Fugu rubrices in order to investigate 1) the relationships between three major lineages of mammals: rodents, artiodactyls and primates, and 2) the rate of evolution and the occurrence of positive Darwinian selection using codon based models of sequence evolution.The availability of abundant sequence data from key model organisms has made large scale studies of molecular evolution an exciting possibility. Here we use full length cDNA alignments comprising more than 700,000 nucleotides from human, mouse, pig and the Japanese pufferfish We provide evidence that the evolutionary splits among primates, rodents and artiodactyls happened shortly after each other, with most gene trees favouring a topology with rodents as outgroup to primates and artiodactyls. Using an unrooted topology of the three mammalian species we show that since their diversification, the pig and mouse lineages have on average experienced 1.44 and 2.86 times as many synonymous substitutions as humans, respectively, whereas the rates of non-synonymous substitutions are more similar. The analysis shows the highest average dN/dS ratio in the human lineage, followed by the pig and then the mouse lineages. Using codon based models we detect signals of positive Darwinian selection in approximately 5.3%, 4.9% and 6.0% of the genes on the human, pig and mouse lineages respectively. Approximately 16.8% of all the genes studied here are not currently annotated as functional genes in humans. Our analyses indicate that a large fraction of these genes may have lost their function quite recently or may still be functional genes in some or all of the three mammalian species.We present a comparative analysis of protein coding genes from three major mammalian lineages. Our study demonstrates the usefulness of codon-based likelihood models in detecting selection and it illustrates the value of sequencing organisms at different phylogenetic distances for comparative studies. Fugu rubrices is used as an outgroup to estimate the branching order of the three species relative to each other.Large scale sequencing projects of many different species allow us to investigate phylogenetic issues in much more detail and to identify whether certain genes have had an extraordinary evolution in one or more species and thus gain insight into the actions of natural selection. Despite the sequencing of an increasing number of mammalian genomes and the implementation of more sophisticated evolutionary models using maximum likelihood and Bayesian methodology, the branching order within the mammalian phylum is still not completely resolved. The main reason for this uncertainty is that the diversification of these orders occurred over a short period of time, making the inference of branching order a difficult problem. One of the highly debated issues concerns the relative order of branching among primates, artiodactyls and rodents -9. Here,Codon based models ,11 allowa in Figure In a recent study of cDNA trios of human, mouse and chimpanzee a codon based branch-site specific model was used to search for human genes that have undergone positive selection since our divergence from other primates . Here, aThe distribution of sequence lengths of the 1120 three-species alignments is shown in Figure The relative branching order of the three mammalian species was investigated with the individual genes as well as with a concatenated super gene. Using the empirical amino acid substitution model of Whelan and Goldman  we maximWe used the baseml program of PAML to compare the three topologies in a nucleotide based framework. Different nucleotide based substitution models were used to maximize the likelihood on the three topologies for each of the three codon positions separately. The results of using different models of nucleotide evolution were highly similar so here we only discuss the results obtained with the HKY85 model . The resThe internal branch is rather short in all cases. Therefore in the remaining analyses we treat the mouse, human, pig split as a trifurcation. Depending on which topology is actually the right one, the only bias introduced by treating the topology as a star tree, as shown in Figure The three-species alignments were used to estimate the synonymous and nonsynonymous substitution rates of the three branches under the free ratio model, see Table The dN/dS ratios on the three different lineages were estimated under the free ratio model Figure . Most geThe one ratio model averages over sites and lineages, which makes this an extremely conservative method of detecting positive selection. Only four of the 1120 three-species alignments have an average dN/dS ratio larger than one, see Table Several studies have shown that averaging over sites is more conservative when searching for positive selection than is averaging over lineages. The branch-site specific model A and model B  were oriThe molecular function of the genes predicted to be under positive selection was determined using the Panther server  and the Several different models have been developed that allow for heterogeneity of ω over sites in an alignment. We used the M4 model  which alThe concatenated super gene was also used to investigate the patterns of codon usage in the three species; the results of this investigation are summarized in Table The phylogeny of the early mammalian radiation has been extensively debated over the last two decades. The classical view based on fossil evidence states that all major orders of placental mammals first appear right after the Cretaceous-Tertiary (KT) boundary approximately 65 million years ago . This suGallus gallus) shows that the average value of dS between human and chicken genes is approximately 1.66 [The incongruence of single gene phylogenies was investigated in a recent study of eight yeast species . The phyely 1.66 , which iely 1.66 ,32. (2) ely 1.66 ,39. (4) ely 1.66 ,36.The rate of synonymous substitution was estimated to be almost three times higher in rodents than in other mammals, in agreement with previous investigations that also showed an elevated rate in rodents -42. ThisPrevious studies of the occurrence of positive selection based on pair wise comparisons have revealed a very low occurrence of positive selection. In a study of 3595 alignments only 17 genes showed evidence of positive selection . The braCodon based branch-site models similar to the ones used here were used in a paper based on a three way comparison among chimpanzees, humans and mice . They reThe multiple testing and the small number of taxa used in a study like this imply that the results presented should not be taken as conclusive evidence for positive selection, but more as an approach to searching among the thousands of genes to look for genes that may have evolved in a biologically interesting manner. Comparative approaches such as the one we use here can only be a first step towards showing that positive Darwinian selection may be a key part in the evolution of many different gene families. Further experimental and computational analyses must then be used to investigate the suggested candidates more thoroughly.During the course of our investigation a large fraction of the genes were re-annotated as putative pseudogenes: 188/1120 ~16.8%. However, all these genes have uninterrupted reading frames in all three species; only a tiny fraction of all codons seems to have evolved in a neutral-like fashion (ω~1), and the distributions of the synonymous as well as the nonsynonymous rates of these putative pseudogenes are almost identical to the distributions of the remaining genes (results not shown). The only difference is a slight increase in the dN/dS ratio in the human lineage, which is actually due to a few genes that experience an unusually high dN/dS ratio. Omitting these genes from the analysis removes the observed differences completely. Thus, if all these genes are indeed pseudogenes in human, the loss of function must have occurred quite recently and they may not be pseudogenes in pig and mouse.The collection of a large set of pig cDNA sequences has enabled us to study long term evolutionary trends in mammalian genes. Our results indicate that the codon models are able to detect evolutionary signals indicating adaptive evolution in several genes. Our phylogenetic investigation of the primate, rodent, artiodactyl split disagree with most recent findings in favouring a primate, artiodactyl clade with rodents as an outgroup. Our study indicates that several genes that are not classified as genes in the most recent human annotation might after all be real genes; or at least they have become pseudogenes very recently, and the orthologous genes in mouse and pig might still be functional. This shows the potential of comparative methods in identifying functional regions of the genome.Sus scrofa was assembled at the Danish Institute of Agricultural Sciences (DIAS) from cDNA libraries from 100 different tissues constructed at DIAS and the Royal Veterinary and Agricultural University in the following way. Total RNA was purified from selected tissues using Rneasy (Qiagen) or Tri ReagentR and poly(A+) mRNA was selected using Oligotex (Qiagene) or PolyATract (Promega). Directional cloneable cDNA was synthezised from Poly(A+) mRNA using the cDNA Synthesis Kit (Stratagene) and was ligated into Eco RI/Xho I digested pTrueBlue (GenomicsOne) or pBluescript (Stratagene) followed by electrotransformation into E. coli XL1-Blue MRF' (Stratagene). 5'-EST sequencing was performed using standard protocols (Applied Biosystem). The sequences were trimmed to the longest open reading frame and the termination codons were removed.Complete cDNA from the domesticated pig Fugu rubrices were obtained with the blastall program with default parameters; the E-score was set to 10-8. We constructed two different datasets, one with and one without Fugu rubrices. Individual alignments were made using ClustalW version 1.83 with default parameters [Sus scrofa have been deposited in Genbank with the following accession numbers: AY609387-AY610506.Homologues sequences from human, mouse and the Japanese pufferfish rameters . We keptNine hundred and eighty-eight four-species alignments were concatenated into a super gene. The three topologies were compared using the super gene as well as each individual gene. Both nonsynonymous nucleotide substitutions and amino acid substitutions were investigated with PAML v. 3.14 . The nonUsing the 1120 three species alignments, the synonymous and nonsynonymous rates of evolution were estimated with the codeml program (seqtype = 1) using the free ratio model (model = 2) with the transition/transversion ratio estimated from the data (fix_kappa = 0).i substitutes into another codon j during the time interval t is determined by the rate matrix Q = (qij) with entriesThe different tests for positive Darwinian selection are all based on extensions of the basic codon based likelihood model . Likelihi ≠ j, with corresponding substitution probability matrix given by exp(Qt). Here πj is the equilibrium codon frequency of codon j, κ is the transition/transversion ratio and ω is the dN/dS ratio. All parameters are estimated independently for each gene. The star topology of the three species is used to estimate the branch lengths  for synonymous and non-synonymous substitutions.for 2 distribution with 2 degrees of freedom. The genes found in one or more lineages evolving with a dN/dS ratio > 1 are compared to a nested model where the dN/dS ratio is fixed at 1 in the lineages shown to have a dN/dS ratio larger than one to see whether the result can be attributed to natural selection or just relaxation of selective pressures.Positive selection was tested in two different ways. Test 1 averages over sites but differentiates among lineages. The LRT compares the free ratio model where all three lineages have a different value of ω estimated from the data with the one ratio model where all three lineages share a common value of ω . We notep1 of sites where ω1 are estimated from the data and is forced to lie between 0 and 1, and a proportion p2 of neutrally evolving sites where ω1 = 1 (p1 + p2 = 1). Model A furthermore allows a pre-specified branch to have a proportion of sites that evolve with a different value of ω estimated from the data. This value cannot be smaller than 1. The LRT follows a χ2 distribution with 2 degrees of freedom. If the value of ω in the foreground lineage is estimated to be equal to one the model collapses to the neutral model.Test 2 is based on a new and improved version of the branch-site method presented in . We willPAML v. 3.14  was usedA simple extension of the F3 × 4 codon equilibrium frequency model can incorporate CpG avoidance by adding an extra parameter that penalizes a C followed by a G in the second and third codon position. The new model is parameterised as followsi11 represents the frequency of nucleotide i1, at codon position 1, and ψ(0 < ψ < 1) is a CpG penalizing parameter. The scaling factor cψ ensures that the codon frequencies sum to one.Here πFGJ carried out the analyses and was the primary writer of the text. AH and FGJ together implemented some of the analysis tools. FGJ, AH and MHS together developed the ideas and discussed the interpretation of the results. MF and CB gathered the EST data used. HHJ assembled the cDNAs and carried out Blast searches. All authors read and approved the final manuscript.


Clinical descriptions of Parkinson's disease remain remarkably similar to those first described by James Parkinson nearly 200 years ago. Patients with “shaking palsy” experience a progressive loss of muscle control, increased muscle rigidity, inhibited movement, and tremors. These symptoms, it was later discovered, result from the loss of dopamine-producing neurons specifically in an area of the ventral midbrain called the substantia nigra. Midbrain dopamine neurons relay chemical signals that regulate motor control and less quantifiable attributes like mood and motivation, and therefore the loss of these cells is predicted to lead to the symptoms of Parkinson's.Despite the well-characterized cellular basis of Parkinson's disease, the molecular mechanisms responsible for dopamine neurodegeneration remain unknown. There is evidence that both genetic and environmental components are involved. That a person with Parkinson's disease is three to four times more likely than an unaffected individual to have a close family member with “parkinsonian” symptoms suggests a genetic factor; furthermore, several genes have been associated with relatively rare, familial forms of the disease. For example, mutations of the protein alpha-synuclein (α-synuclein), which is found to aggregate in the brains of patients with Parkinson's, lead to a familiar parkinsonism syndrome. Mutations in a second gene called DJ-1 were recently found in two families with an inherited form of Parkinson's. Importantly, mutations in DJ-1 have previously been linked to the pesticide paraquat in unrelated research on cell stress and reactive oxygen species, and have been linked to dopamine neuron toxicity. Reactive oxygen species are molecular byproducts of oxygen metabolism that react with and damage cellular components like proteins and DNA, and there is evidence from postmortem studies that reactive oxygen species may play a role in Parkinson's disease.PLoS Biology, Asa Abeliovich and colleagues make the case that a model based on mouse embryonic stem cells offers a promising platform for dissecting the disease mechanism of Parkinson's. Working with these cells, the researchers report that DJ-1-deficient cells—and especially DJ-1-deficient dopamine neurons—display heightened sensitivity to oxidative stress. In a second paper, they link DJ-1 dysfunction to alpha-synuclein aggregation.Part of the challenge of untangling the relative contributions of all these components stems from the difficulty in finding a model that can adequately mimic the loss of dopamine cells. In two papers published in Oxidative stress has long been associated with neuronal cell death and neurodegenerative diseases like Parkinson's. Proving a causal relationship between oxidative stress and neurodegeneration, however, requires establishing a molecular mechanism.In the first paper, to explore the hypothesis that DJ-1 contributes to the cellular response to oxidative stress, Abeliovich and colleagues created mouse embryonic stem cells lacking functional copies of DJ-1 and exposed them to hydrogen peroxide, a powerful oxidizer. Compared to normal cells, DJ-1 mutants showed signs of greater toxicity and higher levels of cell death. These defects were corrected when the researchers reintroduced the protein in the mutants, confirming DJ-1's responsibility for the defects. DJ-1 protects against oxidative damage, the results show, not by inhibiting the accumulation of the reactive oxygen species associated with hydrogen peroxide, but by mitigating the damage created by them.Abeliovich and colleagues then explored DJ-1's function in dopamine neurons by inducing mutant and control embryonic stem cells to differentiate in cell cultures. Production of dopamine neurons was significantly reduced in the DJ-1-deficient cultures relative to the control cultures. And like DJ-1-deficient embryonic stem cells, DJ-1 dopamine mutants were vulnerable to oxidative stress. “DJ-1 deficiency,” the authors conclude, “leads to reduced dopamine neuron survival and predisposes these cells to endogenous and exogenous insults.” Inhibiting DJ-1 activity in neurons from the embryonic mouse midbrain produced the same results.In the second paper, Abeliovich and colleagues go on to probe the molecular basis of DJ-1's activity. There have been several leads regarding how DJ-1 functions, based on homology to related genes, including a potential role as a molecular protein chaperone; protein chaperones assist in the folding and refolding of damaged proteins, and thus play a central role in the cellular response to oxidative stress. Abeliovich and colleagues found that DJ-1 acts as an unusual molecular chaperone that is specifically induced under oxidative conditions, and acts to prevent the aggregation of cellular proteins. Interestingly, the researchers go on to show that one substrate of DJ-1 activity is alpha-synuclein, thus providing a possible mechanism linking these two molecules implicated in Parkinson's disease. Altogether, these results support a link between toxin-induced oxidative damage and disease, and provide a tractable model for studying the molecular mechanisms of neurodegenerative disease.


H. pylori.Horizontal gene transfer is central to evolution in most bacterial species. The detection of exchanged regions is often based upon analysis of compositional characteristics and their comparison to the organism as a whole. In this study we describe a new methodology combining aspects of established signature analysis with textual analysis approaches. This approach has been used to analyze the two available genome sequences of ftsK, xerD and polA. In some cases it was possible to determine which strain represented the 'parent' and 'altered' states for insertion-deletion events. Different signature component lengths showed different sensitivities for the detection of some horizontally transferred genes, which may reflect different amelioration rates of sequence components.This gene-by-gene analysis reveals a wide range of genes related to both virulence behaviour and the strain differences that have been relatively recently acquired from other sequence backgrounds. These frequently involve single genes or small numbers of genes that are not associated with transposases or bacteriophage genes, nor with inverted repeats typically used as markers for horizontal transfer. In addition, clear examples of horizontal exchange in genes associated with 'core' metabolic functions were identified, supported by differences between the sequenced strains, including: New implementations of signature analysis that can be applied on a gene-by-gene basis for the identification of horizontally acquired sequences are described. These findings highlight the central role of the availability of homologous substrates in evolution mediated by horizontal exchange, and suggest that some components of the supposedly stable 'core genome' may actually be favoured targets for integration of foreign sequences because of their degree of conservation. Helicobacter pylori is a bacterial pathogen associated with gastritis, peptic ulcers, gastric adenocarcinoma, and rare lymphomas [E. coli from Salmonella can be attributed to point mutation genes but are broadly attributable to horizontal exchange [H. pylori is relatively unusual in that it is a naturally transformable Gram-negative species that does not appear to have a species-specific DNA uptake sequence and appears to rely upon its niche separation as a transformation barrier [H. pylori strains have been divided into two types, type I being those that carry the cag pathogenicity island [cag PAI), which has a foreign species origin, and are associated with more severe disease.ymphomas . It has ymphomas . The acqymphomas -5. In thexchange . H. pylo barrier . Diseasey island   and longer signatures. In addition, use of a new approach based upon classical text analysis that allows analysis of genomes gene-by-gene is described. Analysis of cag pathogenicity island (6 and 5), vac and related toxins (3 and 4), and restriction-modification genes (2 and 4). On the basis of the similarities determined in the H. pylori strain J99 sequence annotation, 7 of the most divergent genes as determined by DNS are not present in strain 26695. Likewise, 2 of the 50 most divergent genes in strain 26695 are not present in strain J99. This is consistent with the identification of genes acquired from other species that have not extended to both sequenced strains. It also suggests that a significant proportion of the 6 to 7% of genes unique to one or other strain [Helicobacter gene pool, but are variably present in different strains rather than reflecting recent foreign origins. Comparisons of a selection of identified orthologous genes in the two strains are shown in Figure Genes were ranked on the basis of their divergence from the mean genome composition. The degree of divergence that is indicative of acquisition from other species is not an absolute. The frequency with which genes are acquired, the untypicality of the donated material, and the rate at which they are ameliorated to the host sequence composition influence it. Strains J99 and 26695 had 53 Table  and 60  genes rer strain  are inhecag PAI gene  has a highly complex repetitive structure and the size of the large divergent peak associated with this island using previous methods is largely due to the presence of this gene.It cannot be assumed that all genes identified in this manner have been recently acquired. It is necessary to assess the nature of the sequence to determine if its divergence might be accounted for on the basis of features of the encoded protein. For example, JHP0476/HP0527, JHP1300/HP1408 and JHP0074/HP0080 include repetitive sequences likely to account for their DNS divergence. This type of analysis cannot be used to determine the possible foreign origin of such genes. Notably, the most divergent While a significant proportion of the genes identified in this analysis are associated with regions including several such genes and which share characteristics of islands of horizontal transfer or pathogenicity islands, this is far from universally true. There are many instances of single genes or small numbers of genes that are present that are not associated with any features that might otherwise have been used as indicators of horizontal acquisition such as transposases and flanking repeats.mutS, ftsK, xerD, and polA. The comparisons of the latter three between the sequence strains are shown in Figure Our initial goal was to identify recently acquired and exchanged genes as candidates likely to be important in niche-adaptation, host interactions, and alterations in bacterial fitness. It has been argued that essential genes are unlikely to be transferred successfully since recipient taxa would already bear functional orthologues, which would have experienced long-term co-evolution with the rest of the cellular machinery. In contrast, it is proposed that those under weak or transient selection – like those associated with nonessential catabolic processes, new operons, and those providing new niche-adaptive changes are likely to be successfully transferred and retained . This leTetranucleotide composition has been used for the consideration of the presence of palindromic sequences that might be substrates for restriction systems and Chi sites and the presence of unstable repeats mediating phase variation , but therd, 13th and 41st most divergent genes by HNS, were 319th, 857th and 750th most divergent by DNS, respectively. In some instances the TNS gave intermediate results and in others identified other genes as more divergent than the other methods. The TNS was most sensitive for the detection of rpoB (HP1198 / JHP1121) which is associated with a significantly different gene length in the two strains  that were not in the 53 (>2 SD) most divergent by DNS, these included 11 restriction-modification system genes and 6 others that were not annotated within the strain 26695 genome sequence. The identification of genes of a type known to be horizontally exchanged, and different between the gene complements of the strains, is strong corroboration for the foreign origin of the additional genes identified by HNS. In several instances  and the 5' 26695 gene , and the high divergence of the 3' 26695 gene , indicate that the most likely event is an insertion into strain 26695 . When the homologous genes were compared between the strains, extensive divergences were evident between more than one region of each protein. That these genes have divergent signatures in both strains suggests that neither has a wholly native composition. This observation is consistent with the models of rapid evolution which suggest that transient competitive advantages are enjoyed by organisms that are hypermutators under conditions of environmental stress and transitions, and that these states which can be produced by mutations in DNA repair genes [mutS and recN suggest that H. pylori is another species that can make use of this strategy for diversification under stressful conditions.The inclusion of two DNA metabolism genes associated with recombination and repair is notable. Both ir genes -26. Howeir genes -29. The The identification of RNA polymerase genes, with associated differences between the strains, is striking. The divergence of phylogenetic trees based upon different sequences has been highlighted, and particularly the differences between the trees associated with RNA polymerase genes and rRNA ,31. It hThe spectrum of recently horizontally acquired sequences identified emphasizes the two driving forces of horizontal exchange: the transfer of a phenotype which alters or enhances bacterial fitness resulting in increased competitive fitness or altered niche adaptation, and the presence of a substrate for homologous recombination. Because of the focus upon, and relative ease of identifying, large islands associated with readily identifiable features and phenotypes, the importance of the latter component has perhaps been underestimated. The genes that have been considered to code for 'core metabolic' 'house-keeping' functions are amongst those most likely to be changed by horizontal transfer events because of the presence of homologous substrates, and changes are likely to persist even when the change is phenotypically neutral. Equally, changes in the genes involved in core functions such as gene expression and DNA metabolism may have pleotropic effects and there may be significant differences in strain behaviour, that are not simply the consequence of differences in their respective gene complements. The selection of genes for phylogenetic analysis on the basis of their coding for conserved core functions is also problematic because these are also frequently the genes most likely to share the high homology that facilitates recombination and horizontal exchange.k equal-sized subsequences (or 'windows'). The mathematical basis for the signature is an odds ratio – pi – calculated by dividing the frequency of a length-L oligonucleotide by its expected frequency. The odds ratios for each of the 4L oligonucleotides in each window (w) are compared with the odds ratios for the overall sequence (s) [δ is plotted and thus a nucleotide signature consists of a k-length sequence of δ values: δ = (1/4L)Σ|pi(w) - pi(s)|, where x is the set of all permutations of length L and i is one such permutation.A traditional nucleotide signature is generated by segmenting a sequence of DNA into ence (s) ,10,33. Tweighted cusum (cumulative sum) [, is modified so that n denotes the number of ORFs in the genome, Xi the number of oligonucleotides in ORF i, and wi the number of nucleotides in ORF i. The results are scaled according to ORF size using the standard error σ = √(*#ORF). In this way false positives are abrogated by normalizing for over-representation of lower order peptides.There are interesting parallels between signature-style genome analysis and stylometric techniques previously used to determine the authorship of controversial literary texts. This is analogous with the biological problem and it is from this that our method is derived. Rather than using a fixed-window signature, signature scores are calculated for each coding open reading frame (ORF) and weighted with variance estimates so that the scores for shorter ORFs confer with their longer counterparts. Bissell's ive sum) , , is moO(N) (N = sequence length) refinement: probabilities for the complete sequence are calculated in O(N) steps for any length-L oligonucleotide, and maintain O(N) when 4L>N through a hashing function; the second part of the program calculates σ for each ORF using a loop flattening technique, thereby avoiding the program having to recalculate overlapping sub-expressions. The program is available from  and .The method is implemented in Java and efficiency is maintained through an Sequence alignments, as shown in Figure ORF, Open Reading Frame; DNS, Dinucleotide Signature; TNS, tetranucleotide signature; HNS, hexanucleotide signature.NJS initiated the project, performed the genome sequence analyses, compared the two strains, interpreted the results, and prepared the biological aspects of the manuscript. PB was a DPhil student who worked on the coding aspects of the new methodology. JFP contributed to the bioinformatics discussions and planning stage of this project. SAJ directed and primarily developed the analysis strategy and the implementation of the new computational basis of the methodology, and prepared the computational aspects of the manuscript.


One of the major interests in our laboratory has been to develop a scientific framework for dietary advice for patients with diabetes. Knowledge regarding the metabolic consequences and potential effects on health of protein in people with type 2 diabetes has been a particular interest.We recently have completed a study in which dietary protein was increased from 15% to 30% of total food energy. The carbohydrate content was decreased from 55% to 40%, i.e. dietary protein replaced part of the carbohydrate. This resulted in a significant decrease in total glycohemoglobin, a decrease in postprandial glucose concentrations and a modest increase in insulin concentration. Renal function was unchanged.Currently we also are determining the metabolic response to a diet in which the carbohydrate content is further decreased to 20% of total food energy. The %tGHb decrease was even more dramatic than with the 40% carbohydrate diet.From these data we conclude that increasing the protein content of the diet at the expense of carbohydrate can reduce the 24-hour integrated plasma glucose concentration, at least over a 5-week period of time. The reduction was similar to that of oral agents. Renal function was not affected significantly. Thus, increasing the protein content of the diet with a corresponding decrease in the carbohydrate content potentially is a patient empowering way of reducing the hyperglycemia present with type 2 diabetes mellitus, independent of the use of pharmaceutical agents. Our research group has been and continues to be interested in the metabolic response of people with type 2 diabetes to macronutrients in the diet in general. More recently, we have been particularly focused on the metabolic response to a high protein diet. The reason for this is three fold: First, for several years, one of our major goals has been to develop a scientific framework for dietary advice based on sound metabolic principles. Second, we have data that suggest that an increase in dietary protein may be salutary for people with diabetes. And lastly, knowledge regarding the metabolic consequences and potential effects on health of dietary protein has lagged far behind that of dietary fats and carbohydrates.In this paper we will focus on the concept that an increase in dietary protein may be salutary for people with diabetes, and particularly for the control of blood glucose.The concept that an increase in dietary protein may be useful in controlling the blood glucose would appear to be counterintuitive, since amino acids derived from ingested or endogenous proteins are major net gluconeogenic substrates.2 to form urea. The remaining deaminated product is largely converted into glucose through gluconeogenesis, although a small amount is converted into other products. .The reason that 5 weeks was chosen for the study is because this is the time required for the % total glycohemoglobin to decrease 50% of its ultimate value after a rapid stable decrease in blood glucose concentration  .Urine urea, normalized to the urine creatinine, was measured as an index of compliance. Since the protein content of the diet was doubled, one would expect that the urine urea:creatinine ratio also would approximately double if the subjects were compliant. The ratio on the standard diet was ~7 and was stable throughout the 5 weeks. When the same subjects were given the 30% protein diet, the urine urea:creatinine ratio was ~13–14, i.e. a value that one would expect with good compliance with the diet.The fasting glucose concentration did not change when the subjects received the 30% protein diet. However, the postprandial glucose concentrations were lower throughout the day .Although the differences in postprandial glucose values were not very large, when integrated over the 24-hour period, there was a 38% decrease in postprandial glucose area response. If the 24-hour integrated area is considered to be 100% when the subjects ingested the 15% protein diet, when they ingested the 30% protein diet it was 62%  the %tGHb would have decreased by only 50% of the expected final decrease  .None declared.Both authors were equally responsible for designing the experiments, evaluating the statistics, interpreting the data, writing the manuscript, and organizing the figures and tables.


Likewise, we determined that cushion formation is blocked in −/−)cardiofunk . We show that in yeast this mutation negatively affects actin polymerization. Because the lack of cushion formation in sih- and cfk-mutant embryos could be due to reduced myocardial function and/or lack of blood flow, we approached this question pharmacologically and provide evidence that reduction in myocardial function is primarily responsible for the defect in cushion development. Our data demonstrate that early myocardial function is required for later steps of organogenesis and suggest that myocardial function, not endothelial shear stress, is the major epigenetic factor controlling late heart development. Based on these observations, we postulate that defects in cardiac morphogenesis may be secondary to mutations affecting early myocardial function, and that, in humans, mutations affecting embryonic myocardial function may be responsible for structural congenital heart disease.Function of the heart begins long before its formation is complete. Analyses in mouse and zebrafish have shown that myocardial function is not required for early steps of organogenesis, such as formation of the heart tube or chamber specification. However, whether myocardial function is required for later steps of cardiac development, such as endocardial cushion (EC) formation, has not been established. Recent technical advances and approaches have provided novel inroads toward the study of organogenesis, allowing us to examine the effects of both genetic and pharmacological perturbations of myocardial function on EC formation in zebrafish. To address whether myocardial function is required for EC formation, we examined s cardiofunkCardiac anomolies can result from very early defects in heart development. In zebrafish, such defects have been shown to be caused by a new gene called  The genetic programs and developmental processes that lead to organ formation are still poorly understood. We are currently witnessing an expansion in research that aims to identify the genes responsible for the structural development of organs and their later function. Among the organs of the body, the heart is unique because it begins to function mechanically before structural development is complete, begging the important question of whether myocardial function is required for the morphogenetic events that occur after the heart begins beating. One of the late steps of heart development is the formation of the endocardial cushions (ECs), which are tissue swellings that develop in characteristic locations along the anterior–posterior (AP) extent of the heart tube and contribute to valves and, in four-chambered hearts, to septae. Because of the clinical significance and prevalence of EC defects in humans , an undeDuring the process of EC and valve development, specific endocardial cells undergo multiple poorly understood specification, differentiation, and migration events en route to becoming functional heart valves. The genes involved in one substep of this process, epithelial–mesenchymal transformation (EMT), are gradually being identified. Analysis of EMT during cardiac-cushion development has implicated molecules such as Fibronectin, Transferrin, ES-130, hLAMP-1, TGF-β2, TGF-β3, BMP-2 silent heart , but do undergo looping morphogenesis  boundary. This process is easily visualized at 48 h postfer-tilization (hpf) by examining the endocardial cells expressing green fluorescent protein (GFP) under the control of the mouse promoter  , that fails to accumulate tie2::GFP-positive cells at the AV boundary by 48 hpf  extent of the heart tube before becoming localized to the AV boundary at 42 hpf  zC202M22 , and fv49b10  showed a change of arginine 177 to histidine. The three embryos recombinant at either end of the 52 kb region were homozygous at the site of the R177H lesion, suggesting that we had isolated cfk.To understand better the molecular basis for the d Z10582 . AnalysizC202M22 A. Follow GenScan  and BLAS fv49b10 B. Compar fv49b10 C and hum fv49b10 D and concfk mutation supported our hypothesis that cfk corresponded to an actin gene. Analyses of Cfk indicate that it is a sarcomeric actin by virtue of its homology to zebrafish α-cardiac and α-skeletal actins and lack of homology to zebrafish cytoplasmic actin, as well as its conservation of synteny with the human skeletal actin. Further evidence that cfk encodes a sarcomeric actin and not a cytoplasmic actin includes the presence of an extra residue at the N-terminus of Cfk, which is seen in all sarcomeric actins but no cytoplasmic actins, as well as the presence of residues that are stereotypic for sarcomeric actins at all 20 locations where sarcomeric and cytoplasmic actins have unique amino acids , which is distinct from the 3′ UTR sequences of zebrafish α-cardiac and α-skeletal actins. These data showed that cfk is expressed in the myocardial but not endocardial cells of zebrafish embryos from 24 to 48 hpf  leads to unstable filaments, occasionally giving rise to a phenotype in heterozygous embryos.During our work with e cfk+/− A, suggese cfk+/−  demonstr−/−cfk embryos have a dilated heart and lack blood flow and because subsequently approximately 50% of these embryos fail to form ECs, we wondered whether these two phenotypes were causally related or whether lack of EC formation could occur independently of poor early myocardial function. To address this question, 379 embryos from multiple cfk clutches were assayed for cardiac dilation and lack of blood flow at 36 hpf and for lack of endocardial ring formation at 48 hpf. Embryos that were phenotypically wild-type at 36 hpf invariably developed endocardial rings over the next 12 h, whereas all embryos that failed to form endocardial rings had previously shown a functional phenotype at 36 hpf  and fail to generate blood flow, it is impossible to conclusively state whether EC formation is affected in these embryos directly as a result of poor myocardial function or indirectly as a result of the perturbation in blood flow and shear stress caused by poor myocardial function. n = 74) formed an endocardial ring, indicating that blood flow is not required for the initial steps of cushion formation. When myofibril function was further decreased by treatment with 10 mM 2,3-BDM, the percentage of embryos with an endocardial ring decreased to 13% (n = 68). Studies with the anesthetic tricaine confirmed the observation that ECs may form in the absence of blood flow and that the likelihood of forming cushions is inversely proportional to the concentration of tricaine (data not shown). In summary, the results from the 2,3-BDM and tricaine treatments suggest that it is poor myocardial function, and not lack of blood flow, which is primarily responsible for the loss of EC formation in −/−cfk and −/−sih embryos.Because −/−sih embryos fail to beat yet undergo looping morphogenesis and AV boundary specification. The specific absence of EC formation in these embryos clearly demonstrates that myocardial function is required for EC formation. Precisely how myocardial function is required for EC formation is unclear. Prior work has demonstrated a requirement for AV boundary myocardium in the formation of ECs, and several signaling molecules emanating from the myocardium at the AV boundary have been identified , abc-b10 (5′-TCATTCGCAACATTGTCCCATACAT-3′), fv49b10.y1 (5′-CCGACCTAATTCGCTTGGT-CACCAT-3′), and cfk (5′-CATCTTGATGTATTCTTTCTCTGCT-3′). The morpholinos were injected at 4 ng and 8 ng into tie2::GFP embryos at the one-cell stage and examined at 24 to 72 hpf for myocardial function and EC phenotypes. Morpholinos against abc-b10, rab4a, and fv49b10.y1 did not affect myocardial function or EC formation. The cfk morpholino caused no phenotype, an expected result given the ability of actin genes to compensate for each other when down-regulated  to bind to the initiation codon and flanking sequences of the following zebrafish genes: sih , rab4a (egulated  and the s11cfk-AB/SJD hybrid strain using SSLP with various CA repeat markers and SSCP with various ESTs. SSCP was performed by denaturing PCR products at 95°C for 10 min in 0.7 mM EDTA and 36 mM NaOH and placing on ice before loading on nondenaturing acrylamide gels. BAC library filters for the CHORI-211 (zC) library were obtained from P. de Jong at BACPAC . Linkage of the R177H mutation to the s11cfk allele (through genotyping of mutant embryos) was accomplished by performing PCR across the mutation (forward primer = 5′-ATCGTGCTGGACTCTGGTG-3′ and reverse primer = 5′-GAAAGAATAACCGCGCTCAG-3′) and digesting with NsiI, which cuts only the H177 allele. Synteny analysis was performed through http://genome.jgi-psf.org/fugu3/fugu3.home.html (fugu) and http://genome.ucsc.edu (human).We genotyped diploid mutant embryos from a s11cfk mutant embryos to extract mRNA  and synthesize cDNA . PCR was then performed from the 5′ UTR to 3′ UTR and multiple clones sequenced. To confirm that the amino acid change we saw was a mutation and not a polymorphism in the strain used for mutagenesis, we examined the DNA of four F1 females from the screen .We used a pool of 25 e screen  in whichClutches of dechorionated embryos were placed in 2,3-BDM  at 24 hpf at concentrations between 2 and 20 mM. This concentration range has been shown to affect myofibrillar ATPase in a dose-dependent manner, with 25% of activity remaining at 10 mM . 2,3-BDM2, and 0.1 mM DTT) and used within 2 d. Actin polymerization was induced by the addition of MgCl2 and KCl to final concentrations of 2 mM and 50 mM, respectively, and polymerization was assessed by the increase in light scattering as a function of time at 25°C in a 120 μl volume in a thermostatted cuvette using a SPEX Fluorolog 3 fluorimeter with excitation and emission wavelengths set at 360 nm.Site-directed mutagenesis of the yeast-actin coding sequence in a centromeric plasmid and construction of haploid cells producing only the mutant actin were carried out as described previously . Wild-tyVideo S1−/−cfk hearts have complete regurgitation of blood from the ventricle to the atrium and therefore no forward blood flow.Wild-type hearts propel blood through the vasculature, whereas (2.91 MB MOV).Click here for additional data file.Video S2−/−cfk embryos (two different embryos shown) have dilated hearts with weak contractions and are not capable of generating circulation.Wild-type embryos have a narrow, strongly contracting heart and generate effective blood flow. (2.9 MB MOV).Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank/index.html) accession numbers discussed in this paper are for cfk (AY222742), zebrafish α-cardiac actin (AF116824), zebrafish α-skeletal actin (AF180887), and zebrafish cytoplasmic actin (AF057040).The GenBank (


Bordetella pertussis, the causative agent of whooping cough. PT consists of an active subunit (S1) that ADP-ribosylates the alpha subunit of several mammalian G proteins, and a B oligomer (S2–S5) that binds glycoconjugate receptors on cells. PT appears to enter cells by endocytosis, and retrograde transport through the Golgi apparatus may be important for its cytotoxicity. A previous study demonstrated that proteolytic processing of S1 occurs after PT enters mammalian cells. We sought to determine whether this proteolytic processing of S1 is necessary for PT cytotoxicity.Pertussis toxin (PT) is an exotoxin virulence factor produced by Protease inhibitor studies suggested that S1 processing may involve a metalloprotease, and processing does not involve furin, a mammalian cell protease that cleaves several other bacterial toxins. However, inhibitor studies showed a general lack of correlation of S1 processing with PT cellular activity. A combination of replacement, insertion and deletion mutations in the C-terminal region of S1, as well as mass spectrometry data, suggested that the cleavage site is located around residue 203–204, but that cleavage is not strongly sequence-dependent. Processing of S1 was abolished by each of 3 overlapping 8 residue deletions just downstream of the putative cleavage site, but not by smaller deletions in the same region. Processing of the various mutant forms of PT did not correlate with cellular activity of the toxin, nor with the ability of the bacteria producing them to infect the mouse respiratory tract. In addition, S1 processing was not detected in transfected cells expressing S1, even though S1 was fully active in these cells.S1 processing is not essential for the cellular activity of PT. This distinguishes it from the processing of various other bacterial toxins, which has been shown to be important for their cytotoxicity. S1 processing may be mediated primarily by a metalloprotease, but the cleavage site on S1 is not sequence-dependent and processing appears to depend on the general topology of the protein in that region, indicating that multiple proteases may contribute to this cleavage. Bordetella pertussis, a bacterial pathogen of the human respiratory tract that causes the disease whooping cough. PT holotoxin is a multi-subunit complex with an AB5 structure [i proteins) in mammalian cells [Pertussis toxin (PT) is a complex exotoxin and an important virulence factor produced by tructure ,2: the ean cells ,4, and tan cells . The evean cells -9. Subcean cells , providean cells -9. Traffan cells -13. Aftean cells .Pseudomonas exotoxin A and shiga toxin are all activated after cleavage by the endogenous eukaryotic protease furin [Escherichia coli heat-labile toxin (LT) A subunits are cleaved at a protease-sensitive loop to promote maximal activity [Another observation that may bear on the cell biology and cytotoxicity of PT is that the S1 subunit appears to be proteolytically processed to a lower molecular weight form upon interaction of PT with mammalian cells . This prse furin , a subtise furin , or by cse furin . Choleraactivity ,20, and activity .In this study we extend the analysis of proteolytic processing of cell-associated S1 and conclude that S1 processing is not essential for the cellular activity of PT.125I-labelled PT was used for analysis of S1 processing in mammalian cells [In a previous study, an cells . As an aan cells , which, an cells , so it iWe first ruled out the possibility that furin, a protease that cleaves several other bacterial toxins in mammalian cells, is responsible for S1 cleavage, by finding that S1 processing occurs normally in furin-deficient FD11 cells  data no. The bro1, which inhibits vacuolar proton ATPase and therefore prevents endosome acidification [1 had a significant inhibitory effect on S1 processing, with 51% of cell-associated S1 in the unprocessed form  suggested a cleavage site close to either the N-terminus or the C-terminus of S1, although processing at both termini was also a possibility. Processing at the N-terminus of S1 seems unlikely in view of the fact that the arginine at amino acid 9 (R9) is crucial to enzymatic activity . A proteB. pertussis (data not shown) so that we were unable to determine whether processing was affected. However, other changes at this residue  had no effect on PT secretion, processing or activity (data not shown). In an attempt to locate the processing site, we constructed mutant forms of PT with deletions of various stretches of residues in this region. Each of 2 adjacent deletions of 8 residues  with an insertion of 8 amino acids between residues Q205 and A206 in S1 was processed to the same size S1p as wild type PT  and chymotrypsin W215A)  for 3 h to determine cellular activity. As shown in Fig. B. pertussis virulence [B. pertussis, it is possible that S1 could be processed in vivo by alternative proteases absent from CHO cells.We also tested several strains expressing mutant PT constructs in our mouse infection model of irulence , and nonPreviously we constructed stable CHO cell transfectants expressing S1 either with a signal peptide  or without one , and showed that S1 fully ADP-ribosylated target G proteins in each transfectant . In thatThe data from these transfectants do not rule out the possibility that S1 processing may contribute to the trafficking of PT to the ER. However, the data are consistent with the idea that S1 processing is not essential for its activity in mammalian cells, and therefore that translocation of the full-length S1 across the ER membrane occurs. This would distinguish PT from several other bacterial toxins of similar subunit structure, such as cholera toxin, heat-labile toxin and shiga toxin, for which processing of the A subunit is apparently important for cellular activity . The difIn this study we have further characterized the cellular processing of the S1 subunit after PT interacts with mammalian cells. Our major conclusion is that this processing event is not essential for PT activity in mammalian cells, based on several lines of evidence: (1) protease inhibitor studies showed a general lack of correlation between S1 processing and PT cellular activity; (2) mutant forms of PT in which S1 processing was apparently abolished retained significant (>75% of wild type) cellular activity; and (3) no S1 processing was apparent in transfected cells expressing active S1. Although we have not definitively ruled out a contribution of S1 processing to the cellular activity of PT due to the imperfect nature of our unprocessed mutants, it is possible that the processing event is completely unrelated to PT cytotoxicity and instead is an irrelevant activity occurring, possibly in lysosomes, on the large majority of the intracellular pool of PT molecules that do not enter the putative retrograde transport pathway to the ER and then on to the cytosolic target proteins.B. pertussis strain used in this study was a streptomycin- and nalidixic acid-resistant derivative of W28 (Wellcome). The PT9K/129G (PT*) derivative of this strain was constructed as previously described [B. pertussis was grown on Bordet-Gengou agar (Difco) plates containing 15% defibrinated sheep blood and the following antibiotics at the indicated concentrations where necessary: streptomycin 400 μg ml-1, nalidixic acid 20 μg ml-1, gentamicin 10 μg ml-1; or in Stainer-Scholte liquid medium [Escherichia coli strains used were DH10B [B. pertussis, and these were grown on LB agar plates containing 10 μg ml-1gentamicin where necessary or in LB broth containing 100 μg ml-1ampicillin.The escribed . The othd medium  containire DH10B  for stanre DH10B  for conjEcoRI-BsrGI fragment (containing the PT S1 and S2 genes) into EcoRI/Acc65I-digested allelic exchange vector plasmid pJHC1 [E. coli S17.1 and introduced into the B. pertussis chromosome by conjugation and allelic exchange as described previously [Plasmid pJ-PT was obtained by subcloning a 2.3 kb id pJHC1 . Mutatioeviously . Deletioeviously , and theeviously .Samples were run on 12% SDS-PAGE gels and transferred to nitrocellulose filters. To detect S1, blocked filters were incubated with S1-specific monoclonal antibody X2X5 or 1C7, followed by peroxidase conjugated anti-mouse IgG secondary antibody (Amersham). Blots were developed using ECL Plus (Amersham) and exposed to X-ray film.B. pertussis culture supernatants by the fetuin affinity method of Kimura et al. [E. coli DH10B containing the plasmid pGEX-αC20. For induction of the fusion protein, the strain was grown in LB to A600 1.0 and then IPTG was added to a concentration of 0.5 mM. 2 h after IPTG addition, cells were centrifuged, lysed in BPER lysis reagent (Pierce), cleared by centrifugation, and passed through a GSTrap column (Amersham-Pharmacia). Fusion protein was eluted in reduced glutathione buffer, dialyzed against PBS and analyzed by SDS-PAGE and BCA assay (Pierce) to determine protein concentration.PT and mutant derivatives were prepared from a et al. , resuspe3, and 0.5% Triton X-100) or RIPA buffer  and incubated 30 min on ice. The lysate was then centrifuged 15 min at 13,000 rpm at 4°C in a microfuge, the supernatant was removed to a fresh tube and the pellet was resuspended in sample buffer. Samples were boiled 5 min and loaded onto an SDS-PAGE gel.CHO cells were grown in 6-well plates to near confluency and then PT was added and incubated for the indicated times at 37°C. Cells were then collected by trypsinization, washed in PBS, resuspended in 50–100 μl of either NET/Triton lysis buffer . After SDS-PAGE and western blotting of samples, band intensities were measured by densitometry and used to calculate the extent of inhibition of S1 processing.Inhibitors were added to CHO cells 30 min prior to the addition of PT. For protease inhibitors we followed the suggestions of Barrett  to deter2. Trypsin was present at 70 μg/ml. Sample buffer was added and the samples were then boiled 5 min before loading onto an SDS-PAGE gel.PT (100 or 200 ng) was digested with trypsin (Sigma) in a volume of 20 μl at room temperature for 1 h in 50 mM Tris, pH 8, and 2 mM CaClSamples were run on 12% SDS-PAGE gels and transferred to nitrocellulose filters. To detect S1, blocked filters were incubated with S1-specific monoclonal antibody X2X5 (3) (a generous gift from Drusilla Burns) or 3CX4, followed by peroxidase conjugated anti-mouse IgG secondary antibody (Amersham). Blots were developed using ECL Plus (Amersham) and exposed to X-ray film.32P-NAD , 10 ng PT, and an aliquot of the lysate sample. For assessment of in vitro enzymatic activity of PT samples by ADP-ribosylation assay, reactions contained, in 25 μl, 0.1 M Tris, pH 7.5, 20 mM DTT, 0.5 mM ATP, 1 μM 32P-NAD , 10–50 ng PT, and 0.5 μg GST-αC20 protein as substrate. The mixture was incubated 90 min at room temperature, sample buffer was added, and the sample was boiled 5 min and loaded onto 15% SDS-PAGE gels. After electrophoresis, gels were fixed, dried and exposed to X-ray film. Band intensities were measured by densitometry and used to calculate the extent of ADP-ribosylation of target proteins.To determine the cellular activity of PT samples, PT (0.5–2 nM) was added to near confluent CHO cells in 12-well culture plates, and after 3 h at 37°C cells were recovered from plates, washed in PBS and lysed in NET/Triton lysis buffer 30 min on ice. The lysate was then centrifuged 15 min at 13,000 rpm at 4°C and the supernatant was stored at -20°C until the assay. The ADP-ribosylation assay contained, in 25 μl, 0.1 M Tris, pH 7.5, 20 mM dithiothreitol (DTT), 0.5 mM ATP, 1 μM CHO cells were incubated overnight with PT and then lysates were made using Triton X-100 buffer at room temperature (to maximize the proportion of S1 in the soluble fraction). Purified 1C7 monoclonal antibody was allowed to bind to a PS10 chip (Ciphergen Biosystems) for 2 h at room temperature, which was then washed with PBS. The CHO cell lysates (soluble fraction) were diluted 1:1 with PBS and 100 μl of each lysate was added to a spot on the chip and incubated at 5°C for 4 days. The chip was washed 3 times with PBS containing 0.1% Triton X-100, 3 times with PBS, and twice with 5 mM HEPES (pH 7.0). After the chip was air-dried, matrix (Sinapinic acid) was added to each spot and allowed to dry. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry analysis of these samples was performed in a model PBS-II machine (Ciphergen).Six-week-old female BALB/c mice (Harlan) were used for infection experiments. Bacterial inocula were prepared and intranasal inoculation of mice was performed as previously described . Seven dNHC conceived of the study, performed some experiments, supervised personnel and drafted the manuscript. RMM performed most of the plasmid and strain constructions, protein purification, processing assays and cell culture work. GVA performed the mouse infection experiments, some plasmid and strain constructions, protein purification, processing assays and ADP-ribosylation assays. RDP performed some of the plasmid and strain constructions and processing assays. ZEVW performed the GST fusion constructions and purification, some ADP-ribosylation assays, cell culture work and contributed to supervision of the other personnel.


Colorectal cancer is a common malignancy of the gastrointestinal tract. It is the second cancer cause of death in females and third in males. Production of prolactin has been reported with several tumours. However, elevated prolactin plasma levels in colorectal cancer patients remained unclear.In this cross sectional study serum prolactin and carcinoembryonic antigen (CEA) concentrations were assayed using immunoradiometric assay kits, preoperatively in 47 patients, and the results were compared with 51 age and sex matched controls.Prolactin and CEA concentration in patients were significantly more as compared with controls. Hyperprolactinemia was found in 36 (76.6%) patients, while 28 (59.6%) had high level of CEA.Prolactin may be a better tumour marker than CEA in patients with colorectal malignancy. Colorectal cancer is the third highest cause of cancer mortality . In AustCarcinoembryonic antigen (CEA) has been recognized as a serum marker for colorectal cancer in the past three decades  and has Prolactin (PRL) is a hormone with multiple biological actions, synthesized by the anterior pituitary gland  and is bThe aim of this study was to see whether PRL levels are increased in colorectal cancer and to compare the preoperative serum levels of PRL and CEA concentrations in colorectal cancer patients; also we intended to find the possible correlation between hyperprolactinemia and the stage of colorectal cancer.This cross-sectional study included 47 patients with colorectal cancer  and 51 non-cancer patients , who were candidates of colectomy. All patients were admitted in Shariati general hospital affiliated to Tehran University of Medical Sciences (TUMS) from April 2001 to January 2003. All women in the study were postmenopausal. None of the patients were on any drugs known to increase plasma prolactin levels in the last 6 months prior to the study and were not affected by endocrine, renal, and psychiatric disorders. There was no history of rectal bleeding and family history of colorectal cancer. Peripheral venous blood samples were daily collected from seven days before the operation to measure serum PRL and CEA levels. Blood samples were collected between 07:30 a.m. and 09:30 a.m. to avoid diurnal variation of PRL. Serum prolactin levels were measured with immunoradiometric assay (IRMA) kits produced by Kavoshian co (Tehran-Iran). Serum CEA levels were assayed by IRMA, who used kits from IMMUNOTECH co (Marseille-France). The PRL and CEA assays were carried out within 10 days of sampling. The cut off levels for PRL and CEA were 20 ng/ml and 5 ng/ml serum respectively ,18. StagThe study was approved by the Vice-Chancellor of ResearchEthic Committee of TUMS.The data evaluated by Chi-square test and T test using the Statistical Package of Social Science  for Windows version 11.5. A P-value of <0.05 was considered statistically significant.The number of male and female participants was 29 and 18 in case and 30 and 21 in control group.We compared serum PRL and CEA level of 47 patients with 51 healthy controls. There was no significant difference between the age and the sex distribution of the patients and control groups.Mean PRL serum level in colorectal cancer group was 21.6 ± 8.1versus 10.5 ± 4.6 in control group, which was significantly different (p < 0.0001). In other words 36 patients showed hyperprolctinemia (76.7%) while there was only one such patient in the control group (1.9%).CEA level in 59.6% of the cancer group was above the normal compared with 1.9% of controls (P < 0.0001).Out of 47 patients in the cancer group 9 were in stage I 19.1%), 19 in stage II (40.4%), 10 in stage III (21.3 %) and 9 in stage IV (19.1%). Hyperprolactinemia and high CEA concentrations were found in all stages  declare that they have no competing interests.Dr Ahmad Reza Soroush and Dr Hosein Mahmood zadeh participated in the design of study, acquisition of data and interpretation of data. Dr Mehrnush Moemeni participated in the design of study, acquisition of data, interpretation of data, and drafting the article. Behnam Shakiba participated in acquisition of data, interpretation of data, drafting the article and final approval of this version. Sara Elmi participated in analysis and interpretation of data, drafting the article and final approval of this version. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Avidity determination of antigen-specific immunoglobulin G (IgG) antibodies is an established serological method to differentiate acute from past infections. In order to compare the avidity of varicella-zoster virus (VZV) IgG in pairs of serum and cerebrospinal fluid (CSF) samples, we developed a new technique of avidity testing, the results of which are not influenced by the concentration of specific IgG.The modifications introduced for the new VZV IgG avidity method included the use of urea hydrogen peroxide as denaturing reagent, the adaptation of the assay parameters in order to increase the sensitivity for the detection of low-level VZV IgG in CSF, and the use of a new calculation method for avidity results. The calculation method is based on the observation that the relationship between the absorbance values of the enzyme immunoassays with and without denaturing washing step is linear. From this relationship, a virtual absorbance ratio can be calculated. To evaluate the new method, a panel of serum samples from patients with acute and past VZV infection was tested as well as pairs of serum and CSF.For the serum panel, avidity determination with the modified assay gave results comparable to standard avidity methods. Based on the coefficient of variation, the new calculation method was superior to established methods of avidity calculation.The new avidity method permits a meaningful comparison of VZV IgG avidity in serum and CSF and should be of general applicability for easy determination of avidity results, which are not affected by the concentration of specific IgG. Toxoplasma gondii. Therefore, avidity testing has been particularly useful for these pathogens.In addition to the determination of immunoglobulin M (IgM) antibodies, the avidity of immunoglobulin G (IgG) antibodies is an important parameter for the diagnosis or exclusion of acute infections. Testing of IgG avidity has been applied for a large variety of pathogens . A coDetermination of antibody avidity is usually based on the separation of low and high avidity antibodies by denaturing agents in enzyme immunoassays (EIA) or immunofluorescence assays. Several agents such as guanidine hydrochloride , diethylDiagnosis or exclusion of acute infection is the most common but not the only application of avidity determination. Determination of antibody avidity in pairs of serum and cerebrospinal fluid (CSF) samples has been suggested as a diagnostic means to detect intrathecal antibody synthesis  and to dThe aim of our study was to establish an avidity assay that can be used for comparison of the avidities of varicella-zoster virus (VZV) IgG in serum and CSF. In order to achieve this aim, several modifications of a commercial VZV IgG assay were introduced. These modifications include the use of a novel denaturing agent and a new method to calculate the avidity results.The serum and CSF samples used in this study had been sent to the virological laboratory at the University of Würzburg for routine VZV testing and were stored in aliquots at -20°C. Two groups of serum samples were analyzed for the evaluation of the standard and modified VZV avidity assay. Group 1 consisted of 28 samples from 19 patients with acute or recent VZV infection and included 5 follow-up samples of patient F19, spanning a period of 11 months. The cases of this group fulfilled the following criteria: the presence of clinical symptoms suggestive of varicella, information on the disease onset, and a positive VZV IgM . Group 2 consisted of 37 samples from 37 subjects with infection in the distant past. In the subjects of this group, VZV IgG antibodies had been detected in previous samples taken at least 8 months earlier. For initial evaluation of the VZV avidity assay for CSF samples, three sample pairs of serum and CSF were tested, two from patients with VZV encephalitis (V2.1 and V9.2) and one from a patient with multiple sclerosis (M22).VZV IgG avidity determination of serum samples was performed in a semi-automated fashion using the Enzygnost Anti-VZV/IgG test kit (Dade Behring) with some modifications. Each of the sera diluted 1:231 in sample buffer was placed in two antigen coated microtiter plate wells. After 60 min at 37°C, one well was washed according to the instructions with the supplied washing buffer. The other well was washed with a solution of urea, or urea hydrogen peroxide, in the supplied washing buffer  and once with the washing buffer only. The subsequent steps were carried out according to the instructions in an automated fashion using the Behring Elisa Processor III . Peroxidase-conjugated anti-human IgG was added in a 1:50 dilution and the plate was incubated at 37°C for 60 min. After three further washing steps with the supplied washing buffer, tetramethylene benzidine dihydrochloride was added as substrate and kept at room temperature for 30 min. The reaction was stopped with 0.5 N sulfuric acid. Control samples of acute and past VZV infections were included in each run. Where appropriate, VZV IgG antibodies were quantified by an one-point-quantification method  according to the instructions of the manufacturer.To increase the sensitivity of the VZV IgG EIA in order to detect low-titer VZV IgG antibodies in CSF, the standard serum assay was modified as follows. The sample incubation time was increased to 180 min; the anti-human IgG was used in a 1:30 dilution and its incubation time was increased to 90 min. All incubations for the CSF assay were performed at room temperature because variation was found to be lower compared to incubation at 37°C. For the serum samples tested for evaluation purposes, the standard dilution of 1:231 was increased by a factor of 6 to yield a final dilution of 1:1386.Determination of VZV IgG avidity in CSF was always done in parallel with serum samples from the same time-point. Both serum and CSF were tested in at least four dilutions of a two-fold titration series. The starting dilution for each sample was derived from routine determinations of VZV IgG titers. For CSF samples, the starting dilution was at least 1:6.VZV IgG avidity was calculated by various methods. First, it was calculated as the ratio of EIA absorbance values obtained with and without the denaturing washing step. Alternatively, one-point quantification titers (see above) instead of absorbance values were used for avidity calculation.ref) and y the absorbance with the denaturant (absdenat). The m- and c-values of the equation for each sample were calculated with the software Excel (Microsoft) from two or more experimentally derived data points. It is essential, that the absorbance values obtained without denaturant (absref) fall in the linear range of the enzyme immunoassay. For the VZV IgG assay with modified assay conditions, the linear range of the absorbance values without denturant extended from approximately 0.200 to 2.800. Avidity was determined from the linear equation for various virtual x-values as the percent ratio of the absorbance values with and without denaturing agent, i. e. avidity = (absdenat/absref) = (m × x + c)/x. The result was referred to as virtual absorbance ratio.Two additional methods were used for determination of VZV IgG avidity in CSF and the corresponding serum samples. One method involved the use of the software "Avidity 1.2", based on curve-fitting analysis of serial dilutions . SecondlIn order to evaluate the usefulness of the Enzygnost VZV IgG EIA for avidity testing, a panel of serum samples from patients with acute VZV infection of defined onset group 1) and from controls with VZV infection in the distant past (group 2) was tested. In a preliminary experiment with three samples from each group, two different denaturing conditions were employed. The separation of the two sample groups with one 3 min washing step using 4 M urea hydrogen peroxide was superior to three 5 min washing steps with 5 M urea . Because it had been shown previously for the Epstein-Barr virus (EBV) assay that avidity index calculations based on one-point-quantification titers gave results better than calculations using absorbance values , the aviCSF avidity assay.In general, IgG concentrations in CSF are by a factor of 1:200 to 1:1000 lower than in the corresponding serum. Therefore, the detection sensitivity of the VZV IgG assay was increased by prolonging incubations times and increasing the concentration of the anti-human IgG conjugate in order to reliably detect VZV IgG in all CSF samples from patients with positive serum VZV IgG. To study the effect of these assay modifications on avidity determination, all serum samples of group 1 and 17 randomly chosen samples of group 2 were retested with the modified assay. All the serum samples were tested in dilution 1:1386 instead of 1:231 used in the standard serum assay. The results are shown in Figure For meaningful interpretation of avidity values in CSF, it is necessary to test serum samples obtained in parallel with the CSF. Because of the large difference of the IgG concentrations between serum and CSF, comparison of the avidity results necessitates independence of IgG concentration. To study this issue, CSF and serum samples were tested in twofold dilution series with the CSF avidity assay. Avidity was calculated as percent ratio of absorbance values from each dilution. The results for representative CSF and serum samples are shown in Figure Because the two methods for avidity calculation described above were significantly influenced by the chosen working dilutions, we developed a new calculation method of antibody avidity. It is based on our observation that the relationship between absorbance values without and with denaturing agent is nearly linear. This relationship can therefore be described by the linear equation y = m × x + c, where m is the slope, c the intercept, x the absorbance without denaturant and y the absorbance with denaturant. Representative examples from patients with acute and past VZV infection and of serum and CSF pairs are shown in Figure Further dilution series of CSF and corresponding serum were tested with the CSF avidity assay in order to confirm that the influence of the IgG concentration on the avidity indices derived from the virtual absorbance ratio was minimal. Table We established a VZV avidity assay that is suitable for comparative evaluation of antibody avidity in serum and CSF samples. In order to achieve this aim several modifications of a serum VZV avidity assay were necessary. Though there have been a few studies on the use of avidity assays for VZV IgG antibodies -18, no sUrea hydrogen peroxide appeared to be superior to urea in the optimization experiments for the VZV avidity assay and presents a novel option for a denaturing agent in avidity measurements. The denaturing effect of one washing step with a 4 M solution of urea hydrogen peroxide was more pronounced than that of three washing steps with a 5 M urea solution. Thus, the denaturing potential of urea hydrogen peroxide appears to be greater than that of urea. Furthermore, because of better solubility, handling of solutions of urea hydrogen peroxide is easier than handling solutions of urea in high molarity. Nevertheless, the optimal denaturing agent may vary between different antigens necessitating careful evaluation of denaturing conditions for each avidity assay.Under normal conditions, virus specific IgG is present in the CSF in very low concentrations. With an intact blood-CSF-barrier, the concentration gradient between serum and CSF is 200:1 to 1000:1. Thus, the usual serological methods for antibody quantification in serum are not sensitive enough to routinely detect specific IgG in CSF. Therefore, we modified the VZV IgG EIA to increase its sensitivity. This modification made it possible to detect and quantify VZV IgG in CSF samples from virtually all patients with measurable VZV IgG in serum. Corresponding serum samples were always run in parallel with CSF in the same assay. The CSF and serum dilutions were chosen individually for each sample pair in order to achieve similar absorbance values in the modified EIA. This assay formed the basis for avidity determination of CSF and the corresponding serum.For meaningful comparison of avidity values in serum and CSF, it is necessary to apply a calculation method that is independent of the concentration of specific IgG in the samples examined. One-point determination represents the simplest method for avidity index calculation, but such methods did not yield satisfactory results in this respect. The avidity technique based on end-point-titration is not influenced by the IgG concentration and is considered the reference method for avidity determination ,7. Howevdenat) for any virtual absorbance value without denaturation (absref). The avidity index is then calculated as the ratio of the two virtual absorbance values (absdenat/absref). Thus, if a fixed absref is chosen for all samples to be compared, the avidity index becomes independent of the concentration of specific IgG in the samples. These theoretical considerations have been confirmed by the results obtained from dilution series in this study. It will be interesting to test the general applicability of this method for standard avidity assays by comparison with single-point and end-point avidity determinations.Searching further for a simple calculation method of avidity indices independent of IgG concentration and sample dilutions, we observed a linear relationship between the absorbance values of the assays without and with denaturing washing conditions. After performing an avidity assay of two dilutions of a given sample, this relationship can be exploited to calculate a linear equation from the two pairs of absorbance values (with and without denaturation). The only requirement is that all absorbance values should fall in the linear range of the EIA. Once for a given sample the linear equation is experimentally determined, the equation can be used for calculation of the virtual absorbance value with denaturation  using the Avidity 1.2 software in infectious-disease diagnosis, and is a shareholder of an SME (Headman Ltd.) with commercial interest in it.RHK, JS and FT carried out the immunoassays and participated in the design of the study. RHK carried out the avidity index calculations. WZ participated in establishing the standard VZV IgG avidity assay. KH provided the software "Avidity 1.2" and participated in the data analysis. BW conceived of and coordinated the study and drafted the manuscript. All authors contributed to the final editing of the manuscript.The pre-publication history for this paper can be accessed here:


The topic of the paper is to study the stability of gene clusters, defined by various hierarchical clustering algorithms, of microarrays experiments including or not MVs.Microarray technologies produced large amount of data. The hierarchical clustering is commonly used to identify clusters of co-expressed genes. However, microarray datasets often contain missing values (MVs) representing a major drawback for the use of the clustering methods. Usually the MVs are not treated, or replaced by zero or estimated by the k-Nearest Neighbor  are highly sensitive to MVs, and surprisingly, for a very tiny proportion of MVs (e.g. 1%). In most of the case, the MVs must be replaced by expected values. The MVs replacement by the kNN approach constitutes one efficient method for restoring the missing expression gene values, with a low error level. Our study highlights the need of statistical treatments in microarray data to avoid misinterpretation.The presence of MVs (even at a low rate) is a major factor of gene cluster instability. In addition, the impact depends on the hierarchical clustering algorithm used. Some methods should be used carefully. Nevertheless, the  We observe a drastic loss of the clustering stability since τ = 1% of MVs. For instance, with 5% of genes with MVs, i.e. 40 missing data, the mcquitty, Ward and complete-linkage methods have a CPP of 62%, 57% and 52%, respectively. Beyond a rate of τ equal to 10%, the decrease becomes lower.The clusters defined from the reference data and the data with MVs are compared using H2O2 and GSHEAT and all the generated sets from 1/2 to 1/7. These last results show that the quality of the gene clustering is not disturbed by the reduction of the number of genes.Similar results are observed for all the sets OS, GS, GSCPP values of single-and the centroid-linkage methods remain too stable to observe a clear decrease as seen in Figure CPP values and to compare the different aggregation methods.It must be clearly noted that to limit the effect of the topology of each algorithm, we have fixed that the 10 most populated clusters must represent 80% of the genes. The number of the most populated clusters is fixed at 10 due to the Ward linkage method that gives a very limited number of clusters. Then, the percentage of genes belonging to the 10 most populated clusters have been tested ranging from 70% to 90% (data not shown). For example with a percentage equal to 90%, the kNN method has been described by Troyanskaya and co-workers  are approximated with a mean error rate within the range . Conversely the values more than 1.5 or less than -1.5 are approximated with a mean error rate superior to 1.8. The misestimating of extreme values has an impact on the clustering. One can notice that the unweighted kNN (mean of the k observations) exhibits worse results compared to the weighted kNN used in this study (data not shown).Figure CPP was computed for the seven agglomeration methods. We have compared the HC results obtained with the reference sets and the generated sets without replacement of MVs, with kNN replacement or with zero-value replacement methods. Table kNN and zero-value replacements both improved the mean CPP whatever the clustering method used, except for the Ward method with the zero value replacement. The kNN approach is the most relevant method to replace MVs. In 55.2% to 66.3% of the simulations, kNN is better and globally gives a mean increase of the CPP within the range . The centroid-and single-linkage methods have better increase in 74.8% and 99.3% of the simulations respectively due to their particular topologies. The zero value replacement is clearly less efficient.The CPPf to consider the f closest clusters of the selected cluster. This index is similar to the previous one and takes into account that the genes may be relocated in close clusters (see section Methods). It allows the evaluation of the topology conservation. We used f = 5. We observed that the co-associated genes in the reference sets are often displaced to close clusters in the simulated sets. As observed for the CPP, the kNN approach improves the CPPf for all the clustering methods in 51.9% to 60.2% of the simulations within the range . Due to their high initial CPP values (97.7% and 98.8%), single-and centroid-linkage methods do not have a gain as previously observed for the CPP.Nevertheless, as a slight variation can displace one gene into a close cluster, we have characterized another index named H2O2 have CPP and CPPf close to the one of OS. Conversely, the CPP and CPPf of GSHEAT are better than the ones of OS and GSH2O2 for the complete-linkage and Ward methods, but lower for the others. The GS set has higher CPP and CPPf for single-likage to mcquitty method due to a lower influence of the MVs in a vector with a higher number of experimental conditions. Nevertheless, the complete-linkage and Ward linkage still remain at a very low CPP (close to 50%).Similar results are obtained with the other sets with slight variations. For example, GSCPP values. In addition we have tested the consequences of using k values different of kopt values in the range  and we observed a decrease of CPP within the range  (data not shown).Moreover, we have tested the influence of the number of MVs per gene by introducing more than one missing value per gene. We obtained similar results showing less than 0.2% of variations of the i.e. values superior to 1.5 and lower than -1.5. Table CPP values are superior to the CPP values obtained previously, because only the genes with important variations have MVs and are members of small clusters. We observe that MVs replacement has little effect. Indeed, the kNN and zero – value replacement cannot restore a correct distribution .We have followed the same methodology to analyze the extreme values, MVs are a common trait of microarrays experiments. Few works had been reported about MVs replacements -20 and nkNN replacement method was the most efficient approach to compensate the MVs effects compared to the classical replacement by zero. The kopt depends on the sample size. It is important to keep in mind that the MVs corresponding to extreme values are difficult to estimate with the kNN method. The impact of their approximation upon the clustering is significant. Hence, new approaches like the Bayesian Principal Component Analysis (BPCA) may overcome this problem. In a recent work Liu and co-workers suggest to potentially eliminate the incomplete series of data by using robust Singular Values Decomposition [We showed that the position . In addiposition .algo) a specific number of clusters . This point is one of the main difficulties noted by Yeung and co-workers [Contrary to Yeung and co-workers  and Gibb-workers  to evalu-workers  which sh-workers ,34 or Gr-workers  have comN = 6013 genes and n = 8 experimental conditions about the phosphate accumulation and the polyphosphate metabolism of the yeast Saccharomyces cerevisiae [S. cerevisiae [N = 6153 genes and n = 178 experimental conditions. Due to the diversity of conditions in this set, we focused on two experimental subsets corresponding to heat shock and H2O2 osmotic shock respectively.We used 8 public data sets from the SMD database ; see Tab. Two setrevisiae . The secrevisiae . This seN = 5783 genes and n = 8 experimental conditions. The second set without MVs was taken from Gasch et al. and called GS. The experimental conditions (column) containing more than 80 MVs were removed. The resulting GS matrix contains N = 5843 genes and n = 42 experimental conditions. Two subsets were generated from GS and has been noted GSHEAT and GSH2O2. They correspond to specific stress conditions as described previously. GSHEAT and GSH2O2contain respectively N = 3643 genes with n = 8 experimental conditions and N = 5007 genes with n = 10 experimental conditions.To evaluate the incidence of MVs on hierarchical clustering, we built complete datasets without MVs. All the genes containing at least one missing value were eliminated from the Ogawa set (noted OS). The resulting OS set contains i.e. the number of genes, we built six smaller sets corresponding to 1/2, 1/3, 1/4, 1/5, 1/6 and 1/7 of OS, GS, GSHEAT and GSH2O2. To obtain representative subsets, we can not use a random generation which can bias the results. So, we searched for each subset the series of genes which reflect at best all the genes of the complete set. First, the distance matrices between all the genes were computed. Then, we performed an iterative process by: (i) computing the sum of the distances for each possible t-uplets (t=2 to 7) of the set, (ii) choosing the t genes which have the minimal distance, (iii) selecting 1 representative gene upon the t selected genes, this gene is chosen as the closest to the barycenter of the cluster, (iv) eliminating from the process the t genes. Step (i) to step (iv) are repeated until all the genes are used. All the representative genes constitute the subset (1/t). This procedure allows one to reduce the redundancy of similar genes and to maintain approximately a number of gene clusters constant.To test the influence of the matrix size, τ of genes containing one MV (τ = 1 to 50.0%), these MVs are randomly drawn. Each random simulation is generated at least 100 times per experiment to ensure a correct sampling.From the sets without MVs, we introduced a rate vi, a missing value i for a given expression vector v (i.e. a gene), with the kNN method, the k vectors w corresponding to the k most nearest vectors to v (without taking the ith elements of the w-vectors into account) are searched. The missing value vi is then estimated by a weighted value of the k retained wi values.To fulfill d between the vector v and every vector w. The k minimal distances d are selected and the estimated value  is computed as follow [The similar vectors are identified by calculating the Euclidean distance s follow ,19:kNN, s = 1 / d, this similarity measure s is deduced from the distance d between v and its neighbors w. In equation (1), more a vector w close to v, more it contributes to the estimation of the missing value. The kNN approach has no theoretical criterion to select the optimal k value (kopt) [kopt using the sets without MVs to ensure a minimal bias in the comparison of the hierarchical clustering results. The determined kopt value is associated with a minimal global error rate as defined by Troyanskaya and co-workers [In the weighted e (kopt) . We have-workers .hclust package in R software [The hierarchical clustering (HC) algorithm allows the construction of a dendogram of nested clusters based on proximity information . The HC software . Seven hsoftware .i.e. genes) is calculated by using an external module written in C language. We used the normalized Euclidean distance d* to take account of the MV:The distance matrix between all the vectors  is not computed if vi and/or wi is a missing valueTo assess the influence of missing data rates and different replacement methods into clustering results, we have analysed the co-associated genes of an original dataset (without MVs) compared to these genes location in a set with MVs.reference clusterings (RC). In a second step, we generated MVs in data. The MVs are replaced by using the different replacement methods. Then we performed the hierarchical clustering for each new set. The results obtained by these second analyses are denoted generated clusterings (GC). We compared the resulting clusters defined in RC and GC. We assessed the divergence by using an index named Conserved Pair Proportions (CPP). The CPP is the maximal proportion of genes belonging to two clusters, one from the RC and the other one from the GC :The procedure for computing the index algo), we defined Kalgo, the number of clusters. As every type of hierarchical clustering algorithm gives a particular topology, we cannot use the same number of clusters to compare each aggregative method. So, we defined Kalgo such as its 10 most important cluster must represent 80% of the genes. For this purpose, we defined Kinit, an important initial number of clusters , and counted the number of occurrences associated to the 10 most populated clusters. Then we diminished Kinit by one unit and counted again. We stopped the process when the 10 most important clusters represent 80% of the occurrences . We denote by  the jth cluster for a given clustering algorithm with j = {1, ..., Kalgo}. The  clusters are associated with their corresponding gene list .i) For each reference clustering based on a given clustering algorithm  is used -, the second one after estimating the missing data by the kNN method (Eq.1), and the third one after replacing the missing data by zero. For each resulting tree, Kalgo clusters are defined. The  clusters are associated with their corresponding gene list , with j' = {1, ..., Kalgo }.ii) Three hierarchical clusterings are performed after generating MVs in proportion CPP index, we searched for each  cluster the closest  cluster. For each clustering algorithm , the corresponding  cluster is selected as the maximum number of genes  from the gene list  found in . Then, the Conserved Pairs Proportion (CPP) is computed as follow for one simulation:iii) Finally, to estimate the . The term  is the Kronecker symbol, i.e. it is equal to 1 when the genes i and i' in the two gene lists are identical, otherwise 0. G denotes the total number of genes. This index takes the maximal value 1 when the clusterings RC and GR are identical.where CPP-variation. If the remaining genes of a cluster are in the direct neighbour clusters, the use of CPP can bias the analysis. Thus, we characterized the CPPf ratio to consider the f closest clusters of the  cluster. The computation of CPPf ratio is based on the previous ratio and corresponds to the f clusters which are the closest to the winning cluster. From a selected , the upper node of the dendrogram is examined. If the number of clusters linked to this node is inferior to f, the upper node is selected. This process is performed until the number of clusters is inferior or equals to f. The last node tested  is used to compute the CPPf ratio.In addition, a variation of the tree topology may induce a AdB conceived of the study and carried out the MVs generation and the hierarchical clustering results. SH worked on the statistical analysis. AM has the initial idea of evaluating the MV influence on clustering, and participated in its design and coordination. All authors read and approved the final manuscript.


NDRG1 gene is induced by nickel, a transition metal sharing similar physical properties to cobalt. Nickel may create hypoxia-like conditions in cells and induce hypoxia-responsive genes, as does cobalt. Therefore NDRG1 is likely to be another gene induced by hypoxia. HIF-1 is a transcription factor which has a major role in the regulation of hypoxia-responsive genes, and thus it could be involved in the transcriptional regulation of NDRG1 gene. Hypoxia is such a common feature of solid tumours that it is of interest to investigate the expression of Ndrg1 protein in human cancers.The expression of NDRG1 gene and cause the accumulation of Ndrg1 protein. Protein levels remain high even after cells revert to normoxia. Although HIF-1 is involved in the regulation of NDRG1, long term hypoxia induces the gene to some extent in HIF-1 knock-out cells. In the majority of human tissues studied, Ndrg1 protein is overexpressed in cancers compared to normal tissues and also reflects tumour hypoxia better than HIF-1 protein.Hypoxia and its mimetics induce in vitro expression of NDRG1 gene, and nickel probably causes the induction of the gene by interacting with the oxygen sensory pathway. Hypoxic induction of NDRG1 is mostly dependent on the HIF-1 transcription factor, but HIF-1 independent pathways are also involved in the regulation of the gene during chronic hypoxia. The determination of Ndrg1 protein levels in cancers may aid the diagnosis of the disease.Hypoxia is an inducer of the  Cancer is a leading cause of death in humans, and current studies in the clinical area focus on either the early detection of this disease or on the development of new selective treatment tools. New tumour markers can provide aid in cancer diagnosis and create novel treatment opportunities.NDRG1) in different laboratories, is a stress responsive protein which shuttles between cytoplasm and nucleus upon certain insults . MCF-7, PW, HIF-1α+/+, and HIF-1α-/- cells were maintained in DMEM (Dulbecco's modified Eagle's medium) with the same supplements. HOS and HTE cells were grown in α-MEM  additionally supplemented with 2 mM L-glutamine. For Northern and Western blot experiments, 5 × 105 cells in 10 ml of media were plated in each of 10-cm dishes . Cell numbers were determined using the ZM Coulter Counter . To render cells hypoxic, dishes were placed in an incubator chamber flushed with 95% N2 and 5% CO2. This resulted in approximately 0.1–0.5% O2 after several hours. After 20 hours, cells were released from hypoxia and quickly scraped in ice-cold phosphate-buffered saline (PBS), and analyses were performed as described below.The cell lines were maintained at 37°C as monolayers in a humidified atmosphere containing 5% CONDRG1 and actin probes were labelled with [α-32P]dCTP by using a randomly primed-DNA labelling kit (Promega). Cloning of NDRG1 gene was as described previously  containing protease inhibitors . 40 μg or 25 μg of protein was loaded into each lane of a 10% SDS-PAGE gel and separated by electrophoresis. Proteins were then transferred to PVDF membranes , and the membranes were first incubated with rabbit anti-Ndrg1 polyclonal antibody at the dilution of 1:1000 in 5% non-fat dry milk for one hour at room temperature. The antibody production is described elsewhere [4 in 5% milk for one hour at room temperature. At the end the membranes were treated with chemiluminiscent substrate ECL  for 1 min at room temperature, and Biomax MR-1 films  were exposed to the membranes. The molecular weight of Ndrg1 was determined using prestained molecular weight markers .Cells were lysed, and proteins were harvested in 125 μl of TNES buffer [50 mM Tris-HCl (pH: 7.5), 2 mM EDTA, 100 mM NaCl, 1 mM sodium orthovanadate NaVO4, 10 m2O2 for a further 30 min. After incubation with the antibody against Ndrg1 protein overnight, the slides were processed with a second anti-rabbit peroxidase-conjugated antibody . Identification of the protein was then achieved using Avidin-Biotin horseradish peroxidase complex and 3,3-diaminobenzidine (DAB) as the chromogen. Negative controls were performed using nonimmune serum instead of primary antibodies.For in vivo detection of Ndrg1 protein in human cancer and normal tissues, immunohistochemical (IHC) staining was exploited, using a rabbit polyclonal antibody against a 30-aminoacid-sequence at the C-terminal end of the Ndrg1 protein [Immunohistochemical detection of HIF-1α protein was achieved by using Catalyzed Signal Amplification System  which is based on streptavidin-biotin-horseradish peroxidase complex formation. Antigen retrieval was done by 10 mM sodium citrate buffer (pH 6.0). The specimens were incubated overnight at +4°C with monoclonal anti-HIF-1α antibody  in a dilution of 1:1000. After the amplification of the signal according to manufacturer's instructions, the slides stained with chromogen DAB, counter-stained with hematoxylin, dehydrated, and then mounted.