The model, its strengths and its weaknesses are discussed with reference to four examples of functional ncRNA sequences: a riboswitch (guanine), a zipcode (nanos), a splicing factor (U4) and a ribozyme (RNase P). As shown by our visualisations of posterior probability matrices, the selected examples illustrate three different signatures of natural selection that are highly characteristic of ncRNA: (i) co-ordinated basepair substitutions, (ii) co-ordinated basepair indels and (iii) whole-stem indels.Although all three types of mutation "event" are built into our model, events of type (i) and (ii) are found to be better modeled than events of type (iii). Nevertheless, we hypothesise from the model's performance on pairwise alignments that it would form an adequate basis for a prototype multiple alignment and genefinding tool. For each of these scenarios, we can construct a probabilistic model Mx, My, Mz... and compare the likelihood of the observed data under each of these models. The model with the best fit indicates the type of functional element present in the sequence.One of the promises of comparative genomics is to annotate previously undetectable functional signals in genomic sequence, by identifying and characterising evolutionarily conserved elements. A principled way to extract such signals is by fitting the data to probabilistic models of the molecular evolutionary process. The logic runs as follows: suppose there are various kinds of conserved element RNA), protein-coding exons , and unidentified DNA homology . The former (RNA) was modeled using a Pairwise Stochastic Context-Free Grammar (Pair SCFG); the latter two (COD and OTH) using Pairwise Hidden Markov Models (Pair HMMs). The noncoding RNA predictions generated a high yield of experimental hits, and offered an information-theoretic glimpse into a modern-day RNA world [A groundbreaking example of how this probabilistic approach can be used is the QRNA program, designed as a comparative RNA gene predictor . The thrNA world .ad hoc number of bins based on the percentage sequence identity. Alignments in the same bin (i.e. having comparable sequence identity) then represent pairs of sequences at approximately equivalent distances. For example, the BLOSUM62 substitution matrix was estimated from pairwise alignments with at least 62% identity. This sort of approach is used by the RIBOSUM basepair substitution matrices developed for RSEARCH [It is natural to consider how such an approach might be applied to a pairwise comparison where the evolutionary "distance" between the two sequences can vary. One approach, analogous to the BLOSUM series of BLAST matrices for proteins , is to p RSEARCH , recent time measurement, by postulating an underlying evolutionary stochastic process or continuous-time Markov chain whose mutation rate parameters are constant over time . This evolutionary rate approach uses fewer parameters – and makes fuller use of the data – than the dividing-into-bins approach, since it postulates an infinitesimal generator for all time-scales of the process. For the PAM series, this generator takes the form of an instantaneous substitution rate matrix; for a primary-sequence model, the generator is a conditionally-normalized Pair HMM or transducer [evolutionary rate-based models, although  they're less suited to quick software prototyping than the "bin-by-percent-ID" approach.An alternative approach, analogous to the PAM series of BLAST matrices , is to tansducer ; for an ansducer . It's thOTH model, for noncoding DNA sequence, is a Pair HMM with affine gaps; the closest evolutionary equivalent is the "long indel" model [COD [RNA [structure to be described within an evolutionary rate framework.With this in mind, we can consider the evolutionary rate-based equivalents of the three pairwise grammars used in QRNA. The l" model ,9. The ll" model . In contdel [COD  and RNA COD [RNA  models dCOD [RNA ,13 suggeπ-orbitals between adjacent stacked bases in RNA double helices. The fact that the model appears to work reasonably well, despite the exclusion of such features, suggests that very simple models of RNA evolution may turn out to be sufficient to uncover a surprisingly large proportion of RNA sequence homology.A computer program for simultaneous pairwise alignment and secondary structure prediction using the TKF91 Structure Tree has been developed in C++. The potential of the model for RNA sequence alignment has been demonstrated by testing the pairwise aligner on four functional elements from the RFAM database : the purWe begin by reviewing the TKF91 model . This moet al, is time-reversible. This has the implication, called the pulley principle by Felsenstein, that the position of an ancestral node in a phylogenetic tree can be slid around like a pulley without changing the likelihood of the observed data [The TKF91 model, as defined by Thorne ved data . AligninThe TKF91 model can be analysed algebraically , and theTKF91 link sequence: a permanent immortal link at the left end of the sequence, followed by zero or more mortal links. Over time, mortal links can be deleted, and new mortal links can be inserted to the right of either immortal or mortal links. This can be treated as a birth-death process  with constant immigration (λ0), where "births" are identified with single-link insertions occuring to the immediate right of the parent mortal link. and "immigration" with insertions immediately right of the immortal link.The state of the TKF91 process is described by a singlet nucleotide alphabet, Ω = {A, C, G, U}. Each site's alphabet label evolves as an independent four-state reversible continuous-time Markov chain (RCTMC) with substitution rate R0 from state i to state j. Labels for newly inserted mortal links are drawn from the equilibrium distribution p0(i) of this substitution process. By reading off the labels of mortal links, the state of the TKF91 process can be equated to a sequence in Ω*.A further site-independent labeling is introduced on mortal links using the λn, μn) arise in analyses of equilibrium and transition probabilities in the TKF91 model [t is a time parameter.The following functions of (91 model . Here t Rnt) ≡ exp(A) is the exponential of the matrix with elements Aij = Rnt.Here exp an insertion and a deletion; κn is the probability of continuing the ancestral sequence; and Mn is the conditional substitution probability from i to j. Note (1 - γn)κn(1 - αn) = βn . Note also limt→∞ βn = κn.The meaning of the above functions is as follows. κ0. The residues at individual positions of the sequence are independently, identically distributed at equilibrium and are sampled from the equilibrum distribution of the point substitution process.The equilibrium probability distribution over sequences in the TKF91 model is a geometric distribution with parameter P(d|a), while the joint probabilities can be found by multiplying the expressions in columns P(a) and P(d|a) to obtain P.The TKF91 singlet grammar is shown in Figure Various extensions to TKF91 have been proposed ,9,15. ThTKF91 Structure Tree, and which is shown in Figures singlet, paired, loop or stem node. The tree can be broken into overlapping loop sequences and stem sequences, which correspond to strands of unpaired RNA (loops) or double helices of basepaired RNA (stems). Loops are allowed to contain unpaired nucleotides, and can also serve as a branching-off point for nested stems. Stems, on the other hand, are allowed to contain paired nucleotides, and are terminated by a loop . The tree is rooted by a loop sequence. The above description will now be made more precise.Consider the following extension to the TKF91 model, which we call the The state of the TKF91 Structure Tree is described by a rooted tree where each node has degree ≤ 3.There are four basic kinds of node in the tree: singlet, paired, loop and stem.2) represent covariant basepairs.Singlet and paired nodes correspond to observable nucleotides. Singlet nodes (labeled from Ω) represent independently evolving nucleotides, as in TKF91. Paired nodes (labeled from ΩL), of which the root node is one, are present at the beginning of loop sequences, which contain singlet and stem nodes and are written horizontally. Stem nodes (labeled S) are present at the beginning of stem sequences, which contain paired nodes, are terminated by a loop node, and are written vertically.Loop and stem nodes determine the tree structure Figure . Loop no2 ∪ Φ.The set of loop and stem node labels is written Φ. The full set of node labels is Ω ∪ ΩL, S}Φ = {A, C, G, U}Ω = {2 = {AA, AC, AG, AU, CA, CC, CG, CU, GA, GC, GG, GU, UA, UC, UG, UU}Ωimmortal loop link followed by zero or more mortal loop links. The mortal links are inserted and deleted with rates λ1 and μ1, in the style of TKF91. Each link is also a node in the Structure Tree.A loop sequence is very similar to a TKF91 link sequence: as with TKF91, we have a leftmost L, while the mortal loop links are labeled from {A, C, G, U, S}. As with the TKF91 model, the alphabet labeling of each mortal link evolves as an independent five-state RCTMC with substitution rate R1 from i to j and equilibrium probability p1(i) of being in state i, plus the additional restriction that R1 = R1 = 0 for all X ∈ Ω: in other words, embedded stems can't interconvert with singlet nucleotides. See step 1 → 2 of Figure Links are labeled from Ω ∪ Φ: the immortal loop link is labeled S-labeled links possess an independently evolving embedded stem sequence that can be considered to "nest" inside the loop sequence. If the S-link is deleted, then the embedded stem  is deleted with it. Conversely, when a new S-link is inserted, it is inserted with a complete subtree that is sampled from the equilibrium distribution over Structure Trees. See steps 6 → 7 and 7 → 8 of Figure The S link), it obeys the same statistics as a TKF91 sequence. In particular, the probability distribution over loop lengths at equilibrium is a geometric distribution with parameter κ1.Since a loop sequence is effectively a TKF91 sequence with a special "fifth nucleotide" character representing an embedded stem  in Figure 2 ∪ Φ: the immortal stem link is labeled S (this is the node in the parent loop sequence), the mortal links are labeled with the paired nucleotide alphabet Ω2  and equilibrium p2(i)), and the terminating loop link is labeled L. The mortal stem links experience TKF91-style insertion and deletion with rates λ2 and μ2  but possesses an independently evolving loop sequence. See step 2 → 3 of Figure Links are labeled from ΩS, is only immortal from the point of view of the stem sequence beneath it. The S is itself a mortal link in a parent loop sequence, and may be deleted as that sequence evolves. In this event, the loop link L will also be deleted, along with all its children  and the memory complexity O(L2), while for Figure O(L3M3) and the memory complexity O(L2M2), where L and M are sequence lengths. This is also the complexity of the single-sequence and two-sequence Sankoff algorithm [Dynamic programming alignment of sequences to these grammars has the typical complexity for single-sequence  and pairlgorithm , for whilgorithm ,21, thatlgorithm .We now describe some transformations of Figures The presence in a grammar of "null cycles" – sequences of production rules which cause no net change – complicates the parsing algorithms for that grammar. Generally, null cycles are avoided by programmers designing SCFGs or HMMs for sequence analysis . HoweverThere are several classes of null cycle in the grammars for the Structure Tree model, shown in Table degenerate rather than unique. Most stochastic grammars useful for bioinformatics are degenerate in the sense that there are always many folds or alignments consistent with the observed sequence data; this sort of degeneracy is technically called ambiguity. We are more concerned with other forms of degeneracy, such as structural degeneracy (multiple parses denote a single pattern of basepairing) and alignment degeneracy .As well as null cycles, there are other undesirable degeneracies in the Structure Tree grammars. Grammatical degeneracy occurs when more than one parse has the same meaning, so parses are L → LL and the "silent bulge" S → S (a null cycle). A full list of degeneracies for the singlet and pair grammars is shown in Table TKF91, in effect, skirts alignment degeneracy by assigning meaning to the ordering of deletions and insertions in an alignment, but alignment degeneracies arise in the Structure Tree model because there are multiple ways to delete and insert things . There are also structural degeneracies arising from "silent" (i.e. non-emitting) loops or stems. In addition to the null cycles described above, these include (for the singlet grammar) the undesirable "loop bifurcation"  before the corresponding Sk, copying all outgoing rules except the nonemitting Sk → Lk. This also removes the loop bifurcations, but leaves silent bulges of the form Sk → . The silent bulges can be removed by adding nonterminals  before Lk, copying all outgoing rules except Lk → ε, changing  to  so as to prevent escape from  without an unpaired emission, and adding new rules of the form  to allow escape if there is a genuine bifurcation.The null cycles all involve zero-length stems and can be broken  by adding extra nonterminals A more careful analysis, marginalising null cycles and silent bulges rather than simply ignoring them, is almost certainly possible. for pair) the grammar includes nonterminals dedicated to bifurcations  and emissions . The separation of the nonterminals into null, bifurcation and singlet/pair emission sets puts the grammar in the form understood by the DART library [1234 for pair states.Figures  library . The fulO(L3) and the memory complexity O(L2), while for Figure O(L3M3) and the memory complexity O(L2M2), where L and M are sequence lengths. Again, the complexity may be reduced by the use of "banding" [The asymptotic complexity of the dynamic programming recursions implied by these grammars is unchanged by the transformation to DART form. For Figure banding" ,21 or otbanding"  constraiThe Expectation Maximisation (EM) algorithm is often used for training BLOSUM-like models, e.g. estimating emission and transition probabilities for Pair HMMs  or Pair Rn using the EM algorithm, given the following sufficient statistics:The EM algorithm for the TKF91 Structure Tree can be separated into two parts, one for the substitution process and one for the indel process. Earlier work  showed h, the expected number of insertions of state d;, the expected number of aligned sites with ancestral state a and descendant state d.λn, μn for a TKF91 model using the EM algorithm, given the following sufficient statistics:A forthcoming paper describes how to estimate the maximum-likelihood indel rates , the expected number of deleted links not followed by an insertion;, the expected number of surviving links not followed by an insertion;, the expected number of deleted links followed by an insertion;, the expected number of surviving links followed by an insertion.P, and suppose that  is the posterior expectation of the number of times rule m of Figure  be the expected number of times rule m was used to emit the specific nonterminals X, Y ... ∈ Ω. ThenWe can calculate all the above update statistics simultaneously from data (the E-step) using a constrained version of the Inside-Outside algorithm  for the P(d|a), is used.The terms in parentheses are to be omitted if the conditional normalisation, ,  and the expected start, wait and transition usage  of the previous work [The relationship between the expected insert and match usage ous work  is as fo is defined as in the previous work [where ous work The pairwise aligner for the TKF91 Structure Tree is distributed as part of the DART package at the following URL:The aligner is based on the Stochastic Context-Free Grammars (SCFGs) shown in Figures Drosophila , (1B) For each of the two sequences  taken individually, the secondary structure was predicted without the aid of comparative information from the other sequence, using the single-sequence SCFG of Figure (2) The two sequences  were then aligned using the TKF91 model, without making use of any model of RNA structure, using the Pair HMM of Figure (3) Finally, the two sequences  were aligned using the TKF91 Structure Tree model introduced in this paper, using the Pair SCFG of Figure These computations allow a comparison between the TKF91 model, the single-sequence SCFG of Figure (a) This triangular dotplot illustrates the single-sequence structure prediction for sequence A of computation (1A). The pixel color at co-ordinates  represents the posterior probability that residues x and y of A are base-paired, in the absence of any information from sequence B.(b) This triangular dotplot illustrates the single-sequence structure prediction for sequence B of computation (1B). The pixel color at co-ordinates  represents the posterior probability that residues x and y of B are base-paired, in the absence of any information from sequence A.(c) This rectangular dotplot illustrates the structure-free pairwise alignment of computation (2). The pixel color at co-ordinates  represents the posterior probability that residue x of A is homologous to residue y of B, in the absence of any structural information from the two sequences.(d) This triangular dotplot illustrates the comparative structure prediction for sequence A of computation (3). The pixel color at co-ordinates  represents the marginal posterior probability that residues x and y of A are base-paired, summed over all alignments to sequence B.(e) This triangular dotplot illustrates the comparative structure prediction for sequence B of computation (3). The pixel color at co-ordinates  represents the marginal posterior probability that residues x and y of B are base-paired, summed over all alignments to sequence A.(f) This rectangular dotplot illustrates the structural pairwise alignment of computation (3). The pixel color at co-ordinates  represents the marginal posterior probability that residue x of B is homologous to residue y of A, summed over all secondary structures of sequences A and B. Note that the orientation of this plot is flipped  relative to (c).In addition, the "true" (published) structures and alignments are overlaid on the computational results as blue squares .The rate parameters used for the TKF91 Structure Tree were obtained by maximum likelihood training from a random selection of structurally-annotated RFAM alignments, as follows:λ1 = 0.027, μ1 = 0.03; λ2 = 0.007, μ2 = 0.01; p1(S) = 0.01. The substitution rate parameters were taken from the PFOLD program [ program . The evoThe posterior probabilities of folding and alignment (dotplots a-f) obtained by DP on these three classes of element are shown in Figure cis-acting regulatory elements that specifically bind adenine or guanine and are involved in the post-translational regulation of purine transport and biosynthesis [Bacillus halodurans and Streptococcus pneumoniae, which was taken from the RFAM database [The purine riboswitches are a class of ynthesis . Figure database . The twoFigure Drosophila nanos gene, involved in post-translational degradation and transport of nanos mRNA, which localises to the posterior of oocytes and other cell lines [Drosophila virilis and Drosophila melanogaster, which was curated by hand from the description by Gavis et al [The translational control element (TCE) is a regulatory sequence from the 3' untranslated region of the ll lines . Figure is et al . The twoFigure Tetrahymena thermophila and Leptomonas collosoma, was taken from the RFAM database [Leptomonas U2 has a deletion of roughly 35 bp that eliminates an entire stem  covariant substitutions and indels in RNA sequence contingent upon a particular secondary structure, and (ii) changes in the underlying RNA secondary structure, corresponding to gain and loss of substructures. A pairwise alignment algorithm based on the model has been implemented in C++ and tested on four homologous pairs of RNA functional element from RFAM . As with.It should be noted that the present implementation of the TKF91 Structure Tree is not designed to be a direct competitor to programs like FOLDALIGN , DYNALIGThe results of our tests on pairwise alignments from RFAM reveal the strengths and weaknesses of our model. When RNA structure is very strongly conserved and indels are few, as with the purine riboswitches selected for this comparison Figure , the TKFIn view of the room for improvement, we can identify a number of weaknesses of the TKF91 Structure Tree that could be improved in future models:• Sources of degeneracy such as zero-length stems and loops were removed "by hand" from the Pair SCFG Table . These d• Indel rates for whole stems/multistems are same as for unpaired residues. In nature, stem gain and loss is much slower than unpaired residue insertion/deletion, since the former is a structural change while the latter is not.• Multiple-residue indel events, and hence affine gap penalties, are not allowed. Again, the poor performance on the RNase P alignment may in part be due to this: the alignment generated has many small gaps scattered throughout, whereas the "true" alignment has fewer, longer gaps. This is a characteristic artefact of using a point indel model  where a multi-residue indel model  would be more appropriate.• Stems cannot be deleted without deleting all their "children" as well . Empirical inspection of alignments in RFAM, however, reveals many structures where an outer stem has been deleted or truncated, while inner stems are preserved. Again, perhaps an affine gap penalty for covariant indels (i.e. indels in stems) would address this. Alternatively, one might contrive some kind of "ragged-end" local alignment model, e.g. by embedding the finite TKF91 Structure Tree in an infinite, unobserved Structure Tree (c.f. ), thoughπ-orbital conjugation. Mathematically, the complexities of modeling such effects are somewhat similar to those involved in modeling nearest-neighbor substitution biases in DNA (such as methylation-induced CpG deamination). Since recent progress has been made with such models [• The equilibrium distribution over structures is highly simplified. For example, there is currently no modeling of fine-scale energetics such as basepair stacking propensities due to h models ,30 we miS → L → S transition, where the L is empty. To fix this, L nodes could be allowed in stem sequences, just as S nodes are allowed in loop sequences . However, this would increase the potential for degeneracies.• Bulges cannot be inserted into stems, except via the following awkward mechanism: the insertion of a bulge into a stem requires the pre-existence of a null • We have assumed that all stems and loops evolve at the same rate, whereas empirical inspection of RFAM of suggests otherwise. It is known that the analogous assumption in proteins  can skew phylogenetic distance estimation , and perL- or S-branch.• There is no special treatment of structural features such as triloops, tetraloops, triple-A platforms, U-turns and the like. Such features are often observed to be evolutionary conserved ,34 and s• While the lengths of stem sequences are geometrically distributed in the TKF91 Structure Tree, due to their roots in the TKF91 model, empirical observations of real RNA structures suggest that real stem lengths follow a fairly tight length distribution. Such approximations in modeling stem lengths could conceivably contribute to poorer performance of the model. Despite these drawbacks, the results of our preliminary benchmark suggest that the TKF91 Structure Tree may be useful for aligning (at least the better-conserved) RNA functional elements. Given the recent growth of RNA sequence and structure databases such as RFAM  and SCOR


Two new books on speciation update the classic texts by Mayr and Grant and help set the stage for a renaissance of research into one of the most important processes in evolution Plant Speciation [Animal Species and Evolution [Despite the biases that a single author inevitably brings to a subject, only one or a few closely interacting authors can bring coherence, synthesis, and vision to a broad and complex topic. A symposium volume just doesn't do the job. Few topics in biology are as simultaneously encompassing, complex, and controversial as the origin of species, i.e., speciation. Speciation is, after all, the process responsible for biological diversity, at least of sexual organisms, so it is hardly a minor topic. But even though recent years have seen the publication of symposia on speciation and books on the ever-contentious issue of species concepts, it has been 23 years since Verne Grant's authoritative eciation  and 41 yDrosophila, so it is hardly surprising that their treatment of speciation bears a strong genetic emphasis and draws heavily on Drosophila work . Even the most drosophilophobic readers, however, will be pleased by the extent to which Coyne and Orr have conscientiously scoured the literature on nonmodel animals and plants.These books are very different, but wonderfully complementary. Gavrilets reviews and adds to mathematical theories and simulation studies of speciation and related issues, such as fitness landscapes and selection in heterogeneous environments. A deep reading of his book will require considerably more mathematical competence than most evolutionary biologists (including this reviewer) have, but Gavrilets provides excellent verbal explanations of the models' assumptions and conclusions, as well as comparisons and critiques of related models. Gavrilets cites empirical studies (with which he has very broad familiarity) plentifully, but as a theoretician, he does not evaluate them or describe them in depth. That task is undertaken by Coyne and Orr, who introduce most topics with a verbal overview of theory, review empirical evidence and its bearing on hypotheses, and conclude with incisive assessments of what they think we know and what remains uncertain or unexplored. Like Gavrilets, they offer a number of novel ideas or suggestions about how to proceed. Coyne and Orr have both worked mostly on speciation genetics in To appreciate the landmark status of these books, consider what has happened in speciation studies since Mayr and Grant published theirs. Mayr and Grant articulated positions on species and speciation that had developed during and soon after the Modern Synthesis of the 1930s and 1940s, when modern evolutionary theory developed from a reconciliation of genetics, systematics, and paleontology. Mayr and Grant drew on abundant systematic data on patterns of divergence and experimental data on genetic differences between related species. They rightly identified reproductive isolation (RI) as a critical, even defining, property of species, and allopatric divergence  as the major geographic mode of speciation. They recognized that trait differences between species, including RI, usually have a polygenic basis, and that different coadapted  sets of genes underlie incompatibility . They emphasized the role of ecological selection as a driving force in speciation, largely by extrapolation from the primacy of selectionist thinking that developed during the Synthesis. They accepted that natural selection can reinforce prezygotic isolation  between species and thereby reduce production of unfit hybrids, even if Mayr did not share Dobzhansky's belief that this was the norm. Mayr combined selection with genetic drift in his theory of founder-effect speciation , which became widely accepted.  Molecular studies have provided important data on such issues as the absolute dates of speciation events, the duration of speciation, and the time course of the evolution of RI. The field of molecular phylogeography, which documents the history of spatial isolation and geographic expansion of populations, has developed. The relation between range overlap of related species and their molecularly dated time of divergence provides some evidence on the role of geographic versus sympatric speciation . In all these areas and others, our knowledge has increased steadily. For instance, molecular markers enable more detailed dissection of the genetic architecture of species differences, and support the conclusion that they are usually rather highly polygenic, but that much of the variance can be explained by a few major gene substitutions. We now have good evidence, as Coyne and Orr emphasize, that at least in animals, hybrid infertility is caused by differences in gene action, not by structural chromosome differences or failure of meiosis.Regarding the mechanisms of speciation, evidence for the role of divergent ecological selection in allopatric speciation is sparse, because this crucial topic has been unaccountably neglected until recently. Very different kinds of data, ranging from DNA sequences to correspondence between RI and ecological divergence, support natural selection, but there is hardly enough evidence, in my opinion, to support Coyne and Orr's strong conclusion that “at least one important debate has been settled: selection plays a much larger role in speciation than does drift” (p. 410). Even more astonishing than the paucity of studies of the role of ecological selection in speciation is the fact that the likely role of sexual selection was not even recognized until almost 20 years after Mayr's book. I agree with Coyne and Orr that the theory and evidence for speciation by sexual selection is one of the most important advances in speciation studies, but it is important to recognize that the evidence consists mostly of correlations between diversification rates and indices of the likely strength of sexual selection; as Coyne and Orr note, there are no cases in which we understand just how sexual selection has caused speciation. This is a rich, largely unexplored area. Gavrilets feels that divergent evolution by sexual conflict  is a potentially important process, whereas Coyne and Orr are skeptical that this will prove widespread. Coyne and Orr remark that populations may diverge in male signals because of intrasexual selection , and that female mate preference may follow. Quite so, but even though Berglund et al.  summarizDespite their very different approaches, Coyne and Orr and Gavrilets arrive at rather similar conclusions on some of the most controversial issues in speciation. One such is the role of genetic drift in speciation. Gavrilets analyzes founder-effect speciation (which combines drift and selection), agrees with most other theoreticians   that it not responsible for an evolutionary event  is, of course, far heavier than the burden of demonstrating selection; indeed, Coyne and Orr do not address the difficult question of what would constitute evidence for drift. Having, perhaps, stacked the deck, Coyne and Orr find almost no evidence that drift has contributed to speciation in nature, but conclude that there is “considerable evidence” that selection has done so. However, the amount of evidence is hardly on a par with, say, the evidence for allopatric speciation. It consists of only about eight studies of ecological selection, indications that diversification rates are associated with greater scope for sexual selection, selection signatures in a few genes that underlie genetic incompatibility, and a paucity of molecular evidence for bottlenecks  in the history of recently formed species. But the evidence on the role of sexual selection is very indirect, and the high levels of genetic variation revealed in molecular studies argue against past bottlenecks only if this is ancestral variation, rather than variation generated anew since a possible bottleneck—a question that has been addressed in only a few cases. Assuming that experiments with laboratory populations can be validly extrapolated to natural speciation processes, founder-effect speciation may indeed be a moribund hypothesis, but I do not believe long-term genetic drift can yet be ruled out, and cannot agree that this “important debate has been settled” (p. 410).Coyne and Orr appear to adopt selection as the null hypothesis for speciation, whereas drift is generally taken as the null hypothesis in much of evolutionary genetics, for the simple reason that drift operates at all loci in all finite  populations, whereas selection need not. The burden of demonstrating that selection is The geography of speciation continues to be one of the most difficult and contentious topics, and undoubtedly will remain so despite the careful analyses by these authors. They agree that parapatric speciation (evolution of RI between neighboring populations that exchange genes) is theoretically plausible, but Gavrilets notes that although it has become clear that its likelihood is sensitive to several model parameters, parapatric speciation is difficult to model and has been shamefully neglected. Coyne and Orr do not doubt that it is a fairly common mode of speciation, yet “it is almost impossible to demonstrate parapatric speciation in nature” (p. 118), and no cases have been well documented.Gavrilets provides an exhaustive analysis of the many models of sympatric speciation, and identifies some key issues that have been underemphasized. For example, the sympatric evolution of behavioral isolation by “matching traits”  is generally much more difficult than “similaritybased” mating . Just how common the latter is in animals is an open question that Coyne and Orr unfortunately do not address. Gavrilets also identifies the cost of female choosiness as a critical issue: many models of sympatric speciation depend on the assumption that females always succeed in mating even if the male type they prefer is rare, so their choosiness has no cost. Gavrilets criticizes some popular models of sympatric speciation on these and other grounds, and while granting that sympatric speciation by divergent habitat or host preference is plausible, he concludes that it need not be faster than allopatric speciation and that “contrary to common claims in recent theoretical papers, conditions for sympatric speciation are not wide and sympatric speciation does not occur easily” (p. 404).in statu nascendi. Coyne and Orr's conclusion echoes Gavrilets's: “It is hard to see how the data at hand can justify the current wave of enthusiasm for sympatric speciation” (p. 178). Bravi!For their part, Coyne and Orr feel that the prevalence of sympatric speciation is an empirical issue (but a very difficult one), and undertake a broad, detailed review. They identify three examples of completed speciation in which a sympatric scenario “seems plausible.” I see no reason to accept one of these cases, a pair of sister species of “parasites” (fig wasps) on the same host species, since allopatric speciation of a widespread parasite need not be accompanied by speciation of its host. Moreover, Coyne and Orr note weaknesses in all three cases, as well as in examples of “host races” that have been advanced as species Speciation are not only Coyne and Orr's comprehensive, scholarly coverage of an exceedingly broad subject, but also, and especially, their rigorous, incisive analysis, coupled with strongly stated conclusions and suggestions for how to resolve controversies. Many readers will have a visceral reaction against their position on sympatric speciation, reinforcement, founder-effect speciation, or other issues—but can these readers counter Coyne and Orr's arguments with equally cogent analysis? Or are these subjects that simply require more, and perhaps more imaginative, research?I have indicated some disagreements with Coyne and Orr, and could certainly cite others. But whatever weaknesses their book may have  are much less important than its strengths. The strengths of Speciation and the nonmathematical final chapter  of Fitness Landscapes and the Origin of Species to all evolutionary biologists, students, and professionals alike. It may not take another two decades for the next foundational books on speciation to appear, but these books will fill that role for a long time to come.Together, these books provide a comprehensive, thoughtful synthesis of our current understanding of one of the most important processes in evolution. They are required reading for anyone who studies species and speciation. I recommend


The mystery of a sometimes debilitating speech disorder is examined by cognitive neuroscientists Stuttering, with its characteristic disruption in verbal fluency, has been known for centuries; earliest descriptions probably date back to the Biblical Moses' “slowness of speech and tongue” and his related avoidance behavior . Stuttering occurs in all cultures and ethnic groups , althougstuttering usually refers to both the disorder and symptom.Stuttering is a disruption in the fluency of verbal expression characterized by involuntary, audible or silent, repetitions or prolongations of sounds or syllables . These aDevelopmental stuttering evolves before puberty, usually between two and five years of age, without apparent brain damage or other known cause (“idiopathic”). It is important to distinguish between this persistent developmental stuttering (PDS), which we focus on here, and acquired stuttering. Neurogenic or acquired stuttering occurs after a definable brain damage, e.g., stroke, intracerebral hemorrhage, or head trauma. It is a rare phenomenon that has been observed after lesions in a variety of brain areas .The clinical presentation of developmental stuttering differs from acquired stuttering in that it is particularly prominent at the beginning of a word or a phrase, in long or meaningful words, or syntactically complex utterances , and thePDS is a very frequent disorder, with approximately 1% of the population suffering from this condition. An estimated 3 million people in the United States and 55 million people worldwide stutter. Prevalence is similar in all social classes. In many cases, stuttering severely impairs communication, with devastating socioeconomic consequences. However, there are also many stutterers who, despite their disorder, have become famous. For instance, Winston Churchill had to rehearse all his public speeches to perfection and even practiced answers to possible questions and criticisms to avoid stuttering. Charles Darwin also stuttered; interestingly, his grandfather Erasmus Darwin suffered from the same condition, highlighting the fact that stuttering runs in families and is likely to have a genetic basis.The incidence of PDS is about 5%, and its recovery rate is up to about 80%, resulting in a prevalence of PDS in about 1% of the adult population. As recovery is considerably more frequent in girls than in boys, the male-to-female ratio increases during childhood and adolescence to reach three or four males to every one female in adulthood. It is not clear to what extent this recovery is spontaneous or induced by early speech therapy. Also, there is no good way of predicting whether an affected child will recover .The presence of affected family members suggests a hereditary component. The concordance rate is about 70% for monozygotic twins , about 3th century, abnormalities of the speech apparatus were thought to cause stuttering. Thus, treatment was based on extensive “plastic” surgery, often leading to mutilations and additional disabilities. Other treatment options were tongue-weights or mouth prostheses  is the method of choice to investigate fine-grained temporal sequence of brain activity. Consequently, MEG was used to investigate stutterers and fluent controls reading single words . ImportaThus, functional neuroimaging studies have revealed two important facts: (i) in stutterers, the right hemisphere seems to be hyperactive, and (ii) a timing problem seems to exist between the left frontal and the left central cortex. The latter observation also fits various observations that have shown that stutterers have slight abnormalities in complex coordination tasks, suggesting that the underlying problem is located around motor and associated premotor brain areas.Are there structural abnormalities that parallel the functional abnormalities? The first anatomical study to investigate this question used high-resolution MR scans and found abnormalities of speech–language areas (Broca's and Wernicke's area) . In addiAnother recent study investigated the hypothesis that impaired cortical connectivity might underlie timing disturbances between frontal and central brain regions observed in MEG studies . Using aThese new data also provide a theory to explain the mechanism of common fluency-inducing maneuvers like chorus reading, singing, and metronome reading that reduce stuttering instantaneously. All these procedures involve an external signal . All these external signals feed into the “speech production system” through the auditory cortex. It is thus possible that this external trigger signal reaches speech-producing central brain areas by circumventing the frontocentral disconnection and is able to resynchronize frontocentral decorrelated activity. In simple terms, these external cues can be seen as an external “pacemaker.”There are numerous outstanding issues in stuttering. If structural changes in the brain cause PDS, the key question is when this lesion appears. Although symptoms are somewhat different, it would be interesting to find out to what extent transient stuttering (which occurs in 3%–5% in childhood) is linked to PDS. It is possible that all children who show signs of stuttering develop a structural abnormality during development, but this is transient in those who become fluent speakers. If this is the case, it is even more important that therapy starts as early as possible if it is to have most impact. This question can now be answered with current methodology, i.e., noninvasive brain imaging using MRI.Given that boys are about four times less likely to recover from stuttering than girls, it is tempting to speculate that all stutterers have a slight abnormality, but only those that can use the right hemisphere for language can develop into fluent speakers. Language lateralization is less pronounced in women  and mighIt is unlikely that stuttering is inherited in a simple fashion. Currently, a multifactorial model for genetic transmission is most likely. Moreover, it is unclear whether a certain genotype leads to stuttering or only represents a risk factor and that other environmental factors are necessary to develop PDS. Again, this question might be answered in the near future, as the National Institutes of Health has recently completed the data collection phase of a large stuttering sample for genetic linkage analysis.


In a retrospective study of HIV-infected patients, we investigated the influence of the MDR1 genotype (G2677T/A and C3435T) on the virological and immunological response of treatment naïve patients.The MDR1 genotype was analysed from 72 patients in whom antiretroviral therapy was initiated between 1998 and 2004. Data were obtained at week 4, 12, 24 and 48 and were analysed by the Kruskal-Wallis test.During the first 48 weeks of antiretroviral therapy, there were no significant differences in the virological and immunological response with respect to the MDR1 2677 and 3435 genotypes and the 2677/3435 haplotype.In view of different results from several studies concerning the influence of MDR1 polymorphisms on the immunological and virological response to antiretroviral therapy, further studies with larger patient groups and longer follow-up are necessary in order to resolve conflicting issues. P-gp isgenotype . Howevergenotype -12. Howegenotype .The genetic variant C3435T in exon 26 is a synonymous polymorphism that does not alter the amino acid sequence. How this variant could affect P-gp expression is still unknown [Because of the unresolved issues surrounding the potential effects of MDR1 polymorphisms and P-gp function in HIV infection, we investigated whether there was an association between the MDR1 polymorphisms 3435 and 2677 and the immunological response in HIV infected individuals after initiation of antiretroviral therapy.Of the HIV 1 infected patients seen at the Department of Infectious Diseases of the University of Würzburg, Germany, 72 patients , started antiretroviral therapy between 1998 and 2004. The therapy consisted of three nucleoside reverse transcriptase inhibitors (NRTI) (n = 12), two NRTIs plus at least one PI  or two NRTIs plus one NNRTI (n = 20). HIV load and CD4 cell count were determined four weeks after initiation of therapy and approximately every three months thereafter. In patients who received NNRTI or PI drugs as part of their therapy, plasma levels were monitored and adjusted to therapeutic drug levels when necessary as previously described ,15. The The study was in accordance with the Helsinki Declaration and was approved by the local ethics committee. Patients gave informed consent for the study.DNA was extracted from 200 μl blood and the MDR1 3435 genotype was determined with genotype specific hybridisation probes and melting curve analysis on the LightCycler  as previously described . The 267P-value of 0.05 was considered to be significant. Analogous to [Data were analysed by the Kruskal-Wallis test, which as a nonparametric test compairs three or more unpaired groups. Because of the small number of samples a nonparametric test was needed. SPSS version 12.0  was used for statistical analysis. A ogous to , the SNPAt initiation of antiretroviral therapy 8 patients had viral loads of <10.000 copies/ml, 24 patients between >10.000 and <100.000 copies/ml, and 40 patients >100.000 copies/ml. 39 patients had a CD4 cell count of <200 cells/μl, 16 patients between >200 and <350 cells/μl, and 17 patients >350 cells/μl. Determination of NNRTI- and PI drug levels indicated compliance of the patients with a NNRTI or PI containing regimen, because the measured drug levels were in the therapeutic range. Genotype analysis of the MDR1 gene at position 3435 in exon 26 revealed 20 patients with the CC genotype, 33 with the CT genotype and 19 with the TT genotype . Analysis of the 2677 polymorphism in exon 21 demonstrated that 24 patients had the GG-, 23 the GT-, 18 the TT-, 4 the AG-, and 3 the AT genotype; the AA genotype was not found in this group . Detailed genotype and haplotype results with respect to the initial viral load and CD4 cell count are presented in table Table In an analysis of the virological and immunological response of treatment naïve patients with respect to the MDR1 G2677T/A and C3435T polymorphisms, virus load and CD4 cell count were assessed longitunally after initiation of antiretroviral therapy. We did not find an association between the CD4-cell increase or the HIV load decline and the MDR1 2677 or 3435 genotype during the first year of therapy.et al. that showed a significantly greater mean CD4-cell rise in patients with the MDR1 3435TT genotype during an observation period of 24 weeks [et al. selected part of the patients on the basis of long-term viral suppression. In both studies, a variety of antiretroviral regimens was used. While all of the patients in the study of Fellay et al. received either nelfinavir or efavirenz, the treatment regimens in our study were more heterogenous and did not allow a meaningful separate analysis.Our data are in contrast to a report of Fellay 24 weeks . The num24 weeks , 20CC/19et al. [The results of our study are supported by Nasi et al.  who analet al. .et al. [The MDR1 3435 polymorphism is a synonymous polymorphism. The TT genotype may have a reduced translation efficiency or lead to post-translational differences -23. It het al. , who shop = 0.07) with no effect of the C3435T polymorphism in the MDR-1 gene on immunological failure. However, the difference in the virological response was not observed during the first 10 months. Further follow-up of our patient group is ongoing in order to detect long-term effects that may not have been apparent during the observation period analysed in this report.In a retrospective study of 455 treatment naïve patients initiating antiretroviral therapy with 40 months of follow-up , there wDuring a follow-up of 48 weeks, we found no evidence for an association between the MDR1 G2677T/A and C3435T polymorphisms and the virological and immunological response to therapy in HIV-positive drug-naïve patients. The individual response to antiretroviral therapy is a complex phenomenon, which is influenced by a large number of biological variables. Further studies on the role of polymorphisms of the MDR1 and other transporters and enzymes involved in drug metabolism are necessary in order to elucidate the role of pharmacogenetic effects in HIV therapy.None declared.RW participated in the design of the study and the molecular genetic studies, performed the statistical analysis and drafted the manuscript. PL participated in the design of the study, obtained and reviewed clinical data and helped in the data analysis. MZ participated in the clinical management of the study patients. FT and JS participated in the molecular genetic studies and the virological and immunological analysis. HK participated in the study design and coordinated clinical management of the study patients. BW participated in the study design and coordination and made contributions to the manuscript. All authors read and approved the final manuscript.


Epidemiologic Perspectives & Innovations, provide a partial list of specific analyses and topic areas they would like to see submitted to the journal.As a supplement to our lead editorial, the editors of the new journal,  Epidemiologic Perspectives & Innovations (EP&I) present the underlying goals and philosophy of the journal, along with the types of articles we hope to publish. Here we clarify our goals with some specific ideas about articles we would like to see submitted to EP&I and with a "wish list" of methodological research and selected other innovations and perspectives we believe the field needs.In our lead editorial , the ediEP&I welcomes submissions of any papers in and of epidemiology, with the exception of those that solely report research results. "In and of" epidemiology includes papers about epidemiology and those grounded in other disciplines that might be useful for epidemiology. Many of our articles will be highly quantitative, but non-quantitative analyses are also welcome. We welcome submissions whose premises about how the field should be advancing differ from those of this editorial.This list is far from complete, and we want to emphasize that our intention is not to discourage submissions on topics not specifically mentioned here. EP&I hopes to help bridge the gap from best-practice methods to epidemiologic practice and teaching, and from there to epidemiology-based decision making.There is a gap between what methodologists know about doing epidemiology and standard epidemiologic practice. We believe that narrowing this gap is as important as the development of new methodological ideas. Papers that would narrow this gap are encouraged. These could take different forms, including: clear presentations of advanced methods that are underutilized , surveys of epidemiologic practice to assess where the gaps might be, papers that function as advanced textbook chapters, and teaching articles. We will give the highest priority to papers that achieve excellence in explanation.On a similar note, we would like to publish methods articles that fall into the category we call, "Don't Reinvent the Wheel." Other fields have different sets of methods that could be useful to epidemiologists, including observational methods from econometrics and related social sciences, uncertainty analysis methods from engineering and business, and historical perspectives from sciences with a longer history.Also on this theme, we plan to commission updates to classic methods papers from the original authors. Methodological knowledge is ever advancing, and it would be interesting and useful to see what changes an author would make to a must-read paper if that paper were written today. We welcome suggestions of candidates for this series.At the other end of the bridge, we invite methods and teaching papers that show how quantitative health researchers can present results that are more decision-relevant. We are especially interested in methods that go beyond statistical measures that emphasize merely whether an elevated risk has been found to ones that emphasize how much we ought to care about that elevated risk.EP&I have identified several specific methodological areas that we believe need attention. Without intending to restrict methodology submissions to these topics, we are particularly interested in submissions that deal with: (1) full and proper disclosure of uncertainty in study results, (2) decision-making in the face of this full disclosure, (3) identification and control of confounding, (4) quantifying the effect of random error on study results, and (5) specification error .The editors of Papers that fall into category (1) have begun to appear in the epidemiologic literature -11 but mWhile some fields have many papers that fall into category (2), including clinical decision making, there are very few in the epidemiologic literature.As a fundamental challenge for causal inference and a potential source of error in all observational study results, confounding is, and perhaps will always be, a thorn in the side of observational epidemiology . Papers Questions of how to think about, and quantify the effect of, random error in nonrandomized studies remain unresolved. A standard analysis pretends that researchers or nature randomized the study exposure to study subjects ,10. Is tStudy results are a function of both the data being analyzed and assumptions made in the process of the analysis -18. But EP&I. Epistemology in health science is often treated as a matter of statistical rules of thumb, with litigation serving as the ultimate arbiter. Neither statistical rules nor the results of lawsuits that go to trial are a particularly good source of knowledge.There is a substantial amount of epistemologic discussion in epidemiology, mostly relating to how we know if we are seeing a causal relationship and, to a lesser extent, what causation means. We hope that scientists in the field will become more actively engaged in practical epistemology , and we invite them to submit their work to Also falling under the broad category of philosophy is analysis of ethics. In the health sciences, such discussion is dominated by the issue of protecting human research subjects and patients. What is largely missing, and what we would like to invite submissions about, is the ethics of the core of our science: study design, data analysis, and result reporting. Since it is impossible to report every result that might be gleaned from a dataset, what constitutes an ethical choice of what to present? This question is central to most everything that most epidemiologists do, and yet there is remarkably little discussion about it . How manper se to include policy advocacy based on health science findings. Whether or not one takes that position, discussions of and about epidemiology should address questions of what society ought to do with our findings.  One interesting and timely set of questions involves the relative merits of decision-analysis or risk-analysis based decisions, "precautionary-principle" based decisions, and actual current practices.Many epidemiologists consider the field We seek to acknowledge the best epidemiologic studies and papers with articles that discuss what sets these studies apart from the rest: a "Best of Epidemiology" series. Much of methods training involves finding the worst in a study. We believe it would be useful to give students and other epidemiologists examples of how to do epidemiology well. What studies should we most want to emulate, and why?In a similar vein, we hope to receive submissions that remind us about the historical foundations of epidemiology: a "History of Epidemiology" series. What historical studies, insights, or methodology are important for understanding the field, and why, and which are simply interesting enough to be worth highlighting? History-of-science type analysis of our field, almost completely absent from the history-and-philosophy-of-science literature, would be particularly welcome.EP&I of such papers, whether or not a letter to the editor has been published. We encourage authors to take advantage of the speed of online publishing to submit re-analyses of important findings for publication while the implications of the original study are still being debated.  Readers of the health science literature may often find themselves asking a question about a study's results beyond the few specifically addressed in the published reports. A new analysis might be more relevant to a particular decision, or even out-and-out more informative than the original publication.A complement to the "best of" and recounting of historical high-points is reanalysis of particular study results or aspects of the field's literature more generally. Many substantive letters to the editor about published papers reflect a full article's worth of analysis by the letter authors. But those authors have little opportunity to publish that analysis, even when it has substantial practical or methodological importance; we encourage submission to EP&I article are encouraged to contact us in advance to see if we might solicit other related submissions to create a larger collection. Topics of particular interest to us, where we would like to encourage the starting of point-counterpoint series, include the usefulness of causal inference in epidemiology and the scientific basis for a particular regulatory decision . We especially encourage students to suggest potential topics.Finally, we hope to publish "Point-Counterpoint" collections on single topics. Authors considering writing a counterpoint (or simply a related point) to an We will grant that this is an ambitious research agenda. Perhaps it will be years before journals (this one or others) see articles on all these topics. But it is also possible that the next 100 dissertations in the field will include chapters that cover most of this ground. And perhaps those of us well past our dissertations can contribute a few also. We hope that asking (and providing a venue for publication) will make this much more likely.


Firstly, we will determine the additional benefit of true acupuncture when used by physiotherapists alongside advice and exercise for older people presenting to primary care with knee pain. Secondly, we will evaluate sham acupuncture in the same way. Thirdly, we will investigate the treatment preferences and expectations of both the participants and physiotherapists participating in the study, and explore the effect of these on clinical outcome. We will thus investigate whether acupuncture is a useful adjunct to advice and exercise for treating knee pain and gain insight into whether this effect is due to specific needling properties.Acupuncture is a popular non-pharmacological modality for treating musculoskeletal pain. Physiotherapists are one of the largest groups of acupuncture providers within the NHS, and they commonly use it alongside advice and exercise. Conclusive evidence of acupuncture's clinical effectiveness and its superiority over sham interventions is lacking. The Arthritis Research Campaign (This randomised clinical trial will recruit 350 participants with knee pain to three intervention arms. It is based in 43 community physiotherapy departments in 21 NHS Trusts in the West Midlands and Cheshire regions in England. Patients aged 50 years and over with knee pain will be recruited. Outcome data will be collected by self-complete questionnaires before randomisation, and 6 weeks, 6 months and 12 months after randomisation and by telephone interview 2 weeks after treatment commences. The questionnaires collect demographic details as well as information on knee-related pain, movement and function, pain intensity and affect, main functional problem, illness perceptions, self-efficacy, treatment preference and expectations, general health and quality of life. Participants are randomised to receive a package of advice and exercise; or this package plus real acupuncture; or this package plus sham acupuncture. Treatment details are being collected on a standard proforma. Interventions are delivered by experienced physiotherapists who have all received training in acupuncture to recognised national standards. The primary analysis will investigate the main treatment effects of real or sham acupuncture as an adjunct to advice and exercise.This paper presents detail on the rationale, design, methods, and operational aspects of the trial. Knee pain in older adults is a common disabling problem. Approximately 25% of the population aged over 55 years are affected at any one time and half of these will have some restriction of normal daily activities ,2. AfterPatients with musculoskeletal pain often choose methods of treatment that are not widely available within the NHS, such as complementary medicine . ReportsInternational guidelines suggest that the best package of care for this patient group is one that includes patient education, advice and exercise . There iThe physiological properties of acupuncture have been well described in the laboratory. Acupuncture activates central mechanisms of pain control and elicits release of specific neurotransmitters (mainly opioids) in the central nervous system ,35. EffeAppropriate sham interventions for acupuncture have been widely debated and several placebo needles have been introduced and tested ,48. A trWe have designed, and are currently implementing, a prospective sham controlled randomised trial within the primary care setting addressing the important clinical question: is acupuncture a useful adjunct to physiotherapy care (advice and exercise) for treating knee pain in older adults? Research and development in primary care is important to public health and necessary to support the decisions and treatments in this setting .The primary objective is to compare, at 6 months, the clinical outcomes of true acupuncture plus advice and exercise, with advice and exercise alone for treating people aged 50 years and over referred directly from primary care with knee pain. Our secondary objectives arei) to compare, at 6 weeks and 12 months, the clinical outcomes of adding true acupuncture to advice and exercise alone, in the same patient group.ii) to compare, at 6 weeks, 6 and 12 months, the clinical outcomes of sham acupuncture plus advice and exercise, with advice and exercise alone, in the same patient group.iii) to measure patients and physiotherapists beliefs, preferences and expectations about the treatments being tested and to explore their effect on clinical outcome.This multicentre, three-arm sham-controlled randomised trial will be conducted in 43 individual Physiotherapy Centres that provide services for Primary Care Physicians located in 21 NHS Trusts situated in the Midlands and Cheshire regions of the UK. Multi-centre ethical approval has been obtained from the West Midlands Multicentre Research Ethics Committee and local approval was given by 12 ethics committees . The trial was designed by a steering group with expert input from physiotherapists, an acupuncture specialist and trial methodologists. Information will be collected from the individual participating physiotherapists  prior to the commencement of the trial and after the trial has been completed  advice and exercise alone, (ii) advice and exercise plus true acupuncture, (iii) advice and exercise plus sham acupuncture. Follow up will be at 2 weeks (by telephone), 6 weeks, 6 months and 12 months after randomisation, by postal questionnaire. Non-responders will be followed up.Eligible patients are male and female subjects aged 50 years and above with pain (with or without stiffness) in one or both knees presenting to primary care. They must be naïve to acupuncture treatment (i.e. have never experienced acupuncture before for their present or any past complaints), and considered suitable for referral to a physiotherapy outpatients department by their general practitioner. Participants must be able to read and write English, be willing to consent to participation, and able give full informed consent. They must also be available for telephone contact.Patients with potentially serious pathology  on the basis of general practice or physiotherapy diagnosis or from past medical history, those who have had a knee or hip replacement on the affected side(s), are already on a surgical waiting list for total knee replacement, or for whom the trial interventions are contraindicated are excluded from the trial. Those who have received an exercise programme, from a physiotherapist, for their knee problem within the last 3 months  or an intra-articular injection to the knee in the last 6 months are also excluded.Eligible patients referred by their GP to the physiotherapy departments will be invited to take part. Recruitment will take place over 18 months and will operate in one of two ways  to further screen eligibility. Information will be recorded on a standard proforma. For those patients either not eligible or not willing to be recruited, the proforma will be used to detail the reason for ineligibility, or reason for decline. Where patients are willing, two additional questions are asked to those that decline to participate in the trial to capture their treatment preference and expectations with respect to acupuncture and an advice & exercise treatment package. This information will be anonymised.For patients willing to be recruited to the trial, an appointment is arranged for a research assessment at the patient's local physiotherapy department. After gaining verbal consent, the patient will be posted the baseline questionnaire to complete prior to their research appointment. At the research assessment visit the local physiotherapy assessor/trial nurse will perform a more detailed eligibility screen, explain the study, gain informed written consent to randomisation and conduct a baseline research interview and examination. Following consent to the study, the participant will be registered with the Research Centre by fax and allocated a unique trial number. The baseline assessment will be carried out blind to subsequent treatment allocation. An appointment will be made for the treating physiotherapist to begin treatment within 10 working days of the research assessment. Consent to treatment will be gained from each treating physiotherapist prior to commencing treatment, as is current physiotherapy practice. All participants will have an initial clinical physiotherapy assessment and treatment session of up to 40 minutes duration. During this session the physiotherapist will identify and record potential acupuncture points to be used should the participant be randomised to receive acupuncture (true or sham). A minimum of 6 and maximum of 10 points will be selected, based upon the participant's presentation and the clinical opinion of the physiotherapist. This will be carried out as part of the overall physical examination of the knee – the therapist will not draw the participant's attention to the localisation of acupuncture points to avoid raising their expectations about the possibility of receiving acupuncture. The advice and exercise package will then be started during this initial treatment visit.Randomisation will take place after this initial physiotherapy session. The treating physiotherapist will telephone the Research Centre at Keele University, during normal working hours. This methodology ensures that the initial physiotherapy assessment and advice and exercise package provided is performed blind to subsequent treatment allocation. The specific trial interventions will commence during the participant's second treatment visit. During the randomisation telephone call the physiotherapist will be asked to identify the selected acupuncture sites to check that the participant has received their first pre-randomisation treatment session. The physiotherapists will also be asked questions about their own expectations of the individual participant's likely clinical outcome and their beliefs about which treatment they would like the participant to receive. Participants recruited to the trial will then be randomised to one of the three trial interventions in a 1:1:1 ratio based on their unique trial number. Computerised third-party randomisation will be performed using random permuted blocks of 12 (blocked by treatment centre).The interventions will be delivered within 10 working days of randomisation by experienced physiotherapists, trained in acupuncture to at least the minimum standard for basic membership of the Acupuncture Association of Chartered Physiotherapists (AACP) (35 hrs of training). The participant's GP will be contacted at the time of randomisation and asked to avoid co-interventions for the period of the trial wherever possible, but especially until the 6-week follow up has been completed. However, if the GP feels that symptoms are sufficiently troublesome to need further treatment this will be at the GPs discretion. Information about co-interventions will be collected in the follow-up questionnaires and by review of a sample of participants' clinical records.arc Knee OA publication. This leaflet contains standard advice on the use of analgesia. If already using non-steroidal anti-inflammatory drugs, participants will be permitted to continue their stable dose. Participants will have the opportunity to discuss elements of the advice leaflet and exercise programme with their physiotherapist. In line with current practice, a maximum of 6 × 30-minute treatment sessions will be given over a period of 6 weeks. The advice and exercise programme has been developed through the use of reviews of current best evidence [Advice will be supplemented by a leaflet based on the evidence ,27,55, cevidence , a surveevidence , consensThe exercise programme will include concentric, eccentric, isometric and balance exercises. Specificity of training, particularly in the first 6 weeks, is important and therapists will aim for a mix of functional exercises, open & closed kinetic chain exercises and accelerated walking elements. They will also clearly identify a home exercise programme with set targets. The intensity of the exercises will be progressively increased at each session. Previously sedentary individuals who are relatively untrained will initially be prescribed exercise of a low intensity . Progression to medium and high intensity exercise will occur only once adaptation to the current level of training has occurred., a frequently used software package in physiotherapy. Hydrotherapy, group-based work, electrotherapy, additional acupuncture outside of the protocol and intra-articular injections will not be permitted. Participants may receive advice on the use of walking aids, and hot and cold applications. The key messages within the advice to participants include the common nature of knee problems and that rest for more than a day or two usually does more harm than good.Exercises will be prescribed and individualised for each participant by the treating physiotherapist from "Physio Tools" In addition to advice and exercise as detailed above, participants randomised to this group will receive 6 × 30-minute treatments of acupuncture, delivered over a period of 3 weeks. The acupuncture protocol is based on the concept of "treatment adequacy" which has been introduced by Ezzo et al  and MelcTreatment will be performed with sterilised disposable steel needles, 30 × 0.3 mm. The depth of the needle insertion should be between 0.5 – 2.5 cm depending on the points selected for treatment and the needles will be manipulated until de-qi sensation is achieved. Therapists allow 25 (min) to 35 (max) minutes between insertion of the last needle and cessation of treatment and during that time they are to revisit the needles as appropriate. If the sensation is maintained, they should manipulate each needle lightly, if the de-qi sensation is no longer there, they use stronger manipulation in order to elicit it.Participants will be informed that they may or may not experience an aching, warm or `tingling' sensation from this type of stimulation. Therapists question participants at each session to ask them to describe the sensation they feel on needling, which is then recorded on a standard proforma. This also collects information on attendance, failed appointments, physiotherapist's diagnosis and whether any additional treatment modalities or specialist referrals are made.In addition to advice and exercise as detailed above, participants randomised to this group will receive sham acupuncture which involves the placement of mock needles  upon a pThe points chosen for the sham intervention receive no stimulation and participants will be told, as for the true acupuncture group, that they may or may not experience any particular sensation from this type of stimulation. The same parameters as for true acupuncture apply: placement of a minimum of 6 needles, and 6 × 30 minutes sessions within 3 weeks, with monitoring of elicited sensations.Using a standard proforma, the physiotherapists record the number and duration of treatment sessions each participant receives, plus details about the advice and exercises prescribed, the location and number of acupuncture points (where applicable) and any adverse reactions. The sensation that needling (true or placebo) evokes has been shown to be a significant correlate of acupuncture-induced analgesia . Hence, the sensation evoked from each treatment in the acupuncture groups will also be recorded. Acceptability and credibility of the interventions will be evaluated using a telephone follow-up at 2 weeks from the beginning of treatment and a questionnaire administered at 6 weeks .Participants who give verbal consent to participate in the trial are posted a baseline questionnaire which they are asked to complete and bring with them to the research interview and examination. Information collected on this questionnaire is detailed in Table Outcome measures will be performed at 2 weeks Table , 6 weeksThe primary outcome measure for this trial is the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain sub-scale . We haveCollection of data and statistical analysis will be performed blinded to treatment allocation. Analysis will be performed on an intention to treat basis and the primary outcome will also be analysed on a "per protocol basis".Univariate analysis will be performed using t-tests to analyse numerical data and chi-square tests for categorical data. The clinical and demographic data collected at baseline will be inspected and if there are any important differences between the trial groups, these factors will be used as covariates. These analyses will be performed using ANOVA and logistic regression as appropriate.The analysis of the secondary outcomes will be exploratory. A univariate analysis with respect to the different treatment will be performed for the WOMAC pain sub-scale at 6-weeks and 12-months and for the WOMAC functioning sub-scale at 6-weeks, 6- and 12-months. The global outcome assessment with be analysed both as an ordinal and a dichotomous variable (categories 1–3 defined as "success"). Moreover, area under the curve slopes will be calculated for each treatment group over the whole treatment period and compared.Statistical significance will be set at the 5% level (two-tailed). Statistical analysis will be performed using Stata 7.0. The trial will be monitored by an independent Data Monitoring and Ethics Committee. No interim analysis of the primary or secondary outcomes will be undertaken during the trial period.The APEX trial is a major trial of physiotherapy treatment for knee pain. Obtaining participation by physiotherapists, across the regions of the West Midlands and Cheshire in 21 NHS Trusts, to work to agreed treatment protocols has been an important achievement. We have presented the rationale, design, and strategy for implementation of a multi-centre RCT examining whether acupuncture is a useful adjunct to usual physiotherapy care of advice and exercise for treating knee pain in older adults. The primary objective of the trial is to compare the clinical outcomes of true acupuncture plus advice and exercise, with advice and exercise alone for treating people aged 50 years and over referred directly from primary care with knee pain. The secondary objectives are to compare, at 6 weeks and 12 months, the clinical outcomes of adding true acupuncture to advice and exercise alone; to compare, at 6 weeks, 6 and 12 months, the clinical outcomes of placebo acupuncture plus advice and exercise, with advice and exercise alone; and to measure patients and therapists beliefs, preferences and expectations about the treatments being tested and to evaluate the association between these variables with clinical outcomes. The results of this trial will be presented as soon as they are available.The authors declare that they have no competing interests.All authors participated in the design of the trial and drafting the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Optimal pancreatic β-cell function is essential for the regulation of glucose homeostasis in both humans and animals and its impairment leads to the development of diabetes. Type 2 diabetes is a polygenic disease aggravated by environmental factors such as low physical activity or a hypercaloric high-fat diet.Free fatty acids represent an important factor linking excess fat mass to type 2 diabetes. Several studies have shown that chronically elevated free fatty acids have a negative effect on β-cell function leading to elevated insulin secretion basally but with an impaired response to glucose. The transcription factors PPARα, PPARγ and SREBP-1c respond to changing fat concentrations in tissues, thereby coordinating the genomic response to altered metabolic conditions to promote either fat storage or catabolism. These transcription factors have been identified in β-cells and it appears that each may exert influence on β-cell function in health and disease.The role of the PPARs and SREBP-1c as potential mediators of lipotoxicity is an emerging area of interest. Fatty acids are physiologically important both structurally, as components of phospholipids and glycolipids, as well as functionally, as fuel molecules. Metabolites of fatty acids, such as leukotrienes or prostaglandins, act as potent mediators in many biological processes. Fatty acids provide energy ,2, partiDiabetes affects 6 % of the adult population and, with a growth rate of 6% per year, it is estimated that 200 to 300 million people worldwide will be afflicted by the end of this decade , resultsType 2 diabetes, which accounts for 90 to 95 % of diabetes cases worldwide, is a heterogeneous disorder and its prevalence is rising. Type 2 diabetes is accompanied by chronic insulin resistance and a progressive decline in β-cell function  and is bFatty acids, not glucose, are the major endogenous energy source for unstimulated islets . This is2+-adenosine triphosphatase, leading to an increase in intracellular calcium concentration and augmentation of insulin secretion; and closure of the K+- ATP channel with resultant depolarization of the β-cell membrane, which causes an increase in intracellular Ca2+ and stimulation of exocytosis of insulin-containing granules [de novo fatty acid synthesis in the β-cell is very low [Short-term (2–6 hours) elevation of the plasma FFA concentration in human subjects  and animgranules . In addigranules ,21. The very low  indicatevery low . The totvery low , howeverin vitro and in vivo in animals [In contrast to the acute effect of elevated plasma FFA to enhance insulin secretion, longer-term (> 48 h) exposure results in an impaired β-cell response to glucose both  animals ,26 and h animals -31. The  animals , thus a  animals ,33. The  animals . In the  animals . Incubat animals .Free fatty acid metabolism responds to varying metabolic states partially by induction of enzymes that promote either catabolic or anabolic processes. There are two major classes of transcriptional regulators of enzymes involved in fatty acid metabolism, the peroxisome proliferator-activated receptors (PPARs) and the sterol regulatory element binding proteins (SREBPs), which both exist in several isoforms. In general, PPARγ and SREBP-1c regulate processes involved in lipogenesis whereas lipolytic enzymes are induced by PPARα .The PPARs form a subfamily in the nuclear receptor superfamily. PPARs, like other nuclear receptors, regulate gene expression in response to specific ligands through their actions as transcription factors. Peroxisomes contain PPAR-regulated enzymes involved in fatty acid β-oxidation . GeneticPPARα was the first member of this nuclear receptor subclass to be described. PPARα is expressed in numerous metabolically active tissues including liver, kidney, heart, skeletal muscle, brown fat -43, and PPARα plays an important role in the regulation of cellular uptake, activation and β-oxidation of fatty acids. The natural, preferentially-binding ligands of PPARα are long chain unsaturated fatty acids including arachidonic acid, linoleic acid, and oleic acid but saturated fatty acids like palmitic acid can also act as ligands . In hepaXenopus laevis [PPARδ was initially reported as PPARβ in s laevis . Subsequs laevis  as well s laevis  and was s laevis . Saturats laevis ,59. PPARs laevis . In skels laevis .PPARγ stimulates fatty acid storage in adipose tissue by increasing both the storage capacity and the fatty acid flux into adipocytes. PPARγ is expressed in many cell types, including epithelial cells, B and T cells, macrophages, endothelial cells, smooth muscle cells ,63 and pde novo, favours the formation of small adipocytes that tend to replace the large adipocytes normally constituting white adipose tissue [The natural ligands of PPARγ are several unsaturated fatty acids such as oleate, linoleate, eicosapentaenoic and arachidonic acids . Memberse tissue .PPARγ also negatively regulates transcription of several genes that impair insulin action, including tumor necrosis factor-α (TNFα) and leptin, proinflammatory cytokines produced by adipocytes and associated with insulin resistance -72. Thusin vitro on isolated islets may not apply to the in vivo situation. Therefore, the mode of delivery of the agents (directly onto islets versus in diets) and the time frame of study are important considerations.Both PPARα and PPARγ have been detected in pancreatic β-cells ,77. One in vitro or hyperglycemia in vivo suppresses expression [In pancreatic islets, exposure to long chain fatty acids (mixed unsaturated and saturated) induces PPARα expression  whereas pression ,80. Artipression ,81,82. Spression ,81, pyrupression ,81.The question arises as to the role of PPARα in the physiological regulation of insulin secretion. Its induction by long chain fatty acids and its ability to augment the insulin response to low glucose  suggestsIn addition to these postulated mechanisms of PPARα control over β-cell glucose and lipid metabolism, it has also been proposed that amino acid metabolism might be affected. In the liver, an increase in PPARα is associated with a decrease in amino acid catabolism . BecauseIn pathophysiological conditions involving deranged glucose and lipid metabolism, altered expression of PPARα may be important in the β-cell's lack of glucose responsiveness. In Zucker diabetic fatty rat islets, despite chronic hyperlipidemia, expression of PPARα, acyl-CoA oxidase and CPT-I mRNA is reduced . It has in vitro models are employed. Notably, culture of islets or INS-1 cells with high glucose (6–20 mM) for 48 hours strongly suppresses PPARα protein expression by 80%. As predicted, fatty acid oxidation and glucose-stimulated insulin secretion are attenuated, while islet triglyceride and lipid esterification are increased [in vivo, leading to the conclusion that activation of PPARα in tissues other than β-cells causes indirect effects on insulin secretion secondary to changes in peripheral insulin sensitivity [Glucolipotoxicity is a state in which β-cells are exposed to elevated plasma concentrations of both glucose and FFA, as is the case in insulin resistance. Several signalling pathways of the β-cell may be affected by altered PPARα expression and the overall outcome is predicted to depend upon whether fat or glucose has the dominant effect. In cases where glucose is elevated relative to lipid, a chronic reduction in PPARα would be expected to decrease the lipid oxidizing capacity of the β-cell ,89, elimncreased . Conversncreased . Howeversitivity . Thus, tsitivity . Similarsitivity . In bothChronic induction of PPARα may influence also insulin secretion indirectly because PPRE have been found in the promoter region of uncoupling protein-2 (UCP2) . In genein vitro [db/db mice [+/-) on a high fat diet had blunted glucose-stimulated insulin secretion in isolated islets that was associated with an islet-specific accumulation of triglyceride [PPARγ may also be an important transcriptional regulator of both normal and abnormal metabolism in pancreatic β-cells. In hyperglycemic, pancreatectomized rats the expression of PPARγ mRNA is increased  but othein vitro . In adipin vitro ,67. Howein vitro , resultiin vitro ,102. Thiin vitro . Glucose/db mice . Consistlyceride  even tholyceride .The TZD increase glucokinase and GLUT2 expression and activity via interaction with PPRE in the respective gene promoters ,108. A PPPARγ activation also regulates some β-cell functions that have not been ascribed to PPARα. PPARγ activation by TZD may relieve oxidative stress in β-cells of diabetic animals , leadingThe family of SREBPs governs transcriptional activation of a large number of genes involved in regulation of lipid metabolism, including lipogenesis, cholesterol transport and synthesis . Of inteSREBP-1c appears to have a similar function in lipogenesis in pancreatic β-cells as in hepatocytes, but the effects on glycolytic enzymes have received little attention. Notably, blockade of SREBP-1c expression attenuates the glucose-induced increase in acetyl-CoA carboxylase activity seen in control β-cells  whereas FFA exert dual effects on insulin secretion, dependent on the duration of exposure. Acute exposure to FFA increase glucose-stimulated insulin secretion whereas chronic exposure attenuate glucose sensitivity of pancreatic β-cells. The coordinated control of these processes by lipid-sensing transcription factors and its relevance to β-cell dysfunction in type 2 diabetes mellitus is increasingly a subject of investigation.PPARs  are involved in the long-term regulation of lipid metabolism and their activity is modulated by endogenous lipid-derived ligands. PPAR agonists have positive effects on glucose homeostasis and lipid metabolism and can reduce cardiovascular events in obese-diabetic patients. PPARα is a fasting lipid oxidation-glucose sparing regulator whereas PPARγ is post-prandial lipid storing-glucose utilizing regulator. In islets, however, both PPARα and -γ appear to have some functions more consistent with PPARα, particularly induction of lipid oxidizing enzymes, which is potentially particularly important for maintaining basal insulin secretion. Growing evidence suggests that PPARγ is a regulator of β-cell proliferation and that PPARγ agonist-mediated anti-oxidative effects may also contribute to anti-diabetic activity.SREBP-1c up-regulates lipogenic enzymes in β-cells as it does in liver. Its chronic induction in islets of obese-diabetic rodents may therefore contribute to lipotoxicity by promoting triglyceride accumulation and removing fatty-acid derived signalling factors from the cellular pool. SREBP-1c and PPAR functions appear to be closely linked through cross-talk between the pathways that control their own expression, and may function in concert to affect not only fatty acid metabolism but also glucose metabolism, β-cell proliferation and apoptosis.Drugs given orally to activate PPARs can improve insulin sensitivity of peripheral tissues and generally appear to enhance β-cell function secondary to their insulin-sensitizing effects. However, it remains possible that specific effects on β-cells are also important contributors to the positive metabolic effects of PPAR agonists in type 2 diabetes treatment.The author(s) declare that they have no competing interests.ZF-H and CBC contributed equally to the writing of this review.


A marked expansion of the connective tissue population and an abnormal deposition of extracellular matrix proteins are hallmarks of chronic and acute injuries to liver tissue. Liver connective tissue cells, also called stellate cells, derived from fibrotic liver have been thoroughly characterized and correspond phenotypically to myofibroblasts. They are thought to derive from fat-storing Ito cells in the perisinusoidal space and acquire a contractile phenotype when activated by tissue injury. In the last few years it has become evident that several peptide growth factors such as PDGF AA and TGF-β are involved in the development of fibrosis by modulating myofibroblast proliferation and collagen secretion. The fact that during the development of chronic fibrosis there is concomitant deposition of collagen, a known inhibitory factor, and sustained cell proliferation, raises the possibility that stellate cells from chronic liver fibrosis patients fail to respond to normal physiologic controls.In this study we address whether cells from fibrotic liver patients respond to normal controls of proliferation. We compared cell proliferation of primary human liver connective tissue cells (LCTC) from patients with liver fibrosis and skin fibroblasts (SF) in the presence of collagens type I and IV; TGF-β, PDGF AA and combinations of collagen type I and TGF-β or PDGF AA.Our results indicate that despite displaying normal contact and collagen-induced inhibition of proliferation LCTC respond more vigorously to lower concentrations of PDGF AA. In addition, we show that collagen type I synergizes with growth factors to promote mitogenesis of LCTC but not SF.The synergistic interaction of growth factors and extracellular matrix proteins may underlie the development of chronic liver fibrosis. In normal liver, connective tissue cells are rare and mostly restricted to the periportal and pericentrovenular spaces and to the Glisson's capsule. A minor population of connective tissue cells is present inside the hepatic lobule, in the perisinusoidal space. They are known as hepatic stellate cells (HSC) and are considered to be one of the major contributors for fibrogenesis in liver . Chronicin vivo and in vitro studies indicated that TGF-β has a relevant role in the development of fibrosis, being a potent regulator of proliferation and of extracellular matrix protein synthesis -thymidine incorporation. These methods correlated well with autoradiography results in previous studies -thymidine. When assessing the effect of extracellular matrix proteins, serum-free medium was replaced by medium containing 10% serum. Cells were collected 24 or 32 hr after the stimulus and lysed in 10 N NaOH. The trichloroacetic acid precipitable material was then spotted on a filter and counted on a scintillation counter.Cell proliferation was assayed by cell counting and -thymidine (1 μCi/ml) was added at 6, 24, 32 and 40 hr and incubated for 2 hr. Cells were harvested and processed as described above.Cells were plated at a concentration of 2.5 × 10To determine cell adhesion on different substrata plates were prepared with 0.3 mg/ml collagen type I or IV. Cells were plated on each substratum and the supernatant was removed at 0, 10, 20, 40 and 60 min and the cells remaining in suspension counted in a hemocytometer. To determine cell recovery, cells plated after 2 hr were trypsinized and counted.Difference between the means of various subgroups was assessed using the Mann-Whitney U-test.in vitro in order to identify potential mechanisms to explain the excessive proliferation of LCTC during fibrosis. We observed no differences among the cell lines obtained from patients with schistosomal fibrosis or alcoholic cirrhosis, and all the studied primary LCTC lines were included in a single experimental series. We established and characterized 16 primary human cell lines. The experiments performed were done at or before the tenth passage and every experiment was performed with at least two cell lines from each type (LCTC or SF) isolated from different individuals.Chronic liver fibrosis is characterized by abnormal proliferation of connective tissue cells. Therefore, we hypothesized that connective tissue cells from fibrotic lesions have lost normal proliferation controls. By using a series of primary connective tissue cell lines from skin and from fibrotic livers, we investigated several parameters of cell growth 3]-thymidine incorporation in various cell densities  and a marked decline at higher densities. We also examined the response of both cell types to increasing concentrations of fresh human serum. Both SF and LCTC reached maximum proliferation, as measured by [H3]-thymidine incorporation at 20% serum (not shown). In conclusion, primary SF and LCTC were similarly responsive to normal contact inhibition of proliferation and serum concentration, although SF seemed to respond more efficiently to contact inhibition.To assess whether connective tissue cells from fibrotic livers (LCTC) and normal skin fibroblasts (SF) responded to contact inhibition of proliferation, we measured cell proliferation during eight days. Both LCTC and SF showed a comparable pattern of growth and reached a plateau at a similar cell density Figure . Reprodus Figure . Both LC3]-thymidine incorporation in LCTC and SF  was observed in SF derived from some of the studied cases (not shown). We did not observe any change in cell morphology dependent upon the substrate.Despite the abnormal deposition of extracellular matrix in chronic fibrotic livers, LCTC continuously proliferate suggesting that these cells are not responsive to normal inhibition of proliferation by collagen I. To determine the influence of the extracellular matrix on LCTC, we analyzed DNA synthesis in the presence of collagen type I and type IV, two major components of extracellular matrix in fibrotic livers. Collagen type I inhibited DNA synthesis in a dose-dependent manner, as measured by -thymidine for 2 hr, at 6, 20, 26, 32 and 40 hr. [H3]-thymidine incorporation reached its peak at 32 hours after activation in control experiments (plated on plastic), and cells plated on collagen-coated plates did not show any delayed peak, demonstrating that the effect of collagen was not delaying the progression of cell cycle but blocking DNA synthesis , collagen type IV did not induce any significant change on [H3]-thymidine incorporation in two LCTC cell lines, neither in two SF cell lines (not shown). Again, no difference in morphology was noted.Next, we asked whether collagen type IV could also influence the proliferation of the studied cells. Even at high concentrations . In conclusion, LCTC appear to be more sensitive to lower concentrations of PDGF AA.To investigate the effects of PDGF AA we starved cells for 18 hr and stimulated them with increasing concentrations of PDGF AA. As shown in Figure To test the responsiveness of LCTC and SF to TGF-β, cells were starved overnight and stimulated with increasing concentrations of acid-activated TGF-β. In Figure 2; Figure Next, we assessed the interplay between growth factors and the extracellular matrix on the stimulation of proliferation. We compared proliferation of cells cultured on collagen film using the highest inhibitory concentration . On the other hand, it is largely unclear which are the other physiologic determinants for disease evolution. Genetic background is known to influence the development of hypertrophic cheloid skin scars as well as the outcome of schistosomal liver fibrosis . In expeThe results obtained in this study using human primary liver connective tissue cells suggest a plausible scenario for the development of liver fibrosis. Stellate quiescent cells are triggered to proliferate by high molecular weight serum factors  and may HEPES: N-[2-Hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid]; HSC: hepatic stellate cells; LCTC: liver connective tissue cells; SF: skin fibroblasts; PDGF: platelet-derived growth factor; TGF-β: transforming growth factor-β.The author(s) declare that they have no competing interests.AG established primary cell line cultures, carried out the cell biological studies, participated in designing and interpreting the experiments. MC provided technical support for the cell cultures and participated in the writing of the manuscript. RB participated in the design of the study and in the interpretation of the results. AM established primary cultures, conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: