Silene which have an X-Y sex-determining system (S. latifolia, S. dioica, and S. diclinis). We used the X-linked copies to build a genetic map of the X chromosomes, with a marker in the pseudoautosomal region (PAR) to orient the map. The map covers a large part of the X chromosomes—at least 50 centimorgans. Except for a recent rearrangement in S. dioica, the gene order is the same in the X chromosomes of all three species. Silent site divergence between the DNA sequences of the X and Y copies of the different genes increases with the genes' distances from the PAR, suggesting progressive restriction of recombination between the X and Y chromosomes. This was confirmed by phylogenetic analyses of the four genes, which also revealed that the least-diverged X-Y pair could have ceased recombining independently in the dioecious species after their split. Analysis of amino acid replacements vs. synonymous changes showed that, with one possible exception, the Y-linked copies appear to be functional in all three species, but there are nevertheless some signs of degenerative processes affecting the genes that have been Y-linked for the longest times. Although the X-Y system evolved quite recently in Silene (less than 10 million years ago) compared to mammals (about 320 million years ago), our results suggest that similar processes have been at work in the evolution of sex chromosomes in plants and mammals, and shed some light on the molecular mechanisms suppressing recombination between X and Y chromosomes.To help understand the evolution of suppressed recombination between sex chromosomes, and its consequences for evolution of the sequences of Y-linked genes, we have studied four X-Y gene pairs, including one gene not previously characterized, in plants in a group of closely related dioecious species of  Similar processes have been at work in the evolution of sex chromosomes in plants and mammals. A recently evolved plant X-Y system is helping to shed light on these processes Drosophila sex chromosomes and some plants [Silene [Drosophila miranda [Drosophila are excellent for studying the rate and causes of degeneration, but cannot shed light on question (i).Newly evolved sex chromosome systems, such as those in plants  and fishe plants , but note plants ? Second,e plants , and emp [Silene ,7 and fr miranda . Recent Silene. This genus is a model for the study of plant sex chromosome evolution, since the sex chromosomes evolved recently [Studies of the evolutionary divergence of gene pairs on mammalian X and Y chromosomes suggest that recombination between the X and nonrecombining parts of the Y was successively suppressed. In many X-Y systems, including that in mammals, there is a “pseudoautosomal” region (PAR) where the X and Y recombine, and it has been found that DNA sequence divergence between homologous X- and Y-linked genes increases with distance from this region. This pattern has been termed “evolutionary strata” ,10. Partrecently ,13.Silene species (i.e., species with separate sexes) includes S. latifolia, S. dioica, and S. diclinis, which have an X-Y sex-determination system with a male-determining Y [Silene species are hermaphroditic or gynodioecious . Dioecy and sex chromosomes thus probably evolved within this genus [Silene species have n = 12 chromosomes [One group of closely related dioecious mining Y ,14, whilis genus . All dipomosomes , so theromosomes .S. latifolia have recently been identified and sequenced  ceased recombining long before the three dioecious species split, whereas the X and Y copies of SlX1-SlY1 (termed locus 1) continued to recombine until recently. We discuss the implications of these results for the mechanism of recombination arrest between the sex chromosomes.Several sex-linked genes from equenced , allowinS. latifolia cDNA. The SlX3 open reading frame of 575 amino acids encodes a protein sequence similar to calcium-dependent protein kinases (CDPKs) from tobacco, rice, and Arabidopsis thaliana (the best BLAST hits had 75%–80% amino acid identity, based on more than three-fourths of the length). CDPKs are associated with various kinds of stress responses [S. latifolia [Locus 3 was identified from esponses . Thus, latifolia ,19.S. latifolia and S. dioica [latifolia-dioica, latifolia-diclinis, and dioica-diclinis , but for gene 1 no such sites were found. If, on the other hand, X1 and Y1 diverged after the species split, some sites should differ between the species, but not between X and Y of the same species. This is found for mammalian and bird sex chromosomes, and phylogenetic analysis suggests that some X and Y (or, in birds, Z and W) genes ceased recombining independently in different taxa [Silene species are very closely related [X1 sequences. However, some Y variants are exclusive to each species; we found five nucleotide variants fixed only in the S. latifolia Y (plus nine indel variants), and ten fixed only in S. dioica Y (plus one indel). Since only 11 S. dioica Y sequences were analyzed, the number of fixed Y variants is probably overestimated, however . Furthermore, in a tree estimated excluding these sites with fixed differences in the Y-linked sequences (as is appropriate for such closely related species), the Y sequences are nested within those of the X of each species  between the X and Y sequences of S. latifolia and S. diclinis (dSX-Y) correlates with the gene's distance from the PAR in the X chromosome genetic map . X-Y synonymous divergence also differs significantly between genes 1 and DD44 (p = 0.01). These results suggest progressive suppression of the recombination between X- and Y-linked alleles of different genes. In S. dioica, the same correlation exists, using the S. latifolia or S. diclinis gene order; thus, the rearrangement probably arose recently in S. dioica, consistent with its absence in the other dioecious species. A recent rearrangement, such as an inversion, after the DD44-X and -Y sequences had diverged for some time, would not affect this gene's X-Y divergence relative to that of gene 1. In mouse species, where rearrangements have occurred, evolutionary strata corresponding to those on other mammalian X chromosomes are still plainly discernible [Synonymous divergence etic map B. X-Y sycernible .(dN) was less than dS for divergence between X and Y sequences  tests for these two genes, the dN/dS difference between Y and X is highly significant (χ2 = 11.7, with 4 degrees of freedom). Our observation of similar dS values contrasts with previous analyses [S. diclinis Y3 gene also seems to evolve faster than the other Y3 genes  and moving toward the current PAR. This pattern resembles the “evolutionary strata” for mammalian X-Y gene pairs based on Ks values, a measure of divergence per site similar to dS [dSX-Y values among our four gene pairs is 26% for locus 3 in S. diclinis. This overlaps the values for the mammalian stratum 4 and 3 genes ; these strata are inferred to have ceased recombining between the X and Y 30–50 million years ago (MYA) for stratum 4, and 80–130 MYA for stratum 3, whereas strata 1 and 2 diverged 130–320 MYA [The correlation of ar to dS ,10. Howe–320 MYA ,11.S. latifolia, S. dioica, and S. diclinis X-Y sequence divergence data show that X-Y differentiation was already advanced in the common ancestor of these species, except for locus 1. The maximum synonymous X-Y divergence observed for our genes is approximately 25%, including SlAp3, which probably transposed from an autosome onto the Y soon after the sex chromosomes evolved [MROS3-X/Y, whose Y-linked copy is degenerated [Silene species, the Silene sex chromosomes must have evolved much more recently than mammalian sex chromosomes.The  evolved ; all theenerated . Unless Silene. For the nuclear genes Chs and Adh in the family Brassicaceae, estimated rates are, respectively, 1.4 × 10–8 to 2.2 × 10–8 substitutions per synonymous site per year [Ipomoea [S. latifolia group of species. However, substitution rates for some plant Adh genes are almost ten times slower, particularly for plants with long generation times [DD44 differentiated before the S. dioica–S. latifolia–S. diclinis speciation, whereas gene 1 may have ceased recombining after this, perhaps independently in S. latifolia and S. dioica; no analysis can be done in S. diclinis without diversity data for this species, but suppression of X-Y recombination within this species after its split from the other dioecious species is also possible  suggest that the S. latifolia Y may carry genes suppressing recombination, and should help test whether mechanisms other than inversions contributed to reduction of the PAR.The mechanism suppressing X-Y recombination is unknown. Recombination could be reduced either by inversions (or other major recombination rate changes), and/or by modifiers reducing local crossover rates. The “strata” of different divergence in mammalian sex chromosomes may have resulted from a series of Y inversions disrupting X-Y recombination . Inversinversion . Thus, gn Silene ,33, requSilene. Recombination suppression may be selectively favored to preserve advantageous Y-linked combinations of alleles at different loci, such as genes that are advantageous in males but not in females [The mechanism of recombination reduction between X and Y chromosomes is important for understanding the diversity in loci that recently ceased recombining, such as gene 1 in  females , althoug females ,22.Silene sex chromosomes. Degeneration is likely, since genotypes with a Y but no X chromosome are inviable [MROS3-Y in S. latifolia [Silene Y is uncertain, because most currently known sex-linked genes in these plants were ascertained from a cDNA-based search for Y-linked genes. Bacterial artificial chromosome clone sequencing may provide unbiased comparisons of homologous X- and Y-linked regions, and this has been started in papaya [Silene can also be inferred when dN values in the Y are elevated compared with X lineages. This is seen for the two “old” Silene Y-linked genes, locus 3 and locus 4 . Thus the higher dN in the Y-linked alleles is not due to a higher mutation rate (higher dS) in the Y than the X. Moreover, the Y-linked copy of locus 3 fails to amplify in RT-PCR experiments, and may be degenerated.Our analyses suggest that both reduction of recombination and Y degeneration may be in progress for inviable ,35, but atifolia . The extn papaya . Some de locus 4 . DiffereDD44, and no diversity data have yet been obtained for gene 3. The result of this test was nonsignificant; there is thus no evidence that Y1 and Y4 evolution is driven by selection. There is, however, very low polymorphism in the Y copies, so the test has low power [These observations, plus those for gene 1 , suggestow power .Genetic degeneration is supported by low levels of polymorphism of Y- compared with X-linked genes, taking into account the lower Y effective population size ,22. ThisSilene Y-linked genes? Our analyses suggest that degeneration of the genes studied here is partial, at most, consistent with a recent origin of the Silene sex chromosomes. However, there has probably been enough time for degeneration, since this occurred rapidly for genes on the neo-Y chromosomes of D. miranda [Silene Y. Silene sex chromosomes are more advanced in sex chromosome evolution than in some other plants. The papaya sex-determining region is just a small nonrecombining part of one chromosome, yet there is evidence for considerable differentiation, including addition of repeat sequences and some evidence for gene loss [D. miranda neo-Y chromosome [Buchnera [D. miranda neo-Y [Why is degeneration so slight for our  miranda , which aene loss . More liene loss ) may thuromosome , the birromosome , and in Buchnera . In all da neo-Y ), suggesS. latifolia plants were from Edinburgh  and from Fontainebleau forest (France). S. dioica plants were collected in Corrèze (France). S. dioica plants from the Sherringham population (Sherringham, England), used for isolation of the ScOpa09 marker, were kindly provided by D.L. Mulcahy (Department of Biology, University of Massachussetts). S. noctiflora and S. vulgaris were obtained from the seed collection of the Lyon Botanical Garden (Lyon, France). Seeds of S. diclinis were obtained from the seed collection of the Institute of Biophysics in Brno (Czech Republic). Interspecific hybrid Silene diclinis × latifolia plants were generated by pollination of a S. diclinis female with pollen of an MAV line male (S. latifolia) kindly provided by S. Matsunaga (Department of Biotechnology, Osaka University). The S. latifolia U9 line, which was used for pollination of the interspecific hybrid, was kindly provided by S. Grant (Department of Biology, University of North Carolina).Genomic DNA was extracted from leaves as described . For RT-S. latifolia by the approach that yielded loci 1  and 4 (lX4/SlY4 ). From alX4/SlY4 , and the walking , using t walking , SoutherS. dioica, S. diclinis, and S. noctiflora or S. vulgaris using primers designed from S. latifolia sequences (Y3, which was amplified from genomic DNA. The PCR conditions, using Taq polymerase (Amersham Pharmacia, Piscataway, New Jersey, United States), were as follows. One incubation at 94 °C for 5 min; 35 cycles of: denaturation at 94 °C for 30 s, annealing at a temperature that depended on the primers for 30 s, and elongation at 72 °C for a time depending on the length of the amplicon  being the probable ortholog of SlY/X1. In S. latifolia, some of the copies have been shown not to be sex-linked . Southern blots were not done in S. dioica or S. diclinis, but RT-PCR reactions in all the dioecious species always amplified a single sequence in the females plus another very similar sequence in males, clearly representing the expected X and Y copies. Thus, for the analyses presented later, this gene can be treated as a single-copy gene, as was also the case in previous mapping work [All genes, except gene 1, are single-copy see . In S. lrn blots , and theing work .S. dioica, S. diclinis, and S. vulgaris were amplified, either by RT-PCR (X copies) or from genomic DNA (Y copies). PCR products were cloned into pGEM-T Easy vector (Promega, Madison, Wisconsin, United States), and multiple clones were sequenced for each gene. Sequencing reactions were carried out with ABI Big Dye Terminator V1.1 DNA sequencing kit, on an Applied Biosystems 3100 sequencer .For locus 3, the 3′ two-thirds of the coding sequences of S. dioica or S. latifolia. We have now confirmed sex linkage of all four loci by segregation analysis in all three dioecious species  were designed from this sequence. In plants grown from seeds of this family, presence or absence of the expected amplified fragment accords exactly with results using the original pseudoautosomal RAPD marker primer OPA09. With our primers, S. dioica plants from the Corrèze population were genotyped by digesting PCR products with the restriction enzyme TaqI; the recombination frequency between the marker locus and sex was approximately 2.5%, confirming the pseudoautosomal location. In S. latifolia, genotyping was done using the same primers and an AluI site polymorphism. Genotype data were analyzed by both three-point and multipoint mapping, using JoinMap version 1.4 [To orient the X genetic map, we used a pseudoautosomal marker. For this, we cloned and sequenced a RAPD fragment incompletely linked to the X chromosome of the pollen donor of the eveloped . The seqsion 1.4 . Thus thhttp://pbil.univ-lyon1.fr/) [The primer sequences were removed before sequence analyses. For each gene, the nucleotide sequences were aligned using the corresponding amino acid sequences as a guide, using ClustalW with the Seaview interface (on1.fr/) . Alignmehttp://pbil.univ-lyon1.fr/) [http://pbil.univ-lyon1.fr/) [http://taxonomy.zoology.gla.ac.uk/rod/treeview.html) [Phylogenetic trees were estimated including all sites except those with gaps by neighbor joining (NJ), maximum parsimony, and maximum likelihood (ML), using Phylo_Win (on1.fr/) . For NJ on1.fr/)  and Treeew.html) .dS and dN site divergences were estimated using PAML 3.13 (http://abacus.gene.ucl.ac.uk/software/paml.html) [http://pbil.univ-lyon1.fr/) [dS and dN are similar under various substitution models (namely, Goldman and Yang 1994 [Both ml.html)  and JaDion1.fr/) . Estimatang 1994 , Yang anang 1994 , and Neiang 1994 , implemeang 1994  using Jaang 1994 .dS or dN of X and Y sequences were compared using HyPhy 0.99  expresses dS values for Y lineages as multiples of values for X lineages: Rsyn = dSY/dSX. In model 2 , Rsyn was constrained to be equal to 1 (dSY = dSX). We compared the ML values by a LR test with model 2 as the null hypothesis. We used the same approach to compare dN values (with dN/dS replacing Rsyn). To compare dN/dS using LR tests, we again defined two models. Model 1 assumed two global variables (dN/dS)X and (dN/dS)Y so that the nonsynonymous rates of branches of the X lineage were expressed in terms of the synonymous rate (dN/dS)X, and similarly using (dN/dS)Y for Y branches (“shared dN/dS”), while model 2  assumed (dN/dS)X = (dN/dS)Y.Values for  species  to polarS. diclinis Y3 evolves faster than other Y3 sequences, we assumed a common Rsyn for S. dioica and S. latifolia, as in model 1 above, but added a further parameter, the dSY/dSX ratio for S. diclinis (model 1*). We compared models 1 and 1* using a LR test as above; we tested dN and dN/dS differences similarly.To test whether DD44, and no diversity data are yet available for gene 3.McDonald-Kreitman tests were done using DNAsp software, version 3.95 . The divS. latifolia by contingency tests, using DNAsp. To infer fixed differences rigorously, we used diversity data within S. latifolia for genes 1, 4, and DD44. For gene 3 no diversity data are yet available; however, because this gene pair has high X-Y divergence, raw divergence values should suffice, so for this gene we estimated numbers of differences from single X and Y sequences.To test for differences in divergence between the X and Y sequences of different genes, we compared numbers of fixed X-Y differences in S. latifolia and S. dioica Y and X sequences, plus one sequence from each of two outgroup species, S. vulgaris and S. conica; this enables us to identify whether the changes were in the X or Y lineages, using parsimony. With global gap-removal, the program unambiguously distinguishes fixed differences, including insertions and deletions, from polymorphisms within species. The outgroup sequences are shorter than the other sequences, so some fixed differences in the S. dioica Y could not be analyzed.To test whether X and Y sequences of gene 1 continued recombining and started to diverge after the dioecious species split, a C program was written to find fixed differences in a set of multiple http://www.lirmm.fr/guindon/phyml.html) [As this dataset includes the first approximately 2,000 sites of gene 1, including coding and intron sequences , a more ml.html) , excludiTable S1(36 KB DOC).Click here for additional data file.http://www.ncbi.nlm.nih.gov/) accession number for AtMSI4 is AF028711; the UniProt/TrEMBL (http://www.ebi.ac.uk/trembl/) accession number the ScOpa09 marker is Q9FW98.The GenBank (


Natural killer (NK) cells represent a first line of defence against a developing cancer; however, their exact role in colorectal cancer remains undetermined. The aim of the present study was to evaluate the expression of CD16 and CD57 [immunohistochemical markers of natural NK cells] in colorectal adenocarcinoma.Presence of NK cells was investigated in 82 colorectal adenocarcinomas. Immunohistochemical analysis was performed, using 2 monoclonal antibodies . The number of immunopositive cells (%) was evaluated by image analysis. The cases were characterized according to: patient gender and age, tumor location, size, grade, bowel wall invasion, lymph node metastases and Dukes' stage.NK cells were detected in 79/82 cases at the primary tumor site, 27/33 metastatic lymph nodes and 3/4 hepatic metastases; they were detected in levels similar to those reported in the literature, but their presence was not correlated to the clinical or pathological characteristics of the series, except for a negative association with the patients' age (p = 0.031).Our data do not support an association of NK cell tissue presence with clinical or pathological variables of colorectal adenocarcinoma, except for a negative association with the patients' age; this might possibly be attributed to decreased adhesion molecule expression in older ages. Colorectal adenocarcinoma is a neoplasm in which prognosis is mainly determined by the histological stage. However, the prognosis of certain patient groups, especially those of intermediate stages, remains vague . The maiNatural killer (NK) cells play a pivotal role in innate immunity and immunological surveillance. Their cytotoxic effects may be initiated without prior immunization and thus NK cells have been considered as a first line of defence of the host against a developing cancer ,3. In spThe aim of this study was to evaluate tissue presence of NK cells in a series of colorectal adenocarcinomas and to attempt to correlate their presence with clinical and pathological variables and prognostic markers of colorectal cancer.From June 1997 to May 2000, 82 patients from our Department underwent colectomy, due to a diagnosis of colorectal adenocarcinoma of conventional histologic type. NK cell presence was examined via standard 3-step immunohistochemical analysis (ABComplex), which was performed on formalin-fixed, paraffin-embedded tissue sections. Briefly, two appropriate monoclonal antibodies were used  at dilutions of 1/200 with overnight incubation. Antigen retrieval was necessary and was performed by usual microwave treatment. In the examined samples, tissue identification of NK cells was based on strong CD16 immunostaining, which was frequently accompanied by CD57 immunoreactivity. Tissue sections from hypertrophic tonsils were used as positive markers. Diaminobenzidine tetrahydrochloride 0.06% in phosphate-buffered saline buffer containing 0.03% hydrogen peroxide was used as a chromogen. Images were acquired using a Zeiss Axiolab microscope (Carl Zeiss GmbH, Jena, Germany) with a mechanical stage, fitted in a Sony-iris CCD video camera (Sony, Tokyo, Japan). The video camera was connected to a Pentium II PC, loaded with the appropriate image analysis software (Sigma Scan Pro, Science, Erkrath, Germany). Slides were examined at a ×200 magnification. The ratio, expressed in percentile proportion (%), between the number of immunohistochemically positive-stained cells and the total number (stained and unstained) of lymphocytes was calculated. All tumors had been characterized according to the following classical clinical and pathological variables: patient gender  and age (mean: 75.9, median: 76, ranging between 35–95 years), tumor location (rectum 27, 32.9%, sigmoid 26, 31.7%, descending colon 4, 4.8%, transverse colon 5, 6%, ascending colon 10, 12.1%, cecum 10, 12.1%), size (mean diameter: 4.4 cm, median diameter: 4 cm, ranging between 1.2–10 cm), grade (I: 24 cases, 29.3%, II: 48 cases, 58.5%, III: 10 cases, 12.2%), bowel wall invasion (present in 66 cases, 80.5%), lymph node metastases (present in 33 cases, 40.2%) and Dukes' stage (A: 5, 6.1%, B: 44, 53.7%, C: 29, 35.4%, "D": 4, 4.9%). All "D" Dukes' stage cases involved hepatic metastases. The tumors were categorized according to their location into two larger groups: those involving the rectum as well as the left colon (57 cases, 69.5%) and those involving the right colon (25 cases, 30.5%). This was performed for reasons of statistical analysis.Moreover, positive cases were divided into two groups: those in which immunopositive cells ranged from 0–9% of the total number of lymphocytes ("weak" tissue presence of NK cells) and those in which they were ≥10% ("strong" NK cell presence). This was performed somewhat arbitrarily, as we considered that the cutoff level of 10% is in accordance to what a pathologist would consider to be the cutoff level between "negative" and "positive" cases in a qualitative examination, and in order to perform statistical analysis. Associations of NK cell presence with the patients' sex, tumor location, grade, and presence of bowel wall invasion, as well as lymph node metastases and Dukes' stage were examined using chi-square statistics; associations with the patients' age and tumor size were determined using the Mann-Whintey U test.NK cells were detectable in the primary tumor site of 79 cases (96.3%); their percentages were up to 32% of the primary site lymphocytes . Of the 33 cases with metastatic lymph nodes, NK cells were found in 27 cases (81.8%); moreover, NK cells were found among liver lymphocytes in 3 of the total 4 cases with hepatic metastases (75% of stage "D" cases).Forty-nine of the 82 primary tumors (59.8%) had a "weak" tissue presence of NK cells (0–9%), whereas the remaining 33 primary tumors (40.2%) had a "strong" NK cell presence (≥10%). Of the 33 cases with metastatic lymph nodes, 18 cases (54.5%) had "weak" and 15 "strong" NK cell presence.NK cell presence in the primary tumor site was not associated with the patients' sex (chi-square, p = 0.462), tumor size (Mann-Whintey U test, p = 0.377), tumor location (chi-square, p = 0.262), tumor differentiation (chi-square, p = 0.556), wall invasion (chi-square, p = 0.999), the presence of metastatic lymph nodes (chi-square, p = 0.720), or Dukes' stage (chi-square, p = 0.992). However, a negative association between the presence of NK cells at the primary tumor site with the patients' age was noticed (Mann-Whitney U test, p = 0.031).Moreover, the presence of NK cells in the metastatic lymph nodes was not associated with the patients' sex (chi-square, p = 0.999), age (Mann-Whintey U test, p = p = 0.212), tumor size (Mann-Whintey U test, p = 0.729), tumor location (chi-square, p = 0.442), tumor differentiation (chi-square, p = 0.872), wall invasion (chi-square, p = 0.999), or Dukes' stage (C or D) (chi-square, p = 0.308).Although colorectal adenocarcinoma has been characterized as a model neoplasm in which clinical prognosis is mainly determined by the histological stage, pathologic markers often fail to predict the prognosis of a great number of patients. This has necessitated the evaluation of several alternative prognostic markers . ImmunohCD16 and CD57 were chosen in the present study, as they are markers of the presence of NK cells, a cellular line which could reasonably play a significant role in the pathophysiology of colorectal adenocarcinoma. NK cells were considerably present in the primary tumor site, as well as the metastatic lymph nodes of many cases in our series. This observation is in line with data deriving from the literature -14; thisHowever, what seems to be pivotal for rapid and efficient migration of NK cells from the circulation to the tumor stroma, is the expression of appropriate adhesion molecules . As exprIn conclusion, our findings suggest that less NK cells are found in the stroma surrounding the primary tumor site in older patients with colorectal adenocarcinoma. This could possibly be attributed to decreased adhesion molecule-mediated migration; however, this hypothesis needs to be further investigated through more studies.The pre-publication history for this paper can be accessed here:


Ras induces expression of p19Arf, which, in turn, activates p53 and growth arrest. Here, we used a multistage model of squamous cell carcinoma development to investigate the functional interactions between Ras, p19Arf, and p53 during tumor progression in the mouse. Skin tumors were induced in wild-type, Arfp19-deficient, and p53-deficient mice using the DMBA/TPA two-step protocol. Activating mutations in Hras were detected in all papillomas and carcinomas examined, regardless of genotype. Relative to wild-type mice, the growth rate of papillomas was greater in Arf-p19deficient mice, and reduced in p53-deficient mice. Malignant conversion of papillomas to squamous cell carcinomas, as well as metastasis to lymph nodes and lungs, was markedly accelerated in both Arf-p19  and p53-deficient mice. Thus, p19Arf inhibits the growth rate of tumors in a p53-independent manner. Through its regulation of p53, p19Arf also suppresses malignant conversion and metastasis. p53 expression was upregulated in papillomas from wild-type but not  Arfp19-null mice, and p53 mutations were more frequently seen in wild-type than in  Arfp19-null carcinomas. This indicates that selection for p53 mutations is a direct result of signaling from the initiating oncogenic lesion, Hras, acting through p19Arf.Ectopic expression of oncogenes such as  A squamous cell carcinoma model shows Ras mutation not only initiates tumor development but, through Arf and p53, directly influences the subsequent evolutionary trajectory of the tumors Ras, and two tumor suppressors, Arf,p19  and p53, using a multistage epithelial tumor model.Tumor development and metastasis is a multistep process of somatic cell evolution that includes uncontrolled proliferation, impaired apoptosis, loss of differentiation, immortalization, neovascularization, invasion, and metastatic spread . This evRas is among the most frequently mutated oncogenes in human cancer, with approximately 30% of tumors carrying an activating mutation in one of three family members, Hras, Kras, or Nras  seen in human tumors ; p = 0.010) and  Arf+/−p19 mice had an average of 2.60 more papillomas (95% CI (0.05, 5.14); p = 0.045) in weeks 18–30 after DMBA administration. Average papilloma size was also greater in both  Arf−/−p19 and  Arf+/−p19 mice compared to wild-type mice ( Arf−/−p19 mice were greater than 8 mm in diameter versus 14% (38/267) from wild-type mice (p < 0.0001). Papillomas from  Arfp19-deficient mice measured up to 16 mm in diameter while very few papillomas on wild-type mice measured more than 9 mm. Thus,  Arfp19 deficiency resulted in faster growing papillomas, indicating a role for  Arfp19 in regulating the early stages of benign tumor growth.Both in vitro and in vivo studies have demonstrated that p19ppressor . However5 wk see . Papilloype mice A. Relatiype mice B. This eHras are found in more than 95% of DMBA/TPA-induced skin tumors (. In vitro studies showed that mutant Ras induces p19Arf, which, in turn, inhibits Ras-induced proliferation (Arf might reduce or eliminate the need to mutate Ras. All papillomas from  Arf+/+p19 (5/5),  Arf+/−p19 (4/4), and  Arf−/−p19 (5/5) mice contained the identical A→T transversion at codon 61 of Hras, resulting in an amino acid change from glutamine to leucine and a constitutively activated Ras protein ; p = 0.005) and 3.11 times higher for  Arf+/−p19 mice (95% CI (0.90, 10.77); p = 0.073) compared to wild-type mice. The reduced carcinoma latency and increased conversion frequency in the  Arfp19-null mice implicate loss of p19Arf as a critical rate-limiting step in malignant SCC progression.The rate of malignant conversion of papillomas to carcinomas is greatly increased in the absence of p53 function . To deteer 22 wk C. By 28  Arf+/−p19 and  Arf−/−p19 mice also showed a range of grades but a significant number (9/12) were characterized as spindle cell carcinomas. These were characterized by packed and spindle-shaped cells with elongated pleiomorphic nuclei and abundant abnormal mitotic figures. These cells grew in a homogenous pattern with very little evidence of the cellular organization typical of low-grade tumors. These tumors showed focal areas of squamous differentiation, indicating that they were derived from squamous epithelium.Histologic analysis revealed that the carcinomas from control mice ranged in grade from well-differentiated to poorly differentiated SCCs. Carcinomas from  Arfp19 deficiency also increased dissemination and establishment of metastatic SCCs. Carcinoma-bearing p19Arf-deficient mice frequently presented with enlarged lymph nodes, and in several cases tumors were noted on the lungs.  should be seen in the remaining wild-type  Arfp19 allele in carcinomas. Alternatively,  Arfp19 could be recessive, in which case LOH or reduction to a homozygous null state would be expected. The fate of the wild-type allele of  Arfp19 in tumors from heterozygous mice was assessed by semiquantitative PCR analysis of genomic DNA. Three of 15 (20%) papillomas examined showed evidence of loss of the wild-type  Arfp19 allele, compared to ten of 15 (67%) carcinomas (p = 0.0027)  A, indicamal skin B. IncreaArf and p53 were upregulated in papillomas, that p53 expression was reduced in  Arp19f-null papillomas, and that loss of p19Arf had a similar effect on tumor progression as that of loss of p53, provide strong in vivo support of the model whereby p19Arf regulates p53 in response to mutational activation of Hras. However, enhanced tumor growth in  Arfp19-null mice, in contrast to reduced tumor growth in p53-null mice (Arf, independent of p53. To examine the effect of the combined loss of p53 and p19Arf tumor suppressors, skin tumors were induced in  Arfp19 and p53 single and compound mutant littermates. Relative to wild-type mice, p53-null mice developed fewer tumors, averaging 4.05 fewer papillomas (95% CI (−6.10, −2.00); p = 0.0001) 10–16 wk after the DMBA administration, while  Arfp19-null mice averaged 2.68 more papillomas (95% CI (0.52, 4.84); p = 0.015) 18–40 wk after the DMBA administration  (unpublished data). Thus, p53-regulated apoptosis does not appear to play a major role in SCC development, at least at the papilloma stage.To determine whether tumor growth in mice lacking p19ng index C. The p1Arf and p53 cooperate during tumor progression, papilloma to carcinoma conversion was evaluated in Arf−/−, p53−/−,p19 and Arf−/−p53−/−p19 littermates. Tumor progression was accelerated in all  Arf-p19 and p53-deficient genotypes compared to wild-type littermates ( Arf−/−p19 mice regardless of p53 genotype +/+, p53+/−,(p53 or −/−)p53  A, indicaints see B. This c+/−p53 mice there is strong selective pressure to lose the wild-type allele during conversion to malignancy p19 were assessed for LOH of p53 by semiquantitative PCR analysis of genomic DNA. Two out of seven papillomas from  Arf+/+p53+/−p19 mice show loss of the remaining p53 allele, while all papillomas examined from  Arf+/−p53+/−p19 (n = 8) and  Arf−/−p53+/−p19 (n = 8) mice show retention of wild-type p53 ( Arf+/+p53+/−,p19 and three of 14 (21%) from Arf+/−p53+/−,p19  but only one of 12 (8%) from  Arf−/−p53+/−p19 (p = 0.036) showed loss of p53. Thus, deletion of p19Arf disrupts the activation of p53 and thereby reduces selection for mutations in p53 during malignant progression.In lignancy . As p19Atype p53 B. Seven Hras and the tumor suppressors Arfp19 and p53. Somatic mutation of Ras is an early and frequent event in this model of tumor development. Against this backdrop, p19Arf has at least two distinct tumor suppressor properties, which act at different stages of tumor development and which show a range of gene dosage effects. Loss of one or both  Arfp19 alleles leads to accelerated growth of benign tumors, indicating  Arfp19 is partially haploinsufficient for suppression of this early growth phenotype. Although p19Arf regulates p53 at this stage, suppression of tumor growth per se by p19Arf does not appear to be mediated through p53. p19Arf also inhibits the benign to malignant transition and subsequent tumor cell dissemination and metastasis, and this effect of p19Arf is, in contrast, mediated through p53. LOH of Arfp19  occurs preferentially in malignant tumors, indicating complete loss of p19Arf is favored during progression. Thus, p19Arf inhibits several stages in Ras-driven tumor progression. Furthermore, Ras is connected to both p19Arf and p53 through a signaling pathway, indicating that selection for mutations in the p19Arf / p53 pathway are a direct consequence of the initial Ras mutation.Using a multistage model of tumor progression, we have examined the functional interactions between the oncogene Hras mutations are found at high frequency in papillomas from wild-type and both p19Arf- and p53-deficient mice indicates that squamous epithelial cells harboring Hras mutations have a strong selective advantage, with or without the presence of p19Arf or p53. This permits analysis of the effects of Ras on defined genetic backgrounds in the natural setting of tumor cell evolution. The expression levels of both p19Arf and p53 were increased in wild-type papillomas but not in  Arf−/−p19 papillomas, indicating that p19Arf regulates p53 in response to activated Ras in vivo. However, other signals to induce p53, such as those stemming from DNA damage, remain intact in the absence of p19Arf, as shown by the rapid increase in p53 in irradiated  Arfp19-null tumors. Thus, of the many stimuli that have been shown to activate p53 using a variety of experimental systems  or exogenous stimuli (e.g., DNA damage inducers). However, it remains possible that these other modes of p53 activation might predominate in other tumor types or in other circumstances.The observation that  systems , the RasArf and p53 were induced in papillomas, loss of p19Arf or p53 had opposite effects on early tumor growth. p19Arf deficiency resulted in increased tumor cell proliferation and tumor growth while p53-deficient mice had reduced tumor cell proliferation, tumor numbers, and tumor size. The observation that tumors harboring mutant Ras grew faster in the presence of p53 than in the absence of p53 differs from in vitro studies, in which ectopically expressed Ras induces p53-dependent growth arrest or senescence  were provided by Martine Roussel and Charles Sherr ( Arf−/−p19 mice were backcrossed to the susceptible NIH/Ola strain (Harlan Olac, Oxfordshire, United Kingdom), and carcinogenesis studies were performed on the F3 littermates of this cross. 20 mice of each genotype,  Arf−/−, p19 Arf+/−,p19 and  Arf+/+p19 were treated. The backs of 8-wk-old male and female mice were shaved and treated with a single application of DMBA  followed a week later by twice weekly applications of TPA (200 μl of 10−4 M solution in acetone) for 15 wk. The number and size of papillomas on each mouse were recorded every 2 wk. Mice were sacrificed if moribund or following detection of carcinomas. Tumors were frozen for DNA extraction and/or fixed in formalin to be processed and stained with hematoxylin/eosin (H&E) for histological examination.p19es Sherr . To incrp53 (7 backcross to NIH) were crossed to  Arfp19-deficient (F4 backcross to NIH) mice to generate   Arf+/− p53+/−p19 mice. These mice were intercrossed to generate all nine possible  Arf /p53p19 genotypes. Some 20–30 mice of each genotype were subjected to the same DMBA/TPA protocol and monitored as described above.Mice deficient for p53   and p53 (Novocastra Laboratories, Newcastle-upon-Tyne, United Kingdom).In order to remove abundant keratin present in papillomas and carcinomas, nuclear extracts were prepared as described  with modHras was amplified with standard PCR , with 5′-GACTCCTACCGGAAACAGGT-3′ and 5′-CTGTACTGATGGATGTCCTC-3′ primers. We used the internal primer 5′-TGGTCATTGATGGGGAGACA-3′ to sequence exon 2, using PE Biosystems  Dye-Terminator and Big-Dye cycle sequencing.Genomic DNA was prepared from tumor and normal tissue by QIAamp DNA Mini Kit . A 400-bp PCR fragment containing exon 2 of Sections of normal skin, papillomas, carcinomas, and other organs were removed and fixed in 10% normal buffered formalin for 4 h. After fixation, tissues were processed and then embedded in paraffin. From the tissues, 4-μm sections were cut and stained for either p53 (CM5, Novocastra) or pan-keratin (AE1/AE3, Novocastra) using high-temperature antigen retrieval in 10 mM citrate buffer (pH 6), or for BrdU (Dako, Glostrup, Denmark) after treating with 2N HCl followed by 0.1% trypsin. After staining with the primary antibody, the sections were stained with a biotin-conjugated secondary  followed by StreptABComplex/HRP (Dako). Slides were developed with DAB/NiCl and counterstained with methyl green. Control sections with no primary antibody were run concurrently. Other sections were cut and stained using a standard H&E method. Proliferation index was determined by counting the number of BrdU-stained cells per 40× field. The apoptotic index was determined by counting the H&E slides for the number of apoptotic figures per 40× field. All counts were done on a Nikon (Tokyo, Japan) Labophot-2 microscope.Arfp19 wild-type allele in Arf+p19/− tumors, wild-type and knockout alleles were amplified by PCR separately then combined for electrophoresis. Primers 5′-AGTACAGCAGCGGGAGCATGG-3′ (Arf1), 5′-TTGAGGAGGACCGTGAAGCCG-3′ (Arf2), and 5′-ACCACACTGCTCGACATTGGG-3′ (ArfN) were used to amplify wild-type (Arf1 and Arf2) and knockout alleles (Arf2 and ArfN) from 100 ng of genomic DNA using 68 °C for annealing and extension at 90 s for 30 cycles. Equal amounts of each PCR product were then combined for electrophoresis on a 2% TAE agarose gel. Wild-type and knockout alleles of p53+/− tumors were amplified in a separate reaction as described . To measure LOH of the escribed  for 30 cArfp19 (or Arf /p53p19) genotypes on the development of papillomas, longitudinal profiles of papilloma counts were analyzed using the generalized estimating equation (GEE) approach (Arfp19 (or Arf /p53p19) genotypes. Estimates of relative odds were adjusted for sex effects. Fisher's exact test was used for comparing two proportions such as comparing LOH proportions between papillomas and carcinomas. All statistical tests were two-sided.In order to assess the impact of approach . GEE is approach  was used


Low adherence is a key factor in explaining impaired effectiveness and efficiency in the pharmacological treatment of hypertension. However, little is known about which factors determine low adherence in actual practice.The purpose of this study is to examine whether low social participation is associated with low adherence with antihypertensive medication, and if this association is modified by the municipality of residence.1288 users of antihypertensive medication were identified from The Health Survey in Scania 2000, Sweden. The outcome was low adherence with antihypertensives during the last two weeks. Multilevel logistic regression with participants at the first level and municipalities at the second level was used for analyses of the data.Low social participation was associated with low adherence with antihypertensives during the last two weeks (OR = 2.05, 95% CI: 1.05–3.99), independently of low educational level. However, after additional adjustment for poor self-rated health and poor psychological health, the association between low social participation and low adherence with antihypertensives during the last two weeks remained but was not conclusive (OR = 1.80, 95% CI: 0.90–3.61). Furthermore, the association between low social participation and low adherence with antihypertensives during the last two weeks varied among municipalities in Scania (i.e., cross-level interaction).Low social participation seems to be associated with low adherence with antihypertensives during the last two weeks, and this association may be modified by the municipality of residence. Future studies aimed at investigating health-related behaviours in general and low adherence with medication in particular might benefit if they consider area of residence. The effectiveness ,2 and efLow adherence is one important cause of uncontrolled hypertension ,10. Yet,Social participation is an important concept for understanding the influence of social factors on individual health and behaviour, and can be viewed as a feature of individual social networks . Good soMultilevel analysis handles both the micro-scale of people and the macro-scale of context simultaneously within one model . This anThe first aim of this study is to examine whether low social participation is associated with low adherence with antihypertensives during the last two weeks, independently of low educational level and health status . The second aim is to analyse whether the hypothesised association between low social participation and low adherence with antihypertensives during the last two weeks varies between municipalities in Scania.The Health Survey in Scania 2000 (HSS-2000) was a postal self-administered questionnaire sent out to a random sample of 23 437 individuals born from 1919 to 1981 living in Scania. The purpose of the HSS-2000 was to obtain information about health conditions and different types of health hazards among the inhabitants of Scania . The proThe Ethical Committee at the Medical Faculty of Lund University approved the study proposal of The HSS-2000, and all of the participants received written information about the survey.Use of antihypertensives was based on an affirmative answer to the question "Have you during the last year used medicine, which was bought at the pharmacy...?" and indicating "Medication for high blood pressure"Low adherence with antihypertensives during the last two weeks (dichotomised) was based on the question "Have you used (this) medicine during the last year, but not during the last 2 weeks?" Those participants who answered yes were considered to have low adherence.Age was categorised in five groups: <35 (reference), 35–44, 45–54, 55–64 and ≥ 65 years.Low social participation (dichotomised) was assessed after the respondent stated involvement in three or fewer activities (lowest quartile) of 13 formal or informal activities , which the respondent might have participated in during the previous 12 months [2 months .Low educational level (dichotomised) was defined as having nine years of education or less.Poor self-rated health (dichotomised) was defined as a value of ≤ 3 on an ordinal self-rated health scale ranging from 1 ("Very bad") to 7 ("Very good") [y good") .Poor psychological health (dichotomised) was determined by giving three or more affirmative answers to the 12 items composing the Standardised General Health Questionnaire (GHQ-12) [(GHQ-12) .Because of the hierarchy in the data, with individuals nested in municipalities, we used multilevel logistic regression  with indi was created to study the influence of low social participation on low adherence with antihypertensives during the last two weeks, adjusting for age and sex. The second model ii was extended to also include low educational level, because low educational level could be a confounder in the association between low social participation and low adherence with antihypertensives during the last two weeks. The third model iii additionally contained poor self-rated health and poor psychological health. Impaired health may affect both low social participation and low adherence with antihypertensives.The first model The results are shown as odds ratios (OR) with 95% confidence intervals (CI)We calculated the second level variance  regarding prevalence of low adherence with antihypertensives during the last two weeks (i.e., the intercepts in the multilevel regression), and the second level variance regarding the association between low social participation and low adherence with antihypertensives during the last two weeks (i.e., the slope variance in the multilevel regression). We also calculated the covariance between intercept and slope residuals. The covariance gives information about whether the association between low social participation and low adherence with antihypertensives during the last two weeks depends on the prevalence of low adherence in the different municipalities (i.e., cross-level interaction).Parameters were estimated using the Restricted Iterative Generalized Least Squares (RIGLS) and penalised quasilikelihood (PQL). Extra-binomial variation was explored systematically in all models and we found no evidence for under- or over-dispersion. The MLwiN, Version 1.1 software package  was usedLow adherence with antihypertensives during the last two weeks was found among 11% (145/1 288) of the participants and 49% (635/1 288) were classified as having low social participation. The participants mean age was 63 years. Those participants classified as having low social participation more often reported low adherence with antihypertensives during the last two weeks, low educational level, poor self-rated health and poor psychological health than those who were not classified as having low social participation   and in the association between low social participation and low adherence with antihypertensives during the last two weeks (slope variance) Table . The negOur results suggest that low social participation is associated with low adherence with antihypertensives during the last two weeks, independently of low educational level. In other words, the association between low social participation and low adherence withstood adjustment for socio-economic position  in our analyses. Social participation might therefore be considered as a real construct, and not only a proxy for socio-economic position. However, the association between low social participation and low adherence with antihypertensives during the last two weeks was weakened after additional adjustment for poor self-rated health and poor psychological health. Furthermore, the association between low social participation and low adherence with antihypertensives during the last two weeks may vary between municipalities in Scania.The weakening of the association between low social participation and low adherence with antihypertensives during the last two weeks after we adjusted for poor self-rated health and poor psychological health may be an expression for confounding. Impaired health may negatively affect both social participation and adherence with antihypertensives. On the other hand, the observed reduction of the association may instead be telling us that physical and mental health are in the pathway between low social participation and low adherence with antihypertensives during the last two weeks . Social Our present finding that the association between low social participation and low adherence with antihypertensives during the last two weeks may vary between municipalities in Scania gives empirical support to the existence of cross-level interactions  associated with health-related behaviours, such as low adherence with medication. These behaviours may be a result of the interaction between a person and his or her area of residence . In a prThe present study shows that multilevel regression analysis can be used for investigation of geographical disparities in health and health-related behaviour (e.g, adherence with medication), without analysing any specific area characteristic . In multThe rather low participation rate (59%) may increase the risk of selection bias and reduce the ability to generalise the results and compare them to other populations. Nevertheless, the participation rate for participants aged 51–80 was about 65% and the participants using antihypertensives had a mean age of 63 years.The low adherence question we used was stated in a non-threatening manner, which might facilitate for participants to give an honest response and not underreport low adherence . Self-rePeople with low social participation and low adherence with antihypertensives during the last two weeks may have been less inclined to respond to the HSS-2000 questionnaire. This possible selection bias could lead to an underestimation of the association between low social participation and low adherence with antihypertensives during the last two weeks.Our results suggest that low social participation is associated with low adherence with antihypertensives during the last two weeks, independently of low educational level. In addition, the association between low social participation and low adherence with antihypertensives during the last two weeks seems to vary between the municipalities in Scania, which gives empirical support to the existence of cross-level interactions  associated with health-related behaviours, such as low adherence with medication. We have recently showed that factors related to the area of residence influence the individual blood pressure level, especially in people using antihypertensive medication , which iFuture studies aimed at investigating health-related behaviours in general and low adherence with medication in particular might benefit if they consider that area of residence may modify associations between individual variables.The author(s) declare that they have no competing interests.JM and KJ developed the original idea, participated in the design of the study, performed the statistical analyses and drafted the manuscript. LR and JS participated in the design of the study and revised the manuscript. TL participated in the design of the study, helped to collect the data and revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/5/17/prepub


Identifying regions of the human genome that have been targets of natural selection will provide important insights into human evolutionary history and may facilitate the identification of complex disease genes. Although the signature that natural selection imparts on DNA sequence variation is difficult to disentangle from the effects of neutral processes such as population demographic history, selective and demographic forces can be distinguished by analyzing multiple loci dispersed throughout the genome. We studied the molecular evolution of 132 genes by comprehensively resequencing them in 24 African-Americans and 23 European-Americans. We developed a rigorous computational approach for taking into account multiple hypothesis tests and demographic history and found that while many apparent selective events can instead be explained by demography, there is also strong evidence for positive or balancing selection at eight genes in the European-American population, but none in the African-American population. Our results suggest that the migration of modern humans out of Africa into new environments was accompanied by genetic adaptations to emergent selective forces. In addition, a region containing four contiguous genes on Chromosome 7 showed striking evidence of a recent selective sweep in European-Americans. More generally, our results have important implications for mapping genes underlying complex human diseases. An analysis of 132 human genes suggests that the migration of modern humans out of Africa into new environments was accompanied by genetic adaptations to emergent selective forces Despite intense study and interest, a detailed understanding of the evolutionary and demographic forces that have shaped extant patterns of human genomic variation remains elusive. An important goal in studies of DNA sequence variation is to identify loci that have been targets of natural selection and thus contribute to differences in fitness between individuals in a population. Identifying regions of the human genome that have been subject to natural selection will provide important insights into recent human history , the funThe neutral theory of molecular evolution , which pOne way out of this conundrum is to recognize that population demographic history affects patterns of variation at all loci in a genome in a similar manner, whereas natural selection acts upon specific loci . Thus, bHere, we describe an extensive analysis of the molecular evolution of 132 genes that were comprehensively resequenced in 24 African-Americans and 23 European-Americans. In total, over 2.5 Mb of baseline reference DNA was sequenced, spanning 20 autosomal chromosomes and the X chromosome. The sampling of a large number of loci dispersed throughout the genome has allowed us to clarify the relative contributions of demography and selection to patterns of genetic variation at individual genes. Specifically, we developed a rigorous computational approach for taking into account multiple hypothesis tests and demographic history, and we found that while many apparent selective events can instead be explained by demography, there is also strong evidence for positive or balancing selection at eight genes in the European-derived population. In addition, we describe a striking example of a previously unreported recent selective sweep in European-Americans that spans four contiguous genes on Chromosome 7. More generally, our data provide insight into the demographic histories of African-American and European-American populations and have important implications for genetic association studies of complex diseases, as several of the genes showing evidence of selection have been implicated in susceptibility to complex human diseases.p < 0.05) in one or more tests of the allele frequency distribution ((ABO, IL1RN, and TNFRSF1B).We resequenced 132 genes primarily involved in inflammation, blood clotting, and blood pressure regulation and discovered a total of 12,890 SNPs . We firsribution . Thus, tThe direction of Tajima's D, Fu and Li's D*, and Fu and Li's F* is potentially informative about the evolutionary and demographic forces that a population has experienced. For example, negative values reflect an excess of rare polymorphisms in a population, which is consistent with either positive selection or an increase in population size. Positive values indicate an excess of intermediate-frequency alleles in a population and can result from either balancing selection or population bottlenecks. In the European-American sample, we observed eleven significantly positive and five significantly negative values for one or more of these three test statistics . In the The observations of both significantly positive and significantly negative values of Tajima's D, Fu and Li's D*, and Fu and Li's F*, combined with the largely nonoverlapping set of significant genes, could reflect selective pressures unique to one population , different demographic histories, spurious results, or most likely some complex combination of all of these factors. Although these results are intriguing, their interpretation is confounded by two issues: (1) We have not corrected for multiple hypothesis tests, and (2) rejection of the standard neutral model can result from either selective or demographic forces. In the subsequent sections, we develop approaches to address these issues with the dual goals of identifying genes that possess strong evidence of natural selection and of inferring population demographic history.p values (–8) distribution  for one or more tests of the allele frequency distribution  and S2. Although neutrality tests of the allele frequency distribution reveal many significant deviations, it is impossible to unambiguously interpret these data as evidence for natural selection, because the null model used to assess significance makes unrealistic assumptions about population demographic history. In principle, it is possible to distinguish between demography and selection, because demography affects all loci in the genome, whereas selection acts upon specific loci. Thus, by sampling a large number of loci dispersed throughout the genome, we can begin to construct a more realistic null hypothesis by which to evaluate the evidence for or against selection .To this end, we used the empirical data to explore four different demographic models A, which We reestimated the significance of Tajima's D, Fu and Li's D*, Fu and Li's F*, and Fay and Wu's H in each population for each of the four demographic models using the best-fit parameter values. All simulations included recombination and correction for multiple tests using the FDR method (with a FDR of 5%) as described above. Population history can clearly have a profound effect on tests of natural selection , and givEPHB6, TRPV6, TRPV5, and KEL;EPHB6, TRPV6, TRPV5, and/or KEL possess specific alleles that have conferred local adaptation to a unique environmental pressure in European-derived populations. Consistent with this hypothesis, we observed strong levels of population subdivision , which An alternative explanation for why we observed fewer significant results in African-Americans than in European-Americans is that African-Americans are an admixed population , and theABO in African-Americans  of accelerated evolution in either the human (TRPV6 and EPHB6) or chimp (DCN) lineage. In addition, Recently, EPHB6, TRPV6, TRPV5, and KEL. To our knowledge, this is the largest footprint of selection that has been described in the human genome, and likely reflects the combination of strong and recent selective pressures and reduced recombination in this region . Based on our current data it is impossible to identify which gene (or perhaps genes) has been the target (or targets) of selection. However, TRPV6 is a particularly interesting candidate, as it possesses three nonsynonymous amino acid substitutions (C157R, M378V, and M681T) that are each nearly fixed for the derived allele in European-Americans, show significant frequency differences between European-Americans and African-Americans, and are located in the most significant regions of both Tajima's D and reduced nucleotide diversity  to determine the derived allele for Fay and Wu's H test. These data were generated under the auspices of the SeattleSNPs Program for Genomic Applications, which resequences candidate genes involved in inflammatory processes in humans. In general, we resequenced the complete genomic region for each gene, including introns and approximately 2 kb 5′ of the gene and 1 kb 3′ of the gene using Big-Dye terminator chemistry on an ABI 3700 or ABI 3730XL . For several exceptionally large genes, such as F13A1, less than complete coverage was obtained (see http://pga.gs.washington.edu/).Human DNAs were obtained from the Coriell Institute (Camden, New Jersey, United States). We analyzed DNA from 24 African-Americans from the Human Variation Panel, African-American Panel of 50 (HD50AA) and DNA from 23 European-Americans derived from various CEPH pedigrees. We also sequenced each gene in a common chimpanzee . In order to correct for multiple tests, we repeated the coalescent simulations as described above, but included recombination. Following 4 coalescent realizations, we sampled the recombination rate from a Gamma(2, 0.5 × 10–8) distribution whose expectation equals the average genome-wide recombination rate of 10–8/generation .We initially assessed the significance of these statistics by comparing the observed values to 10ulations , conditineration . The posST as described in We quantified the allele frequency differences between the European- and African-American samples by the statistic FTRPV6 is the target of selection and the selected haplotype is defined by the C157R, M378V, and M681T polymorphisms. If mutations are Poisson-distributed, the expected number of segregating sites in a genealogy is E[S] = μE[T], where S, μ, and T denote segregating sites, neutral mutation rate of the locus, and total branch length of the genealogy, respectively. Assuming a star-shaped genealogy, E[T] = n × t, where n is the number of selected haplotypes. Thus, the time back to the selective sweep, t, can be estimated by S/(nμ). For TRPV6 in European-Americans, n = 45 (i.e., 45 out of 46 haplotypes carry C157, M378, and M681), S = 11, and μ = 2.5 × 10–5.We estimated the time since the selective sweep for the Chromosome 7q region in European-Americans by analyzing the amount of nucleotide diversity that has accumulated on the selected haplotype as described in Wfor each population. The bottleneck model is specified by the parameters F (the inbreeding coefficient) and t (the time in years measured from the present) at which the bottleneck occurred. Values of F and t considered were F = [0.05, 0.075, … , 0.40] and t = [10,000, 20,000, … , 100,000]. The exponential expansion model is determined by the parameters α (the growth rate/generation) and t (the time, in years measured from the present, at which the population began increasing in size). Values considered for α and t were: α = [0.0005, 0.001, … , 0.01] and t = [10,000, 20,000, … , 100,000]. The population structure under an island model is specified by the population migration rate between demes, M = 4om,N where oN and m are the effective subpopulation size and fraction of migrants in each subpopulation per generation, respectively. Values of M considered were M = [1, 2, … , 10]. The structure model assuming population splitting with no subsequent migration is determined by the parameter t (the time in years since the populations diverged). Values of t considered were t = [1,000, 2,000, …, 10,000]. In all simulations we assumed an effective population size of 10,000 and a generation time of 25 y in order to facilitate comparisons to a previous study .Click here for additional data file.Table S2(534 KB DOC).Click here for additional data file.Table S3(87 KB DOC).Click here for additional data file.http://www.ncbi.nlm.nih.gov/LocusLink/) for the genes discussed in this paper are ABO (28), ACE2 (59272), APOH (350), BDKRB2 (624), BF (629), C2 (717), CCR2 (1231), CD36 (948), CEBPB (1051), CRF (10882), CRP (1401), CSF2 (1437), CSF3 (1440), CSF3R (1441), CYP4A11 (1579), CYP4F2 (8529), DCN (1634), EPHB6 (2051), F10 (2159), F11 (2160), F12 (2161), F13A1 (2162), F2 (2147), F2R (2149), F2RL1 (2150), F2RL2 (2151), F2RL3 (9002), F3 (2152), F5 (2153), F7 (2155), F9 (2158), FGA (2243), FGB (2244), FGG (2266), FGL2 (10875), FSBP (10646), GP1BA (2811), ICAM1 (3383), IFNG (3458), IGF2 (3481), IGF2AS (51214), IL10 (3586), IL10RA (3587), IL10RB (3588), IL11 (3589), IL12A (3592), IL12B (3593), IL13 (3596), IL15RA (3601), IL17B (27190), IL19 (29949), IL1A (3552), IL1B (3553), IL1R1 (3554), IL1R2 (7850), IL1RN (3557), IL2 (3558), IL20 (50604), IL21R (50615), IL22 (50616), IL24 (11009), IL2RB (3560), IL3 (3562), IL4 (3565), IL4R (3566), IL5 (3567), IL6 (3569), IL8 (3576), IL9 (3578), IL9R (3581), IRAK4 (51135), ITGA2 (3673), ITGA8 (8516), JAK3 (3718), KEL (3792), KLK1 (3816), KLKB1 (3818), KNG (3827), LTA (4049), LTB (4050), MAP3K8 (1326), MC1R (4157), MMP3 (4314), MMP9 (4318), NOS3 (4846), PFC (5199), PLAT (5327), PLAU (5328), PLAUR (5329), PLG (5340), PON1 (5444), PON2 (5445), PPARA (5465), PPARG (5468), PROC (5624), PROCR (10544), PROS1 (5627), PROZ (8858), PTGS2 (5743), SCYA2 (6347), SELE (6401), SELL (6402), SELP (6403), SELPLG (6404), SERPINA5 (5104), SERPINC1 (462), SERPINE1 (5054), SFTPA1 (6435), SFTPA2 (6436), SFTPB (6439), SFTPC (6440), SFTPD (6441), SMP1 (23585), STAT4 (6775), STAT6 (6778), TF (7018), TFPI (7035), TGFB3 (7043), THBD (7056), TIRAP (114609), TNF (7124), TNFAIP1 (7126), TNFAIP2 (7127), TNFAIP3 (7126), TNFRSF1A (7132), TNFRSF1B (7133), TRAF6 (7189), TRPV5 (56302), TRPV6 (55503), VCAM1 (7412), VEGF (7422), and VTN (7448).LocusLink ID numbers (http://coriell.undmj.edu/) repository numbers for human genomic DNAs sequenced for this study are as follows. DNAs from African-Americans were NA17101–NA17116 and NA17133–NA17140. DNAs from European-Americans were NA06990, NA07019, NA07348, NA07349, NA10830, NA10831, NA10842–NA10845, NA10848, NA10850–NA10854, NA10857, NA10858, NA10860, NA10861, NA12547, NA12548, and NA12560.Coriell (


Subcutaneous leiomyosarcoma is a rare condition that accounts for 1% to 2% of all superficial soft tissue malignancies. Approximately 10% of cases arise in the trunk, although the extremities are the most commonly affected.We report herein the case of a 31-year-old man with a subcutaneous leiomyosarcoma, measuring 124 × 105 mm, arising in the left inguinal region. A wide local excision (with a resection margin ≥ 20 mm) was performed. Histological examination of the resected specimen revealed a leiomyosarcoma with high cellularity and two mitoses per 10 high-power fields. The patient remains well with no evidence of disease 5 years and 8 months after the operation.This is the first reported case of subcutaneous leiomyosarcoma arising in the inguinal region and also one of the largest tumors reported. The experience of this case and a review of the English-language literature  suggest that a resection margin of ≥ 10 mm is recommended when excising this rare tumor. Subcutaneous leiomyosarcoma arises from smooth muscle in the walls of arterioles and veins. It is a rare tumor accounting for 1% to 2% of all superficial soft tissue malignancies . It usuaA 31-year-old man presented with a painless left inguinal tumor, which had gradually grown during the past six months. Physical examination on admission revealed a fist-sized subcutaneous tumor in the inguinal region. The overlying skin appeared normal without ulceration Figure . With a The resected tumor, measuring 124 × 105 mm, was solid, encapsulated, and a homogeneous yellowish white in color, without central necrosis and hemorrhage on its cut surface. Routine histological examination with hematoxylin-and-eosin revealed that the tumor comprised spindle-shaped cells with high cellularity in parts Figure  and two The patient had an uneventful recovery and was discharged on the 9th postoperative day. As the resection margin was negative, no adjuvant treatment was given. He remains well with no evidence of disease 5 years and 8 months after excision.Although subcutaneous leiomyosarcoma commonly arises in the lower extremities, it occasionally affects the trunk ,2,4. A ret al., [As subcutaneous leiomyosarcoma is resistant to radiotherapy and chemotherapy ,3,9, suret al.,  demonstrThe author(s) declare that they have no competing interests.KY, YS, NF, and DS took part in the operation, performed the literature search and drafted the manuscript for submission. HU performed histological examination. KH supervised the preparation of the manuscript and edited the final version for publication. All authors read and approved the final manuscript.


To investigate the genetic underpinnings of a particular biological process, geneticists screen large collections of mutant organisms to characterize their physical defects. By comparing the genetic makeup of nonmutant  organisms to mutants, it's possible to tease out the genes responsible for a defective appearance, or phenotype. In a classic study in the fruitfly, Christiane Nüsslein-Volhard and Eric Weischaus bred many lines of flies with mutations that were lethal: the fly embryos died, but not before displaying a wide range of developmental defects. Since it was known that the fruitfly needed only a single wild-type copy of these genes to survive, the mutations in these “embryonic lethals” had to be recessive, meaning that both copies, or alleles, of the gene had to be mutated for the lethal defect to appear. Nüsslein-Volhard and Weischaus's work revealed many such recessive genes crucial to early development and earned them a Nobel Prize.Among the model systems for studying development, the zebrafish has become prized because its transparent embryo develops outside the mother's body. The zebrafish has helped biologists identify many genes involved in embryogenesis and, because it's a vertebrate animal, has become a valuable resource for identifying genes involved in human disease. Now, a team led by Nancy Hopkins of the Massachusetts Institute of Technology, has created over 500 lines of zebrafish with lesions in key embryogenic genes and used them to identify a group of genes that predispose the fish to cancer, with some surprising results.(rp)—that is, each line carried one healthy version and one defective version of a different rp gene. These proteins are components of ribosomes—the massive molecular complexes within cells that mediate protein synthesis—and are essential for embryonic development.All of the 500 lines created by the researchers carried a recessive embryonic lethal mutation; for about 400 of the lines, mutations in 300 distinct genes were identified as the cause of the embryonic phenotype. During the process of cultivating some of these mutant lines, the Hopkins team noticed that an abnormally large percentage of fish experienced early mortality (in some cases, over 50% compared to the 10%–15% seen in nonmutant fish), while the surviving fish in these lines developed large, highly invasive malignant tumors; both phenotypes persisted over successive generations. The tumors resembled malignant peripheral nerve sheath tumors (MPNSTs) that have been found in other fish species as well as in mammals. Suspecting that these mutant lines had elevated rates of cancer, the researchers investigated the genetic makeup of the fish and discovered to their surprise that each line was heterozygous for a mutation in a different ribosomal protein gene rp mutations, the researchers report, either reduced or eliminated expression of the corresponding rp gene. In the case of “classic” tumor suppressor genes, the wild-type allele must be lost for the defective allele to set the stage for cancer. Here, the wild-type allele appeared to remain intact in the tumor cells, implicating the proteins as “haploinsufficient” tumor suppressors—a reduction from two gene copies to one functional copy seems to be enough to increase the risk of cancer. Apart from the mutations in rp genes, the authors also found a loss-of-function mutation in a gene  that acts as a tumor suppressor in mammals—establishing the soundness of this approach for identifying mammalian cancer genes.All of the rp genes whose mutations caused cancer from the five other rp genes whose mutations did not, the authors raise a number of possibilities for future study. And given the high degree of conservation of genes and pathways among vertebrates, it's likely that rp mutations also raise cancer risk in humans. Together, these results demonstrate that the tiny freshwater workhorse of developmental biology has a promising future as a model system for human cancer.While these experiments do not explore how these mutations lead to cancer, the results suggest that some shared, ribosome-associated function allows these genes to act as tumor suppressors and that disrupting this function somehow leads to tumor formation. Though it's not clear what distinguishes the 11


With billions of cells in the adult human body, all replicating and dividing in an environment laden with toxins, radiation, and free radicals, a certain amount of DNA damage is guaranteed to occur. Fortunately, all organisms have built-in checkpoints throughout the cell cycle that prevent such mistakes from propagating. At the G1 checkpoint during cell division, for example, molecules survey nuclear DNA for errors and breaks before the cell is deemed fit to undergo S phase, the DNA replication stage. If damage is found, enzymes either work to repair it or, in some cases, trigger programmed cell death, or apoptosis. But when checkpoints fail, and DNA damage is left unrepaired, disease such as cancer can result. A better understanding of these events, as provided, for example, by Vincenzo Costanzo and colleagues in this issue, will consequently lead to a better understanding of the mechanisms that give rise to cancer.A serious form of DNA damage, called a double strand break (DSB), cuts the helix clean through—a far worse scenario than if just one strand slips free. In response to a DSB, the cell recruits a signaling protein called ATM and a three-protein complex called MRN, whose components selectively bind to broken DNA ends. A malfunction of this signal and repair pathway is dire. People who suffer from the genetic disease ataxia-telangiectasia (A-T) lack a functioning ATM molecule and therefore cannot properly handle DSBs or successfully navigate the G1 checkpoint. This condition leads to a host of problems, including abnormal chromosomes, deficient immune function, and a predisposition to cancer. A-T-like disease (ATLD), another rare genetic condition, has very similar symptoms. The only difference is that the protein missing is Mre11, a subunit of MRN. While recent work on the cellular level has indicated that MRN activates ATM, the biochemical relationship between these proteins has yet to be fully understood.Studying these two molecules using traditional biochemical assays is difficult because knocking out the activity of these proteins is lethal to many cells. Costanzo and colleagues used a novel test system of cell-free frog extracts and found that Mre11 is necessary for both ATM activation and for the formation of large protein–DNA complexes apparently responsible for triggering the cascade of signaling molecules underlying the DNA damage response at the G1 checkpoint.The frog extract system allowed the team to manipulate the presence or absence of Mre11 and accurately measure the response triggered by the addition of fragmented bits of DNA . As predicted, without a functional Mre11 protein, ATM was not activated and there was no response. By simply adding the protein Mre11 back to the mixture, the damage response was restored. But when the researchers added a mutant form of Mre11, still capable of performing its essential tasks in another stage of the cell cycle—DNA replication—the G1 damage response remained suppressed. This mutant form of the Mre11 protein lacks the C-terminal, or DNA-binding, end.Costanzo and colleagues also found that this DNA-binding end is required for the assembly of DNA–ATM–MRN complexes in the presence of fragmented DNA and seems to direct the entire damage response. This work helps to explain the similarity between patients with A-T and those with ATLD, and hints at the formation of a large “signaling” complex that helps to orchestrate the crucial response to DBSs in DNA.


Endothelial cell dysfunction may be implicated in the development of multiple organ failure (MOF) by a number of mechanisms. Among these, altered fibrinolysis promotes fibrin deposition, which may create microvascular alterations during inflammation. Elevated concentrations of C-reactive protein (CRP), especially when these persist over time, are correlated with an increased risk of MOF and death. CRP may inhibit fibrinolysis by inducing plasminogen activator inhibitor-1 (PAI-1) release from human aortic endothelial cells. Moreover, the administration of recombinant CRP in volunteers may increase circulating PAI-1 levels.In this study, we tested the hypothesis that CRP is associated with hypofibrinolysis in intensive care patients with and without sepsis.We studied the association of inflammation and abnormal fibrinolysis in intensive care unit (ICU) patients with (n = 11) and without (n = 21) sepsis. The inflammatory response was assessed by serum concentration of C-reactive protein (CRP), a marker of the acute phase reaction, which increase rapidly in the inflammatory response, and the plasma fibrinolytic capacity was evaluated by the Euglobulin Clot Lysis Time (ECLT), determined by a new semi-automatic method.2 = 0.45; p < 0.001). In a multivariate analysis, CRP was the strongest predictor of ECLT (R2 = 0.51, F = 25.6, p < 0.001). In addition, the overall ICU length of stay was significantly correlated with CRP (R2 = 0.264, p = 0.003) and ECLT (R2 = 0.259, p = 0.003).ECLT was significantly higher in septic than non-septic patients  and was significantly correlated with CRP concentration (RIn critically ill patients a significant correlation thus exists between plasma fibrinolytic capacity and serum CRP levels. Our data were obtained in the first 24 hours of ICU admission or of sepsis, thus, the relation between CRP and hypofibrinolysis appeared very quickly. This finding is compatible with a link between inflammation and abnormal fibrinolysis, and may explain the negative prognostic value of CRP in critically ill patients. Endothelial cells have a key role in the control of vascular permeability and vessel tone, coagulation and fibrinolysis, and inflammatory response . There iEndothelial dysfunction/or activation is associated with an imbalance in hemostatic functions. Endothelial cells are responsible for the release of tissue plasminogen activator (t-PA) and contribute to the release of plasminogen activator inhibitor-1 (PAI-1). Inhibition of the fibrinolytic system amplifies the pathogenic role of fibrin deposition during severe inflammation . MultiplIn infected patients, elevated concentrations of serum CRP are correlated with a risk of MOF and death , especiaCRP can act directly on endothelial cells, inducing, for example, the expression of intercellular adhesion molecule (ICAM)-1  and the In this study, we tested the hypothesis that CRP is associated with hypofibrinolysis as measured by ECLT in intensive care patients with and without sepsis.After approval by the A. Vésale hospital ethics committee, we studied 32 ICU patients with severe sepsis n = 11) or other diagnoses (n = 21). Infection definition required isolation of a microorganism from a normally sterile body site, concurrent with accompanying signs and symptoms of sepsis and decision of antibiotic therapy. Criteria for severe sepsis included signs of at least one organ dysfunction attributed to sepsis . All pat or other®. Fibrinogen was determined by thrombin time on a STA® automate (STAGO). Leukocyte and platelet counts were determined on a hemocytometer (CELL-DYN4000®, ABBOTT). All tests were performed on blood obtained from the same venipuncture.Blood samples were obtained during the first 24 hours of sepsis or on the first day of admission for non-septic patients. Serum samples were collected in vacuum tubes without anticoagulant. Plasma samples were harvested in citrated vacuum tubes and put in melting ice. Whole blood was collected on EDTA-treated tubes. CRP was evaluated by antibody-binding and turbidity measurement on SYNCHRON LXThe Euglobulin Clot Lysis Time (ECLT), which is the most common test used to estimate the plasma fibrinolytic capacity, represents the balance between t-PA and PAI-1 activities . ECLT wa® software package (Jandle Scientific). The data are presented as mean ± SD. Correlation between variables was analyzed using a Pearson correlation test. A multivariate analysis was used with stepwise backward selection of the explicative variables. Sepsis was considered as a dichotomous variable while all other data were considered as continuous (CRP, fibrinogen, leukocyte, monocyte and platelet counts). ECLT was the dependent variable. A probability level of p < 0.05 was considered as statistically significant.We used SigmaStatThe major cause of severe sepsis (9 patients) or septic shock (2 patients) was pneumonia (8 patients); angiocholitis was the cause in 1 patient, and in 2 patients the cause was not identified. In 8 patients (7 with pneumonia and 1 with angiocholitis), the infection was due to a Gram negative bacteria. Only 1 patient had documented bacteremia. Non-septic patients were admitted for postoperative surveillance (7 patients), intracerebral hemorrhage (3 patients), heart failure (3 patients), drug intoxication (3 patients), or aggravated chronic obstructive pulmonary disease (5 patients).2 = 0.45; p < 0.001) with no perceptible threshold . In multivariate analysis, ECLT was best predicted by the CRP level  and not significantly by sepsis or the fibrinogen concentration. Interestingly, the ICU length of stay was significantly correlated with CRP (R2 = 0.264, p = 0.003) and ECLT (R2 = 0.259, p = 0.003) in all patients, and in the survivors (R2 = 0.13, p = 0.05 and R2 = 0.3, p = 0.003, respectively).As expected, inflammatory parameters such as white blood cells and CRP levels, the SAPS II score and ECLT were higher in the septic than the non-septic population Table . In an uMany studies have demonstrated that sepsis is associated with endothelial cell dysfunction and promotes coagulation activation . Althougin vivo. Our data suggest that CRP could itself be involved in the processes leading to endothelium dysfunction. The observed relationship does not prove a direct biological link between increasing CRP and hypofibrinolysis; however, indirect arguments exist in support of the concept.Inhibition of the fibrinolytic system may contribute to MOF, as fibrin can activate endothelial cells leading to a disorganization of the monolayer and the release of inflammatory cytokines . Increasin vitro studies have reported the direct effects of CRP on endothelial cells [In vivo, Cleland et al. [G-monomethyl-L-arginine (L-NMMA), reflecting endothelial dysfunction. Bisoendial et al. reported that the administration of CRP in volunteers impairs the fibrinolytic balance [Several al cells -8. In vid et al.  reported balance . In addi balance . In a no balance .in-vitro and in-vivo. This mechanism seems to be important in sepsis, as high plasma levels of PAI-1 are associated with poor outcome [CRP could also act indirectly on endothelial cells via the action of monocytes and the release of tumor necrosis factor-α (TNF-α). TNF-α is a strong inducer of PAI-1 production  outcome . Moreove outcome . CRP can outcome .CRP also has essential biological functions. No polymorphism of either the gene coding sequence or of the protein itself has been described in humans . Also, hThis work is a pilot study. We have chosen to include patients in the first day of ICU admission to limit the possible rapid effect of inflammatory response on fibrinolysis, especially in septic patients. Indeed, this particular patient population has inflammatory reaction before signs of severe sepsis and thus before their admission to ICU. In fact, we have studied ECLT test at the onset of the organ dysfunction and the inflammatory reaction. Other studies with serial measurement of ECLT in patients who developed nosocomial ICU infections are needed to study the time course of these events. Moreover, we could not definitively exclude that all patients in the non-septic groups were non infected. For example, some non-septic patients with decompensated COPD may have minor infections, despite the negative microbiology cultures and the absence of antibiotic therapy. Viral infections were also possible in some patients.Moreover, it would be of great interest to determine the interactions between CRP and ECLT with IL-6, TNF-α and endothelium dysfunction markers such as soluble thrombomodulin and soluble von Willebrand factor.Despite accumulating evidence that the inflammatory and coagulation systems are activated in sepsis, little is known about the mechanisms that ultimately lead to organ dysfunction and death. Our data were obtained in the first 24 hours of ICU admission or of sepsis, thus, the relation between CRP and hypofibrinolysis appeared very quickly. Prospective studies including the time course of CRP and hypofibrinolysis would provide additional information about this relationship.KZB: Laboratory analysis, writing of the manuscript and design of the study.MP: patients recruitment and design of the study.DB: coordination and design analysis of the results.MG: design of the study.PC: laboratory analysis.JLV: design of the study and analysis of the results.CR: design of the study and analysis of the results.YB: patients recruitment.MV: statistical analysis and coordination.