDuring the follow-up process of 6 months, participants in the BNT162b2 vaccine clinical study revealed that effectiveness towards infection declined by around 6% every 8 weeks . While vThe Arab world\u2019s vaccine acceptance level is lower than that worldwide . A studyAccording to Bartsch et al., achieving public herd immunity requires vaccinating 70% of the population, and that in turn relies on the willingness of the public to accept it .Thus, assessing willingness to receive a COVID-19 booster dose is vital. To the best of our knowledge, no such reports or studies have been conducted in the MENA region. Hence, this study aimed to assess the attitudes of fully vaccinated adults in the MENA region toward receiving a COVID-19 booster dose and identify the related hesitation variables that contributed to their decisions.An online survey was conducted in the MENA region in November and December 2021. After translating from English to Arabic, we used a validated survey  originally designed by Rzymski et al.  after obThe main survey questions included: (\u2160) Are you fully vaccinated against COVID-19? (\u2161) Which COVID-19 did you receive? (\u2162) Please rate the severity of the side effects after receiving the first dose of the COVID-19 vaccine. (\u2163) Please rate the severity of the side effects that occurred after receiving the second dose of the COVID-19 vaccine. (\u2164) Are you willing to receive a booster dose of the COVID-19 vaccine if it is available? (\u2165) Please assess the level of fear associated with receiving the possible additional dose of the COVID-19 vaccine. (\u2166) Have you been infected with COVID-19? Moreover, fear and side effects were reported on a 10-point Likert scale (1 is the lowest and 10 is the highest).Participants had to be Arabic speakers, have received the first and second dose of COVID-19 vaccination (or only one dose in case of Ad26.COV2.S), be living in any MENA region country , and be at least 18 years old. The online survey was available in Arabic and was designed and hosted on Microsoft Forms. Participation was voluntary, and before filling out the survey, each participant had to confirm their consent to participate. We distributed the survey using social media and mailing lists.U test. Chi-squared test with Bonferroni correction was used to identify significant predictors for fear and willingness to receive the booster dose. Kruskal\u2013Wallis ANOVA was used to associate the fear level with the type of COVID-19 vaccine. Moreover, univariate and multifactorial logistic regression were performed to confirm the results and reported as odds ratios (ORs) and 95% confidence intervals (95% CI). p-value < 0.05 was considered statistically significant for all the results.Data were analyzed using TIBCO Statistica  and PQStat Software (version 1.8.2). OpenEpi was used to calculate the sample size according to the proportion. The following parameters were used: the population size was 100,000,000, and 5% was the confidence limit. The hypothesized frequency was set to 50%, and the design effect was 1. Therefore, for a 95% confidence level, the estimated minimum sample size was 384 per country. Missing data were treated by listwise and pairwise deletions. Categorical data were reported as frequency/percentage, and continuous data as median . Normality was calculated using Shapiro\u2013Wilk tests. Differences between side effects and fear among those willing to receive the booster dose were calculated by Mann\u2013Whitney A total of 4056 responses were collected, and only 3041 (75.0%) met the study criteria for further assessments (see n = 1520); (iii) were settled in urban areas; (iv) had received a bachelor\u2019s degree ; (v) were not infected by SARS-CoV-2; and (vi) had never been administered the influenza vaccine. Approximately 11.9% of respondents (n = 361) had at least one chronic disease, with asthma the most common , and there were  immunosuppressed patients. The majority of participants were vaccinated with BNT162b2 , followed by AZD1222  and BBIBP-CorV . The frequency of the administered vaccines is shown in A majority of the participants (i) were below 50 years old; (ii) had higher BMI  of respondents were willing to receive the COVID-19 vaccine booster dose, while 20.4% (n = 619) were hesitant. The principal reasons to refuse the vaccine were safety uncertainties , belief in the non-necessity of the booster dose , and side effects associated with previous COVID-19 vaccine doses .Overall, 60.2% (n = 133), followed by those vaccinated with Ad26.COV2.S  and BBIBP-CorV , while among those vaccinated with BNT162b2, 29.5% (n = 267) stated no desire to be vaccinated again.Individuals earlier vaccinated with AZD1222 were more willing to accept the booster dose , individuals with obesity (p < 0.02), those who regularly or irregularly receive influenza vaccination (p < 0.001), and who have not been infected by SARS-CoV-2 (p < 0.0001). Saudi citizens were more likely not willing to receive the booster dose compared with citizens in other countries in the region , n = 144; respectively; p = 0.01, z = 2.59).More serious side effects after the first and second dose were significantly higher among participants unwilling to receive the booster dose than those willing to receive it \u2014includinn = 263) with a fear level above 5. No significant differences were found between the type of COVID-19 vaccine and the fear of booster dose (p = 0.15). Participants aged <50 years, females, rural residents, immunosuppressed patients, and Saudi citizens reported higher fear levels . In general, 9.5% (n = 218) said they had no specific interest in the type of vaccine, while 17.8% (n = 410) said they could not decide at the time of the study. Nevertheless, the majority of individuals that fulfilled their first BNT162b2 and Ad26.COV2.S vaccination regimens wanted to get a booster dose with the same type of vaccine, if possible . In the case of mRNA-1273, 54.1% (n = 47) of the individuals questioned expressed interest in getting it as a possible booster dose. In contrast, the survey question about the AZD1222 type showed that only 46.6% (n = 342) of those questioned expressed interest in receiving it as a booster dose. Overall frequencies of top-selected booster doses appear in The participants willing to receive the COVID-19 booster did not prefer the same type of vaccine as previously. The individual responses regarding a specific COVID-19 vaccination they preferred as a future booster dose are shown in Several factors increase the willingness to accept a booster dose, such as being fully vaccinated with Ad26.COV2.S, AZD1222 or BBIBP-CorV. Those who received the mRNA-1273 vaccine showed less likelihood of accepting the booster dose. Regardless of the received COVID-19 vaccine, the side effects of previous doses and fear were associated with declining the booster dose. Participants infected with SARS-CoV-2 after the first dose were not likely to accept the booster dose. Males, Sudanese, Egyptians, or those with an influenza vaccination history were willing to accept the booster dose. The binary and multifactorial backward stepwise logistics of the significant predictors of accepting a COVID-19 vaccine booster dose are shown in The majority of the participants (60.2%) were willing to receive a COVID-19 booster dose, according to our findings. Fortunately, it seems that vaccine hesitancy is declining as more information on the vaccine\u2019s safety and effectiveness becomes available. However, there are still certain factors affecting individuals\u2019 decisions whether to receive the COVID-19 booster dose, including fear, side effects, several demographic factors, and previous vaccination with influenza vaccine.n = 2925) of the population of some Middle Eastern countries agreed to be vaccinated against COVID-19, while 32.6% were hesitant and 42.5% objected [It was reported that the COVID-19 vaccine acceptance rate in Middle Eastern countries was relatively low. Abu-Farha et al. reported that only 24.9%  showed that age variations were significant in Brazil, Ecuador, Mexico, and South Africa, where older people were more inclined to follow their employer\u2019s vaccination recommendations. However, younger people were much more likely to do so in France and the USA. Results were similar when compared to respondents from Italy, Poland, and Russia, in which younger people were more likely to express their intention to accept an employer\u2019s vaccine recommendation [p = 0.003, respectively). This can be explained as the elderly are considered a high-risk population for serious COVID-19 infection, suggesting that a booster dose might be advantageous in providing them with further infection protection [In many countries, younger generations are much more vaccine-hesitant, which impedes immunization progress ,36,37,38endation . Our stuotection .p <0.000001, respectively).Furthermore, vaccination reluctance has been higher among younger women in various nations ,42,43. In = 162). Several publications show that relying on social media can be dangerous, as they create an environment for spreading misleading information that may affect public mental health [Moreover, Thomas and Darling reported that the educational level more strongly influences people\u2019s willingness to obtain the vaccine. People of Asian heritage may be the sole exception, since they showed a high willingness to be vaccinated regardless of their educational level. In addition, people with lower levels of education were less trusting of the vaccines . Moreovel health ,46,47,48Regarding vaccine hesitancy and its association with the area of residence , the results from a survey of 1676 adults living in the United States, including Alaska and Hawaii (including interviews from 298 Hispanic adults and 390 non-Hispanic Black adults), showed that those who live in rural areas are still more likely to be vaccine-hesitant than those who live in suburban and urban areas . MoreoveSARS-CoV-2 infection status has also influenced people\u2019s decisions. After at least one vaccination dose, infected participants had an almost 50% lower probability of accepting a booster dose. At the same time, those who were regularly or irregularly vaccinated with the influenza vaccine had higher odds  of accepting a booster dose. These findings imply that previous vaccination experience dramatically influenced people\u2019s decisions to receive a booster dose.The immunosuppressed and chronic patients were willing to receive the vaccine but reported a higher fear level than normal individuals. However, immunosuppressed patients with a fear level above 5 had higher odds of rejecting the vaccine, as shown in A tendency to favor American-based vaccines was observed in a survey released in Saudi Arabia, Lebanon, Jordan, and Iraq, since 45.2% of the participants favored BNT162b2, whereas 30% were unfamiliar with different types of COVID-19 vaccines . Almost Finally, it is worth mentioning that this study exhibits some strengths and potential limitations. First, regarding time constraints, the timing of the study was essential to achieve proper interventions since people in the MENA region had started receiving the booster dose. Second, we implemented strict inclusion and exclusion criteria to obtain accurate results. Third, the sample size covered five different MENA region countries to provide a general overview of this geographic location. Concerning the limitations, self-reporting of the fear scale might not be accurate and may reflect misreporting. Additionally, we asked participants to rate the side effects on a Likert scale; however, we did not know what these side effects were \u2014a topic that can be further addressed in future studies. Some groups were underrepresented, such as people with different educational backgrounds (primary and secondary). Additionally, the anonymity of the survey may have attracted individuals interested in manipulating the data; even though we removed all responses below 1 min in length to prevent any usage of Macros (survey filling bots), this point still needed to be declared for transparency. Even after extending the collection time, the response rate was low, allowing only a 5% error margin, which could have been improved if we had a higher response rate. Moreover, the national coordinators distributed the survey using each country\u2019s most commonly used social media channels; hence, the design effect should have been considered to calculate the sample size per country. Further research can benefit from mailing lists or by promoting their surveys on popular local websites. Finally, the responses represented personal opinions at a given point in time that did not fully reflect the respondents\u2019 future decisions. As we mentioned, several factors could affect that, including the prospective status of the COVID-19 pandemic and the spread of the infodemic.The main reasons to refuse the booster dose were uncertainties over their safety, belief that the booster dose is unnecessary, and side effects associated with previous COVID-19 vaccine doses. Since the introduction of booster doses has been firmly evidence-driven, efforts to improve public awareness should target the population groups in greatest need through effective scientific communication to overcome vaccine hesitancy and fear."}
{"text": "Background: Given the prevalence of the omicron variant and decreased immunity provided by vaccines, it is imperative to enhance resistance to COVID-19 in the old population. We planned to explore the hesitancy rate toward the booster dose of the COVID-19 vaccine and the association between risk perception and the abovementioned rate among people aged 60 and older. Methods: This national cross-sectional study was conducted in mainland China from 25 May to 8 June 2022, targeting people who were 60 years old or above. Four dimensions were extracted from the Health Belief Model (HBM) to assess participants\u2019 perceived risk levels, including perceived susceptibility, perceived severity, perceived barriers, and perceived benefit. An independent Chi-square test was used to compare the vaccine hesitancy rates among different groups stratified by characteristics. Univariable and multivariable logistic regression models were performed to explore the associations between risk perception and hesitancy rate. Results: Of 3321 participants, 17.2% (95% CI: 15.9\u201318.5%) were hesitant about booster shots of COVID-19 vaccines. Believing that they were ineligible for vaccination due to certain illnesses (38.3%), concern about vaccine safety (32.0%), believing the booster shots were unnecessary (33.1%), and their limitation on movements (28.0%) were the main reasons for vaccine hesitation. Adjusted by all the selected covariates, people with low perception level of susceptibility  and benefit  were less likely to receiving the booster dose, and the same results were found in people with higher perceived barriers . Our estimates were stable in all four models. Conclusions: In total, 17.2% of the people aged 60 years and older in China were hesitant about booster dose of COVID-19 vaccines, and it was closely associated with a lower level of perceived susceptibility and benefit, as well as a higher level of perceived barriers. Concerns about contraindications, vaccine safety, and limited movements were the main reasons for vaccine hesitancy. Targeted public health measure is a priority to improve the understanding of the elderly on their own susceptibility and vulnerability and clear the obstacles to vaccination. As one of the worst plagues in nearly a century, coronavirus disease 2019 (COVID-19) has caused an incalculable disease and economic burden across the globe . UndoubtVaccine hesitancy, one of the ten major threats to global health in 2019, refers to the reluctance or refusal of people to be vaccinated when available, especially among the older population . AccordiThe health belief model (HBM) is widely used to assess public attitudes toward vaccines and predict their behavior toward vaccination, revealing significant perception factors such as perceived susceptibility, perceived severity, perceived barriers, and perceived benefit ,19,20. RUp to now, no study has been performed to specifically investigate the hesitancy toward the booster dose of the COVID-19 vaccine and the association between risk perceptions among the Chinese elderly (\u226560 years old). Accelerating global coverage of vaccination in the old population, especially the booster dose, is indeed a priority for achieving herd immunity against COVID-19 . To bettThis national cross-sectional survey was conducted based on an online platform called Wen Juan Xing . Owing clear personal information  of nearly 3 million registered members in China, it could accurately deliver questionnaires to the representative respondents we expect. The target participants of this study were senior citizens aged 60 or above in China. For users who have trouble answering questions using electronic devices (without cognitive impairment), they can enlist the help of those around them, and questions could be relayed to the old people by those young adults living with them. Each question was followed by a reminder that the purpose of the questionnaire was to find out the true thoughts and situations of people aged 60 and above.p = 0.13) among old people, calculating the sample size with \u03b1 as 0.05 and the confidence interval width as 0.1p (0.013). In total, 2647 valid questionnaires were expected when using the exact (Clopper\u2013Pearson) method for calculation by the PASS software 15.0 . Anonymous questionnaires were randomly allocated among 31 provinces from 25 May to 8 June 2022. The minimum number of valid questionnaires for each province was allocated according to the proportion of the older adults aged 60 or above reported in the Seventh National Census [According to previous studies that examined the willingness of Chinese adults to receive booster shots, 10.56% (95% CI: 6.99\u201314.14%) were unwilling to receive booster vaccination among those aged 60 and older . Thus, wl Census . A totalp < 0.001). Then the expressions of some items were modified according to the feedback of the respondents, making them easier to understand. The reliability of this questionnaire was confirmed by Cronbach\u2019s alpha coefficient by different dimensions.This structured questionnaire was divided into 4 sections in total, which consisted of the following parts: (1) sociodemographic characteristics and health status, (2) knowledge of COVID-19 and COVID-19 vaccines, (3) four dimensions of risk perception based on health belief model (HBM), and (4) attitude toward the booster dose of COVID-19. All items in our questionnaire were stated by a panel of experts, including one public health expert and two epidemiologists specializing in infectious diseases. We conducted a pilot study involving 40 old people before it was officially released to test the questionnaire\u2019s validity  is a conceptual framework based on motivation theory, cognitive theory, and expectancy-value theory, which has been widely used to assess public attitudes toward vaccines and predict their behavior toward vaccination ,20,21. FTo assess their attitude toward the booster dose of the COVID-19 vaccine, all eligible participants were required to answer the question, \u201cAre you willing to receive the booster dose of COVID-19 vaccine if available?\u201d The hesitancy rate was defined as the proportion of participants who answered \u201cNo\u201d or \u201cNot sure\u201d, and if the participants had any concerns about the booster dose, the specific reasons for the reluctance were further asked at the same time.Frequencies and percentages were used to describe all categorical variables, consisting of sociodemographic characteristics, health status, knowledge about COVID-19 and the COVID-19 vaccine, and four dimensions of risk perception. An independent Chi-square test was used to compare the vaccine hesitancy rates among different groups stratified by the abovementioned characters. Logistic regression analyses were performed to explore the associations between risk perception and hesitancy rate toward the booster dose among old people (\u226560 years old), and we finally constructed four models to examine the robustness of the estimations. Model A is a univariable logistic regression model. Region, age group, sex, marital status, education, and monthly household income were adjusted in model B. All covariates were included along with the other three dimensions of risk perceptions in model C. Additionally, only covariates with significant differences and the other three dimensions of risk perception were contained in model D. Crude odds ratios (cORs) and adjusted odds ratios (aORs) with 95% CIs were calculated to explain the effect size in different risk perception groups.p-value less than 0.05 was regarded as statistically significant.All statistical analyses were conducted by SPSS 26.0  in this study, and a two-sided A total of 3331 people aged 60 years and older in China were recruited to fill in this questionnaire, of which 3321 participants were eligible for the final analysis . Among tp < 0.05). People with lower perceptions of susceptibility (21.6%) and benefit (45.2%) were less likely to receive a booster dose of vaccination. Perceived barriers had a negative impact on vaccination willingness in older adults. However, no significant difference in vaccination hesitancy was found among groups with different degrees of perceived severity. A total of 571 old people were reluctant about the booster dose of COVID-19 vaccines. Believing that they were ineligible for vaccination due to certain illnesses (38.3%), concern about vaccine safety (32.0%), believing the booster shots were unnecessary (33.1%), and their limitation on movements (28.0%) were the main reasons for vaccine hesitation  were hesitant about the booster shots of COVID-19 vaccines . People sitation .As shown in p for interaction > 0.05). People who perceived low susceptibility in western China  or having lower income  were less likely to accept a booster dose of COVID-19 vaccine  was negatively correlated with hesitation, while perceived barriers  were positively correlated [According to our research, vaccination hesitancy toward the booster dose in the older population was closely associated with low perceived susceptibility, low perceived benefit, and high perceived barriers, but not with perceived severity. As people\u2019s subjective judgment of the characteristics and severity of a particular risk, Risk perception will eventually affect people\u2019s behavior ,21. Somerrelated . The assrrelated ,37,38,39rrelated . Chen etrrelated . At diffrrelated ,42,43,44Unlike young people, older people are more worried and concerned about booster shots. In this survey, 38.3% of the elderly worried that they were not eligible for booster shots for their existing diseases. Evidently, advanced age and comorbidities such as hypertension, diabetes, cardiovascular disease, and chronic respiratory disease were risk factors for SARS-CoV-2 infection and poor prognosis ,7. PeoplAs with other online cross-sectional surveys, our study also has some limitations. First, selection bias may exist. Considering the accessibility of online surveys for the elderly, questionnaires were answered only by Internet users. Moreover, some elderly people who had difficulty answering questions using electronic devices may seek help from young people around them and answer questions orally through young people\u2019s dictation. This may also increase the selection bias of this study. Although we have set up tips at the end of each question and repeatedly emphasized that the subjective questions were supposed to be answered by the elderly orally, we cannot completely rule out proxy answers. However, under the current situation, it is urgent to understand the vaccination willingness toward the booster shots and promote vaccination coverage among the elderly. In addition, people\u2019s acceptance of the booster dose of the COVID-19 vaccine was measured only by self-report, and we were unable to develop a standard scale to assess their willingness. Third, this survey was only conducted in China and based on a specific theoretical model, so results need to be interpreted carefully when extrapolated to other countries or compared with other models. Since this was an online survey, our depth was limited to some extent. Our results were only a crude supplement to the current research gap, and we hope that large-scale offline surveys with more participants will be implemented as soon as feasible. Moreover, this is only a cross-sectional study, which cannot fully verify the relationship between risk perception and vaccination hesitancy from a causal perspective.Of 3321 participants, 17.2% were hesitant about the booster dose of the COVID-19 vaccine, and it was closely associated with a lower level of perceived susceptibility and benefit, as well as a higher level of perceived barriers. Concerns about contraindications, vaccine safety, and limited movements were the main reasons for vaccine hesitancy. Under this circumstance, it is urgent to improve the understanding of the elderly on their own susceptibility and vulnerability, reasonably judge whether they could be vaccinated, and clear the obstacles to vaccination. Therefore, our results have important theoretical and practical implications for the subsequent promotion of the coverage of booster doses in the elderly population."}
{"text": "The coronavirus disease 2019 (COVID-19) vaccines are considered to be an effective way to prevent the spread of the infection. Our previous study has shown that about 75% of healthcare workers (HCWs) in China were willing to receive the vaccine when it became available. Here, we examined the acceptance of a third booster dose among Chinese people and identified the influencing factors.A cross-sectional online survey was conducted and the snowball sampling method was utilized. An online questionnaire was provided to all the participants in the form of a quick response (QR) code. The questionnaire included general demographic information, views on vaccines, the General Health Questionnaire-12 (GHQ-12), and the Depression, Anxiety, and Stress Scale-21 (DASS-21). The univariate analysis was done between all the variables and our dependent variable. Then, we used the multivariate logistic regression model to examine the influencing factors of the third booster dose acceptance.n = 960) declared that they would accept the booster dose. Knowing more about the vaccine and recognizing the efficacy of vaccines were significantly associated with greater acceptance of the booster dose. People willing to take the booster dose had better psychological health. A belief that the booster dose could prevent severe infection caused by COVID-19 and enhance the effectiveness of the first two doses were the main contributing factors to vaccine acceptance. Vaccine hesitancy was mainly due to a low perceived risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and rapid mutation of SARS-CoV-2.We collected 1,062 complete answers. Of these, 90.39% (This study revealed that Chinese people were very receptive to the third booster dose, which is an inspiring result. More positive attitudes regarding COVID-19 vaccination were supported by its efficacy and few side effects. The ongoing pandemic of coronavirus disease 2019 (COVID-19) has caused extensive damage worldwide. Despite established supportive therapies and the development of new antiviral drugs, vaccination is considered an effective method to prevent infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), particularly for at-risk populations. In China, the COVID-19 vaccines started to be used in priority groups on 15 December 2020. The Chinese government accelerated free vaccination for all the Chinese citizens starting in late March 2021 , regardless of region, occupation, or status, were eligible to participate in our study.The study protocol was approved (2022-S-27) by the Ethics Committee of Third People's Hospital of Chengdu . Informed consent was obtained before study enrollment. This was a cross-sectional online survey using a social media platform (WeChat\u2122)-based survey program \u201cQuestionnaire Star\u201d between 1 December and 31 December 2021. The online questionnaire was provided to all the participants in the form of a quick response (QR) code Before initiating the study, we first consulted psychologists working at the Third People's Hospital of Chengdu and the psychologists recommended two commonly used scales, as described below, suitable for measuring psychological status. The questionnaire gathered information on: (i) general demographics ; (ii) whether or not to accept the third booster dose; and (iii) the reasons for being willing or not willing to be vaccinated. Moreover, this questionnaire included the General Health Questionnaire-12 (GHQ-12) and the Depression, Anxiety, and Stress Scale-21 (DASS-21) to investigate the psychological health of respondents.Participants were asked if they would accept the third booster dose of the COVID-19 vaccine. They rated the item regarding their attitudes toward a booster dose on a four-point scale . Those answering 1 or 2 were identified as the vaccine-agree group. Those who answered 3 or 4 were identified as the vaccine-disagree group. Then, we listed common or possible causes of people's willingness or unwillingness to be vaccinated. People who agreed to take the vaccine could voluntarily select factors promoting their willingness to be vaccinated. Those who did not agree to take the vaccine could voluntarily choose the factors causing their resistance. The percentage of people who chose different factors was calculated.With the help of a consultant psychologist, we chose the scores for the GHQ-12 and the DASS-21 to measure participants' mental health. The GHQ-12 is widely used to identify common psychiatric conditions , 16. TheOnly complete questionnaires could be collected and incomplete data could not be submitted through the Questionnaire Star. The Questionnaire Star automatically collected data. We could convert all the data into text format and numeric form and export them to spreadsheets.n), and percentage. We used the univariate analysis with the t-test or the chi-squared test to compare the two groups (agree and disagree) to identify the factors associated with vaccine hesitancy. Then, the multivariate logistic regression model was employed to examine and identify the factors associated with the acceptance of the COVID-19 vaccine. Statistical analyses were performed using SPSS version 23.0 . p \u2264 0.05 was considered statistically significant.Descriptive analyses were conducted on all the study variables, which were reported as the mean, SD, number  declared that they would accept a booster dose. Of those who were willing to accept the third booster dose, 884 responders (83.2%) were strongly willing and 76 responders (7.1%) were willing but waiting to review more data. Of the 1,062 participants, only 102 (9.6%) participants were not willing to accept the third booster dose. Of them, 71 (6.7%) participants did not plan to receive the COVID-19 vaccine and 31 (2.9%) participants were strongly against taking the booster dose. We found that 95.6%  of respondents had completed the two-dose regimen and 97.3% (935) of respondents who had completed the two doses were willing to receive the third dose .2 = 5.577, p = 0.018), had a better understanding of how the vaccine works , had nearly completed a two-dose regimen, were more likely to think that the vaccine works , and had better mental health based on the GHQ-12 . However, the two groups did not differ significantly in terms of sex, age, occupation, educational level, marital status, contact with a person with COVID-19, living in a high-risk area, or income.The univariate analysis was used to decide significant differences between the two groups . People who had completed the two-dose regimen were more likely to accept the third dose compared with those who had not completed the two-dose regimen . People who believed that the vaccine was effective had 7.949 times greater odds of accepting the third dose compared with those who believed that the vaccine was not effective . People with the lower GHQ-12 score were more likely to accept the third dose  compared to those with the higher GHQ-12.To determine the factors associated with the willingness to be vaccinated, we used the multivariate logistic regression models. Results are given in Of 960 respondents who were willing to receive the third dose, 927 (96.6%) respondents chose the reasons for accepting the third dose. Of 102 respondents who were not willing to receive the third dose, 88 (86.3%) respondents chose the reasons for rejecting the third dose. The top two reasons for accepting the third dose were that the booster dose can prevent severe infections (69.6%) and enhance the effect of the first two doses 63.2%; . The topDespite newly approved antiviral drugs, the role of vaccination remains crucial. However, the efficacy of vaccination diminishes over time. Cohn and their colleagues have demonstrated that the efficacy of the COVID-19 vaccine against infection declined from 87 to 48% from February to October 2021 . ProtectMost surveys on COVID-19 have focused on the safety and immunogenicity of the third dose. Only a few studies have assessed the acceptance of the third booster dose. Suman and their colleagues have found that nearly two-thirds of respondents were concerned that vaccination may be ineffective against new strains of SARS-CoV-2 and that booster doses may be required. However, acceptance by vaccine-hesitant respondents of a hypothetical booster dose was only 14.3% . AnotherIn our study, 90.39% of participants declared that they would accept the booster dose. This acceptance rate was higher than that of the first two doses in our previous study (76.63%)  among HCOur study indicated that people with a high perceived risk of being infected had twice the odds of vaccine acceptance compared with those without a perceived risk of being infected. This concern is not surprising given that vaccines remain the main protection method against COVID-19. This result is similar to the findings noted by Harapan and collaborators and Rajamoorthy and coworkers , 34. AnoOur study indicated that learning more about the COVID-19 vaccine might have contributed to people being more willing to take the vaccine compared with those who had known less about the vaccine. This finding is consistent with data from our previous study, suggesting that greater education efforts toward vaccination against COVID-19 should be considered to increase public understanding of vaccines. People who believed that the COVID-19 vaccine was effective had 7.949 times greater odds of accepting the third dose compared with those who believed that the COVID-19 vaccine was not effective. Harapan and colleagues have reported similar findings . We founOur study had two major strengths. First, it was the first study to evaluate the acceptance of the third booster dose among the general population in China. Second, we evaluated mental health to investigate if it influenced vaccine hesitancy.Our study had two main limitations. First, we employed an electronic questionnaire to collect data (instead of a face-to-face interview), which resulted in uncontrolled conditions during questionnaire completion. Second, as we used a snowball sampling method, some individuals in this sample have still not been found and some individuals are probably omitted by a provider, leading to a biased sample. Third, the sample size was small, limiting the generalizability of our findings.In China, about 1.2 billion people have completed the two-dose regimen. The safety and efficacy of the COVID-19 vaccines have been recognized. People's acceptance of the booster dose has also been improved. Our study findings make us optimistic about the COVID-19 vaccination. We believe that the COVID-19 vaccination campaign will progress smoothly and will eventually provide herd immunity against COVID-19.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by the Ethics Committee of Third People's Hospital of Chengdu.YS, TX, PW, and XZ conceived and designed the questionnaire. JZ, DC, and YH recruited participants. YS, TX, and HD analyzed the data. YS wrote and revised the manuscript. All authors have approved the final version of the manuscript and agreed with submission to your esteemed journal.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": "Some HCWs broadly accepted the value of vaccination, and believed that boosters were necessary to effectively combat emergent new virus strains, which contrasted with peers who believed that boosters offered little defence against virus mutations. Fear prohibited some HCWs from getting the booster, with some having experienced adverse side effects from their initial vaccination, whilst others were concerned about future complications. Waning booster uptake rates could be arrested through invigorated communication strategies, while effective evidence-based training can potentially create positive normative vaccination practices amongst HCWs.This study aimed examin the factors associated with the uptake and non-acceptance of COVID-19 vaccine booster doses among healthcare workers (HCWs) in South Africa. We used a mixed-methods design with data from a web-based self-administered survey followed by semi-structured in-depth interviews (IDIs) with selected participants. Of the 6235 HCWs included in our analysis who had fully vaccinated, 3470 (56%) had taken their booster dose with a further 17% intending to get the booster. HCWs aged 35 to 49 years (OR = 1.30 [95% CI: 1.15\u20131.46]), and those aged 50 years or older (OR = 2.66 [95% CI: 2.32\u20133.05]) were more likely to get the booster dose. Females were less likely to have received the booster dose (OR = 0.88 [95% CI: 0.79\u20130.98]) with doctors more likely (OR = 1.58 [95% CI: 1.35\u20131.84]) than Nurses to have received the booster dose. HCWs in direct contact with patients (OR = 1.17 [95% CI: 1.00\u20131.38]) and who had previously received a flu vaccine (OR = 1.99 [95% CI: 1.56\u20132.55]) were more likely to have received the booster dose. Four themes emerged from the qualitative data analysis:  Healthcare Workers (HCWs) remain susceptible to SARS-CoV-2 infection, making this a priority group for vaccination and access to booster options, as outlined in the WHO\u2019s Strategy to Achieve Global COVID-19 Vaccination by mid-2022 .Research among HCWs in SA revealed high initial COVID-19 vaccine uptakes rates . These dStudies of the acceptance and uptake of COVID-19 vaccine booster doses have focused on HCWs in high-income settings, with little evidence from the African continent and South Africa specifically. This study, therefore, aimed to gain a better understanding of the factors associated with vaccine booster uptake among HCWs in SA. Survey data were augmented by qualitative data revealing HCW perspectives on COVID-19 vaccine booster doses, with the summative evidence providing not only guidance for COVID-19 vaccine booster uptake interventions, but also other routine immunisation programmes.The study was granted ethical clearance by the University of KwaZulu-Natal Biomedical Research Ethics Committee (BREC/3970/2022) in compliance with all regulations and policies regarding ethical conduct of research. Written consent was obtained at the beginning of the online survey by providing formal paragraph-wise information about the study, with the participant requiring to click on a button for providing consent on the same online survey platform before moving on to filling the rest of the online survey questionnaire.This study used a mixed-methods design with data from a web-based self-administered survey followed by in-depth interviews (IDIs) with selected participants. Mixed methods research allows for combining elements of qualitative and quantitative research approaches for the broad purposes of providing depth of understanding and corroboration on a particular research topic , 13. Datrandbetween formula in Microsoft Excel for IDIs and scheduled for a virtual interview. In total we interviewed 30 HCWs, 10 vaccinated and 20 unvaccinated.The Foundation for Professional Development\u2019s (FPD) database, which comprised contact details of 88 000 HCWs at the commencement of the study, was used to recruit HCWs for this study. Participants were offered compensation in the form of an entry into a draw for one of ten ZAR500 (~US$33) cash vouchers. The draw was not linked to participants\u2019 survey responses. Participants interested in participating in IDIs were asked to provide their contact details following completion of the self-administered survey. A total of 7 763 HCWs participated in the survey. For this paper, we excluded all participants that were either unvaccinated or did not disclose their vaccination status. Thus, final analysis was undertaken on 6 325 vaccinated participants. As part of the survey, participants had the option to indicate their willingness to be contacted for a follow-up interview (IDI). Participants who had provided their contact details were organised into two groups (vaccinated and unvaccinated), then randomly selected using the The survey captured socio-demographic information, COVID-19 history, chronic conditions, and questions on vaccination behaviour. Survey questions were derived from a review of studies evaluating HCW hesitancy towards COVID-19 vaccines, described elsewhere .Two outcome measures were created based on the question; \u201cAre you planning to get a booster vaccination against COVID-19?\u201d with respondents indicating either \u201cI have already received the booster dose\u201d, \u201cyes, as soon as possible\u201d, \u201cyes, but in a few months up to a year\u201d, \u201cyes, but in a year or more\u201d, \u201cI am unsure\u201d, \u201cno, but I might consider it in the future\u201d, and \u201cno, never\u201d. The first outcome measure was coded as a binary variable where: 1 = \u201cI have already received the booster dose\u201d (Received Booster), and 0 = any other response to the question (No Booster). The second outcome measure was coded as a binary variable where: 1 = \u201cI have already received the booster dose\u201d or \u201cyes, as soon as possible\u201d or \u201cyes, but in a few months up to a year\u201d or \u201cyes, but in a year or more\u201d (Received and Intend), and 0 = \u201cI am unsure\u201d or \u201cno, but I might consider it in the future\u201d or \u201cno, never\u201d (Hesitant and Against).For the IDIs, two interview guides were developed, one for vaccinated HCWs and another for unvaccinated HCWs, with open-ended questions and probes regarding; (1) HCWs\u2019 vaccination behaviour (including vaccine booster doses); (2) HCWs\u2019 experience with administering vaccines; (3) HCWs\u2019 perspectives of the vaccine programme; (4) how HCWs gather and appraise information sources; and (5) HCWs\u2019 perspectives on educational resources that can be used to support them. Interviews were conducted by authors PBN and GG, who are experienced in conducting qualitative interviews using interview guides. IDIs lasted approximately 30 minutes and were recorded and transcribed.Personal information from study participants is available to the study principal investigator (PI) and co PI\u2019s, this data was transmitted via a password-protected secure web-based file sharing dashboard service. Study personnel also transferred all data to password-protected computers.For the quantitative data, we ran a series of univariate logistic regression models to determine; how significantly a number of measures influenced a participant\u2019s likelihood of having received a booster compared to those who hadn\u2019t, and how significantly the same measures influenced a participant\u2019s likelihood of having received or intended to receive a booster dose, compared to those who were hesitant or against receiving a booster dose. Finally, we conducted descriptive analysis (frequencies and proportions) of the underlying reasons for hesitancy towards vaccination boosters, stratified by gender.Qualitative data were analysed thematically using an inductive approach as prescribed by Braun and Clarke  six-stepIn this study, the point of integration was at the data analysis phase where the qualitative data provided in-depth understanding of the quantitative findings, revealing trends on HCWs vaccination attitudes and behaviours. The quantitative and qualitative data were kept analytically distinct where a statistical technique was used to analyse the survey data while thematic analysis was used to analyse interview data, as explained by Tariq and Woodman . FindingThere were 7 763 participants in the study. Of these, 6 235 participants were vaccinated, with 3 470 (56%) having received at least one booster dose. See Doctors were more likely to have received the booster dose than nurses (OR = 1.58 [95% CI: 1.35\u20131.84]) while all other HCWs  were less likely to have received the booster dose when compared to nurses (OR = 0.81 [95% CI: 0.72\u20130.92]). HCWs who were in direct contact with patients were more likely to have received the booster dose than those not in direct contact with patients (OR = 1.17 [95% CI: 1.00\u20131.38]). HCWs who had worked for 10 or more years were more likely to have received the booster dose (OR = 1.79 [95% CI: 1.52\u20132.11]) than their less experienced colleagues.In In the following tables, we conducted univariate logistic regression analysis with our second outcome variable and our demographic measures , and sevThe data further revealed that 15.84% of females had not received their booster dose because they had experienced adverse effects after initial vaccination, compared to 8.24% of males who listed this as a reason.(1) Vaccination as a routine practice among HCWs; (2) Emergence of new COVID-19 variants necessitating vaccine boosters; (3) Fear of potential side-effects from COVID-19 vaccine boosters; and (4) Limited value of COVID-19 vaccine boosters. Overall, participants held positive views towards COVID-19 vaccine boosters, with most highlighting the importance of boosters in maintaining population immunity. However, some HCWs remained both hesitant and against getting the booster, questioning its value, while raising safety concerns, which largely emanated from personal and observed experiences of adverse events following initial vaccination or after receiving a booster dose. These are discussed more fully below.We identified four themes regarding HCWs\u2019 uptake and experiences of COVID-19 vaccine boosters: Participants recognized the value of regular vaccination, often citing that the influenza vaccine was part of their regular health routine. These participants were easily able to accept the importance of regular COVID-19 vaccine boosters, both to provide ongoing protection against COVID-19 disease and severe illness, and for protecting against potential virus variants or mutations.I actually said to my family, this is going to end up being like a flu vaccine. It\u2019s something that we\u2019re going to have to get annually, like we all get our flu vaccines annually. So for me it was never a question of maybe getting only one or two vaccines, and then it\u2019s going to stop. This is a virus, and it changes, and it mutates, and we have to try and keep up with it.Another participant went on to explain:Personally, I think it is important. I think that again I\u2019m a believer, so I have taken flu vaccine annually, and so if I believe that you know I don\u2019t take a flu vaccine once and expect it to cover me for the rest of my life [\u2026] I think boosters are important in terms of coverage, to be fully vaccinated.HCWs continue to feel susceptible to infections, with older HCWs and those with chronic diseases feeling particularly vulnerable to COVID-19.I\u2019ve been boosted yes\u2026Because it\u2019s a virus so that\u2019s what this virus job is to infiltrate and cause chaos, so you need to be one step ahead, just like the flu, so you need to get boosted, especially my age, I\u2019m over 50.Participants recognised the limited effect vaccines had on the new emerging variants, relying therefore on boosters to provide protection against virus mutations.I\u2019ve had the booster. I think it is important because of the variants [\u2026] it\u2019s changing.Participants felt that the booster dose improved individual\u2019s immune response.I did get a booster. I think you know, it\u2019s probably quite important just to help your immune system to create memory cells adequately, and to make sure that you know, you\u2019re actually able to bolster an adequate response.Some HCWs experienced adverse side-effects following their initial vaccination. This, coupled with observing colleagues and others suffering from side-effects after receiving the vaccine or booster shots, dissuaded them from returning for their booster doses.There were many stories about the vaccine and one ended up not getting the booster [\u2026] I was concerned that maybe one would have side effects and getting a booster and I thought, maybe I also would have side effects as well.Another HCW added:Most of my colleagues have not yet gone for the booster but I think most of them had\u2026a few of them still have that fear of, like I mentioned experiencing severe side effects of the vaccine [\u2026]I think, with the booster dose, I delayed it due to the side effects that I had with the second dose of the vaccine.In addition to the side-effects, participants raised concerned around the safety of booster doses for those that were pregnant.The few colleagues that I spoke to, they said to me that they felt so bad after the second vaccination, they\u2019re not going for the boosters. And I also have a colleague now that\u2019s pregnant and she\u2019s not sure what the effects might be on the foetus.Some participants questioned the value of additional booster doses, suggesting that the initial vaccine provided adequate protection against infection and severe illness.I had COVID, I think, last year December, and it was not bad, so I don\u2019t think the booster is going to make any difference, because the symptoms were not bad, so I\u2019m not doing it.Some participants did not feel particularly vulnerable to COVID-19 infection, suggesting that their risk profile did not warrant getting booster doses.I\u2019ve not gone on to receive the booster dosages. I personally, for my own personal risk perspective, I didn\u2019t think it was that important. I don\u2019t have particularly high-risk factors for severe COVID-19, and I didn\u2019t feel that it was necessary for me personally.Even among unvaccinated participants, getting the initial vaccine and the booster dose was often expressed as unnecessary.I would opt for my natural response\u2026 you know development of antibodies etcetera, and see what will happen. Actually, my option was natural response versus COVID-19 vaccines and boosters.In our study, 56% of HCWs had received their initial COVID-19 booster vaccination while a further 17% reported their intention to get the booster dose within 12 months of completing the survey. Of those that had received their first booster dose, 23% had already received their second at the time of the study. These percentages are higher than those observed among the general South African public, where only approximately 24% had received a vaccine booster by March 2023 . These pPreviously published data on primary COVID-19 vaccination among the same study sample highlighted significant disparities in vaccine uptake by age, gender, race, job roles and presence or absence of a chronic health condition . This stThere were, however, some notable incongruities with respect to findings from initial vaccine uptake behaviour , with feTwo further dominant reasons for not getting the booster doses\u2013for both males and females\u2013centred around the belief that the initial vaccination provided adequate protection and that subsequent boosters were unnecessary or ineffective. The IDIs revealed contrasting perspectives, with some believing that boosters were necessary in combating the new virus strains whereas others questioned the efficacy of available vaccine boosters. HCWs operating in other contexts also reported that boosters would be required to counter new emerging strains of the virus .This study also found disparities in COVID-19 booster vaccination uptake among HCWs who were patient facing, which can be viewed as a proxy for increased perceived risk and fear of transmitting to patients, both factors correlating with increased uptake of boosters in another study . HCWs whVaccination booster rates, along with the intention to get the booster dose, remain high among HCWs in South Africa. However, not all HCWs who had vaccinated were committed to receiving boosters. Enthusiasm for further boosters appears to be waning as indicated by the low percentage 23%) who had received their second booster, even though most would have been eligible. The slowing uptake of boosters is evident not only among the South African general population  but amon% who hadOur study adds to the limited available data regarding COVID-19 booster vaccination uptake among HCWs, especially data available from Africa. Findings are expected to guide future vaccine campaigns and public health strategies to build vaccine confidence among HCWs and the general population.The study is limited by using an unrestricted self-administered survey that was dependent on the online reachability of HCWs on selected databases. Limitations in study design may have introduced selection bias and may have limited generalizability amongst HCWs in South Africa.This is the first study examining the uptake of vaccine booster doses among South African HCWs, offering valuable insights into drivers of hesitancy, revealing the perspectives of HCWs, all of which will hopefully influence the design of future vaccination programmes. This study shows that key demographics influence uptake of boosters, as was the case with primary vaccine doses. Age, race, gender, job roles and chronic health conditions were all factors contributing to the uptake of vaccine booster doses. Booster rates in our sample of HCWs were high, with the majority having had or intending to get their booster doses. The data revealed that females were concerned about the potential side effects and future complications which may arise from booster doses. Hesitancy was also fuelled by those who questioned the value of boosters relative to the protection afforded to them from their primary vaccination, or whether these boosters were effective against the emerging virus strains. Efforts are required to ensure that vaccination against disease becomes normative practice amongst HCWs, with this study showing participants who had historically taken the influenza vaccine more willing to get the COVID-19 booster doses. These normative practices can only be inculcated amongst HCWs through effective evidence-based training and communication strategies.S1 Text(DOCX)Click here for additional data file."}
{"text": "This cross-sectional survey explored the attitudes and the reasons, as well their associated factors, for receiving the second booster dose of the COVID-19 vaccine among a sample of all old adults and of people with chronic medical conditions attending two randomly selected immunization centers in Naples . A total of 438 questionnaires were collected. The majority were male (55.1%) and the median age was 71 years. A higher perception of the vaccine\u2019s utility, measured with a 10-point Likert type scale, has been observed among males, individuals with a higher perception that COVID-19 is a severe illness, with a higher self-awareness of being at risk of infection, and with a higher trust in the information received. The most reported reasons for receiving the second booster dose included protection of themselves and of their family members from getting COVID-19, fear of acquiring the disease, and having a physician\u2019s recommendation. Younger participants, married/cohabitant, and with a higher perception that COVID-19 is a severe illness were more likely to have indicated protecting themselves and their family members as reason for receiving the booster dose. Respondents with a chronic medical condition, with a higher perception that COVID-19 is a severe illness, with a lower trust in the information received, and informed by physicians were more likely to have received the vaccine because they perceived of being at risk of getting a severe form of the SARS-CoV-2 infection. Physicians should play a pivotal role in stressing the importance of the second booster dose and in helping individuals to make decisions. The coronavirus disease 2019 (COVID-19) continues to determine global public health concern with the emergence of the Omicron variant as the dominant strain around the world. As of 7 March 2023,there were more than 755 million confirmed COVID-19 cases and almost 7 million deaths in the world . Since 1Understanding the attitudes regarding the second booster dose and the reasons for receiving this dose are crucial for developing specific interventions to increase vaccine uptake and to generate support for health policy makers in the prevention activities. However, no literature is available on this topic. Therefore, to address this gap, this present cross-sectional survey was designed to characterize the attitudes regarding the second booster dose of the COVID-19 vaccine and the reasons for receiving it among a large sample of vaccinated adults and people with chronic medical conditions in Southern Italy, as well as to identify the associated factors.This survey was conducted as part of a larger project aimed at investigating perceptions and behaviors towards the COVID-19 vaccination among different groups of people in Southern Italy ,14,15,16The minimum target sample size of 427participants was determined assuming an expected proportion of 50% of respondents reporting the protection of themselves and their family members from getting COVID-19 as reason for having received the second booster dose of the COVID-19 vaccine, with a margin of error of 5%, considering a confidence interval of 95%, and allowing for an expected response rate of 90%.Before enrollment, well-trained research investigators, with professional skills in recruiting respondents and knowledge on the topic, approached each potential participant or parent/guardian for those younger than 18 years of age who had been registered for the administration of the second booster dose of the COVID-19 vaccine in the waiting rooms of the centers. The research investigators illustrated for each participant the study objectives and procedures, that the survey was answered on a voluntary basis, that no subject specific identifiers were recorded, that information was confidential, that they had the option to withdraw their participation at any stage without justification, and that by answering the questionnaire they gave the consent to take part in the survey. Informed written consent was obtained from each parent/guardian for the participants younger than 18 years of age. The research investigators asked each participant to complete the questionnaire and to return it immediately once filled. Individuals who had difficulties writing had the option to be interviewed face-to-face by the research investigators. No compensation or incentive was given to the individuals completing the questionnaire.The Ethics Committee of the Teaching Hospital of the University of Campania \u201cLuigi Vanvitelli\u201d approved the study protocol and the questionnaire.The questionnaire was designed based on those used in similar previously published surveys carried out by some of us among a variety of populations ,9,10,11.The questionnaire, with instructions that aided self-administration by participants, consisted of three sections . In the p-value less than or equal to 0.25 were selected for inclusion into the multivariable analysis. Multivariable linear and logistic regression models with the stepwise variable selection procedure with a threshold of p = 0.2 to retain and of p = 0.4 to exclude the variables were used to explore the association between several variables and the following outcomes of interest: perception of the utility of the second booster dose of the COVID-19 vaccine (Model 1); protection of themselves and of their family members from getting COVID-19 as reason for having received the second booster dose of the COVID-19 vaccine (Model 2); and perception of being at risk of getting a severe form of SARS-CoV-2 infection as reason for having received the second booster dose of the COVID-19 vaccine (Model 3). The following independent variables have been included in all models: sex , age (continuous), marital status , at least one chronic medical condition , baccalaureate/graduate degree , having been infected by SARS-CoV-2 , at least one family member/colleague/friend who has been infected by SARS-CoV-2 , level of perception that COVID-19 is a severe illness (continuous), level of trust in the sources of information used about the second booster dose of the COVID-19 vaccine (continuous), self-awareness of being at risk of getting the SARS-CoV-2 infection (continuous), physicians as source of information about the second booster dose of the COVID-19 vaccine , and need of additional information about the second booster dose of the COVID-19 vaccine . The variable level of the perception of the utility of the second booster dose of the COVID-19 vaccine (continuous) was included in Models 2 and 3.The following descriptive statistics were computed to summarize the answers of the respondents: frequencies, proportions, means, median, and interquartile range. The chi-square test or Student\u2019s t-test were used to test the association between each variable and the continuous or dichotomous outcome. Variables associated in the bivariate analysis with a p-value equal to or less than 0.05. STATA software version 15.1 was used for performing the statistical analysis.Odds ratios (OR) with a 95% confidence interval (CI) and standardized regression coefficients (\u00df) for potential determinants associated with the study outcomes were estimated respectively in the logistic and in the linear regression models. Statistical significance was considered with a two-sided Of the 452 subjects approached, 438 agreed to participate for an overall response rate of 96.9%. Survey participants were asked to rate their level of agreement, using a 10-point Likert type scale, with statements regarding attitudes towards the COVID-19 infection and the second booster dose of the COVID-19 vaccine. Regarding the perception that COVID-19 was a serious illness, most respondents (42%) reported the value of 10 with an overall mean value of 8.4. Regarding the self-awareness of being at risk of getting the SARS-CoV-2 infection, the overall mean score was 7.4, and 35.9% of respondents had the higher value of 10. The perception towards the usefulness and utility of the second booster dose was very high with an overall mean value of 8.4 and 8.2, respectively. The final multivariable linear and logistic regression models displayed in Most of the sample (91.6%) had learned about the second booster dose of the COVID-19 vaccine. More than half of the respondents indicated mass media (57.1%) as the main source of information. Additional sources were physicians (36.9%) and institutional organizations (24.9%). Only a small number (11.2%) stated that they needed additional information on the second booster dose of the COVID-19 vaccine.The current findings are important as, to the best of our knowledge, this was the first and largest survey that evaluated vaccinated adults and people with chronic medical conditions about their attitudes regarding the second booster dose of the COVID-19 vaccine and the reasons for receiving it, and quantified the relative contribution of several factors.The present survey sheds light on the main reasons why adults and people with chronic medical conditions in Southern Italy received the second booster dose of the COVID-19 vaccine. Protection of themselves and of their family members from getting COVID-19, fear of acquiring the disease, having the vaccine recommended by a physician, and the perception of being at risk of getting a severe form of infection were the primary reasons cited by the survey participants. A possible explanation for indicating the protection as the main reason is the extensive and persistent diffusion of SARS-CoV-2 infection that may have contributed to the awareness among the sample with a consequent high sense of urgency regarding this vaccination. Earlier studies in different geographic areas showed comparable results ,18,19,20A substantial number of survey participants (91.6%) stated that they had learned about the second booster dose. The most important source of information was the mass media and it is interesting to observe that the physicians ranked second. The heavy use of mass media is an issue of concern. Indeed, even though during the pandemic mass media routinely reported the epidemiological data, some misinformation has been generated especially on the vaccines. It is important to note that physicians as a source of information have a positive significant impact. Indeed, individuals who had received information from a physician were more likely to receive the second booster because they perceive to be at risk of having a severe form of COVID-19 than those who did not use this source. This result highlights that physicians, as all healthcare providers, are a reputable source for disseminating health information and for health promotion and prevention of diseases. They play a pivotal role with a direct impact in helping individuals to acquire and to understand COVID-19-related information and, therefore, in influencing individuals\u2019 decision-making regarding healthcare issues including COVID-19 vaccination. This finding is aligned with the mounting evidence showing that advice or recommendation by physicians is very effective for increasing the level of knowledge, awareness, and vaccination uptake in different groups ,26,27,28In addition to the association with the sources of information, the multivariable linear and logistic regression analysis revealed several other significant independent predictors of the three outcomes of interest. It is important to note that among all the socio-demographic and general respondent\u2019s characteristics, age, sex, relationship status, and health status have been identified as important determinants. Males were more likely to have a higher perception of the utility of the second booster dose, whereas young and married/cohabitant respondents were more likely to have received the second booster dose for protection of themselves and of their family members from getting COVID-19 than individuals older and unmarried/not cohabiting. The association with sex may be explained by the number of deaths in Italy, which was higher among males despite the lower numbers of cases compared to females. Regarding the age, younger respondents may be more exposed to SARS-CoV-2 and more worried about the risk of acquiring and transmitting it and, therefore, decide to receive the second booster dose for preventing this infection. Individuals with at least one chronic medical condition were more likely to have received the second booster dose because they perceive to be at more risk of getting a severe form of SARS-CoV-2 infection than healthy individuals. This association may be explained by the fact that they put more emphasis and interest in health issues and thus they are more interested in this vaccination. The associations with socio-demographic and general respondent\u2019s characteristics observed in this survey are consistent with the literature ,31,32,33The present survey has some potential methodological limitations that should be considered when interpreting the findings. First, as in all cross-sectional study design, there is no evidence of a temporal relationship between independent variables and outcomes of interest. Second, the survey has been conducted in immunization centers in a single geographic area, thus the findings should be interpreted carefully as they may not be generalized to the overall populations of adults and people with chronic medical conditions in Italy. Third, respondents using self-reporting questionnaires may have introduced response and social desirability bias. However, these types of bias, common disadvantages of this data collection method, have been reduced by using a completely anonymous questionnaire. Although this survey has these limitations, there are several advantages of this type of data collection that should be emphasized, such as the low cost, the rapidity, the easier data collection, and the high response rate. Moreover, this is the first survey about the reasons for receiving the second booster dose of the COVID-19 vaccine in adults and in people with chronic medical conditions in Southern Italy and the results have potential intervention implications for the health policy makers.In sum, this survey provides useful information about the second booster dose of the COVID-19 vaccine for adults and for people with chronic medical conditions in Southern Italy and underlines the need for implementation and policymaking in order to expedite the achievement of an optimal coverage rate. Lack of recommendations for receiving the second booster dose from physicians is of great concern, since they should play, as with all public health professionals, a pivotal role in stressing the importance of this vaccination and in advising and helping individuals to make decisions, while self-protection and protection of their family members from infection were the main reasons for this sample to receive the booster dose."}
{"text": "Background: Recent evidence has shown that the updated COVID-19 bivalent booster is effective in preventing COVID-19 compared with no previous vaccination and prior monovalent vaccination. Despite its effectiveness, uptake has been poor, and a minority of eligible recipients have received the booster. Understanding healthcare worker (HCW) attitudes for and against voluntary uptake of the bivalent booster dose against COVID-19 can help guide communication strategy to maximize uptake. In this survey study, we investigated attitudes toward updated and/or bivalent booster uptake in a behavioral health hospital shortly after a COVID-19 outbreak. Methods: A survey tool was developed and sent to all HCWs at the Yale New Haven Psychiatric Hospital in December 2022. The survey queried demographic data, job category, history of COVID-19, prior COVID-19 vaccinations, perception of COVID-19 exposure, and updated and/or bivalent booster doses. The survey was administered several weeks after a COVID-19 outbreak on multiple inpatient behavioral health units. Receipt of the COVID-19 primary vaccination series and the first booster dose were mandated for HCWs; however, receipt of the bivalent booster was voluntary. Results: The survey was sent to 664 HCWs with primary assignments in behavioral health settings. In total, 182 (27.4%) provided complete responses to the survey and are included in these data. Moreover, 91 HCWs (50.0%) reported previously having COVID-19 at least once. Overall, 100 HCWs (55.0%) received the bivalent booster. The most identified reasons for receiving the bivalent booster were wanting to protect family and friends (n = 113), importance of staying healthy (n = 112), and protecting colleagues and patients (n = 103). The most identified reasons for not wanting to receive the bivalent booster dose were not thinking it provides additional protection (n = 33), \u201ctoo many\u201d shots already received (n = 31), and concern about side effects (n = 30). Discussion: Bivalent booster dose uptake in HCWs on behavioral health units shortly after a COVID-19 outbreak was greater than the general population. HCWs reported varying reasons for and against receipt of the bivalent booster dose, with the most common being protection of family and friends and perceptions of no additional protection, respectively. A limitation of this study was voluntary response bias, in which results are biased toward individuals more likely to receive a bivalent booster vaccine. It is unclear whether reasons for declining the vaccine are representative of HCWs who did not complete the survey. Assessing attitudes for the bivalent booster dose can assist in guiding communication and outreach strategies to increase vaccine uptake by HCWs.Disclosures: None"}
{"text": "This study aimed to investigate factors influencing the uptake of first and second COVID-19 booster vaccines among adults in Belgium, particularly age, sex, region of residence and laboratory confirmed COVID-19 infection history.A binomial regression model was used with having received the first or second booster as outcome and age, sex, region of residence and infection history as fixed variables. Among adults, there was generally a higher uptake to receive the first booster among older age groups compared to younger ones. Females, individuals residing in Flanders and those with no previous COVID-19 infection were more likely to receive the first booster. For the second booster, the same age trend was seen as for the first booster. Males, individuals residing in Flanders and those who tested positive for COVID-19 once after first booster were more likely to receive the second booster. Individuals with multiple positive COVID-19 tests before and after primary course or first booster were less likely to receive the subsequent booster dose compared to COVID-na\u00efve individuals. This information could be used to guide future vaccination campaigns during a pandemic and can provide valuable insights into booster uptake patterns.The online version contains supplementary material available at 10.1186/s13104-023-06608-4. In the second half of 2021, the World Health Organization (WHO) recommended administering booster vaccines against COVID-19 to adults who had already completed their primary course of COVID-19 vaccination (PC). Several studies have aimed to identify socio-demographic, socio-economic and perception-related factors associated with booster vaccine intention and hesitancy. Findings indicated that older age was positively associated with the intention to receive both first and second booster doses  while thAlthough these studies provided insight into booster acceptance among different populations, they did not delve into actual booster uptake nor its influencing factors. Retrospective cohort studies utilizing registry-linked data could provide more comprehensive insights into the determinants of booster uptake. We identified only few articles that described registry-based studies examining the association between socio-demographic and economic factors, and COVID-19 (booster) vaccination. These studies showed a higher vaccine uptake among older individuals, females and those without prior COVID-19 infection while lower uptake was observed among socially deprived individuals and those with a migrant background \u201316. EvenIn Belgium, the first booster campaign, for which the entire adult population was invited , took plSeveral COVID-19 waves preceded and coincided with the Belgian booster campaigns, resulting in breakthrough infections after PC. These individuals may harbor scepticism about vaccine effectiveness compared to those infected before vaccination or uninfected individuals , 6. ThisTwo databases were used in this analysis: (1) the COVID-19 national vaccination registry (VaccinNet\u2009+) which contains demographic information  and COVID-19 vaccination details , and (2) the COVID-19 Health Data test database which contains individual COVID-19 test data (PCR and antigen test results and testing dates). These databases were linked using the unique Belgian national registry number. Data were pseudonymised before conducting the analyses.\u00ae (Pfizer/BioNtech), Spikevax\u00ae (Moderna), Vaxzevria\u00ae (AstraZeneca-Oxford) or 1 dose of Jcovden\u00ae (Johnson & Johnson)\u2014were included in the analyses focusing on first booster uptake. Persons with incomplete PC (only one dose over two) or a heterologous PC  were excluded from the analyses. For analyses regarding the second booster uptake, those who received one booster dose after a PC and were aged 50\u00a0years or older were included (as only persons over 50 were actively invited for a second booster during the Belgian vaccination campaign). Only individuals who received a booster with Comirnaty\u00ae (Pfizer/BioNtech) or Spikevax\u00ae (Moderna) were included in the analyses. First boosters administered until March 1st 2022 and second boosters until January 31st were considered. We only included persons alive on the 31st of January 2023.All adults (\u2265\u200918 years) who had PC in Belgium\u20142 doses of ComirnatyInfection history of individuals was based on their laboratory-confirmed COVID-19 infection status, utilizing PCR and antigenic COVID-19 tests. This was considered relative to the last preceding COVID-19 vaccination. As shown in Table We fitted a logistic regression model, assuming a quasi-binomial data distribution (number of boosted over number of people who received previous vaccination), with having received a first or a second booster as outcome (number of successes) and with age, sex, region of residence and infection history as categorical fixed effects. Additionally, a sensitivity analysis was performed based on stratification by age, by fitting logistic regression models with the same fixed effects apart from age. The model coefficients were used to calculate the adjusted odds ratio (aOR). All statistical calculations were performed in R (version 4.1.3).The study population comprised 7 857 113 individuals, of whom 51.42% were female. The mean age was 50.6\u2009\u00b1\u200918.8\u00a0years. Among those who received the PC, 82.55% received the first booster and 46.57% received the second booster. Among the first booster recipients, 84.00% were COVID-na\u00efve, 8.00% had at least one prior infection before PC and 7.68% had one infection after PC. Less than 1% of the study population had multiple infections before and after, or only after PC. Among second booster recipients, 74.16% were COVID-na\u00efve, 11.99% had at least one infection before the first booster and 12.73% had one infection after the first booster. Less than 2% had multiple infections before and after or only after the first booster , older age groups showed higher likelihood of receiving the first booster, with the 75\u201384 age group being the most likely . Men had a slightly lower likelihood to receive the first booster compared to women. Residents of Flanders had the highest odds of receiving a first booster compared to Wallonia and Brussels.Compared to COVID-na\u00efve individuals, those with one infection after first booster were slightly more likely to receive the second booster . However, individuals with other infection history patterns were consistently less likely to receive one, compared to COVID-na\u00efve individuals .Compared to the youngest age group (50\u201354 years), higher age groups were more likely to receive the second booster, with the over 85 age group having the highest likelihood . Men had a slightly higher likelihood to get the second booster compared to women. Individuals residing in Flanders had the highest likelihood of receiving the second booster compared to Wallonia and Brussels , but also to changes in testing strategies and non-pharmaceutical interventions during booster campaigns. During the first booster campaign, Belgium was still in a \u201ccrisis mode\u201d with various restrictions and a more elaborate testing strategy. However, as 2022 progressed, the testing capacity and test prescriptions decreased, resulting in a decrease in registered PCR tests. This led to an underestimation of individuals\u2019 infection history relative to the first booster uptake, which causes a vast overestimation of COVID-na\u00efve individuals. The COVID-na\u00efve group was used as reference for infection history in both uptake models, considering its heterogenous nature in the second booster uptake model, it will influence the model outcome directly. Individuals with all other infection history patterns were consistently less likely to have received the second booster, following the trends observed in the first booster uptake. Nevertheless, given the aOR, infection history had a more sizable effect on vaccine uptake for the first booster campaign compared to the second. Bennett et al. and Della Polla et al. reported on the possible effect of previous COVID-19 infection on first and second booster intention respectively, based on self-reported surveys with limited study samples, but the results were not significant . A studyWe observed an increasing likelihood of first and second COVID-19 booster uptake with increasing age, consistent with studies on booster acceptance , 11, 12.Older age, residing in Flanders (compared to the other Belgian regions) were positively associated with both first and second booster uptake. Having had multiple confirmed COVID-19 infections before or after preceding vaccination, were negatively associated with the first and second booster uptake. These findings highlight the potential influence of being previously infected with COVID-19 on individuals\u2019 booster vaccine uptake. This information could give a better estimation of expected booster vaccine uptake in future booster vaccine campaigns.We acknowledge three main limitations. Firstly, the infection history data is limited due to the evolving testing strategy. The incomplete testing of symptomatic individuals and the absence of antigenic self-test results, particularly during the period in between the first and second booster (start of Omicron dominance), resulted in an overestimation of COVID-na\u00efve individuals. However, we believe that this limitation has minimal impact on the overall direction of our estimates given the repeated negative effect of having received multiple infections on booster uptake. Secondly, we cannot asses the influence of individuals perception regarding booster vaccines. Several studies have shown the inherent link between perception, regarding politics, religion, vaccine safety and effectiveness, risk of (severe) disease and peer behaviour, and booster hesitancy , 6, 10. Additional file 1:Figure S1. Comparison of drivers booster uptake in general models and age group-stratified models. A Odds ratio for the general model for first booster uptake. B Odds ratio for the general model for second booster uptake. C Odds ratio for the age group-stratified model for first booster uptake. CI is not shown for \u2018>1 infect. after\u2019 due to out of bounds. D Odds ratio for the age group-stratified model for second booster uptake"}
{"text": "In Krabbe disease (KD), mutations in \u03b2-galactosylceramidase (GALC), a lysosomal enzyme responsible for the catabolism of galactolipids, leads to the accumulation of its substrates galactocerebroside and psychosine. This neurologic condition is characterized by a severe and progressive demyelination together with neuron-autonomous defects and degeneration. Twitcher mice mimic the infantile form of KD, which is the most common form of the human disease. The Twitcher CNS and PNS present demyelination, axonal loss and neuronal defects including decreased levels of acetylated tubulin, decreased microtubule stability and impaired axonal transport.We tested whether inhibiting the \u03b1-tubulin deacetylase HDAC6 with a specific inhibitor, ACY-738, was able to counteract the early neuropathology and neuronal defects of Twitcher mice.Our data show that delivery of ACY-738 corrects the low levels of acetylated tubulin in the Twitcher nervous system. Furthermore, it reverts the loss myelinated axons in the sciatic nerve and in the optic nerve when administered from birth to postnatal day 9, suggesting that the drug holds neuroprotective properties. The extended delivery of ACY-738 to Twitcher mice delayed axonal degeneration in the CNS and ameliorated the general presentation of the disease. ACY-738 was effective in rescuing neuronal defects of Twitcher neurons, stabilizing microtubule dynamics and increasing the axonal transport of mitochondria.Overall, our results support that ACY-738 has a neuroprotective effect in KD and should be considered as an add-on therapy combined with strategies targeting metabolic correction. As a consequence of GALC deficiency, the metabolism of several galactosphingolipids is impaired and GALC substrates - galactosylceramide and galactosylsphingosine , accumulate , or globoid cell leukodystrophy, is a lysosomal storage disorder caused by mutations in the cumulate . Whereascumulate  and leadcumulate . Consequcumulate . Among tcumulate . The Twinal GALC , mimics nal GALC . Similarnal GALC .Hematopoietic stem cell transplantation (HSCT) emerged as one of the most valuable treatments for KD, delaying disease progression and extending life time of KD patients if transplantation is performed before the onset of symptoms . In spitWith respect to neuroprotection, it is important to note that in addition to demyelination, neuronal-autonomous defects are also central players in KD progression . In fact50\u20131,7 nM) with a high selectivity of 60-1,500-fold over class I HDACs. Although this compound has a rapid elimination from plasma (T1/2\u20130.2\u2009h), upon systemic administration, it leads to a ratio brain-plasma exposure of 1.22:1, demonstrating a higher capacity to cross the blood\u2013brain barrier than other HDAC6 inhibitors such as tubastatin A  and a specific histone deacetylase, HDAC6, that has a cytoplasmic location and thereby does not interfere with histone acetylation . Studiesstatin A . As stabstatin A . Currentnditions , and in gression , whereasgression . In the lifespan . ACY-738lifespan . In viewGiven the impairment caused by the defect in tubulin acetylation in Twitcher nerves, we tested whether ACY-738 delivery could counteract the early neuropathology of this KD model. Here, we show that the early systemic delivery of ACY-738 robustly increases tubulin acetylation, stabilizes microtubule dynamics, increases axonal transport, and rescues the early loss of myelinated axons in the optic and sciatic nerves of Twitcher mice. Our results thus support that ACY-738 has neuroprotective effect in KD and should be considered as an add-on therapy in KD.2.2.1.Galc allele possesses the mutation, as described  mice and wild-type (WT) littermates were obtained from heterozygous breeding pairs (Jackson Laboratory). Genotyping of Twitcher mice was based on the use of a PCR mismatched primer that creates a restriction site for EcoRV if the escribed . Mice we2.2.Animals were monitored twice per week until P25 and then daily until humane endpoints were reached. Parameters including weight loss, natural/provoked behavior, and activity level were scored. Natural behavior was assessed by observation of the animals in their home cage; animals with normal behaviour, i.e., moving, grooming and feeding, and alert received the highest score 3, animals with minor changes to these parameters were scored as 2, animals with severely decreased mobility but that were still alert received a score of 1, whereas less motile and not alert animals scored 0. The animals were then touched to provoke a response, which was scored with 3 if the animal reacted normally , 2 if minor changes to normal behavior were found, 1 if moderate changes to the escaping and freezing responses were seen or 0 is the escaping and freezing responses were absent. Activity level was evaluated by observation of the animals in their home cage during 5\u2009min and recording the time spent ambulating and exploring; animals active for more than 4\u2009min received the highest score (3), and animals with less than 1\u2009min of activity scored (0). Twitching frequency was accessed also twice a week using a similar scoring system, with animals with rare twitching scoring 4 and animals with severe tremors soring 1.2.3.n\u2009=\u200910 for each condition) or maintained until humane endpoints were reached . WT mice were also injected subcutaneously daily with ACY-738 (n\u2009=\u20098\u201310) or vehicle (n\u2009=\u20098\u201310) to serve as controls. Either at P9 or when animals reached humane endpoints, brains, sciatic nerves, optic nerves and dorsal root ganglia (DRG) were collected and processed for morphometric analysis, western blot analysis or primary neuron cultures, as described below. During the delivery period, no signs of toxicity of the drug were detected.ACY-738  was prepared in 0.5% hydroxypropyl methylcellulose (HPMC) diluted in PBS at a final concentration of 0.5\u2009mg/mL. Starting at P0, Twitcher mice were injected subcutaneously every day (3\u2009mg/kg) with either ACY-738 (in 0.5% HPMC) or vehicle (0.5% HPMC) and euthanized at P9 (2.4.n\u2009=\u200910) or control Twitcher mice (n\u2009=\u20098\u20139) were homogenized in lysis buffer (PBS containing 0.3% Triton X-100), 1\u2009mM sodium orthovanadate and protease inhibitors . Total protein of each sample was determined using the Bio-Rad DC kit  and protein lysates were run on 10% SDS-PAGE (5\u2009\u03bcg/lane) and transferred to supported nitrocellulose membranes . Membranes were incubated overnight at 4\u00b0C with mouse anti-acetylated tubulin , rat anti-tyrosinated tubulin  and mouse anti-\u03b1-tubulin . Given the difficulty to perform stripping of anti-tubulin antibodies from the membranes, two separated gels were loaded and transferred simultaneously to probe for either acetylated- or \u03b1-tubulin. Secondary antibodies were HRP-conjugated anti-mouse IgG  or the IRDye\u00ae 800CW Goat anti-Mouse IgG . Proteins were detected using Luminata Forte  and quantification was performed by densitometry using QuantityOne software (Bio-Rad). Alternatively, the Odyssey CLx (LI-COR) imaging system was used for detection of membrane total protein  and dye-conjugated labelled secondary antibodies .Sciatic nerves, optic nerves and brains from ACY-738-treated Twitcher mice (2.5.n\u2009=\u20098 ACY-738-treated and n\u2009=\u20098 control Twitcher mice), the right sciatic and optic nerves were used for neuropathological analyses. All tissues were isolated and fixed by immersion in 4% glutaraldehyde in 0.1\u2009M sodium cacodylate buffer (pH 7.4) for 5\u2009days and processed for electronic microscopy as previously described  until humane endpoints  supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin . DRG were digested with collagenase IV-S  for 1\u2009h and 30\u2009min. Neurons were isolated centrifuging the cell suspension into a 15% bovine serum albumin  cushion, for 10\u2009min at 1000\u2009rpm. Cells were resuspended in DMEM:F12 supplemented with 1% P/S, 2\u00d7 B27 , 2\u2009mM\u2009L-glutamine  and 50\u2009ng/mL nerve growth factor .2.7.For the analysis of microtubule dynamics in the growth cone, DRG neurons from Twitcher and WT mice from animals euthanized humane endpoints were isolated as described above and nucleofected with a plasmid encoding pEGFP-eEB3 (Addgene #190164). After transfection, cells were left in suspension for 24\u2009h. At DIV1, cells were plated at a density of 10,000 cells/well in glass-bottom 8-well \u03bc-dishes. At DIV5, 2\u2009h before imaging, half of the medium was replaced with complete media without phenol red and supplemented with 100\u2009nM ACY-738 in 0.1% DMSO or 0.1% DMSO alone. Transfected cells were visualized using the 100x objective of a spinning disk confocal system Andor Revolution XD with an iXonEM+ DU-897 camera and IQ 1.10.1 software (ANDOR Technology). Time-lapse recordings  were acquired in three planes separated by 0.2\u2009\u03bcm each at 37\u00b0C and 5% CO2. For the quantification of EB3 comet growth speed, kymographs were performed using the Fiji KymoResliceWide plugin . Starting and end positions of the traces were defined using the Fiji Cell Counter plugin.2.8.For live imaging of axonal transport, DRG neurons from ACY-738-treated and control mice were isolated as previously described from animals euthanized at humane endpoints. Dissociated DRG neurons  were cultured in DMEM:F12 supplemented with NGF, Pen/Strep, B27 and L-glutamine for 16\u2009h in 8-well \u03bc-slides ibiTreat (Ibidi) coated with PLL and laminin. DRG neurons isolated from ACY-738-treated WT and Twitcher mice were maintained in culture in the presence of 100\u2009nM ACY-738 in 0.1% DMSO. Control WT and Twitcher DRG neurons were maintained in 0.1% DMSO. For the analysis of axonal transport of mitochondria, DRG neurons were incubated with Mitotracker  in DMEM:F12 for 45\u2009min at 37\u00b0C. Axonal transport was visualized in a confocal SP5II Leica microscope. Photomicrographs were taken for 2\u2009min with 1\u2009s frame intervals with 5 Z-stacks per frame. For each condition, the movement of at least 50 organelles was tracked. Analysis of axonal transport (velocity and flux) was done using the Fiji software.2.9.t-test, one-way or two-way ANOVA followed by multiple comparison tests or Welch ANOVA test if unequal variances were observed between the groups. Sample sizes are indicated in figure legends and significance was defined as p value*\u2009<\u20090.05, p**\u2009<\u20090.01, p***\u2009<\u20090.001, p****\u2009<\u20090.0001, ns: not significant.Data is shown as mean and standard error of the mean (SEM). The statistical analysis for all experiments was performed with GraphPad Prism 9. Unless elsewhere stated, the following statistical tests were used as indicated in figure legends: two-tailed unpaired 3.3.1.To test the possible neuroprotective effect of ACY-738 in KD, the drug was delivered subcutaneously daily to Twitcher mice. Since Twitcher mice present axonal loss that can be detected readily at P9, prior to the onset of demyelination , we expl3.2.Twitcher neurons exhibit decreased acetylated tubulin levels, decreased microtubule cytoskeleton stability , and impAs axonal transport efficiency is highly influenced by microtubule organization and dynamics , we hypo3.3.Twitcher mice develop tremors at around 18\u201320\u2009days of age and muscle weakness and weight loss at P20-25 and usually reach the humane endpoints established in our facility (and recommended by FELASA) at 30\u201335\u2009days of age. To determine the possible effect of ACY-738 treatment in disease progression, animals were monitored daily for their functional performance. ACY-738-treatment resulted in a substantial improvement of natural  and prov3.4.Given the functional improvement of Twitcher mice treated with ACY-738, we further investigated its potential to correct the early neuropathology of this model. In the optic nerve, as previously reported , P9 TwitSimilar to the optic nerve, P9 Twitcher sciatic nerves displayed a reduced density of both myelinated and unmyelinated axons in comparison with WT sciatic nerves \u2013H, as pr3.5.The results obtained from the subcutaneous delivery of ACY-738 from P0-P9 warranted the analysis of treated Twitcher mice for extended time periods, i.e., until humane endpoints were reached. Detailed analysis of control and ACY-738-treated Twitcher mice showed that the number of small caliber myelinated axons was increased by ACY-738 treatment . Specifi4.Galc, in long-term surviving AAV-treated Twitcher mice, multiple demyelinating areas are still detected in the brain, suggesting a possible late onset reduction of AAV efficacy , University of Porto, Porto, Portugal.MS and JN-R coordinated research. MJ, MS, and JN-R designed the study. SB, PB, MJ, MS, and JN-R analysed experiments. SB, MM, MP, AM, and JN-R performed the experiments. SB, MS, and JN-R wrote the manuscript. All authors contributed to the article and approved the submitted version.The Nerve Regeneration group was supported by a Sponsored Research Agreement with Acetylon Pharmaceuticals Inc. that provided ACY-738. The work was also supported by Portuguese funds through FCT - Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia/Minist\u00e9rio da Ci\u00eancia, Tecnologia e Ensino Superior in the framework of the project EJPRD/0002/2019. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication.OG and MJ were employed by Acetylon Pharmaceuticals Inc.The remaining 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": "This paper presents an improved solution for the airflow energy harvester based on the push\u2013pull diamagnetic levitation structure. A four-notch rotor is adopted to eliminate the offset of the floating rotor and substantially increase the energy conversion rate. The new rotor is a centrally symmetrical-shaped magnet, which ensures that it is not subjected to cyclically varying unbalanced radial forces, thus avoiding the rotor\u2019s offset. Considering the output voltage and power of several types of rotors, the four-notch rotor was found to be optimal. Furthermore, with the four-notch rotor, the overall average increase in axial magnetic spring stiffness is 9.666% and the average increase in maximum monostable levitation space is 1.67%, but the horizontal recovery force is reduced by 3.97%. The experimental results show that at an airflow rate of 3000 sccm, the peak voltage and rotation speed of the four-notch rotor are 2.709 V and 21,367 rpm, respectively, which are 40.80% and 5.99% higher compared to the three-notch rotor. The experimental results were consistent with the analytical simulation. Based on the improvement, the energy conversion factor of the airflow energy harvester increased to 0.127 mV/rpm, the output power increased to 138.47 mW and the energy conversion rate increased to 58.14%, while the trend of the levitation characteristics also matched the simulation results. In summary, the solution proposed in this paper significantly improves the performance of the airflow energy harvester. With the rapid development of wireless sensor networks , microelAirflow is a kind of widespread source of clean energy in nature, and it has many sources and the widest range of applications. Xin et al.  propose Diamagnetic levitation was first investigated experimentally by Cansiz and Hull  in 2004.This paper focuses on improving the airflow energy harvester based on the new diamagnetic levitation structure to enhance output performance and energy conversion rate. A centrosymmetric rotor is proposed to address the shortcomings of the three-notch rotor. Simulation models are built in COMSOL to compare and verify various floating rotors\u2019 output performance and determine the optimum rotor parameters. A joint COMSOL and MATLAB simulation was carried out to compare the levitation characteristics of the new rotor with those of the three-notch rotor. Experimental prototypes and platforms have been built based on theoretical and simulated models, and the results show that the airflow energy harvester with the new rotor has higher output performance and better levitation characteristics.The three-dimensional model of the airflow energy harvester is shown in PusF and PulF. Diamagnetic force UpF and LowF are exerted on the floating rotor from the upper and lower HOPG sheets. In G represents the gravity of the floating rotor, and RF denotes the axial resultant force. The potential energy of the floating rotor can be expressed by Equation (2).M is the magnetic dipole moment of the floating rotor, PusB denotes the magnetic flux density of the pushing magnet, PulB shows the magnetic flux density of the pulling magnet, m shows the mass of the floating rotor, and z shows the distance from the ground. Equation (3) can be obtained by substituting the diamagnetic influence exponents [zC and rC into Equation (2).The push\u2013pull diamagnetic levitation structure is shown in xponents  Cz and CThe formula for the stability of the floating rotor that can be derived from Equations (3)\u2013(5) are the equations for vertical and horizontal stability.Compared with the old structure , the pusThe schematic diagram of the three-notch rotor is shown in dF1 and dF2, respectively.When the airflow collides with the floating rotor, the motion state of the airflow can be simplified as shown in \u03b8 represents the angle between the driving force and the line connecting the two. When \u03b8 is closer to 90\u00b0, the driving effect on the rotor is more prominent, while the radial force on the rotor is smaller. It can be seen from \u03b81 is greater than \u03b82, and the tangential component of the driving force dF1 is larger, while the radial component of the driving force dF2 is larger.Connecting the collision point of the airflow and the center of the floating rotor, \u03b1 of the yellow and green areas is 32\u00b0, and the angle \u03b2 of the red and cyan areas is 28\u00b0.According to the relative positions of the nozzle and itself, the floating rotor can be divided into three parts, red, green, and blue areas shown in RdF and LdF, respectively. The two forces are decomposed into two tangential forces RdtF and LdtF, and radial forces RdrF and LdrF. As can be seen from rF, which makes the floating rotor unbalanced in the horizontal plane and thus begins to offset. When the pushing magnet, the pulling magnet, and the floating rotor are not coaxial, the pushing magnet and pulling magnet exert a horizontal magnetic force on the floating rotor, and the sum of the two horizontal magnetic forces is the horizontal recovery force HF of the floating rotor. In HF also increases. As expressed in Equation (8), when it reaches O\u2019, the value of the horizontal recovery force HF is equal to that of the radial force rF, the floating rotor reaches its equilibrium in the horizontal direction, and the offset also reaches the maximum value. O\u2019 is denoted as the maximum right side offset point of the floating rotor, where the floating rotor continues to rotate and enters the next stage, as shown in The first stage is shown in RdF is significantly greater than the left driving force LdF. In the horizontal direction, the floating rotor will return to the original central O point under the combined action of the horizontal recovery force HF, the right-side radial force RdrF and the left radial force LdrF. In O, the left and right-side nozzles are at an equal distance from the floating rotor, the radial and tangential components of the left and right-side driving forces LdF and RdF are equal, as shown in Equation (9). The floating rotor enters the third stage after 30\u00b0 rotation around O.In rF is exactly opposite to the horizontal resultant force in the first stage, the floating rotor will be offset to the left, finally reaching the maximum left offset point O\u201d. The floating rotor will enter the fourth stage with another 30\u00b0 rotation. The fourth stage is like the second one in that the floating rotor returns to the initial point O again under the action of the horizontal recovery force HF and the two radial forces. The entire period is the completion of the rotation of the floating rotor before entering the next rotation period.In the third stage, the airflow from the left nozzle blows toward the notch surface, while the right nozzle blows toward the circular surface, which corresponds to the rotation of the floating rotor by 180\u00b0 relative to the first stage. At this time, the direction of the horizontal resultant force During one rotation cycle, the floating rotor is offset to the left and right in the horizontal plane in turn because radial forces cannot be fully counteracted. After reaching a maximum speed of 20,000 rpm, the floating rotor undergoes more than 900 periodic offsets per second. Such a high frequency of periodic offset consumes substantial energy, reducing the energy conversion rate. If the floating rotor deflection is to be eliminated, it must be ensured that the airflow from both nozzles is blowing simultaneously toward the circular surface or the notch surface. The three-notch rotor cannot eliminate the offset due to its structure. When the number of notches is even, the rotor has a centrosymmetric structure, as shown in ECF\u03c1. The quotient of the peak voltage and the speed of the rotor is the value of the energy conversion factor. The peak voltage PE can, therefore, be expressed by Equation (10).In the airflow energy harvester, two factors influence the magnitude of the peak voltage. One is the rotation speed \u03c9 of the floating rotor. Higher speed led to higher induction electromotive force in the coil. The other one is the energy conversion factor 3. The simulation model shown in The energy conversion part of the energy harvester consists of two shaped coils made up of three circular coils connected in series, which are arranged above and below the floating rotor, as shown in TCE (BCE) in the top coils (bottom coils) is less than the sum of the voltages of the three coils, as expressed in Equation (11). Moreover, the value of TCE (BCE) is always zero when using the four-notch rotor. In contrast, as seen in TCE (BCE) of both the three-notch and six-notch rotors are three times the electromotive force in a single coil, as expressed in Equation (12). Furthermore, the total voltage of the three-notch rotor is significantly higher than that of the six-notch rotor by a factor of approximately two. The energy conversion factors ECFS2\u03c1, ECFS3\u03c1, ECFS4\u03c1 and ECFS6\u03c1 of the four rotors in a single coil are calculated from Equation (10), and the values of them are 0.0260 mV/rpm, 0.0263 mV/rpm, 0.0272 mV/rpm, and 0.0146 mV/rpm, respectively. According to Equation (13), the centrosymmetric rotors may have a higher output performance. The coil arrangement of the three-notch rotor is not suitable for the centrosymmetric rotors, which results in their low total voltage.As seen in SCE of the two-notch rotor, three-notch rotor, four-notch rotor, and six-notch rotor are 0.3392 V, 0.4344 V, 0.5415 V, and 0.2839 V, respectively. The output voltage E is calculated according to Equation (14), where N is the number of notches in each rotor. In With the centrosymmetric rotors, the existing coil arrangement must be changed to avoid the induction electromotive force canceling each other out. The improved coil arrangement is shown in The simulation gives a resistance of 2.42 \u03a9 for a single circular coil. The power of four rotors was calculated from the total output voltage and resistance, as shown in The levitation characteristics of the three-notch and four-notch rotors were calculated using a joint MATLAB and COMSOL simulation. The magnetic and diamagnetic forces of the four-notch rotor are smaller compared to the three-notch rotor due to the lower magnetic induction strength, which makes the axial combined forces and the potential energy of the two types of floating rotor follow approximately the same trend, with slight differences, as shown in PusL values of 59 mm, 58.5 mm, 58 mm, 57.5 mm, and 57 mm, the axial magnetic spring stiffness increases by an average of 7.886%, 8.328%, 9.915%, 10.939%, and 11.263%, respectively. The overall average increase in axial magnetic spring stiffness is 9.666%.The axial magnetic spring stiffness of the floating rotor represents the ability of the energy harvester to cope with external axial excitation. The larger the axial spring stiffness, the higher the axial stability. As seen in PusL represents a levitation point, and at each levitation point, there is a maximum monostable levitation space HmaxL. When using the four-notch rotor, PusL takes on a larger range of values and more levitation equilibrium points can be selected. For the same value of PusL, the maximum monostable levitation space of the four-notch rotor is somewhat enhanced, with an overall average enhancement of 1.67%. Furthermore, the maximum monostable levitation space\u2019s maximum value is increased from 4.88 mm to 5.14 mm. The larger maximum monostable levitation space facilitates observation of the levitation state of the floating rotor and reduces the possibility of friction between the floating rotor and the coil due to axial excitation.In As shown in The experimental platform of the improved airflow energy harvester is built according to PusL decreases, the rotor can be kept levitation by reducing PulL. When the value of PusL is reduced from 55.365 mm to 51.365 mm, the maximum monostable levitation space Hmax3L is reduced from 4.73 mm to 3.89 mm for the three-notch rotor and Hmax4L is reduced from 4.81 mm to 3.97 mm for the four-notch rotor. Meanwhile, Hmax4L is consistently greater than Hmax3L, with an average improvement of 2.05%, in line with the simulation results in Experiments were first carried out on the maximum monostable levitation space at multiple levitation equilibrium points for the three-notch rotor and the four-notch rotor. The axial positions of the pushing magnet, the pushing magnet, and the two HOPG sheets are changed so that the floating rotor is at different levitation equilibrium points. The thickness of each component and the axial distance between them and the base plate are measured using Vernier calipers. The distance between the upper and lower HOPG plates was calculated from the measured data to be the maximum monostable levitation space and recorded in PusL is 55.365 mm. The airflow from the nozzle is increased from 0 sccm to 3000 sccm in 200-sccm increments. The horizontal displacement of the floating rotor is measured by a laser displacement sensor ; the larger the horizontal displacement value, the smaller the horizontal recovery force. The data are further processed and plotted in Both rotors are tested for horizontal recovery force when In The experimental platform for testing the output performance is shown in In The four-notch rotor significantly increases the power output and energy conversion rate of the energy harvester, which means that more electronic devices can be loaded and the collected wind energy can be converted into electricity more efficiently. The energy harvester can harvest the wind energy in the environment more effectively and offer workable output with a limited airflow input.This paper investigates the airflow energy harvester using the push\u2013pull diamagnetic levitation structure. Instead of using the three-notch rotor, a four-notch rotor was introduced into the energy harvester, and a corresponding comparison study was carried out to verify the improvement. The centrosymmetric rotors are required to eliminate the periodic offset. Simulations in COMSOL Multiphysics 5.6 of the output performance of multiple floating rotors, have determined that the four-notch rotor has the best output performance. After the improvement, the levitation characteristics of the four-notch rotor were changed due to its reduced magnetic induction strength and mass. The axial magnetic spring stiffness increased by an overall average of 9.666%, and the maximum monostable levitation space increased by an average of 1.67%. However, there is a reduction in horizontal response force of approximately 3.97%, which does not affect horizontal stability due to the elimination of the floating rotor horizontal offset. An experimental platform was set up to verify the output and levitation characteristics. According to the experimental results, when the airflow is 3000 sccm, the peak voltage of the four-notch rotor can reach 2.709 V, and the maximum speed can reach 21,360 rpm. The peak voltage, maximum rotation speed, output power, energy conversion factor, and energy conversion rate of the four-notch rotor are respectively increased by 40.80%, 5.99%, 41.40%, 32.68%, and 41.39%, compared to the three-notch rotor. At the same time, the variation in levitation characteristics is consistent with the simulation results. The use of the four-notch rotor also substantially improves output performance, particularly by increasing the energy conversion rate from 41.12% to 58.14%, while increasing the maximum monostable levitation space and axial magnetic spring stiffness, demonstrating that this is a proven improvement solution."}
{"text": "Why do some zoonotic diseases receive priority from health policy decision-makers and planners whereas others receive little attention? By leveraging Shiffman and Smith\u2019s political prioritisation framework, our paper advances a political economy of disease prioritisation focusing on four key components: the strength of the actors involved in the prioritisation, the power of the ideas they use to portray the issue, the political contexts in which they operate, and the characteristics of the issue itself . These components afford a nuanced characterisation of how zoonotic diseases are prioritised for intervention and highlight the associated knowledge gaps affecting prioritisation outcomes. We apply this framework to the case of zoonoses management in India, specifically to identify the factors that shape disease prioritisation decision-making and outcomes.We conducted 26 semi-structured interviews with national, state and district level health policymakers, disease managers and technical experts involved in disease surveillance and control in India.Our results show pluralistic interpretation of risks, exemplified by a disconnect between state and district level actors on priority diseases. The main factors identified as shaping prioritisation outcomes were related to the nature of the zoonoses problem  as well as political, social, cultural and institutional environments , and challenges in organisation leadership for cross-sectoral engagement.The findings highlight a compartmentalised regulatory system for zoonoses where political, social, cultural, and media factors can influence disease management and prioritisation. A major policy window is the institutionalisation of One Health to increase the political priority for strengthening cross-sectoral engagement to address several challenges, including the creation of effective institutions to reconcile stakeholder priorities and prioritisation processes. Expanding human populations and sustained land-use change have brought humans into close proximity to wildlife, amplifying the risk of potential disease spillover from wildlife to livestock and humans , 2. At tAgainst this background, defining disease priorities for resource allocation and intervention is complex and deeply political \u201314 and aAddressing questions like these requires an integrated approach that provides the analytical lens or platform to reconcile different stakeholder priorities, available evidence and institutional processes that shape prioritisation outcomes. Recent prioritisation efforts have focussed on bridging the gap between animal and human health sectors through the agency of collaborative, cross-sectoral expert-driven prioritisations , 15. Altwhy do some zoonotic diseases receive priority from policy decision-makers and health planners (responsible for disease surveillance and control) whereas others receive little attention? Specifically, we address the following sub-questions: (i) how are zoonotic diseases prioritised for interventions and (ii) what are the knowledge gaps affecting disease prioritisation?In the face of limited resources for disease prevention activities  and control measures, a better understanding of the political, institutional, and socioeconomic factors that affect prioritisation decision-making and outcomes across different contexts remains paramount in guiding defensible resource allocation and optimising interventions. While some previous studies have identified factors that shape political prioritisation and responses to non-communicable diseases , 16, to 2.We conducted a qualitative study using semi-structured interviews with policy decision-makers, disease managers and technical experts involved in zoonoses management in India across national, state and district levels. The selection of key-informants was based on a prior One Health stakeholder mapping exercise conducted to identify different organisations involved in disease surveillance and control. Stakeholders in this context refer to individuals or actors whose interest (or mandate) significantly affect or are impinged by management decisions regarding zoonoses prevention and control. We purposively selected key-informants to cover the primary roles in the policy agenda setting and implementation of interventions across scale. Other actors and institutions were recruited through snowballing. Recruitment of key-informants for the in-depth interviews continued until data saturation was reached, stratified by sector  to capture population heterogeneity , 16. We We adopted Shiffman and Smith\u2019s political prioritisation framework to organise interview data into themes for evaluating factors shaping the political prioritisation of zoonoses in India . The modThe anonymised interview transcripts were analysed thematically within NVIVO 12 using a combination of deductive and inductive analysis, with the individual as the unit of analysis . We firs3.3.1.n\u2009=\u200914) were affiliated to the human health sector, indicative of the dominance of human health actors in India\u2019s health policy arena. Three informants were affiliated with the animal health and two with the wildlife sectors, respectively. The remaining six were experts and other informed actors, with extensive knowledge having spent their careers working on zoonoses in academic institutions  and civil society organisations focussed on disease interventions. In total, 26 interviews were conducted and categorised into four sectoral domains. The majority of informants were male and had over a decade of experience working in the field of zoonoses. Despite efforts to achieve a balanced representation of relevant sectors in zoonoses decision-making across levels, over half of informants .\u201cWe are able to prioritise ourselves as per the local need but common diseases are prioritised at national level. Local diseases are prioritised at local level. That liberty is given to the states. Some states have added diphtheria, others have added pertussis. Karnataka, for example, has added Kyasanur forest disease (KFD) as a priority disease.\u201d .On the rationale for the state-centric prioritisation of diseases, another state-level informant explained that this was mainly to avert any potential dilution effect  and to foster strong state ownership of the process:\u201cIf it [prioritisation] is done at the national level, then dilution will be there. Suppose there are 5 diseases, I will make a plan for 5 diseases. My priority, now at present because of KFD outbreak and H1N1, my top priority is H1N1 and second is KFD. So I have to prioritise my action plan for the control of morbidity and mortality of these two diseases. So better to give at the state level \u2026\u201d .3.3.To the extent that guiding institutions, actor relationships and power shapes the disease system and prioritisation outcomes, it was important to assess the strength/authority and positionality of actors and organisations within the policy arenas responsible for zoonoses agenda-setting and interventions. From the interview data, three types of authority \u2013 institutional, delegated and expert authority \u2013 were identified as linked to zoonoses management . The anaThe key actors within the health system identified as wielding institutional authority regarding zoonoses control and prevention include the National Centre for Disease Control (NCDC), Indian Council for Medical Research (ICMR), Indian Council for Agricultural Research (ICAR), Department of Animal Husbandry and Dairying (AH&D) and state departments of animal, human health and wildlife . State-lConcerning the degree of collaboration between various actors involved in zoonoses prioritisation, national and state-level informants generally concurred that the disease prioritisation process is largely sectorally-driven, i.e., the emphasis of a focal zoonosis that is considered of prime importance for animal health may not be the same for human health. On the rationale for the siloed-approach to prioritisation decision-making, a national-level animal health actor said:\u201cGlanders, for example, is a category B bioweapon which from my perspective is a priority disease. Okay you go to the human health side, the rate of transmission to human is very less. Now, how do you prioritise from that side?\u201d .On the question over what opportunities exist for joint disease prioritisation, the interviews revealed mixed views as to the extent to which decision-making process was indeed collaborative. For instance, animal health actors particularly highlighted that existing collaborations were largely tokenistic as they often played second fiddle to their human health counterparts with respect to zoonoses prioritisation. Two critical views expressed by a state-level actor and an expert from the animal health sector about collaborative prioritisation in the current state of affairs are instructive:\u201cThey [human health actors] will not be at all ready to listen to you. If you go to them and you tell them this is what we need to do, they will not at all be willing to listen to you. Actually the policy matters have been directed towards protection of mankind. We are working to protect humankind, aren\u2019t we [sic]? And the animal health side is completely side-lined. .\u201cThere is missing link between human health and animal health sectors [in respect to collaborative decision-making]. They will not take our ideas and we will not take theirs. It is recently only we came together \u2026\u201d .every sector remains overloaded. For example, if I talk to colleagues in the veterinary sector, I know that human resources is lesser than what is required. So that is a limiting factor. So people remain bounded with their own departmental work that they do not have time, energy and resources to engage other sectors, except during disease emergencies. It may not necessarily be only in India\u201d . Nevertheless, other informants downplayed the contested authority of animal health actors in joint prioritisation commonly citing examples of successful cross-sectoral engagement  and sustained informal relationships which contributed significantly in circumventing otherwise \u2018formal barriers\u2019 to collaborations .From the human health perspective, some state-level informants concurred that their animal health counterparts had circumscribed authority with respect to zoonoses decision-making since the supposedly joint-prioritisation platform was largely human health centred. Interviewees contended that the power asymmetries shaping collaborative arrangements between animal and human health actors are reflective of a broader social stratification, in which human doctors exerted high societal influence relative to veterinarians . Besidesnobody is talking about these neglected disease (say KFD or scrub typhus) and there is no funding to incentivise related interventions. Unlike rabies or TB, national health control and civil society engagement has been weak for other diseases\u201d . Correspondingly, a national-level bureaucrat  recounted how commercial livestock and dairy farms constituted a strong lobbying group that influenced the political prioritisation of brucellosis for eradication by 2030, culminating in the announcement of funding for the National Animal Disease Control Programme (NADCP) by the Prime minister\u2019s office for 2019\u20132024 . As one bureaucrat lamented, \u201c\u201cI think because of the fact that the animal welfare lobby has usurped the agenda for dog population control. It is really to do with the animal welfare lobby \u2026 That\u2019s why there has been no strong move to dog control by the health department to come up with any policy even though it is their mandate to reduce the burden of disease\u201d .3.4.Underpinning the disease prioritisation context is the resonating set of ideas \u2013 i.e. framing of the issue publicly in ways that attracts political support \u2013 that determine the attention accorded to the candidate priority disease. In essence, how a disease is portrayed and understood by actors involved in zoonoses management matters for prioritisation and intervention . We thus3.4.1.A shared understanding of an issue and its solutions by a policy community is fundamental to its coalescence. Despite the lack of formally ratified disease prioritisation criteria, several interviewees highlighted morbidity, case fatality, mortality and economic impact as the common \u2018internal framing\u2019 used to characterise candidate diseases for prioritisation. Two typical observations by a state and national level official regarding the determinants of priority accorded to focal diseases are instructive:\u201cThe most prominent diseases are linked to high case fatality and mortality which informs their high priority status. Everywhere [in the country] importance will come to a [candidate] disease when any death happen from it, mortality is the primary criteria. Until then we do not take it seriously. Once the deaths happen State, national, international, and locally everyone will be alert\u201d .\u201cI guess in most part of the community it is very pathogen-focused but there are a lot of similarities between different kinds of zoonotic diseases. Obviously the kind of visibility they get in the policy agenda and the kind of responses depends on who gets affected in terms of population, types of animals, economic or political visibility and also how visible the diseases are.\u201d .3.4.2.On the question of external framing of candidate priority diseases, a recurring theme across the interviews was that public attention and media publicity featured prominently in determining the political visibility or importance accorded to respective disease problems. Juxtaposing the case of two priority zoonoses (Anthrax and Brucellosis) in elucidating how public perception affects prioritisation outcomes, an expert informant and disease manager, respectively, said:Anthrax causing deaths causes human outcry whereas you know Brucellosis might not in the same way for example, despite having a much larger burden. Rabies having smaller cases is much more visible compared to other silent diseases with larger burden\u201d .\u201c\u201cIf there is a huge number of animal deaths happening in the short span of time, for example the case of an anthrax outbreak people are bound to get incensed they are bound to inform their leadership so chances are that it will be high on the political ladder. But if a disease which is not causing huge deaths or is causing silent disability for a long time then people might or might not be aware about it even if the scientists are aware people might not be aware about it but it is a political issue\u201d .per se but largely the political visibility shaped by narratives about diseases and their portrayal in the media space. In separate interviews, a disease manager and surveillance officer addressed the effect of public portrayals or disease narratives on eventual priority setting outcomes:A number of national and state level actors (responsible for disease surveillance and control) also corroborated the foregoing observations, implicitly suggesting that prioritisation of diseases is not necessarily based on epidemiological characteristics of diseases  Right now, the dengue positivity [for example] is more compared to that of COVID-19, but people are not much bothered about dengue. Also the health department is not giving that much importance, what COVID-19 is getting. The government prioritisation shifted based on how much news that disease is making or how much fear that is made in people\u2019s minds\u201d .\u201c\u201cMost of the things are driven by the press also. Some press persons will magnify few diseases which are not so important. If highlighted in the media, once the world comes to know that is a very serious, these kind of people are really getting troubled then it is highlighted [within policy circles]. But unfortunately, the effort [political visibility] is sustained only initially that euphoria will be there \u2026\u201d .3.5.3.5.1.\u201cso obviously you know the setup is bureaucratic \u2026 most senior people heading relevant committees are not the technical people [but political actors] so other members are going to defer to that person\u2019s you know politics. So there\u2019s only so much you can achieve in an environment like that \u2026\u201d .A general consensus across the interviews was the critical role the political environment \u2013 that is the overall institutional and cultural context in which actors operate  \u2013 plays Another informant had this to say:Whatever the activities that are implemented, the decision-maker should be a technical person. Not the bureaucrats as far as my knowledge is concerned \u2026 That is how we eradicated smallpox, polio and guinea worm because the technical persons were the heads of department in those days. That is the need of the hour!\u201d .\u201cthey are not actually coming up with the truth! Some of the states are absolutely silent and saying like we do not have this disease although our informal surveillance networks suggest otherwise\u201d . In juxtaposition, several state and district level informants disclosed some implicit assumptions that shaped the existing disease reporting regime as some interviewees cited fear of victimisation/scapegoating, punitive-transfers and/or overstretched technical manpower as some probable reasons for the supposed reticence in full disclosure of \u2018actual\u2019 disease outbreaks to higher-ups at the national and state levels. In fact, some informants particularly observed that the reticence/behaviour of under-reporting has consequences for degree to which they can rely on existing disease reporting systems like the IDSP and/or Integrated Health Information Portal (IHIP) for early warning and targeting interventions, understanding factors driving disease emergence, as well as for overall disease prioritisation. A typical perspective by a state-level surveillance officer is illustrative:A cross-section of interviewees also conceded that disease under-reporting or misreporting as a function of wider state-level politics significantly eroded the credibility of the existing prioritisation process. A state-level bureaucrat, in defence of the seemingly heavy political influence in disease prioritisation decision-making, argued that the notion of controlling the narrative was equally important as a disease itself. The said official further noted that the quest to safeguard the country\u2019s reputation, avert unsolicited surveillance, economic losses and public outcry (as in the case of the 1994 plague outbreak) largely incentivised decision-makers\u2019 attempts to control narratives by suppressing news of disease outbreaks. Relatedly, a national bureaucrat voiced out, \u201cIn outbreak situations, it is the state or district level officers whose necks are on the line if they are not able to [in the eyes of political actors] properly control the situation. Technical experts sitting at national level institutions like National Centre for Disease Control [NCDC], you know, they will come and do outbreak investigations but if something happens they really do not have to face that level of accountability because their job is to provide technical advice and the decision-making and the political fallout of that is going to be restricted to state level officers.\u201d .\u201cReflecting on the current state of play, some state and district level informants believed technical officers in some government departments and ministries (responsible for zoonoses decision-making) become \u2018pawns\u2019 often providing seeming scientific justifications for otherwise political decisions . A case \u201cManaging media and elected representatives that becomes a very big burden. In a way it is good but sometimes it is very detrimental [for disease management]. Their [political actors] pressure is always reflected in the eventual decisions we make in the area of our work \u2026\u201d .inter alia, that the high political visibility of brucellosis is largely a product of the religious and economic significance of the \u2018cow\u2019 in Indian society. This has meant that culling as an intervention is frowned upon in Indian political discourse and a silent policy of \u2018non-identification of diseased cattle\u2019 has been adopted, given the potential to trigger distress selling and stigmatisation. A typical view in this regard was given by a zoonoses expert:Aside from the political contexts, the cultural environment  impacted significantly on disease prioritisation process and outcomes. For example, a number of interviewees noted, The cow is a political icon in Indian political discourse so you cannot cull cows and if they are diseased and you cannot mark them as diseased as you end up spreading distress selling \u2026 So the policy of the government now from what I understand is not to identify individual animals but to do pooling of sampling at the farm-level and if they do identify individual animal they ask for quarantine measures and not for culling or not for selling because they do not want the farmer to be stigmatized or to resort in distress selling of the animal \u2026\u201d .\u201c3.5.2.n\u2009=\u200917 key-informants, interviewed after the COVID-19 lockdowns in December 2021) were in agreement that the COVID-19 pandemic raised awareness about zoonoses and provided a good political window of opportunity for galvanising and sustaining cross-sectoral action. Indeed, contrasting the observations across interviews highlight a common refrain \u201cCOVID-19 has given impetus to the need of the hour\u201d , suggesting the importance of leveraging joint technical and surveillance capacities as an avenue to bridge the long-standing gap between animal health and human health actors and decision-makers with respect to zoonoses management. Reflecting on their pandemic experiences, several informants recounted for example how the spontaneous collaboration of functionally disparate ICAR and ICMR institutional laboratories proved instrumental in the turnaround times for the processing of human COVID-19 samples and surveillance for speedy decision-making and intervention. The observations of national human health and state-level veterinarian during separate interviews are instructive:While discussing political moments that shifted policy and facilitated stronger engagement among key actors responsible for zoonoses decision-making, several informants .\u201cDue to the [COVID-19] pandemic, the people [decision-makers] have become sensitized to the One Health agenda and they do give importance to One Health, but having an implementable One Health model with clear-cut guidelines and outcomes, uh still is difficult to develop and implement on the ground\u201d .Some informants were unenthused or sceptical about the so-called \u2018COVID-19 opportunity\u2019 arguing that the seemingly spontaneous cross-sectoral engagement of animal and human health actors was not surprising as it was reflective of the often reactive approach to cross-sector collaborations during outbreak situations. Indeed, to buttress their argument some local-level informants noted the missed opportunity in capitalising the learnings and experiences of the 2008 Avian Influenza outbreak management  to inform a concrete and holistic policy on cross-sectoral collaboration for improved zoonoses management. Other informants noted that the focus on COVID-19 response relegated otherwise equally urgent zoonoses problems to the background due to overstretched personnel and surveillance infrastructure. Two typical views in this regard by a disease expert and surveillance officer are illustrative:\u201cI definitely feel that there is traction in understanding [about cross-sectoral action for zoonoses control] but again at the national level, the picture is much more complicated because there are so many different players and they are pushing so many different kinds of agendas that is difficult to make sense of.\u201d .\u201cWe were just about to take off in terms of Integrated Health Information Platform (IHIP) but when COVID-19 came, other diseases came into the background. So now that COVID-19 has come down to a tremendous extent, we want to bring this general disease surveillance to the forefront now\u201d .3.6.per capita rate of infection, morbidity or mortality of candidate diseases) and effective interventions ranked highest across the interviews. Several interviewees cited the lack of credible indicators \u2013 clear measures that demonstrate the severity of candidate diseases impacted significantly on which candidate diseases gets prioritised \u2013 due to limited scientific evidence particularly on the multi-scale burdens, affected populations and socio-economic impacts of focal zoonoses. The foregoing observation further buttresses the earlier argument that politically induced reticence to record some problematic diseases within disease reporting systems and the state level prioritisation of which diseases are surveilled makes it difficult to judge overall burdens and compare data between states (see Section 3.5). As one interviewee, a state-level bureaucrat, openly admitted:Across the interviews, the biological, social, and economic characteristics of focal zoonoses were highlighted by participants as significant determinants of the degree of political visibility. Of the several disease-specific attributes identified, credible indicators , disease severity .per se constituted the key hurdle for prioritisation efforts as they often resorted to passive surveillance  to inform decision-making rather than active, systematic surveillance mechanisms . Nuancing this narrative, some experts and state-level respondents further iterated that data challenge was largely due to the limited technical and surveillance infrastructural capacity at the district levels. Reflecting on how the limited surveillance capacity on disease reporting  affects data quality and prioritisation, an expert informant said:Corroborating this assertion, other national and state level bureaucrats who were interviewed further clarified that the data quality rather than data availability \u201cWe still lack data in terms of animal diseases under surveillance because it\u2019s just the veterinarians who whatever they detect without much of a laboratory base, they just report it. On the human side, we do have infrastructure and we do have some manpower which I think still lacks some of the skilled manpower to actually do a decent reporting.\u201d .A district level surveillance officer also argued that the limited technical capacity of field officers hampered their effective use of collated data. The said official observed:Even if data is there, I am not able to make the right use of the data and I\u2019m not analysing. Because there is a gap in the building capacity at the field functionary level as we have not inculcated the habit of analysing data to my field functionaries, so they never look at the data.\u201d .\u201cCorroborating observations about data limitations and quality, a top-level national bureaucrat  admitted that while the IDSP has been useful for collating data on disease incidence and outbreak occurrence, a major drawback is the periodicity issues stemming from delayed/ and or non-submission of data by field functionaries:\u201cThe integrated disease surveillance programme [IDSP] has been very useful in terms of data collection, but there are some kind of you know periodicity issues with data as some reporting units are not submitting timely data. Besides, data picked by IDSP is not appropriate because most of the times, the diagnostic facility itself is not available. So we cannot at all be assured of the data quality.\u201d .\u201cnot in Karnataka! No challenges like that. Our staff and medical officers are well aware of these diseases \u2026\u201d . The conflicting view of top-level bureaucrats and district-level implementers on data considerations highlights a potential disconnect between policymakers and implementers on the ground. The deep-seated sensitivities around open communication about challenges and need to safeguard their positions has meant senior-level bureaucrats often play to the \u201cpolitics of the game.\u201d Alluding to this assertion in separate interviews, a state-level surveillance officer and district-level disease manager, respectively, stated:Yet some state-level informants, particularly from the health department did not perceive disease surveillance and data considerations to be a challenge. In fact, one interviewee, a senior state bureaucrat, in his explanation retorted You have to realise that whenever we talk about zoonoses management [in India] is much easier to talk when you are not [directly] involved, it\u2019s very complicated \u2026\u201d .\u201c\u201cPeople who are sitting at the state/national level and who are capable of taking these decisions, sometimes they are also more burdened. At the same time, they do not really know the practical difficulties that we [disease managers] face at the primary health sector. If they get tuned to this kind of situation that\u2019s there, I think they might agree with us. At the same time, district- level officers who are there they need to keep on escalating these questions to the people [decision-makers] who are there at the state level\u201d .\u201cit\u2019s mainly fatality! Otherwise even if a disease is causing any morbidity, it should cause a severe disability or mortality then only the district and state level actors start noticing\u201d . Echoing similar sentiments, another expert informant argued: \u201cBird flu, for example, comes like an epidemic whereas rabies and brucellosis are endemic (day-to-day problem). Whereas bird flu, it strikes and a lot of birds die, there is a lot of news in the newspaper, on the TV and the scare that it can spread to humans \u2026 In that case you know very fast action is to be taken by animal husbandry department so they also become very active\u201d . It therefore follows that existing prioritisation often tends to privilege exotic and more prominent diseases  at the expense of some high burden endemic diseases. Moreover, some local-level informants suggested that who a disease affects in terms of population and/or types of animals tended to significantly influence prioritisation outcomes. A case in point is KFD  which until 2019 had little visibility on the policy radar in Karnataka state. Citing the example of brucellosis, an expert informant observed different risk perception of the same disease by actors in the animal and human sectors leading to differential prioritisation:On the question of how the degree of disease severity affected prioritisation outcomes, several informants suggested that the framing and measurement of severity is often quite ambiguous (due to complexity of zoonoses), but generally acknowledged that disease burdens, case fatality and mortality are critical determinants of political visibility of candidate diseases. As a district-level disease manager summarily noted, \u201cI would say it depends on the pathogen and disease involved. The same disease might affect different people differently. For example, if a disease affects cattle (say brucellosis) then the dairy producers might have a different take (interested in productivity) compared to veterinarians who might perceive differently based on the risk of getting the disease. So I think there is a tension at play here between productivity and risks. Sometimes it is easier to tease them apart \u2026\u201d .The above observation might explain the seeming neglect of human anthrax outbreaks  often reported amongst marginalised tribal populations and workers handling animal carcasses , 29. Fur4.inter alia, that the complex and compartmentalised regulatory regime for zoonoses creates a policy milieu where political, socio-cultural and media influences can dominate prioritisation and management decisions.Understanding the political economy of factors that shape zoonoses prioritisation for resource allocation and intervention necessitates a contextual characterisation of the complex web of actors and networks. This includes their different constraints, priorities, mandates, and sources of information , 23, 30.Our findings highlight a challenging paradox. Several national and state-level interviewees acknowledged some drawbacks of the existing prioritisation process, including the sectorally-driven approach and the over-emphasis on exotic diseases  at the expense of high burden of silent ones . At the same time, the same informants equally recognised that uncertainties of data , lack of sensitive/point of care diagnostics hindering reporting, and the fact that different zoonoses impact populations differently renders the prioritisation of silent zoonoses  difficult. Synonymous with observation in the wider literature , 30, ourWith respect to the characteristics of individual disease problems, the disease severity exemplified by mortality and case fatality significantly determined political prioritisation. The lack of standardised indices for measurement of severity given that different zoonoses affected populations differently and the unevenness in the evidence base for focal diseases renders collaborative-prioritisation  difficult to achieve. For instance, whereas the seroprevalence of brucellosis is very high in animals, the corresponding human risk is low. Moreover, the lack of standard metrics in the animal health system to estimate disease burden (like the disability-adjusted life years (DALY) for humans) makes it difficult to estimate burden for chronic zoonotic diseases. The foregoing observation is also reflected in the contest of authority with respect to avenues for collaborative prioritisation, as animal health actors generally felt short-changed by the current state of play. Thus in facilitating collaborative-prioritisation, improvement in the ways burdens are captured in existing information systems across endemic as well as exotic diseases cannot be overemphasised. In furtherance to this, the Integrated health Information Portal (IHIP) is being developed (as a replacement for the existing IDSP) to afford an enhanced, collaborative and timely disease reporting platform. Besides, further understanding, investigating and developing indicators for the nature and extent of non-clinical, wider societal impact of zoonotic diseases, particularly for marginalised communities, rather than relying on mortality and morbidity metric only is paramount. In any event, the design and implementation of cross-sector action plans for priority diseases  backed by strong political will at the highest decision-making strata remain critical in generating the requisite financial and non-financial incentives to foster and sustain collaboration of different actors in zoonotic disease management . Despite4.1.n\u2009=\u200917) were interviewed after the COVID-19 pandemic could have raised their awareness about zoonoses, and therefore potentially \u2018skewed\u2019 their observations. In any event, India\u2019s federal character implies a need for caution in the generalisability of the findings to other states of India due to the inherent political and culturally and economic diversity that exist in the 29 Indian states and the fact that health is a state subject, with disease prioritisation operationalised at the state level.This study is not without limitations. We acknowledge that the use of a snowball sampling approach in the recruitment of key-informants could have potentially skewed the representation of interviewees in favour of a particular sub-group of interest in this study, noting that actors affiliated with the human health sector constituted the majority of our study sample. Nevertheless, our prior stakeholder analysis  and purp5.Looking ahead, the COVID-19 and One Health platforms could present a window of opportunity to galvanise political support for zoonoses. Proponents argue that One Health partnerships could alleviate information asymmetries and create opportunities for knowledge exchange on candidate zoonoses critical for effective prioritisation , 32. In fesasa@ceh.ac.uk).The datasets presented in this article are not readily available because of sensitive participant information. Request to access the datasets should be directed to UK Centre for Ecology and Hydrology Institutional Data Access contact via FA (email: The studies involving humans were approved by the Research Ethics Committees of the Ashoka Trust for Research in Ecology and the Environment (IRB/CBC/0003/ATV/07/2018), Institute of Public Health (IPH) Bangalore (IEC-FR/04/2017) in India, and the The Liverpool School of Tropical Medicine Research Ethics Committee  in the United Kingdom. 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.FA developed the conceptual focus, reviewed the literature, synthesised its contents, analysed data, and wrote the manuscript. AS contributed to data gathering and curation, data analysis, and reviewed the manuscript. JY, SH, and BP contributed to the conceptual focus, data interpretation, and reviewed the manuscript. MC contributed to the data interpretation and reviewed the manuscript. All authors contributed to the article and approved the submitted version.The IndiaZooSystems project that led to these results is supported by the Medical Research Council (MRC), the Foreign, Commonwealth and Development Office (FCDO), the Economic and Social Research Council (ESRC) and Wellcome under the Health Systems Research Initiative (grant numbers MR/S012893/1 & MR/S012893/2) awarded to BP, MC, SH, and JY. Additional support was provided to FA and BP from the UK Research and Innovation Global Challenges Research Fund funded IndiaZooRisk project (grant number MR/T029846/1). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the study reported.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": "Herpes Simplex Virus Encephalitis (HSVE) can have lifelong sequelae. Presentation can be nonspecific in cancer patients. Cerebrospinal fluid testing may be falsely negative early in illness [1]. New immune-based or targeted radiation (RT) modalities may change risk, incidence of HSVE in cancer patients.Retrospective study at Memorial Sloan Kettering Cancer Center (MSK). Review of records of patients with CSF meningitis/encephalitis (ME) PCR panels from 8/1/2016 to 7/31/2022. All unique patients who received brain RT at MSK during those dates were used to calculate incidence rates.2749 patients had 5497 CSF analyses with ME panel. Of these, 12 tests from 10 patients showed HSV-1 or -2; 1 patient had relapsing HSVE and 3 positive HSV2 tests each done at least 6 months apart.Table 1 shows 10 patients with HSVE. 8 had solid tumors, 6 with receipt of chemo- or immunotherapy within 30 days. All solid tumor patients with HSVE had brain RT; 6 received steroids. 1 patient with relapsing HSVE was on immune checkpoint inhibitor (ICI). 4/8 patients with MRIs (Table 2) lacked typical findings; 4/8 patients with EEGs had focal temporal seizure activity. CSF pleocytosis was absent in 4/10 cases. 5 patients had residual deficits. 4 died due to progression of disease.Incidence of HSVE with brain radiation was 2.6/10,000 patient-years.General population incidence of HSVE ranges from 1-4 per million population per year [2]. Those receiving cancer treatment and brain RT have elevated HSVE risk. We found the risk after brain RT is much higher: 2.6/10,000 person-years. Only 1 of 10 patients in our series made a full recovery.Other key findings include:1) One patient with recurrent HSVE in the setting of nivolumab: ICI combined with brain RT may increase risk of HSV reactivation. Such patients should be considered for high dose antiviral prophylaxis after a first HSVE episode.2) Normal MR imaging and lack of CSF pleocytosis does not preclude HSVE diagnosis in clinically compatible illness.3) No cases occurred among solid tumor patients treated with chemo- and/or immunotherapy only.Limitations:1. Retrospective design.2. Inclusion of lab confirmed cases only.Mini Kamboj, MD , ASCO: speaker fee|Medscape: speaker fee|MJH life sciences: speaker fee|Regeneron: Advisor/Consultant|Web md: speaker fee"}
{"text": "The use of non-occupational post-exposure prophylaxis (nPEP) to prevent HIV acquisition among those exposed as an approach to HIV prevention has expanded in Uganda. Although there are increased efforts to avail nPEP services among most at-risk populations, the usage of nPEP medicines remains low. Therefore, this study examined the risk perception and usage of non-occupational post-exposure prophylaxis (nPEP) among fisherfolk in the Ggulwe fishing parish, Bussi sub-county, Wakiso district.A cross-sectional study among adults was carried out from October 2020 to January 2021 in Ggulwe parish, Bussi sub-county, Wakiso district, to examine the usage of nPEP and factors influencing the usage. Data were collected using semi-structured questionnaires, and key informants' interviews were conducted among healthcare providers and the local leadership. The quantitative data were summarized using bivariate and multivariate logistic regression, while the qualitative data were analyzed thematically to enrich the quantitative results.p < 0.001), lack of knowledge of the existence of nPEP , the perception that nPEP can effectively prevent HIV infection after exposure , and the community's opinion affecting the willingness to take nPEP .Overall, 248 fisherfolk encountered an event that required the use of nPEP, and of these, 55/248 (22.2%) were able to use nPEP to prevent them from acquiring HIV. The usage of nPEP among adults in the Bussi sub-county, Wakiso district, was associated with not knowing that HIV can be prevented using nPEP medicines . The low usage of nPEP was associated with a lack of knowledge and awareness about nPEP. This effort to improve the usage of nPEP should include community sensitization and HIV infection prevention using nPEP to raise awareness about HIV infection exposures and the risk of HIV infection during non-occupational exposures. Globally, 76 million people have acquired HIV, and 33 million people have died of HIV/AIDS since the beginning of the HIV epidemic. In addition, 38.0 million (31.6\u201344.5 million) people were living with HIV at the end of 2019. An estimated 0.7% (0.6\u20130.9%) of adults aged 15\u201349 years worldwide are living with HIV, although the burden of the epidemic continues to vary considerably between countries and regions (HIV/AIDS is a leading cause of death in sub-Saharan Africa (SSA), accounting for 71% of the global burden of the infection . DiffereIn the Uganda Population-based HIV Impact Assessment 2016\u20132017), the prevalence of HIV infection is lowest (0.2%) among the 15\u201319-year-old age group and highest (13.6%) among the 50\u201354-year-old age group , the pre comparedThe fisherfolk community is one of the high HIV-risk groups due to their frequent mobility, transactional and commercial sex, multiple sexual partners, high consumption of alcohol, poor health infrastructure, and limited access to health services \u201315. In aHowever, there is limited information on the factors associated with the usage of PEP in this community. Therefore, the study aimed to describe factors influencing the usage of non-occupational PEP in a fisherfolk community located on the shores of Lake Victoria in central Uganda.This study used a cross-sectional study design employing both qualitative and quantitative methods of data collection . For theIn Uganda, HIV-PEP is provided to all eligible clients within 72 h of exposure. Non-occupational exposure is defined as non-occupational exposures including sexual assault (rape and defilement), road traffic accidents, unprotected sex with an HIV-infected person, and unprotected sex with a person of unknown HIV status . The MinA population of 5,011 spread across the five villages was used in the study based on the Uganda National Bureau of Statistics Population Census 2014. A cross-sectional mixed-method study was carried out among fisherfolk communities located along the shores of Lake Victoria in the Ggulwe parish, Wakiso district, from October 2020 to January 2021. To attain quantitative data, we included participants aged 15\u201349 years selected using purposive and simple random techniques, and for qualitative data, we used healthcare workers and local leaders due to their high level of knowledge on HIV prevention measures and interventions in the area. The sample size was determined using Krejcie and Morgan's  formula,Our outcome variable was the usage of non-occupational post-exposure prophylaxis, defined as receipt of the following: \u201cYes\u201d for participants who had taken nPEP within 72 h of exposure and \u201cNo\u201d for those who had not. The non-occupational exposure was measured by asking whether the participant had had sexual assault (rape and defilement), road traffic accidents, unprotected sex with an HIV-infected person, or unprotected sex with a person of unknown HIV status prior to the use of nPEP. The usage in this study was taken to be whether a participant utilized PEP obtained from either a government or private health facility after accidental or suspected exposure to HIV. The independent variables consisted of individual factors such as demographics, knowledge about PEP, risk perception, and knowledge of other HIV prevention measures; community-related factors including stigma, perceived effectiveness, and associated negative effects; and health facility-related factors consisting of attitude of health workers and availability of nPEP, and finally, nPEP knowledge among health workers and adherence to prescription guidelines.p < 0.02 at bivariate analysis were included in the final multivariable model. Factors specified as important based on previous literature were included in the final model. A two-sided significance p < 0.05 and a 95% confidence interval were considered statistically significant for the analysis. Measures of association were reported as crude odds ratios at bivariate analysis and adjusted odds ratios at multivariable analysis.Data were analyzed using STATA version 14.0. Frequencies and their corresponding percentages were used for categorical variables, and mean and standard deviation were used for continuous variables that were normally distributed. The dependent variable was modeled as a binary outcome. Bivariate and multivariable analyses were performed using logistic regression with odds ratios as a measure of association. All variables with a To enrich and triangulate the quantitative results, we conducted qualitative interviews with men and women in Ggulwe fishing village, Bussi sub-county, Wakiso district. In particular, we held three FGDs, each consisting of six to eight people who were selected randomly from among those community members while maintaining COVID-19 SOPs. The group consisted of both men and women who had encountered or not encountered a situation that exposed them to HIV in the 12 months prior to the study. The FGDs were held within the village meeting rounds in the local language, \u201cLuganda,\u201d by two research assistants (DH and MN), both students trained in qualitative research methods. One research assistant (DH) moderated all the FGDs, while the other (MN) audio-recorded the responses and probed where necessary. Each FGD lasted for ~45 min on average. The moderator encouraged all the group members to ask questions and to provide comments as much as possible on HIV-PEP, knowledge, and usage. For key informant interviews (KIIs), four healthcare providers engaged in the provision of PEP services were purposefully selected and interviewed to elicit their expert opinions on the usage of PEP for HIV prevention. Both FGDs and KIIs were held until saturation was reached. Codes, subthemes, and themes were developed using NVivo V12 Pro, where the transcripts were uploaded and exported to Excel, which helped develop results.The study obtained ethical approval from the research and ethics committee of Uganda Martyrs University. Administrative permission was sought from Ggulwe Parish. We obtained written informed consent from all the eligible participants who were above 18 years. Permission to include participants below the age of 18 was obtained from one or both of the parents/guardians depending on their accessibility. We then sought the child's assent to participate in the study. Those who declined to participate despite permission from their parents/guardians were not included in the study. The study obtained written informed consent from the adult participants. Participants under 18 years, who are considered emancipated minors, had to provide assent. Participation was voluntary and the study ensured maximum confidentiality considering the intricacy of the study topic. Furthermore, the team was mindful of the anticipated emotional discomfort from the survivors of rape and defilement. In such cases, psychosocial support in the form of confidential counseling and post-trauma support was offered by trained healthcare workers who were members of the study team, and further referrals where required were directed to the available community services.p = 0.044). Those with a single exposure are less likely to use PEP services. Being exposed multiple times to HIV infection was not statistically significant for nPEP usage.p < 0.001). Similarly, individuals who did not know about non-occupational PEP were 70% less likely to use the services . Furthermore, those individuals who did not know how non-occupational PEP was supposed to be used were less likely to use the services . The results are summarized in In the study, the assumption was that an individual who encountered a non-occupational event with an increased likelihood of HIV infection exposure used the non-occupational PEP. At multivariable analysis, the social demographic factors did not significantly influence the usage of non-occupational PEP. The study found that individuals who did not know that HIV infection could be prevented using nPEP were 90% less likely to use the nPEP services .\u201cAnother participant cited that there were sensitization campaigns conducted by several organizations to educate communities about PEP.UVRI did a lot of work to educate people but has never heard anyone say you can find that drug here or there. It is only UVRI that has done commendable work to educate the people about PEP in both Kava-enyanja and Kituufu sub islands but has since not heard of similar community awareness on the PEP. The awareness has not sufficiently sunk into the community. People don't know -how does it work; how does it help. There is a lot of awareness on HIV but it is lacking on PEP. People don't usually use it-I have never heard someone use it, yet as you see, this is an island with many prostitutes, so the awareness is lacking, it's not enough\u201d KI Councilor, Ggulwe parish.\u201cThe various HIV/AIDS prevention methods cited were male circumcision, abstinence, the use of male condoms, avoiding multiple sexual partners, regular screening for HIV before having sex, being faithful to one sexual partner, and avoiding sharing sharp objects.The study findings also revealed that the fishing community was given information and supplied with condoms. The health workers also encourage young people to abstain until marriage, although it proves fruitless. One of the key informants explained as follows:The community knows other prevention but young girls are attracted by money to indulge in unprotected sex. We also give out free condoms but currently out of stock for 1 month. We can even spend 3 months without condoms, we tell clients to get condoms somewhere else or even from VHTs who usually have. On abstinence, people know \u2013 but it can't work here. Most of these people leave their families in Kampala and other areas, someone can't abstain from sex for a long period including the youths,\u201d KI enrolled nurse/M&E officer.\u201cFollowing HIV preventive measures is futile in fishing communities since people get easy money and are driven to engage in risky sexual behavior.Also, women in these communities are few and are shared among the available male. Again, there is a lot of alcoholism, a drunkard person can't easily have self-control on who to have or not to have sexual intercourse with, using barriers like condoms,\u201d KI Councilor, Ggulwe parish.\u201cThe following explanations were obtained. One key informant explained the following:Even one lady I gave nPEP in August 2020 after the rape was not aware, she was supposed to receive PEP because she was not aware of it yet the rapist was on ART,\u201d KI, ART in-charge Bussi S/C.\u201cFrom the arguments above, the indications are that little dissemination of WHO guidelines on administering PEP to clients exists. Most of the KIs reported that the community education programs of PEP are few, and they must educate clients on ART. One of the participants explained the following:One may go to the health facility and find someone with no expertise in dealing with PEP, told to go back. The client may not go back for fear of being known by many people who may disclose one's status in public,\u201d KI, VHT coordinator, Bussi S/C.\u201cp = 0.025). Other factors hindering nPEP service usage, such as fear of social stigma, distance to the health facility, refusal by a partner or spouse, and lack of knowledge about nPEP, were found to influence the usage of nPEP. Although 10/55(14.9%) of the clients reported that they had been denied nPEP at the health facilities in the Bussi sub-county, health workers denying clients nPEP because they may not qualify to take it were found to affect its use, and at times it was out of stock.The time spent while waiting to receive nPEP was found to be significant. The odds of an adult waiting <30 min to take nPEP were 0.05 times more likely than their counterparts that had not  said he gave it to someone who tested negative at the end of 3 months. However, one of the key informants explained the following:Some say that once the health workers give you PEP medicine; you are already HIV positive. This is because it is HIV medicine. Differentiating between PEP and ARVs is a problem. This may be aggravated by disclosure to the partner about one's HIV status so that the negative partner can take PEP after sexual intercourse,\u201d KI enrolled nurse.\u201cHowever, despite the community myths, most of the respondents did not have adequate knowledge of the use of non-occupational PEP in the prevention of HIV infection after exposure.The study findings show that the level of knowledge on nPEP to prevent the acquisition of HIV was associated with the usage of nPEP. Conversely, while assessing the use of non-occupational PEP in MSM in the USA, Donnell et al.  found thThe gap in knowledge about nPEP in the study area is not a special case as compared to the cited areas of Nigeria, Boston, and San Francisco. Similar approaches to enhance the uptake of this preventive therapy could be applied.The perceived risk of acquiring HIV/AIDS was not significant in the non-occupational PEP. Similarly, in a study conducted in the USA among MSM, no association was found between the risk of HIV and nPEP use after exposure. Three seroconversions occurred at 384 visits (1.56 per 100 person-years) with nPEP use, compared to 210 seroconversions at 25,550 visits (1.64 per 100 person-years) with no nPEP use . The use of nPEP occurred more frequently in men with high-risk sexual behaviors. Those who had reported 10 or more partners had almost triple the adjusted odds of nPEP use  relative to those who had reported zero to one . Signifip = 0.06). A study on factors associated with the usage of nPEP among Thai men revealed that a higher proportion of them who intended to take nPEP answered \u201cyes\u201d when asked whether nPEP would reduce their concerns about becoming infected with HIV . There was no difference in the sense of stigmatization between those who did and those who did not intend to take nPEP  among fisherfolk in Ggulwe parish on the shores of Lake Victoria in central Uganda had several limitations that need to be noted.The LGBTQ concept was neither reported nor given the desired focus because there was no reference legal document with literature on the subject. This limited the extent to which this population was included and discussed in the study.The sample size of the study was relatively small, which may have limited the generalizability of the findings to a broader population of fisherfolk in different regions. The limited scope of participants might have led to an underrepresentation of diverse perspectives and experiences within the fishing community.The study heavily relied on self-reported data from the participants, introducing the potential for recall bias and social desirability bias. It is likely that participants provided responses that they believed were more socially acceptable, leading to an inaccurate representation of their actual risk perceptions and PEP usage.Relatedly, due to resource constraints and logistical challenges, the study was conducted over a relatively short period of time. This timeframe might not have captured seasonal variations or long-term trends in risk perception and PEP usage among the fisherfolk. A more extended study period could have provided a more comprehensive understanding of this interesting area of study.Furthermore, while efforts were made to ensure cultural sensitivity and local context adaptation, the study's design and data collection methods might not have fully captured the distinctions of the fisherfolk's beliefs, practices, and perceptions related to HIV prevention and PEP. Cultural factors that were not adequately accounted for could have influenced the results.Finally, the researchers faced challenges in accessing some sub-island villages with limited health facilities, potentially leading to an underrepresentation of these areas in the study. This limitation might have affected the comprehensiveness of the findings and recommendations, particularly in terms of equitable PEP access.In conclusion, the 22% nPEP usage study sheds light on the critical need for comprehensive strategies to enhance the adoption and effective utilization of PEP within the fisherfolk communities of Ggulwe parish. The low usage was associated with limited awareness and knowledge about the non-occupational PEP.There is a need to put together new strategies that publicize non-PEP in the general population, especially the sexually active age group.There is a need to develop a collaborative approach that involves government bodies, beach management units, community-based organizations (CBOs), development partners, fisherfolk associations, local leaders, and village health teams (VHTs). This can be complemented by conducting health education outreach and employing peer educators from within the fisherfolk community.Relatedly, the timely dissemination of consolidated guidelines for the prevention and treatment of HIV in Uganda is vital as a reliable resource for both healthcare providers and the fisherfolk. This information can immensely contribute to informed decision-making and reinforce the importance of PEP as an integral part of the overall strategy to combat HIV/AIDS.Equally crucial is the need for equitable distribution of PEP within fisherfolk communities, especially in remote sub-island villages where health facilities are scarce. By extending PEP availability to the last mile, PEP will not only be accessible but will also be effectively utilized. This approach aligns with the overarching goal of leaving no one behind in the fight against HIV/AIDS.Bottom-line, a community that understands the importance of PEP and its role in preventing HIV transmission is more likely to embrace the treatment and integrate it into their healthcare-seeking behaviors. By fostering a sense of ownership and empowerment within the fisherfolk community, the broader goal of eradicating AIDS becomes more achievable.dbahikire1990@gmail.com.The datasets presented in this article are not readily available because date sets are restricted to the authors. Requests to access the datasets should be directed to DB conceived and designed the research study, collected data, wrote the initial draft of the manuscript, and responded to reviewers' comments. MN validated the data collection tools, supervised the study, provided guidance and general oversight during the entire course of the study, and also provided input in the review process. CA supported report compilation and performed statistical analyses. CM and LN contributed to the review of the manuscript. MB provided guidance and critically reviewed and revised the manuscript for important intellectual content. All authors contributed to the article and approved the submitted version."}