The stabilization of host–symbiont mutualism against the emergence of parasitic individuals is pivotal to the evolution of cooperation. One of the most famous symbioses occurs between legumes and their colonizing rhizobia, in which rhizobia extract nutrients (or benefits) from legume plants while supplying them with nitrogen resources produced by nitrogen fixation (or costs). Natural environments, however, are widely populated by ineffective rhizobia that extract benefits without paying costs and thus proliferate more efficiently than nitrogen-fixing cooperators. How and why this mutualism becomes stabilized and evolutionarily persists has been extensively discussed. To better understand the evolutionary dynamics of this symbiosis system, we construct a simple model based on the continuous snowdrift game with multiple interacting players. We investigate the model using adaptive dynamics and numerical simulations. We find that symbiotic evolution depends on the cost–benefit balance, and that cheaters widely emerge when the cost and benefit are similar in strength. In this scenario, the persistence of the symbiotic system is compatible with the presence of cheaters. This result suggests that the symbiotic relationship is robust to the emergence of cheaters, and may explain the prevalence of cheating rhizobia in nature. In addition, various stabilizing mechanisms, such as partner fidelity feedback, partner choice, and host sanction, can reinforce the symbiotic relationship by affecting the fitness of symbionts in various ways. This result suggests that the symbiotic relationship is cooperatively stabilized by various mechanisms. In addition, mixed nodule populations are thought to encourage cheater emergence, but our model predicts that, in certain situations, cheaters can disappear from such populations. These findings provide a theoretical basis of the evolutionary dynamics of legume–rhizobia symbioses, which is extendable to other single-host, multiple-colonizer systems. Symbiosis is an ecological interaction in which two or more species exchange mutual benefits. This cooperative interaction promotes the fitness of both species, and thereby reinforces their symbiotic relationship. However, symbiotic systems are vulnerable to emerging selfish cheaters that extract benefits from the system without paying costs. Such parasitic cheaters may potentially disrupt the symbiotic relationship.One of the well-known symbioses occurs between legume plants and rhizobia (nitrogen-fixing soil bacteria). Rhizobia establish symbiotic organs termed root nodules on the roots of their host, and proliferate by extracting nutrients from the host plant. In turn, they supply their host plants with nitrogen resources produced by nitrogen gas fixation. This mutual nutrient exchange should promote the fitness of both organisms and thereby strengthen the symbiotic relationship. This beneficial effect, known as “partner fidelity feedback”, is assumed as a stabilizing factor for the mutualistic relationship On the other hand, naturally occurring rhizobium strains vary in their nitrogen fixation activity, and ineffective rhizobia that colonize their host plants without undertaking nitrogen fixation in their root nodules are ubiquitous Despite the widespread presence of ineffective rhizobia, the legume–rhizobia symbiosis is evolutionarily stable. Potential mechanisms that stabilize the symbiotic interaction include partner fidelity feedback The evolution of cooperation has been theoretically modeled by game theory, in particular, the prisoner's dilemma game and the snowdrift game  Various mathematical models have been proposed for the evolution of the legume–rhizobia symbiosis. Akcay et al. In contrast, the legume–rhizobia mutualism is more directly reflected by evolutionary dynamics in which rhizobia proliferate according to the quantitative trait of nitrogen fixation with a frequency-dependent selection. However, this situation has yet to be fully investigated, although such a model may explain the effect of mixed colonization In the legume–rhizobia symbiosis, host plants cannot extract benefits (i.e. nitrogen fixation products) without paying costs (i.e. nutrients or energy) to rhizobia, because the symbionts are confined in root nodules and thereby cannot directly interact with the external environment. This fact suggests that host plants cannot develop a cheating strategy, whereas ineffective rhizobia are ubiquitously distributed in nature. Thus, the evolutionary dynamics of the mutualism are most likely driven by the strategies of the symbionts, while the host plants play a minor role. Accordingly, our model is based on the strategies adopted by the rhizobia.n of rhizobia, which correspondingly form n root nodules  and 1 (maximum activity or full cooperation) and which is genetically transmitted to its progeny. Each host plant is assumed to be colonized by a constant number  nodules . Thus, en rhizobia of nitrogen fixation activity kx  according to the probability distribution of rhizobia in the soil, and subsequently generates n corresponding root nodules. The productivity of this focal plant (pφ) is promoted by the total nitrogen resource supplied by the root nodules, represented by the average of their strategies: pφ increases, Thus rhizobial growth rate (pφ: B(0) = 1, where φ0 is the growth rate in the absence of nitrogen fixation (B(x) is an increasing function of x.Because rhizobia are considered to grow much more efficiently in root nodules than in the soil, we consider only their proliferation in root nodules. Proliferation is linked to the benefit (or promoting force) and the cost (or destabilizing force) of nitrogen fixation activity. The benefit encourages cooperative growth between the host plant and its symbionts. Now consider that a host plant is infected with ix) increases; C(0) = 1, where the cost function C(x) is a decreasing function of x. Therefore, when benefit and cost compete, the growth rate of a focal infecting rhizobium adopting strategy ix can be expressed as B(x) and C(x) are strictly increasing and decreasing functions of x, respectively, (i.e. B′(x)>0 and C′(x)<0).On the other hand, the cost (or destabilizing force) exerts an inhibitory effect on symbiont fitness, such that the growth rate of each infecting rhizobium (or its corresponding root nodule) decreases as its own nitrogen fixation activity ). This form differs from that of conventional game theory, in which the cost is subtracted from the benefit. However, qualitatively, both forms of the fitness function yield the same evolutionary dynamics of the symbiotic system, because the evolutionary consequences are similar in the two models; namely, full defection, full cooperation, partial cooperation, and coexistence of cooperators and defectors created by “evolutionary branching” (corresponding to cases (i), (ii)/(vi), (iii), and (iv)/(v), respectively, in this paper) b and Nb correspond to the strength and nonlinearity, respectively, of the benefit, and c and Nc are the corresponding parameters of the cost (B(x)≥0, B′(x)≥0, C(x)≥0, and C′(x)≤0, which are satisfied when 0≤b, 0≤c≤1, 0≤Nb≤1/2, and 0≤Nc≤1/2. In this paper, C(x) is not regarded as an increasing function of x, because nitrogen fixation obviously strengthens if both the benefit and cost functions are increasing functions of x (see The benefit and cost functions are simply defined as:the cost . These fof x see .b) increases as partner fidelity feedback strengthens, while the cost (c) decreases with increasing host sanction.Partner fidelity feedback and host sanction are widely-discussed stabilizers of symbiosis t-th cycle:i  corresponds to strategy x = i/N. The initial distribution is a population of non-fixing rhizobia: p0(0) = 1 and p0(i) = 0 for i≠0. This strategy distribution is iteratively changed in the infection, proliferation/mutation, and release steps . We denote the probability distribution of the M×n infecting rhizobia by j/N to i/N by mutation at transition rate jiμ. Thus the probability distribution after the proliferation/mutation step can be described as a time-discretized form of the replicator–mutator equation:j/N. In this paper, mutation rates are given byμ specifies the mutation rate between neighboring strategies, and μ0 specifies the rate of the null mutation. Parameters are set to N = 200, M = 2000, μ = 0.04, and μ0 = 0.0 or 0.01.In the numerical calculations, we first introduce the discrete probability distribution of the rhizobia strategies in the se steps . We consBy investigating our model, we hope to understand the evolutionary consequences of the legume–rhizobia symbiosis in terms of adaptive dynamics  [38], [3n of rhizobia, each of which adopts a nitrogen fixation activity 0≤x≤1 as its evolutionary strategy  gives the fitness of the mutant strain asIn our model, a host legume plant is infected with a constant number strategy . Now conw>0, the mutant can invade the population, but cannot if w<0. The theoretical basis of adaptive dynamics is described in Note that, as described in section 2.1.1, the fitness is the product of the benefit and cost functions, unlike conventional game theory, in which these quantities are subtracted. We now introduce the relative fitness of the mutant to the resident:B(x) should be a strictly increasing function of x (i.e. B′(x)>0). In contrast, the cost function C(x) is not necessarily a simple decreasing function of x because it may be affected by partner choice and host sanction. If such stabilizing effects are sufficiently strong that C(x) becomes an increasing function of x, the maximum activity of nitrogen fixation (x = 1) is predicted to evolve and be stably maintained (see C(x) is a decreasing function of x (i.e. C′(x)<0) in this paper. Note that under this condition, more cheating rhizobia proliferate more efficiently when they inhabit the same plant.Because legumes and rhizobia provide mutually-enhancing nutrients to each other, thereby increasing the fitness of both, the benefit function ined see . AccordiNc = 0), the selection gradient x (i.e. D′(x)<0 for 0<x<1). Accordingly the evolutionary behavior is classified into the following three cases depending on the cost–benefit balance (for details see In the linear cost function (i.e. D(0)<0 (i.e. c>c0 = b/n), we have D(x)<0 for 0<x<1  but not by those with higher activities (y>x)  , and a sD(1)>0 (i.e. D(x)>0 for 0<x<1. In this scenario, the nitrogen fixation of the resident population always increases, because only mutants with higher strategies can invade and subsequently replace the resident (x = 1. We refer to this scenario as the “Maximum evolution” case. A population that has evolved to x = 1 cannot be invaded by cheating bacteria (x = 0) possessing the null mutation <0; see Conversely, if  (i.e. ) , magentaresident . Therebyc1<c<c0)  and ESS-stable (i.e. D′(x*)<0 and x* and become stably maintained with no invasion of nearby mutants. In addition, similar to case (ii), a population that has evolved to x* cannot be invaded by cheaters possessing the null mutation <0; see The intermediate condition between cases (i) and (ii) (i.e. 1<c<c0) , blue, p<0 and )  (see Tex<0 and ) .3. Thus x = 0) falls into one of three evolutionary outcomes: case (i) “No evolution” when the cost (c) is relatively stronger than the benefit (b) (b) is relatively stronger than the cost (c)  evolves to the maximum nitrogen fixation (x = 1) (In case (ii), an initial population of non-fixing rhizobia ( (x = 1) . The evo (x = 1) .Nb) does not qualitatively affect evolutionary behaviors, but influences the parameter conditions such that, as Nb increases, the parameter region of case (ii) reduces  “No evolution” <0 and E(x*)>0) >0) . This six = 0 evolves to the singular strategy (x*) and then splits into two subpopulations  while the other reduces to the minimum  under most of the investigated parameter conditions. Consequently, nitrogen-fixing and cheating bacteria evolve to stably coexist.This theoretical prediction is confirmed by numerical simulations, in which an initial population with D(0)>0 and D(1)<0; see This coexistence is predicted to be a co-dependent relationship, in which cooperators and cheaters require each other for their own survival because each monomorphic population is invaded by nearby mutants (i.e. x*<1 exists that is CS but not ESS-stable (D(1)≥0). This prediction is also confirmed by numerical simulations, in which cooperator bacteria can stably exist even after removal of the cheaters >0 for 0<x<1). However, unlike case (ii), the evolved population of nitrogen-fixing symbionts (x = 1) will likely be invaded by cheaters (y = 0) possessing the null mutation >0) >0) .This type of coexistence is also confirmed by numerical simulations. Nitrogen-fixing rhizobia first evolve as in case (ii), followed by transient introduction of a null mutation, which produces cheating rhizobia (Nc) promotes symbiosis evolution, because the parameter regions of case (ii) and (iii) increase and decrease, respectively, as Nc increases –(vi)) is absent under weak nonlinearity of Nc . First, symbiotic evolution depends on the cost-to-benefit ratio in the following order: (i) “No evolution” (b) relative to the cost (c), the stronger the symbiotic relationship. Furthermore, Nb inhibits the evolution of nitrogen-fixing bacteria; for example, the parameter region of case (ii) tends to decrease as Nb increases. In contrast, Nc promotes the emergence of cheating rhizobia, as shown by the expansion of cases (iv)–(vi) as Nc becomes stronger.Finally, we examine the effects of nonlinear benefit and cost functions. Similar to section 3.2, numerical calculation yields six evolutionary behaviors depending on the properties of the singular strategy. Under this generalized condition, the results are similar to those obtained in sections 3.1 and 3.2  (which defines the direction of evolution) (x = 0 if the cost is sufficiently strong (case (i)), (B) full mutualism with x = 1 if the cost is weakened (cases (ii)/(vi)), and (C) partial mutualism with x = x* at intermediate cost (cases (iii)–(v)). The evolution of these monomorphic populations is affected and classified by their various evolutionary features. The emergence of a fully mutualistic population depends on whether cheaters evolving by null mutation can invade the population : invasibility of cheaters). That is, a population of mutualists is stably maintained without invasion under weak cost conditions <0; case (ii)). As the cost is strengthened, mutualists and cheaters can coexist because cheaters can successfully invade under this condition >0; case (vi)).As discussed above, the cost–benefit balance determines the evolutionary behavior of the symbiotic system. Here we summarize the evolutionary outcomes obtained in our model . AccordiE(x*): ESS-stability of partial mutualists; see E(x*)<0; case (iii)). As the cost is weakened, evolutionary branching creates a population of co-existent mutualists and cheaters (E(x*)>0; cases (iv)/(v)). The cheaters in this type of coexistence can establish either a co-dependent or parasitic relationship with their mutualistic counterparts (i.e. D(1): ESS-stability of mutualists). In the absence of cheaters, a population of mutualists can stably exist under weaker cost (D(1)>0; case (v)) but cannot maintain nitrogen fixation activity under stronger cost (D(1)<0; case (iv)).However, the emergence of a partial mutualistic population depends on the occurrence of “evolutionary branching” (or sympatric speciation) (i.e. effx) relates to the average strategy adopted by the rhizobial population. Because a monomorphic population evolves in cases (i)–(iii), the efficiency in the linear cost function is described byx* is described in c increases (The efficiency of nitrogen fixation (ncreases .x = 1) and full cheaters (x = 0) coexist in cases (iv)–(vi), the efficiency of nitrogen fixation in the linear benefit function depends on the proportion of cooperators, i.e.*x is described in c increases the efficiency, but an interesting discontinuity occurs at the transition between case (i)/(iii) and case (ii)/(iv)/(v)/(vi) (When full cooperators ((v)/(vi) .B(x) weakens as the nodule number (n) increases (Eq. (1)). Supporting this prediction, the parameter conditions of case (i) expand with increasing n . These results suggest that, as nodule number increases, the symbiotic relationship destabilizes and is less easily established, while cheating rhizobia readily emerge.Overcrowding of nodules on a host is expected to impede the mutualistic relationship, because the effect of an individual rhizobium on the benefit function easing n , gray whα(x): the probability of accepting rhizobial colonization (where 0≤α(x)≤1 and α′(x)>0). We then consider the invasibility of rare mutants with nitrogen fixing ability y into the resident population with x. The mutant colonizes the host at a α(y)/α(x)-fold different rate than in the absence of partner choice (xF(y)) by a factor of α(y)/α(x), so thatPartner choice is often described as the preferential selection of colonizers by the host plant r choice . Equivalx* is an evolutionarily singular strategy (i.e. D(x*) = 0). This result indicates that a singular strategy (satisfying D(x*) = 0) and its CS and ESS stabilities (which depend on the signs of D′(x*) and E(x*), respectively) can result from a modified fitness function Accordingly, we obtain:β (0≤β≤1), specifying the frequency of mixed nodules (or doubly colonized nodules). Each nodule is colonized by two rhizobial strains with probability β; otherwise it is colonized by a single strain (with probability 1–β).Mixed nodules colonized by multiple symbionts are common in the laboratory x1 and x2 would proliferate at rates depending on their relative fitness C(ix) (i = 1 or 2). Thus, we assume their proportion in the population as ip) in the mixed nodule and is simply defined as ip = 1 and ip = 0, respectively.In a mixed nodule, two strains adopting strategies y invade a resident population with ability x. If the mutant is rare, the probability that the host plant is colonized by multiple mutants is very low and can be neglected. Thus, root nodules will harbor single y colonies and colonies containing x and y with relative frequencies of (1–β) and 2β, respectively (y isNow consider that mutants with nitrogen fixation ability ectively . The fitβ = 0). The second term on the right-hand side of Eq. (14) is always negative because C′(x)<0, implying that mixed nodule populations inhibit the evolution of nitrogen fixation.Thus we obtain the selection gradientβ increases, while that of “Maximum evolution” decreases . In this scenario, cheating rhizobia will either emerge or disappear in mixed nodule populations, depending on the parameter conditions. For example, setting c = 0.2 in β<β1≈0.217; case (ii), magenta), but can emerge when the probability of mixed populations is higher –(vi), green, purple, and orange). Conversely, when c = 0.4, cheaters can coexist with mutualists at lower β  and (v), orange and purple), but are excluded at higher β  and (iii), blue and gray).This prediction is supported by numerical simulations, in which the parameter region of “No evolution” expands as In symbiotic relationships, the participating organisms provide mutual benefits to each other. Such positive fitness feedback reinforces their mutualistic interaction. However, symbiotic systems encourage the emergence of selfish parasitic cheaters, whose performance undermines the system. Thus, symbiotic systems are simultaneously exposed to promoting and destabilizing forces, analogous to benefit and cost in game theory.One of the most famous symbioses occurs between legumes and rhizobia. Here, we intensively investigated how benefit and cost influence the evolution of this symbiosis, and the conditions required for establishing the symbiotic relationship. According to our model, stable mutualism depends on the cost–benefit balance  and 10. Ineffective rhizobia with little or no nitrogen fixation activity are widely distributed in natural environments According to our model, cheating rhizobia can be classified into “co-dependent” or “parasitic”, which exert different effects on the system. The presence of “co-dependent” cheaters stabilizes a symbiotic system , while “C(x) (or destabilizing force). Therefore, our model suggests that these factors strengthen the symbiotic interaction in opposite ways; partner fidelity feedback reinforces the benefit while host sanction and partner choice ameliorate the cost <0), implying that more cheating rhizobia proliferate more efficiently if they infect the same host plant, because the fitness is given by In this paper, we assumed a decreasing cost function (i.e. volution .C′(x)≥0), our theory predicts that rhizobial populations always evolve to the maximum activity of nitrogen fixation  (Eq. (1)), here denoted by “systemic” and “local” effects, respectively. Both effects are essential for coexistence of nitrogen-fixing and cheating rhizobia, because coexistence cannot emerge without their mutual cooperation (for details see S(x) and L(x) respectively with B(x) and C(x), Eq. (15) takes the fitness form of Eq. (1); hence, the evolutionary dynamics described by Eq. (15) can be understood within the framework described in this paper. For example, the benefit (or partner fidelity feedback) might include both local and systemic effects. The local beneficial effect (i.e. LB(ix)) would eventually reinforce the symbiotic relationship by affecting the local function L(x) of Eq. (15), similarly to the weakening of the cost function C(x) by host sanction and partner choice in Eq. (1).In our model, rhizobia fitness is influenced by both the average nitrogen fixation of the rhizobia infecting a host plant  and destabilizing force (or cost). We then determined a comprehensive set of conditions under which the symbiotic relationship is established and cheating bacteria emerge. Our findings provide a theoretical basis for understanding how the legume/rhizobium symbiosis evolves. However, as mentioned above, our model is extremely simplified, and evolutionary behaviors in the natural environment are expected to be much more complicated than those seen in our model. By incorporating such effects into our model, we could better understand the evolution of symbiotic systems.Figure S1Effect of Nb, assuming a linear cost function. If a linear cost function (Nc = 0) is assumed, nonlinearity in the benefit function (Nb) hinders symbiotic evolution. As Nb increases, the parameter region of case (ii) “Maximum evolution” (magenta) decreases while that of case (iii) “Intermediate evolution” (blue) increases. This prediction is consistent with numerical simulations; cases (i), (ii) and (iii) are indicated by crosses, squares, and circles, respectively. Parameters are: b = 5.0, Nc = 0.0, n = 5.(TIF)Click here for additional data file.Figure S2Effect of Nc, assuming a linear benefit function. If a linear benefit function (Nb = 0) is assumed, nonlinearity in the cost function (Nc) promotes symbiosis evolution. As Nc increases, the parameter region of case (ii) “Maximum evolution” (magenta) increases, while that of case (iii) “Intermediate evolution” (blue) decreases. In addition, Nc promotes the emergence of cheaters. The parameter region in which nitrogen-fixing and cheating rhizobia coexist (cases (iv)–(vi); orange, purple, and green areas, respectively) is absent when Nc<Nc* = b/(n+(n+2) b), but otherwise increases as Nc increases. These predictions are consistent with numerical simulations; cases (i), (ii), (iii), (iv), (v) and (vi) are indicated by crosses, closed squares, closed circles, open circles, diamonds, and open squares, respectively. Parameters are: b = 5.0, Nb = 0.0, n = 5.(TIF)Click here for additional data file.Text S1Theoretical framework of adaptive dynamics.(PDF)Click here for additional data file.Text S2Increasing cost function (C′(x)>0).(PDF)Click here for additional data file.Text S3Linear cost function (Nc = 0).(PDF)Click here for additional data file.Text S4Linear benefit function (Nb = 0).(PDF)Click here for additional data file.Text S5Systemic and local effects of nitrogen fixation.(PDF)Click here for additional data file.


Slc6a8) gene lead to absence of brain Cr and intellectual disabilities, loss of speech, and behavioral abnormalities. To date, no mouse model of CrT deficiency exists in which to understand and develop treatments for this condition. The purpose of this study was to generate a mouse model of human CrT deficiency. We created mice with exons 2–4 of Slc6a8 flanked by loxP sites and crossed these to Cre:CMV mice to create a line of ubiquitous CrT knockout expressing mice. Mice were tested for learning and memory deficits and assayed for Cr and neurotransmitter levels. Male CrT−/y (affected) mice lack Cr in the brain and muscle with significant reductions of Cr in other tissues including heart and testes. CrT−/y mice showed increased path length during acquisition and reversal learning in the Morris water maze. During probe trials, CrT−/y mice showed increased average distance from the platform site. CrT−/y mice showed reduced novel object recognition and conditioned fear memory compared to CrT+/y. CrT−/y mice had increased serotonin and 5-hydroxyindole acetic acid in the hippocampus and prefrontal cortex. Ubiquitous CrT knockout mice have learning and memory deficits resembling human CrT deficiency and this model should be useful in understanding this disorder.Mutations in the creatine (Cr) transporter (CrT;  SLC6a8) is a member of the solute carrier 6 family that transports Cr into cells in a Na+, K+-dependent manner where it is used as a readily available phosphate pool to replenish ATP levels The creatine (Cr) transporter  gene, which corresponds to the 2nd–4th transmembrane domain of the protein, were flanked by loxP sites (flox/flox. Cre recombinase driven by the CMV promoter (B6.C-Tg(CMV-cre)1Cgn/J; Jackson Laboratory, Bar Harbor, ME) was utilized to create ubiquitous CrT knockout mice. The Cre and Flp were subsequently removed via breeding. As the CrT is X-linked only males have a single copy of the CrT gene therefore WT male mice are referred to as CrT+/y and knockout mice as CrT−/y. Genotyping was performed using a touchdown PCR protocol: 1) 95°C-5 min; 2) 2 cycles: 95°C-30 s, 60°C-30 s and 72°C-1 min; 3) 20 cycles: 95°C-30 s, 60°C-30 s Δ-0.5°C each cycle, 72°C-1 min; 4) 10 cycles: 95°C-30 s, 50°C-30 s and 72°C-1 min 5) 72°C-10 min. Primers  of each genotype were used in the behavioral experiment. Litters were only represented once per genotype. As CrT deficiency is X-linked only males are consistently affected, therefore only the male mice were tested. Mice were weighed once prior to testing. The testing order consisted of: locomotor behavior (1 day duration), Morris water maze (MWM) cued (6 days) and hidden platform phases (21 days), novel object recognition (5 days), sociability (1 day), acoustic startle (1 day), and conditioned fear (3 day). Mice were tested in one task at a time with the next starting 1 day after the completion of the previous task for a total of 38 days of testing. In order to minimize circadian effects, animals were tested during the same time interval each day (1200–1600 h).2 with 16 LED-photodector beams across each side of the chamber in the X and Y-planes (mounted 2 cm above the floor) and another row to detect vertical movements positioned 8 cm above the floor. Photocells were spaced 2.5 cm apart. Animals were placed in the chamber for 1 h and allowed to explore. Dependent measures for locomotor are measured in number of times the beam is broken.The task assesses exploration and general locomotor activity The MWM is a test of spatial learning and reference memory The hidden platform portion was conducted in three phases  consisting of 4 trials per day for 6 days for animals to learn the location of the hidden platform followed by a single probe trial (no platform) on day 7 Novel object recognition (NOR) is a test of short-term memory Mice were tested for social preference as described by Crawley et al. max) was recorded in units of voltage change (mV).Acoustic startle combined with prepulse inhibition (PPI) was used as a test of sensory gating Cued and contextual fear were assessed as described previously with modification A group of animals not behaviorally tested was assayed for tissue and serum Cr levels using a colorimetric Cr assay . Cr levels were normalized to tissue weight or serum volume. For CNS tissue, whole brain was used.A subset of animals was euthanized following behavioral testing and the prefrontal cortex (PFC), hippocampus, and neostriatum were assayed for serotonin (5-HT), dopamine (DA), their metabolites, and norepinephrine (NE) using HPLC-ECD as described previously Data were analyzed using general linear model analysis of variance (ANOVA) . For data with a repeated measure factor, data were analyzed using mixed models (Proc Mixed) with Kenward-Rodger degrees of freedom that can be fractional. Covariance structures were checked for best fit and the autoregressive (AR(1)) covariance structure used. Significant (P<0.05) interactions were analyzed using slice-effect ANOVAs at each level of the repeated measure factor. Data are presented as means ± standard error of the mean (SEM) or as least square (LS) Mean + LS SEM.−/y mice showed reduced body weights compared to CrT+/y mice  = 205.3, P<0.001; In order to determine the effects of the deletion of CrT on body and brain weights, animals were weighed prior to testing and brains were weighed after behavioral testing. CrT−/y model, Cr levels were measured by colorimetric assay in various tissues. In brain and muscle, Cr levels were below detection limits in CrT−/y mice  = 2.30, P<0.01; +/y mice after the first interval . For the probe trial, CrT−/y mice had a greater average distance from the platform [genotype  = 8.59, P<0.01; +/y mice  = 5.84 P<0.05; LS Means ± SEM: CrT+/y: 38.5±5.0% vs. CrT−/y: 24.8±2.7%). The data from the shift phase indicate that neither the CrT+/y or CrT−/y mice were able to learn well under these more demanding conditions and the lack of difference between genotypes appears to be caused by this final phase being excessively difficult. This can be seen by comparing the path length of CrT+/y mice across phases. It is evident that both genotypes showed reduced performance with each subsequent phase until during the third phase even CrT+/y controls showed poor learning.No main effect of genotype was observed during the shift phase of testing . CrT−/y −/y mice spent less time attending to the novel object than CrT+/y mice [genotype  = 7.30, P<0.05, −/y mice investigated the objects and reached the 30 s criterion, therefore, the visual system of CrT−/y mice are sufficiently intact that they can see and recognize the objects adequately to respond to them appropriately.NOR is a test of short-term, hippocampally-dependent memory. CrT−/y mice, body weight was used as a covariate in the analysis. There were no differences in acoustic startle response with or without prepulse inhibition using weight as a covariate  on day-2 [genotype  = 1.78, P<0.05, −/y mice spent less time freezing in response to tone (cued fear) on day 3 than CrT+/y mice [genotype  = 4.71, P<0.05, CrT+/y and CrT−/y mice. In the PFC, 5-HT levels were increased in CrT−/y mice  = 6.0, P<0.05; +/y mice.In order to characterize general brain systems, monoamine neurotransmitter levels were assessed in brain regions of CrT−/y mice compared with CrT+/y mice. NE levels were unchanged.In the hippocampus, 5-HT  = 5.81, P<0.05), 5-hydroxyindoleacetic acid (5-HIAA)  = 25.5, P<0.001), and the 5-HIAA/5-HT ratio  = 7.48, P<0.05) were increased in CrT−/y mice showed a trend towards increased DA  = 3.64, P<0.10); they had increased levels of 5-HIAA  = 12.41, P<0.01) and 5-HIAA/5-HT  = 11.63, P<0.01) compared with CrT+/y mice. No changes were observed for 5-HT, DOPAC, DOPAC/DA, HVA, or NE in the neostriatum.In the neostriatum, CrT−/y mice. The lack of Cr in many tissues of the CrT−/y mice shows that the recombination resulted in a successful disruption of the CrT gene.In humans, loss of function of the CrT leads to intellectual impairment, loss of language, and autistic-like behavioral abnormalities, and no treatment is available −/y mice. The results of this study suggest that the mouse brain is unable to synthesize Cr despite having the requisite mechanisms −/y mice show Cr reductions in muscle, which is in contrast with case reports of two CrT deficient patients who had either the presence of Cr Human CrT deficient patients show no brain Cr using MRS −/y mice show cognitive impairments across a variety of learning and memory tests. CrT−/y mice show spatial learning and memory deficits during the first two phases of the MWM. While the CrT−/y mice were slower swimmers than CrT+/y mice, swim speed does not affect path length; therefore this measure is not confounded by performance effects. CrT−/y mice showed no deficit during the shift phase of the MWM; however, this was because even the CrT+/y control mice could not learn this phase adequately, precluding the detection of differences between genotypes. Nonetheless, on probe trials during this third phase, the CrT−/y mice were impaired, therefore, even the little they were able to learn during this phase they could not remember as well as CrT+/y.Similar to human CrT patients, CrT−/y mice. The improvement in the cued version of the MWM as well as the animals' readily observing and responding to objects during the NOR task  suggests that the visual system is not disrupted in the CrT−/y mice sufficiently to impair visually-mediated tests. In addition, the CrT−/y mice acquired the platform's location as shown by CrT−/y mice having an average latency of 46 s out of a 90 s trial during the acquisition phase, suggesting that distal visual cues were used by the CrT deleted mice to navigate to the platform. This suggests that neither sensorimotor nor motivational impairments contributed to the observed deficits.It is conceivable that sensory deficits play a role in the learning and memory deficits. For example, CrT is highly expressed in the retina −/y mice showed impaired reference memory compared with CrT+/y mice. In addition to the probe trials, mice were assessed for memory in the NOR and conditioned fear tests. CrT−/y mice showed reduced memory in both of these tasks. The deficits in memory across tasks combined with the learning trials in the MWM suggest that CrT−/y mice have a general cognitive impairment. In humans, the type of learning deficit has not been characterized in detail because the phenotype prevents systematic assessment .During the probe trials, CrT−/y mice as evidenced by increases in 5-HT in hippocampus and prefrontal cortex along with increased 5-HT turnover in the neostriatum and hippocampus. It has been shown that serotonergic drugs such as fluoxetine and paroxetine increase Cr kinase activity 1A receptor agonist 8-hydroxy-N,N-dipropyl-2-aminotetralin, or fluoxetine increases the speed of anterograde mitochondrial transport in hippocampal neurons, while serotonergic antagonism slows mitochondrial transport −/y mice spend more time in the periphery of the locomotor chamber with no change in central time; since central movement reflects anxiety, the data suggest that anxiety is not altered in these mice. Additional tests of anxiety will be required to address this in greater depth, including testing for a relationship between these behaviors and changes in 5-HT.There appears to be a disruption of the serotonergic system in CrT−/y mouse provides a tool for studying the relationship between Cr and 5-HT. More broadly, CrT−/y mice exhibit cognitive deficits similar to those seen in CrT deficient patients and as such provides a model with good fidelity to the human condition suitable to test potential therapies for this currently untreatable disorder.In sum, the CrT


There are gender differences in global and domain-specific self-esteem and the incidence of some psychiatric disorders related to self-esteem, suggesting that there are gender differences in the neural basis underlying one's own self-esteem. We investigated gender differences in the brain activity while subjects  performed an implicit self-esteem task, using fMRI. While ventromedial prefrontal cortex (vmPFC) was significantly activated in females, medial and dorsomedial PFC (dmPFC) were activated in males in the incongruent condition (self = negative) compared with the congruent condition (self = positive). Additionally, scores on the explicit self-esteem test were negatively correlated with vmPFC activity in females and positively correlated with dmPFC activity in males. Furthermore, the functional relationships among the regions found by direct gender comparisons were discussed based on the somatic-marker model. These showed that, compared to males, females more firmly store even the incongruent associations as part of their schematic self-knowledge, and such associations automatically activate the neural networks for emotional response and control, in which vmPFC plays a central role. This may explain female cognitive/behavioral traits; females have more tendency to ruminate more often than males, which sometimes results in a prolonged negative affect. Self-esteem is commonly defined as an individual's sense of self-worth There are gender differences in some aspects of self-esteem. Previous meta-analyses found that, in adolescence, males showed higher global self-esteem than females Explicit measures such as Rosenberg scores have ever been widely used in the studies on self-esteem Here, we investigated gender differences in brain activities, measured by event-related functional magnetic resonance imaging (fMRI) of subjects performing the self-esteem IAT, by comparing incongruent situations  and congruent situations . While the congruent association is based on self-positivity, the incongruent association contradicts it and would cause certain conflicts based on self-esteem. Specifically, we investigated the differential brain activity between the incongruent and congruent situations in each gender group, focusing on the relationships between the brain activity and the explicit and implicit self-esteem, and we performed the direct comparison between males and females in the incongruent vs. congruent situations. The present hypothesis is that there would be some gender differences in the neural substrates underlying self-esteem when responding to the incongruences, and the brain activity in females would be more sensitive and cautious to the incongruences than that in males as suggested by the previous psychological and behavioral findings.t = 0.58, p = 0.57, df = 24). They were healthy undergraduate or graduate students with no history of significant medical, psychiatric, or neurological disorders. All were right-handed, as determined by the handedness scale Fourteen males  and 12 females  participated in the present study. There was no significant difference in age between the two groups (t-test).Before fMRI scanning, we assessed the explicit self-esteem in each subject using the Rosenberg self-esteem scale After the scanning, the personality for each subject was indexed by using the NEO Five-Factor Inventory (NEO-FFI), self-report questionnaire that assesses the five personality dimensions of neuroticism, extraversion, openness to experience, agreeableness, and conscientiousness. The 60 items of the NEO-FFI were rated on a five point scale. We examined the two sample t-test between males and females for each personality dimension.During fMRI scanning, subjects completed a computer-administered version of the IAT, with stimuli developed  for estimating levels of implicit self-esteem. Subjects were asked to sort stimuli representing four concepts  into one of two response categories, each of which included two of the four concepts. The usefulness of the IAT in measuring association strength depends on the assumption that when the two concepts that share a response are strongly associated, the sorting task is considerably easier than when the two response-sharing concepts are either weakly associated or bipolar-opposed self and positive words shared a response key, and other and negative words shared a key . These IAT scores reflect the differential ease with which subjects associate the “self” and “other” recognition with positive concepts compared with negative concepts. The IAT scores were compared between males and females using the Welch's t-test.All response times were recorded using a personal computer. The mean latency for the C condition was subtracted from that for the IC condition for each subject (IAT scores). We analyzed the differences between IC and C conditions at the 5% level of significance for males and females  2D sequence was performed .Event-related fMRI was conducted using a 1.5-Tesla whole-body superconducting scanner system  equipped with a quadrature detection head coil of the birdcage type and an actively shielded gradient coil. The functional scans were performed using a gradient-recalled echo, echo-planar imaging MR sequence and T2*-weighted images were acquired. The BOLD-sensitive single-shot EPI sequence parameters were as follows: TR = 4000 ms, TE = 90.5 ms, flip angle = 80°, matrix size = 128×128 pixels, FOV = 240×240 mmhttp://www.fil.ion.ucl.ad.uk/spm) implemented in MATLAB 7.0.1 . The functional images were realigned to correct for head movements and were coregistered with the Montreal Neurological Institute (MNI) template. All functional images were spatially filtered by a low-pass Gaussian filter (FWHM = 10 mm) and smoothed to conform to the Gaussian assumptions of SPM and to improve the signal-to-noise ratio. The data were temporally convolved with the hemodynamic response function and high-pass filtered with a cutoff period of 128 s.All functional imaging data were preprocessed and analyzed using SPM2 . Moreover, a region was considered significant only if its volume was more than 63 mm3. This double threshold corresponds to a 5% multiple comparisons adjusted probability of falsely identifying one or more activated voxel clusters on the basis of Monte Carlo simulations ). Moreover, brain activity differences between gender groups were investigated based on a two-sample t-test with a volume threshold of 63 mm3 .Fixed effects analysis was performed on each subject's data, and regressors were included as factors that accounted for the IC condition . Furthermore, correlational analyses for the male and female groups were performed, at p<0.05, between the IAT and Rosenberg scores and the averaged parameter estimates in the spherical region of interest , the center of which was the peak voxel in each cluster showing significant activity in the IC condition compared with the C condition at t>4.12 .To assess the relationship between explicit self-esteem and implicit self-esteem, we analyzed the correlation between the IAT scores and the Rosenberg scores for each group at t = −0.51, p = 0.61, df = 24, t-scores ranged from 25 to 55 for neuroticism (mean ± SD =  41.0±8.11), from 41 to 61 for extraversion (51.8±6.76), from 40 to 60 for openness (47.9±7.97), from 31 to 63 for agreeableness (50.5±10.0), and from 28 to 69 for conscientiousness (53.9±11.3) in males. On the other hand, in females, scores ranged from 25 to 65 for neuroticism (45.5±11.0), from 37 to 71 for extraversion (53.9±9.27), from 28 to 71 for openness (51.6±12.2), from 43 to 72 for agreeableness (54.3±9.45), and from 36 to 75 for conscientiousness (55.1±10.6).The Rosenberg scores ranged from 14 to 22 in males (mean±SD = 18.2±2.67) and from 14 to 28 in females (18.9±4.25), with no significant gender difference , extroversion , openness , agreeableness , and conscientiousness .t = 4.25, p = 0.02<0.05, df = 13; females: 652±100 ms, t = 9.70, p = 0.0009<0.001, df = 11) than in the IC condition , demonstrating that on average subjects more strongly associated self with positive than with negative adjectives. In short, the subjects exhibited an IAT effect .T effect . In addiBrain regions that were significantly activated  in males in the IC vs. C condition were as follows: the left middle frontal gyrus (MFG), medial prefrontal cortex (mPFC), posterior cingulate cortex, posterior superior temporal gyrus, parahippocampal gyrus, right dorsomedial prefrontal cortex (dmPFC), and cerebellum. On the other hand, the regions that showed a significant differential BOLD response to IC relative to C conditions in females included the left ventromedial prefrontal cortex (vmPFC), inferior parietal lobule (IPL), precuneus, middle occipital gyrus, fusiform gyrus, right superior frontal gyrus, anterior cingulate cortex (ACC), superior parietal lobule, and superior occipital gyrus .r = 0.67, p<0.01, df = 12, r = −0.66, p<0.05, df = 10, In males, activity in the right dmPFC in the IC vs. C condition positively correlated with the Rosenberg score , IPL, postcentral gyrus, and lateral occipital cortex , Fig. 4.p = 0.0009; males, p = 0.02). Moreover, we did not find any significant correlation between the two scores in either group, in partial agreement with a previous study showing no significant gender difference and only weak correlation between two scores Although we did not find any significant differences between males and females in the IAT scores and the Rosenberg scores, the averaged IAT effect was more remarkable in the female than male groups  corresponded to those that exhibited significant differences in the IC vs. C condition in the female group . As discp = 0.0009; males, p = 0.02).Several neuroimaging studies showed that the dACC is sensitive to goal conflicts, expectation violations, and errors in general Studies show that the right postcentral cortex is involved in one's own emotional feelings The somatic-marker hypothesis proposes that decision-making depends in many important ways on neural substrates that regulate homeostasis, emotion and feeling. Imaging studies have shown that decision-making is associated with functioning of a distributed neural network critical for the processing of emotional information, including the vmPFC, amygdala, striatum, ACC, and insular/somatosensory cortices , as well as non-specific neurotransmitter systems that modulate activities of neural processes involved in decision-making The neural processes that were significantly activated in our female subjects may explain female cognitive/behavioral traits; females tend to ruminate more often than males, which sometimes results in a prolonged negative affect Behaviors depend on the brain, and so gender differences in behaviors imply gender differences in brain structure or function. On the other hand, numerous studies report gender differences in neural activity despite no behavioral differences between males and females


Do the integration of semantic information and that of world knowledge occur simultaneously or in sequence during sentence processing? To address this question, we investigated event-related brain potentials elicited by the critical word of English sentences in three conditions: (1) correct; (2) semantic violation; (3) world knowledge violation . Critically, we opted for low constraint sentence contexts . The processing of semantic violations differed from that of correct sentences as early as the P2 time-window. In the N400 time-window, the processing of semantic and world knowledge violations both differed significantly from that of correct sentences and differed significantly from one another. Overall, our results show that the brain needs approximately 200 ms more to detect a world knowledge violation than a semantic one. Most people know that the capital of France is Paris, not Barcelona, or that Big Ben can be found in London, not Madrid. Factual information about the world stored in long-term memory—i.e., world knowledge—is constantly retrieved when processing language to make sense of spoken or written content. Comprehenders do not only rely on definitional knowledge of words and expressions , they also form expectancies from and confront semantic content against world knowledge, which enables them to evaluate information plausibility, modify existing representations, and form opinions. It is this information -not conveyed literally- which leads to perceive the following statement “I am going to Madrid next week, so I will visit Big Ben” as a lie, a confusion, or perhaps a joke. Understanding the cognitive mechanisms underlying language comprehension therefore requires a detailed understanding of the way in which literal semantics and world knowledge are accessed and integrated.Here, we investigated whether readers retrieve and integrate literal semantic and world knowledge information simultaneously or in sequence during sentence comprehension.This question is important because two mainstream theories predict opposite results: According to the “dissociation theory” Forster, , literalTo study the time-course of semantic and world knowledge integration, we recorded event-related brain potentials (ERPs) in English readers presented with sentences containing either literal semantic or world knowledge violations. Hagoort et al.  previousOne of the problems inherent to the study of world knowledge is that each individual has a different and unique knowledge of the world. To address this issue we analyzed ERP data taking into account participant's knowledge as tested by our experimental sentences. After the ERP recording session, each participant was presented again with the experimental material and asked to make true/false/don't know judgments on each sentence. This information was then used to select the trials included in the averaging to generate three ERP: (a) true, (b) false, and (c) don't know.There were two main reasons for taking into account participant's individual knowledge. First, as in Hagoort et al. 's study,Sentence context has a major impact on word processing and word-sentence integration processes . Thus, in this particular case, any critical word that is not the “only possible completion” is likely to be processed as invalid , and will elicit a larger N400. It is then possible that this large N400 due to expectancy violation could mask more subtle N400 modulations dependent on the type of violation, e.g., semantic vs. world knowledge. Thus, to avoid confusion between the effects of anticipation and those elicited by semantic and world knowledge violations, we chose to use low constraint sentences.Moreover, it is already established that both literal semantic and world knowledge violations elicit N400 modulation  took part in the experiment. All participants gave written consent to take part in the study that was approved by the ethics committee of Bangor University, Wales, UK.bracelets and rings” ; (2) sentences with world knowledge violations as “In a jewellery store one can buy croissants and rings” ; (3) sentences with semantic violations as “In a jewellery store one can buy brains and rings” (see Table Stimuli consisted of three versions of 120 sentences: (1) correct and true sentences such as “In a jewellery store one can buy p = 0.90) and syllables (p = 0.62), log-word frequency (p = 0.17), concreteness (p = 0.23), imageability (p = 0.20) and word class .Importantly, the critical word in correct sentences was neither the only possible candidate nor the most expected candidate to complete the sentence. For instance, the sentence “In a jewellery store one can buy… ” can be completed with the words rings, diamonds, necklaces, pearls, etc. A Cloze probabilitys p = 0.6, log-worEach sentence was presented centrally, one word at a time (200 ms duration and 500 ms stimulus onset asynchrony). Sentences were separated by a fixation cross displayed for 800 ms. The instruction was to read each sentence silently and to answer yes or no to the subsequent comprehension question  by pressing Y or N buttons on a response pad. The latter quiz test ensured that participants processed sentence meaning during silent reading.At the end of the experiment, participants were asked to perform a surprise follow-up test. The 80 true and false sentences were presented on the screen along with a rating scale. Participants had to rate each sentence as true or false by pressing “1” or “2.” They had to press “3” if they did not know if the sentence was true or false and “4” if they could not decide because the sentence was meaningless.t-tests for the contrasts of interest . Unstable differences (remaining below p = 0.05 for less than 30 ms) were discarded  in reference to electrode Cz at a rate of 1kHz from 64 Ag/AgCl electrodes placed according to the 10–20 convention. Vertical and horizontal EOG were recorded simultaneously with EEG. Impedances were kept below 5 kOhm. EEG activity was filtered off-line [0.1–30 Hz]. Eye blink artifacts were mathematically corrected using the Gratton and Coles's , implemeAccuracy in the quiz test was of 85.6% ± 7.9. In the follow-up test, participants rated 80% ±9 of correct sentences as true . They rated 73% ± 11 of WK sentences as false (10% ± 8 as true and 17% ± 10 as “Don't know”). In order to take into account individual world knowledge, four ERP conditions were computed: (1) correct sentences, rated as true in the follow-up test; (2) world knowledge violations (WK), rated as false; (3) “don't know” sentences (DK), corresponding to cases in which participants had insufficient knowledge to make a decision; and (4) semantic violations (SV). Overall, 30% ± 4 of the sentences were considered as correct, 26% ± 4 as WK, 10% ± 6 as DK, and 33% ± 0 as SV. Among the 30% of sentences considered as correct, 89% ± 7 were originally true and 11% ± 7 were false. Among the 26% of sentences considered as world knowledge violations, 93% ± 6 were originally false and 7% ± 6 were true. Among the 10% of sentences of sentences of the Don't know condition, 44% ± 11 were originally true and 56% ± 12 were false.p = 0.58) and syllables (p = 0.29), log-word frequency (p = 0.06), concreteness (p = 0.25), imageability (p = 0.24) and word class (equated within each pair).When each condition was computed taking into account individual knowledge, the critical word of true sentences had an averaged cloze probability of 8.02% ± 1 (range 0–44%). The critical words of sentences with world knowledge violations and semantic violations had an averaged cloze probability of 0.68% ± 0.9 and 0.0% respectively. The critical words of “Don't know” sentences had an averaged cloze probability of 3.55% ± 2. The critical words were still matched across the four conditions on the following criteria: average length in characters  between SV and CS were found at 150 and 240 ms. In contrast, the first sustained significant differences between WK and CS were found only at around 350 ms. WK and SV conditions started to significantly differ at 150 ms and then again at 260 ms.To gain a more fine-grained analysis of these effects, a ms-by-ms paired ps > 0.10). (2) We compared the magnitude of the differences  in the P2 and N400 time-windows using profile analyses. The results revealed a significant time-window effect  and a significant difference effect  showing that the magnitude of the N400 effect was larger than the P2 effect, and that the semantic violation effect was larger than the WK violation effect. The time-window × difference interaction was also marginally significant  showing that the increase in effect magnitude from P2 to N400 was larger for semantic (p < 0.001) than WK (p < 0.01) violations. This profile analysis further supported the idea that P2 effects cannot simply be accounted for by N400 effects and vice-versa, since effect magnitudes increased significantly between time-windows. (3) We performed another ANOVA comparing SV, DK (“Don't know”) and correct sentences  We tested for potential correlations between the P2 and N400 mean amplitudes in the three experimental conditions. If P2 mean amplitude was functionally linked to N400 modulation, we could expect P2 and N400 mean amplitudes to be correlated. However, this was not the case in any of the conditions  correct sentences (true sentences); (2) sentences with semantic violations (impossible sentences); (3) sentences with world knowledge violations .Two main results were observed. First, semantic violations, and world knowledge violations elicited a larger N400 component as compared to correct sentences. This result replicates previous observations by Hagoort et al. . Second,Semantic and world knowledge violations have been shown previously to elicit a larger N400 component as compared to control sentences . By contrast, the N400 component appears sensitive to both literal meaning and veracity. Thus, we argue that the N400 reflects simultaneous integration of word meaning, paralinguistic information, and information stored in long-term memory. This interpretation is compatible with Hagoort et al. 's conclutulips”—palms being the expected exemplar; Federmeier and Kutas, pines”). Several studies have shown that the N400 effect was smaller for within-category as compared to between-category violations, because of the organization of long-term semantic memory  of semantic and world knowledge violations are processed with different time-courses. Readers access literal semantic information ~200 ms before they access factual knowledge about the world. Consistent with previous results, we observed the first significant effects of semantic violations around 200 ms after the critical word onset. Then, further down the line, in the vicinity of the N400, both types of information are processed concurrently.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.


Passer domesticus) populations along an urban gradient. In line with theoretical expectations, FA was inversely correlated with genetic diversity estimated at genome level. However, this relationship was largely driven by variation at a single key locus. Contrary to our expectations, relationships between FA and genetic diversity were not stronger in individuals from urban populations that experience higher nutritional stress. We conclude that loss of genetic diversity adversely affects developmental stability in P. domesticus, and more generally, that the molecular basis of developmental stability may involve complex interactions between local and genome-wide effects. Further study on the relative effects of single-locus and genome-wide effects on the developmental stability of populations with different genetic properties is therefore needed.Fluctuating asymmetry (FA), a measure of developmental instability, has been hypothesized to increase with genetic stress. Despite numerous studies providing empirical evidence for associations between FA and genome-wide properties such as multi-locus heterozygosity, support for single-locus effects remains scant. Here we test if, and to what extent, FA co-varies with single- and multilocus markers of genetic diversity in house sparrow ( Developmental stability refers to the ability of an organism to achieve a phenotypic endpoint, predetermined by its genotype and the environment, along a developmental pathway in the face of random perturbations Gambusia holbrookiTurdus helleriLychnis viscariaPopulation and individual levels of developmental stability are most commonly estimated by corresponding levels of fluctuating asymmetry (FA), i.e. small, random deviations from perfect left-right symmetry in bilateral traits heterozygosity hypothesis states that individuals with high levels of protein heterozygosity are developmentally stable as a result of dominance or overdominance effects genomic co-adaptation hypothesis states that balanced co-adapted gene complexes result in higher developmental stability because natural selection favours alleles at many different loci that ‘harmoniously’ interact during the developmental process to produce stable phenotypes Based on developmental and genetic theory, at least two hypothetical mechanisms underlying the genetic basis of developmental stability have been put forward sensuOncorhynchus mykiss) showed reduced levels of developmental stability in heterozygotes, probably due to a reduction in enzyme activity despite potential beneficial effects of chromosomal heterozygosity Lucilia cuprina) showed that developmental stability in bristle numbers (but not wing characters) initially decreased upon exposure to a new pesticide but restored after modification of the genetic background through natural selection Drosophila melanogaster and Arabidopsis thalianaWhile numerous studies have provided empirical evidence for associations between developmental stability and genome-wide processes such as multi-locus heterozygosity, evidence for single locus effects  populations along an urban gradient. Despite the wealth of analytic tools developed for non-coding neutral markers and their presumed suitability to test relationships with genetic diversity, few studies have applied such markers to model single- and multi-locus relationships with developmental stability Here we study how developmental stability in a metric trait co-varies with indices of genome-wide and single-locus genetic diversity in microsatellite markers, within and among 26 house sparrow .2 on the ground) and ranged between 0–0.10 , 0.11–0.30 (‘suburban’) and larger than 0.30 (‘urban’) (Arcgis version 9.2.). We selected 26 plots along this gradient  and its suburban periphery to the rural village of Zomergem, located ca. 12 km NW of Ghent. Urbanization was measured as the ratio of built-up to total grid cell area  and resulted in strong signal-to-noise ratios . Fourth, we calculated unbiased signed FA values (subject specific slope deviations from the fixed regression represented the amount of asymmetry after correcting for DA and ME). Finally, we calculated absolute values of the signed FA values  for hypothesis testing. These individual estimates were used for individual-based analyses while population mean values were used for analyses conducted at the population level.Fifth, we compared the kurtosis levels of the signed FA values to detect antisymmetry To estimate FA in tarsus length at an individual and population level, we carried out mixed regression analysis with restricted maximum likelihood parameter estimation (REML) to obtain unbiased individual FA estimates Genomic DNA was extracted from ten plucked body feathers using a Chelex resin-based method  e2, Because the genotyping of noninvasive DNA samples is potentially prone to artefacts http://www.r-project.org), which also provides two additional heterozygosity-based indices, i.e. homozygosity by loci    and resup<0.001) , only rehttp://genetics.forestry.ubc.ca/ritland/programs.html) and calculated as i and l represent alleles and loci, respectively; ilS equals 1 if both alleles are allele i or 0 otherwise, ilP is the frequency of allele i at locus l and ln denotes the number of alleles at locus lRitland estimates were obtained from the software program MARK  and the relationship between genetic variability and strength, measured as total variance explained, of these (single-locus) genotype-FA associations was assessed. Positive Spearman rank correlation coefficients imply that more heterozygous markers are more informative We used Pearson correlation coefficients to quantify the strength of associations between d2-MLH: r = 0.52, p<0.001; d2-25,502 = 2.35, p = 0.0003 and F25,502 = 2.00, p = 0.0031) while d2 showed a near-significant trend .All loci were highly polymorphic and most locus by population combinations were in Hardy-Weinberg equilibrium, yet some deviations reached significance after Bonferroni correction . There was no evidence that scoring errors due to large allele drop-out or stutter contributed to this nonequilibrium. To ascertain these deviations did not influence our results we ran all analyses with and without these five markers. Removing these loci did not alter any of the overall conclusions, hence only results based on the total dataset are reported. There was no evidence for linkage disequilibrium between any pair of loci. Standard statistics for each marker are presented in p<0.001) . MLH was1,517 = 6.59, p = 0.01, R2 = 0.049; 1,517 = 4.70, p = 0.03, R2 = 0.046) . As opposed to the weak associations measured at the individual level, mean values of MLH and 1,24 = 12.31, p = 0.001, R2 = 0.34; 1,24 = 7.88, p = 0.009, R2 = 0.25) .Genetic diversity estimated by MLH and = 0.046) . When te = 0.25) ; Table 22≤0.06). When analyzing each microsatellite locus separately, the association between heterozygosity and FA at population level was strongest at loci Pdoµ1, Pdo16 and TG04-012, whereas mean differences in allelic size were strongest at locus Pdo16. After sequential Bonferroni correction for multiple testing, the association at locus Pdoµ1 remained significant . The strength of single-locus FA-d2 relationships (16 loci) were positively correlated with expected heterozygosity at population level  but not at individual level . In contrast, FA-MLH relationships did not significantly vary with genetic diversity   .2,513 = 4.25, p = 0.01): both variables were inversely related in rural populations , but unrelated in urban  and suburban  ones. In contrast, the strength and direction of FA- 2,41.6 = 0.79, p = 0.46) and FA-d2  relationships did not vary with urbanization.The strength of FA-MLH relationships tested at the individual level significantly varied with urbanization  genome and the position of the nearest known zebra finch gene.(PDF)Click here for additional data file.


To investigate the value of diffusion-weighted imaging (DWI) at 3.0 T (3T), and especially the apparent diffusion coefficient (ADC), in discriminating ovarian thecoma from other adnexal solid masses.Eighteen thecomas or fibrothecomas, 14 ligamentous leiomyomas, and 24 other ovarian solid tumors underwent prospective DWI magnetic resonance imaging (MRI) in addition to routine MRI on a 3T MRI machine. The baseline characteristics, components, and conventional MRI and DWI-MRI signals for the thecomas were recorded. The ADC values (ADCs) were measured for each group and compared.1WI; 11/18) or T2WI (11/18) on DWI-MRI, with minor (9/18) or mild (6/18) enhancement. The mean ADC value for thecoma (1.20 ± 0.45 × 10−3 mm2/s) was almost equal to that of the other solid ovarian masses (1.26 ± 0.51 × 10−3 mm2/s), but lower than that for leiomyoma (1.48 ± 0.42 × 10−3 mm2/s), although not significantly so. There was a significant difference (p = 0.043) in the ADCs of the benign ovarian solid masses (1.16 ± 0.47 × 10−3 mm2/s) and leiomyomas (1.48 ± 0.42 × 10−3 mm2/s).The thecomas often appeared as homogeneous isointensity (17/18) on T1-weighted images (TThere is no significant difference in ADC between thecoma and other adnexal solid masses, but the ADCs of thecomas are lower than those of leiomyomas. Thecomas are rare, solid sex-cord stromal ovarian tumors, and account for approximately 0.5%–1% of primary ovarian lesions . BecauseBetween January 2010 and August 2012, 347 consecutive patients with clinically suspected adnexal disease underwent 3T MRI examinations before pelvic or laparoscopic surgery at our institution. The time interval between the MRI evaluation and surgery was less than one month . Of these patients, 18 with histologically proven thecoma or fibrothecoma  [1-weighted images (T1WI), and sagittal FSE T2WI and fat-suppressed T2WI (FS T2WI) in the axial plane. A DWI-MRI sequence included an echo-planar imaging sequence with an array spatial-sensitivity-encoding technique (ASSET). The parameter details of the T1WI MR protocol were: repetition time (TR), 460 ms; echo time (TE), 10 ms; number of excitations (NEX), 2; and thickness, 6.0 mm. The parameter details of the T2WI MR protocol were: TR, 2400 ms; TE, 85 ms; NEX, 1; and thickness, 6.0 mm. The parameter details of the FS T2WI MR protocol were: TR, 3160 ms; TE, 90 ms; NEX, 2; and thickness, 6.0 mm. The parameter details of the DWI-MRI protocol were: TR, 3500 ms; TE, 61 ms; NEX, 6; and thickness, 6.0 mm; and the b value = 0 or 700 s/mm2. Liver acquisition with a volume acceleration (LAVA) sequence was used for contrast-enhanced pelvic imaging, and a power injector  was used to inject the contrast material . The parameter details of the LAVA MR protocol were: TR, 3.4 ms; TE, 1.4 ms; NEX, 1; flip angle, 15°; band width, 125 kHz. The images were acquired in multiple phases of contrast medium enhancement in both the sagittal and axial planes . Details of the parameters of image acquisitions are summarized in Table All MRI examinations were performed on a 3T system  equipped with an eight-channel cardiac array coil. The scan range was from the umbilicus to the pubic symphysis in the caudocranial direction. For any larger lesion that could not be fully accommodated on axial imaging, a sagittal scanning sequence was performed to include as much of the entire lesion as possible. Routine MRI protocols were used for the assessment of the adnexal masses, which included axial fast spin-echo (FSE) T1WI/T2WI and DWI-MRI was evaluated and recorded . On T1WI, hypo-, iso-, and hyperintensity were similar for the pelvic fluid, pelvic wall muscle, and fat signal; on T2WI, hypo-, iso-, and hyperintensity were similar for the pelvic bone, pelvic wall muscle, and fat signal; on b = 700 mm–2/s DWI images, the low, intermediate, high signal intensities were similar for the pelvic bone, myometrium, and endometrium. After the intravenous injection of the contrast medium, the degree of lesion enhancement was graded as follows: 1, minor enhancement (clearly less than the myometrium); 2, mild enhancement (less than the myometrium); 3, moderate enhancement (similar to the myometrium); or 4, severe enhancement (more than the myometrium). Two observers , who were blinded to the histological results, independently analyzed all the MRI datasets for each participant on a PACS terminal server. Consensus was achieved for any interobserver discrepancies in the evaluation of the adnexal lesions or G.F.Z.’s decision was arbitrarily accepted.The MRI characteristics of each thecoma were recorded separately, including the following items: 1) lesion components ; 2) signal intensity on Tb = 700 mm–2/s DWI-MRI images. A circle or ellipsis with an area range of 160–320 mm2 was placed centrally in the targeted region. Only the lowest ADC value was used for the subsequent statistical analysis.The ADCs were calculated by one observer (H.Z.) on a commercially available postprocessing workstation . Regions of interest were drawn manually in both the cystic and solid areas, with no more than three sites in each lesion on t test if normally distributed . SPSS version 13.0  was used to perform all statistical analyses. A p value of less than 0.05 indicated a statistically significant difference.Continuous variables were expressed as means ± standard deviation (SD) or as medians ± numerical ranges and compared with an unpaired The histological results revealed 18 ovarian thecomas in 18 patients (10 patients who underwent laparotomy and eight patients who underwent laparoscopic surgery). Two patients were perimenopausal and all the other patients were postmenopausal. All the thecomas were unilateral lesions, five of which were accompanied by other ovarian etiologies, including uterine fibroids n = 3), endometrial polyps (n = 1), or fibroma and ligamentous leiomyoma (n = 1). All the patients presented to the gynecological department of our institution complaining primarily of a pelvic or abdominal mass, with no previous gynecological disease history, except in one patient with renal carcinoma  incidentally detected on a routine follow-up abdominal MRI examination. There was no postmenopausal bleeding or vaginal discharge in this patient cohort. The baseline characteristics of the 18 thecomas are listed in Table , endomet1WI  and on T2WI . An isointense signal mixed with a patchy high signal (n = 4)  displayed minor enhancement, six mild enhancement, and three moderate enhancement. On b = 700 mm–2/s DWI images, 11 of the 18 lesions  displayed intermediate signal intensity, four high signal intensity, two low signal intensity, and one mixed signal intensity. The mean ADC value for the thecoma group was 1.20 ± 0.45 × 10–3 mm2/s. The details of the MRI characteristics of the ovarian thecomas are listed in Table In this patient population, all the thecomas were round or almost round solid masses with clear margins, except in one patient, who had cystic and solid components  Figure . In most 17/18, 9.4% and op = 0.018), but similar to that of patients with other solid ovarian masses  was almost equal to that of the group of other solid ovarian masses , although not significantly so (p = 0.086). When the ovarian solid masses were categorized into a benign group  and a malignant group (19 lesions), there was no significant difference in their maximum diameters  or ADC values , but age differed significantly , although a wide overlap between the two groups was observed . In this study, the lesions most often accompanying thecoma were uterine fibroids or intraligamentous leiomyoma (4/18). In the 19 cases studied by Li XC et al., endometrial abnormalities  accompanying the thecoma were described as resulting from elevated levels of estrogen [In this study, the mean age at onset of thecoma was 59.9 years and 16 patients (88.9%) were in the postmenopausal period. Our results are consistent with the literature in that thecoma predominantly affects postmenopausal women, with a mean age of 59 years ,3. The peiomyoma /18. In testrogen , we obse1WI  and T2WI  or on DWI-MRI . However, we did find that the larger lesions (four lesions > 100 mm in maximum diameter) displayed a more heterogeneous signal on T2WI  than did the smaller tumors. Our observations are basically consistent with those of the studies in the literature [2WI images (representing cystic degeneration) occurred centrally or eccentrically, whereas the low signal  was more often peripheral [According to the MRI findings in the present study, all the thecomas were solid-component masses, regardless of the lesion size, except for one cystic and solid mass, with a pathological diagnosis of luteinized thecoma, which is described in the literature as a rare condition . In anotterature -5. In a  17/18, 9.4% and Tb values were selected, varying pathologies were examined, and the samples studied had different volumes. In this study, the ADCs for the solid ovarian masses in the benign group (23 lesions) were slightly lower than those of the malignant group (19 lesions), but not significantly so. The benign group, which included thecomas, fibromas, and Brenner tumors, all showed many closely arrayed spindle fibroids and thecal cells, which would obviously reduce the ADCs on DWI images. Our results are consistent with those of other recently reported studies [p = 0.043). This result confirms that ADCs can be used to differentiate benign ovarian solid tumors from ligamentous leiomyomas.DW-MRI is a functional imaging technique that provides information about water mobility (Brownian movement), tissue cellularity, and the integrity of cellular membranes, and is gradually becoming part of the standard imaging protocols used to evaluate obstetric and gynecological diseases . The com studies ,18. It iThere were some limitations to this study. First, there was an inherent selection bias because the study was retrospective. However, all the patients underwent prospective MRI examinations, which may have partly offset this bias. The limited study sample size could also have influenced the final results. Second, we compared our results using 3T MRI with other studies based on 1.5T MRI. The potential influence of this difference in magnetic field may have made our comparison inaccurate. Third, the ADCs were manually measured on the regions of interest based on individual habits. The lack of standardization in calculating ADC may also have influenced the final results.1WI/T2WI and DWI-MRI. There was no significant difference in the ADCs for thecoma and other adnexal solid masses, although the ADCs of the thecomas and fibrothecomas were lower than those of the leiomyomas. A significant difference in the ADCs for benign solid ovarian masses and ligamentous leiomyomas was observed, but this parameter was not useful in differentiating benign from malignant solid ovarian tumors.Ovarian thecoma or fibrothecoma often manifests as a solid mass with homogeneous isointensity on both TThe authors declare that they have no competing interest.Guarantor of integrity of entire study, GFZ.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, GFZ, HZ; approval of final version of submitted manuscript, all authors; literature research, HZ; clinical studies, GFZ, HZ, TPW; statistical analysis, HZ; and manuscript editing, HZ. All authors read and approved the final manuscript.


Aims: The stereotactic brain biopsy is an essential diagnostic procedure in modern neurologic patient management. A side-cutting biopsy needle is one of the most widely used needle types. Recently we found a characteristic tissue artifact named “peripheral compressing artifact” in the brain tissues biopsied using a side-cutting needle of Leksell’s system. We investigate prevalence, possible cause and its clinical implication of this type of artifact. Materials and methods: We examined the biopsies from 80 patients  in the stereotactic biopsy group with a suspected brain tumor, who underwent a stereotactic brain biopsy using side-cutting needle of Leksell’s system. We also evaluated 16 cases of open brain biopsies without Leksell’s system as a control group. Results: The artifact is a semi-circular or band-like tissue compression in the periphery of the biopsied tissue. This artifact was found in 30 (37.5%) out of 80 cases and 57 (11.9%) out of 477 biopsied pieces. It might be produced during rotating of the inner cannula of the biopsy needle. Histologically, it might be misinterpreted as “hypercellular”, “spindle”, “well circumscribed”, or rarely as “pseudopalisading” especially in glioma. Conclusions: Awareness of this artifact would help making the appropriate pathological diagnosis for glioma.  Stereotactic brain biopsy is one of essential diagnostic procedures in modern neurologic patient managements , 3, 4, 5There are variable macroscopic and microscopic artifacts  artifact, artifact from foreign materials, autopsy artifacts, etc.) from the brain tissue processing and evaluation . HoweverThe cases of stereotactic brain biopsy performed between January 2005 and February 2011 using Leksell’s system were retrieved from the Pathology Department archives of our institution. The cases of frozen section biopsy were excluded because of the frozen section artifact. We enrolled 80 patients with a suspected brain tumor, who underwent a stereotactic brain biopsy using a side-cutting needle of Leksell’s system  with heaAll patients underwent a stereotactic brain biopsy in a usual manner. The neurosurgeon advanced a brain biopsy needle with closed side opening toward the target of the lesion with MRI guidance. The side-cutting type brain biopsy needle consists of inner and outer cannulae, both of which contain a side window. When the needle tip reached the target, the surgeon opened the window by aligning the window of both cannulae. Through this opening, a piece of the lesion was aspirated by negative vacuum pressure. Then surgeon rapidly rotated the inner cannula of needle with constantly holding outer cannula to close the window, and a small piece of material was resected within the inner cannula in a guillotine action. Finally, the surgeon withdrew the inner cannula and collected the tissue samples. Next, routine histological processing was done according to conventional protocol. At the time of open brain biopsy, the neurosurgeon used “Yasargil bayonet tumor grasping forceps” . Two pathologists reviewed the pathologic diagnosis of the slides. They counted the numbers of biopsied pieces in each case and evaluated the existence of the peripheral compressing artifact. We defined the “peripheral compressing artifact” as semi-circular or band-like tissue compression in the periphery of biopsy samples . The sevThe mean ages in the stereotactic biopsy group and the control group were 46.24 (11 – 78) and 51.94 (1 – 79) years, respectively. There were 44 cases of gliomas, 13 lymphomas, 7 germ cell tumors, 2 other tumors, 1 metastatic carcinoma, 4 non-tumorous conditions such as demyelinating disease and 8 non-diagnostic in the stereotactic biopsy group. All control group cases were gliomas. The total numbers of biopsy samples in the stereotactic biopsy and control groups were 477 and 87 samples, respectively. The mean numbers of biopsy samples were 5.96 (1 – 14) and 5.44 (1 – 15) per case. Among the 477 samples of stereotactically biopsied tissues, we found a “peripheral compressing artifact” in 57 samples (11.9%). The severities of peripheral folding were “mild” in 21 samples, “moderate” in 28 samples, and “marked’ in 8 samples. However, we found the “peripheral compressing artifacts” in 30 cases among a total of 80 cases (37.5%). When we counted the maximum severity, 12 (15.0%) and 11 (13.8%), and 7 (8.8%) cases showed “mild” and “moderate”, and “marked”, respectively. Among 30 cases that showed “peripheral compressing artifacts”, 19 cases were gliomas Figure 2A, B, C), 3 cases were lymphomas , 3 cases were germ cell tumors and 5 cases were non-diagnostic or non-neoplastic glial tissues A, B, C, B”, respeThere were no “peripheral folding artifacts” in the control cases. However, we observed other tissue artifacts such as pinching in the control cases. The stereotactic biopsy has become one of the standard diagnostic tools for neurosurgical management although there were some limitations , 2, 6 suThe artifact which we reported in this study is a semi-circular or band-like tissue compression in the periphery of the biopsied tissue. This artifact can be observed only when a side-cutting type biopsy needle is used. We presume that this tissue artifact might be caused by the mechanics of the side cutting needle used. As previously mentioned, a side-cutting biopsy needle consists of inner and outer cannulae and in the biopsy procedure, and during the biopsy procedure, a neurosurgeon rotated the inner cannula to close the side window and to cut the tissue. We thought that this artifact might occur during rotating the inner cannula, especially when the neurosurgeon rotated slowly. The artifact’s semi-circular or band-like shape, rather than a whole-circular shape B, F. As mentioned in “Results”, we started this project since we have experienced 2 cases of biopsy that have been nearly diagnosed as higher grade glioma . Because of the compression, the border between the non-compressed portion and artifact of biopsied tissue is well demarcated A. In addIn conclusion, in case of stereotactic biopsies using a side-cutting needle, “peripheral compressing artifacts” can be observed, and it might lead to misdiagnosis of glioma in term of subtyping or grading. Awareness of this artifact would help making the appropriate pathological diagnosis for glioma. This work was supported by the Mid-career Researcher Program and the Basic Science Research Program through a National Research Foundation grant funded by Korean Ministry of Education, Science & Technology to Jong Hee Chang (No. R01-2008-20545-0) and Se Hoon Kim (2010-0021092). The first author (SH Kim) thanks Dr. Gregory N. Fuller  for his inspiring, passionate teaching.