The size distribution of gene families in a broad range of genomes is well approximated by a generalized Pareto function. Evolution of ensembles of gene families can be described with Birth, Death, and Innovation Models (BDIMs). Analysis of the properties of different versions of BDIMs has the potential of revealing important features of genome evolution.In this work, we extend our previous analysis of stochastic BDIMs.In addition to the previously examined rational BDIMs, we introduce potentially more realistic logistic BDIMs, in which birth/death rates are limited for the largest families, and show that their properties are similar to those of models that include no such limitation. We show that the mean time required for the formation of the largest gene families detected in eukaryotic genomes is limited by the mean number of duplications per gene and does not increase indefinitely with the model degree. Instead, this time reaches a minimum value, which corresponds to a non-linear rational BDIM with the degree of approximately 2.7. Even for this BDIM, the mean time of the largest family formation is orders of magnitude greater than any realistic estimates based on the timescale of life's evolution. We employed the embedding chains technique to estimate the expected number of elementary evolutionary events (gene duplications and deletions) preceding the formation of gene families of the observed size and found that the mean number of events exceeds the family size by orders of magnitude, suggesting a highly dynamic process of genome evolution. The variance of the time required for the formation of the largest families was found to be extremely large, with the coefficient of variation >> 1. This indicates that some gene families might grow much faster than the mean rate such that the minimal time required for family formation is more relevant for a realistic representation of genome evolution than the mean time. We determined this minimal time using Monte Carlo simulations of family growth from an ensemble of simultaneously evolving singletons. In these simulations, the time elapsed before the formation of the largest family was much shorter than the estimated mean time and was compatible with the timescale of evolution of eukaryotes.The analysis of stochastic BDIMs presented here shows that non-linear versions of such models can well approximate not only the size distribution of gene families but also the dynamics of their formation during genome evolution. The fact that only higher degree BDIMs are compatible with the observed characteristics of genome evolution suggests that the growth of gene families is self-accelerating, which might reflect differential selective pressure acting on different genes. An extremely broad variety of phenomena in physics, biology, and the social sphere is described by power law distributions. The power laws apply to the distribution of the number of links between documents in the Internet, the population of towns, the number of species that become extinct within a year, the number of sexual and other contacts between people, and numerous other quantities -4. In thP(i) ≈ ciγ -where P(i) is the frequency of nodes with exactly i connections or sets with exactly i members, γ is a parameter which typically assumes values between 1 and 3, and c is a normalization constant. Obviously, in double-logarithmic coordinates, the plot of P as a function of i is close to a straight line with a negative slope. Recently, it has been shown that the distributions of several genome-related quantities are best described by the so-called generalized Pareto function: P(i) = c(i + a)γ -where γ > 0, a are parameters  / [Γ(1 + a) Γ (1 + a2)], andwhere nd order balanced, then pi ~ iρ-2. Note that the asymptotic behavior frequencies pi do not depend on free coefficients r2 and q2 in (2.3), but only on θ and r1 - q1, although the constant c2 could depend on the free coefficients r2, q2.Thus, if the quadratic BDIM is 2nd order balanced, then pi ~ iγ-, .λi = λ(i + a1)/(i + a2)2, δi = δ(i + b1)/(i + b2)2.The rational BDIMs can describe a substantially wider class of birth and death rates compared to polynomial models. In particular, birth rate can have a maximum at some specific value of family size and then decrease with further growth of the size, e.g., as shown in Fig. N, should be finite, although intrinsic features that could determine the value of N so far have not been considered . Under the BDIMs described above, birth rate grows monotonically as the family size increases from 1 to N and then abruptly drops to 0 . However, this behavior is an arbitrary simplification of the model and hardly can reflect the actual process of genome evolution.Evidently, the number of size classes of protein families, In population dynamics models, the finiteness of a population size typically results from the "saturation type" of growth: the growth rate tends to 0 as the population size tends to the maximal possible value . It seeλ (i) = c3(c1 + i)(N-i), δ(i) = c3i(c2-i), ck > 0, k = 1,2,3, c2 >N.i, namely, 1 <<i <<N and N >> 1.This model produces log-normal and log-series distributions; with the appropriate values of parameters, power low distributions of frequencies also appear, but only for intermediate values of We have shown previously  that theλ*i = λig(i), δ*i = δig(i-1)     (2.4)g(i), i = 0,...N, is a positive function, g(0) = 1, results in a BDIM with the same ergodic distribution of the family sizes as the original one. In particular, modifications of a linear BDIM with g(i) = (i + 1)d-1 or g(i) = (i + 1)d-1(1 - i/(N + c)) define, respectively, wide classes of rational or logistic BDIMs with the same stationary distribution as the original linear BDIM, but with manifestly different dynamic properties.where n before reaching state 0 from an initial state i> 0 is given by formula (A.2.2). In terms of BDIM (1.1), this means that the probability of formation of a family of size n starting from a family of size i before getting to extinction is given by (A.2.2).In is known  that theThe random birth-and-death process (1.1) certainly visits state 0 in the course of time; this means that any domain family will eventually get extinct . Below we compute the mean time to extinction of a family of the given size for different versions of BDIM; the mean time to extinction of the largest family in the given genome is of particular interest.S(n)=inf{t:X(t) = 0|X(0)=n} the time to the first passage of state 0 from the initial state n; S(n) is a random variable for each n. The mean time to extinction of the family of initial size n, E(S(n)), is given by the general formula A.3.2.Let us denote nd order balanced BDIM, the probability that a singleton expands to a family of size n before dying, P(1) has the power asymptotics for large n (A.2.5). The values of probabilities P(1) for different species are shown in Table -4 - 10-5. The mean time to extinction, E(S(n)), can be calculated using the relation E(S(n)) = 1/λE(1)n, where E(1)n, the mean time to extinction expressed in the 1/λ time units, is given by formula A.3.3  has,n hasnd Var(S(n)) for the linear 2nd order balanced BDIM is Var(S(n)) = 1/λ2W(1)n, where W(1)n can be calculated using the formula (A.3.7). The plot of the coefficient of variation s(1)n = (W(1)n)1/2/E(1)n versus n for different species is shown in Fig. The variance of extinction time The stochastic behavior of the system and its characteristics also can be investigated within the broader framework of rational BDIMs. We will examine models represented as transformed linear BDIM (2.1), withλi = λ(i + a)(i + 1)d-1, δi = λ(i + b)id-1,     (3.1)d ≥ 1 is the model degree. Let us recall that Theorem 1  ~ iγ-. The power γ of this distribution is completely determined by the degree d and the coefficients at id-1. Thus, the model (1.1), (3.1) is representative of all rational BDIMs of the degree d with a given power asymptotic (γ = b - a + 1) of the stationary distribution. Besides, according to Proposition 1, this distribution for model (3.1) is exactly the same as for the corresponding linear model with λi = λ (i + a), δi = λ (i + b), which was studied in detail in [12].where ndix see ) shows We applied formula (A.2.6) g(i) = (i + 1)d-1, to calculate the probability of formation of a family of the given size from a singleton before getting to extinction for the BDIM of degree d, Pd). For example, the probabilities P(2) and P(3) for the quadratic and cubic BDIMs, respectively, are given by this formula with g(i) = i + 1 and g(i) = (i + 1)2, respectively. Figures P(2) and P(3) on the family size n for different species. The dependence of the probability Pd) of the formation of the largest family on the model degree is shown in Fig. with ,n) and P ford is calculated using the formula (A.3.4)  where The mean time to extinction for the rational BDIM (1.1), (3.1) with a fixed E*n is the mean time to extinction in the 1/λ time units. Figures E(2)n and E(3)n on n for the quadratic and cubic BDIMs, respectively. Fig. Ed))= 1/λ2Wd)((n), d = 2, 3 for the quadratic and cubic BDIMs, and the coefficient of variation sd)(n = (Wd)(n)1/2/Ed). The results are shown in Figs. Here Figures En and E(i + 1)d-1(1 - i/(N + c)), δi = δ (i + b)id-1(1 - (i - 1)/(N + c)).     (3.2)c as saturation boundary. The shape of λi essentially depends on the value of c , (3.2) is a transformation (2.9) of the linear BDIM using the function:i) = (i + 1)d-1(1 - i/(N + c), c = const ≥ 0.     (3.3)g(i + 1)d-1, δi = δ (i + b)id-1, but with small c, we can observe some new effects when the family size approaches N.The stationary distribution of family size frequencies for the logistic model (1.1), (3.2) is exactly the same as that for corresponding linear BDIM but the stochastic properties are different and close to the rational models, and essentially depend on the boundary g(i) is given by (3.3). The dependence of this probability on the model degree d under a fixed large value of the boundary c~N is similar to that for the corresponding rational models but differs under a small c; Fig. c = 1.The probability of formation of a family of a given size from a singleton before getting to extinction for the logistic BDIM is calculated using the general formula (A.2.6) where the function Ed). Fig. T = inf{t: X(t) = n|X(0) = j} the time to the first passage of state n from the initial state j; T is a random variable for each j, n. The mean time to the first passage for BDIM (1.1), m = E), can be calculated using the formula m = m0 + m1. Here the term m0 is the mean time elapsed before the system leaves the 0 state for the last time, and the term m1 is the mean time of formation of a family of size n from a singleton after its last "resurrection" (see formulas (A.4.1) for details). Below we examine only the mean family formation time from an essential singleton (model (1.3)).Let us denote n from a singleton for different species [M(1) = 1/λ M(1)n, where M(1)n, the mean formation time in 1/λ units is calculated using the formula (A.4.6)Previously, we determined the mean time of formation of a family of size  species . For theλ time units to years is considered in s.6 of the Mathematical Appendix [see E) in years is calculated using the formula (A.6.4) and the current empirical estimates of the gene duplication rate [E) for different species are shown in Fig. The transition from the 1/ndix see . The meion rate . Plots omean time of formation of a family of size n for different species, the question arises how accurately is the time T of the first random passage through the threshold n predicted by the mean value. To address this problem, we estimated the variance of the time of family formation, Var) using the general formulas (A.5.2) for model (1.1) and (A.5.3) for model (1.3), respectively. For the linear BDIM, the variance of the formation time for a family of size n from an essential singleton, V(1)n, is given by the formula (A.5.5). A more important and informative characteristic, which is independent on the model parameter λ, is the coefficient of variation, which is equal to . The coefficient of variation of the formation time of a family of size n from a singleton, σd)(n = (Vd)(n)1/2/Md) for the BDIM of degree d is the most relevant value. The plots of σ(1)n versus n for the linear model and for different species are shown in Fig. Once we computed the rdu ≈ 2 × 10-8 duplications/gene/year [λ units, we obtain M(1) ~ 1013 - 1014 yrs, a completely unrealistic time estimate. The mean family formation times given by the linear BDIM would become realistic only if the recent analyses underestimated the gene duplication rate by a factor of ~104, which does not seem plausible. Thus, the linear BDIM cannot provide an adequate description of genome evolution, at least when only the mean time of family formation is considered. The variance of the family formation time is extremely large (the coefficient of variation is ~102), and, accordingly, large deviations from the mean time, more to two orders of magnitude, are possible. However, even taking this into account, the family formation times predicted by the linear BDIM are far longer than the time allotted for life's evolution of earth. In the next section, we consider non-linear, higher order models that have the potential to yield shorter mean times of family formation.The coefficients of variation were very large for all species  to exprn for the polynomial BDIMs is E) = 1/λ M*n where M*n, the mean time of formation in 1/λ units can be calculated using the formulas (A.4.9)The mean time of formation of families from an essential singleton (or after the last "resurrection" of a family) depending on the family size Fig. n can be calculated using the formula (A.5.6), with g(j)=j+1 for the quadratic BDIM and g(j) = (j + 1)2 for the cubic BDIM, respectively. The dependence of the coefficient of variation σ(2)n = (V(2))1/2/M(2) on the family size for the quadratic BDIM is shown in Fig. The variance of formation time of a family of the size Although the variance of family formation times for the quadratic BDIM is approximately 5 orders of magnitude less than that for the linear BDIM, the values of the coefficient of variation for quadratic BDIM are about 1.3–1.5 times greater than those for the linear BDIM. Thus, the actual formation time for the largest family could differ from the mean value by several orders of magnitude with a high probability. Figures under the same value of the parameter λ [11 yrs. This value is close to the minimum possible time of family formation that can be calculated using the duplication rate estimates of Lynch and Conery [We have shown previously that the cubic model shows extremely high evolution rate comparatively with the linear and even quadratic models ameter λ . On the d Conery  and non-n from a singleton under a fixed model degree d, Md), for the rational BDIM (1.1),(3.1), is calculated using the formula (A.4.9). A comparison of the mean time of formation and extinction for rational BDIMs reveals an interesting property of non-linear BDIMs: for any given family size n, there exists such a model degree that the times of family formation and extinction are equal ,.1, is cad results in indefinite decrease of the family formation time expressed in 1/λ time units , does not decrease indefinitely, but has a minimum at the model degree between 2 and 3  e units  and (A.5.6). Although the variance of formation times for the quadratic and, especially, for the cubic BDIM is several orders of magnitude less than that for the linear BDIM, the coefficients of variation for both formation and extinction time increase with the model degree Table . These cλ units) of a family of size n from an essential singleton for the logistic BDIM (1.3), (3.2) under fixed d is calculated using formula (A.4.9). Fig. Md), on the family size n for different model degrees d under the fixed saturation boundary c = 1, and Fig. λ. However, when this inner model parameter is excluded and the mean time of family formation is expressed in years according to formula (A.6.5), then we again face a restriction that does not allow indefinite shortening of the family formation time, Td)} instead of the original BDIM. The embedding chain {Yn} for a particular BDIM is a random walk with discrete time on the same set of states and transition probabilities pi,i+1 = βi = λi/(λi + δi), pi,i-1 = μi = δi/(λi + δi) and pij = 0 for all other cases .i to the state i+1 (or i-1) corresponds to the duplication (or deletion) of a domain in a family of size i. The only difference between the original birth-and-death process and the embedding chain is that the sojourn time for the embedding chain is equal to 1 for any state i instead of 1/(λi + δi). The ratio βi/μi (= λi/δi) characterizes the trend of family evolution from the state i, i.e., is the family more likely to grow or to shrink; for a symmetric random walk, βi/μi = 1 for all i. The dependence of the ratio βi/μi on i for different rational and logistic embedded chains is shown in Figures βi/μi ≈ 1 for large i; for the logistic models, βi/μi ≈ 1 for 0 <<i <<N . Thus, the behavior of the embedding chain is similar to the behavior of the symmetric random walk in the corresponding subsets of states. Informally, the plots in Figures The transition from the state fn, is computed using formulas (A.7.5)-(A.7.7). The plots in Figures fn on the family size for different species for the linear and quadratic models, respectively. The mean number of elementary events before the extinction of a family of the given size, en, is computed using formulas (A.7.13)-(A.7.15) and Figures The mean number of elementary events before the formation of a family of the given size, ulas A.7.3-, and the optimum values of d was lower by ~0.5 , it is reasonable to view these unique genes as resulting from innovation. For genomes to maintain equilibrium, the combined rates of duplication and innovation over the entire ensemble of gene families should equal the rate of gene loss, at least when averaged over long time spans. The observed distribution of family sizes, which asymptotically tends to a power law, dictates a much more specific connection between the gene birth and death rates, namely, the second order balance. It should be noted that this form of balance does not amount to particularly fine tuning of the gene birth and death rates. The only requirement is that these rates tend to the same value when the family size tends to infinity according to the condition (1.5). In contrast, for small families, the rates may substantially differ, without significantly changing the shape of equilibrium distribution.Here and in the previous publications ,43,50, wThe incentive to examine BDIMs in detail stems from at least two fundamental questions: i) are the above elementary evolutionary mechanisms sufficient to account for the empirically observed characteristics of genomes, ii) what is the contribution of natural selection to the general, quantifiable features of genomes, such as the size distribution of gene families. The analysis of BDIMs starts to provide some answers, albeit preliminary ones. The critical observation made in the course of BDIM analysis was that different versions of these models could be readily distinguished on the basis of goodness of fit to the empirical data. This being the case, we found that the simplest possible model, in which all paralogs are considered independent, is incompatible with the data. Thus, turning to the first of the above questions, we had to conclude that, in addition to the three elementary processes, "something else" was required to model genome evolution. This "something" is the dependence or "interaction" between gene family members which results in self-accelerating family growth. In order to account for the observed stationary distribution of family sizes, it is sufficient to introduce a very weak dependence as embodied in the linear BDIM. However, when we switched from the deterministic to the stochastic version of BDIMs, which provide for the possibility of analysis of the dynamics of the systems evolution, we found that evolution under the linear BDIM was much too slow to account for the emergence of the large families of paralogs found in all genomes during the time of life's evolution. Only higher order BDIMs, with degrees between 2 and 3, i.e., with "strong interactions" between family members were found to provide for sufficiently fast evolution to be compatible with the real biological timescale.Obviously, these findings beg the question: what is the nature of the mysterious "interactions" between paralogs? Although, on some occasions, paralogous protein do form physical complexes or interact functionally, the situation when such interaction does not exist is much more common. Therefore, the "interactions" in our models should not be perceived literally. This brings us to the second of the above major problems. BDIMs do not explicitly include the notion of selection. However, the simplest interpretation of the virtual interactions implied by the higher order BDIMs seems to be that these reflect differential tendencies of genes to form paralogous families of different sizes depending on the intensity of selection. Recent studies have shown that evolutionary fixation of gene duplications is linked to the evolutionary rates of genes. Specifically, duplications of slowly evolving genes, i.e., those that are subject to stronger purifying selection, are fixed more often ,55. The In this work, we extended our analysis of stochastic Birth, Death and Innovation Models (BDIMs) of gene family evolution and showed that:• the behavior of logistic BDIMs models, in which birth/death rates are limited for the largest families, is essentially the same as that of previously investigated BDIMs that included no such limitationd ≈ 2.7, the mean time of the largest family formation is orders of magnitude greater than any realistic estimates based on the timescale of life's evolution;• the mean time required for the growth of large families is limited by the overall number of duplications and does not increase indefinitely with the increase of the model degree but instead passes through a minimum; even under the best-case scenario, which corresponds to a non-linear rational BDIM with • using the embedding chains technique, we estimated the expected number of elementary evolutionary events (gene duplications and deletions) preceding the formation of gene families of the observed size; the mean number of events exceeds the family size by orders of magnitude, suggesting a highly dynamic process of genome evolution;• the variance of the time required for the formation of the largest families is large (coefficient of variation >> 1), which means that some families might grow much faster than the mean rate; thus, the minimal time required for family formation is more relevant for a realistic representation of genome evolution than the mean time;d ≈ 2.2).• Monte Carlo simulations of family growth from an ensemble of simultaneously evolving singletons show that the time elapsed before the formation of the largest family was much shorter than the estimated mean time and approached realistic values (2.2 ± 0.5 Ga for the non-linear rational BDIM with GPK developed most of the mathematical formalism and wrote the draft of the mathematical part of the manuscript; YIW performed the imitation modeling and wrote the draft of the corresponding part of the manuscript; FSB derived some of the mathematical statements; EVK contributed to the inception of the work and the formulation of the models, gave the biological interpretation of the results, wrote the background and discussion sections and extensively edited the entire manuscript.This additional file includes proofs of some of the mathematical statements contained in the main text as well as accessory mathematical formulations.Click here for file


C. elegans. Feeding of this RNAi library to the standard wild-type laboratory strain Bristol N2 detected phenotypes for approximately 10% of the corresponding genes. To increase the number of genes for which a loss-of-function phenotype can be detected, we undertook a genome-wide RNAi screen using the rrf-3 mutant strain, which we found to be hypersensitive to RNAi. Feeding of the RNAi library to rrf-3 mutants resulted in additional loss-of-function phenotypes for 393 genes, increasing the number of genes with a phenotype by 23%. These additional phenotypes are distributed over different phenotypic classes. We also studied interexperimental variability in RNAi results and found persistent levels of false negatives. In addition, we used the RNAi phenotypes obtained with the genome-wide screens to systematically clone seven existing genetic mutants with visible phenotypes. The genome-wide RNAi screen using rrf-3 significantly increased the functional data on the C. elegans genome. The resulting dataset will be valuable in conjunction with other functional genomics approaches, as well as in other model organisms.RNA-mediated interference (RNAi) is a method to inhibit gene function by introduction of double-stranded RNA (dsRNA). Recently, an RNAi library was constructed that consists of bacterial clones expressing dsRNA, corresponding to nearly 90% of the 19,427 predicted genes of  The screen suggested functions for 393 genes for which no RNAi-mediated phenotype was known. The comparison with similar screens in worms has general implications for RNAi experiments Caenorhabditis elegans was constructed (C. elegans using this RNAi library or other techniques (RNA interference (RNAi) is targeted gene silencing via double-stranded RNA (dsRNA); a gene is inactivated by specific breakdown of the mRNA . It is astructed . Upon festructed . Other gchniques . These srrf-3, a putative RNA-directed RNA polymerase (RdRP), resulted in increased sensitivity to RNAi . To explore the variability of global RNAi screens, we performed the rrf-3 screen twice for Chromosome I and carried out a Chromosome I screen with wild-type. These were cross-compared and also compared to the results of the wild-type screen of rrf-3 consistently allowed detection of more phenotypes than wild-type. In addition, we found that there is a significant screen-to-screenvariability (10%–30%).We have conducted a genome-wide RNAi screen using the rrf-3 and found RNAi phenotypes for 456 bacterial clones. We compared these data to those obtained by rrf-3 . These data show that large-scale RNAi screens done within the same laboratory and by the same investigators also give variable results. A few examples of variable RNAi results are shown in It is possible that different laboratories or investigators have slightly different results. However, when we compare the results that we obtained with two independent screens of Chromosome I using e second D. Among In conclusion, we find that RNAi results from different laboratories and from experiments done in the same laboratory vary from 10% to 30%. This appears to be due to a high frequency of false negatives in each RNAi screen, even when the same method is used in the same laboratory.rrf-3 strain, we next screened the complete RNAi library with rrf-3 mutant animals. We obtained results for 16,401 clones and detected phenotypes for 2,079 (12.7%). Of these, we identified phenotypes for 625 clones for which no phenotype was reported in the generating phenotypes in both screens  and F27C8.6 (a member of the esterase-like protein family), we detected sterile progeny (Stp) and embryonic lethality (Emb), respectively; these were also found by gpc-2, hlh-8, and unc-84. This suggests that many of the unconfirmed phenotypes reflect true gene functions.Some of the RNAi phenotypes only found with rrf-3, we first assayed the rate of false positives in the total dataset . In the assay used by rrf-3 data is similarly low (0 of 152 genes).To validate the results obtained using rrf-3 data were confirmed by refeeding only if there was no phenotype detected in the N2 screens by rrf-3 and the N2 screens, we used the Chromosome I datasets, where phenotypes were confirmed independently for the two strains. Of 75 genetic loci on Chromosome I, rrf-3 (rrf-3 dataset (excluding the 202 unconfirmed phenotypes), we detected the published phenotype for 54% of 397 selected loci, compared to 52% for N2 , we detected 31 using rrf-3, compared to 33 for N2. Thus, detection of essential genes was similar in the two strains, but detection of postembryonic phenotypes was improved with rrf-3. Finally, for the whole genome using rrf-3, we reproducibly detected the published phenotypes for 34 genetic mutants for which no RNAi phenotype was reported in the N2 screens . By comparison, published phenotypes were detected for 23 loci only with N2   see . We concrrf-3 dataset is the high number of clones where a slow or arrested growth (Gro/Lva) defect was induced, without associated embryonic lethality or sterility. Overall, 619 clones induced a Gro/Lva defect using rrf-3, compared to 276 for N2, whereas the number of essential genes detected was similar . In addition, in the confirmed set of 423 clones with rrf-3-specific phenotypes, Gro/Lva defects are the largest category (42%), whereas this is only 18% for N2, with the largest category being essential genes (49%). These data suggest that rrf-3 might particularly enhance detection of genes that mutate to a slow-growth phenotype; we cannot easily test this hypothesis, as there are currently few known loci with this mutant phenotype. In some cases, a Gro/Lva phenotype was seen in rrf-3, whereas a different phenotype was seen in N2 . This suggests that some of the Gro/Lva phenotypes detected are due to incomplete RNAi of an essential gene  or by a stronger RNAi effect (where no growth defect was seen in N2). In addition, it is possible that some of the Gro/Lva phenotypes detected are synthetic effects of using the rrf-3 mutant strain.A striking feature of the rrf-3 RNAi supersensitive strain in large-scale screens increases the percentage of clones for which it is possible to detect a phenotype. Detection of postembryonic phenotypes is particularly increased, whereas detection of essential genes is similar in rrf-3 and N2. In addition, using rrf-3, there is a high rate of induction of Gro/Lva defects.To summarise, using the http://www.wormbase.org, the Internet site for the genetics, genomics, and biology of C. elegans). Second, we searched for clones near the defined map positions that, when fed to N2, rrf-3, or both, gave phenotypes corresponding to the phenotypes of the genetic mutants. For most genetic mutants, more than ten clones with a similar phenotype were found in the interval to which the genetic mutant was mapped. However, for 21 genetic mutants, only one or a few candidate clones were found. The genes corresponding to these clones were subsequently sequenced in the genetic mutant to determine whether a mutation was present. In total, we sequenced 42 predicted genes for the 21 genetic mutants  by RNAi causes uncoordinated movement  using rrf-3. For 13 genes, With this genome-wide RNAi screen using the hypersensitive strain rrf-3 strain has an increased incidence of males (Him) and displays slightly increased embryonic lethality and a reduced brood size . In particular, a large number of clones induced a developmental delay phenotype using rrf-3. Synthetic effects cannot be excluded without investigating genetic mutants. Again, variability in the efficiency of RNAi will also contribute to these differences, and a small portion may be false positives. In general, the few false positives that occur in the screen are most likely due to experimental errors, whereas the false negatives are due to reduced efficiency of the RNAi. Finally, differences between rrf-3 and N2 do not only involve the absence and presence of an RNAi phenotype, but also differences in the phenotypes for clones that did induce phenotypes in both screens . For example, we detected for unc-112 a 100% embryonic lethal (Emb) phenotype with rrf-3, whereas gon-1 a 100% Emb phenotype and other phenotypes with N2, while we did not detect an Emb phenotype with rrf-3.When an RNAi phenotype is detected with What could be the source of the interexperimental variation of RNAi? Different phenotypes for the same gene can possibly occur owing to slight differences in the developmental stage at which the animals are exposed to dsRNA and owing to changes in temperature during the experiment. However, this probably does not account for the differences we see, as we always used animals of the same larval stage (L3/L4) and used incubators for constant temperature. It was shown previously that the level of induction of dsRNA production by isopropylthio-β-D-galactoside (IPTG) can modify the penetrance of the RNAi phenotype . TherefoC. elegans, since these mutants have been extensively used as visible markers in linkage studies.The RNAi data can be a useful starting point for many new experiments, such as positional cloning of genetic mutants. By sequencing candidate genes based on the RNAi phenotypes, we identified the causal mutation in seven genetic mutants. Identification of these mutated genes gives insight into the biological process in which they are involved. In addition, cloning of these genes increases the resolution of the genetic map of rrf-3 will be submitted to WormBase, annotated as confirmed or unconfirmed. There the data can be evaluated in the context of information on gene structure, expression profiles, and other RNAi results.The complete set of RNAi phenotypes detected for the 2,079 clones using C. elegans strains: Bristol N2, NL4256 rrf-3(pk1426), CB767 bli-3(e767), MT1141 bli-3(n259), CB518 bli-5(e518), BC649 bli-5(s277), CB1158 dpy-4(e1158), CB1166 dpy-4(e1166), CB14 dpy-6(e14), CB4452, dpy-6(e2762), F11 dpy-6(f11), CB12 dpy-9(e12), CB1164 dpy-9(e1164), BC119 dpy-24(s71), CB3497 dpy-25(e817), MT1222 egl-6(n592), MT1179 egl-14(n549), MT1067 egl-31(n472), MT151 egl-33(n151), MT171 egl-34(n171), egl-34(e1452), MQ210 mau-4(qm45), CB754 rol-3(e754), BC3134 srl-2(s2507dpy-18(e364); unc-46(e177)rol-3(s1040), CB713 unc-67(e713), CB950 unc-75 (e950), HE177 unc-94(su177), HE33 unc-95(su33), HE151 unc-96(su151), unc-96(r291), HE115 unc-100(su115), MT1093 unc-108(n501), and MT1656 unc-108(n777).We used the following rrf-3 strain was used: after transferring L3- to L4-staged hermaphrodites onto the first plate, we left them for 48 h at 15°C instead of 72 h and then plated single adults onto other plates seeded with the same bacteria. Furthermore, we did not remove the mothers from the second plates. The phenotypes assayed are these: Emb , Ste (sterile), Stp (sterile progeny), Brd (low broodsize), Gro (slow postembryonic growth), Lva , Lvl , Adl , Bli (blistering of cuticle), Bmd , Clr (clear), Dpy (dumpy), Egl (egg-laying defective), Lon (long), Mlt (molt defects), Muv (multivulva), Prz , Pvl (protruding vulva), Rol (roller), Rup (ruptured), Sck (sick), Unc (uncoordinated) Thin and Pale. Emb was defined as greater than 10% dead embryos for N2 and greater than 30% dead embryos for rrf-3. Ste required a brood size of fewer than ten among fed N2 worms and fewer than five among rrf-3. Each postembryonic phenotype was required to be present among at least 10% of the analysed worms.RNAi was performed as described elsewhere  with minhttp://primers.niob.knaw.nl/). Sequence reactions were done using the ABI PRISM Big Dye terminator sequencing kit  and were analysed on the ABI 3700 DNA analyser. The coding sequence and the 5′- and 3′-untranslated region (about 500 bp upstream and downstream of the coding sequence) of the predicted genes, as annotated in WormBase, was analysed for mutations by sequencing amplified genomic DNA of the genetic mutants see . Nested C. elegans using the BLAST program (http://www.sanger.ac.uk/Projects/C_elegans/blast_server.shtml) or analysed using the PolyPhred program (available from http://droog.mbt.washington.edu/PolyPhred.html).Sequences were compared to the genomic sequence of Table S1(482 KB PDF).Click here for additional data file.Table S2(188 KB PDF).Click here for additional data file.Table S3(25 KB DOC).Click here for additional data file.http://www.wormbase.org).RNAi data from this study will be submitted to WormBase (


The objective was to describe the causes of initial and overall AIDS-defining disease episodes among HIV patients in Singapore.A retrospective observational cohort study was performed of all adult patients seen at the national HIV referral center between 1985 and 2001. Data were extracted from the patients' records by ten trained healthcare workers. AIDS-defining conditions were established using predefined criteria.Pneumocystis carinii pneumonia (35.7%), Mycobacterium tuberculosis (22.7%) and herpes simplex (7.4%). In total 1742 AIDS-defining episodes occurred. The most frequent causes were Pneumocystis carinii pneumonia (25.1%), Mycobacterium tuberculosis (16.2%) and cytomegalovirus retinitis (9.5%).Among 1504 patients, 834 had experienced one or more AIDS-defining diseases. The most frequent causes of the initial AIDS-defining episode were The most frequent causes of AIDS-defining illnesses in Singapore are similar to those reported in the West, prior to the introduction of anti-retroviral therapy. Opportunistic infections remain the most frequent AIDS-defining illnesses. Mycobacterium avium have decreased from 9% to 2% and 8% to 3% respectively [Pneumocystis carinii, Mycobacterium avium and other opportunistic infections have also been observed in the United States [Cohort studies have provided valuable information on the clinical course of HIV infection in patients from Europe -16, Nortectively . The proectively . Substand States ,21.Pneumocystis carinii pneumonia (7.0%) and cryptococcal meningitis (10.9%) [Relatively few data are available on the course of HIV infection in Asian populations. In Bangkok between 1987 and 1993 the most frequent AIDS-defining diagnoses were extrapulmonary tuberculosis (22.8%),  (10.9%) . Little The Communicable Diseases Centre (CDC) in Singapore is the national reference centre for adult patients with HIV and AIDS and nationwide 94.9% of all Singaporean adult residents, who have ever been diagnosed, have been referred there. Underreporting of HIV does not occur as reporting occurs automatically from the reference laboratory. Records have been kept since the first case was identified in Singapore in 1985. Therefore the Singapore HIV Observational Cohort Study (SHOCS) contains almost the entire country's HIV experience. Nucleoside analogues have been available in Singapore since the early 1990s and protease inhibitors and non-nucleoside reverse transcriptase inhibitors since 1997. The usage of these drugs has been increasing annually and during 2000 and 2001 approximately 70–80% of patients regularly attending CDC were taking some form of anti-retroviral therapy.st December 2001, by ten trained healthcare workers. Probable and confirmed criteria were developed for the diagnosis of category C [The details of the SHOCS cohort have been described previously . The stutegory C  and catetegory C .All disease episodes were initially determined by the data extractors and then checked by an infectious disease physician (RB). Diagnoses were not included if they did not fulfill the specified criteria even if there was a strong clinical suspicion of a particular condition. If there was insufficient evidence to satisfy the criteria for a specific diagnosis, a more general diagnosis was assigned, for example cerebral lesion (cause unknown) would be used if there was insufficient evidence to support a diagnosis of toxoplasmosis of the brain or primary cerebral lymphoma.Pneumocystis carinii pneumonia, Mycobacterium tuberculosis and cytomegalovirus retinitis), comparisons were made between the pre-HAART era (1986 to 1995) and the established HAART era (2000 to 2001) using χ2 tests.Median CD4 counts were calculated based on the sample taken nearest the time of diagnosis of the initial AIDS-defining condition. CD4 counts from 893 patients were used, as in 37 (4.0%) cases no suitable count was available. Data were entered into a computer database (Microsoft Access™) and checked for errors and inconsistencies. Statistical analyses were performed using Stata 7.0™. Each new or recurrent AIDS-defining condition counted as one event (i.e. a patient with three diagnoses would contribute three events). An AIDS-defining condition was counted as a separate additional event if it recurred six or more months after the initial diagnosis or in the case of tuberculosis, six or more months after treatment completion. To examine the effects of highly active anti-retroviral therapy (HAART) on the proportions of AIDS-defining illnesses due to each of the three most common infections , M. tuberculosis (22.7%), herpes simplex (chronic mucocutaneous) (7.4%) and candidiasis  (6.9%)  and Kaposi's sarcoma  than for Pneumocystis carinii pneumonia , herpes simplex  and extrapulmonary cryptococcosis  , M. tuberculosis (16.2%), cytomegalovirus retinitis (9.5%), candidiasis (7.8%), chronic mucocutaneous herpes simplex (8.2%) and disseminated M. avium (8.2%) , but that due to M. tuberculosis had increased to 18.0% . There was no significant change in the percentage of episodes due to cytomegalovirus retinitis   [Pneumocystis carinii pneumonia (4.8%) [Pneumocystis carinii pneumonia caused a much higher proportion of AIDS-defining diseases in Singapore than in Bangkok and cryptococcal meningitis occurred less frequently in Singapore.In the SHOCS cohort the proportion of conditions which were of infectious etiology remained very high throughout the study period. The distribution of conditions was similar to that seen in Western countries prior to the introduction of anti-retroviral therapy . Howevernia (7%) . Betweena (4.8%) . PneumocThe SHOCS cohort provides data on the experience of 94.9% of Singaporean residents who have been diagnosed with HIV since the epidemic began. There are good diagnostic facilities in Singapore and patients with HIV-related illness receive extensive investigations. Therefore Singapore offers an ideal location for collecting information on the causes of morbidity and mortality in persons with HIV in Asia. Medical record keeping is of a high standard in our hospital and this allowed accurate diagnoses to be assigned for AIDS-defining conditions which occurred throughout the AIDS era. To ensure consistency, data was extracted by carefully trained healthcare workers and diagnoses were assigned using predefined criteria. All AIDS-defining diagnoses and all causes of death were reviewed by the same infectious diseases physician (RB). Follow-up rates in our cohort are very high with only 5.8% of patients lost to follow-up at 12 months and 9.3% after 3 years. The SHOCS cohort therefore provides a unique source of information on HIV infection in an Asian country since the start of the epidemic.Mycobacterium tuberculosis". However if a patient receives a successful trial of treatment for disseminated M. avium, the diagnosis is "probable mycobacteriosis (species unidentified)".In a retrospective study it is impossible to ensure that all patients received a full series of investigations in order to satisfy the study criteria for diagnosis of HIV-related conditions. As a consequence the criteria for making a probable diagnosis are unavoidably biased against certain conditions. For example if a patient undergoes a therapeutic trial of tuberculosis treatment he or she can be given the diagnosis of "probable M. avium and M. tuberculosis may also have been affected by improvements in mycobacterial culture techniques.Differences in physicians' awareness of the link between HIV and opportunistic infections can influence diagnosis rates. For example, the increase in the proportion of AIDS-defining disease episodes caused by tuberculosis between 1986–1995 and 2000–2001 may have been due to greater awareness of the link between HIV and tuberculosis. Diagnosis rates for Despite the availability of anti-retroviral medication, opportunistic infections remain a common problem among HIV patients in Singapore. This may partly be explained by the high proportion of Singaporean patients, who present with advanced disease and very low CD4 counts. These patients often already have one or more AIDS-defining opportunistic infections at first presentation. Opportunistic infections may also occur before adequate immune-reconstitution can occur when HAART is commenced when the patient has a very low CD4 count. Studies in the West have shown that AIDS-defining illnesses occur more frequently among patients who have a CD4 count below 50 when anti-retroviral therapy is commenced than among those with higher CD4 counts ,38. ImmuThe current reductions in the cost of anti-retroviral therapy (Eg. due to generic manufacture) may enable high levels of anti-retroviral use to be achieved in many Asian countries. The experiences of the SHOCS cohort are likely to be repeated throughout Asia. Our results suggest that avoidable opportunistic infections may continue to occur even when high levels of anti-retroviral use are achieved. To reduce the burden of morbidity and mortality caused by these infections, efforts must be made to diagnose HIV infection earlier and to make the treatment of HIV and its associated opportunistic infections more affordable.SHOCS = Singapore HIV observational cohort study, CDC = Communicable Diseases Centre, AIDS = acquired immunodeficiency syndrome, HIV = human immunodeficiency virus, HAART = highly active anti-retroviral therapy.The authors declare that they have no competing interests.RB participated in the design of the study, supervised data extraction, performed the statistical analysis and wrote the manuscript. SS designed the database and supervised data extraction. NP participated in the design of the study and contributed to the statistical analysis and production of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Depression and anxiety in patients with coronary heart disease (CHD) are associated with a poorer prognosis. Therefore the screening for psychological distress is strongly recommended in cardiac rehabilitation. The Hospital Anxiety and Depression Scale (HADS) is a widely used screening tool that has demonstrated good sensitivity and specificity for mental disorders.We assessed mental distress in in-patient cardiac rehabilitation in Germany. The factor structure of the German language version of the HADS was investigated in 1320 patients with CHD. Exploratory factor analysis and confirmatory factor analysis were used to determine the underlying factor structure of the instrument.Three-factor models were found to offer a superior fit to the data compared to two-factor (anxiety and depression) models. The German language HADS performs similarly to the English language version of the instrument in CHD patients. The German language HADS fundamentally comprises a tri-dimensional underlying factor structure .Despite of clinical usefulness in screening for mental disturbances the construct validity of the HADS is not clear. The resulting scores of the tri-dimensional model can be interpreted as psychomotor agitation, psychic anxiety, and depression in individual patient data or clinical investigations. Coronary heart disease (CHD) is of profound interest to health and clinical psychology due to the high levels of anxiety and depression observed in patients following the occurrence of a coronary event -5. CHD wA candidate screening tool that has been widely and increasingly used with CHD patients is the Hospital Anxiety and Depression Scale (HADS: ), an easOn the other hand, recent psychometric evaluations of the HADS in a range of clinical populations have suggested that the proposed factor structure of the instrument may indeed be compromised by the physiological aspects of the disease or by changes in health status ,29,30. CDunbar et al.  found a There has been little systematic investigation of the factor structure of the translated HADS in German cardiac patients, though interestingly a German language version of the instrument has been developed in Germany using cardiac patients within the context of the original assumed two-dimensional (anxiety and depression) structure . To dateIdentification of a coherent three-factor underlying structure of the HADS has a number of significant implications in terms of the validity of the tool as a screening instrument. Firstly, referral to mental health services could be undermined based on a two-dimensional (anxiety and depression) interpretation of HADS scores in cardiac patients. Secondly, further replication of a three-factor structure of the HADS in a German cardiac population would be valuable in determining if the HADS should be more effectively used as a screening instrument when comprised of three sub-scales in this group. Thirdly, replication of a consistent three-factor structure in the German-translated version of the HADS would provide strong evidence that the three-dimensional structure is implicit to the instrument and not a language-based artifact. Finally, the widespread international use of the HADS provides a compelling rationale to establish the psychometric properties of the instrument not only in broad diagnostic categories, but also across culturally-diverse groups.The purpose of the present study was to determine whether the three-factor structure of the HADS identified by Martin and colleagues  in myoca1) Do exploratory factor analysis (EFA) and confirmatory factor analysis (CFA) techniques concord to the proposed bi-dimensional structure of the HADS in German CHD patients?2) Does a three-dimensional factor structure provide a superior fit compared to competing bi-dimensional factor structures?The study used a cross-sectional design with all measures taken at one observation. The dependent variables were sum scores obtained on the HADS , and the anxiety (HADS-A) and depression (HADS-D) sub-scales. Exploratory factor analysis (EFA) and confirmatory factor analysis (CFA) methods were used to address the research questions using a pooled HADS data set from all patients. Ethical approval for the study was given by the local ethical committee of the University Hospital of Freiburg. Written informed consent was obtained from all participants prior to the commencement of the study.The study was conducted in three German cardiac rehabilitation hospitals. The patients stay in these hospitals for three to four weeks for a comprehensive cardiac rehabilitation program consisting of medical advice, exercise, patient education, relaxation and psychosocial interventions. All cardiac patients who agreed to take part in the PROTeCD-study (Psychotherapeutic Resource-Orientated Treatment for Cardiac Patients with Depression) were screened for mental distress with the German version of the HADS  at admisExploratory factor analysis was performed on the full 14-item HADS using SPSS 12.0 statistical software. The criterion chosen to determine that an extracted factor accounted for a reasonably large proportion of the total variance was based on an eigenvalue greater than 1. A maximum likelihood factor extraction procedure was chosen since this method of factor condensation is consistent with our previous research  and is p2 indicates a significant proportion of variance remains unexplained by the model [Confirmatory factor analysis was conducted using the Analysis of Moment Structures (AMOS) version 4  statistihe model .t(401.14) = 4.14, p < 0.001) than male patients .1320 patients  enrolled in an in-patient cardiac rehabilitation programme in three hospitals in Germany provided complete HADS data sets for analysis. Inclusion criteria for participation in the study was a confirmed diagnosis of CHD; for details see . The patpossible clinically relevant levels of anxiety and 373 participants (28%) possible clinically relevant levels of depression. Adopting Snaith and Zigmond's [probable clinically relevant levels of anxiety and 161 participants (12%) probable clinically relevant levels of depression.The mean HADS-A sub-scale score was 6.14  and the mean HADS-D sub-scale score was 5.41 . Using Snaith and Zigmond's  cut-off icipants % possibl2 = 7758.34, df = 91, p < 0.001) was highly significant indicating the data satisfied the psychometric criteria for the factor analysis to be performed based on data distribution characteristics. Examination of individual item skew and kurtosis characteristics confirmed the suitability of the maximum likelihood factor extraction procedure [r = 0.82, p < 0.001, explaining 67% of the common variance between factors.The Kaiser-Meyer-Olkin (KMO) measure of sampling adequacy and the Bartlett Test of Sphericity (BTS) were conducted on the data prior to factor extraction to ensure that the characteristics of the data set were suitable for the EFA to be conducted. KMO analysis yielded an index of 0.94, and BTS  indicating that a proportion of the total variance was unexplained by each model. Examination of the fit indices revealed that the best fit to the data was offered by Friedman et al.'s [The factor models tested and accompanying fit indices are shown in Table et al.'s  three-fa p < 0.00 indicatiet al.'s  three-faet al.'s  'tripartet al.'s  three-faet al.'s  and Mooret al.'s  offered et al.'s  was obseThe findings of the current study offer a further important contribution to the evidence base regarding the underlying factor structure of the widely used HADS. It is worthy of note that high levels of HADS assessed anxiety and depression were observed in the study. This finding is consistent with investigators using this instrument in cardiac populations in other parts of the world ,24,26 anThe findings from the factor analyses conducted on the HADS data are of pertinent methodological as well as clinical interest. EFA of the HADS revealed two factors, the loadings of individual items being consistent with the anxiety and depression sub-scale domains. However, it was also observed that the HADS-A item-7 'I can sit at ease and feel relaxed' was jointly loading on both anxiety and depression factors, this split-loading slightly in favour of the depression factor. A recent EFA of the HADS conducted with patients with significant facial disfigurement  also revtri-dimensional instrument, consistent with contemporary research with this instrument across diverse clinical presentations [The findings from the CFA revealed the best model fit to be provided by Friedman et al.'s  three-fantations ,31,33,35In conclusion, the current study found the German language version of the HADS to have an underlying three-dimensional factor structure following CFA in CHD patients, an observation consistent with UK  and ChinJB designed the study, carried out the data collection and clinical assessment. JB drafted part of the manuscript and was involved in the interpretation of the findings. CRM developed the statistical framework, carried out the statistical analysis and drafted part of the manuscript. Both authors have no competing financial or other interest in relation to this manuscript.The study was funded by the Federal Ministry of Education and Research, Germany; Regional Pension Insurance Institute, Baden-Wuerttemberg, Germany, LVA 02 804 and is part of the Rehabilitation Research Network South-West (Germany).


Limb-salvage is a primary objective in the management of extremity soft-tissue sarcoma in adults and children. Wide-local excision combined with radiation therapy is effective in achieving local tumor control with acceptable morbidity and good functional outcomes for most patients.Two cases of deformity after wide-local excision, brachytherapy and external beam irradiation for lower-extremity synovial cell sarcoma are presented and discussed to highlight contributing factors, time course of radiation effects and orthopedic management. In an effort to spare normal tissues from the long-term effects of radiation therapy, more focal irradiation techniques have been applied to patients with musculoskeletal tumors including brachytherapy and conformal radiation therapy. As illustrated in this report, the use of these techniques results in the asymmetric irradiation of growth plates and contributes to the development of valgus or varus deformity and leg-length discrepancies.Despite good functional outcomes, progressive deformity in both patients required epiphysiodesis more than 3 years after initial management. There is a dearth of information related to the effects of radiation therapy on the musculoskeletal system in children. Because limb-sparing approaches are to be highlighted in the next generation of cooperative group protocols for children with musculoskeletal tumors, documentation of the effects of surgery and radiation therapy will lead to improved decision making in the selection of the best treatment approach and in the follow-up of these patients. Limb-salvage is an important treatment objective for adults and children with extremity soft tissue sarcoma and often requires the use of limited surgery and irradiation . LimitinSurgery and radiation therapy both have the potential to cause significant morbidity including loss of function and deformity ,11-15. TIndividual cases of valgus and varus deformity after limited surgery and irradiation for extremity soft-tissue sarcoma are presented and discussed to identify factors that may be responsible for these treatment complications. Pre-existing orthopedic problems, multiple attempts at resection, post-operative infection, the use of chemotherapy and the addition of brachytherapy to external beam radiation therapy appear to be contributory. Because limb-sparing approaches will be an important component of the next generation of cooperative group studies for extremity soft tissue sarcoma in children, the incidence and severity of this and other treatment-related complications should be documented as well as efforts to limit the effects of these treatments and identify solutions for established problems.At the time of diagnosis, this patient was an 8 year-old male with an approximate 12–24 month history of mild pain and swelling in the left popliteal region. There was no complaint of fever or decreased range of motion. He presented to a local orthopedist (April 1995) and was found to have a palpable abnormality on the posterior aspect of the knee consistent with a Baker's cyst. Aspiration was unsuccessful and the patient was treated with ibuprofen for a 10-day course. Nearly one year later (April 1996) the family sought a second opinion and an MR study was ordered that revealed a cystic structure [Figure At the time of his evaluation after referral, he had strong popliteal pulses. There was a 7.0 × 4.5 cm area of swelling and numbness in the left popliteal region. The deep-tendon reflexes were brisk and the motor exam and gait were normal. MR showed residual abnormality consistent with tumor lateral to the joint capsule. Metastatic work-up including nuclear bone scan and CT scan of the chest was negative. Tumor bed re-excision with placement of afterloading catheters was performed in December 1996. All visible residual abnormality was removed without significant disruption of underlying ligaments and tendons. The walls of the tumor bed were biopsied to map the extent of microscopic residual disease. Microscopic residual disease was anticipated given the site of involvement and the limited ability to operate beyond the extent of the abnormal appearing tissues. Eleven afterloading catheters were placed in a parallel array to cover the tumor bed Figure . Radio-o125. The dwell time of the implant was 64 hours and the patient received a total implant dose of 2560 cGy delivered at 40 cGy/hr. Three weeks later, the patient began external beam irradiation at 180 cGy per day and received a total external beam dose of 4860 cGy using 6 MV photons with treatment delivered in a parallel-opposed beam arrangement using a CT based treatment plan [Figure Five days after surgery, the 11 catheters were loaded with a total of 135 seeds representing 408 millicuries of IA decision was made to initiate chemotherapy three weeks into the course of external beam irradiation based on the perceived high-risk nature of his case – longstanding history of symptoms, known residual tumor and size of tumor at presentation. Chemotherapy included vincristine, ifosfamide, and adriamycin was eventually administered for a total of four cycles. There was central dehiscence of the wound prior to the completion of radiation therapy. The wound was colonized with Enterococci sensitive to ampicillin and managed with antibiotics, whirlpool treatment and daily dressing changes. There was a one-week treatment break during the external beam portion of the treatment.At the completion of chemotherapy (June 1997) the patient underwent excision of scar tissue with rotation flap of gastrocnemius and skin and Z-plasty of the semi-membranous and semi-tendinous tendon for a non-healing ulcer in the operative region. There was also contracture of the knee joint without signs of abscess or cellulitis. One month later, a second procedure was required to debride and irrigate the left popliteal fossa wound at the site of previously irradiated tissue and contracture release with muscle and fasciocutaneous flap closure. One year after the completion of all therapy (June 1998) the patient reported full range of motion and softening of previously fibrotic tissue. He was actively playing baseball and had no imposed limitations.Nearly two years after completion of treatment (March 1999), the patient was noted to have Trendelenburg gait after prolonged walking ascribed to poor endurance of weakened hip abductors bilaterally. Hip hiking was also noted on the contralateral side due to leg length and ASIS height discrepancy. A difference of 2.5 cm was noted when measured from the umbilicus to the medial malleolus. He had grown 7.0 cm since the time of diagnosis. He was instructed to stretch the heel cord and strengthening his weak hip abductors. Arrangements were made to provide a shoe lift to accommodate the leg length difference. The discrepancy improved to less than 1 cm over a three month period of time (March-June 1999). He was carefully monitored for growth discrepancy and 6 months later, nearly 3 years after the initiation of treatment, the discrepancy returned to its original value of greater than 2 cm. He also had difficulty with ambulation with slight left-sided limp. Orthopedic surgery was re-consulted. The discrepancy was followed and treated with additional shoe lift. The patient continued to engage in normal activities including sports and reported full range of motion and normal strength. More than four and a half years after initiating treatment (August 2001) Figure , x-ray sAt the time of diagnosis this patient was a 9 year-old female with a one year history of pain and swelling about her left knee. She had experienced a fall and related all symptoms to the fall. She was seen in her local emergency room by her family physician; there was no diagnosis or treatment. Approximately one month prior to her representation, she was struck in the left knee by a basketball and developed worsening pain. She was seen by an orthopedic surgeon (December 1999) and was noted to have a valgus posture of both lower extremities, exaggerated on the left by external rotation and she walked with a mild limp. The left knee had no effusion but was hypersensitive to light touch over the lateral aspect where there was soft tissue swelling just below the knee. There was no obvious mass in the area, although firm palpation was difficult because of patient discomfort. Plain films were normal and an MR was ordered that revealed an apparent meniscal cyst in the lateral aspect of the left knee Figure . Biopsy 125 . The dwell time of the implant was 62 hours and the patient received a total implant dose of 2480 cGy delivered at 40 cGy/hr. Two weeks later the patient began external beam irradiation at 180 cGy/day and received a course of treatment and total external beam dose of 5040 cGy using 6 MV photons with treatment delivered with two beams using a CT based treatment plan [Figure At the time of her evaluation after referral (January 2000), there was a well healed scar with no excessive swelling. There was mild tenderness on the lateral aspect of her left knee. Additional imaging studies showed abnormality at the site of prior surgery equivocal for residual tumor. There was no evidence of abnormality in the joint space. The tumor bed was explored. There was no physical evidence of compromise at the level of the joint space. She underwent wide local excision with placement of afterloading catheters. Six catheters were placed in a parallel array with 1 cm spacing. Radio-opaque clips were placed to delineate the tumor bed and assist in brachytherapy planning Figure . The marn Figure . RadiatiThe patient suffered moist desquamation corresponding to the radiation therapy portal that was predicted based on the treatment and the use of a tissue equivalent bolus material which was placed on the wound on alternating days during her course of external beam irradiation. She was able to return home on the last day of treatment. On routine follow-up, only 4 months after treatment, left leg appearing to be slightly longer than her right leg by less than 1 cm. No corresponding gait problems were reported. Nearly one year after treatment (January 2000) physical examination showed good range of motion at the left knee; however, there was significant valgus angulation. An MR study was reviewed by Orthopedic Surgery and was noted to show growth arrest laterally and predominantly involving the distal femoral physis Figure . Based oDue to its high propensity for local recurrence and metastasis, aggressive treatment of synovial sarcoma is imperative. While many different methods have been used in the treatment of these high-grade tumors, including mono-bloc soft part resection and amputation, the current standard includes local excision and radiation therapy when feasible . Wide loBoth patients in our study underwent two surgical procedures and were treated with brachytherapy and external beam irradiation. Brachytherapy was used to confine the highest doses to the region at risk and minimize the dose received by normal tissues. The use of brachytherapy shortens the overall treatment time and increases the rate of local control in the setting of involved margins of resection. CT-based treatment planning was used to define the volume of irradiation and to spare normal tissue structures. By reducing the amount of radiation dose delivered to normal tissues, the probability of growth deformity, radio-chemotherapy interactions, and even the hypothetical risk of second tumor formation may be lowered. No effort was made to symmetrically irradiate the physes, which would hypothetically lead to symmetrically diminished growth without the added effect of angular deformity. Because of concerns about the effects of total joint irradiation and its possible effects on functional outcome, the inhomogeneous and asymmetric approach was taken. Despite the valgus and varus deformity experienced by these children and the need for intervention, both children and parents were completely satisfied with their functional outcome and indicated that they would chose the same course of treatment if presented again with the same options.The importance of achieving local control with aggressive surgery and high-dose irradiation cannot be overemphasized. Local control is crucial to long-term survival and avoiding the morbidity of local tumor progression. Local control, even in the setting of metastatic disease, is an important endpoint. Radiation therapy is highly successful in achieving local control in soft tissue sarcoma and is standard in the care of children with high-grade tumors such as those reviewed in this report. At our institution high-grade tumors with resection margins of 1 cm has an observed local control rate of 72% (5 of 7 patients) in the absence of radiation therapy and 100% (7 of 7 patients) when radiation therapy was given postoperatively. Among 20 unirradiated high-grade tumors that were completely resected with margins > 1 cm only 15 (75%) were locally controlled for an extended period . Our polDespite efforts to achieve local control and minimize the effects of treatment on normal tissues, damage to bone and soft tissues may be unavoidable. Synovial cell sarcoma commonly arises near tendon sheaths and joint capsules of adolescents and young adults and may be in close proximity to an epiphyseal growth plate during a time of rapid growth. The situation is made worse if the tumor is located around the distal femur or proximal tibia. Among the four epiphyseal plates in the lower extremity that contribute to the growth of the limb, those around the knee make the most significant contribution, with the distal femur and proximal tibia accounting for 50–90% and 57% of limb growth, respectively depending on age . In our There are two non-congenital mechanisms that are known to interfere with growth of the physis: direct trauma and environmental change around the plate. Trauma includes acute injury to the growth plate in manner that affects all or partial growth and results in premature closure or the formation of a physeal bar. Even if the region has retained its ability to grow it is hampered by solid bone formation across the plate . EnvironParalysis is also known to cause of growth inhibition, although the mechanism is poorly understood. Proposed contributors include reduced muscle activity, which indirectly alters the blood supply, and abnormal vasomotor control . AvasculRogalski et al.  observedIt is also conceivable that repeated surgeries to the lateral aspect of the knee in both patients played a contributive role. A change in the environment surrounding the physis, such as muscle atrophy following prolonged bed rest, paralysis, or limb-sparing surgery with muscle loss, is known to cause a significant slowing of growth . AlthougThe patients included in this report had many of the above noted contributions to abnormal growth and development including a pre-existing condition, vascular compromise due to multiple surgeries, loss of muscle mass, limited use of the extremity for a defined period of time, infection and chemotherapy. Chemotherapy may temporarily reduce bone growth through systemic effects that include the direct effects of specific agents or the indirect effects resulting from systemic infections and abnormalities in metabolism and nutrition.It has been known for almost a century that radiation therapy at sufficient levels can affect growing bone. Several factors contributing to the severity of effect including the total dose, dose per fraction, dosimetry (asymmetry and inhomogeneity) and age at the time of irradiation ,30,31. PThe conclusions of Gonzalez and Breur  were sliThe epiphyseal growth plate is the area of the developing skeleton most sensitive to the effect of radiation due in part to its rapidly proliferating stem cell population. Even low doses of radiation have been shown to cause histologic changes including temporary swelling, fragmentation, and degeneration of chondrocytes . When hiCreating a treatment plan for patients with leg deformities who have undergone radiation therapy with or without chemotherapy can pose a difficult challenge. Because the osteocytes of neighboring bone are also destroyed, it may take years for the bone to revascularize and repopulate with healthy osteocytes . The absHistorically, there have been two primary treatments for patients with angular limb deformities: epiphysiodesis and stapling. Both methods seek to achieve the same result while offering different sets of advantages and disadvantages. Partial epiphysiodesis of the knee to correct angular deformity was first described by Phemister in 1933 . It has Newer radiation delivery techniques including high-dose rate brachytherapy, intraoperative radiation therapy ,40 and tLimited surgery and irradiation may result in growth abnormalities and deformity. These effects may have minimal or significant impact depending on functional outcome and the value attached to limb preservation for a particular patient. As mentioned in this report, both patients and families were queried about their decisions regarding treatment and both reported satisfaction with outcome recognizing that side effects were anticipated. Both families attached a high value to limb preservation.None declared.DTF reviewed the patient records and drafted the manuscript. WCW contributed to drafting the manuscript. MDN contributed to drafting the manuscript. TEM conceived of the study, participated in the review of the data, and helped draft the manuscript. All authors read and approved the final manuscriptThe pre-publication history for this paper can be accessed here:


It is commonly held that "a calorie is a calorie", i.e. that diets of equal caloric content will result in identical weight change independent of macronutrient composition, and appeal is frequently made to the laws of thermodynamics. We have previously shown that thermodynamics does not support such a view and that diets of different macronutrient content may be expected to induce different changes in body mass. Low carbohydrate diets in particular have claimed a "metabolic advantage" meaning more weight loss than in isocaloric diets of higher carbohydrate content. In this review, for pedagogic clarity, we reframe the theoretical discussion to directly link thermodynamic inefficiency to weight change. The problem in outline: Is metabolic advantage theoretically possible? If so, what biochemical mechanisms might plausibly explain it? Finally, what experimental evidence exists to determine whether it does or does not occur?Reduced thermodynamic efficiency will result in increased weight loss. The laws of thermodynamics are silent on the existence of variable thermodynamic efficiency in metabolic processes. Therefore such variability is permitted and can be related to differences in weight lost. The existence of variable efficiency and metabolic advantage is therefore an empiric question rather than a theoretical one, confirmed by many experimental isocaloric studies, pending a properly performed meta-analysis. Mechanisms are as yet unknown, but plausible mechanisms at the metabolic level are proposed.Variable thermodynamic efficiency due to dietary manipulation is permitted by physical laws, is supported by much experimental data, and may be reasonably explained by plausible mechanisms. Carbohydrate restriction as a general strategy for weight loss continues to gain in popularity and its utility and generally protective effect in lipid profile and glycemic control continues to be demonstrated, at least in an experimental setting -4. The sWe have previously presented arguments that there is no violation of physical principles ,7 and, iThe misunderstanding that continues to be repeated in the expression "a calorie is a calorie" appears to be exclusive reference to the first law of thermodynamics. The difficulty with this theoretical approach is that it is only part of the relevant physics and its relationship to biologic systems. The first law says that in any transformation the total energy in the system can be accounted for by the heat added to the system, the work done by the system on its environment and the change in energy content of all the components of the system. It is important to understand, however, that the first law does not say what the relative distribution between these effects will be for any process. In fact, the first law does not even allow us to say whether the process will occur at all. To understand the progress of a physical change it is necessary to understand the second law which introduces an entity known as the entropy, S, a measure of disorder in all processes. In all real (irreversible) processes, entropy increases, usually written ΔS > 0. The most common marker of increasing entropy is heat, although it is by no means the only evidence for increased entropy.maximum useful work that can be performed by the process. The actual process however, in general derives less useful work than permitted by the theoretically available ΔG due to inefficiency in energy capture. A proper accounting of entropy and efficiency must be included if we are to understand energy utilization in biological and biochemical systems.In systems at constant temperature and pressure (i.e. biologic systems)), the first and second law are combined in the Gibbs Free Energy, ΔG, which represents the It is also important in the discussion of biological systems to understand that they are open systems, i.e. they take in nutrients and oxygen and excrete carbon dioxide, water, urea and other waste products, as well as heat. The importance with respect to weight considerations is that mass and energy are conserved , but they are not conserved entirely within the organism.To illustrate the proper interpretation of the first law of thermodynamics consider a subject whose resting energy expenditure is met by the production of 95 moles of ATP. Since oxidation of a single mole of glucose provides 38 moles of ATP, 2.5 moles of glucose will be needed to meet this individual's resting energy requirements. It is important to note that the resultant carbon dioxide, water, and heat are not retained within the organism. The useful retained energy is in the 95 moles of ATP Figure . . This value approximates the efficiency for oxidation of carbohydrate as well as lipid, whereas proteins are generally oxidized at a lower value of approximately 30–35%  value for glucose obtained in the calorimeter is approximately 4 kilocalories of energy per gram Figure . By cont% Figure .1. The second law of thermodynamics dictates that there is an inevitable metabolic inefficiency in all biological and biochemical processes with heat and high entropy molecules  as the most common products.excreted and the heat radiated and exported in high entropy molecules.2. The first law of thermodynamics is satisfied in living (open) systems by properly accounting for the mass The most common example of weight loss is reduction of caloric intake. At the risk of oversimplification, if our subject ingests fewer than 2.5 moles of glucose and produces, for example, only 90 moles of ATP from food, then homeostasis would require enlisting endogenous body stores for further oxidation. This oxidation would then provide the additional 5 moles of ATP required. Oxidation of body stores (lipid or lean body mass) will result in production of additional carbon dioxide, urea, water and heat. The excretion of these products will result in weight loss. Figure .uncoupling in mitochondria. ATP is produced in a variety of cellular locations. Glycolysis produces a net of two ATP's per molecule of glucose, in the cell cytoplasm. On the other hand, we recall that 36 additional molecules of ATP are produced from glucose as a result of the mitochondrial TCA cycle and electron transport. A critical part of the process involves the development of a hydrogen ion gradient across the mitochondrial membrane. This concentration gradient provides the energy that is converted into ATP as hydrogen ions pass down the gradient through the ATP synthase particle, entirely analogous to the energy in a high-pressure gas in a cylinder with a movable piston. (The expansion of the gas is like diffusion down a gradient: It does work against the piston). In the mitochondrion the energy of moving down the gradient is captured in ATP, the medium of exchange for the performance of work within cells. This capture of energy, referred to as coupling the energy to the formation of ATP, is the essential process permitting work to be done by living systems.The implication of the first and second laws of thermodynamics is that reduced efficiency has precisely the same result as reduced caloric intake. One conceptually simple means of reducing efficiency involves the process of There are known endogenous and pharmacologic agents, which result in uncoupling the formation of ATP from the dissipation of the gradient. Uncouplers such as 2, 4-dinitrophenol bypass ATP synthase and cause hydrogen ion gradient dissipation without ATP formation that can result in organ dysfunction causing death. More modest degrees of uncoupling may be caused by the class of endogenous compounds we know as uncoupling proteins (UCP's). Three different isoforms, UCP1, UCP2 and UCP3 have been identified thus far in mammalian tissues. While the overall and relative physiologic importance of these proteins remains incompletely understood in human tissues, UCP1 has been shown in mice  to resulguarantees inefficiency in all metabolic processes. However, variation of efficiency is not excluded. In fact, the laws of thermodynamics are silent on the existence of variable efficiency. If efficiency can vary (as in the example of oxidative uncoupling) then "a calorie is a calorie" is no longer a true statement. The role of uncoupling proteins in humans, as indicated, is as yet incompletely defined [The essence of the second law of thermodynamics is that it  defined . HoweverIt is possible that metabolic efficiency may be decreased by oxidative uncoupling as described above. Polymorphisms connecting uncoupling proteins with obesity or propensity to gain weight have been identified in humans ,12 altho2 and water generates 38 moles of ATP with an overall efficiency of about 38.5%. On the other hand, if glucose is first incorporated into glycogen, followed by hydrolysis of the glucose and subsequent oxidation, 2 moles of ATP are lost per mole in this cycle with overall efficiency reduced to 35%. Similarly an amino acid from an "average" protein, when directly oxidized to CO2, produces ATP with an efficiency of about 33%. If the amino acid is first incorporated into a protein and later hydrolyzed and oxidized, four ATP's per molecule are used for synthesis of the peptide bond. This reduces the efficiency to 27%. Smaller degrees of inefficiency are seen for lipid cycles  . If indeth binomial coefficient shows this probability to be p < 0.01.Having established that there is no theoretical barrier to metabolic advantage and that there are plausible mechanisms that could account for such an effect, we must ask whether it can be demonstrated experimentally, that is, whether the proposed effects are of sufficient magnitude to be a practical feature of weight reduction strategies, in particular very low carbohydrate diets. If so there will be increased weight loss for the same caloric intake, or metabolic advantage. A recent animal model provides support for greater metabolic inefficiency in rats fed carbohydrate restricted diets compared with higher carbohydrate, leading to excess weight loss . Human dWhile the above suggests the possibility of metabolic advantage, it does not prove it, nor do we know the magnitude of the effect, or the factors that control it. The studies above were chosen from among those quoted by many of the authors who have disputed the existence of metabolic advantage. Nonetheless, a formal meta-analysis would be necessary to avoid the possibility of conscious or unconscious bias in their selection. Further, it would be necessary to establish evidence that energetically costly metabolic processes are more prevalent in low carbohydrate diets than in diets of higher carbohydrate content. Whereas the proposed mechanisms are plausible, they need to be proven.guarantees inefficiency in all metabolic processes and is silent on the possibility that inefficiency may be augmented in some instances. A familiar example of inefficiency is thyrotoxicosis, with attendant weight loss and heat generation despite unchanged or increased caloric consumption. The theoretical possibility of inefficiency and metabolic advantage due to macronutrient compositional change exists, but demonstration of the phenomenon can only be resolved experimentally. Isocaloric dietary studies with a low vs. a higher carbohydrate arm support the experimental possibility of metabolic advantage. A formal meta-analysis would be required to evaluate this more objectively. Further studies, including tracer methods, would be required to establish mechanisms. The presence of high quantities of dietary protein (often a feature of low carbohydrate diets) is known to stimulate protein turnover, an energetically costly process. However, it is unclear whether this is the only factor, or whether it is necessary for metabolic advantage to occur. In particular, obligate gluconeogenesis from endogenous sources may also contribute to induction of protein turnover.Thermodynamics is not the limiting factor behind the concept of metabolic advantage. On the contrary, thermodynamics The author(s) declare that they have no competing interests.


Cholecystokinin (CCK) concentrations in plasma have been shown to be significantly higher in colectomised subjects compared to healthy controls. This has been ascribed to reduced inhibition of CCK release from colon. In an earlier study CCK in all but one woman who was colectomised, induced release of oxytocin, a peptide present throughout the gastrointestinal (GI) tract. The aim of this study was thus to examine if colectomised women had a different oxytocin response to CCK compared to healthy controls.Eleven women, mean age 34.4 ± 2.3 years, who had undergone colectomy because of ulcerative colitis or constipation were studied. Eleven age-matched healthy women served as controls. All subjects were fasted overnight and given 0.2 μg/kg body weight of CCK-8 i.v. in the morning. Samples were taken ten minutes and immediately before the injection, and 10, 20, 30, 45, 60, 90 and 120 min afterwards. Plasma was collected for measurement of CCK and oxytocin concentrations.The basal oxytocin and CCK concentrations in plasma were similar in the two groups. Intravenous injection of CCK increased the release of oxytocin from 1.31 ± 0.12 and 1.64 ± 0.19 pmol/l to 2.82 ± 0.35 and 3.26 ± 0.50 pmol/l in controls and colectomised women, respectively (p < 0.001). Given the short half-life of CCK-8 in plasma, the increased concentration following injection could not be demonstrated in the controls. On the other hand, in colectomised women, an increase of CCK in plasma was observed for up to 20 minutes after the injection, concentrations increasing from 1.00 ± 0.21 to a maximum of 1.81 ± 0.26 pmol/l (p < 0.002).CCK stimulates the release of oxytocin in women. There is no difference in plasma concentrations between colectomised and controls. However, colectomy seems to reduce the metabolic clearance of CCK. The hyperCCKemia in patients who had undergone colectomy is consequently not only dependent on CCK release, but may also depend on reduced clearance. The gut hormone cholecystokinin (CCK) is synthesised in endocrine I cells in the mucosa of the upper small intestine  and is rOxytocin is synthesised in the supraoptic and paraventricular nuclei of the hypothalamus as part of a larger precursor polypeptide . While tWe have recently found that CCK also leads to oxytocin release in healthy women . HoweverEleven women from the Departments of Medicine and Surgery at Malmö University Hospital  were studied. They had all a history of colectomy. Two were colectomised because of slow transit constipation (STC) and had undergone a subtotal colectomy with the creation of ileo-rectal anastomosis. Proctocolectomy with ileal pouch-anal anastomosis had been performed in seven of them because of ulcerative colitis, and one because of familial multiple polyposis. The last patient has an ileostomy after subtotal colectomy, saving the rectum, because of ulcerative colitis. Thus, the subjects were cured from their original conditions. The time interval between the proctocolectomy/colectomy and this study was 10–149 months, with a mean of 48.5 ± 12.6 months. Eleven age-matched healthy women with preserved GI tract served as controls. Physical examination and laboratory routine screening were all within normal limits in both groups. The body weight was 68.7 ± 5.8 kg in the patients and 73.8 ± 6.1 kg in the controls. No drugs and no oral contraceptives or other hormonal treatments were allowed in either group. The experiments were performed at no specific stage of the menstrual cycle. None of the included subjects had participated in our former study [The protocols were approved by the local Ethics committee at the University of Lund, and written informed consent was obtained from all subjects before the study was started. The possibility of pregnancy was excluded in all women.All subjects were fasted overnight. In the morning they were given 0.2 μg/kg body weight of cholecystokinin octapeptide (CCK-8)  as an intravenous injection. This bolus was chosen as it was the only dose giving raise to a weak, but not significant, oxytocin release in an earlier study . Blood sAll blood samples consisted of 8.0 ml whole blood drawn into iced heparinised tubes. The plasma was separated and frozen at -20°C immediately after the experiment. Oxytocin was measured as described by Balment et al . The limThe values are expressed as mean ± standard error of the mean (SEM). The basal value is the mean of the two fasting values. The peak value is the mean of the highest concentration in every subject after the injection. The total plasma CCK and oxytocin response was assessed by calculating the area under the plasma concentration time curve (AUC). The Kruskal-Wallis followed by Wilcoxon signed ranks test were used for assessment of the significance of the differences within and between the two groups. The Spearman rank test was used for calculating the correlation between CCK and oxytocin concentrations in plasma. Probabilities of less than 0.05 were considered significant.The basal oxytocin concentration in plasma was similar in the two groups. The concentration was stable before the start of the experiments. Injection of CCK-8 led to an increase of the oxytocin secretion compared to basal values in both groups p < 0.001) . Neither was there any difference in CCK and oxytocin concentrations between patients with different diagnosis and those who had rectum saved or resected (data not shown).This study shows for the first time that CCK-8 increases the secretion of oxytocin in women. We have previously shown that exogenous CCK-33 and -39, and a fatty meal with endogenous CCK release, led to enhanced concentrations of oxytocin in plasma . One patCCK has been shown to stimulate oxytocin secretion in mammals in many studies -14. HoweOxytocin is present in plasma in men, although at lower concentrations ,25, and Several studies have reported that after colectomy in different species there are higher concentrations of CCK in plasma, both basal and postprandial, compared to healthy controls -17. It hCCK has a wide range of effects on the GI tract. Three physiological effects on gut motility have been identified; contraction of the gallbladder , relaxatCCK stimulates the release of oxytocin in women, probably via an effect on the neurohypophysial system. There is no difference in plasma concentrations between colectomised women and women with intact GI tract. The hyperCCKemia observed in patients who have undergone colectomy is dependent not only on an increase in CCK release, but may also depend on a reduced degradation. It was beyond the aim of our study to determine the clearance of CCK. However, this should be evaluated further.AUC = area under the curveCCK = cholecystokininGI = gastrointestinalPYY = peptide YYSEM = standard error of the meanBO designed the study, included patients, paid for the most, performed the statistical analysis and drafted the manuscriptJR carried out the radioimmunoassay for CCK and participated in the writing processMF carried out the radioimmunoassay for oxytocin and participated in the writing processAll authors read and approved the final manuscript.The authors declare that they have no competing interests.The pre-publication history for this paper can be accessed here: