The estimation of θ is unbiased only under neutrality and constant population growth. It is well known that the estimation of θ is biased when these assumptions are violated. However, the effects of sample size in modulating the bias was not well appreciated.One of the fundamental measures of molecular genetic variation is the Watterson’s estimator  and the magnitude of selection pressure. For example, θ estimated for the nonsynonymous sites of highly constrained genes (dN/dS < 0.1) using 512 exomes was 3.6 times higher than that estimated using 16 exomes. In contrast this difference was only 2 times for the less constrained genes (dN/dS > 0.9).We examined this issue in detail based on large-scale exome data and robust simulations. Our investigation revealed that sample size appreciably influences D, Fu-Li D and those based on the McDonald and Kreitman test: Neutrality Index and the fraction of adaptive substitutions. For instance, use of 16 exomes produced 2.4 times higher proportion of adaptive substitutions compared to that obtained using 512 exomes (24 % vs 10 %).The results of this study reveal the extent of underestimation owing to small sample sizes and thus emphasize the importance of sample size in estimating a number of population genomic parameters. Our results have serious implications for neutrality tests such as Tajima The online version of this article (doi:10.1186/s12864-016-2441-8) contains supplementary material, which is available to authorized users. Empirically θ could be estimated by the Watterson’s estimator θw [θ hereafter), which is based on the number of segregating sites (S) or by the Tajima’s estimator θπ [π hereafter), which uses the mean pair-wise differences between sequences. The estimation of θ is based on population coalescent theory and its popularity is due to its simplicity. Hence it is widely used in theoretical and empirical population genetic analyses. For instance θ or S is used to model the expected number of mutations, which is fundamental in molecular evolutionary biology [θ is an unbiased estimator when the assumptions such as neutrality and constant population sizes are met [θ is downwardly biased for an exponentially growing population. Similarly, estimates of θ are biased for sequences under purifying selection, which results in an excess of low frequency variants. This is because the theoretical relationship (θ = 4Neμ) assumes that all mutations neutral (or observable), which is not true in reality. Therefore this relationship can be written as θ = 4Neμf, where f is the fraction of neutral mutations and f = 1 and f < 1 for neutral and for selectively constrained sites respectively [Measuring genetic variation is fundamental in population genetics. Molecular genetic variation . In contrast π estimated for the two datasets were identical (3.96). Similarly, another study using exomes from >5,000 Americans showed a negative correlation between sample size and Tajima D estimates and up to fourfold difference between the estimates obtained using various sample sizes [D is based on the difference between θ and π the fourfold difference observed in this study was due to the difference in the θ estimated for various sample sizes. Similar discrepancies owing to sample size bias were also reported in other organisms such as plants [Although the factors influencing stimates –9. Recenn et al.  studied s plants . These sθ estimation is purifying selection. However, whether sample size will modulate the magnitude of this bias is not known. This is an important issue because every gene (and the genome) consists of regions under selection as well as under neutral evolution and most of the population genetic parameters are estimated for both regions. If sample size differentially influences the θ estimates of neutral regions and selected regions, then the estimates obtained for these regions are not comparable. This has serious implications for the tests of neutrality such as Tajima D [D [NI) [α) [Another important factor that is known to bias Tajima D , Fu and ima D [D  and the ima D [D  namely t [D [NI)  and the [NI) [α) .θ and to determine the probable cause for this pattern.To examine the differential effects of sample size bias in neutral and constrained sites we assembled a large dataset consisting over 1000 exomes [obtained from the 1000 genomes project  and estiθ we used 1008 human exomes and grouped them into six categories, each containing non-overlapping 16, 32, 64, 128, 256 and 512 exomes respectively. We then estimated θ at synonymous (θS) and nonsynonymous (θN) sites of >13000 protein-coding genes. Figure θ estimates significantly correlate (P < 0.01) and systematically increase with the sample size for both neutral and constrained sites. However, the rate of increase is much higher for constrained than that of neutral sites. This is evident since the slope of the regression line of the former was 44 % higher than that of the latter (0.26 vs 0.18). To further confirm this we estimated the ratio of θN/θS, which showed a highly significant positive correlation (P < 0.01) with the sample size . The above results suggest that increase in the sample size lead to the identification of more nonsynonymous variants compared to synonymous SNVs. To confirm this, we estimated the proportion of nonsynonymous SNVs (nSNVs) in each dataset and found positive correlation with the sample size . Fig. θN (with respect to the sample size) was much higher for the constrained genes compared to those under relaxed selective constraints. For example, the slope of the regression line for the genes with dN/dS < 0.1 was 0.34, which is 79 % higher than that observed for the genes with dN/dS > 0.9 (0.19). Note that the slope of the latter was close that observed for the neutral synonymous sites (0.18). To further support these results we computed the ratio between θN estimated using 16 exomes and that estimated using 512 exomes (θN(16)/θN(512))  on the genes (P < 0.01).The above results indicate that the purifying selection on constrained sites could inflate the sample size bias in estimating 2)) Fig. . We showπ). Unlike θ this measure is not affected by population growth or purifying selection. Hence we compared these two measures when they are estimated with various samples sizes. This reveals that π is not affected by sample sizes not only for neutral sites (P > 0.05) but also for constrained sites (P > 0.05)  statistic uses the properties of π and θ to test a genomic region for any deviation from neutral evolution. As we have shown that sample size influences only one of these two measures (θ) we examined the extent of its effect on DT. As expected, we observed negative correlation (P < 0.01) between DT and sample size for neutral and constrained sites  and neutral sites (DTS) with increasing sample size. For instance, the difference between DTN and DTS was only 0.22 for a sample size of 16. But this value was 0.54 for a sample size of 512, which is more than two fold higher than the former. We also examined the relationship between the magnitude of selection and extent of bias in estimating DTN. For this purpose, we estimated DTN using the genes under varying levels of selection pressures. We computed DTN for the synonymous and nonsynonymous sites of these genes. We then measured the difference (δDTN) between the DTN value obtained for the lowest (N = 16) and highest (N = 512) sample sizes. This measure δDTN was computed for 10 gene sets with dN/dS ranging between 0–1 with an interval of 0.1  between dN/dS and δDTN for the nonsynoymous sites of protein-coding gene sets. In contrast this relationship was not observed for the synonymous sites of the same gene sets (P = 0.48). This suggests that the level of selective constraint significantly influences the estimation DTN of different genes. On the contrary the bias in the estimation of DTS is similar across the genes under various levels of selection pressures.Apart from the number of segregating sites another popular measure for the extent of variation is nucleotide diversity (05) Fig. . The poptes Fig. . However0.1 Fig. . We obseD test - without using an outgroup (DFL). This test uses the difference between the total number of mutations (η) and the singleton  mutations (ηs). We computed the difference (η – ηs) for synonymous and nonsynonymous sites of protein-coding genes using different sample sizes. While the difference for neutral sites (ηS – ηSs) only slightly varied with sample sizes that (ηN – ηNs) for constrained sites significantly increased with sample size  and nonsynonymous sites (DFLN) using different sample sizes. We found a positive relationship between DFL and sample size (P < 0.01) and the bias in the estimation of DFL was more pronounced for the constrained sites . However, there was no significant relationship between sample size and δDFLS for the neutral sites (P = 0.14)  Fig. . Hence tα). Both these measures use polymorphisms and substitutions in synonymous and nonsynonymous positions. To examine the effect of sample size on these parameters we computed them using different numbers of human exomes. We observed a highly significant positive relationship (P < 0.01) between sample size and Neutrality Index (NI)  Fig. . The valα) using different sample sizes. Our result produced a strong negative correlation (P < 0.01) between the sample size and α   and these estimates varied up to 84 % between the samples sizes of 16 and 512. However, the estimates of π were not affected by sample sizes and this is true for the populations under constant as well as exponential growth (P > 0.05).To investigate whether SFS_CODE . We mode05) Fig. . On the θ varied with sample sizes. The rate of variation was much higher for sequences under high selective constraints (NeS = 2000) than those under relaxed selective pressures (NeS = 2). The slope of the regression line of the former was 2.7 times higher than that of the latter (0.21 Vs 0.08). However, θ estimated for neutrally evolving sequences did not vary with the sample size as there was no significant correlation between the two variables (P < 0.13). These results suggest that for populations under constant growth, purifying selection alone modulate the sample size bias in estimating θ.The results from the sequences simulated under varying levels of selective constraints are shown in Figs. θ is much higher compared to that under constant growth. For instance, the slopes of the regression lines shown in Fig. θ estimates obtained for large (N = 512) and small (N = 16) sample sizes are also much higher for exponentially growing populations than those under constant growth. For instance, this difference was 3.3 times for the highly constrained (NeS = 2000) exponentially growing populations  follows standard coalescence model and Watterson’s θ clearly captures this. However, when a population grows exponentially, a higher proportion of low frequency variants are created due to faster coalescence events near the tip of the genealogies. This is shown in Fig. θ is expected. However, to observe or to identify the vast majority of low frequency variants a larger sample size is required. In the context of mutations, small sample size underestimates the overall mutations in a population as it misses rare mutations and a higher sample size is needed to observe all of them.In this study we showed that there is an effect of sample size in estimating the fundamental population genetic parameter, ulations –10, 17. θ for the constrained regions of exponentially growing populations is much more severely biased by sample sizes because they are modulated by both demographic and selective forces. Since the human population is known to be under exponential growth, the sample size effects on the estimation of θ for neutral synonymous sites are influenced by the demographic factor alone but estimates for the nonsynonymous sites are modulated by both demographic and selective forces. This is clear in the simulation study, which showed that the sample size bias is influenced only by purifying selection in constant populations. Therefore, the extent of sample size bias for the constrained regions of exponentially growing populations was much higher than that observed for the constrained regions of populations under constant growth. Furthermore, humans have a unique demographic history and it is well known that human populations have undergone an explosive population growth, which resulted in much higher fraction of rare deleterious variants [θN/θS (0.5) observed for large sample sizes . In contrast for constrained regions this difference was 26 % (P < 10−7). As expected, π estimates were similar between large and small sample sizes and this was true for neutral (P = 0.48) as well as constrained sites (P = 0.61). Similar results were observed for African (YRI) and Asian (CHB) populations  population and divided the data into two groups, one with small (16 exomes) and another with large (64 exomes) sample sizes  true values will only be obtained for very large sample sizes. When sample size is small Tajima’s D of the two types of sites are apparently similar. Hence use of small number of samples in this analysis will produce erroneous results due to severe underestimation of θ for constrained sites. This also is true for the results of Fu-Li D test , use of large number of samples results in identifying more deleterious (low frequency) nonsynonymous SNVs, which increases Pn in eqn 5 and thus the value of α is reduced. In contrast, small sample sizes identify fewer nonsynonymous SNVs, which leads to an overestimation of the proportion of adaptive substitutions. The other measure based on MK-test, the Neutrality Index, is underestimated using a small number of samples due to the failure to precisely identify some of the low frequency nonsynonymous SNVs.In the case of MK test based statistics, the proportion of adaptive nonsynonymous substitutions . Using the exon-intron boundaries provided in the reference gene annotations we extracted the protein-coding transcripts from the human-chimp alignment. Using the gene annotations from Ensembl (http://www.ensembl.org/) we retained the longest transcript for each gene. For each gene the divergence at synonymous sites (dS) and nonsynonymous sites (dN) were estimated based on the maximum likelihood method employed in the software PAML [dS (SM/SS) is the number of synonymous substitutions (SM) per synonymous site (SS) in a gene dN (NM/NS) is the number of nonsynonymous substitutions (NM) per nonsynonymous site (NS). In estimating dN or dS, the maximum likelihood method tend to overestimate when the actual divergence is large. To avoid such estimation errors (due to the overcorrection of multiple hits) we excluded the genes for which dN or dS estimate was > 0.8. These filters resulted in 13,454 unique protein-coding genes, which were eventually used for further analysis. The ratio of dN and dS (dN/dS) was used as the proxy for the magnitude of selection pressure on a gene.We obtained the genome data for 1092 humans from GenBank, which was originally generated by the 1000 genome project (phase 1-version 3) . Using tare PAML . While dθ, π, Tajima D, Fu-Li D, Neutrality Index and the proportion of adaptive nonsynonymous substitutions using the following equations.We estimated a number of population genetic parameters such as θ) measures the molecular genetic variation as the population scaled mutation rate using the number of segregating sites as [S is the number of segregating sites, n is the number of sequences and θ and S as the number of segregating sites per site. Nucleotide diversity (k) is the average number of pair-wise nucleotide differences between sequences, which was estimated using the following equation [The Watterson’s estimator  is given by [This is another neutrality test similar to Tajima given by :η is the total number of mutations, ηs is the number of singleton mutations in the sequences and uD and vD are given by the equations in page 701 of Fu and Li [η and ηs respectively as suggested [u and Li . We useduggested .Pn and Ps are number of nonsynonymous and synonymous polymorphisms and Dn and Ds are number of nonsynonymous and synonymous substitutions respectively.MK test uses the ratio of nonsynonymous to synonymous polymorphisms and divergence . Under nNI) and the proportions of adaptive nonsynonymous substitutions (α) are based on the principles of the MK test.The popular statistics used in population genetics namely the Neutrality Index  genomic data are available . α can bNe = 10,000 and a mutation rate of 1 × 10−8 per site per generation was used for the simulation [Ne = 9,210. Then only one population undergoes a large exponential growth phase until it reaches Ne = 512,210. The other population undergoes a constant growth phase and thus its number remains at Ne = 9,210. For modelling constrained site evolution, we used the scaled selection coefficient γ = 2Ns with γ following a gamma distribution, which has a mean of α/β. We fixed α as 0.206 based on previous studies [β to model various magnitudes of selection ranging between γ = 2 to 2000. We performed 1000 replicates, obtained the estimates θ and π and computed the mean values. Since the simulation conducted here was only to compare the θ estimates from different sample sizes changing any parameter (eg. mutation rate) does not affect the end results.We conducted an extensive simulation using the program SFS_CODE , which imulation . We condmulation . This momulation . In the  studies  and vari


Stem cell divisions are either asymmetric—in which one daughter cell remains a stem cell and one does not—or symmetric, in which both daughter cells adopt the same fate, either stem or non-stem. Recent studies show that in many tissues operating under homeostatic conditions stem cell division patterns are strongly biased toward the symmetric outcome, raising the question of whether symmetry confers some benefit. Here, we show that symmetry, via extinction of damaged stem-cell clones, reduces the lifetime risk of accumulating phenotypically silent heritable damage (mutations or aberrant epigenetic changes) in individual stem cells. This effect is greatest in rapidly cycling tissues subject to accelerating rates of damage accumulation over time, a scenario that describes the progression of many cancers. A decrease in the rate of cellular damage accumulation may be an important factor favoring symmetric patterns of stem cell division. Recently, highly symmetric patterns of stem cell division have been observed in a variety of adult mammalian somatic tissues. Here we identify conditions under which this behavior serves as a strategy to protect the organism against mutation accumulation. First, we find that a sufficient number of lifetime stem cell divisions must occur, potentially explaining why stem cell pools with the most symmetric divisions are rapidly cycling. Second, we find that late-occurring mutations must occur rapidly, a scenario known in cancer biology as genetic instability. These findings provide a potential explanation for the observation that cancer risks among large, long-lived organisms fail to rise as expected with lifespan and body size. The accumulation of heritable damage—both mutation and epigenetic change—within individual cells is thought to be a major driver of cancer One way to delay damage accumulation in the absence of phenotypic consequences is to employ a lineage hierarchy in which self-renewing stem cells produce “transit-amplifying” cells that proliferate before differentiating into cells that eventually leave the tissue The “immortal strand” mechanism The observation that many stem cell pools undergo symmetric divisions is interesting, given that tissue homeostasis (constancy of stem and differentiated cell numbers) demands that symmetric renewal events (where one stem cell generates two stem cells) be balanced, on average, by an equal number of symmetric differentiation events . Feedback signals from differentiated cells most likely provide such a matching mechanism We were intrigued by the fact that the somatic tissue with the highest degree of symmetric stem cell division observed to date (close to 100%) is the vertebrate intestinal epithelium When a stem cell undergoes an extinguishing division, it and all of its mutations  become fated to leave the body, suggesting that some of the mutation “flushing” enjoyed by short-lived transit-amplifying cells also accrues to symmetrically-dividing stem cells . SymmetrK loci as a stepwise transition of cells through K stages , whereas for larger pools we simulated a simple branching process without imposed size constraints ; K, the number of loci in which mutations of interest may accumulate; 0u,…,K-1u, the mutation rates for acquiring each of the K mutations; and L, the organism lifetime measured in stem cell cycles. For each set of parameters we determined the fraction of simulations of purely asymmetric division in which at least one stem cell had K mutations—the “asymmetric risk”—as a function of time , because a change in the probability of having a deleterious phenotype (K mutations in at least one stem cell) at a fixed time point (e.g. the end of an organism's reproductive period) is directly connected to the pressures of natural selection at the organism level.Simulation parameters consisted of time see . The “syK-fold mutant stem cell. Later, we also consider cases of many small stem cell pools functioning in parallel , for which much smaller asymmetric risks must be used.Care must be exercised in choosing the time at which PF is evaluated since, with enough time, all risks plateau at 100%. Accordingly, PFs were typically ascertained when the asymmetric risk  lay in the vicinity of 50% , i.e. atN∼60,000 stem cells and the number of accumulated mutations was K = 3. The symmetrically dividing stem cell population clearly displays a significantly reduced risk of arriving at the fully mutant state. K) were chosen at random, subject to the condition that the time (in cell cycles) when the asymmetric risk was close to 50% should fall within a reasonable range of organism lifetimes  in individual simulations. Stage size fluctuated more when divisions were symmetric versus asymmetric ; this isFluctuations in one stage are expected to alter the rate of entry of cells into the next stage, depending upon the size and direction of the fluctuation. As long as upward and downward fluctuations balance—which they usually do—they should have no long-term effect on rates of mutation accumulation, and therefore offer no protection. There is one circumstance under which they will not balance, however, which is when fluctuations extinguish all the mutants in a given stage. In that case that stage must await the entry of a new mutant cell from the previous stage before upward fluctuations can resume; in principle, this effect could slow the rate of mutation accumulation. Consistent with this idea, when we visually examined simulations of symmetrically dividing populations that exhibited high PF, we always observed frequent extinctions of one or more mutant stages .i clone that arises during an organism lifetime , color-coding the data by the observed PF; significant protection only occurs when If stage extinctions are the basis for protection, then the magnitude of protection might reflect the propensity of stages to extinguish before they progress (i.e. acquire a subsequent mutation). In other words, for protected cases we expect the average time for a stage to extinguish to be much smaller than the average time to progress. For stages 1, 2 and 3 in chastic” . Parametchastic” . In Fig.isfying  .What sorts of parameter values give rise to such behavior? We established a simple scaling relation (Section 1.5.2 of 6 stem cells (using a Moran model). 3 cell cycles, and 10−6 per cell cycle, respectively). The grey contour marks parameter combinations for which the asymmetric risk is 50% at the organism lifetime (i.e. the conditions under which risks were compared in u1≫u0) is necessary to achieve significant protection.To gain further insight, we focused on cases of mutation accumulation with just two mutant stages . With laor fast terminal mutation rates favor protection are in fact just two sides of the same coin  whereas ulations , in whiculations . In shors is the fraction of divisions that are symmetric) explains why: new clones likely extinguish so long as 1≪su, i.e. provided the mutation rate is slow compared to the symmetric-division rate—not a particularly stringent condition, even with an elevated mutation rate.So far, we have assessed the protection offered by a purely symmetric pattern of stem cell renewal, yet many tissues  can no longer compete with the protected path (genetic stability gene mutated first). Thus protection conferred by division symmetry persists. It should be noted that, for these calculations, mutations in A were treated as neutral (i.e. not by themselves affecting fitness), which is valid as long as the increase in mutation rate is small enough that the added lifetime burden of subsequent deleterious mutations is inconsequential.One important scenario where mutation rates are not independent is the development of cancer, where alterations at “genetic stability” loci—whether by DNA sequence changes or aberrant epigenetic alterations—elevate mutation rates throughout the genome see . Fig. 5C10 stem cell divisions Mutation accumulation is expected to be particularly acute in the mouse small intestine because its calculated lifetime proliferative output—some 10We show here that observed levels of division symmetry in vertebrate tissues Even when we relax the assumption that damage is phenotypically silent , we find that protection can persist, provided that selection coefficients are  . Recent Others have noted that patterns of division symmetry should have an impact on the stochastic dynamics of cancer stem cells . Accordingly, the acquisition of highly symmetric division patterns may be less about implementing special mechanisms than about not implementing mechanisms required to guarantee asymmetric divisions.Although we have evaluated the effect of mutation accumulation in a single cell type, our results are easily generalized to multi-stage lineages, in which intermediate cell stages (“committed progenitors”) may be modeled as products of stem cells whose rates of differentiation exceed those of renewal . This imbalance by itself enhances the flushing of mutants arising at such stages. Any further propensity for symmetric divisions beyond this minimum level would provide further protection against mutation accumulation, in precisely the same way as described above for stem cells.Recently, Roche et al. argued that the documented lack of an expected inter-species correlation between cancer risk and body size/longevity (“Peto's paradox”) implies that large, long-lived species must have evolved strategies to reduce cancer risk K-fold mutant stem cell appeared or the simulation reached the organism's lifetime. For each parameter set, we generated ∼103successful runs in which the mutant appeared within a lifetime, which was sufficient to estimate the lifetime cumulative risk of mutation accumulation to an accuracy of ∼3% ) lay in a defined range (10%<RA<99.996%). For each such parameter set, we then simulated the asymmetric and symmetric risks  Concordance of “Moran” and “Branching” models used to screen large and small populations, respectively. The lifetime cumulative risk of accumulating two mutations in a symmetric population was computed for a variety of stem-cell population sizes and organism lifetimes under both models (right panels). The models predict the same cumulative risk over most of parameter space but differ significantly in small populations at large lifetimes, where extinctions of the entire stem cell population in the Branching model reduce risk by at least a factor of two (white contour in left panel). (B) Parameter sets comprising the numerical screen of (PDF)Click here for additional data file.Figure S2Analysis of stochastic tunneling and sequential fixation regimes.  A single wild-type stem cell was simulated until either one of its descendants mutated (with probability N = 103 stem cells and mutation rates are 0u = 10−6 and 1u = 10−3. (G–I) Protection vanishes for small secondary mutation rate, 1u≪1/L2. In these panels, population size is N = 103 stem cells and mutation rates are 0u = 10−6 and 1u = 10−8. (G) Simulated (symbols) and theoretical ) cumulative risk.  Typical trajectories that generate a double-mutant stem cell by end of life, 2T<L = 103 cc. In both cases, one of the first few single-mutant lineages to arise from the wild-type background produces a double-mutant stem cell that arises improbably early in its parent single-mutant lineage, 2T≪1u. (J–O) Sequential Fixation Regime. In these panels, population size is N = 10 stem cells and mutation rates are 0u = 10−6, 1u = 10−4. (J) Cumulative risk calculated using simulation (symbols) and Eqs. (S48), (S56) and (S59) (lines).  A double-mutant stem cell typically arises in the first single-mutant stem cell in a purely asymmetric population.  Dynamics in a purely symmetric population. (L) At long times, t≫1/1Nu, single-mutant lineages frequently extinguish before one survives drift, fixes in the population, and then rapidly acquires the next mutation. The time taken to fix, N, and to acquire the second mutation once fixed, 1/1Nu, are negligible compared to the time taken for a single-mutant lineage destined for fixation to arise, 1/0u. (O) At short times, t≪1/1Nu, the time between fixation and mutation cannot be neglected. (M) Symmetric extinctions out-compete fixation events to reduce mutation accumulation risk. (P–R) Accuracy of piecewise analytic formula for PF measured by the ratio of analytically computed PF . Panels A and B were generated under the Branching model, Eq. (S1), whereas panels C–R were generated using the Moran model, Section 2.1 of puted PF  to exactputed PF . Grey co(PDF)Click here for additional data file.Figure S3Unordered “fast” and “slow” mutations. (A–C) A “fast-slow” ordered pathway.  The abundance of Ab stem cells is approximated by its mean value, t, 01/u≪1/1u. (C) Simulated cumulative risk (symbols) is approximated by N = 104 stem cells and mutation rates are 10−2 (“fast”) and 10−6 (“slow”). Time courses are plotted until the first double-mutant stem cell appears in the entire stem cell population.(PDF)Click here for additional data file.Figure S4Clonal extinctions out-compete progression in the intestine. (A) Model of mutation accumulation in the intestine. (B) Typical dynamics showing how various patterns of division generate the triple-mutant stem cell in the un-compartmentalized case. (C) Dynamics in a single crypt of a compartmentalized intestine. The purely asymmetric trajectory (s = 0%) is representative of all trajectories examined whereas the mixed (s = 10%) and purely symmetric (s = 100%) trajectories show the most frequently observed type since all four possible combinations of stochastic tunneling and sequential fixation were observed at appreciable frequencies  and 10−3 (fast).(PDF)Click here for additional data file.Figure S5Protection persists when selection acts on stochastic stages. Protection against ordered accumulation of K = 2 mutations after 1000 stem cell cycles for symmetry fractions s = 100% (A) and 10% (B), calculated by Monte Carlo simulation of the generalized model presented in Section 4.1 of 1-w0w)/0w, where iw is the fitness of stage i (see Section 4.1 of 0u = 10−6 and 1u = 10−3 per locus per stem cell cycle.(PDF)Click here for additional data file.Figure S6“Increasing” mutation rates yield a broad distribution of latencies. Probability distributions of times at which the first double-mutant stem cell arose in a population of N = 104 symmetrically dividing stem cells, from simulations of the discrete-time branching process defined by Eq. (S1) (bars) and from the probability mass function (PDF)Click here for additional data file.Figure S7Protection in the human colon. Cumulative risk of ordered accumulation of K = 4 mutations by ages 30  and 60 , assuming mutation rates increase 1000-fold  or 100-fold  during the course of mutation accumulation from an initial rate of 0u = 5×10−7 per locus per stem cell cycle; see Ref M = 107 crypts,  are (A) 5×10−7; 5×10−7; 5×10−6; 5×10−4; (B) 5×10−7; 5×10−7; 5×10−7; 5×10−4; (C) 5×10−7; 5×10−7; 5×10−5; 5×10−5; (D) 5×10−7; 5×10−7; 5×10−6; 5×10−5.ells Ref  dividing(PDF)Click here for additional data file.Figure S8Symmetry protects even when mutations may occur simultaneously in both daughter stem cells. The more accurate ordered mutation accumulation model presented in Section 6 of (PDF)Click here for additional data file.Table S1Numerical screen of large-population model with non-monotonic mutation rates .(PDF)Click here for additional data file.Table S2Numerical screen of small-population model with non-monotonic mutation rates .(PDF)Click here for additional data file.Table S3Numerical screen of large-population model with “monotonic” mutation rates .(PDF)Click here for additional data file.Text S1Formulation and analysis of mathematical models (contains supporting figure legends).(PDF)Click here for additional data file.


Farmland abandonment takes place across the world due to socio-economic and ecological drivers. In Europe agricultural and environmental policies aim to prevent abandonment and halt ecological succession. Ecological rewilding has been recently proposed as an alternative strategy. We developed a framework to assess opportunities for rewilding across different dimensions of wilderness in Europe. We mapped artificial light, human accessibility based on transport infrastructure, proportion of harvested primary productivity , and deviation from potential natural vegetation in areas projected to be abandoned by 2040. At the continental level, the levels of artificial light were low and the deviation from potential natural vegetation was high in areas of abandonment. The relative importance of wilderness metrics differed regionally and was strongly connected to local environmental and socio-economic contexts. Large areas of projected abandonment were often located in or around Natura 2000 sites. Based on these results, we argue that management should be tailored to restore the aspects of wilderness that are lacking in each region. There are many remaining challenges regarding biodiversity in Europe, but megafauna species are already recovering. To further potentiate large-scale rewilding, Natura 2000 management would need to incorporate rewilding approaches. Our framework can be applied to assessing rewilding opportunities and challenges in other world regions, and our results could guide redirection of subsidies to manage social-ecological systems.Mapeo de Oportunidades y Retos para el Retorno de la Vida SilvestreEl abandono de tierras agrícolas ocurre en todo el mundo debido a factores socio-económicos y ecológicos. En Europa, las políticas ambientales y agrícolas tienen el objetivo de prevenir el abandono y frenar la sucesión ecológica. La reintroducción o el retorno de la vida silvestre (“rewilding”) representa una estrategia alternativa a esto. Desarrollamos un marco de trabajo para evaluar las oportunidades de reintroducción en diferentes dimensiones de naturaleza a lo largo de Europa. Mapeamos la luz artificial, la accesibilidad para humanos con base en la infraestructura de transporte, la proporción de productividad primaria  y la divergencia de vegetación natural potencial en áreas que se proyecta estarán abandonadas para el 2040. A nivel continental, los niveles de luz artificial fueron bajos y la divergencia de vegetación natural potencial fue alta en las áreas de abandono. La importancia relativa de las medidas de naturaleza difirió regionalmente y estuvieron conectadas fuertemente a los contextos ambientales y socio-económicos locales. Las grandes áreas de abandono proyectado estuvieron localizadas frecuentemente en o alrededor de sitios Natura 2000. Con base en estos resultados, argumentamos que el manejo debería ser fabricado para restaurar los aspectos de la naturaleza que son carentes en cada región. Todavía quedan muchos obstáculos con respecto a la biodiversidad en Europa, pero las especies de megafauna ya se están recuperando. Para potenciar aún más la reintroducción a gran escala, el manejo de Natura 2000 necesitaría incorporar estrategias de reintroducción. Nuestro marco de trabajo puede aplicarse a la evaluación de las oportunidades de reintroducción y a los obstáculos en otras regiones del mundo, y nuestros resultados pueden guiar la redirección de los subsidios para manejar los sistemas socio-ecológicos. Since the development of agriculture, large areas have been converted into farmland across the world  and the map developed by Bohn et al. (b).We mapped 4 metrics of wilderness in Europe at a 4 kml et al. . Net harl et al. . The dPNl et al. a and then et al.  based onn et al. . The lign et al.  and datan et al. b.We extracted the values of the metrics at the location of projected abandonment from the values calculated at continental level with a bivariate normal kernel function with a radius of approximately 10 km. We split the raster values for all wilderness metrics across the EU27 into quantiles to calculate the amount of farmland abandonment at different ranges of wilderness. We identified the percentage of abandonment areas that fell within the 10%, 25%, 50%, and 75% highest levels of wilderness for accessibility, pHPP, and dPNV. The division into quantiles of the night light data was less precise due to many ties in the values. Therefore, we used the 16.7%, 33.3%, 50%, and 83.3% of the area with the highest levels of wilderness for night light. Because the night light data set was restricted to areas south of 66°N parallel, the quantiles of all metrics were calculated after clipping each data set to this region. We mapped the overlap between dPNV and pHPP by calculating the difference between the normalized values of the 2 metrics. We calculated the projected abandonment around protected areas by measuring Euclidian distance to the borders of protected areas of IUCN category I and II and to Natura 2000 sites. A detailed description of the data sets and methods is in Supporting Information.These wilderness metrics outline human impacts on biodiversity and ecosystem function. The effects of artificial light are documented for invertebrates  categories. Protected areas of category I (strict nature reserves and wilderness areas) and II  directly address the maintenance and support of natural ecological processes and minimum human intervention Dudley  and therNational systems of protected areas coexist with Natura 2000, the European Union system of protected areas. The Natura 2000 network occupies almost 18% of the EU territory Table, 1992. MThe speed at which different dimensions of wilderness will respond to farmland abandonment varies. The pHPP will respond almost immediately  because Other dimensions of wilderness may also have a delayed response to abandonment. Artificial light may decrease soon after abandonment, but due to the presence of public light infrastructure and the development of new activities in the landscape, such as tourism  and brown bears (Ursus arctos) (Enserink & Vogel Bison bonasus) .Rewilding will often result in the increase of forest cover, leading to many specialist species of open areas becoming less abundant and more spatially restricted. Common farmland birds and grassland butterflies are already becoming less abundant . The management of protected areas can also be used to facilitate rewilding in areas of high abandonment. In marginal agricultural regions where agricultural subsidies are politically difficult to remove, subsidies can be shifted to rewilding measures such as the creation of no-hunting zones and wildlife habitat (Merckx & Pereira Conservation management in the face of anthropogenic change represents an issue of global importance. Soulé  argues t


Whistler-mode emissions are important electromagnetic waves pervasive in the Earth’s magnetosphere, where they continuously remove or energize electrons trapped by the geomagnetic field, controlling radiation hazards to satellites and astronauts and the upper-atmosphere ionization or chemical composition. Here, we report an analysis of 10-year Cluster data, statistically evaluating the full wave energy budget in the Earth’s magnetosphere, revealing that a significant fraction of the energy corresponds to hitherto generally neglected very oblique waves. Such waves, with 10 times smaller magnetic power than parallel waves, typically have similar total energy. Moreover, they carry up to 80% of the wave energy involved in wave–particle resonant interactions. It implies that electron heating and precipitation into the atmosphere may have been significantly under/over-valued in past studies considering only conventional quasi-parallel waves. Very oblique waves may turn out to be a crucial agent of energy redistribution in the Earth’s radiation belts, controlled by solar activity. Whistler-mode waves regulate trapped electrons in the magnetosphere, but an accurate determination of their energy budget has remained elusive. This study presents a full analysis of their magnetic and electric field contributions and finds that a large amount of energy is stored in oblique waves. Since whistler-mode waves regulate fluxes of trapped electrons134671012141516No global study on the basis of satellite measurements since then has led to a real revision of this conventional picture. Although some studies161718W of whistler-mode waves is determined by wave electric E and magnetic B field vectors through a complex relationship involving the tensor of absolute permittivity  and refractive index N=kc/ω (with k is the wave vector and c is the velocity of light).To compare the impact of oblique and parallel waves in the formation and evolution of keV to MeV electron fluxes in the inner magnetosphere, a reasonable approach consists in first estimating the energy density of both wave populations. Such a global survey is presented in W clearly shows that the proportion of very oblique waves, propagating near the resonance cone angle , is generally similar to or even larger than the proportion of quasi-parallel waves for L=3 to 6 during moderate geomagnetic activity (defined by index Kp<3) on the dayside. On the nightside or during more disturbed periods such that Kp>3 , the amount of very oblique waves is sensibly reduced. The latter reduction stems probably from the presence of higher fluxes of hot (∼100 eV to 1 keV) plasmasheet electrons injected in the midnight region during disturbed periods1920The present results therefore challenge the conventional assumption of predominantly quasi-parallel whistler-mode waves in the outer radiation belt. A big, missing slice of the wave energy appears to be stored in very oblique waves—which are mainly made up of electrostatic energy1124Φ, which depends also on cyclotron or Landau resonance harmonic number and on wave field components, provides the exact portion of wave energy interacting resonantly with particles132=B2Φ2/N2. 2 of resonant waves plotted in the same manner as the wave energy density previously, using measured wave field components from Cluster. θ-values (between the Gendrin and resonance cone angles) is 5–10 times larger than for parallel waves throughout the region L=3–6. Hence, very oblique waves are expected to play a crucial role in the scattering of electrons in this region of space.What are the consequences of the large energy of oblique waves on the dynamics of energetic particles? As their name suggests, wave–particle resonant interactions are controlled not only by wave intensity, but also by the actual efficiency of the resonant interactions. Waves must be in resonance with particles, implying that a certain relationship must be fulfilled between particle energy, pitch angle, wave frequency and obliquity  are considered  around −50 nT.The remarkable effectiveness of the resonant interaction of very oblique waves with keV to MeV electrons can modify particle scattering and energization processes substantially in the radiation belts as compared with conventional theoretical estimates obtained for quasi-parallel waves alone. This effect should be most pronounced during moderately disturbed periods where oblique waves are more ubiquitous. To estimate the effects of oblique waves on resonant electron scattering during the course of a geomagnetic storm, we use here parameterizations of lower-band chorus wave magnetic intensity and τL of energetic electrons during the course of these two storms has been calculated numerically for various energies ranging from 100 eV to 1 MeV. τL with Dst. Such strong variations can be explained by the combination of two main effects: (1) lifetimes increase when wave intensity decreases (both with and without oblique waves)10Φ is significantly stronger for very oblique waves than for quasi-parallel waves over a very wide energy range , only very oblique waves are still able to resonantly scatter electrons towards the loss-cone.When considering a realistic wave-normal angle distribution, the first clear consequence of the additional presence of very oblique waves is a general reduction of lifetimes during the storm. Most remarkably, however, such a reduction is much less significant during the early recovery period corresponding to θ is a key factor for accurately modelling the evolution of relativistic as well as low-energy electron fluxes under the influence of resonant wave–particle interactions. As noted above, this θ distribution is tightly controlled by the density and temperature of hot electrons. The large energy stored in very oblique waves can be readily tapped by sufficiently hot electrons newly injected from the outer magnetosphere and lead to their further heating via Landau damping. It, therefore, represents an accelerating factor of change for this important population1878Such results definitely show that a precise knowledge of the actual distribution of wave energy as a function of propagation angle DEEτL of the energy diffusion rate DEE and lifetime, because a longer τL leaves more time for electron acceleration to proceed1827Dst) is almost fully compensated in this factor DEEτL by the inverse dependence of the lifetime on the wave intensity. Since DEE varies also much more weakly with wave obliquity than τLBeside determining electron precipitations into the atmosphere, whistler-mode waves are also responsible for the rapid energization of ∼10 keV to 1 MeV electrons to multi-MeVs in the radiation belts during geomagnetic storms35Dst<−75 nT followed by a quick return to Dst>−20 nT should take advantage of high parallel wave intensity and weak overall losses to strongly energize electrons. Conversely, another storm (close to type #2) with a shorter initial period at Dst<−75 nT followed by a much slower recovery back to Dst>−20 nT should generally involve much stronger electron losses induced by larger amounts of very oblique waves during the early recovery phase  profiles. This effect allows to separate precipitation-dominated storms with a fast early recovery further slowing down, from acceleration-dominated storms with a slow initial recovery later on speeding up.Excluding oblique waves from consideration would actually make the dimensionless energization factor The surprisingly high level of very oblique wave energy discovered in 910W of whistler-mode waves in the Earth’s magnetosphere is determined by wave electric field E and magnetic field B vectors through the relation:The energy density E* is the conjugate vector to E, ω is the wave frequency and B. One gets W=B2(1+WE)/8π where WE depends only on the wave characteristics: its frequency ω, wave-normal angle θ (which defines the wave obliquity with respect to the geomagnetic field), and refractive index N=kc/ω (where k is the wave vector). W steeply increases with N, which is itself a rapidly growing function of θ. The refractive index N (as well as θ) can be determined either solely from full three-component wave magnetic field measurements on a given spacecraft, or else by complete wave magnetic and electric field measurements. The dominant contributions to the wave energy distribution can be further assessed on the basis of either method. However, wave electric field measurements on Cluster satellites are often noisy, at least much more than magnetic field measurements, limiting their use in practice to some case studies. Therefore, we have chosen to resort to the just-discussed method of determination of the full wave energy density on the basis of measurements of the wave magnetic components alone. Nevertheless, the accuracy and reliability of this method must first be demonstrated.where N values obtained by the two methods in a series of Cluster observations of chorus waves displayed in ω/Ωc0∼0.35. Waves can be considered as very oblique when θ is comprised between the Gendrin angle θg≈arccos(2ω/Ωc0) (which corresponds to wave propagation at a group velocity independent of frequencyθr∼arccos(2ω/Ωc0) ) consists of such oblique waves. The large ratio W/WB≫1 shows that most of the energy density then comes from the wave electric field. More importantly, evaluations of the wave refractive index N from three-component measurements of the sole wave magnetic field yield values very similar to calculations making use of both magnetic and electric field components, attesting the reliability of the former method (compare panels (e) and (f)). The discrepancy does not exceed 25% on average, showing that this method can be safely used for evaluating the wave energy density.To this aim, we have compared the crucial Φ , pitch angle α and wave obliquity θ. As a result, one gets a normalized estimate Θ2=Φ2B2/N2 of the resonant wave energyprovides the necessary relation between particle energy (Lorentz factor Φ (averaged over latitude) have been performed as a function of wave propagation angle θ and geomagnetic activity index Dst, for various electron energies ranging from 100 eV to 1 MeV. Here, as well as for ωm/Ωc0∼0.35 and frequency width Δω/Ωc0∼0.2 of lower-band chorus wavespe/Ωce=5 corresponding to L∼5. Dst>−60 nT), wave–particle coupling Φ is clearly stronger for very oblique waves than for quasi-parallel waves over a wide energy range. For a given level of wave intensity, the available range of variation of the wave–particle coupling efficiency Φ at small equatorial pitch angles (near the loss-cone where particles are precipitated in the atmosphere) is so large that it could presumably explain any observed fluctuations of electron flux by fluctuations of the wave obliquity only and associated variations of electron scattering. In addition to the increase of Φ for a given resonance, the number of such contributing resonances can moreover increase 10-fold for oblique waves gω(ω) where gθ and gω are normalized distributions of θ and wave frequency. To calculate the gθ normalization, one should determine resonant k and ω for given particle pitch angle and energy. Then, an integration over θ must be performed. The upper limit of this integration is determined by the maximum value of the refractive index NMax. The latter is imposed by the presence of both thermal effects in the dispersion relation and Landau damping by 100–500 eV suprathermal electrons of oblique waves near the resonance cone angle19NMax∼120 to 300 for lower-band chorus waves from low to high latitudes during periods of quiet to moderately disturbed geomagnetic activity, with NMax varying as the inverse of the frequency ω and increasing with latitudeNMax∼min corresponding to a predominant effect of Landau damping.The efficiency of charged particles resonant interaction with waves is determined by diffusion rates proportional to the weighting factor How to cite this article: Artemyev, A. V. et al. Wave energy budget analysis in the Earth’s radiation belts uncovers a missing energy. Nat. Commun. 6:7143 doi: 10.1038/ncomms8143 (2015).Supplementary Figures 1-2


BCL2 translocation were ultimately classified as nodal marginal zone lymphoma (NMZL). This study set out to define recurrent morphological features of these cases. Thirty-three low-grade B cell lymphomas without a BCL2 rearrangement were studied for recurrent morphological features. These features were then applied on 20 randomly selected cases to verify if these criteria are able to distinguish between lymphomas with and without a BCL2 rearrangement, assigning them to one of five categories ranging from “certain FL” to “certain NMZL.” Highly recurrent morphological features were noted in the lymphomas without a BCL2 rearrangement, which were strongly overlapping with the morphological features of NMZL. All six cases that were assigned to the category of certainly FL or most likely FL indeed harbored a BCL2 rearrangement, whereas all 12 cases assigned to the category of most likely NMZL or certain NMZL had no BCL2 break. Of the two cases in the ambiguous category, one had received a final diagnosis of FL and the other of NMZL. This study raises the hypothesis that a subset of low-grade B cell lymphomas with a follicular growth pattern but without a BCL2 translocation actually represents NMZL. This is at present difficult to prove, because no gold standard is available to differentiate between NMZL and FL without a BCL2 rearrangement, so further investigations are needed.In our consultation practice, it was noted that many cases that were considered to represent follicular lymphoma (FL) without a  BCL2 in up to 90 % of cases, resulting in BCL2 upregulation. It usually has a follicular growth pattern with expression of germinal center markers . In most cases, a diagnosis of FL is rather straightforward and can be made by morphology and immunohistochemistry alone. However, in the absence of characteristic morphology, typical immunophenotype, or in the absence of a BCL2 translocation, the diagnosis becomes more difficult. An important difficulty results from the morphological and immunohistochemical overlap between FL and nodal marginal zone lymphoma (NMZL) with a nodular/follicular growth pattern. NMZL is currently defined as “a primary nodal B cell neoplasm that morphologically resembles lymph nodes involved by MZL of extranodal or splenic types but without evidence of extranodal or splenic disease” [Follicular lymphoma (FL) is a low-grade B cell lymphoma that has a translocation involving disease” . The folBCL2 translocation, but display BCL2-positive follicles at low power, were often considered to represent FL. However, careful study of BCL2 at high power in these cases often showed negative cells as a sign of follicular colonization, suggesting a diagnosis of NMZL rather than FL. In this study, we set out to substantiate the hypothesis that a subset of lymphomas that are initially classified as FL without BCL2 translocation (translocation-negative FL), actually represent NMZL.In routine diagnostic practice, it was noted that lymphomas that lack a n = 21) and consultation cases (n = 35), were retrieved from the archive of the Department of Pathology of the Radboud University Medical Center . These were selected based on the fact that they had been tested for a BCL2 translocation by fluorescence in situ hybridization (FISH) between 2004 and 2010. During this period, FISH was used in diagnostic practice in all cases with a differential diagnostic consideration of FL and NMZL.Fifty-six primary nodal B cell lymphomas, both from in-house patients  tissue, dried, deparaffinized, rehydrated, and subjected to heat-mediated antigen retrieval. Specific retrieval procedures for each antigen are indicated in Table BCL2 DNA probe, Dako, Y5407, Glostrup, Denmark; BCL6 DNA probe, Dako, Y5408, Glostrup Denmark). After 5 min of denaturation at 82 °C, hybridization was performed overnight at 45 °C in a Dako S2451 hybridizer (Dako). Sections were then dehydrated, dried, and mounted using medium containing DAPI as a nuclear counterstain . Stained sections were assessed with a Leica DM 4000B fluorescence microscope.FISH was performed on 4-μm sections from FFPE tissue that were dried, deparaffinized, rehydrated, and heated for 10 min in sodium citrate (pH 6.0). Slides were rinsed in 0.01 M hydrochloric acid before 15 min of pepsin digestion. After three rinses of 0.01 M hydrochloric acid and 5 min of fixation in 1 % formaldehyde in PBS, sections were dehydrated and dried after which the probe was added , with subsequent confirmation by one of the hematopathologists . FISH for BCL6 was scored by a single observer (MvdB). Split signals in 20 % of cells or more were regarded as positive, although in most positive cases, the amount of positive cells was far above 20 %.For BCL2 translocation, firstly (immuno)morphological criteria were set for the diagnosis of NMZL .Based on cases lacking a BCL2 translocation) by four hematopathologists  at a multi-headed microscope with all histochemical and immunohistochemical stainings but without clinical information or knowledge of BCL2 translocation status. These cases were scored on a 5-point scale: certainly FL (1), most likely FL (2), ambiguous (3), most likely NMZL (4), or certainly NMZL (5).Subsequently, these criteria were applied on 20 of the 53 cases  effBCL2 rearrangements in this series and the morphological features of NMZL is in line with our hypothesis that a subset of lymphomas that are initially classified as translocation-negative FL actually have features of NMZL.The strong overlap between the morphological features in lymphomas lacking BCL2 rearrangement, they were applied to 20 cases that were selected randomly from the total of 53 cases. These 20 cases were reviewed without knowledge of clinical features or the presence of a BCL2 translocation and assigned to one of five categories ranging from “certain FL” to “certain NMZL,” as indicated in Table BCL2 rearrangement, whereas all 12 cases assigned to the category of most likely NMZL or certain NMZL had no BCL2 break. Of the two cases in the ambiguous category, one had received a final diagnosis of FL and the other of NMZL.To verify if the criteria defined above were able to recognize lymphomas without a BCL2 translocation-negative case was ultimately classified as follicular lymphoma. Because the morphological criteria for the absence of a BCL2 rearrangement showed strong overlap with the morphological criteria for NMZL, and because the large majority of lymphomas without a BCL2 rearrangement were ultimately diagnosed as NMZL, these results suggest that a subset of lymphomas that are classified as translocation-negative FL are actually NMZLs. Although the results are suggestive, establishing a final proof for this hypothesis is currently very difficult if not impossible, because specific criteria for NMZL are lacking. The fact that some lymphomas without a BCL2 rearrangement showed expression of the germinal center markers CD10 and/or BCL6 is an argument in favor of a diagnosis of FL. However, the morphological features outlined above that were present in these lymphomas argue against this. Also, expression of CD10 and BCL6 has been reported in NMZL [As indicated in Table  in NMZL , 6. In oBCL2 rearrangement might have a rearrangement involving BCL6. We studied 22 of the cases without a BCL2 rearrangement for the presence of a rearrangement involving BCL6, using a dual-color break-apart probe locating to BCL6. In 5 out of 22 cases (23 %), a rearrangement of BCL6 was identified. Of these 5 cases with a BCL6 rearrangement, one was weakly positive for CD10, one was positive for CD10 and weakly positive for BCL6, one was weakly positive for BCL6, and two were negative for both CD10 and BCL6. In summary, 3 out of 5 (60 %) cases with a BCL6 rearrangement showed expression either CD10 or BCL6 versus 2 out of 12 (17 %) cases without a BCL6 rearrangement. Although these results suggest some enrichment for germinal center marker expression in cases with a BCL6 rearrangement, the difference between these groups was not statistically significant (p = 0.117 with Fisher’s exact test). This finding is of potential interest, but a definitive conclusion would require the study of additional cases.FLs without a BCL2 translocation with FISH. In general, BCL2 FISH is not performed on cases that are considered to be straightforward FL by morphology and immunohistochemistry, resulting in a bias toward atypical cases in our study. This also means that the group of “straightforward” FLs, that was not included in this study, might contain BCL2 translocation-negative cases, and we therefore cannot make any conclusions on the frequency of BCL2 translocation-negative FL. However, our study does suggest that a proportion of lymphomas regarded as BCL2 translocation-negative FL are in fact NMZLs. Because NMZL is often a diagnosis of exclusion, this suggestion is currently hard to prove. However, previous studies suggest that BCL2 translocation-negative FLs are genetically different from FLs with a BCL2 translocation and have at least some features of NMZL. One study reported specific features of MUM1-positive and CD10-negative follicular lymphomas, including a frequent lack of BCL2 translocations [BCL2 translocations suggest that this series might very well include NMZLs. A comparative genomic hybridization and gene expression study in BCL2 translocation-negative FLs demonstrated less frequent expression of CD10 and enriched NF-κB signatures, both features of NMZL [BCL2 translocation [Cases were included in this study when they had been tested for a ocations . The com of NMZL . This sulocation .To finally resolve these issues, a positive marker for NMZL is highly needed. In addition to proposed new immunohistochemical markers for NMZL –11, bettBCL2 translocation. These criteria largely overlapped with the features of NMZL, and we propose that a subset of lymphomas that is currently often classified as FL without a BCL2 translocation is actually better classified as NMZL.In conclusion, we have established immunomorphological characteristics of a group of low-grade B cell lymphomas that lacked a


N-acetylglucosamine-1-phosphotransferase is an α2β2γ2 hexameric enzyme that catalyzes the synthesis of the mannose 6-phosphate targeting signal on lysosomal hydrolases. Mutations in the α/β subunit precursor gene cause the severe lysosomal storage disorder mucolipidosis II (ML II) or the more moderate mucolipidosis III alpha/beta (ML III α/β), while mutations in the γ subunit gene cause the mildest disorder, mucolipidosis III gamma (ML III γ). Here we report neurologic consequences of mouse models of ML II and ML III γ. The ML II mice have a total loss of acid hydrolase phosphorylation, which results in depletion of acid hydrolases in mesenchymal-derived cells. The ML III γ mice retain partial phosphorylation. However, in both cases, total brain extracts have normal or near normal activity of many acid hydrolases reflecting mannose 6-phosphate-independent lysosomal targeting pathways. While behavioral deficits occur in both models, the onset of these changes occurs sooner and the severity is greater in the ML II mice. The ML II mice undergo progressive neurodegeneration with neuronal loss, astrocytosis, microgliosis and Purkinje cell depletion which was evident at 4 months whereas ML III γ mice have only mild to moderate astrocytosis and microgliosis at 12 months. Both models accumulate the ganglioside GM2, but only ML II mice accumulate fucosylated glycans. We conclude that in spite of active mannose 6-phosphate-independent targeting pathways in the brain, there are cell types that require at least partial phosphorylation function to avoid lysosomal dysfunction and the associated neurodegeneration and behavioral impairments.UDP-GlcNAc:lysosomal enzyme  N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase), an α2β2γ2 hexameric enzyme that catalyzes the first step in the synthesis of the mannose 6-phosphate (Man-6-P) targeting signal on lysosomal hydrolases which is required for their targeting to lysosomes in many cell types GNPTAB and GNPTG, which give rise to the α/β subunit precursor and the γ subunit, respectively. The α/β subunits contain the catalytic function of the enzyme, whereas the γ subunit enhances the phosphorylation of a subset of lysosomal hydrolases GNPTAB give rise to ML II (MIM# 252500) or ML III α/β (MIM# 252600), whereas mutations in GNPTG cause ML III γ (MIM# 252605) −/−)(Gnptab and ML III γ −/−) and mucolipidosis III (ML III) are autosomal recessive lysosomal storage diseases caused by a deficiency of UDP-GlcNAc:lysosomal enzyme −/−Gnptab−/−Gnptg mice −/−Gnptab and −/−Gnptg mice are maintained in a C57Bl/6-129S5/SvEvBrd background. Mice were genotyped as previously described to identify WT or knockout mice for use in this study All mice were housed in a barrier facility at 24°C on a fixed 12-hour light and 12-hour dark cycle and were fed rodent chow #5001  ad libitum. All protocols involving the use of animals were in compliance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals and approved by the Animal Studies Committee in the Division of Comparative Medicine at Washington University School of Medicine in St. Louis (Protocol #20130010). Mice were housed in a barrier facility maintained under standards meeting federal, state and local guidelines and under the supervision of licensed veterinarians. We have previously reported the generation and initial characterization of the −/−Gnptab and −/−Gnptg mice were evaluated on a 1h locomotor activity test, a battery of sensorimotor measures and the rotarod test (in that order) to assess possible age-related impairments in motor/sensorimotor functions. In the cohort of −/−Gnptab mice and their WT littermate controls, the sensorimotor battery was conducted 1 day after the activity test with the rotarod test being conducted 1 day after the sensorimotor battery when the mice were evaluated at 1 month of age. The same was true for the testing conducted at 4–5 months of age except that the 1h activity test was not conducted at this age. The same sequence of tests was used for the cohort of −/−Gnptg and their strain and age-matched WT control mice although the times intervening between tests were different as were the ages at testing (4–6 and 12–14 months). In this latter cohort, the sensorimotor battery was conducted within 48 h of the activity test and the rotarod test was conducted 12–14 days after the activity test.Based on preliminary behavioral observations, Locomotor activity was evaluated in the mice using transparent (47.6 × 25.4 × 20.6 Cm high) polystyrene enclosures and computerized photobeam instrumentation as previously described −/−Gnptab group was deleted from the data set because it appeared to have difficulty moving one of its forepaws and another mouse was deleted from the WT control group because it climbed over different parts of the apparatus making it impossible to record valid times on the rod.Motor coordination and balance were further studied using procedures similar to our previously published methods Analysis of variance (ANOVA) models were used to analyze the behavioral data. Repeated measures (rm) ANOVA models containing two between-subjects variables (Genotype and Sex) and one within-subjects (repeated measures) variable  were used to analyze the ambulatory activity and vertical rearing data. A similar model was used to analyze the rotarod data although two within-subjects variables  were included to analyze the constant speed and accelerating rotarod data. Typically, one-way ANOVA models were used to analyze differences between groups for the other activity variables and measures in the sensorimotor battery. The Huynh-Feldt adjustment of α levels was utilized for all within-subjects effects containing more than two levels to protect against violations of sphericity/compound symmetry assumptions underlying rmANOVA models. Pairwise comparisons were conducted following relevant, significant overall ANOVA effects, which were subjected to Bonferroni correction when appropriate, although planned comparisons were also conducted., diluted to 1∶1500 and applied for 1 h at RT; anti-LAMP-1 , diluted 1∶400 and applied for 1 h at RT; anti-LC3B , diluted 1∶2000 and applied for 2 h at RT; anti-MAC2  diluted 1∶20,000 and applied for 30 min at RT; anti-NFP  diluted 1∶40 and applied for 2 hours at RT; and anti-ubiquitin  diluted 1∶800 and applied for 15 min at RT. Binding of primary antibodies  was visualized using rabbit or rodent polymeric horseradish peroxidase method  on a LabVision stainer . Binding of MAC2 and NFP was visualized using streptavidin horseradish peroxidase (Thermo Fisher), with 3,3′-diaminobenzidine as the chromogenic substrate and a light hematoxylin counterstain. Binding of primary antibody for ubiquitin was visualized using Bond Polymer Refine Red Detection Kit .Mice of the indicated ages were anesthetized and perfused through the heart with 10% neutral buffered formalin  and fixed for 4 days in 10% neutral buffered formalin  at RT. The brain and spinal cords of perfused mice were left in place within the skull and vertebral columns, respectively, and post-fixed by immersion in 10% buffered Formalin for at least 24 h before being decalcified in formic acid . Coronal sections of brain and spinal cord were embedded in paraffin, sectioned at 4 µm, mounted on positively charged glass slides , and dried in a 60°C oven for 20 min. Antigen retrieval on tissue sections was completed in a pressure cooker  in citrate buffer, pH 6 for 15 min at 110°C, followed by incubation with the primary antibodies. The primary antibodies used in this study included: anti-Iba1  diluted to 1∶250 and applied for 2 h at RT; anti-GFAP Mice of ages indicated were anesthetized and perfused through the heart with PBS, brain tissues isolated and frozen at -80°C until used. Sample preparation and quantification of sphingolipid species by tandem mass spectrometry (LC-MS/MS) was performed as previously described g for 10 min. The supernatant was isolated and stored at −20°C. Amino sugar content was determined by the Reissig assay N-Acetylglucosamine  used as a standard. Each sample was normalized to protein concentration and data plotted as nmol amino sugars/mg total protein.Mice of ages indicated were anesthetized and perfused through the heart with PBS, brain tissues isolated and frozen at −80°C until used. Whole brains were homogenized in water, sonicated and an aliquot was saved to determine the protein concentration. The remaining lysate was brought to 4N Hcl , heated in a sand bath at 100°C for 4 h, lyophilized, re-suspended in 1mL water, and insoluble material spun down at 25,000X2SO4 , heated in a sand bath at 100°C for two h, neutralized with barium hydroxide , and the volume adjusted to 1 mL with water. After 30 min at 4°C, samples were spun down at 25,000Xg for 10 min and the supernatant isolated and stored at −20°C. Fucose content was determined by the Dische assay Mice of ages indicated were anesthetized and perfused through the heart with PBS, brain tissues isolated and frozen at −80°C until used. Whole brains were homogenized in water, sonicated and an aliquot saved for protein concentration. The remaining lysate was adjusted to pH 1.0 with 1M Hg. An aliquot of the supernatant was reserved for protein determination and fucosidase activity of the supernatant was determined by fluorometic enzyme assay as previously described Mice were anesthetized and perfused through the heart with PBS, brain tissues isolated and frozen at −80°C until used. Whole brains were homogenized in PBS containing 1% Triton-X  and protease inhibitors , placed on ice for 15 min, sonicated and centrifuged for 10 min at 25,000X−/−Gnptab mice, but not the −/−Gnptg mice, developed a hind limb clasping response between 4–6 months of age, suggestive of brain and/or spinal cord abnormalities −/−Gnptab mice to determine whether more subtle motor/sensorimotor impairments were present during the juvenile period long before the clasping response became apparent during early adulthood. The same cohort of −/−Gnptab mice was retested at 4–5 months to assess the progression of functional impairment. We also focused on tests that did not rely heavily on visual function since the −/−Gnptab mice have progressive retinal degeneration starting at 2–3 months with complete photoreceptor degeneration by 10 months −/−Gnptg mice were assessed on these same tests as young adults (4–6 months) and in middle age (12–14 months).Before starting the behavioral tests to investigate the impact of complete or partial acid hydrolase Man-6-P deficiency on motor and sensorimotor functions, we noted that the −/−Gnptab mice performed similarly to WT control mice in terms of general ambulatory activity, vertical rearing frequency, distance traveled in the periphery, and measures of emotionality including distance traveled, time spent, and entries made into a central zone in the test field (data not shown). In contrast, the −/−Gnptab mice exhibited impairment on 3/7 measures within the battery of sensorimotor tests. Specifically, significant genotype effects derived from ANOVAs conducted on these data revealed the following performance deficits in the −/−Gnptab mice: 1) −/−Gnptab mice remained on the elevated platform for a significantly shorter period of time (p  =  0.026); 2) the −/−Gnptab mice took significantly longer to climb down the pole (p  =  0.0007); and 3) the −/−Gnptab mice were able to remain upside down on the inverted screen for significantly less time . No significant effects involving genotype were found for the ledge  and genotype x sex interaction (p  =  0.041) were also revealed by the ANOVA conducted on the ledge data , while differences were not significant between the females of each group  and sex (p  =  0.026) effects along with a significant genotype by trials interaction (p  =  0.0004) for the stationary rod condition  showed that this depended on the test session, with the greatest differences found during session 1 (−/−Gnptab mice declined with age such that robust (and significant) differences were found relative to the WT group for every trial across test sessions for each component of the rotarod procedure , with impaired performance being found for the male −/−Gnptg mice compared to the males from the WT group (p  =  0.0003), while the performance of the females from the two groups did not differ significantly . Similar to the activity results from the earlier age, no differences were observed between groups with regard to distance traveled in the periphery, or the emotionality variables (not shown). Although the −/−Gnptg mice showed a strong trend towards inferior performance at 12–14 months compared to WT littermate control mice with regard to the 60° inclined screen test, the differences were not significant like they were at the earlier time point  and significant genotype x sessions (p  =  0.002) and genotype x sex x trials (p  =  0.004) interaction (−/−Gnptg mice for the constant speed and accelerating rotarod components (p  =  0.017). Additional contrasts revealed that the female −/−Gnptg mice were significantly impaired on average across trials and sessions relative to the WT females (p  =  0.0001). Performance deficits were also observed in the male −/−Gnptg mice versus the male WT controls on average across trials and sessions, although the effect was smaller compared to that of the female groups (p  =  0.022).The o trials . Additio trial 1 . Gnptg−/ rotarod  where throd test  where aneraction . When theraction  althoughmponents ;Table S3−/−Gnptab and −/−Gnptg mice to evaluate these tissues and then added periodic acid-Schiff (PAS) and immunohistochemistry (IHC) staining methods to more fully characterize the CNS lesions. Tissues were obtained from WT and −/−Gnptab mice at 4, 6, 10 and 12 months of age and from 12-month-old −/−Gnptg mice. At 4 months, no notable changes were seen in the CNS of WT or −/−Gnptab mice by routine H&E staining, but by 6 months of age, −/−Gnptab mice showed widely dispersed swollen dystrophic axons and neurites in multiple foci within the brain, the afferent spinal nerves and the dorsal horn grey matter of the spinal cord. The severity of clinical signs and the extent of associated histopathological lesions in −/−Gnptab mice continued to increase over time. In the 10- and 12-month-old −/−Gnptab mice dystrophic axons/neurites and spheroids were widely dispersed in the brainstem and dorsal grey matter of the spinal cord. These changes were particularly severe in the medial piriform (olfactory) cortex  stained sections of brain and spinal cord from WT, ) cortex , especia) cortex . In bothrebellum . Staininell loss .−/−Gnptab mice, −/−Gnptg mice which do not display any abnormal clasping behavior did not develop any of the swollen dystrophic axons/neurites or white tract lesions that were widespread in the brains and spinal cords of age-matched −/−Gnptab mice. In fact, no CNS lesions of any type were detected in −/−Gnptg mice when using only H&E stained sections of the brain and spinal cord. However, as shown later, special staining methods used to study the tissues in more detail did reveal mild multifocal lesions in −/−Gnptg mice, but these were much more extensive in −/−Gnptab mice.In marked contrast to the widespread pathological findings observed in −/−Gnptab mice stained by the PAS method revealed widely dispersed PAS positive granular staining in macrophages and microglia throughout most areas of brain and spinal cord , ubiquitin (UBQ), and glial fibrillary acidic protein (GFAP) in order to detect microglia, ubiquinated proteins, and astrocytes respectively. These staining methods served to highlight the true extent and progression of lesions in −/−Gnptab and −/−Gnptg mice. For example, staining for Iba1 showed no evidence of increased labeling in 12-month-old WT mice  and dorsal sensory grey matter of the spinal cord, there was no significant labeling of either reactive microglia or neurons for ubiquitin. In contrast, there was only a minimal increase in ubiquitin staining in 12-month-old −/−Gnptg mice  staining in the white tracts in both brain and spinal cord. WT mice were essentially negative for galectin-3 staining  staining in these areas, indicative of lysosomes distended by undigested material. The LAMP-1 labeling that is evident in many neurons of the −/−Gnptab mice  was present in only a few dystrophic axons in the white tracts of the cerebellum and spinal cord of −/−Gnptab mice while the abundant large swollen neurites in the superficial laminae (layers 1 to 3) of the dorsal horn grey matter were consistently NFP-negative  whereas the −/−Gnptg mice had a significant decrease in α-L-fucosidase activity (p = 0.0059)  . One exp−/−Gnptab mice revealed an accumulation of GM2 in the cerebrum and brainstem while −/−Gnptg mice had accumulation of GM2 in the cerebrum, cerebellum and brainstem  may contribute to this. Although differences in the experimental designs of the −/−Gnptab and −/−Gnptg studies make it inappropriate to directly compare the results between the two models, it appears that the −/−Gnptg mice generally showed less severe sensorimotor deficits suggesting more mildly disrupted sensorimotor functions than the −/−Gnptab strain and possibly less extensive neuropathology.In this study we used mouse models of ML II and ML III γ to compare and contrast the course of neurologic and behavioral changes associated with either complete or partial loss of the Man-6-P-targeting signal. We previously reported that both −/−Gnptab mice were associated with progressive neurodegeneration characterized by microgliosis, astrocytosis, Purkinje cell loss, widely dispersed swollen dystrophic axons and neurites filled with granular material along with the accumulation of PAS- and ubiquitin-positive granules in various cell types. The changes were widespread, involving the spinal cord, brainstem, cerebrum and cerebellum. The dystrophic neurites and axons in the spinal cord of −/−Gnptab mice were concentrated in the dorsal horn and extended into the dorsal columns, suggesting that sensory neurons and pathways were affected. Lesions in the ventral and lateral regions of the spinal cord primarily involved the white tracts and were characterized by vacuolization of axons and hypertrophy of associated LC3B-positive oligodendrocytes. Some motor neurons contained increased amounts of PAS-positive material (lipofuscin?). Taken together, these findings suggest that subsets of both sensory and motor neurons were affected in −/−Gnptab mice, in contrast to the almost normal histopathology of the −/−Gnptg mice.The more extreme behavioral disturbances in the −/−Gnptab mice. Lesions involving both white tracts and neurons were widespread but not diffuse in both the brain and spinal cord, indicating that cell subsets in the CNS differ in their susceptibility to lysosomal enzyme deficiencies. Similar lesions were not present in gamma KO mice.The hypertrophy and increased LAMP-1 staining of oligodendrocytes in areas of white tract vacuolization suggests that oligodendrocytes are particularly susceptible to the lysosomal enzyme deficiencies that occur in −/−Gnptab cerebellum showed a striking pattern with the cellular processes of reactive Bergmann glial cells appearing as prominent parasagittal bands. Notably, these bands of reactive GFAP-positive Bergmann glia correlated directly with areas of Purkinje cell degeneration and loss, a pattern also seen in the brains of humans and mice with other lysosomal storage disorders, such as Juvenile neuronal ceroid lipofuscinosis The immunohistochemical staining for GFAP in the It is difficult to compare these findings to those reported for ML II patients since only a few autopsies have been reported and most of them did not include IHC stains. However, for both ML II patients and mice, macroscopic evaluation of the brain and spinal cord revealed little to no abnormalities, but the microscopic examination reported here showed evidence of storage material, vacuolization, Purkinje cell loss and positive PAS staining in neurons.−/−Gnptab mice, the −/−Gnptg mice showed impaired performance on only a few of the behavioral tests and these abnormalities did not become evident until these mice were much older compared to −/−Gnptab mice (4-6 months versus 1 month), although more precise information regarding the age of onset of these behavioral deficits requires testing at earlier ages in the −/−Gnptg mice. Further, changes in the −/−Gnptg mouse brain were slower to develop and much milder in extent and severity than those seen in the −/−Gnptab mice. This correlates well with the finding that ML III γ patients have normal to mildly impaired neurologic functioning In contrast to the −/−Gnptab and −/−Gnptg mice is normal or only moderately decreased and even increased in a few instances −/−Gnptab mice, consistent with defective lysosomal function. PAS positive material and increased fucose-containing glycans were found in the brains of older −/−Gnptab mice but not −/−Gnptg mice whereas both types of mutant mice showed accumulation of GM2 in their cerebrum and brainstem. However, it must be noted that secondary accumulation of GM2 has been reported in a number of lysosomal storage diseases −/−Gnptab mice also had an accumulation of ceramide in all three regions of the brain. Since accumulation of gangliosides has been associated with inflammation and neuronal degeneration −/−Gnptab and −/−Gnptg mice. However, it seems unlikely that this ganglioside accumulation is the predominant factor in the development of the CNS pathology and behavioral alterations seen in the −/−Gnptab mice as GM2 accumulates to similar levels in the −/−Gnptab mice and the more mildly affected −/−Gnptg mice. Since we did not analyze individual subpopulations of cells in the brain, we cannot exclude the possibility that the GM2 accumulation is happening in different cells types or regions of the brain in the two models thus influencing the degree or type of impairment. However, a more likely explanation is the accumulation of different materials or different amounts of undigested material in the CNS of −/−Gnptab mice versus the −/−Gnptg mice.It is interesting that in spite of finding that the activity of a number of acid hydrolases in total brain extracts of the Gnptab containing a mutation originally identified in ML II patients, c.3028insCGnptab−/−Gnptab mice survive to 40 weeks and then either die or have to be euthanized due to disease progression by 60 weeks (data not shown). In contrast, the c.3082insCGnptab mice have a steady decline in survival starting at birth with ∼50% of the mice dead prior to 40 weeks and only a few surviving up to 64 weeks c.3082insCGnptab mice. This abnormality has also not been reported in ML II patient autopsies. The c.3082insCGnptab mice also showed an accumulation of cholesterol in the Purkinje and granule layer of the cerebellum based on filipin staining −/−Gnptab or −/−Gnptg mice. This difference may be due to the methods used to detect cholesterol since filipin staining can detect alterations in the subcellular distribution of cholesterol that don't necessarily involve an increase in the total content of this compound. Finally, we have previously reported that the levels of LC3-II, LAMP-2 and ubiquitinated proteins in the brains of −/−Gnptab mice were unchanged as determined by western blotting c.3082insCGnptab mice, which showed increased LC3-II and p62 using this technique. However, using IHC we did detect increased staining of ubiquitin-positive granules in white tracts, especially in the corpus callosum and cerebellum, and the dorsal sensory gray matter of the spinal cord at 6 months of age with considerable progression at 12 months of age.While this work was in progress, Kollmann et al. −/−Gnptab mice were generated through an insertional mutagenesis strategy −/−Gnptab mice had no detectable Man-6-P phosphorylation (less than 2% in these assays) whereas the same acid hydrolases of WT mice had 41-82% phosphorylation −/−Gnptab mice in C57BL/6-129SvEvBrd versus c.3082insCGnptab mice in C57BL/6-129SvJ) or secondary effects of residual C-terminally truncated α/β precursor protein in the c.3082insCGnptab mice such as activation of the endoplasmic-reticulum-associated protein degradation (ERAD) system. Regardless of these differences in the two mouse models of ML II, the finding that both exhibit severe neurodegeneration confirms that the Man-6-P independent sorting pathway is unable to fully support adequate lysosomal function in the CNS. The more benign course of the −/−Gnptg mice shows that the partial phosphorylation of acid hydrolases is effective in decreasing the lysosomal dysfunction and lessening the associated neurodegeneration and behavioral changes.The modest differences in the two models may be a result of several factors. One possibility is that since the et. al.,  A novel mouse model of a patient mucolipidosis II mutation recapitulates disease pathology. The Journal of biological chemistry, in press. Published on August 8, 2014) reported a new mouse model of ML II, characterized by growth retardation, skeletal and facial abnormalities and reduced life span. These mice exhibited impaired motor function and psychomotor retardation. Histological analysis showed progressive degeneration in the cerebellum. While many of the features are similar to those found in our mutant mice, our mice lack the craniofacial defects found in the new model and the very early onset of death.While this manuscript was in revision, Paton Table S1−/−Gnptab mouse that had an injured paw and one WT mouse that continually climbed onto the apparatus rather than remaining on the rod were dropped from the analyses of the rotarod data.Note that one (PDF)Click here for additional data file.Table S2Note that one WT mouse died between the 4–6 months old and 12–14 months old tests. Abbreviation: Incl.  =  Inclined(PDF)Click here for additional data file.Table S3Significant ANOVA effects involving genotype (Geno) and sex variables from the rotarod test in 4–6 and 12–14 months old Gnptg−/− and WT mice.(PDF)Click here for additional data file.


P < 0.05) but no effect was observed for mean power output (MPO) (P > 0.05). Handgrip (HG) strength was also found to be significantly lower following the upper body exercise. These results demonstrate that the upper body is meaningfully involved in the generation of leg power during intense cycling.It has been reported previously that the upper body musculature is continually active during high intensity cycle ergometry. The aim of this study was to examine the effects of prior upper body exercise on subsequent Wingate (WAnT) performance. Eleven recreationally active males  completed two trials in a randomised order. In one trial participants completed 2 × 30 s WAnT tests (WAnT1 and WAnT2) with a 6 min recovery period; in the other trial, this protocol was preceded with 4 sets of biceps curls to induce localised arm fatigue. Prior upper body exercise was found to have a statistically significant detrimental effect on peak power output (PPO) during WAnT1 ( P < 0.05).High intensity cycle ergometry has been widely employed in sport and exercise science research to assess indices of muscular performance , 2. Amon−] were elevated, via arm-crank exercise, and dynamic performance during two 30 s WAnT was assessed. It was found that prior arm exercise was related to a decline in PPO during the second WAnT with the authors suggesting that the resulting elevated [La−] caused an increased uptake in La− and H+ by the inactive leg muscles, leading to an overall performance decrement. Karlsson et al. (1975) [−] and [H+].The effects of prior upper body exercise on subsequent cycling performance have previously been examined . In this. (1975)  have als− per se does not directly cause muscle fatigue, a rise in other metabolic by-products such as inorganic phosphate (Pi) [i is likely to play a major role in muscular fatigue during high intensity exercise. Potential mechanisms whereby high [Pi] can impair contractile function thus affecting muscle force production include hindering crossbridge transition to the strongly bound high force state; reducing myofibrillar calcium (Ca2+) sensitivity; increasing the opening probability of the sarcoplasmic reticulum (SR) Ca2+ release channels; inhibiting Ca2+ uptake by the SR; and precipitating with the Ca2+ in the SR, so decreasing the amount of Ca2+ available for release  and handgrip strength were obtained at predetermined time points throughout the protocols  trial, participants were required to perform two maximal 30 s sprints (WAnT1 and WAnT2) on a cycle ergometer with a standard handlebar grip, separated by 6 min passive recovery. In the other arm fatigue (ARF) trial, bicep curls were completed prior to WAnT1 and WAnT2. Blood  were taken from the fingertip using standard lancets and capillary tubes. Samples were taken at rest, 3 min following warm-up and 3 and 5 min following each exercise bout  for selected statistically significant t- and F-ratios were also established. These calculations were based on Cohen's (d) classification of a small (0.2 ≤ d < 0.5), moderate (0.5 < d < 0.8), and large (d ≥ 0.8) ES  × 2 [TEST]) ANOVAs were performed to detect main effect and interaction effects. For the blood lactate data, a balanced design was only evident for the postexercise data (3 and 5 min following exercise). For these data, data were analyzed by repeated measures three-way (2 [TRIAL] × 2 [TEST] × 2 [TIME]) ANOVA. For HG data, a balanced design was evident when using the post-warm-up data for the NOF trial and the post-arm fatigue test data for the ARF trial as the preexercise data. Preexercise was then compared to postexercise (1 min WAnT1 versus 1 min WAnT2) using a repeated measures two-way (2 [TRIAL] × 3 [TIME]) ANOVA. For all data variables, specific contrasts were performed to test for mean differences for significant main or contrast effects. Isolated paired mean differences outside of the balanced ANOVA designs were assessed by a paired  0.8) ES . SignifiF1,8 = 19.5, P < 0.01, rdf = 0.84), relative PP0 , and relative MPO  ; however, no significant interaction was found (P > 0.05). No significant differences were found in FI (%) for TEST or TRIAL (P > 0.05) .There was a significant main effect for the TEST for each of PP0 (W:  = 0.91) , revealiF1,8 = 15.8, P < 0.01, rdf = 0.81) and a significant TRIAL × TEST interaction . The three-way interaction effect of TRIAL × TIME × TEST revealed a trend toward statistical significance . Blood lactate was significantly higher 5 min after the arm fatigue exercise in the ARF trial compared to post-warm-up in the NOF trial (P = 0.001). Based on the main effect and interaction ANOVA results, blood lactate was significantly higher throughout the recovery after WAnT2 than WAnT1. In addition, the interaction effect was caused by a net decrease in blood lactate between 3 and 5 min of recovery in WAnT2, whereas blood lactate continued to increase between 3 and 5 min of recovery after WAnT1.The blood lactate response to each of the experimental protocols is displayed in P = 0.004,  rdf = 0.81). Therefore, HG strength was significantly lower at all time points for the ARF versus NOF trial.The HG strength response to each of the experimental protocols is displayed in r = 0.29,  P = 0.38). However, in the ARF where bicep curls were completed before WAnT1, there was a meaningful trend of a positive linear relationship  between PPO from WAnT2 and HG strength after WAnT2  in WAnT1 in the ARF protocol compared to the NOF protocol which suggests that in the absence of leg fatigue, the strength of the grip upon the handlebar may be influencing PPO. This is in agreement with the findings of Baker and colleagues (2001)  through prior arm exercise (arm ergometry) and determine the effects of this on subsequent high intensity cycle ergometry performance. In contrast to our results they found PPO to be significantly lower during the second sprint following prior arm exercise but similarly there were nonsignificant changes in MPO between the two protocols. It is difficult to directly compare results with Bogdanis et al. [In a previous study by Bogdanis and colleagues , a very s et al.  due to dAs with most maximal cycle ergometer tests, prior to the load being applied there was an initial high rpm. This inertia was not accounted for in the calculation; therefore the considerable energy which had already been accumulated before the 30 s test may have resulted in a possible overcalculation of PPO and MPO .All participants were physically active and accustomed to high intensity exercise. However no physiological fitness testing was undertaken prior to data collection which meant there was no control over the exercise capacity of each individual. This is an extraneous variable which may have led variation in power profiles observed among participants which was not related to the exercise protocol.In conclusion, this investigation has shown that prior fatiguing upper body exercise has a statistically significant detrimental effect on PPO during the first of two WAnTs. This can be related to a number of factors, including the decrease in HG strength following the upper body exercise suggesting the upper body is less able to help overcome the high resistive loads, confirming results of previous investigations which suggest that the upper body is crucial in achieving an optimum PPO. It was also found that MPO was able to be maintained, which could be explained by prior intense exercise resulting in faster