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529319

Distinct Genomic Integration of MLV and SIV Vectors in Primate Hematopoietic Stem and Progenitor Cells

Murine leukemia virus (MLV)-derived vectors are widely used for hematopoietic stem cell (HSC) gene transfer, but lentiviral vectors such as the simian immunodeficiency virus (SIV) may allow higher efficiency transfer and better expression. Recent studies in cell lines have challenged the notion that retroviruses and retroviral vectors integrate randomly into their host genome. Medical applications using these vectors are aimed at HSCs, and thus large-scale comprehensive analysis of MLV and SIV integration in long-term repopulating HSCs is crucial to help develop improved integrating vectors. We studied integration sites in HSCs of rhesus monkeys that had been transplanted 6 mo to 6 y prior with MLV- or SIV-transduced CD34 + cells. Unique MLV (491) and SIV (501) insertions were compared to a set of in silico-generated random integration sites. While MLV integrants were located predominantly around transcription start sites, SIV integrants strongly favored transcription units and gene-dense regions of the genome. These integration patterns suggest different mechanisms for integration as well as distinct safety implications for MLV versus SIV vectors.

Introduction Integration of proviral DNA into the host cell genome is an essential step in the life cycle of retroviruses. The process begins after a retrovirus enters the cell and the RNA genome is reverse transcribed into double-stranded DNA. Preintegration complexes (PICs) containing linear proviral DNA associated with several viral and cellular proteins ( Bushman 1999 ) either enter the nucleus of nondividing cells through the nuclear pores (lentiviruses) or gain access to chromosomal DNA after dissolution of the nuclear membrane during mitosis (oncoretroviruses). When the PIC associates with the host chromosome, the virally encoded integrase directs the insertion of the proviral DNA into the cellular chromosomal DNA ( Hindmarsh and Leis 1999 ). The provirus is then stably transmitted to all progeny of transduced cells as an integral element of the host genome. Beyond its importance to the reproduction of the virus itself, this distinctive feature of retroviruses accounts for many of the characteristics associated with retroviral infection, including latency and persistence of infection, insertional mutagenesis, and the usefulness of retroviruses as vectors for gene therapy. Engineered replication-defective retroviruses were introduced over 20 y ago and rapidly became attractive tools for efficient and stable introduction of genes of interest, in particular into hematopoietic stem cells (HSCs). Retroviral gene therapy targeting HSCs has been aggressively pursued because of its potential to treat many congenital and acquired human diseases. Its therapeutic promise was convincingly demonstrated in children with X-linked severe combined immunodeficiency (SCID-X1) and adenosine deaminase deficiency ( Aiuti et al. 2002 ; Hacein-Bey-Abina et al. 2002 ). Unfortunately, elation over this success was recently tempered when lymphoproliferative disease developed in two children who received genetically modified CD34 + cells for treatment of SCID-X1, in association with proviral activation of the LMO2 transcription factor gene ( Hacein-Bey-Abina et al. 2003 ). These serious adverse events have galvanized investigators to further assess the potential risks associated with gene therapy protocols utilizing retroviral vectors. For many years, researchers have been aware that retroviral insertional activation of proto-oncogenes can result in tumors. Administration of replication-competent oncoretroviruses to susceptible mouse strains led to tumor development, the result of a high number of repetitive insertion events in vivo during rapid cell proliferation, with outgrowth of a clone containing one or more proviruses activating growth control genes ( Dudley 2003 ). While the possibility of insertional mutagenesis using replication-defective vectors has been discussed as theoretically possible ( Cornetta et al. 1991 ), such risks have been estimated to be extremely low ( Moolten and Cupples 1992 ) based on the assumption that proviral integration into the genome was random ( Coffin et al. 1997 ). With the readily accessible human genome sequence, mapping studies of retroviral integration sites in cell lines have uncovered nonrandom integration patterns, when studied using wild-type HIV, HIV-derived, or murine leukemia virus (MLV)-derived vectors ( Elleder et al. 2002 ; Schroder et al. 2002 ; Laufs et al. 2003 ; Wu et al. 2003 ; Mitchell et al. 2004 ). However, these integration patterns have not been investigated in the most relevant primary cells for hematopoietic gene therapy, namely HSCs. HSC transduction by retroviral vectors and their subsequent vector-genome integration patterns can unequivocally be assessed only by transplanting these cells and analyzing vector-containing cells in multiple lineages in vivo long-term, since stem and progenitor cell activity are defined by functional reconstitution of hematopoiesis in vivo. Interpretation of such studies may be more complex due to the potential for integration-specific impact on engraftment or functional properties of primitive hematopoietic cells in vivo. However, large-scale analysis of retroviral integration sites in a relevant long-term large animal model is critical to fully assess the potential risks associated with proviral insertion in this population of cells, prior to implementing new gene therapy trials. These studies may also provide further insights into mechanisms of integration targeting and the impact of integration events on the behavior of hematopoietic cells. In order to evaluate the integrating vectors currently being developed for gene therapy applications, we compared the integration patterns of MLV and simian immunodeficiency virus (SIV) vectors. MLV vectors have been utilized for over a decade in clinical trials. However, they have a number of limitations, including inefficient transduction of quiescent cells and difficulty in maintaining stable high-level expression from tissue-specific internal genetic control elements. Thus, lentiviral vectors based on HIV or SIV “backbones” have been pursued and shown to overcome these limitations, and are now moving into clinical trials. Vector-genome junction sequences were retrieved from mature granulocytes and mononuclear cells (MNCs) from rhesus macaques transplanted 6 mo to 6 y prior with mobilized peripheral blood (PB) CD34 + cells transduced with an MLV- ( Schmidt et al. 2002 ) or SIV-derived vector ( Hanawa et al. 2004 ), both containing marker genes with no known impact on proliferation or survival of transduced cells ( Wu et al. 1998 ). This model represents a unique opportunity to analyze retroviral insertion patterns in the engrafted progeny of primitive long-term repopulating cells, without interference from confounding factors such as the impact of transgene expression or an underlying hematopoietic disease. Results Cloning, Sequencing, and Bioinformatic Analysis of Retroviral Integration Sites We used a modification of the sensitive linear amplification-mediated (LAM)-PCR method ( Schmidt et al. 2002 ) to retrieve and clone the genomic regions adjacent to proviral integration sites from circulating granulocytes and MNCs sampled in rhesus macaques engrafted stably long-term, between 6 mo and 6 y after transplantation of transduced CD34 + for the MLV-transduced animals, and 6–7 mo posttransplantation for the SIV-transduced animals. In our extensive prior analysis of 46 rhesus macaques, genetic marking levels and clonal integration patterns are stable by 3–4 mo posttransplantation, and remain stable for up to 6–7 y ( Kiem et al. 2004 ). This approach uses a frequent-cutting enzyme to generate average genomic fragments of 80 bp, thereby circumventing PCR bias against large fragments, while facilitating amplification and cloning. The average length of all analyzed genomic fragments was 159 bp (median 131 bp, range 30–728 bp). Owing to the close phylogenetic relationship between human and rhesus macaques, we were able to directly align our sequences with the human genome assembly. We considered a sequence as a genuine retroviral integration site only if it (a) juxtaposed to the vector long terminal repeat (LTR), (b) yielded a unique best hit by BLAT software (University of California, Santa Cruz [UCSC] Genome Browser, http://genome.ucsc.edu ), and (c) showed at least 90% identity to the July 2003 human genome assembly ( Kent, 2002 ; Karolchik et al. 2003 ). After several analyses using different cutoffs, we decided to use a conservative alignment cutoff of 90% in order to include most of the orthologous regions between human and rhesus genomes, while discarding sequences of technically poor quality. This cutoff eliminated 5 to 10% of the retrieved sequences, and we verified that omission of these sequences with less than 90% identity from our analysis did not change the overall distribution of the integration sites ( Table S1 ). Using these selection criteria, we have retrieved and analyzed 992 independent unequivocal retroviral integration sites ( n = 491 for MLV [ Dataset S1 ], and n = 501 for SIV [ Dataset S2 ]). Of the 992 integration sites analyzed, 232 (23%) were distributed among the four major classes of transposable repetitive elements, and therefore could not be mapped to a unique position in the genome. These insertions accounted for 59 of 491 (12%) and 173 of 501 (34.5%) of the MLV and SIV integration events, respectively. Human transposon-derived repeats encompass at least 45% of our genome and their distribution is highly variable, with density varying from 2% to 98% depending on the location ( Lander et al. 2001 ). Integration Targets Transcription Units We correlated the 760 integration sites (432 for MLV and 328 for SIV) that unequivocally mapped to a unique position in the genome to the locations of annotated genes, using the UCSC Genome Browser Reference Sequence (RefSeq) Genes track, which displays the positions of National Center for Biotechnology Information mRNA Reference Sequences ( Table 1 ). We observed that 212 of 432 (49%) of the MLV integrations and 241 of 328 (73%) of the SIV integrations landed between the transcription start and stop point of a RefSeq gene. As a control, we compared the coordinates of two sets of 1,000 in silico-generated random integration sites, each containing 432 or 328 coordinates (760,000 total) with the positions of known genes. Both MLV and SIV insertion patterns were significantly different from the random integration sites ( Figure 1 ), of which only 32 % were within RefSeq genes, a percentage identical to the average estimation of the human genome content (25.5%–37.8%, median 31.6%) ( Venter et al. 2001 ). Figure 1 Comparison of MLV and SIV Integration Events Shown are integrations that landed within RefSeq gene introns (arrows) in comparison to in silico-generated integration sites (bars). Black indicates MLV and gray indicates SIV. * p < 0.0001 by a Chi 2 test. Table 1 MLV and SIV Integration Sites Distribution (Reported to UCSC RefSeq Genes) Compared to In Silico-Generated Random Integration Sites The random integration sites correspond to two sets of 1,000 sets, each containing 432 or 328 coordinates (760,000 total). Two-sided p -values were obtained by the Chi 2 test a p < 0.0001 compared to in silico-generated random integrations b p < 0.0001 compared to SIV-derived integrations 10.1371/journal.pbio.0020423.t001 We next examined whether specific regions of the transcription units were more likely sites of integration than others. We analyzed the distribution of the integration events within the transcription unit by dividing the distance of each integration site from the transcription start site by the gene length. The resulting ratio, reported as the total number of integration events in RefSeq genes for each vector, provides the percentage of integrations within ten equal sections of transcription units. While SIV targets the entire transcription unit with no noticeable preference, 42 of 212 (20%) of the MLV integration sites that land within RefSeq genes, as compared 18 of 241 (7%) for SIV, are located within the first one-tenth of the transcription unit, indicating MLV's clear predilection for the 5′ portion of transcription units ( p = 0.0002). MLV Vectors Favor Integration around Transcription Start Sites, and SIV Vectors Integrate Predominantly within Transcription Units To further explore MLV preferential integration in the vicinity of transcription start sites, we determined the distance to the nearest 5′ and 3′ ends of a RefSeq gene for each integration site. Interestingly, whereas SIV integration events do not favor locations upstream or downstream of transcription units ( Table 1 ), 48 of 432 (11%) of the total MLV integration sites landed within a 10-kb region upstream of a RefSeq gene, as compared to 5% expected with the random integration sets ( p < 0.0001). The frequency of insertions within 10 kb downstream of the 3′ end is almost identical for the MLV and the in silico-generated random sets (5.3% versus 4.8%). We then looked at the proviral integrations within a 2-kb window on either side of transcription start sites. This survey revealed a strong tendency for MLV vectors to integrate close to transcription start sites, with 46 of 432 (11%) of the total MLV integration events occurring within 2 kb upstream or downstream, as compared to 7 of 328 (2%) for SIV ( p < 0.0001). We broadened this analysis to a 60-kb window centered on transcription start sites ( Figure 2 ). The overall distribution of the 432 MLV integration events upstream and downstream of transcription start sites is almost identical (20% versus 27%, p = 0.02), but their distribution is clearly nonrandom and favors a 10-kb window centered around transcription start sites. This pattern is markedly different from the distribution of SIV sites: Although there is no predilection for integration in the vicinity of transcription start sites, there is a strong preference for integration within transcription units, rather than upstream of them. Of the SIV-derived sites, 122 of 328 (37%) are within 30 kb downstream of the transcription start site, while only 30 of 328 (9%) are within 30 kb upstream ( p < 0.0001). Taken together, these data show a distinct integration pattern between MLV- and SIV-derived vectors ( p < 0.00001 using an omnibus contingency Chi 2 test): While the latter appear to integrate predominantly within transcription units, MLV vectors strongly favor integration within a 10-kb window centered on transcription start sites. Figure 2 Distribution of MLV and SIV Integration Sites within a 60-kb Window Centered on Transcription Start Sites The vertical arrow points to 0 kb. Each gray bar corresponds to the percentage of SIV integration sites within a 5-kb interval, and black bars correspond to the percentages of MLV integration sites in a 5-kb interval. The distribution of a set of 65,000 in silico-generated random integration sites is represented by the dashed line. SIV-Derived Vectors Favor Integration within Gene-Dense Regions of the Genome In order to ask whether the preferential integration of SIV vectors within transcription units might be associated with physical properties of the genome such as gene density, we analyzed the overall distribution of integration sites. The highest density of SIV integration sites per Mbp are on Chromosomes 17, 19, and 22 (0.50, 0.25, and 0.27 respectively), the three most gene-dense chromosomes, with 15, 23, and 17 genes per Mbp, respectively ( Venter et al. 2001 ). Since each chromosome is a patchwork of domains with varying gene density, we determined the number of RefSeq genes within 1 Mbp of every integration site's LTR coordinate ( Figure 3 A). While most (84%) of the random integration sites tended to be within regions of average gene density (0–10 genes per Mbp), MLV displayed a strong tendency to integrate within more gene-dense regions. This was particularly evident for SIV integration sites, 174 of 328 (53%) of which occurred in regions of the genome whose gene density is higher than 11 genes per Mbp, compared to 149 of 432 (34%) and 17% for the MLV and the in silico, random sets, respectively. These data point out another difference between MLV- and SIV-derived vectors, the latter exhibiting a marked tendency to target gene-rich regions of the genome ( p < 0.00001 using an omnibus contingency Chi 2 test). Figure 3 Distribution and Location of Integration Sites Relative to Chromosomal Gene Density (A) Distribution of MLV and SIV integration sites relative to gene density within a 1-Mbp window compared to in silico-generated random integration sites. Each bar corresponds to the percentage of integration sites within the corresponding gene density region. (B) Location of MLV and SIV integration sites and gene density on human Chromosome 6. MLV and SIV integrations were aligned to Chromosome 6 (obtained from the UCSC custom annotation track feature) and shown in relation to RefSeq gene density (blue). 73% of the SIV integration events are within the 20-Mbp unique ridge of Chromosome 6, compared to 29% for MLV. Distance between thick black bars is 20 Mbp; centromere is represented by the black circle. Recent studies have shown that about 30 highly gene dense clusters, called “ridges” (a loose acronym for “regions of increased gene expression”), are distributed among chromosomes. These ridges are characterized by typical expression levels per gene up to seven times higher than the genomic average ( Caron et al. 2001 ). This feature is particularly evident for Chromosomes 3 and 6 ( Versteeg et al. 2003 ). When looking at the distribution of retroviral integration sites on Chromosome 6 ( Figure 3 B), 22 out of the 30 SIV integration events (73%) fall within this unique ridge, a region of 20 Mbp (12% of Chromosome 6) with a density of 24 genes per Mbp, corresponding to the major histocompatibility complex region. This tendency to target gene-rich regions is less obvious for the MLV vector, which had only 7 out of 24 integration sites (29%) within this ridge ( p < 0.005). Of the 22 SIV proviruses within this 20-Mbp ridge, ten were found clustered within a 2-Mbp, extremely gene-dense region (62 RefSeq genes per Mbp). Unexpectedly, only two out of these ten integration sites are inside transcription units, underscoring the strong tendency of SIV vectors to target gene-rich regions of the genome even if not within genes. Another feature of ridges is that they are noticeably enriched for short interspersed element (SINE), but depleted for long interspersed element (LINE) repeats ( Versteeg et al. 2003 ). This correlation between SINE repeat density, GC content, and gene density has been previously reported ( Bernardi et al. 1985 ) and may account for our observation of overrepresentation of integration events in SINE versus LINE elements, with 119 of 501 (24%) of the SIV set of integration sites being within SINE repeats. Common Integration Sites Differ between SIV- and MLV-Derived Vectors Given their distinct patterns of integration, we compared identified common integration sites of MLV and SIV vectors. Using the definition of a common integration site as two or more proviruses integrated within a transcription unit ( Suzuki et al. 2002 ), we have identified 40 genes targeted more than once by MLV and/or SIV vectors ( Table S2 ). Of the RefSeq genes targeted by MLV and SIV vectors, 16 of 199 (8%) and 19 of 222 (9%), respectively, were hit at least twice, and ten genes were identified as common integration sites because they harbor both MLV and SIV proviruses. These genes have been targeted two times ( n = 32), three times ( n = 6), five times ( n = 1), and seven times ( n = 1). Among these 40 genes, seven are known to be involved in oncogenic translocations: ARHGEF12, MDS1, MKL1, MSF, HMGA2, RAD51L1, and RUNX1 . Seven independent integration events have been identified in MDS1 , predominantly within the second intron, 20–180 kb upstream of the first intron of EVI1 . Discussion A better understanding of retroviral integration patterns has evolved due to the availability of the complete murine and human genome sequences. Prior mapping studies have been performed in cell lines or in primary cells cultured short-term in vitro. However, integration site patterns may be cell type–dependent, for instance, if gene activity impacts integration site selection ( Schroder et al. 2002 ), or if specific integrations facilitate engraftment and long-term contribution to hematopoiesis. Our aim was to provide a comprehensive comparative analysis of integration sites distribution of MLV- and SIV-derived vectors in long-term repopulating HSCs. Nonhuman primates have been shown to closely predict results in human transplantation and gene therapy clinical protocols ( Donahue and Dunbar 2001 ) and thus represent the best currently available approach to generate information with relevance to design of future human clinical trials. MLV-derived retroviruses are currently the most widely used vectors in clinical gene transfer protocols. Reports of proto-oncogene activation by replication-defective MLV vectors in mice and humans mandate more detailed evaluation of their potential for insertional mutagenesis. Separating the impact of overexpressing a growth-altering transgene from the insertional events themselves is particularly important to assess in primary repopulating HSCs. The main limitation of murine oncoretroviruses as gene therapy vectors is the requirement that cells pass through mitosis in order for the PIC to reach the nucleus and integrate. Since lentiviruses can transduce noncycling cells, lentivirus-based vectors have been actively developed and a clinical trial using these vectors has commenced. A detailed analysis of lentivector integration patterns is essential to assess the risk of insertional mutagenesis of these vectors compared to MLV vectors. Although HIV-derived vectors can enter Old World monkey cells, they encounter a block prior to reverse transcription that is mediated by the dominant repressive factor TRIM5α, a component of cytoplasmic bodies ( Stremlau et al. 2004 ), and thus are very inefficient at transducing nonhuman primate cells ( An et al. 2000 ; Horn et al. 2002 ). Lentiviral vectors derived from SIV have been generated ( Hanawa et al. 2004 ; Negre and Cosset 2002 ) and are useful for preclinical testing in nonhuman primates. SIV vectors may also be used to transduce human cells, and offer a number of potential advantages over HIV vectors for eventual clinical applications, such as lack of seroconversion to HIV positivity after exposure. Only limited information exists regarding the rhesus monkey genome, but paleontological and genomic sequence data suggests that Macaca mulatta is 92.5%–95% identical to the humans at a DNA level ( Page and Goodman 2001 ; Stewart and Disotell 1998 ). Moreover, the human and macaque karyotypes are virtually identical, with near absence of interchromosomal rearrangements and no detectable segments of nonhomology in euchromatic regions ( Best et al. 1998 ; Muller and Wienberg 2001 ). We believe that this evolutionary information, combined with the characteristics of the sequences obtained in our study, validates our use of the human genome sequence to localize rhesus genomic insertion sites. Analysis of SIV integration shows a striking tendency to integrate within transcription units (73% of the mapped integration events), but no propensity toward integration in any specific region of the transcription units, in contrast to MLV vectors. Although we did not observe regional hot spots for SIV integration, as previously reported in cell lines for HIV ( Schroder et al. 2002 ), we instead noted the clustering of integrations within gene-rich regions. This penchant for integrating in so-called ridges may offer clues to a specific mechanism of integration. Loops of chromatin extending away from chromosome territories are frequently observed on the major histocompatibility complex locus of Chromosome 6, the ridge shown in Figure 3 B, especially when transcription is induced ( Mahy et al. 2002 ; Volpi et al. 2000 ). These data suggest that the formation of decondensed chromatin territories might be driven by transcription ( Chubb and Bickmore 2003 ) to establish a nuclear environment accessible to transcription factors ( Gilbert et al. 2004 ) and, therefore, to lentiviral PICs. This hypothesis is corroborated by the fact that genes targeted by SIV vectors tend to be more highly expressed in human CD34 + Rho lo cells, as compared to the total set of 33,000 expressed sequences analyzed on a standard expression array ( Figure S1 ). Interestingly, functional analysis of genes identified as targets for SIV insertion using the Gene Ontology classification ( Ashburner et al. 2000 ) and the EASE bioinformatics software ( Hosack et al. 2003 ) shows a statistically significant overrepresentation of genes coding for transcription factors and nuclear proteins ( Figure S2 ), suggesting either these genes are more concentrated in targeted areas of the genome or they share common genomic motifs or cellular proteins. This striking tendency was not observed with the MLV-derived set of integration sites. While the ratio of MLV integration sites within transcription units was significantly higher than expected compared to in silico-generated random integration sites, the MLV proviruses displayed a unique and specific affinity for the region surrounding the transcription start site of annotated genes. The finding that among the 491 MLV integration sites, only 12% are within SINEs or LINEs may support the fact that MLV inserts into 5′ regulatory elements where insertions of transposable elements are probably strongly selected against. This also indicates tethering between some transcription factor(s) and MLV PIC protein(s). These observations, consistent with previous comparative analyses in vitro ( Wu et al. 2003 ; Mitchell et al. 2004 ), likely reflect the vectors' distinct mechanisms for accessing DNA and integrating, and may have implications for the relative risk of insertional mutagenesis. While replication-competent oncoretroviruses have been widely used to identify genes involved in cancer ( Dudley 2003 ), insertional oncogenesis has, to our knowledge, never been clearly reported after lentiviral infection. Both vectors have drawbacks: MLV vector integrations near the 5′ ends of genes may be more likely to disrupt transcriptional control and result in dysregulated expression of potentially oncogenic gene products, while SIV vector insertions within transcription units might be more likely to result in frame shifts or other events abrogating production of the normal gene product. Thus, the possibility that SIV vectors are less likely than MLV vectors to induce tumorigenesis needs to be carefully evaluated in relevant animal models. A large number of genes were identified with two or more integration events, and thus were deemed common integration sites, including ten genes that had both MLV and SIV integrations. This suggests either that these genes are particularly susceptible to integration events due to open chromatin or other factors that favor both types of viruses, or that integration events in these particular genes alter expression and favor engraftment and long-term contributions to hematopoiesis. However, the most striking finding was the occurrence of seven independent hits by MLV in the first two introns of the MDS1 gene, whereas MDS1 was not found in the SIV dataset of integration sites. MDS1 is adjacent to the EVI1 locus, which has been implicated as a retrovirally activated proto-oncogene in a number of murine leukemogenesis studies ( Bartholomew et al. 1989 ; Bordereaux et al. 1987 ; Morishita et al. 1988 ; Li et al. 2002 ). This unexpected and highly nonrandom clustering raises several questions since recent mapping analyses in cell lines did not report any common integration site ( Wu et al. 2003 ). This suggest that proviral insertion near a proto-oncogene (MDS1/EVI1) may occur at a much higher frequency than previously expected. Studies are ongoing to better understand the causes and consequences of retroviral integration within this genomic locus. It is important to stress that the very long-term follow-up of a large cohort of nonhuman primates, including all animals in the current study, has revealed completely normal hematopoiesis and lack of any progression towards neoplasia ( Kiem et al. 2004 ). All animals have stable polyclonal hematopoiesis from transduced cells without any progression toward oligoclonality. Despite the nonrandom nature of integration and the possible targeting of certain proto-oncogenes, the use of replication-defective MLV or SIV vectors expressing nontransforming transgenes in the setting of one or very few integrants per cell still likely carries a very low risk of oncogenesis ( Baum et al. 2003 ). Design of safer vectors including insulating elements to decrease the risk of activation of adjacent genes, development of targeted integration systems, or use of novel vectors with different integration patterns, should allow continued progress toward safe and effective gene therapy. However, for serious disorders such as SCID, even current MLV vectors are likely justified. Materials and Methods Rhesus macaque autologous transplantation model Rhesus macaques were handled in accordance with the guidelines set by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources ( National Research Council 1985 ). Protocols were approved by the Animal Care and Use Committee of the National Heart, Lung, and Blood Institute. Details of mobilization, transduction, and transplantation were previously published ( Hanawa et al. 2004 ; Hematti et al. 2003 ; Takatoku et al. 2001 ; Wu et al. 2000 ). Animals were mobilized with stem cell factor (SCF) and granulocyte colony-stimulating factor for five doses, and underwent apheresis on day 5. CD34 + cells were enriched from mobilized PB by immunoabsorption and transduced for 96 h with either amphotropic MLV vectors LNL6 and G1Na containing the neomycin resistance gene ( n = 22) ( Miller and Buttimore 1986 ), or for 48 h with amphotropic SIV vector containing the green fluorescent protein gene ( n = 3) ( Hanawa et al. 2004 ). All transduction cultures were carried out in the presence of 100 ng/ml Flt3 ligand, 100 ng/ml SCF, and either 20 ng/ml interleukin-3 and 50 ng/ml interleukin-6 ( n = 12), or 100 ng/ml megakaryocyte growth and development factor ( n = 10 MLV animals and all SIV animals). All animals received cells transduced on flasks coated with Retronectin (TaKara, Shiga, Japan). In addition, two MLV animals also received cells transduced on autologous marrow stromal cells ( Wu et al. 2000 ). Cells were reinfused intravenously following 1,000 rads of total body irradiation. PB samples were collected at a minimum of 6 mo after transplantation from three animals receiving SIV-transduced cells and 22 receiving MLV-transduced cells. MNCs were isolated by density gradient centrifugation over lymphocyte separation medium (Organon Teknika, Durham, North Carolina, United States), and granulocytes were obtained as previously described ( Tisdale et al. 1998 ). Cloning of the integration sites by LAM-PCR LAM-PCR and cloning of insertion site vector genomic fusion sequences was performed as described ( Hanawa et al. 2004 ; Schmidt et al. 2002 ) using 5′-linker cassettes and 3′-LTR primers designed specifically for MLV- or SIV-based vectors ( Hanawa et al. 2004 ) ( Table S3 ). Amplicons of junctions between genomic regions and 5′-LTRs were purified from agarose gels and cloned with the TOPO TA cloning kit (Invitrogen, Carlsbad, California, United States). Cycle sequencing was performed using an ABI Prism Genetic Analyzer 3100 (Applied Biosystems, Foster City, California, United States). Sequences were analyzed using Lasergene software (Dnastar, Madison, Wisconsin, United States). Creation of a control set of in silico-generated integration sites For statistical comparison to the integration site sets, we computationally generated 1,000 sets of integration sites. For MLV, we made 1,000 datasets, each containing 432 randomly selected genomic coordinates; for SIV, we made 1,000 datasets of 328 points each. All human chromosome sequences were concatenated into a single long sequence. We used the random number generator function in Perl to pick a number between 1 and the total number of nucleotides in the human genome (3,098,026,039), then identified this position in the concatenated sequence and correlated this position back to its chromosomal origin. If this coordinate fell within a sequencing gap, a new number was picked. We performed an ANOVA on the in silico-generated integration sites to demonstrate that 1,000 random sets were sufficient (unpublished data). Genomic analysis of the retroviral and in silico-generated integration sites We used a bioinformatic pipeline ( Crawford et al. 2004 ) to map the position of each retroviral and in silico-generated integration site relative to 20,623 National Center for Biotechnology Information mRNA RefSeqs aligned by the UCSC Genome Browser. For each integration site, we calculated the distance to the nearest 5′ and 3′ end of a RefSeq gene. We disregarded cases in which RefSeq mRNAs aligned only partially to the genome. Genomic location of all LTR coordinates are available through the UCSC Genome Browser Custom Tracks (available at http://research.nhgri.nih.gov/projects/Dunbar/May2004/ ). Two-sided p values were obtained using the Chi 2 test. Functional clustering and over-representation analysis of targeted genes Genes identified as targeted by retroviral insertion were analyzed for significant functional clusters of genes using the EASE bioinformatics software ( http://david.niaid.nih.gov/david/ease.htm ). This software was used to rank functional clusters by statistical overrepresentation of individual genes in specific categories relative to all genes in the same category. The functional clusters used by EASE were derived from the Gene Ontology classification system ( http://www.geneontology.org ). Supporting Information Dataset S1 MLV-Derived Integration Site Sequences (FASTA Format) (78 KB TXT). Click here for additional data file. Dataset S2 SIV-Derived Integration Site Sequences (FASTA Format) (60 KB TXT). Click here for additional data file. Figure S1 Relative Expression of Genes with Identified MLV and SIV Integrations, Using Data from Human CD34 + Rho lo Cells (38 KB PDF). Click here for additional data file. Figure S2 Gene Ontology Categories Statistically Overrepresented in the Genes Targeted by SIV-Derived Vector (16 KB PDF). Click here for additional data file. Table S1 Comparison of Retroviral Integration Sites Distribution within Transcription Units (12 KB PDF). Click here for additional data file. Table S2 RefSeq Genes Targeted More than Once by SIV, MLV, or Both Retroviral Vectors (18 KB PDF). Click here for additional data file. Table S3 Primers Used for the LAM-PCR Experiments (8 KB PDF). Click here for additional data file. Accession Numbers The retroviral integration site sequences larger than 50 bp discussed in this paper have been deposited in GenBank ( http://www.ncbi.nlm.nih.gov/Genbank/ ) under the accession numbers AY728482 to AY728804 for SIV, and AY733679 to AY734083 for MLV. LocusLink ID numbers ( http://www.ncbi.nlm.nih.gov/LocusLink/ ) for the genes discussed in this paper are ARHGEF12 (23365), EVI1 (2122), HMGA2 (8091), LMO2 (4005), MDS1 (4197), MKL1 (57591), MSF (10801), RAD51L1 (5890), RUNX1 (861), and TRIM5α (85363).

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526770

Increased deep sleep in a medication-free, detoxified female offender with schizophrenia, alcoholism and a history of attempted homicide: Case report

Background Psychiatric sleep research has attempted to identify diagnostically sensitive and specific sleep patterns associated with particular disorders. Both schizophrenia and alcoholism are typically characterized by a severe sleep disturbance associated with decreased amounts of slow wave sleep, the physiologically significant, refreshing part of the sleep. Antisocial behaviour with severe aggression, on the contrary, has been reported to associate with increased deep sleep reflecting either specific brain pathology or a delay in the normal development of sleep patterns. The authors are not aware of previous sleep studies in patients with both schizophrenia and antisocial personality disorder. Case presentation The aim of the present case-study was to characterize the sleep architecture of a violent, medication-free and detoxified female offender with schizophrenia, alcoholism and features of antisocial personality disorder using polysomnography. The controls consisted of three healthy, age-matched women with no history of physical violence. The offender's sleep architecture was otherwise very typical for patients with schizophrenia and/or alcoholism, but an extremely high amount of deep sleep was observed in her sleep recording. Conclusions The finding strengthens the view that severe aggression is related to an abnormal sleep pattern with increased deep sleep. The authors were able to observe this phenomenon in an antisocially behaving, violent female offender with schizophrenia and alcohol dependence, the latter disorders previously reported to be associated with low levels of slow wave sleep. New studies are, however, needed to confirm and explain this preliminary finding.

Background Female violent behaviour has been studied less than that of men. This is partly because women commit fewer crimes than men [ 1 ], but also because female aggression is traditionally carried out in private and domestic settings [ 2 ]. Even though the rate of violent crimes among women appears to be increasing [ 3 , 4 ], female homicide can still be seen as a rare phenomenon. A psychotic disorder has been observed in ca 30 % of homicidal women [ 5 ], and schizophrenia has been reported to increase the risk for homicidal violence [ 6 ]. Alcohol is often present in severe violent crimes [ 7 ] and the risk for homicidal behaviour is extremely high in women with both schizophrenia and alcohol dependency [ 8 ]. Schizophrenia patients with antisocial personality disorder represent a special high-risk subgroup that is vulnerable to severe substance abuse, psychiatric impairment and aggression as well as to legal problems [ 9 ]. Human sleep consists of two main components: rapid eye movement (REM) sleep, and non-REM sleep, the latter divided into stages 1–4 (S1–S4). Stage 3 sleep (S3) and stage 4 sleep (S4) in non-REM sleep are defined as slow-wave sleep (SWS), also called delta sleep or deep sleep. In normal sleep, REM and non-REM sleep periods alternate cyclically. Though the exact functions of the different sleep stages are unknown, it is generally accepted that SWS is the physiologically significant, refreshing part of sleep. Feelings of unwellness, either somatic or psychiatric, are frequently associated with decreased SWS. Serious sleep disturbances are associated both with schizophrenia and alcoholism. Typical findings in both disorders are long sleep latency, reduced sleep length, long periods of waking after sleep onset, abnormalities in REM parameters as well as decreased SWS [ 10 - 12 ]. The reduction in SWS tends to persist after the clinical remission of psychotic symptoms [ 13 ], and it has been suggested that reduced SWS is the prevailing alteration in the sleep of patients with schizophrenia [ 14 ], although Hoffmann et al. [ 15 ] challenged this view in their recent study. The aim of the present study was to characterize the sleep architecture in a severely violent, medication-free, detoxified woman with schizophrenia, alcoholism and a history of attempted homicide. She is the first subject in a larger study concerning sleep structure in women with this type of comorbidity. Case presentation Participants The patient The patient was a 23-year-old woman charged with attempted manslaughter. She was ordered by the court to undergo a pre-trial forensic psychiatric examination which took place in a maximum security state mental hospital. The trial records and all available background information were reviewed. Both of her parents were alcoholics and she was taken to protective custody under the age of one year. Her grandmother suffered from schizophrenia and her mother had been in a psychiatric hospital after a suicide attempt. The patient used to shoplift before the age of 15 and later she stole money from time to time. She also worked as a prostitute to earn money. She started to abuse alcohol at the age of 15. She had several boyfriends but has never married and has no children. Her personality was noticed to change before the age of 17. She started to have obsessive-compulsive behaviour, paranoid thoughts as well as depressive symptoms. She tried to kill herself by hanging and by several drug intoxications. She brutally killed her own pet. She was never hospitalized before the forensic mental examination but she irregularly visited an outpatient clinic. Despite psychiatric treatment, she impulsively tried to kill her male friend with whom she was drinking. During the psychiatric examination the diagnoses – schizophrenia paranoid type, alcohol dependence and features of antisocial personality disorder – were made by a senior forensic psychiatrist using the structured clinical interview for DSM-IV, SCID I and II [ 16 , 17 ]. Neither waking EEG nor brain MRI (1.5T) disclosed any abnormality. She had no somatic disorders. She had finished high school and vocational school, and was within average intelligence (WAIS-IQ total 109). She was not sentenced, but was ordered by the Finnish National Board of Medico-Legal Affairs to stay in the state mental hospital as a criminally insane patient. The sleep recordings were performed during the psychiatric examination period and the patient was completely medication-free and had abstained from alcohol and drugs for six months. The controls The control group consisted of three 23-year-old female students without criminal records or a history of physical violence. They were healthy with no signs of somatic, psychiatric, or neurological disorders. As part of a psychiatric interview, the SCID-non-patient version [ 18 ] was filled in. To exclude general diseases that could affect sleep, blood tests (including serum prolactin, thyroid function, kidney and liver function) and electrocardiograms were taken and they were within normal range, both in the patient and in controls. No history of alcohol abuse or dependence was detected in controls. The controls were asked to avoid alcohol, drugs or medication two weeks prior to the sleep examinations. Caffeine and nicotine consumption was neither restricted nor recorded. Written consent was obtained from all participants after the study procedure had been fully explained to them. The study was approved by the local human ethics committee. Sleep examination Polysomnography Polysomnography (PSG) was recorded over two consecutive nights but only the second night was considered for the study. The study patient slept in a single room within the department, while controls slept in the hospital guest room. All participants were allowed to sleep as long as they wanted to. Recordings took place on an ambulatory basis; the participant had a portable recording device (Embla, Flaga hf, Reykjavik, Iceland) that was connected to the recording electrodes. The recordings were performed using the standard Rechtschaffen-Kales method [ 19 ]. The high-pass filter was 0.5 Hz and the low-pass filter 45 Hz, with a sampling rate of 100 Hz. Commercial software (Somnologica, version 2.0, Flaga hf, Reykjavik, Iceland) was used for scoring and calculation of sleep parameters. Sleep onset was defined as the first occurrence of three consecutive epochs (90 sec) of stage 1 (S1) or other sleep stages. The following parameters were calculated: time in bed, sleep latency, sleep period (time in bed – sleep latency), wake after sleep onset, total sleep time (sleep period – wake after sleep onset), sleep efficiency (total sleep time/sleep period), number of awakenings, REM latency and percentage amounts of different sleep stages (S1–S4 %, REM %). All data for the analysis were scored by the same scorer (NL), not blinded to the patient group. Sleep diary A sleep diary was used for one week during the study period to ensure a normal sleep-wake rhythm and to exclude the effects of daytime naps. The participant filled in the time of retiring to bed, estimated time of falling asleep and time of awakening in the morning for each consecutive night as well as daytime naps. Results and discussion The patient's PSG recording with reduced sleep length, long sleep latency, and a high number of awakenings after sleep onset as well as reduced sleep efficiency (Table 1 ) is very typical for patients with schizophrenia or alcoholism. Reduced REM sleep latency has been attributed to cholinergic hyperactivity secondary to increased dopaminergic tone in schizophrenia [ 20 ]. The most striking finding in the sleep recording was the high amount of deep sleep (SWS: 41.8%, S4 sleep: 26.1%). One of the consistent alterations in normal ageing is a decrease of SWS. The sleep patterns of children are typically characterized by high amounts of SWS, but a quantitative decrease occurs during puberty. As ageing proceeds, a gradual decline in SWS is observed [ 21 ]. In normal young adult individual SWS generally constitutes about 13 to 23% and S4 sleep about 10 to 15% of sleep [ 22 ], and the amounts of these sleep stages in controls of the present study are in good agreement with this. Table 1 Polysomnography parameters of a homicidal woman with schizophrenia, alcoholismand features of antisocial personality disorder and of three healthy female controls. REM = rapid eye movement sleep, S1–S4 = sleep stages 1–4, SWS = slow wave sleep. patient controls mean (SD) control 1 control 2 control 3 time in bed (min) 475.5 562.0 (10.00) 552.0 562.0 572.0 sleep latency (min) 56.0 19.0 (7.26) 18.5 26.5 12.0 sleep period (min) 419.5 543.0 (14.76) 533.5 535.5 560.0 awakenings (n) 14 7.7 (0.58) 7 8 8 wake after sleep onset (min) 56.0 30.3 (2.02) 30.0 28.5 32.5 total sleep time (min) 363.5 512.7 (12.97) 503.5 507.0 527.5 sleep efficiency (%) 86.7 94.4 (0.25) 94.4 94.7 94.2 REM latency (min) 52.5 85.8 (5.01) 81.0 85.5 91.0 S1 (%) 3.3 6.9 (0.90) 6.0 7.8 6.8 S2 (%) 33.2 53.1 (1.66) 51.3 53.3 54.6 S3 (%) 15.7 7.8 (1.41) 7.7 6.5 9.3 S4 (%) 26.1 11.6 (2.07) 14.0 10.7 10.2 SWS (%) 41.8 19.5 (2.25) 21.7 17.2 19.5 REM (%) 21.7 20.6 (1.35) 21.0 21.7 19.1 The finding concerning SWS is completely contrary to previous sleep reports in patients with schizophrenia as well as with alcohol dependence. The patient had abstained from alcohol for several months before the PSG, and this raises the question if the result could be explained with a withdrawal state. However, reduced SWS is also associated also with alcohol withdrawal and the phenomenon has been reported to be extremely long lasting [ 11 ]. Even after one or two years of abstinence, the sleep records of alcoholics had partly normalized but the percentage of S4 sleep still remained at lowered levels [ 23 ]. Thus, alcohol abstinence cannot explain the finding. MRI disclosed no post-traumatic signs in the brain substance of the patient. Waking EEG was also within normal limits. Minor head injuries (concussions) are, however, frequent events among people with alcoholism. Kaufman et al. [ 24 ] demonstrated a chronic sleep disturbance several years after a minor head injury in a non-selected population. They found lower sleep efficiency, and more awakenings lasting more than three minutes, but no changes in S3 or S4 sleep compared with healthy controls. The patient had a history of violent acts and her lifestyle was described to be antisocial and unstable and she was diagnosed as having features of antisocial personality disorder in addition to schizophrenia and alcohol dependence. The authors are not aware of previous sleep studies in patients with both schizophrenia and antisocial personality disorder. In a PSG study among habitually violent male offenders with antisocial personality disorder as a primary diagnosis, increased amounts of SWS and S4 sleep were observed as compared with age-matched healthy men [ 25 ], which is in agreement with the present case-study. Furthermore, the male offenders with severe conduct disorder preceding ASP had higher amounts of deep sleep than men with only mild or moderate conduct disorder [ 26 ]. Adult antisocial personality disorder, childhood conduct disorder [ 27 ] and childhood ADHD [ 28 ] are the only psychiatric disorders reported to be associated with increased deep sleep. Whether the phenomenon observed in these three disorders reflects specific brain pathology, or a delay in the normal development of sleep patterns in the course of ageing, is still an open question and needs to be clarified. All patients with schizophrenia should not be considered to be violent, although there are minor subgroups of schizophrenic patients among whom the risk for violence is remarkably high. It has been estimated that this increase in risk could be associated with the paranoid form of schizophrenia, coexisting substance abuse and antisocial behaviour [ 6 ]- the disorders, which our patient has. Clozapine, an atypical antipsychotic with significant anti-aggressive effects [ 29 ] is widely used in institutions like state mental hospitals where habitually violent patients with schizophrenia are treated. Interestingly, clozapine has been shown to significantly reduce the amount of S4 sleep both in healthy controls [ 30 ] and in patients with schizophrenia [ 31 ]. Future research is needed to clarify this association and the mechanisms behind this phenomenon. Conclusions Increased deep sleep has been associated with antisocial behaviour with severe aggression. The authors were able to observe this phenomenon in an antisocially behaving, violent female offender with schizophrenia and alcohol dependence, the latter disorders previously reported to be related to low levels of SWS. New studies are, however, needed to confirm and explain this preliminary finding. Competing interests The author(s) declare that they have no competing interests. Authors' contributions This manuscript was prepared by a multidisciplinary team consisting of: NL, generated the idea for this case report, interviewed the controls, scored the sleep recordings, and prepared the manuscript together with the team. PT, had a substantial contribution in theoretical background and processing of the present study as expert in sleep research PT, reviewed all material concerning the patient as expert in forensic psychiatry ES, participated in processing of the manuscript as expert in forensic psychiatry HP, participated in the processing of the manuscript as expert in forensic psychiatry and female violence ME had a central role in planning the study design as well as in formulating the theoretical background of the present study. He also allocated financial resources to this project and helped solving practical problems of the study project MV, supervised and participated with great impact in all stages of this manuscript Pre-publication history The pre-publication history for this paper can be accessed here:

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526764

Within you, without you: HIV-1 Rev and RNA export

Nucleo-cytoplasmic transport of RNA is one of many cellular pathways whose illumination has progressed hand in hand with understanding of retroviral mechanisms. A recent paper in Cell reports the involvement of an RNA helicase in the pathway by which HIV exports partially spliced and unspliced RNA out of the nucleus. This suggests the ubiquity of RNA helicases in RNA export from the nucleus, and has novel mechanistic implications.

HIV must export to the cytoplasm not only full length genomic and structural gene-encoding RNA species, but also partially and fully spliced RNAs which code for a variety of overlapping structural and accessory genes. Whereas the fully spliced HIV RNA species readily exit the nucleus and undergo translation, the partially spliced and unspliced species require the interaction of the HIV-1 Rev protein with the cis-acting, viral RRE (Rev responsive element) for expression. Simpler retroviruses, such as the type-D Mason-Pfizer monkey virus (MPMV) solve a similar problem by encoding a cis-acting, constitutively active CTE (constitutive transporter element) in the 3'UTR of the viral genome, which uses a distinct pathway [ 1 , 2 ]. Of less clear significance is the existence of CRM-1 dependent nuclear export signals in gag proteins of HIV and RSV. It is theoretical possibility that shuttling gag proteins might contribute to nucleocytoplasmic export of genomic RNA, but it is equally possible that the only need for nuclear export of gag is to overcome the nuclear targeting signals late in viral replication [ 3 , 4 ]. Over the last seven years or so, tremendous progress has been made in characterizing nucleocytoplasmic transport pathways. The Rev/RRE pathway involves the Ran-CRM-1, shuttling system: In the nucleus, Ran-GEF converts Ran-GDP to Ran-GTP. Ran-GTP bound CRM-1 binds the NES (nuclear export signal) domain of Rev, which is in turn bound to the RRE, and enables CRM-1 to transport the resulting RNA/protein complex as cargo into the cytoplasm, presumably through CRM-1 interactions with nucleoporins. In the cytoplasm, Ran-GAP converts Ran-GTP to Ran-GDP, releasing the REV/RNA cargo. The asymmetric distribution of Ran-GEF and Ran-GAP between nucleus and cytoplasm ensures a constant RAN-GTP/GDP gradient to facilitate CRM-1 recycling and continued Rev/RRE export. CTE-mediated RNA export is based on binding to the Tap-NXT complex, and export through an unrelated transport pathway, which, ironically, is responsible for the bulk of mRNA transport [ 2 ]. Enter the laboratories of Kuan-Teh Jeang and Larry Kleiman, who, in a comprehensive study in the October 29th issue of Cell , report the involvement of the RNA helicase, DDX3, in the Rev/RRE pathway [ 5 ]. Interestingly, DDX3 attracted their attention in preliminary screens indicating it was upregulated by the HIV-1 Tat protein, which is directly involved in transcriptional, not post-transcriptional regulation. Studying the effects of DDX3 overexpression, expression of dominant negative DDX3 mutants, or antisense knockdown of DDX3, variously on expression of Rev/RRE dependent reporter genes, Rev/RRE dependent subgenomic HIV constructs, or full length HIV genomes, Yedavalli et al. determined that in all cases, DDX3 had effects consistent with its playing a significant role in Rev/RRE dependent expression. These effects included expression of proteins, and nucleocytoplasmic partitioning of Rev/RRE dependent and Rev/RRE independent RNAs. In parallel studies, they determined that DDX3 had no effect on expression from CTE dependent constructs. A number of findings not only provided evidence consistent with a role for DDX3 in the Rev export pathway, but also provided mechanistic insights. DDX3, though constitutively cytoplasmic, shuttles between nucleus and cytoplasm in a manner dependent on CRM-1, as evidenced by inhibition by leptomycin B (LMB), which inhibits CRM-1 export, but not Tap export. DDX3 also localizes to nuclear outer membranes in a speckle formation and co-immunoprecipitates with nucleoporins, in addition to having a distribution in the cytoplasm. In cell lysates, DDX3 co-immunoprecipitates with either Rev protein or CRM-1. Although the studies on cell lysates fail to distinguish between pairwise binding and ternary complex formation, ternary complex formation is a distinct possibility. DDX3 directly binds CRM-1 in vitro (in a manner independent of its NES). Although no data are presented concerning direct in vitro binding of DDX3 to Rev, for such an interaction to exist and to be significant, DDX3 would have to bind one of the three identified functional regions in Rev. These include the NES, the coincident NLS/RBD (nuclear localization signal/RNA binding domain) and the Rev multimerization domain, which flanks the RBD [ 6 ]. No regions in Rev besides these have been identified by mutagenesis or by any other techniques to be critical for function. Such an interaction would seem unlikely (though not impossible) in a complex of oligomerized Rev bound to RNA and CRM-1. Conceptually it might be easier to accommodate all the binding interactions in some kind of "pass-along" mechanism. This could allow some of the binding interactions to take place sequentially, rather than simultaneously. In the tradition of good research, the results of Yedavalli et al. [ 5 ] pose as many questions as they answer. Though the binding interactions so far discovered, and DDX3's ability to shuttle between nucleus and cytoplasm could be consistent with a quarternary complex of Rev, RNA, CRM-1 and DDX3, the existence of such a complex remains to be proven (and the authors avoid implying its existence). It is possible there is some sort of "pass along" mechanism in which some of the binding interactions are sequential. It is tempting to suggest that DDX3 plays a role in relieving torsional tension to facilitate RNA passage through a confining nuclear pore, or that it helps remove certain proteins from RNA before, as, or after it reaches the cytoplasmic side of the nuclear membrane. Two other helicases have been implicated in RNA nuclear export pathways. In yeast, Dbp5p helicase, binds nucleoporins, localizes to nuclear pore complexes on the cytoplasmic side of the nuclear membrane, and is involved in mRNA export [ 7 - 9 ]. RNA helicase A (RHA) has been implicated in CTE-mediated transport [ 10 - 13 ]. A general requirement for RNA helicases in RNA transport is an attractive concept. Conceivably they could even be a significant molecular motor contributing to RNA transport: some RNA helicases are highly processive [see [ 14 ] and references therein] and may work by translocating along ssRNA, displacing bound RNA and protein (the "snowplow" model). At least one highly processive RNA helicase has been show to displace tightly bound protein from dsRNA [ 15 ]. An energy-dependent, highly processive RNA helicase tethered to the cytoplasmic side of the nucleopore could thus pull a long RNA molecule through the pore (see Figure 1 ). Theoretically this could solve the problem of how to get the rest of the RNA through the pore once the region initially targeted by CRM-1 (the RRE in the case of HIV) is transported and released. Other possibilities could, of course, include other molecular motors such as kinesin, in association with protein/RNA complexes [ 16 ]. Figure 1 Schematic diagram outlining Rev mediated RNA export from nucleus to cytoplasm. a , Viral RNA develops secondary structure before binding critical protein components of the export pathway. b , Rev protein binds the RRE, forming a complex of REV, viral RNA, CRM-1 and DDX3, which begins to unwind secondary structure. c , The export complex enters the nucleopore, where both CRM-1 and DDX3 interact with nucleoporins. d , Even after CRM-1 and Rev are released from the export complex, DDX3 may still pull RNA through the complex by virtue of its processivity. On a highly speculative note, the intimate involvement of a DEAD box helicase in the Rev pathway, and the observation that DDX3 has a cytoplasmic localization, in addition to its nuclear membrane localization, recalls some old data from two independent laboratories, indicating that in some systems Rev may not act on export, but on post export events, to promote Rev/RRE dependent expression [ 17 , 18 ]. The eIF4A family has been called the "godfather" of DEAD box helicases [ 19 ]. EIF4A3 can be targeted to the exon junction complex of spliced mRNA, and it has been speculated that it may have a splicing-dependent influence on mRNA translation [ 20 ]. In cell types where Rev does not act through export, could it act by specifically targeting to the ribosome a related DEAD box RNA helicase, translation initiation factor? Time will tell. Finally there is the possibility that specific helicases will be valid therapeutic targets for antiretroviral chemotherapy. Although targeting a cellular gene involved in a viral pathway risks inhibiting a necessary cellular function, it avoids the overwhelming problem posed by rapid viral mutation to resistance. Abbreviations RRE: Rev Responsive Element MPMV: Mason-Pfizer monkey virus CTE: Constitutive Transport Element NES: Nuclear Export Signal LMB: Leptomycin B NLS: Nuclear Localization Signal RBD: RNA Binding Domain Competing Interests None. The opinions expressed are those of the author and do not necessarily express the opinion of the FDA.

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524167

Method for inducing experimental pneumococcal meningitis in outbred mice

Background Streptococcus pneumoniae is the leading cause of bacterial meningitis. Pneumococcal meningitis is associated with the highest mortality among bacterial meningitis and it may also lead to neurological sequelae despite the use of antibiotic therapy. Experimental animal models of pneumococcal meningitis are important to study the pathogenesis of meningitis, the host immune response induced after infection, and the efficacy of novel drugs and vaccines. Results In the present work, we describe in detail a simple, reproducible and efficient method to induce pneumococcal meningitis in outbred mice by using the intracranial subarachnoidal route of infection. Bacteria were injected into the subarachnoid space through a soft point located 3.5 mm rostral from the bregma. The model was tested with several doses of pneumococci of three capsular serotypes (2, 3 and 4), and mice survival was recorded. Lethal doses killing 50 % of animals infected with type 2, 3 and 4 S. pneumoniae were 3.2 × 10, 2.9 × 10 and 1.9 × 10 2 colony forming units, respectively. Characterisation of the disease caused by the type 4 strain showed that in moribund mice systemic dissemination of pneumococci to blood and spleen occurred. Histological analysis of the brain of animals infected with type 4 S. pneumoniae proved the induction of meningitis closely resembling the disease in humans. Conclusions The proposed method for inducing pneumococcal meningitis in outbred mice is easy-to-perform, fast, cost-effective, and reproducible, irrespective of the serotype of pneumococci used.

Background Bacterial meningitis is an important infection of the central nervous system (CNS), and the three major responsible bacteria are Neisseria meningitidis , Haemophilus influenzae and Streptococcus pneumoniae [ 1 ]. Despite the use of antimicrobial therapy, pneumococcal meningitis (PM) has the highest case-fatality rate (up to 30 %) for bacterial meningitis, and in 27 % of cases, it leads to serious neurological sequelae, including cognitive impairment [ 2 , 3 ]. Development of PM generally starts from pneumococcal colonisation of the nasopharynx, which is the natural reservoir of S. pneumoniae in humans and especially in children [ 4 ]. The pathogenic steps leading to PM include invasion of the bloodstream from the nasopharyngeal mucosa, survival in the blood, and subsequent entry into the CNS by crossing the blood-brain-barrier (BBB) [ 1 , 2 ]. However, PM can also be caused by either contiguous spread of pneumococci infecting the sinuses or the middle ear, or accidental traumatic inoculation of bacteria into the CNS [ 2 ]. A recent paper showed that non-hematogenous invasion of the brain by S. pneumoniae in mice may also occur through retrograde axonal transport along olfactory neurons [ 5 ]. Once the pneumococcus starts replicating in the cerebrospinal fluid (CSF), severe inflammation occurs in cerebral vessels and subarachnoid space, and damage to the brain parenchyma is produced [ 1 , 2 ]. Animal models of PM have been developed in order to: (i) characterise the pathogenesis of meningitis, (ii) analyse the role of pneumococcal virulence factors in the disease, (iii) understand the host immune response to S. pneumoniae infection, and (iv) test the efficacy of novel antibiotics and vaccine candidates. Both infant and adult rats [ 6 - 9 ], and also adult rabbits [ 10 - 13 ] have largely been employed as animal models to characterise PM induced by intracisternal inoculation of bacteria. Infant rats have also been used to study haematogenous meningitis following intraperitoneal infection with S. pneumoniae [ 14 ]. However, models of PM have also been developed in the mouse by using the following routes of infection: (i) intraperitoneal (i.p.) [ 15 - 17 ], (ii) intranasal (i.n.) [ 18 , 19 ], or (iii) intracranial (i.c.) parenchymal [ 20 , 21 ] or cisternal [ 22 , 23 ]. Haematogenous murine meningitis models (both i.p. and i.n.) allow to study PM pathogenesis, and i.n. models are particularly useful as they mimic the natural infection route of S. pneumoniae in humans. However, those models present the disadvantage that PM is induced in about half of the animals, while the remaining mice may die of sepsis without developing meningitis. Models of meningitis induced by the i.p. route were employed to carry out therapeutic studies [ 16 ] and investigations on PM pathogenesis [ 15 , 17 ]. The i.n. model by Zwijnenburg et al. [ 19 ] was employed in interleukin (IL)-10, IL-18, and IL-1 receptor deficient mice to investigate the role of different cytokines in PM [ 24 - 26 ]. Direct induction (i.c. route) of PM mimics meningitis caused by contiguous spread from the sinuses or traumatic entrance of pneumococci into the CNS and allows to study the host-parasite interaction in the brain. Besides an early model of i.c.-induced meningitis in mice [ 20 ] employed in therapeutic studies [ 6 , 20 ], the model by Gerber et al . is an useful and reliable system for causing PM in the mouse [ 21 ]. Following infection of C57BL/6 inbred mice into the right lobe of the brain with type 3 pneumococci, bacterial enumeration in different organs, brain histology, behavioural tests, and clinical scores were performed [ 21 ]. A model of intracisternal infection was described by Koedel et al ., who induced meningitis via inoculation of S. pneumoniae (type 3) into the cisterna magna of C57BL/6 mice and investigated the function of nitric oxide in the disease [ 23 ]. Both models, largely employed in studies on the roles of both pneumococcal [ 27 ] and host factors [ 28 - 32 ] in PM, rely on the use of inbred mouse strains and type 3 pneumococci. In the present study, we describe an experimental model of PM in outbred mice based on the direct inoculation of bacteria into the subarachnoid space through a soft point located 3.5 mm rostral from the bregma. Both the technique employed for infection and the anatomical coordinates of the inoculation site are accurately described. The model was tested with pneumococcal strains of three different capsular serotypes and it was characterised in detail by using TIGR4 (type 4) as a model strain. The proposed method is precise, simple, cost-effective, fast and reproducible, and the disease induced closely resembles PM in humans. Results Inoculation site and technique The bregma is the intersection of the coronal and sagittal sutures of the skull and can be recognised in mice by visual examination of the exposed skull (Fig. 1A ). We inoculated mice by the i.c. route through a soft point located along the skull midline 3.5 mm rostral to the bregma (Fig. 1A ). The stereotaxic coordinates of such inoculation point are 0 mm (× plane), 3.5 mm rostral (y plane) and 2 mm ventral (z plane) from the bregma [ 33 ]. A preliminary study was carried out to trace diffusion of the inoculation material from the point of injection into the brain. Three animals were injected with 30 μl of trypan blue and sacrificed 30 minutes after inoculation. Following decapitation, skulls were sectioned into coronal planes, and diffusion of trypan blue was observed (Fig. 1B ). The dye rapidly spread from the injection site into the subarachnoid and ventricular spaces (Fig. 1B ); hence, this infection route is referred to as i.c. subarachnoidal. Histological analysis of the brain sections confirmed that the inoculation needle crossed the mouse frontal lobes and reached the subarachnoid space (data not shown). In order to assess whether the inoculation technique was traumatic to animals, another experiment was performed by inoculating three control mice with saline. Animals recovered soon after injection and did not present any neurological problem ( i.e. lethargy, paralysis) for several weeks after inoculation (data not shown). These data allowed localisation of the anatomical coordinates of the inoculation site and proved the suitability of the i.c. subarachnoidal infection technique. Meningitis induction by type 2, 3 and 4 S. pneumoniae After characterisation of the inoculation site and technique, mice were infected with pneumococci, and the establishment of PM was evaluated and clearly evidenced by histological analysis (see below). In order to test the model with different pneumococcal serotypes, dose-dependent survival studies were performed, and the lethal doses killing 50% of animals (LD 50 ) were calculated. We chose three commonly used S. pneumoniae strains, such as D39, HB565, and TIGR4. The D39 strain (type 2) is the encapsulated parent of the rough type 2 R36A strain used by Avery [ 34 - 36 ]. The HB565 strain (type 3) is a streptomycin-resistant derivative of the A66 strain used by Avery [ 34 , 36 , 37 ]. The serotype 4 TIGR4 is the genome strain sequenced by the Institute for Genomic Research [ 38 ]. The survival patterns of mice inoculated with D39 and HB565 were almost identical, with LD 50 of 3.2 × 10 and 2.9 × 10 colony forming units (CFU), respectively (data not shown). The TIGR4 strain was less virulent in the i.c. subarachnoidal infection model compared to D39 and HB565, as its LD 50 was 1.9 × 10 2 CFU (data not shown). Animal survival and bacterial titres after infection with the TIGR4 strain To describe the features of PM in detail following i.c. subarachnoidal infection of mice, we chose to characterise PM induced by the TIGR4 genome strain. For this purpose, we performed time-dependent survival studies, bacterial counts in different organs/tissues, and histological analysis of brain and spleen (see below). In order to study animal survival, five groups of MF1 mice were infected with doses of TIGR4 increasing from 10 to 10 5 CFU per mouse. The percentage of animals surviving over time at each bacterial dose was analysed by a Kaplan-Meier curve (Fig. 2 ). After inoculating 10 CFU, only one mouse out of eight died 72 hours after infection (87.5 % survival). At the doses of 10 2 CFU and 10 3 CFU, 40 % of mice survived pneumococcal challenge, whereas the remaining 60 % died within 72 hours from infection. Survival further decreased from 20 % in mice infected with 10 4 CFU to 0 % following infection with 10 5 CFU, which induced severe symptoms and subsequent death of all mice within the first 48 hours after challenge (Fig. 2 ). The median time-to-death of the groups injected with 10, 10 2 , 10 3 , 10 4 , and 10 5 CFU were 240, 70, 72, 72, and 40 hours, respectively. From these results, the median survival time of animals did not vary at intermediate doses (10 2 , 10 3 , 10 4 CFU), while it considerably decreased at the highest dose (10 5 CFU) leading to rapid death of all mice. To determine the number of pneumococci in brain, spleen and blood at the final stages of the disease, moribund mice infected with 10 5 CFU of TIGR4 were sacrificed, samples collected and appropriately treated, and viable counts were carried out. Moribund animals showed comparable bacterial counts in the brain (3.1 × 10 6 ± 1.3 × 10 6 CFU/organ). Similarly, dissemination from the brain to vital organs occurred and was consistent in all animals, with bacterial counts of 3.8 × 10 6 ± 4.8 × 10 6 CFU and 2.1 × 10 8 ± 3.0 × 10 8 CFU in the spleen and blood, respectively (data not shown). Histological characterisation of the PM model In order to prove the establishment of PM and study the features of the disease, we performed histological analysis on the brain of moribund mice inoculated with several doses of the TIGR4 strain and sacrificed at various time-points after infection. Animals at the final stages of the disease showed typical signs of meningitis ( i.e. hunchbacked, photophobic, lethargic), but they did not develop hemiparesis or plegia. Moribund mice that had received 10 2 , 10 3 , 10 4 , and 10 5 CFU were humanely killed at 72, 72, 48, and 24 hours post-infection, respectively. Two animals for each pneumococcal dose and three additional control mice were sacrificed. Brains and spleens were excised and treated for both haematoxilin-eosin and Gram staining. In infected mice, no cerebral abscesses were observed, but only granulocytic infiltrations involving the subarachnoid and ventricular spaces. Differently, control animals injected with saline showed no histological changes following inoculation. Brains of moribund mice following infection with the TIGR4 strain showed different degrees of inflammatory changes. Inflammation was regarded as mild in the presence of marked congestion of leptomeningeal blood vessels with margination of polymorphonuclear cells (PMNs), edema, and wisps of fibrin (Fig. 3A ). Inflammation was considered severe when the subarachnoid space (Fig. 3B,3C ) and/or the ventricular spaces (purulent ventriculitis) (Fig. 3D ) contained cellular exudates composed of PMNs entrapped in a dense fibrin net. No large areas of cerebral necrosis were found; however, in some cases, brain damage represented by neuronal shrinkage was observed in the hippocampus (Fig. 3E ). Gram staining of brain sections of infected mice revealed the presence of short chains (mainly diplococci) of Gram-positive dark blue bacteria in the subarachnoid space; pneumococci were located mainly extracellularly in a background of PMNs (Fig. 3F ). Analysis of the spleen of animals infected with S. pneumoniae revealed histological changes in both the white and red pulp with a massive congestion of the red pulp (Fig. 3G ), compared to the spleen of control mice (Fig. 3H ). These data demonstrate that the i.c. subarachnoidal route of infection is an effective and reliable way for inducing PM in the mouse. Discussion S. pneumoniae is one of the causative agents of bacterial meningitis responsible for death and sequelae worldwide. The mouse has largely proved to be a reliable animal model for studying two major pneumococcal diseases such as pneumonia [ 39 - 44 ] and sepsis [ 45 - 47 ]. In the case of PM, the rat [ 6 - 9 , 14 ] and the rabbit [ 10 - 13 ] have often been preferred to the mouse. With the exception of an initial work describing an i.c. infection procedure for inducing PM in mice in outbred mice [ 20 ], only recently, models of PM based on i.c. inoculation were made available in inbred mice [ 21 , 23 ]. In the present work, starting from an experimental method used to study meningitis caused by Cryptococcus neoformans [ 48 ], we developed a model of PM based on the inoculation of bacteria into the subarachnoid space of outbred mice. This infection route (i.c. subarachnoidal) mimics bacterial entrance into the CNS from the sinuses or the middle ear, or following a trauma. We chose to inoculate mice into the subarachnoid space through a soft point located on the skull 0 mm lateral, 3.5 mm rostral, and 2 mm ventral from the bregma. Such point easily allows inoculation of bacteria from the skull through the frontal lobes into the subarachnoid space, as shown by a preliminary experiment in which trypan blue was used to localise the injection site and trace the inoculum within the brain (Fig. 1 ). The finding that the inoculation technique did not cause any trauma to animals can be explained by the fact that frontal lobectomy is tolerated in both humans [ 49 ] and rats [ 50 ], as frontal lobes are mainly committed to behavioural and cognitive functions. We decided to use MF1 outbred mice because this strain is well-known for its susceptibility to both intranasal [ 40 , 51 , 52 ] and intravenous [ 47 ] challenge with S. pneumoniae , and because the use of outbred strains is cost-effective. Another research group had previously used CD-1 outbred mice in a study on the efficacy of clinafloxacin against PM; however, the authors did not provide a detailed description of the model [ 22 ]. Our i.c. subarachnoidal infection model was tested by using a range of bacterial doses of three different S. pneumoniae strains. The strains chosen are the serotypes routinely employed by researchers in the pneumococcal field, and have proved to be highly pathogenic in different mouse infection models [ 27 , 40 , 47 , 52 ]. The use of several bacterial doses is also important, as it allows a more accurate evaluation of virulence for each strain, as well as establishing the most appropriate dose to be employed in different studies. The model proved to be suitable for use with pneumococci of different serotypes, as type 2 D39, type 3 HB565, and type 4 TIGR4 were all able to cause PM with LD 50 ranging from 2.9 × 10 to 1.9 × 10 2 CFU. Then, PM was further characterised and standardised by using TIGR4 as a model strain. Kaplan-Meier survival analysis of animals inoculated with different doses of TIGR4 showed that 10 5 CFU was lethal for all mice within 48 hours from infection (Fig. 2 ), suggesting it as the appropriate dose inducing PM similar to hyperacute meningitis in humans. Moribund mice with acute meningitis after infection with 10 5 CFU were also septicaemic, as pneumococci could also be recovered from the blood and spleen. Bacterial titres were consistent in each organ/tissue of every mouse examined, underlining the reproducibility of data obtainable by using our model. Dissemination of pneumococci after infection with lethal doses is also in agreement with other i.c. murine models of PM [ 21 , 22 ]. The actual induction and subsequent characterisation of PM caused by the TIGR4 strain was then carried out by histological analysis of the brain tissue from moribund animals. We chose not to analyse PM by viable counting of pneumococci in the CSF, due to the difficulty of sample collection [ 53 ] and to the necessity of using pooled CSF samples [ 21 ]. Histological examination of the brain showed both cases of mild meningeal inflammation and cases of severe granulocytic effusion in the subarachnoid and ventricular spaces (purulent ventriculitis) (Fig. 3 ). Cerebral abscesses were not observed, further confirming that the our i.c. subarachnoidal model is indeed a meningitis and not an encephalitis model of infection. Neither inflammatory changes nor death were observed following injection of saline into control mice. Inflammation and PMNs distribution in the brain of moribund mice closely mimicked the histopathology of meningitis in humans [ 54 ]. Post-mortem examination of brains from patients who rapidly (less than 24 hours) died due to hyperacute meningitis generally reveals the presence of mild lesions consisting of a sparse leptomeningeal exudate with vessel congestion and PMN margination, in contrast to patients who survived for two or more days, who often exhibit a severe inflammation with fibrin and PMNs in subarachnoid and ventricular spaces [ 54 ]. A detailed analysis of PM largely resembling human meningitis was also reported by Gerber et al. , who injected C57BL/6 inbred mice into the right lobe of the brain [ 21 ]. In that model, the inoculation site was characterised by a large purulent infiltrate present in both meninges and ventricula, and necrosis was observed in all investigated brain regions [ 21 ]. In another model, proposed by Koedel et al ., pneumococci were given by transcutaneous injection directly into the cisterna magna [ 23 ]. In that study, brain lesions occurred in all mice 24 hours after infection, and histopathological examination revealed intense granulocytic infiltrations in the subarachnoid and ventricular spaces, and absence of cortical necrosis [ 23 ]. This finding differs from the model by Gerber et al. , who instead reported the presence of extensive cerebral necrotic processes [ 21 ]. In our model, we could not observe large areas of necrosis, but we found some signs of neuronal damage ( i.e. neuronal shrinkage with picnotic nuclei) in the hippocampus of a few animals. Conclusions The present work proposes a method to induce experimental PM in outbred mice by using an i.c. subarachnoidal route of infection. The stereotaxic coordinates of the injection site are provided to allow easy recognition of the inoculation point in the mouse. The model is simple and fast, and the technique assures the development of meningitis, as demonstrated by histological analysis, survival data, and microbiological parameters. No significant differences were observed in the ability of the three pneumococcal strains used to cause disease, emphasising the value of the model. It is worth noting that the use of outbred mice still results in data reproducibility, as replicates in this model closely paralleled each other in terms of survival, CFU counts per organ, and histopathological features. In addition, experiments in outbred mice are cost-effective and can be performed in larger animal groups thereby improving statistical significance. This experimental PM model may be particularly useful for all researchers involved in studies that will investigate the host-pathogen interaction at the cerebral level, with emphasis on both pathogen-associated virulence factors and host-specific brain defences. Methods Pneumococcal strains, media and growth conditions Survival studies were performed with the D39, HB565 and TIGR4 strains. TIGR4 was chosen as a model strain for histological characterisation of PM and CFU counts in organs. S. pneumoniae was cultured in Tryptic Soy Broth (TSB, Difco, Detroit, MI) at 37°C with 5 % CO 2 . Solid media were obtained by addition of 1.5 % agar and 3 % defibrinated horse blood (Biotec s.n.c., Grosseto, Italy) to TSB. When necessary, streptomycin was used at the final concentration of 500 μg/ml. Mice Outbred 9-weeks-old female MF1 mice weighing 25–30 grams were obtained from Harlan Nossan (Correzzana, Italy). Animals were allowed to settle in the new environment for one week before performing the experiments, they were caged and given food and water ad libitum . All animal experiments were approved by the Local Ethical Committee (document no. 754/03, 12.9.03, see Additional file 1 ) and were conducted according to institutional guidelines. Preparation of the challenge dose Mouse-passaged S. pneumoniae strains were prepared by using a modified version of a previously described method [ 40 ]. Briefly, bacteria were injected i.p. into mice and recovered 16 hours later from homogenising the spleens with a screen mesh in 2 ml of ice-cold sterile H 2 O. Passaged bacteria were grown to mid-exponential phase, centrifuged for 20 minutes at 1500 × g , resuspended in fresh TSB containing 10 % glycerol, and stored in aliquots at -70°C. Numbers of bacteria were determined by viable counting of serial dilutions in sterile phosphate-buffered saline, pH = 7.4 (PBS), and plating onto blood-agar plates. Before inoculation, bacteria were thawed at room temperature, harvested by centrifugation, and resuspended in sterile PBS at the appropriate dilutions. Mouse model of meningitis PM was induced in lightly anaesthetised mice (50 mg/kg ketamine and 3 mg/kg xylazine) by modifying a method previously used to establish meningitis by C. neoformans in mice [ 48 , 55 ]. Animals were immobilised by hand and inoculated i.c. at a depth of about 2 mm through a soft point located 3.5 mm rostral from the bregma. A preliminary experiment was carried out by injecting 30 μl of trypan blue i.c. into three MF1 mice. After 30 minutes, animals were sacrificed and decapitated. Their skulls were fixed in 10 % buffered formalin for 24 hours and treated with Decal (Decal Corporation, Tallman, NY) for 24 hours. Coronal sections of about 3 mm were made, and diffusion of trypan blue was observed. Then, to localise the injection site within the brain, the above sections were embedded in paraffin and treated for histological analysis. Standard experiments were performed by injecting the bacterial inoculum in a total volume of 30 μl. Injections were performed by using glass micro-syringes (Hamilton, Bonaduz, Switzerland) with 26 gauge needles. Survival studies Different bacterial doses ranging from 10 to 10 4 CFU per mouse were used to infect mice (n= 4) with strains D39 and HB565. In the case of TIGR4, groups of 6 to 10 animals each were inoculated with doses ranging from 10 to 10 5 CFU per animal. Control mice were inoculated with PBS (30 μl). Mice were closely monitored twice a day for clinical symptoms (starry fur, hunched appearance, photophobia, lethargy, moribund). Mice were humanely killed before reaching the moribund state. Survival was recorded for 10 days (240 hours). Microbiology and histology Infected mice were sacrificed either for microbiological or histological analysis. Animals were humanely killed before being moribund, and various samples were collected. For CFU counts, blood was withdrawn by cardiac puncture before sacrifice and added to a tube containing 3.8 % of sodium citrate. Brains and spleens were excised and homogenised in 2 ml of sterile PBS. Bacterial counts in blood, brain and spleen were performed by plating 10-fold dilutions onto blood-agar plates. For histopathological analysis of tissues after infection with TIGR4, brains and spleens were immediately fixed in formalin for 24 hours and then embedded in paraffin according to standard procedures. The brains were entirely sectioned along a coronal plane. Sections were stained with both haematoxilin-eosin and Gram according to standard techniques. Morphological changes were assessed by using routine light microscopy. The presence and degree of inflammation were carefully evaluated. Statistical analysis Calculations of LD 50 values were performed by using both the method by Reed and Muench [ 56 ] and Probit analysis with 95 % confidence interval [ 57 ]. Survival over time was analysed by the Kaplan-Meier curve. Authors' contributions DC, animal experiments, microbiological analysis, writing of manuscript. ST, histological analysis, writing of manuscript. RP, animal experiments, microbiological analysis. MRO, experimental design. EB, development of methodology for induction of meningitis. MC, co-ordination of histological analysis. GP, co-ordination and design of the study, data evaluation. SR, co-ordination and design of the study, data evaluation, direct supervision of experimental work, writing of manuscript. All authors read and approved the manuscript. Supplementary Material Additional File 1 Document stating the ethical approval for animal experimentation conceded to the Laboratory of Molecular Microbiology and Biotechnology (LA.M.M.B.) from the University Hospital of Siena, the Medical Faculty and the Local Ethical Committee (document no. 754/03, 12.9.03). Click here for file

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423146

Electroencephalographic Brain Dynamics Following Manually Responded Visual Targets

Scalp-recorded electroencephalographic (EEG) signals produced by partial synchronization of cortical field activity mix locally synchronous electrical activities of many cortical areas. Analysis of event-related EEG signals typically assumes that poststimulus potentials emerge out of a flat baseline. Signals associated with a particular type of cognitive event are then assessed by averaging data from each scalp channel across trials, producing averaged event-related potentials (ERPs). ERP averaging, however, filters out much of the information about cortical dynamics available in the unaveraged data trials. Here, we studied the dynamics of cortical electrical activity while subjects detected and manually responded to visual targets, viewing signals retained in ERP averages not as responses of an otherwise silent system but as resulting from event-related alterations in ongoing EEG processes. We applied infomax independent component analysis to parse the dynamics of the unaveraged 31-channel EEG signals into maximally independent processes, then clustered the resulting processes across subjects by similarities in their scalp maps and activity power spectra, identifying nine classes of EEG processes with distinct spatial distributions and event-related dynamics. Coupled two-cycle postmotor theta bursts followed button presses in frontal midline and somatomotor clusters, while the broad postmotor “P300” positivity summed distinct contributions from several classes of frontal, parietal, and occipital processes. The observed event-related changes in local field activities, within and between cortical areas, may serve to modulate the strength of spike-based communication between cortical areas to update attention, expectancy, memory, and motor preparation during and after target recognition and speeded responding.

Introduction The waking brain updates and fulfills intentions through brain processes that operate within and across multiple brain areas to integrate perception, association, and action. Fulfillment of intentions is facilitated by features of these and other processes that support informed anticipation of and selective attention to events and their probable consequences. The dynamics of ongoing electroencephalographic (EEG) activity recorded from the human scalp differ markedly with state of attention and intention ( Makeig and Inlow 1993 ; Worden et al. 2000 ), yet most event-related EEG research has assumed that the effects of events on EEG signals emerge out of a flat baseline, as in the typical averaged event-related potential (ERP). The electrophysiological consequences of stimulus events spread quickly in the brain. By 50–150 ms, sensory stimulus information is widely distributed ( Hupe et al. 2001 ), perturbing ongoing patterns of local field activity in many brain areas ( Klopp et al. 2000 ). There is little chance, therefore, that any but still earlier ERP features occur within single brain areas. The adequacy of time-domain ERP averaging for modeling macroscopic brain dynamics also depends on the assumption that the cortical sources of EEG activity contributing to and not contributing to average ERP waveforms are somehow distinct. The scalp topographies of unaveraged EEG and averaged ERP data may, however, be quite similar ( Makeig et al. 2002 ), strongly suggesting that areas contributing to ongoing EEG signals may also contribute to ERP averages. EEG processes not contributing to response averages may also be affected by experimental events, and several types of dynamic EEG response processes are not reflected in ERP averages ( Pfurtscheller and Aranibar 1977 ; Makeig 1993 ; Makeig et al. 2002 ). A more comprehensive model of event-related brain dynamics is therefore needed to capture features of EEG signals that index the dynamic interplay between spatially coherent brain processes supporting anticipation of, attention towards, associations to, and behavioral responses following experimental events. The above considerations suggest that event-related EEG dynamics may be better modeled as coordinated event-related perturbations in the statistics of multiple, intermittently active local field processes. Since the volume-conducted scalp projections of such processes generally overlap, they cannot be separately identified in the scalp-recorded data. An alternate approach we adopt here is to separate the contributions of local field processes by using distinct differences in their time courses ( Makeig et al. 1996 , 1997 ; Jung et al. 2001a ). Following stimuli belonging to an anticipated but infrequently presented category, the averaged ERP is dominated by a broad vertex-positive peak often called P300 after its earliest appearance in auditory responses ( Sutton et al. 1965 ; for a review see Soltani and Knight, 2001 ). Results of ERP ( Ruchkin et al. 1990 ) and brain lesion studies ( Halgren et al. 1980 ; Knight et al. 1989 ) and of functional imaging experiments ( Ford et al. 1994 ; Ebmeier et al. 1995 ; Ardekani et al. 2002 ) strongly suggest that P300 is actually a late positive-going response complex that sums effects of local field perturbations in several brain regions. A more inclusive model of the event-related EEG brain dynamics occurring in such data should consider, therefore, how target stimulus presentations and subject motor responses perturb the dynamics of ongoing EEG signals both within and across single subjects. Here, we present such a model. Results The independent component analysis (ICA) method provides a complete decomposition of single-trial (or continuous) EEG data, separating the data into distinct information sources. As the results presented below show, the amount of information about cortical dynamics provided by this method is large. Here, we detail for the first time dynamics occurring within single trials of the classes of maximally independent EEG processes whose event-related activities contribute to 31-channel visual target responses recorded during a test of spatial selective visual attention ( Figure 1 ). Figure 1 Task Display Subjects fixated on a cross above which five boxes were constantly displayed. In each 76-s task block, one of these (grey box) was colored differently. The location of this covertly attended box varied pseudorandomly across blocks. A series of disks were presented briefly in any of the five boxes in a random order. Subjects were asked to respond with a thumb button press as quickly as possible whenever a disk appeared in the attended box. Response Dynamics of the Scalp Recordings: ERPs and ERP Images To orient readers used to analyses of the raw scalp-channel data, we first present conventional ERP results at selected channels. As we previously reported, in these experiments performance level was high; more than 95% of targets were followed by a button press within the allotted response window (150–1000 ms). Mean subject-median reaction time (RT) was 352 ms. The average ERP time locked to onset of the target stimulus followed by a subject button press contained the expected late positive complex or P300 positivity following early stimulus-locked peaks conventionally termed P1, N1, P2, and N2 ( Figure 2 A). The scalp topography of the late positive complex varied continuously through its extent ( Figure 2 A, scalp maps). Figure 2 Grand Mean and RT-Sorted Single-Trial Responses at Sites Fz and Pz (Left) Stimulus-locked grand mean and (right) response-locked grand mean responses to target stimuli. (A and B) Grand mean responses at all 29 scalp channels (colored traces), plus scalp maps at indicated latencies. (C–F) Grand moving mean single-trial responses from all 15 subjects, at frontocentral site Fz (C and D) and at central parietal site Pz (E and F), plotted in ERP-image format and sorted by subject RT (curving dashed trace in left column; vertical solid line in right column, plotted at the mean subject-median response time of 352 ms). ERP-image units: z = microvolts divided by root-mean-square microvolts in the (−1000 ms to 0 ms) channel baseline EEG of the same subject after removal of eye and muscle artifact components from the data. Vertical smoothing window: 300 trials. Grand mean normalized responses are shown below each image. In the grand average of the same epochs, each time-locked to the subject response ( Figure 2 B), the early response-locked peaks became smeared out, and the P300 and succeeding negative dip more concentrated. In two-dimensional “ERP-image” plots of the 8,413 single trials from all 15 subjects ( Figure 2 C– 2 F), potential fluctuations in single trials are shown as color-coded horizontal lines, here normalized by channel baseline variability then sorted (across all trials) by RT and smoothed (vertically) with a 300-trial moving average. The ERP images clearly show that the early visual response peaks at central posterior site Pz ( Figure 2 E) were time-locked to stimulus onset, while the late positivity at Pz immediately followed the button press (compare Figure 2 E and 2 F) except in the trials with the quickest RTs. Over half of these were contributed by two fast-responding subjects whose responses, unlike those of the other 13 subjects, preceded P300 onset. At frontocentral channel Fz, however, the late positivity in the stimulus-locked grand average ( Figure 2 C, bottom) was largely composed of two response-locked positive peaks, separated by 200 ms, that, together with intervening and flanking negativities, could be partially modeled by a two-cycle, 5-Hz wavelet ( Figure 2 D). The single P300 peak at Fz in the stimulus-locked ERP ( Figure 2 A) “smears out” the two-cycle pattern that is captured clearly in the response-locked average ( Figure 2 B and 2 D), while highlighting a concurrent, broader, and possibly stimulus-locked positivity in faster-RT trials ( Figure 2 C). Event-related spectral perturbations Figure 3 summarizes the grand mean time course of changes from prestimulus baseline in log spectral EEG power at all the scalp channels time-locked to button presses (solid vertical line) across the EEG frequency range. During and just prior to the button press, an approximately 3-dB increase in low-theta-band power peaked (red area) near 4 Hz in bilateral central and posterior cortex. This increase remained significant ( p < 0.01) for 14 of the 15 subjects even after the subject-mean ERP was subtracted from each trial (data not shown). Figure 3 Changes in Mean Scalp Spectral Power Time-Locked to the Subject Response The solid vertical line indicates moment of the motor response (shown at the grand mean subject median RT, 352 ms). Color scale: decibel change from prestimulus baseline. Image shows signed-RMS power changes across all 29 scalp channels prior to removal of all but the largest eye artifacts. Scalp maps show the scalp topography of the spectral power change in decibels relative to baseline. Note (A and B) the broad posterior low-theta- and anterior higher-theta-band maxima at the button press, (C, D, and F) the bilateral central alpha and beta blocking, (E) the central lateral postresponse beta increase, and (G) the increase in low-frequency eye artifacts at the end of the record. A concurrent but weaker theta power increase near 6 Hz ( Figure 3 B) was maximal at frontocentral and parietal scalp sites. The theta increase at these sites was accompanied by a blocking of mu activity around 10-Hz and 22-Hz, maximal at the left and right central scalp but also widespread over posterior scalp ( Figure 3 C, 3 D, and 3 F). Following the button press, a late central bilateral increase in beta activity (maximal at 16–18 Hz) appeared ( Figure 3 E). Figure 3 was computed prior to performing ICA and removing eye movement artifacts. The diffuse, far frontal increase in 3–10 Hz activity that began 500 ms after the button press ( Figure 3 G) doubtless reflects increased subject eye activity following the target response. ICA Decompositions Conventionally, characterizing the sources of ERP ( Figure 2 ) or event-related spectral perturbation (ERSP) ( Figure 3 ) processes is thought to be difficult because the scalp sensors are relatively far from the actual brain sources and therefore each sums the volume-conducted activities of several source areas. Moreover, the biophysical inverse problem of determining the potential source distribution from a given scalp map is in general severely underconstrained, with many mathematically correct but physiologically different solutions. Nonetheless, infomax ICA, applied to the concatenated single trials for each subject, after removing trials containing out-of-bounds or uncharacteristic artifacts, decomposed the whole set of concatenated EEG signals into 31 spatially fixed, maximally temporally independent component processes, and the scalp maps associated with many of these processes resembled the scalp projections of synchronous activity in either one or sometimes two nearly bilaterally symmetric cortical patches Component contributions to the single-trial EEG signals Figure 4 shows two single trials at site Pz (black traces) from one subject after removal of six eye and muscle artifact components. Projected activities of the three independent components most strongly contributing to each trial are shown as thin colored traces and accompanying scalp maps. Since infomax ICA provides a complete linear decomposition, the observed data (black traces) are in each case the sum of the remaining 25 (31 minus six) component projections, including the three component projections shown. In the upper trial, and typically, the single-trial P300 at Pz was accounted by ICA as summing contributions from at least two independent EEG processes. Component IC1 for this subject (ranked first by amount of total EEG variance accounted for) was later included in the parietal “P3b” component cluster (described below) on the basis of its scalp map and activity power spectrum. Figure 4 Independent Component Decompositions for Two Single Trials Black traces indicate two of 561 single target-response trials from one subject at scalp site Pz (upper right scalp map). Solid vertical lines indicate stimulus onsets; dashed vertical lines indicate button presses. A prominent late positivity occurred in the upper trial. All 561 1-sec, 31-channel EEG epochs time-locked to target stimuli were concate-nated and decomposed by infomax ICA, yielding 31 maximally independent data components. Colored traces show the projections (in microvolts) to this scalp channel of the three (nonartifact) independent components contributing the largest variance to each postresponse data window, linked to (individually scaled) maps of their scalp topographies. Component numbers (IC1–IC6), ranked by total EEG variance accounted for, and cluster affiliations (P3f, P3b, FM, P3b, Rα) are indicated above the scalp maps. Note differences in the time courses of IC1. While component IC1 accounted for the largest part of the P300 peak in the upper trial, in the lower trial the same IC1 process showed mixed low alpha and beta activity with a smaller postmotor response positivity. Note that the positive postresponse contribution of IC1 in this trial (thin blue trace) was sometimes larger than the observed positivity in the whole-channel data (thick black trace). At these times, some of the other 24 components contributed negative potentials to the signal at this scalp channel, partially canceling the IC1 positivity in the recorded data. Thus ICA, applied to the continuous or concatenated single-trial data, may actually recover more of the actual projected signals than are available in the single-channel data. Independent component clusters Cluster analysis, applied to the normalized scalp topographies and power spectra of all 465 components from the 15 subjects (see Materials and Methods ), identified at least 15 clusters of components having similar power spectra and scalp projections. These component clusters also showed functionally distinct activity patterns. Six distinct component clusters (data not shown) accounted for eye blinks, horizontal eye movements, and left and right temporal muscle noises, respectively. These were effectively removed from the activity of the other component clusters by the ICA decomposition and are not further considered here. Equivalent dipole locations Figure 5 shows the results of modeling the grand mean scalp maps for each of the nine independent component clusters as the projection of an equivalent dipole. Residual scalp map variances unaccounted for by these models were relatively small (range: 0.87% to 9.55%; mean: 4.93%), though the equivalent dipole locations for the individual clustered components were not all tightly clustered, as shown by the spatial standard deviation ellipses. Figure 5 Mean Component Cluster Equivalent Dipole Locations The mean scalp map for each of the nine component clusters could be well fit by a single equivalent dipole (mean residual variance: 4.8%). The figure shows the locations and orientations of these dipoles, as determined by BESA, plotted on the spherical head model, with ellipses showing the spatial standard deviations of the locations of the equivalent dipoles for the individual components in the cluster. The location of the equivalent dipole for a radially oriented cortical source patch (or here, effective sum of patches) is typically deeper than the cortical patch itself ( Baillet et al. 2001 ). The equivalent dipoles for individual components in the P3b cluster (data not shown) were scattered across parietal and central cortex (as indicated in Figure 5 by P3b's larger spatial standard deviation). Therefore, the equivalent dipole for the mean scalp map of the P3b cluster was unnaturally deep and represented the center of the active cortical source distribution only symbolically. The mean equivalent dipole location for the cluster designated “P3f” was estimated chiefly from the two periocular electrodes. Moreover, the complicated geometry of the frontal skull cannot be well fit to the spherical head model used here. Thus, the mislocalization of the P3f cluster equivalent dipole below the orbitofrontal brain surface should not be taken literally. The spatial standard deviations of the other component cluster dipoles were smaller. Their equivalent dipoles indicate the respective dominant cortical regions of their source domains. Though the mean cluster scalp map for the central occipital alpha cluster (Cα) could be fit satisfactorily by a single equivalent dipole located in the central occiput, for several of the cluster components a better model of the component scalp map was obtained from a symmetric dipole pair in left and right pericalcarine cortex (data not shown). Component Cluster Dynamics Mean dynamics properties of nine nonartifact component clusters are summarized in Figures 6 – 9 , each of which shows the mean scalp map and response-locked ERP image, activity and ERP spectra, and ERSP for one or more component clusters. Because of the complexity of the results, we report and interpret the nine component clusters in four groups based on shared dynamic features. Figure 6 Far-Frontal and Parietal Component Clusters Contributing to the P300 (A–D) Far-frontal component cluster accounting for the preresponse (P3f) positivity. (E–H) Broad parietal component cluster accounting for part of the postresponse (P3b) positivity. The periresponse energy increase for these processes peaks at below 5 Hz. (A and E) The mean component scalp map. (B and F) The whole-data (black traces) and cluster-accounted (red fill) ERP envelopes (minimum and maximum voltage channel values at each time point), plus (inset) the power spectrum of the whole EEG (black traces) and the whole response-locked average ERP (red fill). The lower edge of the red fill shows actual ERP power, the upper edge, the phase-random EEG spectrum required to produce the observed average ERP spectrum by phase cancellation. The difference between the upper edge of the red fill and the actual EEG spectrum (black trace) reflects phase consistencies across trials in the single trial data. (C and G) ERP-image plot of the color-coded single trials time-locked to the response (solid vertical line) and sorted by RT from stimulus onset (dashed line). Trials normalized by dividing by the standard deviation of component activity in the 1-s prestimulus baseline. (D and H) The component mean ERSP showing mean event-related changes in (log) spectral power across data trials time-locked to the response (solid line). Here, median stimulus delivery time is indicated by the dashed line. Figure 9 Three Posterior Alpha-Rhythm Component Clusters Panels as in Figure 6 . (A–D) Left posterior alpha (Lα) component cluster. (E–H) Central posterior alpha (Cα) component cluster with characteristic trapezoidal scalp projection, consistent with a bilateral, pericalcarine equivalent dipole source model, and demonstrating prolonged phase resetting following stimulus onset (curving dashed trace). (I–L) Right posterior alpha (Rα) component cluster. Note (C) the relative absence of the alpha-ringing pattern in the Lα cluster activity and the (D, H, and L) relatively weak postresponse alpha blocking in these clusters. Two clusters contributing to the late positive response (P3f and P3b) Two component clusters made distinct contributions to the late positive complex of the target ERP. After subtracting the larger back-projected scalp-data contributions of components accounting for blinks and saccadic eye movements, the response-locked ERP at both periocular channels contained a broad, approximately 2-μV positive-going scalp potential peaking on average 39 ms before the recorded button press. Figure 6 A– 6 D shows the mean scalp map and dynamic properties of a cluster of ten independent components from ten subjects that together largely accounted for an ERP feature whose time course was highly similar to the peak we labeled P3f (for P3-frontal) in an earlier report on decomposing the matrix of 25 condition ERPs from these experiments ( Makeig et al. 1999a ). Note, in the ERP image ( Figure 6 C), the absence of sharp excursions not regularly time-locked to experimental events, which would mark blinks or lateral eye movements. Such activity at far frontal and periocular channels was effectively separated out by ICA into artifact components (data not shown). Instead, as shown in Figure 6 B, the P3f component cluster accounted for nearly all the positivity occurring before the button press (designated P3f by Makeig et al. 1999a ), particularly in shorter latency-response trials ( Figure 6 C). The P3f cluster-mean response-locked positivity began near 150 ms, consistent with direct neurophysiological evidence that by 150 ms after stimulus onset, visual information is spread throughout the brain by a complex web of afferent and efferent connections ( Klopp et al. 2000 ; Hupe et al. 2001 ). Subtracting the button travel time (approximately 25 ms, roughly estimated from electromyographic recording during one experimental session) and the neuromuscular conduction time (approximately 15 ms) suggested that the P3f peak at 39 ms before the button press occurred very near the moment of the subcortical motor command. It is thus tempting to speculate that the P3f process should originate in frontal structures involved in motivated decision making and response selection, such as orbitofrontal cortex ( Ikeda et al. 1996 ), though the sparse spatial sampling of the present data does not allow more specific conclusions. The far-frontal (P3f) component cluster response appears similar to the 280-ms “P2a” peak noted in responses to (foveal) visual “oddball” stimuli by Potts et al. (1998) . Potts and Tucker (2001) reported that P2a was maximal near the eyes but can be recorded over most of the face and may also be found in attention conditions involving no subject button press. The scalp map of the ERP-derived P3f component derived by ICA applied to the 25 condition ERPs ( Makeig et al. 1999a ) also included bilateral parietal features not seen here in the P3f component cluster. Evidently, the temporal and far-frontal projections joined in the ERP-derived P3f component map did not cohere in the much more extensive single-trial data and so were separated by ICA applied to the concatenated single-trial data. This detail points to the advantage of decomposing a sufficient number of unaveraged data trials over decomposing even a relatively large set of averaged responses. Figure 6 E– 6 H shows the mean scalp map and activity patterns of a bilaterally distributed cluster of 15 components (from nine subjects) that projected most strongly to posterior and central scalp sites and made a substantial contribution to the slow postmotor P300 or P3b positivity. Examination of the raw ERP waveforms of the six subjects not contributing to this cluster suggested the absence of a typical central parietal positivity in their target responses. The mean cluster scalp map ( Figure 6 E) resembled that of the response-locked parietal ERP peak itself (see Figure 2 B). The ERP image of the normalized single-trial component activity ( Figure 6 G) includes an early series of small, positive and negative, wave fronts following the (sorted) time of stimulus delivery (dashed curve) by fixed delays. These are followed by a large response-locked positivity (red area) accounting for 62% of postre-sponse ERP variance at 300 ms for site Pz. The P3b cluster positivity is clearly smaller in late-response trials ( Figure 6 G, top), consistent with the Pz data ( Figure 2 F). The mean cluster ERSP ( Figure 6 H) reveals a significant (3-dB) post-response low-theta power increase. The significance of the stimulus-locked P300 (or P3b) peak over central parietal cortex has long been debated. Our results clearly show ( Figure 2 E) that in these experiments, this peak was time-locked to and predominantly followed the motor response. P3b onset occurred at about the moment of the motor command, coincident with the P3f peak. It could not, therefore, index activity involved in making the motor decision or action, as this is seemingly associated with the P3f process. The equivalent dipole distribution of the P3b cluster was broad, the strongest commonality being dipole orientation toward the central parietal scalp. Between-subject variability in locations of P3b generators have also been reported by researchers using other source localization methods ( Moores et al. 2003 ). It is possible that more advanced three-dimensional component clustering methods, applied to decompositions from more subjects and more data channels, might allow further distinctions among processes in this cluster. Central midline clusters Figures 7 and 8 show mean properties of four classes of components producing the two-cycle postresponse evoked-response pattern seen clearly in the response-locked data at site Fz (see Figure 2 D). Principal among these were two component clusters ( Figure 7 ) projecting maximally to the frontal midline (FM) and central midline (CM) scalp, respectively. Figure 7 Two Mediofrontal Independent Component Clusters Showing a Postmotor Theta Response Pattern Panels as in Figure 6 . (A–D) FM cluster of components often exhibiting a theta-band peak in their activity spectra. (E–H) CM component cluster projecting maximally to the vertex. Figure 8 Two Mu Rhythm Component Clusters Also Showing the Postmotor Theta Response Pattern Panels as in Figure 6 . (A–D) Left mu rhythm (Lμ) component cluster with mu characteristic 10-Hz and 22-Hz peaks in the activity spectrum. (D) Following the button press, this activity is blocked. (E–H) Corresponding right mu rhythm (Rμ) component cluster. In the RT-sorted FM cluster ERP image ( Figure 7 C), the two negative wave fronts follow the curving trace marking stimulus onsets, the second of these merging with the earlier RT-locked negativity. Though the vertex-maximum CM component cluster ( Figure 7 E– 7 H) also exhibited the postmotor theta feature ( Figure 7 F and 7 G, red arrows) with the mid frontal and mu rhythm clusters (see below), it contributed little to the broader (P3b) positivity produced mainly by the central parietal (see Figure 6 E) and central occipital ( Figure 9 ) component clusters. Though the equivalent dipole locations of components in the two midline clusters were somewhat overlapping (see Figure 5 ), their mean equivalent dipole locations were generally consistent with sources in or near the dorsal anterior cingulate and cingulate motor areas, respectively ( Ullsberger and von Cramon 2003 ). These areas are implicated by fMRI and neurophysiological experiments as participating in motor response selection and anticipation of the consequences of events, including those involving self-perceived errors ( Shima and Tanji 1998 ; Luu and Tucker 2001 ; Manthey et al. 2003 ; Ullsperger and von Cramon 2003 ). The phase/latency of the postmotor theta burst appeared to be consistent across quicker and slower responses. The theta burst appears to resemble other reported FM EEG activity patterns: theta bursts or trains (fmθ) appearing during mental concentration ( Mizuki et al. 1980 ; Gevins et al. 1997 ; Uchida et al. 2003 ) and brief bursts of theta activity linked to and following the error-related negativity ( Luu and Tucker 2001 ; Luu et al. 2004 ), an ERP peak whose latency matches the first negativity in the FM cluster postresponse ERP (at approximately 60 ms). Inverse source modeling has placed the generating cortical domain of the ERN and fmθ in or near the dorsal anterior cingulate. In this ICA decomposition, however, the two-cycle postmotor theta burst pattern appeared not only in the FM cluster, but also in the CM, mu, and parietal clusters (see below). The scalp map of the CM cluster ( Figure 7 E) resembles scalp maps of the “P3a” or “P3novel” ERP peaks seen, e.g., when unique and unexpected stimuli are included in a randomly alternating sequence of target and nontarget stimuli ( Courchesne et al. 1975 ; Polich and Comerchero 2003 ). Here, however, the CM cluster made only a small contribution to the stimulus-locked target ERP. Mu rhythm clusters The left and right mu rhythm component clusters (Lμ and Rμ in Figure 8 ) exhibited the defining feature of mu rhythms—distinct spectral peaks near 10 Hz and 22 Hz that are strongly blocked following movements, with equivalent dipoles located roughly over hand motor cortex (and/or adjacent postcentral somatosensory areas), and oriented roughly orthogonal to the directions of the central sulci. Both the ERP and ERSP peaks were larger in the left mu cluster (contralateral to the response hand) than in the right. In common with the midline clusters, the mu component clusters contained the two-cycle postmotor theta pattern ( Figure 8 D) concurrent with a mean theta power increase ( Figure 8 H). They also made slower, positive-going contributions to the parietal ERP, particularly to the late “slow wave” phase of the stimulus-locked P300 complex that, unlike the main (P3b) peak, exhibits a polarity reversal over the central scalp ( Simson et al. 1977 ). In a previous ICA analysis of ERPs from these experiments, the late slow wave phase of the stimulus-locked P300 complex was confined to a single component ( Makeig et al. 1999a ), but here it was separated into distinct left and right mu rhythm processes in at least ten of the subjects. More detailed source analysis of magnetoencephalographic mu rhythms has assigned their source mainly to somatosensory cortex ( Forss and Silen 2001 ). Thus, the postresponse slow positivity ( Figure 8 C) of the Lμ cluster, larger following slower responses, might index tactile feedback from the hand and button surface ( Makeig et al. 1999a ). Posterior alpha clusters Figure 9 shows the dynamics of three clusters of components projecting to the posterior scalp. Each had a distinct near 10-Hz alpha frequency peak in its activity spectrum, most pronounced in components of the central cluster ( Figure 9 F, inset). The stimulus-locked ERP contributions of the two lateral posterior alpha clusters, shown as sloping wave fronts in Figure 9 C and 9 K, included an early stimulus-locked peak accounting for most of the P1 ERP peak (near 145 ms) and for part of the succeeding N1 peak, which summed contributions from several clusters. In the central alpha component cluster, the initial stimulus-locked response feature was followed by a train of approximately 10-Hz stimulus-locked waves. These can be said to be produced by partial phase resetting of the intermittent alpha activity of these components following stimulus onsets, since they were accompanied by no mean increase in alpha power. The central alpha cluster also made an appreciably broad, triangular contribution ( Figure 9 F and 9 G) to the P300 positivity, while the contributions of the lateral clusters to the response-locked ERP, beginning just before the button press, were small and narrow. The mean response-related ERSPs for these three clusters were weak ( Figure 9 D, 9 H, and 9 L), and their postresponse alpha and beta blocking were brief and weak, compared to the two mu clusters (see Figure 8 ). The lateral clusters, but not the central cluster, exhibited a low beta increase above baseline (near 14 Hz) beginning near the button press. While the existence of multiple alpha rhythms has long been noted, ICA here neatly separated their activities and identified their complete, overlapping scalp maps based on the relative independence of their activity patterns in the unaveraged data. The central posterior alpha processes had a stronger alpha-band peak than lateral posterior alpha components, and showed longer-lasting phase resetting following visual stimulus onsets ( Figure 9 G). The longer phase memory implied by the prolonged phase resetting is compatible with longer bursts of alpha activity in these components. Possibly, the distinct dynamics of the central and lateral posterior clusters may serve different though still unknown purposes. The “trapezoidal” signature of several of the central posterior component scalp maps ( Figure 9 E) is compatible with a model comprising two equivalent dipoles located symmetrically in left and right pericalcarine cortex. To be fused into a single infomax ICA component, activity in both hemispheres must have been largely synchronous with negligible phase delay. Alpha-band activity in two cortical areas can indeed be synchronous if the two areas are densely connected, here most likely via corpus callosum. Synchronization of bilateral generator regions via dense callosal coupling might also support the observed sharp (around 10-dB) alpha peak in the activity spectra of these components. In these data, the lateral posterior alpha components were always unilateral and never bilateral. Possibly this may reflect the lower density of direct connections between these areas. Early ERP features in these experiments appeared predominantly contralateral to the stimulus locations. No doubt this was because these stimuli were presented above and usually lateral to fixation. In other data, we have noted that visual ERPs time-locked to foveally presented stimuli usually contain a bilateral posterior P1/N1/P2 complex ( Makeig et al. 2004 ). Component ERP Contributions Together, the nine component clusters accounted for 91.1% of the variance of the response-locked grand mean ERP at all channels in the 1000 ms following stimulus onset, as well as for 90.8% of the variance of the stimulus-locked grand mean ERP. Figure 10 A and 10 B shows the envelopes (most positive and negative channel values, across all channels, at each time point) of the stimulus-locked and response-locked grand mean ERPs (black) and the envelope (red fill) of the summed back-projections to the scalp of the components comprising the nine clusters. The normalized grand average activity time courses for the nine clusters are shown in Figure 10 C– 10 H, for comparison with the time courses of the grand mean ERP ( Figure 10 A and 10 B). Figure 10 Component Time Courses and Summed Scalp Projections Summed projections (A and B) to the grand mean ERP average of all trials time-locked to stimulus onsets (left) and to subject responses (right), plus (C–H) grand mean normalized activity time courses of each of the nine independent component clusters, scaled and separated into the same cluster groupings as in Figures 6–9 . For comparison with the stimulus-locked responses (left), response-locked data epochs (right) are shown aligned to the mean subject-median response time (352 ms, dashed line in left panels). Note that stimulus-locked component cluster ERP activity first appeared in the lateral posterior alpha clusters (at 100 ms). Onset of the stimulus-locked ERP of the P3b cluster at about the same time was soon followed by the far-frontal P3f cluster onset (near 120 ms, Figure 10 C). The stimulus-locked ERP deflection began at the same moment in the four postmotor theta clusters ( Figure 10 E). Six of the nine clusters had a negative peak in their stimulus-locked ERP average near 200 ms, confirming the spatial complexity of the N1 peak, as indicated by invasive measures ( Klopp et al. 2000 ) and comparable to previous analysis of nontarget epochs from this data set ( Makeig et al. 2002 ). In the response-locked cluster ERPs, note that the P3f cluster activity appeared to begin early, while response-related activity in the P3b cluster ERP diverged from baseline 10–20 ms before the P3f peak, concurrent with a posterior-negative peak in the left mu cluster ERP. The posterior-positive peaks in the response-locked ERPs of both mu clusters, the early shoulder of the P3b cluster ERP peak, the central cluster ERP slow wave, and the negative-going peak of the FM cluster ERP all occurred together, about 100 ms after the P3f peak. Figure 11 shows the individual and summed independent component cluster contributions to the grand mean ERP at sites Fz and Pz. At Pz, no component cluster contributed more than a third of peak parietal P300 amplitude in either the stimulus-locked or response-locked ERPs ( Figure 11 C and 11 D). The largest cluster contribution to the peak at Fz was also from the P3b cluster, which contributed about half its peak amplitude ( Figure 11 A and 11 B). The P3f cluster contributed at best a third. These results cast doubt on claims that the target-response P300 peak at Fz predominantly indexes frontal activity. Figure 11 Cluster Projections to the Scalp ERP Component cluster contributions (in microvolts, thin traces) to the grand mean stimulus-locked (left) and motor-response-locked (right) target ERPs at scalp sites Fz (top) and Pz (bottom), plus their summed contributions (thick traces). Although the P3b cluster makes the largest contribution to the evoked responses at both scalp sites, its contribution does not outweigh the summed contributions of the other clusters. Post-Response Theta Synchronization Phase coherence analysis of consistent phase relationships between the FM and CM clusters, and between the FM and left mu clusters, time-locked to and following the motor response showed that significant theta phase coherence appeared in the data, even after the respective component ERPs were subtracted from each trial, indicating a transient postresponse phase linkage between these otherwise maximally independent processes. Figure 12 illustrates this phenomenon in the time domain using phase-sorted ERP-image plots. Sorting trials by the phase (with respect to the button press) of the postmotor theta activity of the FM cluster ( Figure 12 A) and then imaging the single-trial activities of the CM cluster in the same trial order ( Figure 12 B) induced partial theta phase ordering on the CM data (i.e., the slightly diagonal wave fronts in Figure 12 B). The converse procedure ( Figure 12 C) gave a similar result ( Figure 12 D). Figure 12 Phase Coupling of Theta Components: Time-Domain View (A) ERP-image view of baseline-normalized, response-aligned single-trial activity time series of components in the FM cluster, sorted (top-to-bottom) by phase at 4.87 Hz in a window centered 89 ms after the button press. Vertical smoothing: 400 trials. Units: microvolts normalized by dividing by the standard deviation of component single-trial baseline activity. The curving vertical trace (left) shows a moving mean of stimulus onset times; the central vertical line, the time of the button press. Data band pass in all panels: 0.1–40 Hz. (B) Exporting the same trial sorting order from (A) to CM cluster components (from the nine subjects contributing components to both clusters) demonstrates the significant partial theta phase coherence (r is approximately 0.3) between the two clusters in the postresponse time/frequency window. Note the induced (top-down, left-to-right) slope of the latency of the two (orange) positive-going CM cluster theta wave fronts. (C) Phase-sorted ERP image, as in (A), of the normalized CM cluster trials. (D) FM cluster component trials sorted in the same trial order as (C). Again, the partial theta-band phase coherence of the two clusters in the postresponse period is reflected in the diagonal (blue) negative-going wave fronts of the FM cluster data. Video 1 is an animation representing the joint response-related theta-band dynamics occurring in and between all nine component clusters ( Delorme et al. 2002 ). The figure shows an analysis window centered 89 ms after the button press. Note that the transient theta phase coherence between the mu, parietal, and midline components was selective: phase coherence between FM and CM, FM and Lμ, and Rμ and P3b clusters (indicated by linking cylinders) were significant, whereas no significant phase linkage occurred in this time period between the FM and P3b clusters, nor between the CM and Lμ clusters. This selectivity diminishes the possibility that the observed transient phase linkages were produced by appearance of postresponse EEG activity not separated out by ICA into a separate component and therefore misattributed by the ICA model to nearby independent components. The results presented here, however, do not allow us to completely discount this possibility. Discussion ICA used the temporal information contained in the single-trial EEG time courses to identify and separate maximally independent processes. These were associated with overlapping scalp maps and time courses whose distinctive features were no longer blurred by volume conduction as in the scalp electrode data. The nine independent component clusters here identified by their similar scalp projections and activity spectra resemble classes of EEG phenomena long described by neurologists from observation of paper data displays—central and lateral alpha, left and right mu, and FM theta rhythms. By cleanly separating the EEG contributions of these processes, ICA allowed exploration of their individual and joint event-related dynamics. Our finding of selective theta synchronization between FM, motor, and parietal processes ( Video 1 ) was only possible using ICA. The clear separation of “alpha ringing” in the stimulus-locked response from the other ERP features (see Figure 10 G) also illustrates the power of ICA to separate temporally and functionally distinct activities that are generated in different brain areas but project to the same scalp channels. The nine clusters largely reproduced the component clusters we obtained previously from ICA decomposition of brief (100-ms) poststimulus time windows following nontarget and target stimuli in these experiments ( Makeig et al. 2002 ). The major difference in the two sets of clusters was the inclusion, here, of a parietal P3b component cluster. In both analyses, the clustering omitted many small components with “noisy” scalp maps and time courses, and also omitted “outlier” components specific to single subjects. After removing clear ocular and muscle artifact components from the raw data, however, the nine identified EEG component clusters together accounted for over 90% of the grand mean ERP variance (over all channels) as well as almost 60% of variance in the whole EEG. By contrast, the ERP data themselves accounted for only 6% of poststimulus EEG variance, supporting our claim that this analysis presents a more complete model of the event-related EEG dynamics occurring in these data than the averaged ERP. Cluster Localization The group mean equivalent dipole locations shown in Figure 5 only symbolize the actual distribution of the component source domains. The relation between processes derived by ICA from scalp data and processes seen in invasively recorded cortical data are not yet clear. For example, the equivalent dipole localization of the P3b cluster-mean scalp map does not correspond directly to all the cortical areas noted to generate P3b-like local field potentials in implanted presurgical epileptic patients by Halgren et al. ( 1995a , 1995b )—parts of the superior parietal lobule, inferior frontal, and temporal cortices, as well as the limbic medial temporal lobe. However, in general it is difficult to infer the cortical distribution of a cortical source domain from equivalent dipole location. Clearly, more inclusive methods of ICA component source localization fitted to actual subject cortical geometry ( Dale and Sereno 1993 ) may be useful for further research. It is not clear, however, whether “hot spots” recorded by sparsely implanted intracranial electrodes necessarily map activity that dominates the scalp EEG dynamics; hot spots might arise from focal or more spatially diffuse activities in other areas. ERSPs Production of scalp EEG signals requires partial synchronies in local field activity extending across the relatively large (2-cm or more) domains of neuropile ( Nunez 1981 ). These must be dynamically maintained, and might well be perturbed by biological systems and mechanisms that implement and reinforce “top-down” cognitive decisions such as, as here, to selectively attend and respond to relatively infrequent visual stimuli while ignoring frequent nontargets ( Giesbrecht et al. 2003 ). Posterior alpha, in particular, increases promptly when visual attention shifts between hemifields or between visual and auditory stimulus streams ( Worden et al. 2000 ). Most of the spectral perturbations appearing in this analysis have been previously reported in some form, for instance, the alpha blocking following visual stimuli cueing visual attention and mu blocking accompanying cued finger movements ( Hari et al. 1997 ; Pfurtscheller et al. 2000 ). A late beta increase following target responses has also been reported (e.g., Makeig 1993 ). ERP Influence Traditionally, averaging event-locked EEG data from single scalp channels to form an ERP is assumed to reject, by random phase cancellation, “background” EEG rhythms whose statistics are tacitly assumed to be unaffected by experimental events. To test the effect of the average ERP on the observed spectral perturbations, we computed the cluster ERSPs again after removing the component mean ERP from each trial (data not shown). All the effects shown in Figures 6–9 remained significant. Common ERSP Features Note that many of the cluster ERSPs ( Figures 6 – 9 ) share common features. How is this possible? First, “independent” components returned by infomax decomposition of EEG data are never perfectly independent, but are instead those found by infomax to be maximally independent. This is not a mere play on words, but an advantageous feature of infomax decomposition that allows it to separate activity from different cortical areas even when the independence of synchronous activities within those areas is not unbroken or absolute. Second, the infomax independence metric is weighted toward separation by phase differences rather than by power spectral differences. The observed spectral perturbations may reflect, in part, common modulatory influences of central neurotransmitter-labeled brainstem systems involved in orienting and arousal, which project widely to cortex and are known to change the spectral properties of cortical field activity following novel or meaningful events ( Aston-Jones et al. 2001 ; Fries et al. 2001 ; Hasselmo et al. 2002 ). Functional Significance of the Postmotor Theta Bursts At and after the button press, a mean frontocentral theta power increase appeared in all 15 subjects' data. It was partially phase coherent in four of the component clusters and was not eliminated by removing the subject-mean ERP from each trial. Local bursts of theta-band activity are widely distributed on the human cortex ( Kahana et al. 1999 ) and associated with cognitive function ( Caplan et al. 2003 ). In hippocampus, an association between theta phase and high-frequency “sharp wave” activity has been observed in nonhuman animals ( Csicsvari et al. 2003 ). In turn, high-frequency activity can index the organization of spike timing of similarly tuned neurons into brief near-synchronous volleys more likely to trigger further spikes in common target neurons ( Fries et al. 2001 ; Salinas & Sejnowski 2002 ). Following nontarget stimuli in this experiment, FM components exhibited weak “theta ringing” (partial poststimulus theta phase locking) not accompanied by increased theta power ( Makeig et al. 2002 ). Here, following targets, a two-cycle period of increased theta activity appeared, time-locked to the motor response and weakly phase coherent between FM, parietal, and motor areas. Coherent theta activity might enhance the speed, salience, and reliability of spike-based communication between these and other brain areas connected with them, including hippocampus and related limbic structures such as the amygdala ( Seidenbacher et al. 2003 ). The result might be facilitation of information transfer to and from memory structures about the event and its anticipated consequences, and selective retuning of attentional states in relevant cortical areas based on anticipatory evaluation of the consequences of the cued motor response, including readjusted sensory and motor expectancies. The postmotor theta bursts seen here following correct speeded responses very likely are tightly linked to the ERP feature with strong theta-band energy that follows highly speeded manual (or foot) responses in the Erickson flanker task ( Holroyd et al. 1995 ). Luu and Tucker (2001) have suggested that the so-called error-related negativity in the response-locked ERP following responses the subject knows immediately to be in error partly represents partial phase locking of transiently increased theta-band activity in FM and other sources ( Luu et al. 2004 ). A similar negative-going ERP feature has been reported following negative feedback whose valence is not known in advance ( Gehring and Willoughby 2002 ; Ruchsow et al. 2002 ). Luu and Tucker (2001) also reported the appearance of enhanced theta activity in the ERP above somatomotor cortex following known-error responses. Our animation of the event-related theta-band dynamics in our data (see Video 1 ) demonstrates that correct speeded button presses produce partially synchronized theta-band increases in frontal (but not central) medial and contralateral somatomotor process clusters. Mean coherence phase lag between the midline clusters suggests that the postmotor theta activity in the FM cluster components leads that of the CM components by about 8 ms, a physiologically plausible value whose statistical reliability should be tested on a larger data set. Postmotor Theta and P300 Elbert and Rockstroh (1987) have proposed that cortical surface positivities in general index periods of relative neural depolarization and concomitant insensitivity (“disfacilitation”) of the involved cortex to distal input, possibly explaining the concurrent attentional blink ( McArthur et al. 1999 ; Kranczioch et al. 2003 ) and amplitude decrease in the auditory steady-state response ( Rockstroh et al. 1996 ). von Stein et al. (2000) , on the other hand, have reported that following delivery of visual targets to cats, coherence in the theta band occurred between the output layer of a higher and the input layer of a lower visual cortical area. They did not observe this coherence following nontarget stimuli. Thus, the postmotor P300 and the theta burst response may have complementary functions: to decrease bottom-up environmental sensitivity and to concurrently apply the results of top-down processing to cortical perceptual areas. Although probably not available for observation in scalp data, the postmotor theta-band synchronization might extend to limbic areas (hippocampus, amygdala, and others) and play a role in memory updating (e.g., of learned/remembered “context”) following goal-directed actions (cf. Seidenbecher et al. 2003 ). Thus, we suggest that the postmotor theta burst may reflect directly “context-updating” processes previously proposed to be indexed by the broad P300 positivity ( Dien et al. 2003 ). Evoked Responses In this speeded response paradigm at least, the ERP P300 positivity was nearly strictly time-locked to and predominantly followed the motor response. The P300 positivity was, here, indeed a late positive complex of potentials generated in several brain areas, confirming results of invasive recording ( Smith et al. 1990 ) and clinical group-difference studies ( Potts et al. 1998 ). ICA decomposed the unaveraged EEG signals composing the target-response ERP into several classes of brain EEG processes originating predominantly in frontal, central, parietal, and occipital cortex. This result adds to longstanding doubts about the specificity of ERP peak measures. In particular, it shows that parietal sources may account for less than half of the peak amplitude of the stimulus-locked positive peak at Pz (see Figure 11 C), the most commonly used index of P300 magnitude. Altogether, we found four component clusters contributing to the P300 maximum at Pz—in descending order, central parietal, left and right mu, and central occipital alpha EEG processes. As well, the (3-fold) largest EEG contributor to the stimulus-locked P300 positivity at Fz is volume-conducted from the same parietal (P3b cluster) sources, casting strong doubt on the specificity of peak amplitude at Fz for indexing frontal function. Phase Resetting As in our previous single-trial analysis of some of these data ( Makeig et al. 2002 ), partial phase resetting of ongoing intermittent alpha and theta EEG processes contributes to some features of the average ERP. However, partial phase resetting is not a sufficient model for all the ERP features. For example, the central posterior alpha cluster actually showed a postmotor response decrease in alpha power during its prolonged alpha-ringing ERP contribution (see Figure 9 G and 9H), whereas the monopolar parietal P300 (or P3b) ERP feature was associated with an event-related increase in both whole EEG variance in the central parietal channel (Pz; data not shown) and in component activity variance of the P3b, Cα, and Lμ clusters that contributed the most to it. The central posterior alpha-ringing feature of the stimulus-locked ERP can be parsimoniously described as arising through alpha phase resetting. The P300/P3b feature, on the other hand, might better conform to the usual conception of an ERP as measuring evoked response activity added to ongoing EEG activity. Note in Figure 6 H, however, that the low-frequency energy of the P3b cluster component activities increased by less than 3 dB at 3 Hz, demonstrating that the “baseline” activity of these processes included slow-wave processes with similar spectral characteristics. The postmotor theta burst phenomenon comprised both a frequency-specific power increase and significant (though partial) phase locking through its two-cycle ERP duration. The variety of these ERP features suggests that assuming a strict dichotomy between evoked and phase-reset activities is unproductive. Rather, each ERP feature may be usefully and better characterized as summing event-related perturbations of various sorts in the ongoing activities of one or more localized cortical EEG processes. To produce a reproducible peak or peaks in the average ERP, these perturbations should involve some degree of (partial) phase locking of the contributing process activities to the time-locking events in one or more frequency regions. At very low (near-DC) frequencies, event-related “phase locking” implies event-related “sign locking.” For example, the P300 would never appear in ERP averages at all if it were negative-going in half the trials. Similarly, there need be no strong dichotomy between evoked phenomena that involve partial phase locking and so contribute to average ERPs, and induced phenomena that may involve changes in spectral amplitudes but do not show phase locking and thus do not contribute to average ERPs. Rather, it is important to realize that most event-related EEG dynamics have both induced and evoked aspects. Event-Related Brain Dynamics The results presented here confirm that extensive, complex, and flexible information concerning links between cognitive processes and macroscopic brain dynamics are available in noninvasive high-density EEG data. Availability of more comprehensive analysis techniques, such as that introduced here, should make EEG (and related magnetoencephalographic) data analysis of increasing interest both to cognitive neuroscientists and to neurophysiologists, as event-related EEG dynamic models complement observations of slow-changing hemodynamics while greatly expanding the restricted spatial information available from single- and multineuron spike recordings. Materials and Methods Task design ERPs were recorded from subjects who attended to randomized sequences of filled disks appearing briefly inside one of five empty squares that were constantly displayed 0.8 cm above a central fixation cross (see Figure 1 ) following Townsend and Courchesne (1994) . The 1.6-cm square outlines were displayed on a black background at horizontal visual angles of 0 ° ± 2.7 ° and 0 ° ± 5.5 ° from fixation. During each 76-s block of trials, one of the five outlines was colored green and the other four blue. The green square marked the location to be attended. This location was varied in random, counterbalanced order across blocks. In each block, 100 stimuli (filled white disks) were displayed for 117 ms within one of the five empty squares in a pseudorandom sequence with interstimulus intervals of 250 to 1000 ms (in four equiprobable 250-ms steps). Subjects and task Fifteen right-handed volunteers (ages 19 to 53 y, mean = 30; 12 male, three female) with normal or corrected-to-normal vision participated in the experiment. Subjects were instructed to maintain fixation on the central cross while responding only to stimuli presented in the green-colored (attended) square. Subjects were required to press a thumb button held in their right hand as quickly as possible following stimuli presented in the attended location (see Figure 1 ). Thirty blocks of trials were collected from each subject, yielding 120 target and 480 non-target trials at each location. Subjects were given approximately 2-min breaks between blocks. EEG recordings EEG data were collected from 29 scalp electrodes mounted in a standard electrode cap (Electro-Cap International, Eaton, Ohio, United States) at locations based on a modified International 10–20 System, and from two periocular electrodes placed below the right eye and at the left outer canthus. All channels were referenced to the right mastoid with input impedance less than 5kΩ. Data were sampled at 512 Hz with an analog pass band of 0.01–50 Hz. To further minimize line noise artifacts, responses were digitally low-pass filtered below 40 Hz prior to analysis. Trials containing electrooculographic potentials larger than 70 μV or amplifier blocking were rejected, and brain responses to stimuli presented at each location in each attention condition were stored separately. Responses to target stimuli were analyzed only when (as in nearly all cases) subjects responded 150–1000 ms after target onset. The few targets followed by no such button press were not considered in the present analysis. Previous analysis Analysis of average ERP and some single-trial data from these experiments have been reported previously. Makeig et al. (1999a) first reported ICA decompositions of late (P300) target responses in a 5 × 5 matrix (five stimulus locations by five attended locations) of grand mean visual stimulus ERPs from these experiments. They reported three maximally independent ERP components of interest which they labeled P3f, P3b, and Pmp. Makeig et al. (1999b) applied the same multiple-ERP analysis to the first 250-ms period following stimulus onsets and demonstrated that distinct contributions to the N1 ERP peak were generated in the right hemisphere 9 ms earlier, on average, than in the left. ICA separates component processes mixed in scalp data based on their relative temporal independence, which should be maximally expressed in the unaveraged data. Systematic application of ICA to unaveraged data from these experiments was first demonstrated for non-target stimulus trials ( Makeig et al. 2002 ). In that analysis, a 100-ms poststimulus period (150–250 ms after stimulus onset) was extracted from each of the over 3,000 trials for each subject, and these data were concatenated and decomposed by ICA. Some information about the target stimulus trials was also presented in Jung et al. (2001b) . Here we report the results of comparing ICA decompositions of roughly 600 1-s target-response trials from each of 15 subjects. Independent component analysis Infomax ICA ( Bell & Sejnowski 1995 ; Makeig et al. 1996 ) is one of a family of algorithms that exploit temporal independence to perform blind separation of underlying data sources. Lee et al. (1999) have shown that these algorithms have a common information-theoretic basis, differing chiefly in the form of distribution assumed for the sources, which may not be critical ( Amari 1998 ). Infomax ICA finds, by natural gradient ascent, a square “unmixing” matrix that maximizes the joint entropy ( Cover and Thomas 1991 ) of a nonlinearly transformed ensemble of zero-mean input data vectors. Maximizing joint entropy implies, under reasonable assumptions ( Bell and Sejnowski 1995 ), minimizing mutual information among the component activities. This means that information about the simultaneous activity values of any number of the components gives minimum information about the concurrent activity values of any other components. Independent component activities are minimally correlated, both in standard second-order and in higher-order senses. That is, they each appear to ICA to be “free-running” and in this sense act as separate sources of information in the data. The power of infomax source separation in a growing range of signal processing applications derives from its basic aim to identify information sources in data, in contrast to previous root-mean-square estimation methods that aim simply to model data variance ( Jung et al. 2001a ). Natural-gradient logistic infomax ICA in the automated form we use here (the runica algorithm, Makeig et al. 1997 ) can accurately decompose mixtures of component processes having symmetric or skewed distributions without requiring nonlinearities specifically tailored to them, and can be usefully applied to EEG data from 100 or more channels. The number of time points required for training may be as few as several times the number of unmixing weights (usually the square of the number of channels), though using more (clean) training data are preferable. In turn, the number of channels must be at least equal to (and preferably larger than) the number of interpretable components to be separated ( Makeig et al. 1999a ). The success of ICA applied to EEG data is strictly determined by the degree to which EEG dynamics fit the ICA model. The first requirement, that the underlying sources mix linearly in the electrode recordings, is assured by the biophysics of volume conduction at EEG frequencies ( Nunez 1981 ). The assumption of relative spatial stationarity of EEG sources is compatible, at least, with evidence of brain modularity from anatomy and functional imaging. The assumption of relative independence of the source signals is compatible with physiological models that emphasize local, short-range intracortical and radial thalamocortical coupling in the generation of local electrical synchronies in the EEG range ( Salinas and Sejnowski 2001 ). The ultimate validity of the assumptions above in any data set cannot be guaranteed a priori. The consistency and physiologic plausibility of the results of ICA decompositions, such as we present here, including their often tight linkage to behavioral and cognitive variables, are strong indirect evidence for the workability of the assumptions and of the ICA model. Direct physiological testing of the model and its physiologic assumptions will require development of multiscale recording methods. Meanwhile, yet more flexible (but also more intricate) ICA models of EEG activity are possible (e.g., Anemueller et al. 2003 ). It remains to be seen, however, whether the information gain offered by such models exceeds the loss of statistical power associated with their higher complexity. As first demonstrated by simulations ( Makeig et al. 2000 ), when training data consist of fewer large source components than channels, plus many more small source components, as might be expected in actual EEG data, large source components are accurately separated into separate output components, with the remaining output components consisting of mixtures of smaller source components. In this sense, performance of the infomax ICA algorithm degrades gracefully as the amount of noise in the data increases. For more details about applying ICA to ERP and EEG data, see Makeig et al. ( 1999a , 2002 ) and Jung et al. ( 2000a , 2000b ). Here, the runica algorithm (available for download with the EEGLAB toolbox of Delorme and Makeig [2004a ] from http://sccn.ucsd.edu/eeglab ) was applied to sets of 400 to 600 1-s trials (31 channels, 256 time points) time-locked from −200 ms before to +800 ms after onsets of target stimuli presented at any of the five stimulus locations (see Figure 1 ). Target trials in which the subject did not respond with a button press (fewer than 5%) were removed from the data. Learning batch size was 50. Initial learning rate began near 0.0004 and gradually reduced to 10 −6 during 50–150 training iterations that required about 30 min of computer time. Results of the analysis were relatively insensitive to the exact choice of learning rate or batch size. Reducing the stopping weight change from 10 −6 to 10 −7 did not appear to change the resulting decompositions qualitatively, although when decomposing data from many more channels, we have since noted an advantage to continuing to train until weight change falls below 10 −7 . Component clustering Commonly in ERP research, neural activity expressed in periocular data channels is ignored for fear of mislabeling eye-activity artifacts as brain activity. Some ICA components of EEG records can be clearly identified as accounting primarily for eye movements, line or muscle noise, or other artifacts through their characteristic scalp maps and activity time courses ( Makeig et al. 1996 ; Jung et al. 2000a , 2000b ). Subtracting the projections of artifactual components from averaged or single-trial data can eliminate or strongly reduce these artifacts while preserving the remaining nonartifactual EEG phenomena in all of the data channels. ICA thus makes it possible to examine periocular EEG activity apart from eye movements. Here, the total of 465 (31 times 15) component maps and mean activity log spectra from the 15 subjects were clustered by applying a modified Mahalanobis distance measure ( Enghoff, 1999 see Appendix of Jung et al. 2001b ) to vectors coding differences in the component 31-channel (x, y) map gradients and activity log spectra after reduction to 12 and five dimensions respectively by principal component analysis. Cluster membership was in a few cases then further adjusted by eye for uniformity. Clustering based on scalp map gradients and activity spectra, as reported here, is one of several possible component clustering approaches, whose relative advantages have not yet been explored. Event-related spectral dynamics To examine stimulus- and response-induced changes in the EEG spectrum, we computed ERSP transforms ( Makeig 1993 ) for each channel and each clustered independent data component using the publicly available EEGLAB toolbox ( Delorme and Makeig 2004a , 2004b ). ERSPs show changes in decibels from baseline in spectral power across a broad frequency range (here, 3–50 Hz). The time/frequency analysis used Hanning-windowed sinusoidal wavelets of 3 cycles at 3 Hz, rising linearly to about 15 cycles at 30 Hz. This modified wavelet transform was selected to optimize the trade-off between temporal resolution at lower frequencies and stability at higher frequencies. Constructing surrogate data sets by shuffling the data epoch subwindows used to construct the time-locked spectral average allowed choosing an initial within-subject significance cutoff (not corrected for multiple comparisons) of p < 0.01. To construct between-subject mean ERSPs, we used binomial statistics to select a significance cutoff based on the minimum number of subjects required to have significant differences (in the same direction) from baseline at a given time/frequency point ( p < 0.0001). ERSP transforms of the data were computed at each channel and then for each clustered data component. To test for partial phase locking (i.e., nonrandom phase relationships) between EEG processes and the occurrence of experimental events across trials, we used intertrial phase coherence ( Makeig et al. 2002 ). To test the presence of nonrandom phase relationships (possibly including fixed delays) between activities in different (maximally) independent components, we performed event-related phase coherence analysis ( Makeig et al. 2002 ; Delorme et al. 2002 ), again with a single-subject bootstrap significance threshold of p < 0.01 (uncorrected) between pairs of independent components (from the same subject) included in each pair of independent component clusters (defined below). To exclude the possibility that the observed phase linkages arose only from common phase locking of the portion of the single-trial data constituting the ERP, we also subtracted the concurrent mean ERP from each trial before computing phase coherence. Functions to compute and plot the time/frequency measures used here are also available in the EEGLAB toolbox. Equivalent dipole modeling Both simple anatomic considerations and observed results of ICA decomposition suggest that cortical EEG sources may be usefully modeled as patches of cortex with partially synchronous local field activities. In brief, the high local coupling density of both excitatory and, particularly, inhibitory neurons in cortex means that local field potentials sufficiently synchronous to create measurable EEG signals should tend to extend through a compact spatial domain—roughly speaking, a patch of cortex of unspecified extent. Through volume conduction, partially synchronous activities within cortical source patches produce far-field potentials throughout the brain and on the scalp. The distribution of the scalp electrical field produced by such a source patch is nearly identical to that of a small dipolar potential element whose geometry is like a tiny flashlight battery oriented perpendicular to the cortical surface. This “battery” is termed the equivalent dipole for the cortical source. Here, we used a relatively simple and well-known method for fitting the positions and orientations of equivalent dipoles in a four-shell spherical head model for each component (BESA; Megis Software, Munich, Germany). To reduce the time required to process the 485 component maps, we used a version (3.0) of the BESA software that allowed batch processing to fit single-dipole models to each component scalp map. (A dipfit tool set by R. Oostenveld, producing equivalent results, is now available at http://sccn.ucsd.edu/eeglab/dipfit.html ). Some bilaterally symmetric component maps were better fit with symmetric dual-dipole models. The successful fits of single-dipole models for many of the clustered components is compatible with their generation within a single, compact patch of cortex, while bilateral dual-dipole models are compatible with tightly coupled oscillatory activity (without net phase delay) in two bilaterally symmetric cortical patches densely connected via corpus callosum or common subcortical drive. To distinguish the relative regional locations of the component clusters, the scalp maps for the individual components in each cluster were first oriented similarly (e.g., so as to all be positively correlated), normalized, and averaged. These cluster mean maps were then fit with single-dipole models to roughly illustrate the regional distinctions between the sources of the component clusters. Finally, the cluster-mean dipoles and event-related time/frequency information measured by ERSP, intertrial phase coherence,and event-related phase coherence analysis of all the single trials were visualized together in three dimensions using an animated display developed by Delorme et al. (2002) . Video 1 Postmotor Theta Dynamics An animation representing grand mean patterns of event-related dynamics in the theta band. Black traces in the lower panel show the envelope of the grand mean ERP time-locked to the subject button press; the dotted vertical line shows the median time of stimulus onset (response minus 352 ms). Theta dynamics computed in a window (center frequency 4.87 Hz, 3-cycle Hanning taper) centered (blue vertical line) 89 ms after the button press (red vertical line). Each sphere in the upper panel represents the location of the equivalent dipole for a component cluster. Approximate projections of the equivalent-dipole locations are shown in shadow on three planes from an average magnetic resonance image (Montreal Neurological Institute). Log spectral power changes (relative to prestimulus baseline) are indicated by the sizes of the spheres (see key, bottom right). Nongrey sphere colors indicate consistent intertrial phase locking (intertrial phase coherence). Colored cylinders joining spheres indicate significant event-related phase coherence between cluster components. (4.26 MB MOV)

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524173

Enhancement of blood-tumor barrier permeability by Sar-[D-Phe8]des-Arg9BK, a metabolically resistant bradykinin B1 agonist, in a rat C6 glioma model

Background While it is well known that bradykinin B2 agonists increase plasma protein extravasation (PPE) in brain tumors, the bradykinin B1 agonists tested thus far are unable to produce this effect. Here we examine the effect of the selective B1 agonist bradykinin (BK) Sar-[D-Phe8]des-Arg9BK (SAR), a compound resistant to enzymatic degradation with prolonged activity on PPE in the blood circulation in the C6 rat glioma model. Results SAR administration significantly enhanced PPE in C6 rat brain glioma compared to saline or BK (p < 0.01). Pre-administration of the bradykinin B1 antagonist [Leu8]-des-Arg (100 nmol/Kg) blocked the SAR-induced PPE in the tumor area. Conclusions Our data suggest that the B1 receptor modulates PPE in the blood tumor barrier of C6 glioma. A possible role for the use of SAR in the chemotherapy of gliomas deserves further study.

Background Bradykinin (BK) is a vasoactive peptide released from its high molecular weight precursors, the kininogens, through the action of serine proteases, the kallikreins, playing a crucial role in pathologic processes like inflammation, infectious diseases, and cancer [ 1 , 2 ]. The brain and spinal cord contain all of the components necessary for kinin formation and action. In addition, central nervous system traumas lead to kinin formation [ 3 ]. BK type-1 (B1) receptors are mostly expressed in pathological or stressful situations related to tissue damage [ 1 , 2 ] including tumors [ 2 ]. The BK type-2 (B2) receptor is defined as a constitutive one, being normally encountered in many tissues, including the brain [ 2 , 4 ]. It is well known that the blood-brain barrier (BBB) in brain tumor regions (also called blood tumor barrier, or BTB) shows different plasma protein extravasation (PPE) characteristics when compared with the BBB of normal brain tissue and this effect is particularly related to the increase of B2 receptors [ 5 - 7 ]. In animal models, B2 receptor agonists like Cereport ® (RMP-7) have been used by the intracarotid route to enhance the delivery of chemotherapeutic agents to the brain tumor area [ 7 - 9 ]. B1 receptor agonists with low metabolic resistance (MR) have already been unsuccessfully tested on PPE extravasation through the BTB in brain tumors [ 8 , 9 ]. The B1 receptor agonist, Sar-[D-Phe8]des-Arg9BK (SAR), shows some resistance to enzymatic degradation that is due to the addition of amino acids to the amino-terminal portion of the molecule [ 10 ]. Thus, it is possible that SAR could be successful in promoting PPE through the BTB, having potential uses in enhancing the delivery of chemotherapeutic agents to brain tumors. In order to test this hypothesis, we investigated the effect of SAR on PPE using the rat C6 glioma model [ 11 ]. Results Eighty percent of C6-inoculated animals developed brain tumors after a period of 30 days (data not shown). We determined PPE in experimental groups of animals treated with bradykinin (BK), the BK receptor B1 agonist (SAR) or the BK receptor B1 antagonist (LEU) (Fig. 1 ). PPE in the tumor area of C6-inoculated animals was 22.8 ± 6.4 μg EB/g dry tissue, a value that did not differ from those observed in tumor-free animals inoculated with vehicle only (26.3 ± 16.7 μg EB/g dry tissue), or inoculated with cell vehicle plus SAR (23.2 ± 9.8 μg EB/g dry tissue). Injection of BK into C6-inoculated animals also did not change the tumor PPE (30.6 ± 10.9 μg EB/g dry tissue). It is important to note that in all groups the left (non-inoculated) hemispheres presented PPE values similar to those of the right (inoculated) hemisphere in the control animals. However, SAR significantly increased PPE in the tumor area of C6-inoculated animals (91.9 μg EB/g dry tissue ± 7.9) ( p < 0.01). This effect was abolished, with a return to basal levels, by pre-treatment of the animals with the BK receptor B1 antagonist LEU (35.8 ± 8.6 μg EB/g dry tissue). The effect of SAR in increasing PPE in C6-inoculated right hemispheres (91.9 μg EB/g dry tissue ± 7.9) was also observed (t = 24.9, p < 0.001) when compared to the contralateral non-tumoral hemisphere (21.7 μg EB/g dry tissue ± 3.0, n = 6) (Fig. 2 ). Discussion We demonstrate here that the selective B1 receptor agonist SAR [ 16 ] enhances PPE in C6 glioma without affecting the normal brain parenchyma. It has been previously shown that SAR at 100 nmol/Kg also does not alter the systemic blood pressure of rats [ 17 ]. The effect of SAR was blocked by pre-treatment with the selective B1 receptor antagonist LEU, suggesting that PPE can be modulated by B1 receptors in the glioma model of C6-inoculated rats. It was recently described that gliomas may express both B1 and B2 BK receptors, with B2 being located closer to the periphery of the tumor cells while B1 was observed throughout the cell [ 18 ]. In view of their effects on vascular dilatation and blood flow, bradykinin receptor agonists have been tested in the chemotherapy of brain tumors [ 18 , 19 ], whereas bradykinin itself was less used for this purpose [ 6 , 7 ] due to its lower efficacy and shorter blood half-life. In contrast to other authors [ 6 , 9 ] we did not observe an effect of BK by itself in enhancing BBB or BTB permeability. Fast BK degradation could account for this difference since we used a femoral vein, whereas other authors used more proximal accesses like the carotid artery [ 6 , 9 ]. The B2 receptor agonist RMP-7 (Cereport ® ) has been successfully employed to enhance BTB permeability to chemotherapeutic agents [ 7 , 20 ], but in the animal models already tested only local or regional routes like the carotid artery were used [ 20 , 21 ]. In general, B1 receptors are non-constitutive [ 3 ] and restricted to pathologic processes including tumors or degeneration [ 2 ]. Systemically administered metabolically resistant B1 receptor agonists may be useful to increase chemotherapy levels at the target site, with higher specificity and lower toxicity than B2 receptor agonists. Thus, we believe that SAR injected together with chemotherapeutic agents may be useful to increase drug delivery specifically to the brain tumor with less toxicity to non-affected areas. Additionally, because EB circulates bound to albumin, it is reasonable to think that SAR could increase the delivery of several chemotherapeutic agents of molecular weight similar to, or lower than that of albumin [ 22 , 23 ]. Although the Evans Blue method is less sensitive than other labeling methods like quantitative autoradiography, for example, we used it because it was the only method available to us at the time of this experiment. However, the Evans-Blue method is of low cost, can be easily reproduced by other laboratories, and was sensitive enough to detect differences provoked by SAR in these experiments. BK or BK agonists have been previously used to increase BTB permeability but, due to their faster enzymatic degradation [ 6 , 8 ], they were injected into the carotid artery. However, our findings suggest, that SAR administered intravenously remains stable and thus could be an attractive agent for the treatment of brain or even other tumors [ 24 , 25 ] that express BK B1 receptors. Conclusions Our data suggest that the B1 receptor modulates PPE in the blood tumor barrier of C6 glioma. A possible role for the systemic use of SAR in the chemotherapy of gliomas or other CNS neoplasms deserves further study. Methods C6 glioma cell culture The C6 rat glioma cell line was cultured as previously described [ 12 ]. Briefly, cells were grown in Dulbecco's modified Eagle medium (DMEM, Sigma, Saint Louis, MO, USA) supplemented with 5% fetal calf serum (FCS, Cultilab, Brazil), 100 mg/ ml streptomycin and 100 U/ ml penicillin (all from Sigma) under standard culture conditions. Cells were harvested with 0.125% trypsin/0.78 mM EDTA when they reached confluence. Animals and tumor inoculation Male Wistar rats (aged 3 months and weighing 250–300 g upon arrival from the UFSC Central Animal House) were used. The rats were maintained in a temperature- and light-controlled vivarium (22°C; 12:12-h light:dark cycle; lights on at 07:00 h), with food and water available ad libitum . Animals were treated according to the Guidelines for the Use of Animals of Universidade Federal de Santa Catarina. Before the procedures, the rats were anesthetized with sodium pentobarbital (50 mg/Kg body weight, i.p.) and then injected with 1 × 10 6 C6 cells prepared in 5 ml DMEM/FCS (vehicle) into the right cerebral hemisphere using the following coordinates: 4 mm lateral from right bregma and 4.5 mm deep from the dural surface, as previously described [ 14 ]. Pharmacological treatment and protein plasma extravasation (PPE) studies Seventeen days after C6 inoculation, BBB or BTB extravasations were assessed by the Evans blue (EB, Sigma) method [ 14 ]. EB (20 mg/Kg; 25 mg/ml in 0.9% NaCl) was administered intravenously via a femoral vein with saline ( n = 10, C6-inoculated; n = 6, control) or in combination with 100 nmol/Kg bradykinin (RBI, USA) ( n = 6, C6-inoculated) or with 100 nmol/kg of selective bradykynin B1 agonist Sar-[D-Phe8]des-Arg9BK ( n = 9, C6 inoculated; n = 6, control) (SAR, a gift from Dr Domenico Regoli, Université de Sherbrooke, Sherbrooke, Québec, Canada). An additional group of C6-inoculated animals ( n = 6) were treated intravenously with SAR (100 nm/Kg) 5 min after pre-treatment with 100 nmol/Kg of the B1 receptor antagonist [Leu8]-des-Arg9BK (LEU, Sigma). Previous pilot studies conduced in our laboratory (data not presented) showed that SAR had a maximum effect at the equivalent dose of 100 nmol/Kg. These assays also showed that LEU at the dose used was effective in blocking the effect of SAR effect on isolated brain parenchyma and dura-mater. Fifteen minutes after the treatments, rats were perfused with 4 ml/Kg 0.9% NaCl for 3 min [ 14 ] and the brains were removed. Two cubic centimeters of tissue were removed around the inoculation site (right hemisphere) and from the contralateral homologous region (left hemisphere). EB was extracted with formamide (4 ml/g of wet weight tissue at room temperature for 24 h) and quantified at 620 nm with a Titerk Multiscan Microplate reader by comparison with a standard curve of EB (0.5 to 25 μg/ml of formamide). Extravasation was expressed as microgram of EB per gram of dry tissue. Statistical analysis Differences between treatments were evaluated by one-way ANOVA followed by the Bonferroni post-hoc test (plasma extravasations studies) or by the Student t-test for paired samples (for comparisons between right and left cerebral hemispheres). Values were considered statistically significant when p < 0.05. List of abbreviations used BBB- Blood brain barrier BK- Bradykinin BTB- Blood tumor barrier B1- Bradykinin receptor B1 B2- Bradykinin receptor B2 EB- Evans Blue LEU- [Leu8]-des-Arg9BK MR- Metabolic resistance PPE- Plasma protein extravasation SAR- Sar-[D-Phe8]des-Arg9BK metabolically resistant bradykinin B1 agonist Authors' contributions BLS and RCC contributed equally to this work. BLS and RCC carried out the surgical procedures and cell culture. MMB, RW, and CGCJr participated in the preparation of the manuscript. MAS carried out the absorbance assays. AGT and MN conceived the study and participated in its design and coordination. All authors participated in the analysis of the data and discussion of the results. All authors read and approved the final manuscript.

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529443

Mapping the brain's orchestration during speech comprehension: task-specific facilitation of regional synchrony in neural networks

Background How does the brain convert sounds and phonemes into comprehensible speech? In the present magnetoencephalographic study we examined the hypothesis that the coherence of electromagnetic oscillatory activity within and across brain areas indicates neurophysiological processes linked to speech comprehension. Results Amplitude-modulated (sinusoidal 41.5 Hz) auditory verbal and nonverbal stimuli served to drive steady-state oscillations in neural networks involved in speech comprehension. Stimuli were presented to 12 subjects in the following conditions (a) an incomprehensible string of words, (b) the same string of words after being introduced as a comprehensible sentence by proper articulation, and (c) nonverbal stimulations that included a 600-Hz tone, a scale, and a melody. Coherence, defined as correlated activation of magnetic steady state fields across brain areas and measured as simultaneous activation of current dipoles in source space (Minimum-Norm-Estimates), increased within left- temporal-posterior areas when the sound string was perceived as a comprehensible sentence. Intra-hemispheric coherence was larger within the left than the right hemisphere for the sentence (condition (b) relative to all other conditions), and tended to be larger within the right than the left hemisphere for nonverbal stimuli (condition (c), tone and melody relative to the other conditions), leading to a more pronounced hemispheric asymmetry for nonverbal than verbal material. Conclusions We conclude that coherent neuronal network activity may index encoding of verbal information on the sentence level and can be used as a tool to investigate auditory speech comprehension.

Background One key function of the cerebral cortex involves the integration of elements into a percept that separates them from the background. In this process, changes in cortical networks are formed and modified by experience through the simultaneous excitation of groups of neurons [ 1 - 3 ]. These "long-range connections formed by excitatory cortical neurons" [[ 4 ] p.3] are considered the anatomical substrate of this integrative capability. This integration has been modeled in detail for the visual system [e.g., [ 4 ]] and similar principles should also describe other sensory functions such as auditory speech perception and comprehension. This assumption was tested in the present study by probing patterns of co-activation within and across hemispheres during the processing of verbal and nonverbal acoustic material. Intra-hemispheric co-activation was taken as a large-scale measure of functional network activation, and coherence of oscillatory electromagnetic activity served as measure of co-activation in time. Coherence is defined as the correlated activity between two locations within a distinct frequency range. Event-related brain responses, traditionally used in the study of cognitive processes, have been found to result from regional perturbations in ongoing brain activities in a self-organizing system rather than constituting a response set from an otherwise silent system. For example, Makeig and coworkers [ 5 - 7 ] showed that event-related potentials (ERP) must be viewed as perturbations in the oscillatory dynamics of the ongoing EEG. The response of successively activated groups of neurons is governed by an attractor, which means that different neuron groups, one after the other, contribute to large-scale changes in the magnetic field that move across brain areas, indicating spatio-temporal changes on a macroscopic level. The basin of attraction guarantees robustness of the propagating synchrony. Therefore, the activation of functional cortical networks may best be determined by examining the pattern of dynamic co-activation of groups of neurons [ 8 , 9 ]. As such, whenever neuronal cell assemblies fire 'in phase' the amplitude of oscillatory activity will increase. On a macroscopic level, oscillatory coupling between large neuronal populations can be examined by externally driving the nervous system using oscillatory stimulation and then measuring the regional coherence of the electromagnetic activity [ 10 ]. Amplitude modulation of the stimuli induces the oscillatory pattern of the Steady-State-Response (SSR). For auditory stimuli the SSR is most prominent at modulation frequencies around 40 Hz [ 11 ]. Patel & Balaban [ 12 ] assessed the synchronization of the magnetoencephalographic SSR at this frequency over time (i.e., coherence) in order to investigate neural correlates of musical comprehension. When the stimulus sequences formed a percept (a melody relative to random sequence), coherence increased between left posterior and right frontal nodes. Similarly, Srinivasan et al [ 13 ] found increased inter- and intra-hemispheric coherence in the visual SSR when subjects consciously recognized visual stimuli in their field of view. Coherence measures have also been employed in the investigation of complex networks involved in the processing of nouns [ 14 , 15 ], music [ 16 ], the perception of Necker cube reversals [ 17 ], and in the acquisition of contingencies in a conditioning paradigm [ 18 ]. The present study investigated coherence patterns of the auditory evoked magnetic Steady-State-Field (SSF), specifically coherence among SSF-generators within and across hemispheres, as a measure of neural networks involved in speech comprehension. If, as we hypothesized, the comprehension of speech was related to the activation of neuronal assemblies in the left hemisphere, then we should see increased coherence in this region with the recognition of a meaningful sentence as compared to an incomprehensible string of sounds. We further hypothesized that meaningful verbal stimuli should be processed differently from musical melodies. That is to say, verbal material should affect the coherence of electromagnetic signals more in the left than in the right hemisphere whereas listening to a nonverbal complement of a meaningful sentence like a melody will activate more right- than left-hemispheric neuronal networks and influence coherence patterns involving the right hemisphere. Given that language and music share components, we assumed only a relative dominance in the interconnection of networks toward left- or right-hemispheric activity. Results and discussion The present study studied co-activated cortical networks involved in speech comprehension by using auditory steady-state (41.5-Hz amplitude modulated) stimuli and measuring the coherence of generator activity of the magnetic steady state response. Steady-state stimulus modulation were used for a sentence, which – following a German play-of-words – was first presented as an incomprehensible string of sounds, but became a comprehensible sentence after the sentence's meaning was explained to the subjects and was properly articulated. In addition to verbal stimuli, non-verbal stimuli were also studied which included a 600-Hz tone, a scale, and a melody-like combination of the scales' tones. The present analysis of SSF coherence in the source space (see methods) extended previous approaches [ 12 ], which employed SSR in the signal space to disclose networks involved in auditory perception. Figure 1 (lower part) gives an example of the evoked magnetic 41.5-Hz SSF, averaged for the tone condition at the 148 sensors across the 12 subject. The sinusoidal 41.5 Hz oscillation is evident at all 148 sensors and a change in polarity over temporal areas suggests generator sources in the temporal cortices of each hemisphere. The Fourier Transform confirms the peak at the modulation frequency of 41.5 Hz for all stimulus-conditions in the sensor space (Fig. 1 , upper left graph) and in the source space (mid-right graph in Fig. 1 ; illustrated for a selected dipole in the expected generator structure of the SSF, as indicated by the filled circle). No such peak was observed during the baseline. A comparison of the grand averages of the power spectra in sensor and source space (see Fig. 2A ) demonstrates that conversion using the Minimum Norm Estimate (see methods) preserves the basic profile across conditions. As expected for acoustic stimulation, overall MNE amplitudes were most pronounced in auditory areas of both hemispheres, with a varying degree of laterality. For the Laterality Index (see methods and Fig. 2B ) an interaction of CONDITION × HEMISPHERE (F(4,44) = 3.06, p < 0.05, ε = 0.69) verified that nonverbal conditions as compared to the verbal ones induced a more pronounced asymmetry with more activity in the right compared to the left hemispheres (for the main effect of HEMISPHERE, F(1, 11) = 3.33, p < 0.1, and for the main effect of CONDITION, F(4,44) = 12.65, p < 0.0001, ε = 0.57). Planned comparisons confirmed significant effects of HEMISPHERE only for the nonverbal conditions (tone, t(1,11) = 4.5, p < 0.0001, scale, t(1,11) = 4.3, p < 0.000, and melody-like tone sequence, t(1,11) = 3.8, p < 0.0005). Intra-hemispheric coherence was specifically affected by conditions (CONDITION × HEMISPHERE, F(4, 44) = 3.72, p < 0.05, ε = 0.46): As illustrated in Fig. 3A for the Laterality Index, higher intra-hemispheric coherence in the left than in the right hemisphere was induced when the string of words became a comprehensible sentence (planned comparison: t(1, 11) = 2.7, p < 0.01), whereas the tone induced higher intra-hemispheric coherences in the right as compared to the left hemisphere (t(1, 11) = 2.3, p < 0.05). The main effect of CONDITION was significant for intra-hemispheric coherence (F(4,44) = 8.35, p < 0.001, ε = 0.62) and inter-hemispheric coherence (F(4,44) = 10.79, p < .001, ε = 0.61) indicating higher coherence was induced by nonverbal than by verbal conditions. Since inter-hemispheric coherence may depend on the different generator strength, which was higher in the right than in the left hemisphere, the coherence measures were normalized in order to compensate for an effect of the signal to noise ratio. For normalization, the inter-hemispheric coherence measures were divided by the intra-hemispheric coherence measure of each condition. Still, a main effect CONDITION (F(4,44) = 12.1, p < 0.0001, epsilon = 0.76) indicates that coherence was larger for nonverbal than for verbal conditions. Given that the major goal was to depict network signatures specifically involved in sentence comprehension, we applied an ANOVA to compare the coherence measure of the two verbal conditions. These were identical with respect to the physical stimulation, but differed in meaningful comprehension. For intra-hemispheric coherence a significant interaction involving CONDITION × HEMISPHERE × GRADIENT (F(1, 11)= 7.37, p < 0.05) reflected a relatively higher coherence in the left-posterior area after the string of words had been made comprehensible by explaining the sentence's meaning as opposed to the higher coherence in the right-posterior area for the incomprehensible word string. Profiles of intra- and inter-hemispheric coherence were similar, thereby resulting in similar statistical power for the CONDITION effect. This cannot be explained simply by a reduced signal-to-noise ratio in the verbal conditions, because normalized values show the same effect. We rather assume that increased laterality varies with decreased inter-hemispheric communication (coherence). Inter-hemispheric coherence between dipoles located in the left (left cortical input) and right (right cortical input) auditory cortex and the remaining dipole sites are characterized (Fig. 3B ) by larger coherence of activity across areas including the left auditory, occipital and right-posterior regions in response to the comprehensible sentence relative to the incomprehensible word string. Considering coherent activity, i.e., synchronized oscillations between spatially distributed maps, as the representation of a percept, we followed Makeig et al. [ 6 , 7 ] who discuss evoked activity in terms of oscillatory perturbations, i.e., alteration of synchrony in ongoing activity. The comparison of two conditions with identical physical stimulation but different degrees of integration into a percept revealed that the synchronicity of auditory SSF increased among areas in the posterior left-temporal and right-occipital cortex when a sentence was comprehensible compared to the same material being incomprehensible. This suggests that a network was activated when an intelligible sentence was being processed. This assumption is in line with previous research in which a left-posterior activity focus was found during semantic processing [ 19 - 23 ], a left lateralized auditory-conceptual interface was localized at the temporal-parietal-occipital junction [ 24 ], and an occipital focus of oscillatory activity found for the processing of (visually presented) content words relative to verbs [ 25 ]. Whereas Scott et al. [ 26 ] reported an increase in regional cerebral blood-flow in the anterior part of the left superior temporal sulcus for intelligible sentences compared to acoustically equivalent non-intelligible sentences, the present results indicated such a pattern – enhanced left-anterior coherence – to be induced by the incomprehensible string of words (see Fig. 3B ). At this point, hypotheses to resolve this discrepancy must remain provisional. However, it seems possible, that the speech-like – though incomprehensible – stimuli activated syntactical processing which has been associated with frontal activity [ 27 ]. In addition, the attempt to determine a syntactical structure has been found to activate the right temporal area [ 39 ] which would be in line with the right temporal coherence found for the present condition of incomprehensible word string processing (see Fig. 3B ). Patel and Balaban [ 12 ] discussed increased coherence between the left posterior and right frontal areas for melody-like stimuli as a correlate of integrative processing of local and global pitch information. Thus, it seems possible that in our study the condition of incomprehensible word string similarly activated pitch processing. Finally, there is the possibility that the order of stimulus presentations may have affected the results. While counterbalancing was not possible for the specific verbal stimulus condition (see methods), we would not have expected order effects to be large since similar temporal dynamics were not observed for the nonverbal conditions. However, an effect of time cannot be ruled out as steady state responses and their generator activity were largest for a simple 600-Hz tone which was presented first. SSF were larger for the nonverbal conditions (tone, scale, melody) than for the verbal material, particularly in the right hemisphere. While right-hemispheric processing of tonal perception has frequently been reported [ 28 - 31 ], the general dominance of right-hemispheric SSF remains to be explained. As mentioned before, it seems possible that it reflects a carry-over effect from the sequence of conditions which invariably started with the tone. It may also reflect bilateral processing of verbal material which has been indicated by various imaging approaches [ 19 ]. The combination of verbal and nonverbal conditions within one experimental session may have blurred rather than elucidated the co-activation of material-specific networks. Still, greater right- over left-hemispheric generator activity asymmetry was found in the nonverbal conditions and less asymmetry found in the verbal conditions. Moreover, intra-hemispheric coherence patterns showed distinct, hemisphere-specific patterns for verbal (more pronounced left-hemispheric) and nonverbal (more pronounced right-hemispheric coherence) processing. When lateralized coherence patterns were examined by a laterality index, the clearest left-hemispheric coherence focus emerged for the comprehensible sentence and the clearest right-hemispheric coherence focus emerged for the tone. While we had expected a melody induced dominant right-hemispheric activation, a more bilateral activation was found for the melody-like tone sequence. For the scale, there was a shift towards left-hemispheric asymmetry of coherence. An explanation for this finding might be that the 'melody' was constructed to include the tones of the scale which may have resulted in a melody-like tone sequence even though it did not resemble common melodies or songs. This processing of an unfamiliar 'melody' might have activated temporal (left) and spectral (right) processing, as suggested by [ 28 , 29 ], resulting in a more bilateral activation. While a simple tone contains only spectral information, a melody also contains temporal information. Conclusions In sum, the present study demonstrates that the analysis of the synchronization of evoked magnetic steady-state fields in the source space can map neuronal networks (co-)activated during speech comprehension. Our techniques add spatial information to evidence on left-hemispheric areas involved in language processing, and support co-activation or synchronization within complex neuronal networks as a cortical substrate of integration in perception – like speech comprehension. Methods Subjects Data of twelve German native speaking subjects (7 female, mean age 25.3 ± 6.3 years) were included in the analysis. (From the 14 subjects, who had participated in the study, data from one subject had to be discarded because of frequent movement artifacts and from another one, who recognized the play-of-words, see below.) It was ascertained by interview that the subjects did not suffer from any language, audiological or neurological dysfunction. Right-handedness was assessed by a modified version of the Edinburgh handedness questionnaire [ 32 ] to be 97.1 ± 4.3. Moreover, all subjects reported having first-degree right-handed relatives. None of the subjects reported to be a professional musician and none reported to be particularly involved in hearing or practicing music. Prior to the experimental session, subjects were informed about the procedure and given informed consent forms. After the experiment, each subject received a financial bonus of 15€. Material and design All stimuli were amplitude modulated at 41.5 Hz (sinusoidal amplitude) with a modulation depth of 90%. Verbal stimuli consisted of words composing a sentence. Nonverbal stimuli consisted of tones forming a scale or a tune or a simple tone. A German play-on-words served as the template for the two verbal conditions. In the first case a sentence is spoken without spacing between words and without accents which creates an incomprehensible word string. The German sentence 'Mähn Äbte Heu? Heu mähn Äbte nie! Äbte mähn Gras' means in English 'Do abbots cut hay? Abbots never cut hay, abbots mow lawns'. If pronounced as a string 'MähnÄbteHeuHeumähnÄbtenieÄbtemähnGras' this utterance, due to a lack of non-phonetic context [ 33 ], sounds like speech although meaning cannot be inferred. When the sentence is properly pronounced in the second case, the meaning becomes clear and can be used to parse the information at subsequent trials, allowing a listener to comprehend the sound string as a sentence. For the present study, the incomprehensible string-of-word-version was generated synthetically (software: MBROLA) with a female voice and a fundamental frequency of 200 Hz. None of the 12 subjects included in the data analyses knew the play-of-words and were unable to recognize the meaning of the sentence before it was properly articulated and explained. The three nonverbal conditions comprised of a 600 Hz sinusoidal tone, a descending major scale (C6 B5 A5 G5 F5 E5 D5 C5, 1034 – 517 Hz), and an arrangement of the same tones (C5 E5 G5 C6 A5 F5 D5 B5). All stimuli of all conditions were adjusted to the length of the sentence and lasted for 4419 ms (sample-rate of 16 kHz/16 bit, mono), and each of the five conditions comprised 15 repetitions that were separated by inter-stimulus intervals of 4419 ms. This long inter-stimulus interval allowed the same signal-to-noise ratio for the baseline and the stimulus conditions which should prevent habituation effects on the SSF. Stimuli were adjusted to have the same average loudness by normalizing to root-mean square (RMS) and were presented at 50 dB above the individually assessed hearing threshold balanced for both ears. In each subject, the hearing threshold was assessed by presenting short 600 Hz beeps with ascending and descending intensity. For each subject and ear the mean hearing threshold was determined from the ascending and descending sequence. Task and procedure During the experiment, which lasted about 45 minutes, the subject was seated in a supine position. Subjects were asked to listen carefully to the stimuli, while fixating a point at the ceiling of the chamber in order to avoid head and eye movements. They were further informed that they would be asked questions about the stimuli during the experimental session, and that they should reply by saying 'yes' or 'no'. All stimuli were presented in blocks with 15 repetitions. Conditions were separated by breaks of about 5 min each. For every subject the experimental session started with the 600-Hz sinus tone (15 repetitions, condition 1), followed by the word string (condition 2). After 5 repetitions, the subject was asked whether he/she understood what he/she was hearing and could reproduce the meaning of the speech. (None of the subjects could.) Subsequently, the stimulus presentation was continued, and the subject was asked again after the 10th and the 15 th presentation, whether he/she understood the meaning of the speech (None of them could). Then, the experimenter entered the room and pronounced the sentence properly and slowly, so that its meaning became clear. Each subject was asked to reproduce the sentence, in order to ascertain that it was properly understood. After the experimenter had left the subject chamber, the experiment continued with condition 3, which comprised the identical physical stimulation as condition 2 differing only in that the subject now listened to the string of words knowing its meaning, Again, the subjects were asked after 5 repetitions, if they could reproduce the meaning of the sentence, which now they all could. Given that once the sentence's meaning is obvious, one can easily grasp the sentence, the sequence of condition 2 and 3 could not be reversed and thus, the sequence of presentation could not be randomized across subjects. Condition 4 (scale) and 5 (melody-like tone sequence) were arranged in a similar way, in that the subject was asked after 5 repetitions each, whether or not s/he perceived the sequence of tones as a melody. Eleven of the twelve subjects indicated that the tone sequence sounded like a melody and one was not sure about it. None of them perceived the scale or the simple tone as melodic. Data acquisition and analysis The magnetoencephalogram (MEG) was recorded with a 148-channel whole head system (MAGNES ® 2500WH, 4D Neuroimaging, San Diego, USA) installed in a magnetically shielded room (Vaccumschmelze, Hanau, Germany). Data were recorded continuously with a sampling-rate of 1017.25 Hz and a 0.1–100 Hz band-pass filter. The electrooculogram (EOG) and the electrocardiogram (EKG) were recorded and stored together with the MEG-data for offline artifact control. Silver-silverchloride electrodes were placed on the outer canthi for the monitoring of horizontal eye movements, and above and below the right eye for vertical eye movements. EKG electrodes were placed on the right collarbone and below left costal arch. Prior to data analysis, the trials for each condition were submitted to a noise-reduction procedure that subtracted the external noise recorded by MEG reference channels. These noise-corrected data were then bandpass filtered (28–60 Hz, 48 db/Oct, zerophase) and averaged across epochs separately for each condition (epoch-length: 8838 ms, 4419 ms pre-stimulus baseline). Epochs were visually inspected for EOG and EKG artifacts and epochs with magnetic fields greater than 5 pT were rejected. A minimum 13 (of the total 15) epochs per subject were available for further analyses. The steady state field (SSF) in response to the 41.5-Hz amplitude modulated stimuli was extracted using a moving average procedure. A window of 5 cycles (120.5 ms) of the 41.5 Hz Steady-State signal was shifted 179 times cycle-by-cycle (24.5 ms) across averaged epochs (separately for the 4419-ms baseline and the 4419-ms stimulus duration, the moving average procedure starting 144.5 ms post stimulus). The resulting moving-average epoch was detrended. Figure 1 illustrates that a SSF was successfully induced by the stimulation. The generators of the SSF were determined in the source space for each epoch using the minimum norm estimate, MNE [ 34 - 37 ] using an algorithm implemented in MATLAB-based in-house software developed by Hauk [ 35 , 36 ]. The MNE is an inverse method reconstructing the primary current that underlies an extracranially recorded time-locked magnetic field. The procedure is based on the assumption that the data vector d, which contains the recorded magnetic activity at given sensor sites, can be described as the product of the leadfield matrix L, which specifies the sensor's sensitivity to the sources, the source current vector j [ 34 ] and a noise component ε. Since L and d are known, and ε is treated as if estimated with an accuracy of ~.05, the MNE for j is the mathematically unique solution of the equation which minimizes the squared current density (j 2 = min). This solution is obtained by multiplying the pseudo-inverse of the leadfield matrix L with the data. Given the high number of sensors and the presence of noise, spatial regularization is performed with the factor λ. This algorithm allows sources to be omitted, if they do not contribute to the measured magnetic field. A priori information about the number or locations of cortical sources is not required. Following Hauk et al. [ 35 , 36 ], who evaluated the dependence of the accuracy of inverse solutions on the depth of the source for concentric shells, solutions for a shell at 60% radius were determined as a compromise between blurring and depth sensitivity (ca. average radius of cortex, 77 equidistant dipole locations, covering the lateral surface of the brain, were chosen). That is, voltage data were projected to a source space consisting of 350 evenly distributed dipoles with three orthogonal orientations at each dipole location. For every location two tangentially orientated dipole-components were included in further analysis. The mean MNE amplitude, corresponding to the dipole strength in nAm/cm 2 , was determined as mean vector length of both tangentially orientated dipole-components across 5 cycles. Co-activation of generators was evaluated by all possible pair-wise combinations of the MNE at all dipole locations, according to the algorithm (Matlab, Mathworks): Spectral coherence is a function of frequency with values between 0 and 1 that indicate how well the input x (in the present study MNE at dipole location x) corresponds to the output y (MNE at dipole location y) as a function of frequency (in the present study 41.5 Hz). This algorithm estimates the coherence of two vectors x and y by computing the ratio of the squared cross power spectra (Pxy), divided by the product of the power spectra for each vector (Pxx.*Pyy), where Pxy(f) is the cross power spectrum estimate, Pxx(f) is the power spectrum of the time series at location x, Pyy(f) is the power spectrum estimate of time series at location y and f is the frequency index. The vectors of 4495 points in length were subdivided into 8 overlapping segments of 613 points (603 ms), each of which was submitted to Hanning windowing. For each vector, the power spectra were obtained as the product of the Discrete Fourier Transforms and its complex conjugate, scaled by the number of points used for x and y, and averaged across segments [ 38 ]. For cross spectra the products of discrete Fourier Transforms for vectors x and y were averaged across segments. This algorithm was applied to four pairs of dipole orientations (dpo), normalized (Fisher Z-transformation) and averaged to result in one coherence measure for every pair of locations (dp x -dp y ). As a measure of co-activation or coherence, the first order coherence between a region of interest (ROI) covering 7 locations over Heschl's gyrus and all other 77 locations was determined. Effects of the five conditions on the distribution of MNE amplitudes and on the coherence measure were evaluated by means of repeated measurement analyses of variance (ANOVA) with the factors CONDITION, HEMISPHERE (comparing all left and all right dipole-locations, excluding midline locations), and GRADIENT (comparing left- and right-anterior versus left- and right- posterior dipole-locations, excluding midline locations). For inspection of the hemispheric asymmetry of MNE, the ANOVA was performed on the Laterality Index (LI: left- minus right-hemispheric MNE divided by their sum, resulting in an index without units). For the evaluation of intra-hemispheric coherence, the first order coherence between the respective left- or right-hemispheric ROI and the other 34 locations of the respective hemisphere entered the ANOVA (comparing conditions), for the evaluation of inter-hemispheric coherence, coherence between the left-hemispheric ROI and all other 34 locations of the right hemisphere and between the right-hemispheric ROI and all other 34 locations of the left hemisphere was submitted to the ANOVA comparing conditions. A separate ANOVA of the two verbal conditions with the factors CONDITION, HEMISPHERE and GRADIENT probed the hypothesis of a change in coherence-topography induced by sentence comprehension. Where appropriate, significance levels are reported with Greenhouse-Geisser correction adjusted degrees of freedom. Interactions were verified by planned posthoc comparisons (two-tailed paired t-tests), and displayed in t-maps without additional alpha correction. List of abbreviations ANOVA: Analysis of variance EEG: Electroencephalogram MEG: Magnetoencephalogram MNE: Minimum Norm Estimate RMS: Root-mean square SSF: Steady-State- (magnetic) Field SSR: Steady-State-Response Authors' contributions MH developed the experimental design, carried out the experimental study and developed and accomplished the data analyses, BR supervised the study and composed the paper, AK advised and assisted the SS-design and SSF analysis, CW supervised the MEG measurements and advised the coherence analyses, TE provided the experimental idea, advised the experimental design, the MEG methods and analyses.

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555952

Fluvastatin in the therapy of acute coronary syndrome: Rationale and design of a multicenter, randomized, double-blind, placebo-controlled trial (The FACS Trial)[ISRCTN81331696]

Background Activation of inflammatory pathways plays an important contributory role in coronary plaque instability and subsequent rupture, which can lead to the development of acute coronary syndrome (ACS). Elevated levels of serum inflammatory markers such as C-reactive protein (CRP) represent independent risk factors for further cardiovascular events. Recent evidence indicates that in addition to lowering cholesterol levels, statins also decrease levels of inflammatory markers. Previous controlled clinical trials reporting the positive effects of statins in participants with ACS were designed for very early secondary prevention. To our knowledge, no controlled trials have evaluated the potential benefits of statin therapy, beginning immediately at the time of hospital admission. A previous pilot study performed by our group focused on early initiation of cerivastatin therapy. We demonstrated a highly significant reduction in levels of inflammatory markers (CRP and interleukin-6). Based on these preliminary findings, we are conducting a clinical trial to evaluate the efficacy of another statin, fluvastatin, as an early intervention in patients with ACS. Methods The FACS-trial (Fluvastatin in the therapy of Acute Coronary Syndrome) is a multicenter, randomized, double-blind, placebo-controlled study evaluating the effects of fluvastatin therapy initiated at the time of hospital admission. The study will enroll 1,000 participants admitted to hospital for ACS (both with and without ST elevation). The primary endpoint for the study is the influence of fluvastatin therapy on levels of inflammatory markers (CRP and interleukin-6) and on pregnancy associated plasma protein A (PAPP-A). A combined secondary endpoint is 30-day and one-year occurrence of death, nonfatal myocardial infarction, recurrent symptomatic ischemia, urgent revascularization, and cardiac arrest. Conclusion The primary objective of the FACS trial is to demonstrate that statin therapy, when started immediately after hospital admission for ACS, results in reduction of inflammation and improvement of prognosis. This study may contribute to new knowledge regarding therapeutic strategies for patients suffering from ACS and may offer additional clinical indications for the use of statins.

Background During the past decade, inflammation has often been cited as a major factor in the pathogenesis of atherosclerosis and its clinical sequelae, including ischemic heart disease. It was found that traditional risk factors such as hypertension, hypercholesterolemia, diabetes, and smoking could not fully account for the development of coronary stenosis in all patients suffering from ischemic heart disease. Intensive study of the pathogenesis of coronary plaque development and rupture led to the hypothesis that inflammatory factors contribute to this process. For example, T-lymphocytes and monocytes/macrophages have been repeatedly identified in plaque lesions; elevated levels of acute phase proteins (C-reactive protein, serum amyloid A, fibrinogen), cytokines (interleukin 1, interleukin 6, interleukin 8, tumor necrosis factor), and adhesive molecules (ICAM-1) correlate with the worse prognoses in patients with ischemic heart disease [ 1 - 5 ]. Furthermore, the increased level of C-reactive protein (CRP) is now widely recognized as being an independent risk factor for a higher incidence of non-fatal and fatal coronary events in patients with chronic ischemic heart disease and acute coronary syndromes [ 6 - 9 ]. Activation of the immune reaction in acute ischemic heart disease likely derives from: (i) pathological events occurring in the arterial wall where the lesion develops, leading to plaque rupture and the subsequent clinical consequences and (ii) myocardial necrosis, which triggers processes involved in removal of the necrotic mass and replacement with scar tissue. Whereas activation of plaque inflammation (as noted above) serves as a marker for plaque instability, elevation of inflammatory factors from the second source correlates with the extent of myocardial necrosis. Statins, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, lower cholesterol levels by decreasing the production of low-density lipoproteins (LDL) and up-regulating the expression of the LDL-receptor. These drugs are widely used in patients with hypercholesterolemia for primary and secondary prevention of coronary artery disease because of their efficacy in reducing cardiovascular morbidity and mortality [ 10 - 12 ]. Surprisingly, statin therapy also improves prognosis in patients with normal or low cholesterol levels[ 13 ]. Evaluation of the non-lipid effects of statins reveals a possible beneficial effect mediated by the reduction of inflammatory markers, namely, CRP. This effect seems to be independent of cholesterol level [ 1 , 14 ]. The mechanisms by which statins inhibit inflammation are not fully understood. It has been reported that they suppress production of monocyte chemotactic protein-1 (MCP-1) [ 15 ], as well as matrix metalloproteinases (MMPs) [ 5 , 16 , 17 ]. Statins also decrease macrophage expression of soluble ICAM-1 and secretion of IL-1, IL-6, TNF-alpha [ 5 , 18 - 23 ]. A number of large clinical trials have been designed to investigate the effect of statins in treating acute coronary syndromes (ACS) [ 24 - 30 ] Because patients in these studies were randomized later after being admitted to hospital, often after they had been clinically stabilized, these randomized, double blind trials focused more on early secondary prevention, as opposed to evaluating earlier therapy to target plaque instability in ACS. Furthermore, broad exclusion criteria in some of these trials, including coronary intervention during the index hospitalization visit, deter from the generalizability of their results to the majority of patients treated according to current clinical practice [ 24 ]. Nevertheless, these studies have shown promising results, despite the fact that the statins were administered after activation of the immune mechanisms was completed and after the inflammatory reaction was already fully developed. Little information is available on the efficacy of statins in treating ACS at an earlier phase, i.e., at the time of hospital admission. Recently published data from experimental projects [ 31 - 35 ] and from small clinical trials [ 36 - 38 ]) have shown a positive effect of statins when they are administered in the acute phase of ACS. Our preliminary results with cerivastatin treatment in patients with non-ST segment elevation ACS starting at the time of hospital admission have shown the safety of such a strategy as well as a decrease in inflammatory markers (CRP, IL-6) by 24-hour follow-up, as compared to the non-treated group [ 39 ]. Based on these pilot data, we are conducting a clinical trial to evaluate fluvastatin therapy administered to patients with ACS immediately at the time of admission (Figure 1 ). Figure 1 Design of the FACS trial in comparison to other trials evaluating statins in ACS patients. In the Pravastatin in Acute Coronary Treatment (PACT) trial statin, therapy was initiated within 24 hours of onset of ACS. In the Myocardial Ischemia Reduction With Aggressive Cholesterol Lowering (MIRACL) trial, patients were randomized 24 to 96 hours after ACS. In the Z-phase of the A-to-Z trial, simvastatin therapy was initiated within 5 days of the onset of ACS, after clinical stabilization. In the Pravastatin or Atorvastatin Evaluation and Infection Therapy (PROVE IT) trial, patients were randomized up to 10 days after ACS. In the Lipid-Coronary Artery Disease (L-CAD) trial, statin therapy was initiated up to ten days following the onset of ACS. In the Fluvastatin on Risk Diminishing After Acute Myocardial Infarction (FLORIDA) trial, patients were randomized up to two weeks after ACS. Methods Objectives The objectives of the FACS trial are to determine: (i) Whether initiation of fluvastatin therapy in patients with ACS immediately after hospital admission decreases levels of CRP, IL-6, and pregnancy-associated plasma protein A/ proform eosinophilic major basic protein (PAPP-A/proMBP), which represent indirect markers of plaque instability and indicators of poor prognosis; and (ii) Whether initiation of fluvastatin therapy decreases the occurrence of ischemic events (death, nonfatal myocardial infarction, recurrent symptomatic ischemia, urgent revascularization, cardiac arrest) in patients with ACS. Overview This is a prospective, 30-day, multicenter, randomized, double-blind, placebo-controlled study in 1,000 patients with ACS. Patients are enrolled from 10 sites in the Czech Republic and Slovakia. At each institution, the protocol and the informed consent form are reviewed and approved by the institutional ethics committee before study initiation. Eligible patients are randomized to one of two treatment groups immediately after hospital admission (within one hour). One group is assigned 80 mg/day fluvastatin (Lescol XL), with the other group receiving placebo. Participants are followed on an intention-to-treat basis. The primary endpoint relates to levels of CRP, IL-6, and PAPP-A/proMBP. The secondary endpoint is the occurrence of an ischemic event, defined as death, nonfatal myocardial infarction (MI), recurrent symptomatic myocardial ischemia, cardiac arrest with resuscitation, and urgent revascularization. Study population This study will enroll high-risk patients admitted to the hospital for ACS. Eligible patients with ST elevation ACS must have resting chest pain less than 12 hours before admission and either ≥ 1 mm ST-segment elevation in 2 or more continuous leads or new left bundle branch block on ECG. Those with non-ST elevation ACS must have resting chest pain during the previous 48 hours and either ≥ 1 mm ST segment depression or negative T waves in 2 or more continuous leads. Exclusion criteria Subjects are excluded from study participation if they are <18 years of age or if they have concomitant active liver disease or persistent elevation of transaminases (> 3 times the upper limit of normal), a history of lipid-lowering therapy less than 30 days before the index event or a known allergy to fluvastatin or to any additives present in the drug. Other exclusions include inability to ingest oral medication, unwillingness to be followed for the duration of the study, muscle disease (e.g., myositis), and creatine kinase ≥ 5 times the upper limit of normal due to conditions other than myocardial infarction. Women of childbearing potential who are pregnant, nursing or who are not using effective contraception will also be excluded. Follow-up After obtaining informed consent, blood samples are taken from patients for examination of inflammatory markers (CRP, IL-6, and PAPP-A/proMBP). Patients are then randomized to 80 mg fluvastatin (Lescol XL) or to placebo immediately p.o. Medical history and physical examination, standard 12-lead ECG, blood lipid profile, and liver function tests are performed as part of participants' routine admission care. Fluvastatin 80 mg or placebo are then taken once daily for 30 days. Follow-up measurement of inflammatory markers (CRP, IL-6, and PAPP-A/proMBP) is performed on day 2 and day 30. Follow-up visits are scheduled at pre-discharge, day 30, 90, 180 and 360. Blood liver function and creatine kinase tests are done at pre-discharge and at the 30-day visits. At day 30, the lipid profile is also examined, and study medication is withdrawn. All visits include assessment of ischemic events and recent medical history since the last follow-up visit, including use of concomitant medications (Figure 2 ). Figure 2 Study design of the FACS Trial. Patients admitted with acute coronary syndrome (ACS) are randomized to either fluvastatin 80 mg or placebo for 30 days. Patients are then followed for one year. Assessments of CRP, IL-6, and PAPP-A/proMBP (LAB) are performed at admission, on day 2, and day 30. During follow-up, no specific recommendations are made with respect to diagnostic and therapeutic strategy, except that other lipid-lowering drugs should not be given after randomization until day 30. All management decisions are left to the discretion of each patient's treating physician. Safety The principal safety concerns are hepatic dysfunction and myopathy. If a patient's serum transaminase levels are persistently elevated to > 3 times the upper limit of normal, the study medication is discontinued. Similarly, study medication is stopped if the patient develops muscle pain, weakness, or tenderness in association with a serum creatine kinase level > 10 times the upper limit of normal. Sample size The trial will enroll 1,000 patients, to ensure adequate power to detect significant treatment benefit of 80 mg fluvastatin (Lescol XL) with respect to the primary endpoint (30-day decrease of CRP and IL-6) and the combined secondary endpoint (death, nonfatal myocardial infarction, recurrent symptomatic ischemia, urgent revascularization, cardiac arrest). With 500 patients randomized to 80 mg fluvastatin (Lescol XL) and 500 patients randomized to placebo, the trial will have more than 80 % power to detect a decrease in CRP level by 1.36 μg/L and a decrease in IL-6 level by 1.09 ng/L. Calculations are based on a two-sample t -test. The estimated combined secondary endpoint rate at 30-days is 20 %. Based on comparison of proportions with p = 0.05 test significance, the trial will have more than 80 % power to detect a 33% decrease in the combined secondary endpoint. Conclusion The FACS trial is the first multicenter, randomized, double-blind, placebo-controlled trial investigating the effects of fluvastatin therapy started immediately after hospital admission in patients with ACS. Competing interests The author(s) declare that they have no competing interests. Authors' contributions PO has made substantial contributions to the concept and design of the trial and has drafted the manuscript. DA, PH, JVej, JK, MMat, and JVes have made substantial contributions to the concept and design of the study and have been involved in revising the manuscript. PB, MW, OA, JS, EN, FH, MR, and JC have been involved in the acquisition and analysis of data. MMac and MK have given final approval of the version to be published.

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543473

Good vs complementary genes for parasite resistance and the evolution of mate choice

Background Female mate choice may be adaptive when males exhibit heritable genetic variation at loci encoding resistance to infectious disease. The Hamilton-Zuk hypothesis predicts that females should assess the genetic quality of males by monitoring traits that indicate health and vigor (condition-dependent choice, or CD). Alternatively, some females may employ a more direct method of screening and select mates based on the dissimilarity of alleles at the major histocompatibility loci (we refer to this as opposites-attract, or OA). Empirical studies suggest that both forms of mate choice exist, but little is known about the potential for natural selection to shape the two strategies in nature. Results We used computer simulation models to examine the evolutionary fates of the two forms of mate choice in populations at risk for infection by debilitating parasites. We found that populations exhibiting random mating (no mate choice) can be invaded and replaced completely by individuals practicing CD type mate choice. We also found that an allele encoding OA choice can increase when rare in randomly mating populations, but that it does not go to fixation under selection. A similar result was obtained when the OA strategy was introduced into populations practicing CD mate choice. As before, we found that the OA choice allele will increase when rare, and that it will not go to fixation under selection. The converse however was not true, as CD individuals gain no rare advantage when introduced into an OA population. Conclusions Taken together, the results suggest that, when rare, OA is the best strategy for parasite evasion (of those considered here). The consequence of OA increasing in the population, however, is to reduce the parasite driven genotype oscillations and facilitate the breakdown of linkage disequilibrium at the disease-resistance loci. This leads to a neutrally stable situation in which different strategies have equal fitness, and suggests that multiple forms of mate choice may be expected to occur in populations at risk from infectious disease.

Background Since Darwin published his classic work on sexual selection [ 1 ], biologists have puzzled over the evolutionary consequences of mate choice. As a general rule, evolutionary theory predicts that females rather than males will benefit most from discriminating among potential mates [ 2 ]. A large body of evidence now supports the hypothesis that female mate choice is a potent evolutionary force, but its causes and consequences can vary widely. For example, choosy females may benefit directly through the acquisition of valuable resources [ 3 ], may gain indirect benefits by obtaining better genes for their offspring [ 4 ], or may simply prefer mates due to some preexisting sensory bias [ 5 , 6 ]. In the early 1980's, Hamilton and Zuk hypothesized that females discriminate in favor of males bearing genotypes encoding resistance to coevolved parasites [ 7 ]. A key prediction of this model is that females should select males based on indicator traits that reveal the bearer's status of infection. Investigations of mate choice in birds and fish support the prediction that females may indirectly discriminate against parasitized males by selecting brightly colored males [ 8 - 12 ], but other studies suggest that females may use cues other than condition to identify males with genes that could contribute to disease resistance. For example, results from recent experiments suggest that females of some species select males based on their genetic configuration at the major histocompatibility complex (MHC) loci. The role of the MHC in the vertebrate immune response is well established, and is known to enhance resistance to many species of endoparasites [ 13 , 14 ]. Hence, it would seem that females could benefit from mating preferentially with males having favorable MHC profiles, provided they can identify these males in the population. This seems plausible, as recent studies have documented its occurrence in mice and rats [ 15 - 17 ], humans [ 18 , 19 ], and fish [ 20 - 23 ]. In a previous study, we investigated the potential for active mate choice to indirectly favor the maintenance of sexual reproduction [ 24 ]. We found that sexual populations in which females prefer to mate with males having dissimilar resistance alleles (which we called OA for opposites attract) are more resistant to invasion by parthenogenetic clones than those in which females prefer uninfected males (a condition dependent strategy, CD). In the present study, we investigate the potential for OA and CD mate choice to generate a selective advantage to females at the level of the individual, independent of the incidental effects that such choice may have on the evolutionary stability of sex. Results The results from our study show that when populations are confronted with infectious disease, alleles encoding OA and CD mate choice both increase when rare in randomly mating populations (Figs. 1A and 2A ). Hence the results are consistent with the basic idea that mate choice will be spread in populations engaged in coevolutionary interactions with parasites. However, the two different kinds of mate choice had different effects on the population genetic dynamics for multilocus genotypes associated with disease resistance. Figure 1 Results from a single run of a two-locus, two-allele version of the simulation in which a rare mutant for the opposites attract (OA) strategy was introduced into a randomly mating (RM) population of 20,000 hosts. At generation 200, the mutation rate between OA and RM was set at 0.000001 per individual per generation. (a) Once established, the OA allele spread until its frequency was about 0.5 in the population. (b) The 1,1 host genotype frequency at the two interaction loci before and after the invasion of the OA choice allele. Note the drastic reduction in the amplitude of the oscillations which accompany the spread of the OA allele. Figure 2 Results from a single run of a two-locus, two-allele version of the simulation in which a rare mutant for the condition dependent (CD) strategy was introduced into a randomly mating population of 20,000 individuals. At generation 200, the mutation rate between CD and RM was set at 0.000001 per individual per generation. (a) Once established, the CD allele spread until fixation. (b) The 1,1 host genotype frequency at the two interaction loci before and after the invasion of the OA choice allele. Note the increase in amplitude of the oscillations which accompany the spread of the CD allele. OA-choice vs. random mating As the allele for OA increased in the population, the oscillations in both host and parasite resistance genotypes underwent a dramatic reduction in amplitude (Fig 1B ). In addition, as the oscillations became damped, the spread of the OA allele into the population was slowed. This result makes sense, as rare resistance genotypes have an advantage when OA enters the population, whether or not they are over or under infected (Note that over infection of rare types occurs during part of the coevolutionary cycle). However, the advantage under sexual selection is not lagged in time (as is the advantage due to parasite resistance), and the amplitude of the oscillations becomes damped as the OA phenotype increases in the population. As the oscillations become damped and the genotypes become equally frequent (barring some drift), the value of mate choice is diminished, and the spread of the OA allele is halted. At this point, OA and RM are selectively neutral, and their frequencies in the population change only as the result of drift. Selection for OA, however, is reestablished if the OA allele drifts below 0.5, as this reestablishes conditions favoring the production of rare resistance genotypes. CD-choice vs. random mating In contrast, as the allele for CD choice increased from its rare starting point, the oscillations in genotype frequency increased, rather than decreased (Fig. 2B ). This result also makes sense, as genotypes that are least infected are also more likely to be selected as mates, and both advantages are lagged in time. This increases the selection for genotypes that are currently underinfected, and increasing the selection differential increases the amplitude of the oscillations. It also greatly increases the oscillations in linkage disequilibrium over time [ 24 ]. In addition, unlike the OA allele, the CD allele goes to fixation in an RM population. Thus it would appear that in the presence of parasites, there is sustained selection against random mating, even when CD-choice is common. OA-choice vs. CD-choice Under the conditions studied here, both OA and CD alleles increase when rare in randomly mating populations, but only the CD allele goes to fixation due to sexual selection. How does CD fare against an OA population? We found that when introduced into a population of 20,000 OA individuals, a rare allele encoding CD choice was not favored by selection, but that it could become established and increase in frequency as a result of genetic drift (Fig. 3 ). This result appears to stem from the fact that in a population dominated by OA individuals, the disease-resistance genotypes are virtually equally frequent and equally infected; hence there is little selective value in choosing the least infected individuals as mates. Thus OA seems to be an evolutionary stable strategy (ESS). In contrast, OA did increase when rare due to selection in a population of CD individuals; hence CD is not an ESS (Fig. 4 ). The overall pattern was similar to that obtained from our previous runs of OA against a RM population. As before, the oscillations in genotype frequencies became damped as OA increased in the population, and the OA allele lost its selective advantage and began to drift. The result is apparently for the same reasons as given above: as OA spreads the oscillations become damped and the resistance genotypes become equally frequent and equally infected. At this point CD and OA are selectively neutral. Interestingly, the extent to which OA spread under selection appeared to increase in the three-locus, five-allele version of the simulation (Fig. 5 ). Figure 5 Results from a three-locus, five-allele run of the simulation in which a rare mutant for OA mate choice was introduced into a CD population of 20,000 individuals. At generation 200, the mutation rate between CD and OA was set at 0.000001 per individual per generation. (a) . Once established, the OA allele spread rapidly until it reached a frequency of about 0.8 in the population. (b) Frequency of the 1,1,1 host genotype. Note that in this version of the simulation there are a possible one hundred and twenty five host and parasite interaction genotypes. Conclusions Among the three strategies of mate choice for parasite avoidance considered in this study, only OA is evolutionary stable. However, OA will not eliminate either RM or CD-choice through the action of sexual selection, since OA becomes selectively neutral at intermediate frequencies in both situations by causing reductions in the fitness variance among resistance genotypes. Thus, OA and RM, OA and CD, or all three could coexist in the short term in large populations (until OA goes to fixation by drift). As such, in populations at risk from disease, females might practice more than a single type of mate choice. Methods The methods employed in this investigation are similar to those used in our previous studies of host parasite coevolution [ 24 , 25 ]. We used individual-based computer simulation models to track the dynamics of alleles encoding different strategies of mate choice in host populations at risk of infection by debilitating parasites. Both hosts and parasites were modeled as sexual hermaphrodites with discrete, non-overlapping generations; parasites underwent two generations for each host generation. The antagonistic interaction was mediated by a matching alleles model of infection in which successful parasites were required to match hosts exactly at each of two or three freely-recombining loci (interaction loci). In the simplest form of the model, the interaction was mediated by two alleles at each of two loci, which allowed for a total of four possible resistance genotypes. We also conducted runs for a three-locus version of the model, with five resistance alleles at each locus. This configuration allowed for the production of one hundred and twenty five genotypes. The mutation rate between the alternative alleles at each of the interaction loci was set at 0.03 per generation in the parasite population. This was done to prevent the fixation of parasite alleles under the conditions studied, e.g. where high risks of parasite exposure are coupled with moderate to high levels of virulence. During each parasite generation, hosts were drawn sequentially and exposed to a randomly drawn parasite with a probability of T . For the two locus version of the model, T was set to a value of 0.8, and parasites underwent two generations for each host generation. In the three-locus, five-allele version of the model, higher rates of parasite transmission coupled with a greater asymmetry of host and parasite generation times were required to sustain infection in the host population. For this version of the model, T was set to a Poisson distributed mean of eight, and parasites underwent ten generations for each host generation. In all cases, if a parasite matched a host exactly at all interaction loci, the host was marked as infected and the parasite was placed in a pool of potential reproductives. Once infected, individual hosts were protected against further attack by parasites. Reproduction in hosts and parasites was accomplished by drawing individuals from their respective populations at random with replacement. The parasite life cycle included a "free-living" stage in which reproductive adults emerged from infected hosts to mate. When an individual (host or parasite) was selected for reproduction, a second individual was randomly selected for cross-fertilization. Gametes from the two haploid "parents" were then brought together to form a diploid zygote stage, where free recombination between the interaction loci made possible the production of a genetically diverse brood of haploid offspring. All else equal, sexual individuals (hosts and parasites) produced a lifetime average of 10 haploid offspring. The number of offspring produced by hosts, however, was reduced according to the status of parasitic infection for each of the parents. The reproductive output of each host parent was discounted according to 10(1- E ), where E simulates the detrimental effect of parasitism on host reproduction (virulence). In the present study, E was set to a value of 0.8 to simulate the effect of a moderately virulent parasite. Following reproduction, a maximum of 20,000 parasite and 20,000 host offspring were selected at random to become the next generation of adults. At the beginning of each run, the interaction alleles at each locus in the host and parasite populations were initialized to a frequency of 1/ n , where n was the total number of alleles present at each locus. Prior to data collection, the simulation was allowed to run for 200 host generations; this allowed for establishment of long-term coevolutionary dynamics arising from the host-parasite interaction. Next, we tracked the fates of alleles encoding different types of mate choice in the host population. Mating preferences in hosts were controlled by a single locus encoding one of three strategies: random mating (RM), condition-dependent mate choice (CD), or opposites attract (OA) mate choice. Random mating was simulated by pairing two randomly drawn adults. Condition-dependent choice was implemented by allowing the first reproductive drawn (the "female") to identify and discriminate against parasitized "males". For the opposites attract strategy, females preferred "males" with alleles different from their own at each of the two interaction loci. For both OA and CD choice, "females" were allowed to choose the "best" available male from a sequence of 20 randomly drawn "males". Authors' contributions RSH wrote the computer code and implemented the simulations. Both authors participated equally in all other phases of the work. Figure 3 Results from a two-locus, two-allele version of the simulation in which a rare mutant for CD mate choice was introduced into an OA population of 20,000 individuals. At generation 200, the mutation rate between CD and OA was set at 0.000001 per individual per generation. (a) . The CD allele failed to become established over the course of 4,000 generations (b) Frequency of the 1,1 host genotype. Figure 4 Results from a two-locus, two-allele run of the simulation in which a rare mutant for OA mate choice was introduced into a CD population of 20,000 individuals. At host generation 200, the mutation rate between CD and OA was set at 0.000001 per individual per generation. (a) . Once established, the OA allele spread rapidly until it reached a frequency of about 0.5 in the population. (b) Frequency of the 1,1 host genotype. Note the rapid attenuation of cycling which accompanied the spread of the OA allele into the population.

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555577

Computational model of blood flow in the aorto-coronary bypass graft

Background Coronary artery bypass grafting surgery is an effective treatment modality for patients with severe coronary artery disease. The conduits used during the surgery include both the arterial and venous conduits. Long- term graft patency rate for the internal mammary arterial graft is superior, but the same is not true for the saphenous vein grafts. At 10 years, more than 50% of the vein grafts would have occluded and many of them are diseased. Why do the saphenous vein grafts fail the test of time? Many causes have been proposed for saphenous graft failure. Some are non-modifiable and the rest are modifiable. Non-modifiable causes include different histological structure of the vein compared to artery, size disparity between coronary artery and saphenous vein. However, researches are more interested in the modifiable causes, such as graft flow dynamics and wall shear stress distribution at the anastomotic sites. Formation of intimal hyperplasia at the anastomotic junction has been implicated as the root cause of long- term graft failure. Many researchers have analyzed the complex flow patterns in the distal sapheno-coronary anastomotic region, using various simulated model in an attempt to explain the site of preferential intimal hyperplasia based on the flow disturbances and differential wall stress distribution. In this paper, the geometrical bypass models (aorto-left coronary bypass graft model and aorto-right coronary bypass graft model) are based on real-life situations. In our models, the dimensions of the aorta, saphenous vein and the coronary artery simulate the actual dimensions at surgery. Both the proximal and distal anastomoses are considered at the same time, and we also take into the consideration the cross-sectional shape change of the venous conduit from circular to elliptical. Contrary to previous works, we have carried out computational fluid dynamics (CFD) study in the entire aorta-graft-perfused artery domain. The results reported here focus on (i) the complex flow patterns both at the proximal and distal anastomotic sites, and (ii) the wall shear stress distribution, which is an important factor that contributes to graft patency. Methods The three-dimensional coronary bypass models of the aorto-right coronary bypass and the aorto-left coronary bypass systems are constructed using computational fluid-dynamics software (Fluent 6.0.1). To have a better understanding of the flow dynamics at specific time instants of the cardiac cycle, quasi-steady flow simulations are performed, using a finite-volume approach. The data input to the models are the physiological measurements of flow-rates at (i) the aortic entrance, (ii) the ascending aorta, (iii) the left coronary artery, and (iv) the right coronary artery. Results The flow field and the wall shear stress are calculated throughout the cycle, but reported in this paper at two different instants of the cardiac cycle, one at the onset of ejection and the other during mid-diastole for both the right and left aorto-coronary bypass graft models. Plots of velocity-vector and the wall shear stress distributions are displayed in the aorto-graft-coronary arterial flow-field domain. We have shown (i) how the blocked coronary artery is being perfused in systole and diastole, (ii) the flow patterns at the two anastomotic junctions, proximal and distal anastomotic sites, and (iii) the shear stress distributions and their associations with arterial disease. Conclusion The computed results have revealed that (i) maximum perfusion of the occluded artery occurs during mid-diastole, and (ii) the maximum wall shear-stress variation is observed around the distal anastomotic region. These results can enable the clinicians to have a better understanding of vein graft disease, and hopefully we can offer a solution to alleviate or delay the occurrence of vein graft disease.

Background The complex anatomy of coronary vessels has made the investigation of coronary flow and hemodynamics one of the most difficult and challenging studies until today. Leonardo Da Vinci (generally called as "man ahead of time"), developed insightful sketches (some 550 years ago) of the anatomy of the heart and its blood vessels, as shown in Figure 1 . Figure 1 Leonardo da Vinci's sketches showing the distribution of the heart and blood vessels. The major cause of death in both the developed and developing countries is cardiovascular disease. The study of coronary arterial circulation is important, because it is crucial in maintaining heart perfusion and function. The process of arteriosclerosis involves the formation of atherosclerotic plaques in the coronary tree. With time, this results in stenosis of the blood vessel and in turn decreases coronary flow. When it cannot match the oxygen demand of the myocardium, it results in ischemia and thereafter in an infarct. In order to overcome this problem of diminished perfusion of the affected myocardium, CABG is one of the treatment options that are being used to improve coronary perfusion. In this procedure, new routes around narrowed and blocked arteries are constructed with both arterial and venous conduits, allowing sufficient blood flow to be delivered to the ischemic heart muscles. In spite of CABG being an effective surgical technique to revascularize the myocardium, 20–50% of bypass grafts fail due to the formation of intimal hyperplasia [ 1 - 3 ]. Previous studies have reported anastomotic intimal hyperplasia thickening to be associated with several factors, such as hemodynamic factors [ 4 ], compliance mismatch between graft material and host artery [ 5 ], biomaterials incompatibility [ 6 ]. For considering the influence of hemodynamic factors on graft patency, it is important to replicate the in-vivo geometry of the entire bypass conduit. In other words an arterial bypass flow model must be anatomically accurate, so as to capture the essential hemodynamics of the true geometry [ 7 ]. Relation between wall shear stress and intimal hyperplasia Flow regimes in end-to-side anastomosis, provide pertinent information concerning wall shear stress gradient which affects intimal hyperplasia thickening. The three- dimensional experimental studies of flow in an end-to-side anastomosis by Ojha et al [ 8 ] have revealed a relation between low and fluctuating wall shear stress and intimal hyperplasia. For the distal end-to-side anastomosis model, Hughes and How [ 9 ] have shown that intimal hyperplasia occurs in regions of flow separation at the toe and the heel, and that flow-stagnation is observed on the floor of the anastomosis. Additionally, animal model studies, conducted by Bassiouny et al [ 10 ], provide correlation of regions of low wall shear stress with areas of intimal hyperplasia. Using CFD for wall shear stress determination Although wall shear stress distribution is a major factor in the onset of coronary diseases it cannot be measured directly and is hence calculated from velocity profiles. Even though these velocity profiles can be obtained in vivo using magnetic resonance (MR) or color-flow Doppler ultrasound (CDU), there are some limitations to measurements due to the small dimensions of the arteries. Thus the alternative to measure the wall shear stress is to use CFD to simulate flow in CABG geometry and thereafter compute the wall shear stress. Anastomosis geometry Unfortunately, most of the modeling work in graft-flow is limited to only a part of the total bypass conduit geometry, namely the anastomosis site. As regards to the anastomosis geometry, Song MH et al [ 11 ] developed a Y-Figure anastomotic model for proximal arterial stenosis (at angles ranging from 10° to 30°), in order to analyse the three-dimensional simulation of coronary artery bypass grafting. In this work, in the end-to-side anastomosis model, all the vessels were adopted to be of the same diameter. The simulation results suggest that the more acute the angle of anastomosis, the smaller is the energy loss. Even three-dimensional CFD simulations have been performed on end-to-side anstomosis of a stenosed coronary bypass, by Bertolotti and Deplano [ 12 ]. Herein, the anastomosis geometry was based on the assumption that the graft and the host vessel are of the same diameter, with the graft inclined at 45° to the host vessel. The inputs into the anastomotic domain were flow-rates from the graft and from the occluded proximal artery. The flow features were compared for different flow rates and distance of the anastomosis from the site of occlusion. It was concluded that the risk of intimal hyperplasia could be minimized if the anastomosis was sutured at a sufficient distance of length. Proximal artery flow conditions An important factor is the effect of proximal artery flow condition on the hemodynamics at the distal end-to-side anastomosis. This effect has been analysed by Kute and Vorp [ 13 ], using CFD through the idealized geometry model consisting of equal diameter vessels with the graft inclined at 30°. The boundary conditions for the anastomotic model involved blunt velocity profiles into the graft and from the proximal artery, with a fixed pressure at the distal artery outlet. The velocity vectors for all the proximal arterial flow conditions exhibited skewing towards the floor of the artery, and their distribution varied with the flow conditions in the proximal artery. Graft flow determination The combined use of imaging techniques and CFD may be necessary for meaningful clinical studies. With the availability of imaging techniques like coronary angiography, magnetic resonance angiogram (MRA), computed tomography (CT), several researchers have analysed the flow in realistic bypass graft models. There have also been some studies combining experimental measurements and CFD analyses [ 14 - 17 ]. Doppler analysis is generally preferred since it is safe, accurate and rapid when compared to angiography. Even though angiography certainly gives a definite answer about graft patency, there is more risk involved and it is time consuming and requires the assistance of a cardiologist. Doppler methods have been used by Bertolotti et al [ 18 ] and Lin et al [ 19 ] for graft flow analysis during minimally invasive CABG. The functioning of the graft has also been analysed through methods such as intraoperative angiography, probing of the anastomosis, electromagnetic flow meter, and the transit time flow measurement [ 20 - 22 ]. Limitations of works carried out It is clear from literature review that an important step towards realistic flow simulations concerns the generation of appropriate coronary vasculature geometry (involving the entire flow domain from the aorta to the perfused artery) with physiological boundary conditions. Unfortunately, a lot of work in these areas has been carried out on subsections of this bypass flow domain [ 8 , 9 , 11 - 13 , 15 , 16 , 18 ], and that too involving idealized geometry of the anastomosis section. Therefore in our work, we have addressed this drawback by studying the flow characteristics (by means of a three-dimensional CABG model) in the complete bypass domain under representative physiological conditions. We have thereby analysed realistic physiological simulations, and highlighted the resulting flow patterns and wall shear stress that are deemed to play a major role in intimal hyperplasia. Methods Geometrical Model The bypass models simulating the flow field of the anastomosis in the right and left aorto-saphenous bypass grafts are illustrated in Figures 2a and 2b respectively. The ascending aorta is modeled to be of length 80 mm with diameter 25 mm. The normal coronary artery is modeled to be of circular cross-section. The 100% occluded coronary artery is modeled as a straight tube, with dimensions of length 45 mm and diameter 2 mm. Figure 2 Geometry (plane view) and dimensions (in mm) of the bypass models of (a) The aorto-right coronary artery bypass model. (b) The aorto-left coronary artery bypass model. (PSCA-Perfused Segment of the Coronary Artery; OSCA-Occuluded Segment of the Coronary Artery; T-Toe; H-Heel) In Figures 2a and 2b , the venous graft is shown to have a non-uniform circular cross-section, larger than that of the coronary artery. The intersection between the graft and the coronary vessel has an elliptical shape, which is due to the deformation of the larger diameter graft due to its suturing to the smaller diameter coronary vessel surface. The dimensions are provided by our cardiac surgeon joint-author (TYS) and the model has been constructed with this data. Model Assumptions, Data Input, and Boundary Conditions The blood is assumed to be incompressible, with a Newtonian behaviour having dynamic viscosity ( μ ) of 0.00408 Pa and a density ( ρ ) of 1050 kg/m 3 . The blood vessel walls are assumed to be rigid and impermeable. For a quasi-steady, three-dimensional and laminar flow, the Navier-Stokes equations (for mass and momentum conservation) governing fluid motion is written as follows: where denotes the velocity vector in three dimensions. The distributions of velocity and wall shear stress are obtained by computationally solving the above equations subject to the boundary conditions given below. In accordance with equation (2), the flow field is automatically updated during each time-interval, by adopting the time-varying input data of the aorta (Figures 3a & 3b ), the left coronary artery (Figure 3c ) and the right coronary artery (Figure 3d ). Figure 3 (a) Flow-rate waveform at the inlet of aorta. (b) Flow-rate waveform of the ascending aorta, reverse flow seen due to 4% of the stroke volume that goes into the ascending aorta. (c) Flow-rate waveform at the left coronary artery, derived from the flow velocity waveform obtained using intravascular Doppler ultrasonic flow technique. (d) Flow-rate waveform imposed at the right coronary artery, measured using a Doppler flow meter catheter. Systolic phase This flow is obtained by prescribing a uniform velocity (across the cross-section) at the inlet to the aorta, from the left ventricle (LV). The velocity magnitude is computed from the physiologically representative stroke volume over the ejection period, based on the flow wave form [ 23 ] shown in Figure 3a . Through out this period, the left ventricle contracts and squeezing the coronaries, which are embedded in the cardiac muscle. This allows very little amount of blood to flow into the coronary arteries. During systole, the inputs to the model consist of (i) monitored time-varying flow-rate waveform at the inlet of the aorta, (depicted in Figure 3a ), adopted from Ganong [ 23 ], as well as (ii) monitored time-varying flow-rate waveform at the left coronary artery(depicted in Figure 3c ) obtained from in-vivo intravascular Doppler ultrasonic technique [ 24 ], and (iii) monitored time-varying flow-rate waveform imposed at the exit of the right coronary artery (depicted in Figure 3d ) obtained by means of a Doppler flow-meter catheter [ 25 ]. Diastolic phase At the start of diastole, there exists a small amount of back-flow into the left ventricle through the aortic inlet (Figure 3a ). For the rest of the diastolic phase, the aortic valve remains closed. In all these cases, blood flows back from the ascending aorta into the coronary arteries. Herein, we take into account that a major portion (second/third) of the backflow into the ascending aorta, (flow waveform displayed in Figure 3b ) goes into the left coronary artery (Figure 3c ), allowing the remainder to flow into the right coronary vessel (Figure 3d ). This yields the blood volume flowing into the left-coronary vessel (during a cardiac cycle) to be 2.67% of the stroke volume, with the remaining 1.33% of the stroke volume going into the right coronary artery. From these specified flow-rates at the left coronary artery, and at the right coronary artery, the backflow from the ascending aorta into the aortic root domain is calculated. Hence the data input to the model consists of (i) the calculated uniform velocity at the ascending aorta, and (ii) the flow conditions at the left and right coronaries obtained from the monitored time-varying input flow-rate waveforms (Figures 3c & 3d ). Fluid Dynamics Simulation Setup The fluid dynamics simulations are performed by using a control-volume-based technique, implemented in the computational fluid dynamics (CFD) code Fluent [ 26 ]. The computation procedure of the commercial code consists of (i) construction of the geometry using a pre-processor, Gambit [ 27 ], (ii) meshing the computation domain, (iii) assigning boundary conditions in terms of velocities and flow-rate weightings, (iv) assigning fluid properties, and (v) the solution algorithm. The geometry of the aorto-coronary bypass graft models is constructed in Gambit, using the dimensions provided by our cardiac-surgeon joint-author (TYS). The elements employed to mesh the computational domain consisted primarily of regular structured hexahedral elements as well as wedge elements wherever necessary. In order to carry out the mesh sensitivity analysis, numerical simulations were carried out by varying the number of mesh elements in the computational domain. Initially, the domain was discretized into 120974 hex/wedge elements. The accuracy of the simulation results was then improved by employing a finer mesh that contained 419765 elements. This number was further increased to 623138, but resulted in no significant improvement in the results. Thus to maintain a balance between the computational cost and the numerical accuracy, we concluded (based on the mesh independence test) that the appropriate number of elements for our study is 419765. In the solution algorithm used by Fluent , the governing equations are solved sequentially. Because the governing equations are non-linear (and coupled), several iterations of the solution loop need to be performed before a converging solution is obtained. Using this approach, the resultant algebraic equations for the dependent variables in each control volume are solved sequentially by a point implicit (Gauss Seidel) linear equation solver, in conjunction with an algebraic multi-grid (AMG) method. The governing equations are solved iteratively until convergence of all flow variables is achieved. The solutions of all the flow variables are deemed to have converged once their residuals computed from two successive iterations are below the set desired convergence criteria of 10 -3 . The study was also carried out by setting the convergence criteria as 10 -4 ; this did not alter the results obtained earlier. Results For these numerical simulations we have incorporated the unsteady flow character by dividing the cardiac cycle into a number of small time interval, and analyzing for steady flow within these time intervals. To observe the velocity distribution features of the entire flow field, the computed velocity vectors are illustrated in the plane of symmetry at two different instants of the cardiac cycle. Simulated flow field for the aorto-right coronary bypass (Figures 4 and 5 , for t = 0.13 sec and t = 0.7 sec respectively) Figure 4 (a) Velocity vectors computed at t = 0.13 sec are depicted on the centre plane of the aorto-right coronary bypass model. (b) Enlarged view of the velocity vectors inside aorta. At the start of ejection, blood from the aortic inlet flows into the ascending aorta. The blood flow at a distance of 10 mm from the entrance of the aorta behaves almost like an inviscid flow. Very little amount of flow enters the coronaries; this may be due to the high pressure in the myocardium. (c) Parabolic profiles of the velocity vectors are observed inside the graft. Slight skewing of the flow profiles is seen due to the influence of the graft curvature. (d) A close view of the recirculation region in the artery-graft junction. Major portion of the flow exiting from the graft moves towards the distal portion of the right coronary vessel. (e) The flow pattern shows slight skewing towards the floor of the artery. With increasing distance skewing disappears thus shifting the maximum velocity magnitude 0.37 m/s to the centreline of the host artery. (f) A wide variation in the wall shear stress is observed at the distal anastomotic region. The weak recirculation zone at the proximal portion of the bypassed vessel results in negligible wall shear stress with the peak wall shear stress magnitude, 5.75 Pa appearing at the floor of the artery. Figure 5 (a) Velocity vectors computed at t = 0.7 sec are depicted on the centre plane of the aorto-right coronary bypass model. (b) At the mid-diastolic instant, the aortic valve is fully closed and thus back flow from the ascending aorta enters both the coronary vessels, the left coronary vessel and the bypassed right coronary vessel. (c) Parabolic profiles of the velocity vectors are seen inside the graft. Maximum perfusion of the graft occurs at mid-diastole. (d) Maximum flow velocity approaching the graft exit is around 0.95 m/s. The flow exiting the graft with a higher velocity results in a stronger impingement on the floor of the artery. The strong recirculation region seen at the proximal portion of the distal anstomotic region forces the flow to move towards the right coronary artery exit. (e) The inner wall of the host artery exhibits significant skewing of the velocity profiles. The maximum flow velocity magnitude, 2.04 m/s, seen close to the floor of the artery is shifted to the centre line of the vessel with increasing axial distance. (f) Maximum perfusion occurs at the mid-diastolic instant, t = 0.7 sec. This gives rise to maximum wall shear stress with the peak magnitude being 72.22 Pa. (a) Flow field in the aorto-right coronary bypass model at the onset of ejection, t = 0.13 sec At the onset of ejection, at t = 0.13 sec, Figure 4a depicts the distribution of velocity-vectors in the flow field. The flow-velocity distributions in different sections of the flow field are depicted in Figures 4b–4e . The uniform velocity at the inlet to the aorta is 0.035 m/s. At a distance of 10 mm from the entrance, the computed flow field appears to be like inviscid flow. There is hardly any flow entering the graft at the start of systole as shown in Figure 4c . Figures ( 4d & 4e ) depict the flow-velocity distribution in the anastomosis domain. The flow pattern in the graft anastomed to the aorta at a distance of around 40 mm from the aorta entrance is parabolic, with very slight skewing. The forward flow coming from the graft into the host artery shows a small region of recirculation at the heel of the anatomosis domain (i.e. where the occluded (proximal) bypassed coronary artery connects to the anastomosis domain) as indicated in Figure 4d . The flow coming into the perfused right coronary vessel gets fully developed, with a peak velocity of 0.37 m/s, as depicted in Figure 4e . The computed wall shear stress (product of velocity gradient at the wall and the viscosity of fluid) is depicted in Figure 4f as (i) a low wall shear-stress region in the heel section of the anastomosis where there is flow-stagnation along the floor of the artery and (ii) a high wall shear-stress at the toe of the anastomosis (associated with the disturbed flow patterns, shown in Figure 4d ) which is prone to intimal hyperplasia. (b) Flow field in the aorto-right coronary bypass model at the mid-diastolic instant, t = 0.7 sec At the mid-diastolic instant (at t = 0.7 sec), when the aortic valve is closed, Figure 5a illustrates the velocity vector plots. Some amount of the backflow coming from the ascending aorta enters into the graft, and the rest goes into the left coronary vessel as shown in Figure 5b . Along the graft, the peak velocity is skewed towards the outer wall. Initially the flow is seen to follow the outer wall, and then this slowly turns to the centre as demonstrated in Figure 5c . In the anastomosis domain, a strong region of recirculation is observed near the occluded end of the artery, which forces the flow to move into the perfused right coronary (distal) artery as indicated in Figure 5d . The flow in the perfused distal artery exhibits significant skewing of the velocity profiles towards the inner wall of the perfused (distal) artery, with a peak magnitude of 2.04 m/s as seen in Figure 5e . The flow pattern variations give us insight into the wall-shear distribution. The high velocity gradients in the anastomosis give rise to large spatial variations in the resulting wall shear stress. The largest value of wall shear stress is seen in Figure 5f , near the toe of the graft-artery junction. Simulated flow field for the aorto-left coronary bypass (Figures 6 and 7 , for t = 0.13 sec and t = 0.7 sec respectively) Figure 6 (a) Velocity vectors computed at t = 0.13 sec are depicted on the centre plane of the aorto-left coronary bypass model. (b) The aortic valve is opened and blood flows from the left ventricle down into the aorta. The flow inside the aorta is almost like an inviscid flow. A small amount of the flow enters the right coronary vessel and the remaining moves towards the ascending aorta. An additional flow into the ascending aorta is due to that coming from the left coronary vessel. (c) There is a change in flow direction in the graft. The reverse flow from the left coronary vessel enters the graft. Parabolic profiles of the velocity vectors are seen inside the graft. (d) Very little amount of flow moves towards the proximal portion of the left coronary vessel as it is 100 % occluded. A small region of weak recirculation is seen close to the occluded site. (e) At the start of ejection, there is a reverse flow that comes from the left coronary artery. This flow arises due to the myocardium which is subjected to the intra-myocardial pressure during early systole. As blood flows through the host artery the flow becomes fully developed, this feature no longer exists once the flow reaches the junction. (f) The low velocity gradients in the distal anstomotic junction exhibit low wall shear stress distribution. The maximum wall shear stress magnitude is 1 Pa. Figure 7 (a) Velocity vectors computed at t = 0.7 sec are depicted on the centre plane of the aorto-left coronary bypass model. (b) The aortic valve remains fully closed at mid-diastole and the flow from the ascending aorta enters both the coronary vessels with a high velocity. (c) Significant skewing of the velocity profiles seen at the outer wall of the graft with a high velocity indicating that good perfusion occurs during mid-diastole. (d) A small amount of flow exiting from the graft moves towards the proximal portion of the bypassed left coronary vessel. As this region is 100% occluded the flow reverses thus giving rise to a recirculation region. (e) Maximum perfusion of the bypassed left coronary vessel occurs during mid-diastole. The peak velocity magnitude is 1.84 m/s seen close to the floor of the host artery. (f) The low velocity components in the vicinity of the occluded region show negligible wall shear stress while elevated shear stresses with a magnitude of 59.43 Pa in the junction region where most of the flow moves towards the distal portion of the left coronary artery. (c) Flow field in the aorto-left coronary bypass model at the onset of ejection, t = 0.13 sec The distribution of velocity-vectors in the flow field at the onset of ejection, t = 0.13 sec is shown in Figure 6a . The aortic valve opens, and blood flows into the aorta with an entrance uniform velocity of magnitude 0.043 m/s, with some fluid entering the right coronary vessel. The flow inside the aorta behaves like inviscid flow as can be observed in Figure 6b . During early ejection, as the left coronary vessel is embedded in the myocardium, the myocardium is subject to intramyocardial pressure which causes some reverse flow. The effect of LV contraction on the graft flow is incorporated by adopting the measured flow profile in the left coronary vessel at a point prior to its anastomosis with graft. In this way, we simulate the reversal of flow in the graft at the onset of ejection. A parabolic velocity profile of the back-flow in the graft is seen in Figure 6c . Most of the fluid flows into the graft; a small amount goes towards the heel of the anastomosis, which results in a weak recirculation region at the occluded region of the bypassed left coronary vessel, as demonstrated in Figure 6d . The back-flow in the distal portion of the left coronary vessel (coming into the anastomosis domain) gets fully developed as it approaches the anastomosis domain, with a maximum velocity of magnitude 0.074 m/s seen along the centreline of the vessel as shown in Figure 6e . The low velocity gradients in the distal anastomotic junction result in low wall shear stress, with maximum magnitude of 1 Pa, as shown in Figure 6f . (d) Flow field in the aorto-left coronary bypass model at the mid-diastolic instant, t = 0.7 sec The velocity vectors plots at mid-diastole are displayed in Figure 7a . The aortic valve remains closed, and the flow entering the coronaries is caused by the back flow from the ascending aorta as shown in Figure 7b . Most of the fluid enters the graft, exhibiting significant skewing with high velocity magnitude, resulting in maximum perfusion of the host artery as depicted in Figure 7c . In the anastomosis domain, a strong recirculation region is seen in the heel region of the anastomosis at the distal end of the bypassed vessel, Figure 7d . Maximum perfusion occurs during the mid-diastole phase. The velocity distribution in the perfused host artery segment shows significant skewing towards the floor of the artery (Figure 7e ), with a peak velocity magnitude of 1.84 m/s. In the anastomosis heel region (in the occluded arterial segment) there is negligible shear stress. However, elevated shear stress (of magnitude 59.43 Pa) is seen in the anastomotic toe region (as shown in Figure 7f ), where most of the flow moves towards the distal segment of the host coronary vessel. Discussion Model Geometry Our model differs from other models in the following ways: (i) both the proximal and distal anastomotic regions are included, while other works [ 8 , 9 , 11 - 13 , 15 , 16 , 18 ] have emphasized the distal anastomotic site alone, (ii) the dimensions taken up are close to true dimensions at surgery, unlike the idealized geometries cited in previous investigations[ 11 - 13 , 18 ] that had all vessels of equal diameters, (iii) the anastomosis geometry is also more realistic compared to the idealized junction angles adopted in previous works, and (iv) our model incorporates the varying cross-section of the graft, that alters gradually along its length from a circular to an elliptical shape in order to fit the smaller artery at the distal anastomosis (thus attempting to reproduce the same geometry as obtained with the surgical procedure). Flow patterns Our results for the aorto-right coronary bypass model and the aorto-left coronary bypass model at two different instants of the cardiac cycle, clearly reveal the following features: (i) at the onset of ejection, in the aorto-right coronary bypass model, very little flow enters the graft with a velocity magnitude around 0.0495 m/s; the maximum flow velocity inside the graft is around 0.2 m/s; (ii) at the mid-diastolic instant, the flow profile in the graft is skewed towards the outer wall, with the peak velocity increasing as it travels downstream; close to the graft exit, the maximum flow velocity attained is around 1 m/s; (iii) at the onset of ejection, in the aorto-left coronary bypass model, there is a backflow from the left coronary artery into the graft; the peak velocity of flow at the entrance to the graft is 0.0788 m/s, and very little perfusion is given to the host artery; (iv) at the mid-diastolic instant, the graft perfusion is maximum, with peak velocity magnitude of 1.1 m/s. Features Our study confirms that blood flow through the coronary artery bypass graft primarily occurs only during the diastolic phase of the cardiac cycle. This is in agreement with the physiological observation of coronary blood flow. There is however some difference between the flow patterns in the right and left coronary graft at the onset of ejection, with some backflow from the left coronary artery into the bypass graft which is not obtained in the case of the right coronary arterial bypass. The phenomenon (of reversal of flow during systole) can be explained by the predominant intra-cardiac course of the left coronary artery system. This aspect is also an original feature of our work. Lastly, our study has also shown (i) a low wall shear-stress region near the heel region of the anastomosis domain, and (ii) a high wall shear-stress in the toe region of the anastomosis domain, making it prone to intimal hyperplasia. This may have some clinical significance. We should take this into consideration in designing a coronary anastomotic device, so as to minimize biomechanical injuries to the coronary arterial wall. In doing so, we can alleviate or retard the development of intimal hyperplasia, which is the Achilles heel of the effectiveness of coronary artery surgery with saphenous vein. Conclusion The computed results have revealed that (i) maximum perfusion of the occluded artery occurs during mid-diastole, and (ii) the maximum wall shear stress variation was observed around the toe of the anastomotic region. According to our cardiac surgeon joint author (TYS), this preliminary result can enable the clinicians to have a better understanding of vein graft disease, and hopefully we can offer a solution to alleviate or delay the occurrence of vein graft disease. Authors' contributions MS carried out the computational fluid dynamic studies and drafted the manuscript. DNG and LPC guided the study, helped in interpretation of results and critically reviewed the manuscript. YST provided the surgical aspect of the study, and the dimensions for the model. All authors have read and approved the final manuscript.

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548268

Half versus full vacuum suction drainage after modified radical mastectomy for breast cancer- a prospective randomized clinical trial[ISRCTN24484328]

Background Suction drains are routinely used after modified radical mastectomy and are an important factor contributing to increased hospital stay as the patients are often discharged only after their removal. Amongst various factors that influence the amount of postoperative drainage, the negative suction pressure applied to the drain has been reported to be of great significance. While a high negative suction pressure is expected to drain the collection and reduce the dead space promptly, it may also prevent the leaking lymphatics from closing and lead to increased drainage from the wound. Against this background a prospective randomized clinical study was conducted to compare the amount and duration of drainage between a half negative suction and full vacuum suction drainage in patients following modified radical mastectomy. The associated postoperative morbidity was also compared between the two groups. Methods 85 FNAC (fine needle aspiration cytology) proven cases of locally advanced breast cancer were randomized. (Using randomly ordered sealed envelops, which were opened immediately before the closure of the wound) in to 50 patients with full vacuum suction (pressure = 700 g/m2) and 35 cases in to half vacuum suction drainage (pressure = 350 g/m2) groups. The two groups were comparable in respect of age, weight, and technique of operation and extent of axillary dissection. Surgery was performed by the same surgical team comprising of five surgeons (two senior and three resident surgeons) using a standardized technique with electrocautery. External compression dressing was provided over the axilla for first 48 hrs and following that patients were encouraged to do active and passive shoulder exercises. The outcomes measured were postoperative morbidity and the length of hospital stay. Statistical methods used: Descriptive studies were performed with SPSS version 10 and group characteristics were compared using student t-test. Results Half vacuum suction drains were removed earlier than the full suction vacuum suction drains. There was no significant difference in the incidence of seroma formation in the two groups and there was a significant reduction in the total hospital stay in patients with half vacuum suction drainage systems as compared to the full suction drainage group (p < 0.001) without any added morbidity. Conclusions Half negative suction drains provide an effective compromise between no suction and full or high suction drainage after modified radical mastectomy by reducing the hospital stay and the post operative morbidity including post operative seromas.

Background Suction drainage in the management of mastectomy patients was used for the first time in 1947 [ 1 ] and has been found in various studies superior to other methods of fluid evacuation to minimize the dead space. The mechanism proposed is that the suction helps skin flaps to adhere to the chest wall and axilla sealing off all the leaking lymphatics[ 2 , 3 ]. This reduces the incidence of post-operative seromas, hematoma formation and flap necrosis, which are, recognized complications of modified radical mastectomy [ 2 , 3 ]. When no postoperative suction drains were used the incidence of seromas was found to be unacceptably high in various studies [ 4 ]. Prolonged drainage on the other hand, may increase the hospital stay and increase the risk of infection by allowing retrograde migration of bacteria [ 4 ]. Indiscriminate or premature withdrawl of postoperative drains irrespective of the amount of fluid drained may be accompanied by an increase in the incidence of axillary seromas [ 4 - 6 ]. If kept for longer periods it has been observed that drain itself might contribute to increased drainage and the risk of infection in addition to the increased hospital stay resulting in to wasteful utilization of the hospital resources. The amount of postoperative drainage is influenced by various factors like the clinical profile of the patient including the body mass index, extent of axillary lymph node dissection, number of lymph nodes dissected, use of elctrocautery, co morbid conditions and also the negative pressure on the suction drain [ 4 - 10 ]. The amount of postoperative fluid drained has been found to be significantly influenced by the negative pressure on the suction drainage. While the negative suction drain is logically expected to drain the fluid, a high negative suction drain may prevent the leaking lymphatics from sealing off thus leading to prolonged drainage leading to increased hospital stay [ 4 ]. The present prospective randomized clinical trial compared the postoperative wound drainage in patients with full suction drain (high) and those with half vacuum drainage system (low). The study also compared the drain volume, average hospital stay and postoperative morbidity between full vacuum and half vacuum suction groups. Methods The study was conducted in one surgical unit of a tertiary care center over a period of two years.85, FNAC (fine needle aspiration cytology) proven cases of locally advanced breast cancer were randomized (using randomly ordered sealed envelops, which were opened immediately before the closure of the wound) into full vacuum suction (pressure = 700 g/m2) group – (A) and 35 cases into half vacuum suction (pressure = 350 g/m2) group – (B). The two groups were comparable in respect of age, weight and type of operation i.e. modified radical mastectomy (MRM). Following complete routine and metastatic work up, all patients received three cycles of Neoadjuvant chemotherapy (NACT) using CAF regime (Cyclophosphamide, Adriamycin, 5-Fluorouracil) and underwent Patey's modified radical mastectomy after three weeks of the last cycle. Surgery was performed by the same surgical team comprising of five surgeons (two senior and three resident surgeons) using a standardized technique with electro cautery. Axillary dissection was done up to level- III in all the cases. The boundaries of axillary dissection were defined by superior limit as the posterolateral border of the Pectoralis major muscle and axillary vein, medial limit being clavipectoral fascia or Hallstead's ligament, lateral limit as the anterior border of lattismus dorsi and the inferior limit being the angular vein joining the thoracodorsal vein. The long thoracic and thoracodorsal nerves were identified, dissected and preserved. Two silicone tube drains (12Fr) (one axillary and pectoral) were inserted in all the patients. All resected specimens were examined and the lymph nodes dissected, counted and assessed histo-pathologically for metastases. No patient received intra-operative blood transfusion. Both the drains were connected to a single 600 ml suction bottle (Romovac -Romson). In-group A (n = 50), drainage was performed using complete vacuum negative suction (700 g/m2) and in-group B (n = 35) with half vacuum suction drainage (350 g/m2). The pressure was also measured by attaching a manometer to the exit opening of the drainage bottle. The two groups were comparable with respect to age, weight (body mass index), type of operation indicating the success of randomization (Table. 1 ). The drain was emptied every 24 hours to reset suction at the respective pressures and to measure the daily drain out put. External compression dressing was provided over the axilla for first 48 hrs and following that the patients were encouraged to do active and passive shoulder exercises. The outcomes measured were morbidity and the length of hospital stay. The total drain output was measured and recorded daily in both the groups, the drains were removed once the output was less than 30 ml in 24 hrs and the patients were discharged on the same day. The mean total drain output was measured in each group and compared. The mean hospital stay in both the groups was calculated and compared. The associated morbidity in the form of seroma formation, flap necrosis and wound infection during the postoperative period was recorded and compared in both the groups Statistical methods used Descriptive studies were performed with SPSS version 10 and group characteristics were compared using student t-test (Tables. 2 & 3 ). Results 1. Half vaccum suction drains were removed earlier than full vacuum suction drains without any significant addition to the postoperative morbidity. 2. The use of half vacuum drains after modified radical mastectomy reduced the hospital stay significantly without any increase in the postoperative morbidity. 3. The high negative suction is an important contributory factor to the amount of drainage following breast surgery along with axillary dissection. While negative suction helps in prevention of postoperative seroma formation, a high negative suction may affect adversely by increasing the amount and duration of drainage. This probably is on account of persistent drainage from the lymphatics, which do not close due to high negative suction. Discussion Seroma formation is the most frequently observed early complication after breast and axillary surgery. The use of closed suction drainage is a common practice that has been shown to reduce the incidence of seroma formation [ 1 - 6 ]. These drains are generally removed once the lymph production falls to less than 35–50 ml/24 hours, a level generally reached between 3–17 days after surgery [ 1 ]. The length of postoperative axillary drainage is a major cause of morbidity after axillary dissection as the patients are usually discharged once the drains are removed. The patients with suction drains in situ are normally managed in the hospital (although some authors advocate discharge with the drains in situ)[ 13 ]. Migration of bacteria along these drains has also observed to increase the risk of infection if the drains stay in situ for a long time [ 7 ]. Early or premature removal however has been found to be associated with an unacceptably high incidence of seroma formation and its continuation until fluid discharge is acceptably low leads to a prolonged stay in the hospital, which has a bearing on the cost of surgical management of breast cancer [ 1 , 11 - 13 ]. Shortening the hospital stay has been shown to be an effective way of reducing the costs in the case of surgery for breast cancer and axillary drains are the main obstacles in achieving it [ 1 , 8 - 10 ]. To reduce the hospital stay after MRM, early discharge with the drains in situ has been reported but discharging patients with drains in situ has an inherent difficulty faced by the patients in management of drains besides higher incidence of wound infection [ 13 , 14 ]. The other disadvantages are discomfort for the patients, with difficulties undressing or using the toilet. It may be feasible with patients of higher cultural and social standing, but not all the patients have the required background. In a third world country where the patients are poor, uneducated coming from far and remote areas with limited medical facilities, there is an added difficulty in management of the drains away from the hospital. As most of our patients come from far flung rural areas with limited education, poor medical and communication facilities they were managed indoors until the drains were removed. There are other solutions proposed for prevention or reduction of fluid accumulation and early discharge after axillary dissection e.g by Patrek et al [ 15 , 16 ] where several parallel drains were used. Suture obliteration of axillary space under skin flaps with sutures to the chest wall, approximation of the pectoralis major and the latissimus dorsi muscle in the form of axillary padding has been suggested by some authors [ 9 , 17 ]. The incidence of seroma formation had reduced but the length of drainage was not specified in these studies. Further more suture approximation of the muscles may limit movement of the arm leading to shoulder dysfunction. Harada et al [ 14 ] used fibrin glue in rats to occlude transected lymph channels and obliterate the subcutaneous cavity. The association of seroma formation with large amounts of drainage before removal of the drain has already been established [ 18 - 20 ]. In one study it was observed that when the amount of fluid drained before removal of the catheter was less than 250 ml in three days no seromas developed and they concluded that it is safe to remove drains if the total amount of fluid drained during the first postoperative days is low. Yii et al [ 20 ] reported that removal of drains after 48 hours did not result result in seroma formation if the total amount of fluid drained before removal was less than 150 ml Proposed factors contributing to the increased drainage and seroma formation [ 2 , 3 , 8 - 15 ] Patrek et al [ 15 ] examined 13 factors influencing fluid drainage. Only two (a large number of positive lymph nodes and previous biopsy) predicted greater drainage. 1. Body mass index . A significant linear relation exists between BMI and increased seroma formation was reported by (Boonman et al) 2. Technique : Use of elctrocautery has been reported to be associated with increased incidence of seroma formation as compared to cold knife. It has also been reported that tissue ligation around the axillary vein rather than mere transection with knife or diathermy may reduce the amount of postoperative discharge, the technique was followed in the presented study [ 8 ]. 3 Drains themselves encourage drainage by stimulating tissue reactions or by suction [ 20 ] 4. Early shoulder exercises have been implicated but were not observed to be a factor in the present study [ 10 , 11 ] Although early mobilization of the shoulder did not increase fluid discharge in various studies but it was reported to be an additional factor leading to increased drainage after axillary dissection [ 10 , 11 ]. 5 The negative suction applied may prevent the lymphatics from closing leading to continuous leakage and discharge [ 18 ]. 6. Extent of axillary dissection . More seromas were seen when more lymph nodes were dissected from the axilla. The higher lymph node yield may well be an indirect measure of more extensive dissection performed. [ 3 ]. The drainage may also reflect the damage to the lymph vessels and therefore the number of lymph nodes dissected may have a bearing on the amount of drainage. In our study also, it was observed that patients with higher lymph node yield had a higher volume and duration of drainage although it was not found to be significantly different in both the groups because they were matched in all respects except the negative suction pressure of the drainage. Negative suction and the drainage It is an accepted fact that negative suction prevents seroma collection and helps in the adherence of the walls of the axilla thus reducing the dead space and allowing the lymphatics to close. High negative suction pressure generated by the drain can maintain lymph drainage by a negative pressure gradient [ 18 ]. It is also reported that the high negative suction pressure does not allow the lymphatic channels to close leading to continuous drainage and a higher incidence of seroma formation [ 18 - 20 ]. There are studies to suggest that high negative suction may be beneficial in the sense that the amount of drainage would be more thus allowing an early adherence of walls of the axilla to the chest wall and reduction in the seroma formation [ 18 ]. However in the present study it was observed that high suction caused prolonged drainage, which can possibly be explained by the hypothesis that high negative suction may not allow, leaking lymphatics to close. Therefore no suction or high suction drainage both may contribute to the same result that is higher incidence of seroma formation and longer hospital stay. To strike a balance between not having suction at all and having a very high or full negative suction, half negative suction drainage was used in the present study to achieve a shorter hospital stay without any increase in the rate of post operative seroma formation. The external compression dressings in the first forty-eight hours perhaps helped in the adherence of the flaps and reduction of dead space without compromising on the shoulder mobility. This was found to effectively reduce the Hospital stay and also did not increase the postoperative morbidity as compared to high (full) negative suction group. Conclusions Reducing the negative suction pressure applied to the drain (making it half suction) along with external compression dressings applied for first 48 hours can significantly reduce drainage from the axilla following modified radical mastectomy without increasing the incidence of seroma formation as was observed in this randomized prospective clinical study. The hospital stay was reduced considerably compared to a matched group with full suction drain (p < 0.001). Half suction drain following axillary dissection in patients with carcinoma breast may thus be recommended as an effective approach to reducing the hospital stay and the cost of treatment without adding to the morbidity. Competing interests The author(s) declare that they have no competing interests. Authors' contributions CM was the surgeon in charge of the project and prepared the study design. VS was the first surgical assistant and did the data processing and statistical analysis. JP, the second surgical assistant assisted in the preparation of the manuscript. SS the Director of ICMR and AB the research officer, Tumor Biology Lab ICMR were responsible for the histopathological analysis and contributed to the preparation of the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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543467

Multiple synaptic and membrane sites of anesthetic action in the CA1 region of rat hippocampal slices

Background Anesthesia is produced by a depression of central nervous system function, however, the sites and mechanisms of action underlying this depression remain poorly defined. The present study compared and contrasted effects produced by five general anesthetics on synaptic circuitry in the CA1 region of hippocampal slices. Results At clinically relevant and equi-effective concentrations, presynaptic and postsynaptic anesthetic actions were evident at glutamate-mediated excitatory synapses and at GABA-mediated inhibitory synapses. In addition, depressant effects on membrane excitability were observed for CA1 neuron discharge in response to direct current depolarization. Combined actions at several of these sites contributed to CA1 circuit depression, but the relative degree of effect at each site was different for each anesthetic studied. For example, most of propofol's depressant effect (> 70 %) was reversed with a GABA antagonist, but only a minor portion of isoflurane's depression was reversed (< 20 %). Differences were also apparent on glutamate synapses-pentobarbital depressed transmission by > 50 %, but thiopental by only < 25 %. Conclusions These results, in as much as they may be relevant to anesthesia, indicate that general anesthetics act at several discrete sites, supporting a multi-site, agent specific theory for anesthetic actions. No single effect site (e.g. GABA synapses) or mechanism of action (e.g. depressed membrane excitability) could account for all of the effects produced for any anesthetic studied.

Background General anesthetics have been shown to depress neuronal responses in virtually all brain areas studied and this depression has been proposed to result from actions at GABA A -mediated inhibitory synapses and postsynaptic chloride channels [ 1 - 4 ], potassium channels [ 5 - 7 ], or calcium channels [ 8 - 11 ], and/or at glutamate-mediated excitatory synapses [ 12 - 17 ]. The last decade has seen a major shift in our understanding of general anesthetic mechanisms of action, away from a non-specific Unitary theory of action, towards a detailed view of anesthetic actions at membrane receptor and ion channel targets for these agents [ 18 , 19 ]. It is likely that several anesthetic actions occurring at independent sites contribute in additive ways to depress neuronal circuits in higher brain structures. Alternatively, anesthetic effects could result from actions at only a few sites and this should become evident by studying overall effects on the CA1 neural circuit and 'chasing down' the underlying actions. In the present study, the effects produced by five general anesthetics were studied at several possible sites of action within the well characterized Schaffer-collateral to CA1 neuron circuit using electrophysiological recordings from rat hippocampal slices. The CA1 circuit has previously been shown to be depressed by anesthetics from several chemical classes [ 20 - 26 ] at concentrations which alter hippocampal electrical activity in chronically instrumented rats during anesthesia [ 27 - 29 ]. The five agents chosen for this study are all clinically used anesthetics and provide a good representation from unique chemical classes: a halocarbon (halothane), halogenated ether (isoflurane), barbiturate (pentobarbital), sulfonated-barbiturate (thiopental), and a newer di-isopropylphenol compound, propofol. Results and discussion Anesthetics enhance GABA-mediated inhibition All five anesthetics depressed synaptically evoked discharge, measured as a block of population spike (PS) responses recorded from CA1 neurons (Fig. 1 ). The two volatile anesthetics, halothane and isoflurane, produced a nearly complete depression (to 3.3 ± 3.5 and 5.6 ± 7.1 % of control respectively) at clinically effective concentrations: halothane (1.0 rat MAC; 1.25 vol % ~ 250 μM) and isoflurane (1.0 rat MAC; 1.55 vol % ~ 350 μM; for Sprague-Daley rats [ 30 ]; Minimum Alveolar Concentration – the expired anesthetic gas concentration for a 50 % loss of a tail clamp response – motor reflex in rats). The three intravenous agents, pentobarbital (400 μM), thiopental (80 μM) and propofol (30 μM), also depressed PS responses to a comparable degree: 1.7 ± 3.1, 3.4 ± 2.8 and 6.2 ± 5.8 % of control responses, respectively (p < 0.001, n ≥ 5 slices from individual rats, for all five agents compared with pre-anesthetic control responses, using ANOVA-Tukey). All anesthetic effects were reversible on washout of the agent with drug free ACSF. It should be noted that the more lipophilic intravenous anesthetics produce lower effect site concentrations in these brain slices than the applied concentrations shown, especially for these short time periods of application, because it can take several hours for these agents to diffuse 200 to 300 μm into brain slices and achieve steady-state levels [ 31 ]. For example, an applied concentration of 30 μM propofol would be expected to produce only ~ 1.0 to 3.0 μM at a recording depth of 250 microns within 30 minutes [ 32 ]. The volatile anesthetics, in contrast, rapidly equilibrate throughout the brain slice due to their relatively high aqueous solubility. These equi-effective applied concentrations for PS depression were used in subsequent experiments to determine whether this depression resulted from enhanced GABA A -mediated inhibition. A GABA A receptor antagonist, bicuculline, was applied in the continued presence of each anesthetic to attempt to reverse the anesthetic-induced PS depression. Bicuculline (10 μM) reversed anesthetic-induced PS depression to varying degrees for each agent: isoflurane – 16.2 ± 7.4 %, halothane – 22.3 ± 18.4 %, pentobarbital – 56.2 ± 12.4 %, thiopental – 64.9 ± 12.9 % and propofol – 69.5 ± 14.3 %. Similar degrees of reversal were observed using the GABA-chloride channel blocker, picrotoxin (100 μM; a supra-maximal blocking concentration). A GABA B receptor antagonist, CGP 55845A (10 μM) did not reverse PS depression for any of the anesthetics studied (see Table 1 ). None of the anesthetics produced a significant depression of antidromically stimulated PS responses (± 5 % depression, p > 0.15) indicating that CA1 neuron axonal conduction was not appreciably altered. Thus, enhanced GABA A -mediated inhibition appeared to play a major role for the PS depression produced by propofol and thiopental (~ 75 %), less so for pentobarbital (~ 50 %), and contributed only partially to the depressant effects of the volatile anesthetics (< 25 %; Fig. 1E ). Anesthetics depress glutamate-mediated excitatory synapses To determine whether anesthetic-induced PS depression resulted from depressed glutamate-mediated excitatory synaptic inputs to CA1 neurons, field excitatory postsynaptic potentials (EPSPs) were recorded from dendritic regions in stratum radiatum. All five anesthetics depressed EPSP responses (e.g. Fig. 1C and 1D ): isoflurane to 52.2 ± 7.6 (p < 0.001), halothane 61.3 ± 8.4 (p < 0.001), pentobarbital 54.5 ± 4.8 (p < 0.001), thiopental 75.5 ± 9.8 (p < 0.01) and propofol 72.7 ± 23.5 (p < 0.05) % of control responses. Bicuculline did not reverse volatile anesthetic-induced EPSP depression, but did partially reverse the effect for pentobarbital (11.4 ± 3.6 %) and completely reversed the EPSP depression produced by thiopental and propofol (Fig. 1F ). Thus, depressed glutamate-mediated synaptic excitation appeared to play an important role for PS depression produced by isoflurane, halothane and pentobarbital. The thiopental and propofol-induced EPSP depression would also contribute to PS depression for these agents, but appeared to occur via enhanced GABA-mediated inhibition at a dendritic level, since this depression was reversed by bicuculline. Pre- and postsynaptic sites of action at GABA A synapses Whole cell voltage clamp recordings from CA1 neurons were used to examine more closely anesthetic effects on membrane currents at GABA synapses. Spontaneous GABA-mediated inhibitory postsynaptic currents (IPSCs) were observed in all CA1 neurons studied (n = 15) and were completely blocked by bicuculline (10 μM; Fig. 2A ). In the presence of glutamate receptor antagonists (CNQX 17.2 μM and APV 100 μM) the anesthetics produced agent-specific effects on holding currents needed to clamp neurons at the control resting membrane potentials (-60 to -70 mV). Propofol was most effective at increasing holding currents (376 ± 83 pA, n = 3), followed by thiopental (320 ± 72 pA, n = 4) and pentobarbital (127 ± 65 pA, n = 3). Halothane (n = 6) and isoflurane (n = 3) produced weaker and more variable responses (< 50 pA). The changes in holding currents produced by propofol and the barbiturates were reversed by bicuculline (10 μM) or picrotoxin (100 μM), indicating that they involved activation of GABA A -mediated chloride channels. The most dramatic effect produced by all five anesthetics was observed on IPSCs (e.g. Fig. 2B ). Membrane charge transfer, for example, was increased by 3 to 4 fold in the presence of halothane and came about by at least two separate mechanisms. The first mechanism was a prolongation of IPSC time course (Fig. 2C ) resulting in nearly a 3 fold increase in charge transfer for each IPSC (284 ± 33 % of control, p < 0.005, n = 6). This result was in good agreement with previous findings showing that anesthetics prolong IPSCs by increasing the open time of GABA-gated channels in the postsynaptic membrane [ 33 - 35 ]. The second mechanism appeared to involve presynaptic sites, observed as an increase in frequency of IPSCs (143 ± 28 % of control, p < 0.005, n = 6 neurons from separate slices) and occurred with a small, but significant, depression in IPSC amplitudes (92 ± 6 % of control, p < 0.05, n = 6). The anesthetic-induced IPSC frequency increase was also observed in the presence of tetrodotoxin, used to block action potentials (n = 5 for halothane, n = 4 for propofol), indicating a direct action on GABA nerve terminals. This confirms earlier findings that anesthetics can increase IPSC frequency and the release of GABA from nerve terminals [ 36 - 39 ]. This presynaptic effect combines with postsynaptic prolongation of IPSCs to account for the marked increase in membrane charge transfer observed, and would contribute to the anesthetic-induced postsynaptic hyperpolarization of CA1 neurons previously reported [ 4 , 40 - 42 ]. All of the anesthetics studied increased inhibitory charge transfer and the degree of enhancement corresponded well with the ability of bicuculline to reverse the anesthetic-induced depression of population spike responses (Fig. 1 and Table 1 ). For halothane and isoflurane, this enhanced inhibitory charge transfer played a relatively minor role in population spike depression compared with their ability to depress glutamate-mediated excitatory inputs to the CA1 neurons. Anesthetics increase paired-pulse facilitation To determine whether presynaptic actions also contribute to anesthetic effects at glutamate synapses, paired pulse (120 ms) facilitation of Schaffer-collateral evoked EPSPs were studied. In the presence of either halothane or isoflurane no apparent change in EPSP rise time or decay kinetics were observed (Fig. 3A ), contrasting with the marked prolongation of IPSC decay time produced by the anesthetics. Facilitation was increased to nearly 115 % of control and this effect was independent of GABA A -mediated actions, since they persisted in the presence of the antagonist – bicuculline (Fig. 3B ). This increase in facilitation is consistent with a presynaptic depression of glutamate release from nerve terminals, perhaps via depressant actions on voltage activated calcium or sodium channels which couple axon spike depolarization to the release of transmitter [ 8 , 9 , 17 , 43 , 44 ]. Anesthetics increase paired-pulse inhibition Agent-specific effects were observed for paired pulse inhibitory responses (120 ms separation) recorded from CA1 neurons (Fig. 3C ). Halothane and isoflurane produced no apparent change in paired pulse responses, both the first and second population spike following a pair of stimuli were depressed to a similar degree by these anesthetics. In contrast, propofol, thiopental and pentobarbital increased paired pulse inhibition, evident in a greater degree of depression for the second of a pair of responses. To quantify these increases in paired pulse inhibition, effects on second pulse responses were compared at concentrations that produced a half maximal depression of first spike responses. At a level of 50 % depression of first pulse responses, pentobarbital produced a 134 ± 8 % increase in second pulse inhibition, thiopental produced a 156 ± 15 % increase and propofol produced a 149 ± 13% increase (p < 0.001, n = 5 for each agent compared to first pulse responses). This effect is consistent with in vivo findings [ 45 ] and is thought to reflect a greater degree of GABA-mediated inhibition contributing to the second of a pair of stimuli, via recurrent (feedback) activation of inhibitory interneurons caused by the first pulse [ 26 ]. Anesthetics depress CA1 neuron excitability To determine whether the anesthetics could alter postsynaptic membrane excitability, effects on action potentials evoked by direct current injection into CA1 neurons were studied. Differences in effect were apparent across anesthetic agents – hardly any effect was evident for halothane and isoflurane, but the barbiturates and propofol produced a significant depression of action potential discharge (Fig. 3D ). When measured as a reduction in the number of action potentials produced in response to a one second long depolarizing current step, halothane produced an 8.2 ± 2.2 % depression and isoflurane an 11.6 ± 6.1 % depression (p < 0.01 for both agents compared to control responses). Propofol was much more effective at depressing CA1 discharge, producing a 93.5 ± 6.1 % depression (p < 0.001). Thiopental produced a 90.3 ± 9.9 % depression and pentobarbital a 79.5 ± 7.4 % depression (p < 0.001 for both anesthetics compared with control). The anesthetic-induced depression of spike discharge activity was accompanied by decreases in membrane resistance and to a lesser extent by small changes in membrane resting potential. In spite of the marked depressant effects observed for the intravenous anesthetics on spike discharge, none of the anesthetics appeared to alter action potential amplitude, rise time or decay profiles (Fig. 3D ), suggesting that the major depressant effect was accounted for by actions on spike threshold – not on the sodium currents which underlie action potentials per se . Conclusions Two conclusions can be drawn from these results: 1) for a given anesthetic, like halothane, multiple sites of action contributed in an additive manner to produce an overall depression of transmission through the CA1 neuronal circuitry (Fig. 3E ); 2) for each anesthetic the degree of effect was agent specific at some of these sites. Together the results support a Multisite Agent Specific (MAS) mechanism of action for general anesthetics. This represents a departure from traditional Unitary theories of action in several important respects. Unitary theories posit that all anesthetics act via a common molecular mechanism, such as to change the fluidity of nerve cell membranes, or to enhance a potassium current, or most recently to enhance GABA-mediated inhibition [ 2 , 3 ]. With the MAS theory, no common site of action is required (nor apparent) for anesthetics. This is consistent with observations at the molecular, [ 46 - 50 ] cellular [ 22 , 51 ] and behavioral levels [ 52 - 55 ]. Differing degrees of action (efficacy) were evident at both glutamate and GABA synapses for each anesthetic. For example, our results demonstrate that the two barbiturates studied appear to have differing degrees of effect at GABA synapses since thiopental's depressant effects were reversed ~ 65 % by a GABA antagonist, but pentobarbital's effects were only reversed by ~ 55 %. Similarly, these two barbiturates exhibited differing degrees of depression for glutamate-mediated excitatory inputs to the CA1 neurons, pentobarbital produced a 45 % depression in contrast to thiopental with only a 25 % depression. It was interesting that opposite actions were seen at presynaptic sites (GABA release was increased by anesthetics, while glutamate release was depressed) and at postsynaptic sites (GABA-mediated synaptic currents were prolonged, glutamate-mediated currents were not). The MAS theory can readily account for the unique agent-specific profiles of effects observed in various experimental models, and also seen clinically – a long standing weakness of Unitary theories [ 56 ]. Finally, the MAS theory predicts that agents which selectively target GABA and glutamate synapses could lead to the design of safer and more effective therapeutic agents for anesthesia, that exhibit fewer undesirable side effects. Glutamate and GABA synapses in the hippocampus are among the best characterized synapses in the brain and appear to utilize receptor subtypes which are similar to those in neocortex, thalamus and other higher brain regions. Thus, the effects described in the present study would be expected to occur in these other brain regions as well, but it should be noted that different GABA and glutamate receptor subtype distributions are known to occur in cerebellum, spinal and some brain stem nuclei, and it remains to be determined whether anesthetics alter these synapses in a similar manner to their hippocampal counterparts. Ted Eger's group at UCSF has recently found that enhanced GABA-mediated synaptic transmission at the spinal level plays an important role for propofol-induced immobility in response to a noxious stimulus [ 54 ], but this was not the case for isoflurane-induced immobility [ 57 ]. This agrees well with our findings that the volatile anesthetic-induced depression of synaptic signaling involves mechanisms other than enhanced GABA inhibition (see also [ 58 ]), while the depression produced by the barbiturates and propofol are more dependent on enhanced GABA-mediated inhibition. Additional in vivo support comes from studies utilizing a GABA beta 3 receptor mutant mouse model – proprofol-induced anesthesia was blocked in these mice, while volatile anesthetic effects were not [ 59 ]. Taken together with these in vivo findings, our results indicate that effects on GABA synapses play a role in anesthetic actions, especially for propofol, thiopental and pentobarbital; but the results also indicate that effects on glutamate synapses and postsynaptic membrane excitability contribute to the CNS depression produced by all anesthetics. Given the multiple effects observed for anesthetic actions on the two types of synapses studied here, it is likely that effects on other neurotransmitter systems also contribute to anesthetic-induced depression of the CNS. Methods Male Sprague-Dawley rats were anesthetized with ether (22 vol % in air) and the brain was rapidly removed and placed in ice cold (5°C) and pregassed (95/5 % O 2 /CO 2 , carbogen) artificial cerebral spinal fluid (ACSF). The ACSF had the following composition (in mM): Na 151.25; K 2.5; Ca 2.0; Mg 2.0; Cl 131.5; HCO 3 26.0; SO 4 2.0; H 2 PO 4 1.25; and glucose 10. Whole brain coronal slices (450 μm) were cut using a vibratome (Campden Instruments), following careful removal of the dura and pia membranes. Hemisected brain slices were equilibrated for at least one hour at room temperature in an incubation chamber filled with ACSF and continually bubbled with carbogen. Individual slices were transferred to a recording chamber and equilibrated for an additional 10 minutes prior to electrophysiological recording. Oxygenated ACSF solution was continuously perfused through the chamber at a flow rate of 3.0 ml/min and maintained at 22 ± 1°C. The present studies were carried out at room temperature because synaptic responses recorded from cooler brain slices exhibit considerably better baseline stability and the tissue remains viable for many more hours in vitro compared to slices maintained at physiological temperatures. Room temperature also facilitates the use of submerged preparations (oxygen solubility and delivery to slices is increased), which allows the use of 60× optics to visualize single neurons for the patch clamp recordings used in some experiments. Previous studies comparing both volatile and intravenous anesthetic effects at physiological and cooler temperatures in brain slices found that there were no apparent differences in effects [ 43 , 61 , 66 ]. The most important effect of lower temperature is to increase the aqueous solubility of the volatile anesthetics and previous work from our laboratory has described in detail the solubility changes observed at 22 vs . 35°C and our methods for measuring and compensating for changed aqueous solubility, as well as the remarkably similar physiological responses recorded from brain slices at these two temperatures [ 43 , 66 ]. To measure population spikes, bipolar tungsten microelectrodes were placed on Schaffer-collateral fibers to electrically stimulate inputs to hippocampal CA1 pyramidal neurons. Glass recording electrodes filled with ACSF (2 to 5 KOhm) were placed in stratum pyramidale to record stimulus-evoked population spike field potentials, or in stratum radiatum to record field EPSPs. Single stimulus pulses (0.01 to 0.05 ms duration; 10 to 80 μA @ 1.0 to 5.0 V) were delivered via constant current isolation units (Grass Instruments, SIU 6D) from a Grass S8800 two channel stimulator; at stimulus rates of 0.05 Hz. Field potential signals were amplified (× 1000), filtered (1 Hz to 10 KHz, bandpass), conditioned (DC offset), and digitally stored for later analysis (A/D with 20 μs resolution on a 486, and 50 MHz microcomputer using Data Wave Systems Corp. or Strathclyde Electrophysiological software). Whole cell patch-clamp recordings were made using thin-walled borosilicate capillaries (1.5 mm O.D.) pulled in two stages on a Narishige PP83 pipette puller. Patch electrodes were filled with the following intracellular solution (in mM): potassium gluconate or CsCl2 – 100, EGTA – 10, MgCl2 – 5, HEPES free acid – 40, ATP disodium salt – 0.3, and GTP sodium salt -0.3. The electrode solution also contained the local anesthetic QX 314 (1.0 mM) in some experiments, to prevent action potential discharge that would contaminate recordings of IPSCs. Electrode solutions were filtered and pH adjusted to 7.2 using KOH or CsOH and had a final osmolarity of 260 to 270 mOSM. Patch electrodes with a DC resistance of 4 to 5 MOhm were used. Recordings were made using an Axoclamp 2A preamplifier (Axon Instruments) in single electrode voltage clamp mode with > 80 % series resistance compensation and > 5 GOhm seals. Patch-clamp current signals were filtered (0.1 Hz to 10 KHz, bandpass), amplified (× 100) and digitized (10 KHz) for storage and analysis. Frequency and amplitudes of IPSCs were analyzed using Data Wave Technologies and Strathclyde Electrophysiological software in a continuous data recording configuration. The intravenous anesthetics (propofol and pentobarbital) were made fresh for each experiment, solubilized using 0.5% dimethyl sulfoxide (DMSO) and sonicated immediately prior to test administration in stock solutions and serially diluted into ACSF to achieve the final concentrations for testing. Volatile anesthetics (halothane and isoflurane) were applied in the perfusate at equilibrated concentrations, delivered from calibrated vaporizers and bubbled into the perfusate for at least 10 min prior to switching from control ACSF, to ensure steady-state concentrations were achieved. The concentration of volatile anesthetics in the gas phase were continually measured using a Puritan-Bennett anesthetic monitor. Only a single concentration of a given anesthetic was tested on each brain slice. Data are expressed as the mean ± standard deviation and statistical analysis (ANOVA with post Tukey test) was performed using Instat from GraphPad Software. For drug effects on paired pulse inhibition, the percent change was first calculated as: Drug 1st/Control 1st = (0.5 × 100) - 100 % = 50 % depression; and Drug 2nd/Control 2nd = (X × 100) - 100 % = X % depression. Then the percent increase in paired pulse inhibition was = (X / 50) × 100 %. This approach has the advantage of normalizing paired responses with respect to varying degrees of population spike facilitation observed during control recordings, i.e. differing degrees of EPSP facilitation on a background of differing degrees of inhibition from preparation to preparation. Authors' contributions SP and AMH contributed equally to this study by conducting most of the experimental work and they also contributed to data analysis and helped with figure preparation. MBM conceived of the study and contributed to experimental design, data analysis and interpretation of results; as well as wrote and prepared the manuscript.

/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC543467.xml

535532

Biotechnology approach to determination of genetic and epigenetic control in cells

A series of studies aimed at developing methods and systems for analyzing epigenetic information in cells are presented. The role of the epigenetic information of cells, which is complementary to their genetic information, was inferred by comparing the predictions of genetic information with the cell behaviour observed under conditions chosen to reveal adaptation processes and community effects. Analysis of epigenetic information was developed starting from the twin complementary viewpoints of cells regulation as an 'algebraic' system (emphasis on the temporal aspect) and as a 'geometric' system (emphasis on the spatial aspect). The knowlege acquired from this study will lead to the use of cells for fully controlled practical applications like cell-based drug screening and the regeneration of organs.

Review 1. General background Knowledge about living organisms increased dramatically during the 20th century and has produced the modern disciplines of genomics and proteomics. Despite these advances, however, there remains the great challenge of learning how the different living components of the cell are integrated and regulated. As we move into the post-genomic period, the complementarity of genomics and proteomics will become apparent and the connections between them will be exploited. However, neither genomics nor proteomics alone can provide the knowledge needed to interconnect the molecular events in living cells. The cells in a group are individual entities, and differences arise even among cells with identical genetic information that have grown under the same conditions. These cells respond to perturbations differently. Why and how do these differences arise? Cells are the minimum units containing both genetic and epigenetic information which are used in response to environmental conditions such as interactions between neighbouring cells and of changes in extracellular conditions. To understand the rules underlying the possible differences occurring in cells, we need to develop methods for simultaneously evaluating both the genetic information and the epigenetic information (Fig. 1 ). In other words, if we are to understand adaptation processes, community effects, and the meaning of network patterns of cells, we need to analyze the epigenetic information in cells. Thus we have started a project focusing on developing a system that can be used to evaluate the epigenetic information of cells by observing specific cells and their interactions continuously under controlled conditions. The importance of the understanding of epigenetic information will become apparent in cell-based biological and medical fields like cell-based drug screening and the regeneration of organs from stem cells, fields in which phenomena cannot be interpreted without taking epigenetic factors into account. Figure 1 Epigenetic information: complementary to genetic information. In 1999 the author moved to the Univ. of Tokyo and began his research on the "determination of genetic and epigenetic cellular control processes". To understand the meaning of the genetic variability and the epigenetic correlation of cells, we have developed the on-chip single-cell-based microcultivation method. As shown in Fig. 2 , the strategy consists of a three step process. First we purify cells from tissue individually in a nondestructive manner. [ 1 ] Then we cultivate the cells and observe them under fully controlled conditions ( e.g ., cell population, network pattern, or nutrient conditions) by using the on-chip single-cell cultivation chip [ 2 - 10 ] or by using an on-chip agarose microchamber system [ 11 - 14 ]. Finally, we do a single-cell-based expression analysis using the photothermal denaturation method and a single-molecule level analysis [ 15 ]. In this way, we can control the spatial distribution and interactions of cells. Figure 2 Our strategy: on-chip single-cell-based analysis. 2. Aim of the project The aim of our project is to develop methods and systems for analyzing the epigenetic information in cells. The project is based on the idea that, although genetic information makes a network of biochemical reactions, the history of the network as a parallel-processing recurrent network was ultimately determined by the environmental conditions of cells, which we call epigenetic information. As described above, if we are to understand the events in living systems at the cellular level, we need to keep in mind that epigenetic information is complementary to genetic information. The advantage of this approach is that it bypasses the complexity of underlying physicochemical reactions which are not always completely understood and for which most of the necessary variables cannot be measured. Moreover, this approach shifts the view of cell regulatory processes from the basic chemical ground to the paradigm of a cell as an information-processing unit working as an intelligent machine capable of adaptation to changing environmental and internal conditions. It is an alternative representation of the cell and can bring new insight into cellular processes. Moreover, models derived from such a viewpoint can directly help in the more traditional biochemical and molecular biological analyses of cell control. The basic part of the project is the development of on-chip single-cell-based cultivation and analysis systems for monitoring the dynamic processes in the cell. In addition we have employed these systems to examine a number of other processes eg; the variability of cells having the same genetic information, the inheritance of non-genetic information between adjacent generations of cells, the cellular adaptation processes caused by environmental change, the community effect of cells and network pattern formation in cell groups (Figs. 3 and 4 ). After making extensive experimental observations, we can understand the meaning of epigenetic information in the modeling of more complex signaling cascades. This field has been largely monopolized by physico-chemical models, which provide a good standard for the comparison, evaluation, and development of our approach. The ultimate aim of our project is to provide a comprehensive understanding of living systems as the products of both genetic information and epigenetic information. Figure 3 Aim of our project (1): temporal aspect. Figure 4 Aim of our project (2): spatial aspect. 3-1. Single-cell cultivation chip system [ 2 - 10 ] To understand the variability of cells having the same genetic information and to observe the adaptation processes of cells, we need to compare the sister cells or the direct descendant cells directly (Fig. 3 ). For that purpose, we have developed the system for an on-chip single-cell cultivation chip. The system enables excess cells to be transferred from the analysis chamber to the waste chamber through a narrow channel and allows a particular cell to be selected from the cells in the microfabricated cultivation chamber by using a kind of non-contact force, optical tweezers (Fig. 5 ). Figure 6 depicts our entire system for the on-chip single-cell microculture chip. The system consists of a microchamber array plate, a cover chamber, a phase-contrast/fluorescent microscope and optical tweezers. The cover chamber is a glass cube filled with a buffer medium and is attached to the array plate so that the medium in the microchambers can be exchanged through a semipermeable membrane. Figure 5 Single-cell cultivation in microchambers for measuring the variability of genetic information. Figure 6 System for on-chip single-cell microculture chip. Using the system, we examined whether the direct descendants of an isolated single cell could be observed under the same isolation conditions. Figure 7(a) plots the variations in interdivision times of consecutive generations of isolated E. coli cells derived from a common ancestor. The four series of interdivision times varied around the overall mean value, 52 min (dashed line); the mean values of the four cell lines a, b, c, and d were 54, 51, 56 and 56 min, showing differences rather small compared with the large variations in the interdivision times of consecutive generations. This result supports the idea that interdivision time variations from generation to generation are dominated by fluctuations around the mean value, and it was evidence of a stabilized phenotype that was subsequently inherited. To explore this idea, we examined the dependence of interdivision time on the interdivision time of the previous generation. We grouped the interdivision time data into four categories and determined their distributions (Fig. 7(b) ). Comparison of these distributions showed that they were astonishingly similar to one other, suggesting that there was no dependence on the previous generation. That is, there was no inheritance in interdivision time from one generation to the next. Figure 7 Genetic variability of direct descendant cells of E. coli . 3-2. On-chip agarose microchamber system [ 11 - 14 ] One approach to study network patterns (or cell-cell interactions) and the community effect of cells is to create a fully controlled network by using cells on the chip (Fig. 4 ). We have therefore developed a system consisting of an agar-microchamber (AMC) array chip, a cultivation dish with a nutrient-buffer-changing apparatus, a permeable cultivation container, and a phase-contrast/fluorescent optical microscope with a 1064-nm Nd:YAG focused laser irradiation apparatus for photothermal spot heating (Fig. 8 ). The most important advantage of this system is that we can change the microstructures in the agar layer even during cultivation, which is impossible when using conventional Si/glass-based microfabrication techniques and microprinting methods. Figure 8 On-chip agarose microchamber system. As explained above, the agarose-microchamber cell-cultivation system includes an apparatus for photothermal etching. Photothermal etching is an area-specific melting of the agarose microchambers by spot heating using a focused laser beam and a thin layer made of a light-absorbing material such as chromium (since agarose itself has little absorbance at 1064-nm). We made the three-dimensional structure of the agar microchambers by using a photo-thermal etching module. Figure 9 is a top-view micrograph of the agar microchambers connected by small channels. The space on the chip was colored by filling the microchambers with a fluorescent dye solution. Also shown are cross-sectional views of the A-A and B-B sections, in which we can easily see narrow tunnels under the thick agar layer in the A-A section and round tunnels in the B-B section. These cross-sectional micrographs show that we can make narrow tunnels in the agar layer by photothermal etching. The left micrograph in Fig. 9 is a top view of the whole microchamber array connected by narrow tunnels. Figure 9 Three-dimensional structure of agarose microstructures. By using this photothermal etching method, we can change the neural network pattern on a multi-electrode array chip during cultivation. Figure 10 shows the time course of the axon growth of rat hippocampal cells. After 5 days of cultivation (5DIV), when the cells in six microchambers had been connected by axons grown through the four existing tunnels (arrows in Figs. (a) and (b)), two new tunnels (arrows in Figs. (c) and (d)) were created by photothermal etching. After five more days of cultivation (10DIV), connecting axons had grown through them as well. Figure 10 Stepwise formation of neuronal network of rat hippocampal cells. The agarose microchamber system can also be used to observe the dynamics of the synchronizing process of two isolated rat cardiac myocytes. Figure 11 shows an example of the synchronizing process of two cardiac myocytes. After the cultivation had begun, the two cells elongated and made physical contact within 24 hours, followed by synchronization. It should be noted that, as shown in the graph, the synchronization process involved one of the cells following the rhythm of the other, and that the 'copy cat' cell stops beating prior to acquiring the new beat rhythm. Figure 11 Dynamics of the synchronizing process of two isolated rat cardiac myocytes. Conclusions We have newly developed and have just started to use a series of methods for understanding the meaning of genetic information and epigenetic information in a simple cell model system. The most important expected contribution of this project is to reconstruct the concept of a cell regulatory network from the 'local' (molecules expressed at certain times and places) to the 'global' (the cell as a viable, functioning system). Knowledge of epigenetic information, which we can control and change during their life, is complementary to genetic information, and those two kinds of information are indispensable for living organisms. This new kind of knowlege has the potential to be the basis of a new field of science. Authors' contributions KY conceived of the study, its design and coordination.

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555946

Glutamate-induced apoptosis in primary cortical neurons is inhibited by equine estrogens via down-regulation of caspase-3 and prevention of mitochondrial cytochrome c release

Background Apoptosis plays a key role in cell death observed in neurodegenerative diseases marked by a progressive loss of neurons as seen in Alzheimer's disease. Although the exact cause of apoptosis is not known, a number of factors such as free radicals, insufficient levels of nerve growth factors and excessive levels of glutamate have been implicated. We and others, have previously reported that in a stable HT22 neuronal cell line, glutamate induces apoptosis as indicated by DNA fragmentation and up- and down-regulation of Bax (pro-apoptotic), and Bcl-2 (anti-apoptotic) genes respectively. Furthermore, these changes were reversed/inhibited by estrogens. Several lines of evidence also indicate that a family of cysteine proteases (caspases) appear to play a critical role in neuronal apoptosis. The purpose of the present study is to determine in primary cultures of cortical cells, if glutamate-induced neuronal apoptosis and its inhibition by estrogens involve changes in caspase-3 protease and whether this process is mediated by Fas receptor and/or mitochondrial signal transduction pathways involving release of cytochrome c. Results In primary cultures of rat cortical cells, glutamate induced apoptosis that was associated with enhanced DNA fragmentation, morphological changes, and up-regulation of pro-caspase-3. Exposure of cortical cells to glutamate resulted in a time-dependent cell death and an increase in caspase-3 protein levels. Although the increase in caspase-3 levels was evident after 3 h, cell death was only significantly increased after 6 h. Treatment of cells for 6 h with 1 to 20 mM glutamate resulted in a 35 to 45% cell death that was associated with a 45 to 65% increase in the expression of caspase-3 protein. Pretreatment with caspase-3-protease inhibitor z-DEVD or pan-caspase inhibitor z-VAD significantly decreased glutamate-induced cell death of cortical cells. Exposure of cells to glutamate for 6 h in the presence or absence of 17β-estradiol or Δ 8 , 17β-estradiol (10 nM-10 μM) resulted in the prevention of cell death and was associated with a significant dose-dependent decrease in caspase-3 protein levels, with Δ 8 , 17β-E 2 being more potent than 17β-E 2 . Protein levels of Fas receptor remained unchanged in the presence of glutamate. In contrast, treatment with glutamate induced, in a time-dependent manner, the release of cytochrome c into the cytosol. Cytosolic cytochrome c increased as early as 1.5 h after glutamate treatment and these levels were 5 fold higher after 6 h, compared to levels in the untreated cells. Concomitant with these changes, the levels of cytochrome c in mitochondria decreased significantly. Both 17β-E 2 and Δ 8 , 17β-E 2 reduced the release of cytochrome c from mitochondria into the cytosol and this decrease in cytosolic cytochrome c was associated with inhibition of glutamate-induced cell death. Conclusion In the primary cortical cells, glutamate-induced apoptosis is accompanied by up-regulation of caspase-3 and its activity is blocked by caspase protease inhibitors. These effects of glutamate on caspase-3 appear to be independent of changes in Fas receptor, but are associated with the rapid release of mitochondrial cytochrome c, which precedes changes in caspase-3 protein levels leading to apoptotic cell death. This process was differentially inhibited by estrogens with the novel equine estrogen Δ 8 , 17β-E 2 being more potent than 17β-E 2 . To our knowledge, this is the first study to demonstrate that equine estrogens can prevent glutamate-induced translocation of cytochrome c from mitochondria to cytosol in rat primary cortical cells.

Background High concentrations (mM) of the excitatory neurotransmitter glutamate can accumulate in the brain and are thought to be involved in the etiology of a number of neurodegenerative disorders including Alzheimer's disease [ 1 - 4 ]. A number of in vitro studies indicate that at high concentrations, glutamate is a potent neurotoxin capable of destroying neurons [ 5 , 6 ]. The mechanisms by which glutamate-induced neurotoxicity or excitotoxicity is mediated, has not been established, however, a substantial body of evidence suggests that glutamate toxicity involves oxidative stress and apoptosis (programmed cell death) [ 2 , 7 - 9 ]. This latter form of cell death is characterized by DNA degradation that results by cleaving DNA at internucleosomal sites [ 10 ]. Apoptosis is a gene-directed process and an increasing number of genes and their proteins are involved in this process [ 11 , 12 ]. We have previously reported that in a stable mouse hippocampal neuronal cell line (HT22), glutamate-induced cell death is associated with DNA fragmentation and up-regulation of the pro-apoptotic protein Bax and down-regulation of the anti-apoptotic protein Bcl-2, however, in this cell line, the apoptotic process did not appear to involve caspase-3 [ 13 ]. In contrast, recent studies demonstrate that a family of cysteine proteases (caspases) play an important role in apoptotic cell death observed in some neurodegenerative diseases [ 14 - 16 ]. Caspase-3 is considered to be the central and final apoptotic effector enzyme responsible for many of the biological and morphological features of apoptosis [ 15 - 17 ]. Caspase-3 usually exists in the cytosolic fraction of cells as an inactive precursor that is activated proteolytically by cleavage at a specific amino acid sequence to form the active enzyme [ 18 ] which is capable of cleaving several proteins that culminate in apoptotic cell death [ 19 ]. Although these observations strongly indicate that caspase-3 is essential for apoptosis in mammalian cells, the mechanisms involved in caspase-3 regulation of the neuronal system remain to be elucidated. Many signal transduction pathways such as Fas receptor-mediated signaling pathway via caspase-8, via activation of granzyme B, or the damage of mitochondria that results in cytochrome c release, have been implicated in the initiation of caspase-3 cascade [ 20 - 25 ]. A number of studies have demonstrated that estrogens are potent antioxidants capable of inhibiting some of the neurotoxic effects of oxidative stress [ 7 , 26 , 27 ]. We, and others have shown that estrogens can increase cell survival and attenuate in vitro cell death induced by various neurotoxins [ 28 - 31 ]. We have also previously demonstrated in HT22 cells and in the neuronal-like PC12 cell line derived from rat adrenal pheochromocytoma cells that the neurotoxic effects of glutamate can be inhibited differentially by various equine estrogens [ 31 ]. The data further indicated that the less estrogenic (uterotropic) Δ 8 -estrogens were the most effective neuroprotectors [ 31 ]. We further suggested that the increased potency of these Δ 8 -estrogens may to some extent, be due to their greater antioxidant properties [ 28 , 29 , 31 ]. However, the mechanism(s) involved in estrogen mediated neuroprotection are not fully understood. In the present study, we have determined whether glutamate-induced neuronal apoptosis in primary cortical cells and its inhibition by estrogen, involves changes in caspase-3 protease and whether this process is mediated by Fas receptor and/or mitochondrial signal transduction pathways involving release of cytochrome c. The estrogens selected for this study were 17β-estradiol (17β-E 2 ) which has high affinity for both estrogen receptors ERα and ERβ and Δ 8 , 17β-estradiol (Δ 8 , 17β-E 2 ), an estrogen that is a more potent antioxidant than 17β-E 2 and whose activity appears to be mediated to a greater extent via ERβ [ 31 - 34 ]. Results Effects of various concentrations of glutamate on cortical cell viability Cortical cells (2.5 × 10 4 ) cultured for 7 days in 96-well plates were treated with increasing concentrations (0.2 – 100 mM) of glutamate for 18 h. As depicted in Figure 1 , increasing concentrations of glutamate resulted in a dose-dependent decrease in cell viability measured by the MTS (3-[4,5-dimethylthiazol-2 yl]-5 [3 carboxymethoxyphenyl] 2 H-tetrazolium, inner salt) cell proliferation assay as described under "Methods". Generally as the amount of glutamate increased, cell death increased progressively, however, there was no significant difference between 1 and 25 mM glutamate in 18 h. Similar results were obtained after treatment with glutamate for 6 h (data not shown). Based on these preliminary data, all subsequent experiments were carried out for 6 h at glutamate concentrations between 1 to 20 mM. At these concentrations, the mean percent cell death was approximately 20%. Demonstration of apoptosis in cortical cells treated with glutamate Cortical cells cultured in poly-lysine coated 6-well plates for up to 7 days were treated with 1 mM glutamate for 18 h. DNA was extracted, purified and subjected to agarose gel electrophoresis as described under "Methods". The results indicate (Figure 2 ), that in DNA isolated from untreated cortical cells prior to culture (Figure 2 , lane 2), untreated cells in culture for 1 day (Figure 2 , lane 3) no DNA fragmentation was detectable. However, after 2 and 7 days in culture, DNA fragmentation was detectable (Figure 2 , lanes 4 and 6). The extent of DNA fragmentation was potentiated in cells treated for 18 h with 1 mM glutamate (Figure 2 , lanes 5 and 7). These results indicate that cortical cells in culture for longer than 2 days display characteristic DNA fragmentation or laddering that is associated with apoptosis mediated via caspase-3 activation. Glutamate treatment as can be seen in Figure 2 , lane 7 induced a much greater DNA fragmentation than untreated cortical cells. However, these are qualitative data and should be interpreted with caution, particularly since some DNA fragmentation occurs in untreated cells. Glutamate-induced enhancement of apoptosis was also detectable by characteristic morphological changes observed by using phase contrast microscopy (Figure 3 ). After 6 h in culture, untreated cortical cells retained normal morphology of neuronal cortical cells and their cellular extensions (dendrites) and membranes were clearly visible. An occasional degenerated cell was also visible (Figure 3A ). In contrast, after a 6 h incubation with 1 mM glutamate degenerated, dead or apoptotic cells were clearly visible in these cultures (Figure 3B ), and the cellular extensions seen in untreated cortical cells (Figure 3A ), were retracted, and cells appeared rounded. Similar changes were also observed with 5 mM glutamate (data not shown). In the presence of 1 mM glutamate and 1 μM, 17β-E 2 (Figure 3C ) and Δ 8 , 17β-E 2 (Figure 3D ), the cells retained the normal morphology of untreated cells, and only an occasional degenerated cell was visible. These results clearly confirm that glutamate induces and enhances apoptosis. These morphological changes and cell death were prevented by both estrogens. Taken together, these data indicate that cortical cells in the presence of glutamate undergo apoptotic changes in culture. Effects of glutamate and estrogens on cell death Cortical cells were cultured in 6-well plates (1 × 10 6 cells/well) for 7 days. The medium was then changed and the effects of various concentrations (1–20 mM) of glutamate on lactate dehydrogenase (LDH) release were measured for 6 h. The results indicate that glutamate induced a significant (30–40%) increase in LDH release compared to the control untreated cells. No significant difference in LDH release was observed in 6 h at glutamate concentration between 1 to 20 mM, (data not shown). Based on these observations and the cell viability data (Figure 1 ), all subsequent experiments were done in the presence of 1 or 5 mM glutamate to avoid potential necrosis which may occur at higher concentration of glutamate. In order to follow the kinetics of glutamate cytotoxicity, the effect of 5 mM glutamate on LDH release as a function of time was measured for up to 24 h. The results shown in Figure 4 indicate that glutamate toxicity varied markedly during the course of culture. Significant increase (40–60%) in cytotoxicity was observed after 6 to 24 h exposure of cells to glutamate. During this period, LDH release in untreated control cells remained the same for up to 8 h and significantly increased (P < 0.05) after 24 h (Figure 4 ). Next, cortical cells were treated with 5 mM glutamate for 6 h in the presence or absence of various concentrations (0.01–10 μM) of 17β-E 2 and Δ 8 , 17β-E 2 . Release of LDH from cells was measured and the results are summarized in Figure 5 . Both 17β-E 2 and Δ 8 , 17β-E 2 inhibited glutamate-induced cell death in a dose-dependent manner, with Δ 8 , 17β-E 2 being more potent. Thus, 0.1 μM Δ 8 , 17β-E 2 and 1 μM 17β-E 2 significantly reduced cell death compared to glutamate alone (Figure 5 ). However, even at the highest concentration of estrogens tested, cell death induced by 5 mM glutamate was not fully preventable. In contrast, when cell death was induced by lower concentration (1 mM) of glutamate, 10 μM of Δ 8 , 17β-E 2 completely inhibited cell death and the release of LDH returned to control levels (data not shown). Effects of glutamate and estrogens on caspase-3 protein levels Following measurement of LDH, cortical cells were harvested, lysed and processed for Western blot analysis as described under "Methods". The results (Figure 6 ) indicate that both anti-caspase-3 antibodies detected the presence of caspase-3 Mr 32 kDa protein band (precursor protein), but not the p 20 and p 11 active fragments of caspase-3. The results further indicate that the exposure of cells to glutamate resulted in an increase in caspase-3 precursor protein in a dose (Figure 6 ) and time (Figure 7 ), dependent manner. Thus, 1 to 5 mM glutamate increased caspase-3 protein levels by 45 to 66% respectively, however, higher concentrations of glutamate (10–20 mM) did not result in any further increase in caspase-3 protein levels (Figure 6 ). The kinetics of glutamate effects on caspase-3 protein levels indicate that a significant increase in the levels occurred by 3 h of glutamate (5 mM) exposure and reached maximum levels observed at 6 h (54% increase) (Figure 7 ). The levels were significantly lower at 24 h (Figure 7 ), most likely due to decreased transcription of caspase-3. These results further indicate that changes in levels of caspase-3 occur soon after induction of apoptosis. Lack of further increase in caspase-3 protein levels suggest that cell death at this late stage may be due to necrosis and therefore all subsequent experiments were carried out for up to 6 h only. These glutamate-induced changes were reversed in the presence of estrogens. Thus, increasing concentrations of 17β-E 2 and Δ 8 , 17β-E 2 in the presence of 5 mM glutamate for 6 h resulted in a decrease in caspase-3 levels in a dose-dependent manner (Figure 8 ). As depicted in Figure 8 , glutamate (5 mM) alone increased the levels of caspase-3 by 60% and in the presence of 1 to 10 μM 17β-E 2 , the levels of caspase-3 protein decreased gradually and returned to control values with 10 μM 17β-E 2 . Similar results were obtained with Δ 8 , 17β-E 2 , however, significant decrease (30%) in the levels of caspase-3 occurred at 10 times lower concentration (1 μM) (Figure 8 ). When cell death was induced with lower concentration (1 mM) of glutamate, 0.1 μM 17β-E 2 and Δ 8 , 17β-E 2 completely inhibited the changes in caspase-3 protein and the levels returned to control values (Figure 9 ). Whether the ability of low concentrations of estrogens to fully protect the cortical cells against neurotoxicity induced by 1 mM glutamate was due to the cell death resulting via predominantly apoptosis rather than necrosis or a mixture of the two processes remains to be investigated. Effects of caspase inhibitors, z-DEVD and z-VAD, on glutamate-induced cell death To further confirm the role of caspase-3 in glutamate-induced cell death, cortical cells were pre-incubated with 100 μM of z-DEVD or z-VAD prior to induction of cell death by 1 mM glutamate. Glutamate alone caused a significant increase in cell death over control (Figures 10 A, B ), while pre-incubation with the caspase-3 inhibitors prior to glutamate exposure resulted in a 50% reduction in cell death (Figures 10 C, D ). In absence of glutamate, both inhibitors had no effect on cell viability (Figures 10 E, F ). Similarly, DMSO, the vehicle used in the preparation of the inhibitors had no effect on cell viability (Figure 10 G ). These data confirm that in primary culture of rat cortical cells glutamate induces cell death via the apoptotic pathway that involves changes in caspase-3 protease. Activation of caspase-3 (a) Effects of caspase inhibitors on proleolytic cleavage of protein kinase c (PKC) in untreated primary cortical cells and cells treated with glutamate During apoptosis, activation of caspases including caspase-3 can result in the generation of breakdown products (BDPs) of PKC [ 35 , 36 ]. Primary cortical cells were either treated with glutamate alone or first pretreated with caspase-3 specific inhibitor z-DEVD or pan-caspase inhibitor z-VAD as described under "Methods". The immunoblots are depicted in Figure 11 . Glutamate treatment resulted in an increase in the formation of two BDPs (48 kDa and 45 kDa) in both the cytosol and cell lysates. Pretreatment with z-DEVD and z-VAD reduced the amounts of the two BDPs to levels seen in the untreated primary cortical cells (Figure 11 ). These results clearly indicate that pro-caspase-3 is activated and that active caspase-3 is present in primary cortical cells and cells treated with glutamate. (b) Detection of active caspase-3 in primary cortical cells treated with glutamate Western blot analysis had indicated the presence of pro-caspase-3 in primary cortical cells treated with glutamate (Figure 12 ). To detect the presence of active caspase subunits, highly specific active caspase-3 antibody raised against amino acids 163 to 175 (p 18 subunit) of murine caspase-3 was obtained from Stratagene. Primary cortical cells were treated with 1 mM glutamate for 3 and 6 h and processed as described under "Methods". The results (Figure 12 ) indicate that glutamate treatment for 3 h, did not significantly change the levels of caspase-3 active form. However, as can be seen (Figure 12 ) after 6 h of glutamate treatment, the levels of caspase-3 active form increased over 2 fold compared to untreated cells (Figure 12 ). These results further confirm that caspase-3 is activated in apoptosis induced by glutamate in primary cortical cells. Effects of glutamate on Fas receptor protein expression To determine whether the regulation of caspase-3 in glutamate-induced cell death in cortical cells is modulated by Fas receptor mediated apoptotic pathway, expression of Fas receptor protein was evaluated by Western blot analysis. The results indicate (Figure 13 ) that anti-Fas receptor antibody reacted specifically with Fas receptor protein, however, no significant changes in the levels of this protein were observed following treatment with various concentrations (1–20 mM) of glutamate (Figure 13 ) for up to 24 hours (Figure 14 ) compared to control levels. Effects of glutamate and estrogen on the release of mitochondrial cytochrome c into the cytosol To determine whether the activation of caspase-3 in glutamate-induced cell death requires cytochrome c, primary cultures of cortical cells were first incubated with 1 mM glutamate for up to 6 hours. The cells were then processed for Western blot analysis as described under "Methods". The results depicted in Figure 15 clearly indicate that prior to glutamate treatment, the bulk of cytochrome c is localized in the mitochondria and barely detectable levels were observed in the cytosol. In contrast, exposure of cells to glutamate resulted in a rapid release of cytochrome c from the mitochondria into the cytosol (Figure 15 ). Significant increase in cytosolic cytochrome c occurred as early as 1.5 h, and after 3 h, the levels of cytochrome c were higher in the cytosol compared to the mitochondria (Figure 15 ). By 6 h, the levels of cytochrome c in the cytosol were 2.5 times higher than those in the mitochondria. The cytosolic levels of cytochrome c between 1.5 hours to 6 hours were 2 to 5 fold higher in the cytosol prepared from glutamate treated cells compared to untreated cells (Figure 15 ). Concomitant with these changes in the cytosol, the mitochondrial levels of cytochrome c decreased significantly. Similar results were also observed when 5 mM glutamate was used (data not shown). Exposure of cortical cells to 1 mM glutamate and 1 μM 17β-E 2 or Δ 8 , 17β-E 2 for 6 hours resulted in a significant decrease in the release of cytochrome c into the cytosol (Figure 16 ). Thus, the levels of cytochrome c in the cytosol from estrogen-treated cells were 30 to 50% lower than the corresponding levels in the glutamate alone treated cells (Figure 16 ). Discussion In the present study, we used primary fetal rat cortical cell cultures to demonstrate that glutamate can induce neuronal cell death by apoptotic mechanisms and that the process can be reversed or inhibited by equine estrogens such as 17β-E 2 and Δ 8 , 17β-E 2 . Our results further indicate that glutamate-induced cell death appears to result, to some extent, by a mechanism that involves DNA fragmentation and morphological changes characteristic of apoptosis. These changes are similar to those we and others have reported previously with other neuronal cell models [ 7 , 13 ]. Unlike the mouse hippocampal cell line HT22 [ 13 ], the primary cultures of rat cortical cells after day two in culture appear to undergo some degree of apoptotic cell death in absence of glutamate (Figure 2 ), however, the extent of DNA fragmentation is significantly enhanced after treatment with glutamate. Moreover, compared to the HT22 cells, the primary rat cortical cells appear to be resistant and require higher concentration of glutamate to induce apoptosis. Similar differences were also observed between HT22 cells and PC12-neuronal cells derived from rat adrenal pheochromocytoma [ 31 ]. The glutamate induced DNA fragmentation and subsequent cell death was associated with characteristic morphological changes also noted previously in HT22 cells following treatment with β-amyloid and glutamate [ 13 , 37 ]. The results from the present study indicate that these morphological changes associated with apoptosis were prevented by 17β-E 2 and Δ 8 , 17β-E 2 (Figure 3 ). To quantitatively estimate the extent of cell death induced by glutamate, we measured cell viability by MTS assay and cell death by LDH activity released in the media during culture. Results from the MTS assay clearly indicated that cell viability was dependent on the dose of glutamate (Figure 1 ). Thus, increasing concentrations of glutamate resulted in a dose-dependent decrease in cell viability. Because in the MTS proliferation assay, cells cannot be reused, subsequent quantitative experiments were carried out using LDH release assay. The LDH assay had previously been validated using the MTS assay [ 29 , 31 ]. In the present study, we further observed that glutamate induces cell death of primary rat cortical cells and involves changes in caspase-3 protease. Exposure to glutamate up-regulated caspase-3 protein levels while caspase inhibitors blocked this apoptotic process. These observations indicated that cell death induced by glutamate in primary cortical neurons was via apoptosis. Caspase-3 was one of the cysteine proteases that played an essential role in apoptosis by cleaving several key cellular proteins such as poly (ADP-ribose) polymerase (PARP), sterol-regulatory element-binding protein (SREBPs), PKC, DNA-dependent protein kinase, DNA-fragmentation factor (DFF) and a number of others [ 16 , 19 , 21 , 24 , 25 , 35 , 36 ]. Our studies show that up-regulation of caspase-3 expression preceded neuronal cell death, supporting the possibility that glutamate-induced apoptotic cell death was the consequence of up-regulation of caspase-3 gene in cortical neurons. These observations are consistent with up-regulation of precursor caspase-3 in frontal neuronal cortex of subjects with Alzheimer's disease [ 5 ]. This enzyme has been proposed to activate death effector molecules resulting in the fragmentation of genomic DNA and was associated with morphological and structural changes characteristic of apoptosis [ 16 , 19 , 21 , 25 , 38 ]. When cells undergo apoptosis, caspase-3 is initially present as pro-enzyme (32 kDa precursor protein) that is subsequently transformed into the active heterodimeric complexes through a cascade of proteolytic events [ 14 , 18 ]. The active form of caspase-3 is composed of two subunits of Mr 20 kDa (p20) and 11 kDa (p11), which are derived from proteolytic processing of the 32 kDa precursor during apoptosis [ 21 , 24 , 25 ]. In our study, these p 20 and p11 forms of caspase-3 were not detected in glutamate-induced cell death by immunoblotting analysis with three different kinds of antibodies: i. a polyclonal goat antibody caspase-3 p11 (K-19) against the p11 fragment of caspase-3, ii. a polyclonal rabbit antibody against caspase-3 (H-227) and iii. a monoclonal mouse antibody (E-8) (data not shown with this latter antibody). These observations suggest that either our antibodies are incapable of detecting these fragments or that cells may clear them rapidly as been observed in other studies [ 39 ]. Since we observed that glutamate-induced cell death was effectively blocked by both caspase-3 specific inhibitor z-DEVD and pan-caspase-inhibitor z-VAD, these data further provide evidence that caspase-3 protease is not only up-regulated, but is also activated during glutamate-induced cell death. That activation of pro-caspase-3 does indeed occur in glutamate-induced apoptotic cell death of primary rat cortical cells was confirmed by the demonstration that PKC, an endogenenous substrate of active caspase-3, was cleaved into two 45 kDa and 48 kDa BDPs [ 35 , 36 ]. The formation of these BDPs is characteristic of caspase-3 like protease activity [ 35 , 36 ]. The formation of the two BDPs was inhibited by caspase-3 specific inibitor z-DEVD and pan-caspase-inhibitor z-VAD). These results strongly suggest that proleolytic cleavage of PKC involves active caspase-3. These observations were further supported by the direct immunodetection of activated caspase-3 in the primary cultures of rat cortical cells treated with glutamate (Figure 12 ). The primary antibody was raised against amino acids 163 to 175 of murine caspase-3 and this neo-epitope is present on the p18 subunit of cleaved caspase-3. This antibody recognizes the p18 subunit but not the inactive pro-caspase-3 [ 40 ]. Taken together these data strongly indicate that in our neuronal cell cultures, glutamate induces apoptosis that involves active caspase-3. In contrast, it has been suggested that glutamate-induced cell death in HT22 mouse hippocampal cells appears to occur by apoptotic mechanisms that are independent of caspase-3 [ 13 ]. Since in HT22 cells, glutamate also induced DNA laddering, mitochondrial proteins such as apoptosis inducing factor (AIF) and endonuclease G (Endo G) released in response to death signals may also play a role in the HT22 neuronal cell line [ 13 ]. Whether similar caspase-3 independent pathways are also involved in glutamate-induced apoptosis in primary cultures of rat cortical cells remains to be investigated. Several signaling pathways are implicated in the initiation of the caspase-3 cascade; one of the well defined pathways for activation of apoptosis is Fas receptor-mediated pathway [ 41 - 43 ]. Fas is a cell surface antigen and a member of tumor necrosis factor (TNF) receptor family [ 44 ]. Activation of Fas by its ligand or an agonistic anti-Fas antibody can transmit apoptotic signal by activation of caspase-3 and induces apoptosis in T-lymphocytes and malignant cells [ 26 , 45 , 46 ]. In vitro, cross-linking of Fas antibody to its antigen increases caspase-3 activity and induces apoptotic cell death in Jurkat cells [ 47 ]. However, in primary cortical cells, we did not observe any change in Fas receptor protein levels following glutamate treatment. These results suggest that activation of caspase-3 in neuronal cortical cells and their apoptotic demise induced by glutamate is independent of Fas receptor mediated pathway. However, the Fas mediated pathway is complex and further studies are required to establish whether this pathway is or not involved in glutamate-induced cell death in cortical cells. In contrast to glutamate, chronic morphine administration has been shown to induce up-regulation of Fas receptor and caspase-3 precursor protein in the rat neuronal cells [ 48 , 49 ]. Thus, whether Fas receptor mediated pathway plays a role in up-regulation of caspase protein or caspase-3 activation and apoptotic cell death may depend on the nature of neurotoxic agents being used. Another recently characterized mechanism for pro-caspase-3 activation involves translocation of the respiratory chain protein, cytochrome c (Apaf-2), from mitochondria to the cytosol during the induction of apoptosis by a variety of different agents in non-neuronal and neuronal cells such as cerebellar granule cells [ 21 , 50 ]. Cytosolic cytochrome c binds to Apaf-1 in the presence of dATP and this leads to the recruitment and activation of caspase-9 and subsequent activation of caspase-3 [ 23 , 25 ]. In mitochondria cytochrome c resides in the inter-membrane space and matrix as a soluble protein and it functions as an electron carrier in oxidative phosphorylation [ 21 ]. The mechanism responsible for the translocation of cytochrome c from mitochondria into cytosol is not known. Cytochrome c release can occur upon interaction of pro-apoptotic protein with the outer mitochondria membrane protein such as Bax [ 51 , 52 ] or can be induced by interaction with elevated calcium or reactive oxygen species (ROS) with mitochondria. This results in mitochondrial dysfunction and a reduction in mitochondrial trans-membrane potential [ 53 - 55 ]. Our present studies demonstrate that glutamate-induced cell death in primary cortical cells is accompanied by the early release of cytochrome c into the cytoplasm that precedes changes in caspase-3 protease. Although we were unable to directly detect by Western blot analysis, the active caspase-3 fragments p20 and p11, the presence of active caspase-3 was confirmed as discussed above. Since there was a close correlation between the time course of cytochrome c release from the mitochrondria and changes in caspase-3 protease levels; it provides a possible mechanism whereby caspase-3 protease is not only up-regulated, but also activated by cytochrome c during glutamate-induced apoptotic cell death of primary cortical cells. Whether cytochrome c release from the mitochondria to cytosol is involved in the regulation of caspase-3, remains to be investigated. We have previously reported that a number of equine estrogens, which are components of the drug CEE (conjugated equine estrogen) used extensively for management of vasomotor symptoms and osteoporosis in postmenopausal women, are potent antioxidants and protect neuronal cells against cell death induced by oxidized LDL and glutamate [ 28 , 29 , 56 , 57 ]. In keeping with these observations, the results from the present study indicate that in primary cortical cells, these equine estrogens prevent cell death by reducing glutamate-induced cytochrome c release from mitochondria and caspase-3 protein levels. To our knowledge, this is the first study that demonstrates that equine estrogens can prevent glutamate-induced translocation of cytochrome c from mitochondria to the cytosol in the rat primary cortical cells. Previous studies have shown that 17β-E 2 induces release of cytochrome c from heart mitochondria [ 58 ] and also decreases cytosolic cytochrome c levels in hippocampus following global ischemia [ 59 ]. These observations indicate that equine estrogens protect against glutamate-induced apoptosis of primary cortical cells at least in part by inhibiting caspase-dependent apoptotic pathway. However, the mechanism by means of which estrogens reduce cytochrome c release from mitochondria is not yet fully understood. Some have suggested that the mechanism of estrogen-mediated neuroprotection involves regulation of mitochondrial calcium (Ca 2+ ) and Bcl-2 expression [ 59 ]. Exposure of glutamate has also been associated with an increase of cytosolic Ca 2+ in cortical cells [ 3 , 53 ] and up-regulation of pro-apoptotic protein Bax in neuronal cell line, HT22 [ 13 ]. Whether these changes induced by glutamate are involved in the mitochondria release of cytochrome c into the cytosol, remains to be investigated. The cortical neuronal cells used in the present study contain both estrogen receptors ERα and ERβ (Bhavnani and Zhang, unpublished data), and therefore whether the mechanism by means of which estrogens exert their neuroprotective effects remain to be elucidated. In general, the current evidence indicates that there are at least two mechanisms for estrogen action: i) the genomic mechanism mediated via two nuclear receptors ERα and ERβ, and ii) the non-genomic mechanism mediated via putative membrane receptors and these have been recently reviewed [ 32 ]. Although the concentrations of estrogens that protect the cortical neurons from glutamate toxicity are pharmacological doses, these levels can however, be potentially attained in a postmenopausal women taking daily 0.625 mg of (CEE) [ 61 ]. These estrogen effects are most-likely mediated to some extent by the non-genomic mechanism, and may be related to the estrogens' antioxidant property [ 28 , 29 ]. This is in keeping with our previous observations that although 17β-E 2 has higher affinity for ERα and ERβ, it is possible that some of these effects are due to the antioxidant properties of estrogens. However, we have also demonstrated [ 33 ] that Δ 8 , 17β-E 2 expresses its biological effects to a two fold greater extent via ERβ than 17β-E 2 [ 33 ]. Since the localization of ERα and ERβ in the brain is differential [[ 32 ] and references therein] further detailed studies are needed to elucidate the molecular mechanisms involved in the neuroprotective aspects of estrogens. Recent studies further report that estrogen receptor (ERβ) is localized in the mitochondrial membrane and estrogen can directly modulate the mitochondrial content of Ca 2+ and mitochondrial trans-membrane potential [ 61 , 62 ]. The role of the novel estrogen membrane receptors in apoptotic cell death induced by glutamate remains to be investigated. We along with others, have previously demonstrated that estrogens prevent cell death induced by glutamate [ 13 ] or β-amyloid [ 63 ] via modulation of Bcl-2 family of proteins. Thus, it is possible that in the neuronal cell model, estrogens decrease glutamate-induced cytochrome c release from mitochondria by protecting mitochondrial trans-membrane potential via binding estrogen receptor on mitochondrial membrane as well as up-regulation of anti-apoptotic protein Bcl-2. Studies to further delineate these mechanisms are being initiated. Conclusion In the primary cortical cells, glutamate-induced apoptosis is accompanied by up-regulation of caspase-3 that can be blocked by caspase protease inhibitors. These effects of glutamate on caspase-3 appear to be independent of changes in Fas receptor, but are associated with the rapid release of mitochondrial cytochrome c, which precedes changes of caspase-3 protein levels leading to apoptotic cell death. This process was differentially inhibited by estrogens with the novel equine estrogen Δ 8 , 17β-E 2 being more potent than 17β-E 2 , To our knowledge, this is the first study to demonstrate that equine estrogens can prevent glutamate-induced translocation of cytochrome c from mitochondria to cytosol in rat primary cortical cells. Methods Materials All primary and secondary antibodies and other reagents were purchased from commercial sources: rabbit polyclonal antibody to caspase-3 (H-227: sc 7148 ) and goat polyclonal antibody to caspase-3 p11 (K-19: sc-1224 ). Both of these antibodies react specifically with precursor protein: Mr 32 kDa and the active caspase-3: Mr 20 (p 20) and 11 kDa (p 11) fragments. Rabbit polyclonal antibody to protein kinase c (P4334) (PKC, crossreacts with PKCα /and PKC δ) purchased from Sigma. Goat polyclonal antibody to Actin (I-19: sc1616 ) (all from Santa Cruz, California), mouse monoclonal antibody to Fas raised against Fas extracellular domain (CD95: AF435 ) (R&D System, Inc. Minnepolis, MN), mouse monoclonal antibody cytochrome c (7H8.2C12) (BD PharMingen, Mississauga, ON). Secondary antibodies were obtained from Sigma (Saint Louis, Missouri). SuperSignal ® West Pico Chemiluminescent Substrate (Pierce, Rockford, IL). Z-Val-Ala-DL-Asp (OMe)-fluoromethylketone (z-VAD-fmk) and z-Asp (OMe)-Glu (OMe)-Val-DL-Asp (OMe)- fluoromethylketone (z-DEVD-fmk) (Bachem Bioscience Inc., King of Prussia, PA) and the Cytotox 96 Non-radioactive Cytotoxicity Assay Kit (Promega G1780, from VWR, Toronto, Ontario, Canada). Neurobasal, B27, Hanks' buffered salt solution (HBSS) and antibiotics were from Gibco Life technologies (Burlington, Ontario, Canada). Active Caspase-3 Detection FITC kit # 280000 was obtained from Stratagene (La Jolla, CA). Animals Timed pregnant Sprague-Dawley rats were obtained from commercial sources. All procedures were reviewed and approved by the Institutional Animal Care and Use Committee at St. Michael's Hospital, University of Toronto, in accordance with the guidelines of the Canadian Council on Animal Care. Cell culture Eighteen to twenty day old pregnant Sprague-Dawley rats were sacrificed by cervical dislocation. The fetuses were delivered and fetal skulls were carefully opened and the meninges were removed and the brain processed essentially as described by Brewer et al. [ 64 ]. Briefly, two pieces of cortex (0.5–1.5 × 1–2 mm) were cut from the posterior dorsal surface and dissociated in HBSS (Ca 2+ and Mg 2+ free). Following dissociation, cells were suspended in 2 X volume of HBSS with Ca 2+ and Mg 2+ and allowed to settle for 3 to 5 minutes. The supernatant was transferred to new tubes and centrifuged at 1100 rpm (200 g) for 1 min. The pellets were suspended in HBSS buffer. Fresh cell suspensions were plated onto poly-L-lysine-coated 96-well plates or 6-well plates containing neurobasal medium supplemented with 2% B27, 0.5 mM L-glutamine, 25 μM glutamic acid, 120 mg/L penicillin and 260 mg/L streptomycin and incubated at 37 C in a humidified incubator with 5% CO 2 /95% air. Culture medium was replaced every other day. Experiments were performed on days 7 to 8 of cultures in neurobasal medium containing 2% antioxidant free B27 supplement. Under these serum-free culture conditions, only neuronal cells survive. Determination of glutamate-Induced cell cytotoxicity During the development of this work, the lactate dehydrogenase (LDH) cytotoxicity assay (Promega G1780, VWR Toronto, Ontario, Canada) and the Cell Titer 96 ® Aqueous Non-radioactive cell proliferation assay (MTS assay, Promega G5430) were used to assess cell death and cell viability respectively [ 31 ]. In MTS (3-[4,5-dimethylthiazol-2 yl]-5 [3-carboxymethoxyphenyl]-2H-tetrazolium, inner salt) the formation of a formazan product occurs only in live cells, however, once treated with MTS, the cells are not useable for other measurements. We have previously demonstrated [ 31 ] a strong correlation between the MTS and LDH assays [ 31 ]. Since we wanted to use the cells for various determinations, the LDH assay for cell death was selected as in this assay only the supernatant is used and the cells can be used for other measurements. Thus after the initial determination of the glutamate dose response curve, only the LDH assay was used in all subsequent experiments. Cell death was measured by the Cytotox 96 Assay kit, which quantitatively measures the release of lactate dehydrogenase into the medium following cell lysis or cell death as described previously [ 13 ]. In all experiments, cultures were also treated with 0.1% Triton X-100 to lyse the cells, and LDH levels measured under these conditions, were taken as the maximal LDH release (100% cell death). The results were expressed as a percentage of maximum LDH release. Assessment of apoptosis by DNA fragmentation Apoptotic cells produce characteristic DNA ladders made up of nucleotide fragments which are visualized by staining with ethidium bromide following DNA-agarose gel electrophoresis. We used DNA fragmentation as one of the criteria for apoptotic cell death and this was determined as described previously [ 13 ]. Briefly, primary cortical cells isolated from 18 to 20 day old fetal rat brains were cultured for up to 7 days and then treated with 1 mM glutamate for 18 h. The cell monolayers were washed with ice cold TBS (20 mM Tris-HCl, pH 7.6, 137 mM NaCl) and then DNA was isolated as described previously [ 13 ]. The DNA (5 μg) was electrophoresed on 1.5% agarose gel for 1.5 h at 100 V. The DNA fragments were visualized by staining with ethidium bromide and detected under UV transillumination. Western blot analysis Protein levels of Caspase-3, Fas or PKC were determined by Western blotting using a polyclonal and monoclonal antibody raised against caspase-3 or Fas or PKC as described previously [ 13 ]. In brief, cells were washed twice with cold PBS, harvested using a cell scraper, and lysed in buffer (9 mM Na 2 PO 4 , 1.7 mM NaHPO 4 150 mM NaCl, pH 7.4), containing 1% Nonidet P-40 (Sigma, Toronto, Canada), 0.5% sodium deoxycholate, 0.1% SDS and 1 mM phenyl methyl sulfonyl fluoride) for 20 min on ice. Cell lysates were centrifuged at 10,000 g at 4 C for 10 min. The protein concentration was determined by Bradford method (BioRad, Toronto, Canada). Cell lysates containing 10 to 20 μg protein were added in equal volume of 2 X reducing sample buffer (100 mM Tris-CI pH 6.8, 200 mM dithiothretiol, 4% SDS, 20% glycerol, and 0.2% Bromophenol blue) and heated at 100 C for 3 min. The samples were electrophoresed on discontinuous 10% polyacrylamide gel electrophoresis under constant current (14–15 mA). Separated proteins were electrotransfered onto a Protan nitrocellulose membrane (Schleicher & Schuell Inc., Keene N.H). The blots were blocked with 5% non-fat milk in TBST (20 mM Tris-HCl pH 7.6, 137 mM NaCl, 0.05% Tween-20) at 4 C overnight and then incubated with primary antibody for 1 to 2 h at room temperature, washed three times with TBST and incubated (1–2 h) with appropriate horse radish peroxidase (HRP)-conjugated second antibody. The membranes were washed three times with TBST and the immunoblots were visualized on X-ray films after exposure to enhanced chemiluminescence reagent (ECL) (Amersham, Toronto, Canada). Actin bands were monitored on the same blot to verify consistency of protein loading. Briefly, the Immunoblots were stripped with TBST containing 0.04% sodium azide for 30 min at room temperature. The blots were probed with anti-actin primary antibody and second antibody (anti-goat) as described above. The molecular size of protein was determined by running pre-stained protein markers in an adjacent lane, omitting primary antibody as a procedural control. The band intensity was measured from scanned images using UN-SCAN IT Gel Automated Digitizing system, Version 5.1 (Silk scientific, Inc, Orem, Utah, USA), software. Statistics Each experiment was repeated at least three times and combined data were compared using the Student's paired t- test. Analysis of variance (ANOVA) when appropriate, was used for multiple comparisons. Significance was set at P < 0.05. Measurement of cytochrome c translocation The cell homogenates, mitochondria and cytosolic fractions were prepared essentially as described by Atlante et al. [ 54 ]. The cells were washed twice with ice-cold PBS, pH 7.4 and then suspended in cold isotonic buffer (250 mM sucrose, 20 mM Hepes-KOH, 10 mM KCI, 1.5 mM MgCI 2 , 1 mM Na-EDTA, 1 mM Na-EGTA, 1 mM dithiothretiol and protease inhibitors-complete cocktail). After 15 min incubation on ice, cells were homogenized using a glass homogenizer (25 stokes) at 4 C. Cell homogenates were spun at 750 g for 10 minutes and the pellets containing the nuclei and unbroken cells were discarded. The supernatant was then centrifuged at 10,000 g for 15 minutes. The pellets containing mitochondria was stored at -80 C until processed. The 10,000 supernatant was further centrifuged at 100,000 g for 1 hour at 4 C to obtain the cytosolic fraction (S-100). The intactness of the mitochondrial membranes was checked by assaying mitochondrial specific enzyme glutamate dehydrogenase in mitochondria, mitochondria treated with Triton X-100 and in cytosol (S-100) as described previously [ 54 ]. GDH was only detectable in mitochondria and mitochondria treated with Triton X-100. It was undetectable in cytosol. Thus, the cytosol fraction was free of mitochondrial contamination. Following analysis of protein concentration, the levels of cytochrome c in these two fractions were analyzed by Western blot on 12% SDS-polyacrylamide gel. Cytochrome c was detected using murine anti-cytochrome c antibody as described above. Assessment of the effects of caspase inhibitors Caspase inhibitors were used as described previously [ 65 ] caspase-3-inhibitor z-DEVD-fmk and a pan-caspase-inhibitor z-VAD-fmk. The concentrations of inhibitors tested were 25, 50 and 100 μM. Both were prepared as 20 mM stocks in DMSO and stored at -80 C. Primary cultures were incubated with the inhibitors for 30 min prior to the addition of glutamate. Effects of inhibitors on glutamate-induced cell death were measured with LDH release assay as described above. Detection of active caspase-3 by immunofluorescence The primary cortical cells were cultured for 7 days in 96-well microplates and then treated with or without glutamate for 3 h and 6 h. Cells were then processed essentially as described by the manufacturer of the assay kit (Stratagene). Briefly, the microplates were centrifuged (200 × g) for 2 minutes and the supernatant discarded. The cells were fixed for 15 minutes following which the fixative was aspirated and the cells washed twice with phosphate buffered saline (PBS) containing 1% saponin. The cells were then incubated with the primary anti-cleaved caspase-3 antibody in PBS + 1% saponin for 30 minutes at room temperature. The cells were then rewashed and incubated for 30 minutes with secondary FITC-goat anti-rabbit antibody and then washed twice and the relative fluorescence measured using a microplate reader (excitation 490 nm; emission 520 nm). Authors' contributions YMZ is a postdoctoral fellow who participated in the development of the hypothesis, study design and carried out most of the experimental work and preparation of the manuscript. BB conceived the study and participated in the development of the hypothesis, the study design, and overall direction of the study and preparation of the manuscript.

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Dengue fever: new paradigms for a changing epidemiology

Dengue is the most important arthropod-borne viral disease of public health significance. Compared with nine reporting countries in the 1950s, today the geographic distribution includes more than 100 countries worldwide. Many of these had not reported dengue for 20 or more years and several have no known history of the disease. The World Health Organization estimates that more than 2.5 billion people are at risk of dengue infection. First recognised in the 1950s, it has become a leading cause of child mortality in several Asian and South American countries. This paper reviews the changing epidemiology of the disease, focusing on host and societal factors and drawing on national and regional journals as well as international publications. It does not include vaccine and vector issues. We have selected areas where the literature raises challenges to prevailing views and those that are key for improved service delivery in poor countries. Shifts in modal age, rural spread, and social and biological determinants of race- and sex-related susceptibility have major implications for health services. Behavioural risk factors, individual determinants of outcome and leading indicators of severe illness are poorly understood, compromising effectiveness of control programmes. Early detection and case management practices were noted as a critical factor for survival. Inadequacy of sound statistical methods compromised conclusions on case fatality or disease-specific mortality rates, especially since the data were often based on hospitalised patients who actively sought care in tertiary centres. Well-targeted operational research, such as population-based epidemiological studies with clear operational objectives, is urgently needed to make progress in control and prevention.

Introduction Dengue is the most important arthropod-borne viral disease of public health significance. Compared to nine reporting countries in the 1950s, today the geographic distribution includes more than 100 countries worldwide. Many of these had not reported dengue for 20 or more years and several have no known history of the disease. The World Health Organization (WHO) estimates that more than 2.5 billion people are at risk of dengue infection. Most will have asymptomatic infections. The disease manifestations range from an influenza-like disease known as dengue fever (DF) to a severe, sometimes fatal disease characterised by haemorrhage and shock, known as dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), which is on the increase. Dengue fever and dengue haemorrhagic fever/dengue shock syndrome are caused by the four viral serotypes transmitted from viraemic to susceptible humans mainly by bites of Aedes aegypti and Aedes albopictus mosquito species. Recovery from infection by one serotype provides lifelong immunity against that serotype but confers only partial and transient protection against subsequent infection by the other three. First recognised in the 1950s, it has become a leading cause of child mortality in several Asian and South American countries. The average number of DF/DHF cases reported to WHO per year has risen from 908 between 1950 and 1959 to 514,139 between 1990 and 1999. The real figure is estimated to be closer to 50 million cases a year causing 24,000 deaths. Of an estimated 500,000 cases of DHF/DSS requiring hospitalisation each year, roughly 5% die according to WHO statistics. Regional distribution of dengue and its serotypes are described elsewhere [ 1 , 2 ]. In summary, DF/DHF/DSS is an immediate problem in south and southeast Asia and Central and South America. Although DF is present in the African region, there are no cases or outbreaks reported to WHO [ 3 ]. Half the world's population lives in countries endemic for dengue, underscoring the urgency to find solutions for dengue control. The consequence of simple DF is loss of workdays for communities dependent on wage labour. The consequence of severe illness is high mortality rates, since tertiary level care required for DHF/DSS management is beyond the reach of most of the persons at risk. This paper reviews the changing epidemiology of the disease, focusing on host and societal factors and drawing on national and regional journals as well as international publications. It does not include vaccine and vector issues. Although each one of the issues taken up below merits an independent, in-depth treatment, we have selected only those issues where the literature raises challenges to prevailing views and therefore require further research, particularly given that most of these issues are key for improved service delivery in poor countries. Analysis Clinical presentation Dengue infection can cause a spectrum of illness ranging from mild, undifferentiated fever to illness up to 7 days' duration with high fever, severe headache, retro-orbital pain, arthralgia and rash, but rarely causing death. Dengue Haemorrhagic Fever (DHF), a deadly complication, includes haemorrhagic tendencies, thrombocytopenia and plasma leakage. Dengue Shock Syndrome (DSS) includes all the above criteria plus circulatory failure, hypotension for age and low pulse pressure. DHF and DSS are potentially deadly but patients with early diagnosis and appropriate therapy can recover with no sequelae. Case management for DF is symptomatic and supportive. DHF requires continuous monitoring of vital signs and urine output. DSS is a medical emergency that requires intensive care unit hospitalisation [ 4 ]. The increase in dengue mortality is considered to be a reflection of the increase in the proportion of DF patients who develop DHF/DSS. The pathogenesis of DHF/DSS is widely considered to be antibody-dependent enhancement in secondary infection with a virus of different serotype [ 5 ]. Evidence in support of this comes from many studies including from the Cuban epidemics of 1981 and 1997 [ 5 , 6 ] and a five-year study of Yangon (Myanmar) [ 7 ]. However, absence of a significant association between secondary infection or co-circulation of different serotypes and DHF/DSS has also been noted [ 8 , 9 ]. The disease is widely considered to be associated with secondary infection and co-circulation of several serotypes. Alternative or additional factors associated with severe illness, such as high viraemia titres, have also been suggested [ 10 ]. So far, this has been associated with secondary infection as demonstrated by Vaughn et al ., and Libraty et al . [ 11 , 12 ]. On the other hand, one expression of higher viral virulence could be higher viraemia leading to greater severity, but this has not not yet been demonstrated (Guzmán, 2003 personal communication). Viral virulence [ 13 ], immunological responses and increased pathogenicity of specific serotypes [ 14 ] have been implicated as critical for the appearance of DHF. This has been found for the three serotypes DEN1, [ 13 ] DEN 2 [ 15 ] and DEN 3 [ 8 , 10 , 16 , 17 ], but so far not for DEN 4 [ 18 ]. The evidence from different studies also shows that the pathogenesis of DHF/DSS may be multi-factorial and understanding remains incomplete. Epidemiological changes Demographic, economic, behavioural and social factors are often keys for effective communicable disease control and underpin successful public health programmes. Despite promising indications in the literature, these factors have remained poorly understood in the case of dengue. Furthermore, recent field evidence raises some questions regarding widely accepted characteristics of dengue that need review and confirmation. Shift in modal age DF is typically acknowledged to be a childhood disease and is an important cause of paediatric hospitalisation in southeast Asia. There is, however, evidence of increasing incidence of DHF among older age groups. Since the early 1980s, several studies in both Latin America and southeast Asia have reported a higher association of DHF with older ages. The earliest studies were by Guzmán (1981) in Cuba and Rigau-Pérez in Puerto Rico [ 6 , 19 ]. Later on similar observations were noted in Nicaragua and Brazil. In some southeast Asian countries where dengue has been epidemic for several years, this age shift is clearly observed, indicating an epidemiological change in dengue infection in those locations [ 20 - 22 ]. Three studies in Asia using surveillance data report increasing age of infected patients. In Singapore, surveillance data showed a shift in peak dengue mortality from paediatric ages (1973–1977) to adults in 1982, since which year more than 50% of the deaths occurred in patients older than 15 years. From 1990–96, the highest age-specific morbidity rates were in the 15 to 34 year age groups [ 23 ]. In Indonesia, surveillance data from 1975 to 1984 showed an increase in incidence rates among young adults in Jakarta as well as in the provincial areas [ 24 ]. Adults have accounted for proportions as high 82% of all cases in the hospital-based surveillance study during the 2000 epidemic of dengue in Bangladesh [ 25 ]; the highest proportion of cases occurred in the 18 to 33 year age group. All deaths in the Bangladesh outbreak in 2000 were in persons older than 5 years. In Puerto Rico, surveillance data analysis showed the highest incidence rate (11.8/1000) in the 10–19 year age group during an outbreak in 1994 and 1995 [ 26 ]. Hospital-based studies have similarly reported increasing infection rates among adults, mentioning that it is contrary to the popular belief that dengue is a paediatric disease [ 27 , 28 ]. The trend for increased incidence among young adults has important implications for control and prevention. Whether these are real increases (based on population distributions), increases in the proportion of DHF/DSS (and, hence, the proportion hospitalised) but not DF, or the result of improved classification and diagnosis needs clarification. Comparative incidence and case fatality ratios (CFRs) of severe illness in adults and children and the economic implications are discussed later. Racial predisposition Race-related susceptibility to dengue has been observed in a few studies and merits further investigation. In a retrospective seroepidemiologic study Guzmán reported that blacks and whites were equally infected with DEN-1 and DEN-2 viruses during the Cuban epidemics of 1977 and 1981, while severe dengue disease was observed less frequently in dengue-infected black persons than whites [ 5 , 6 ]. A study in Haiti observed that despite virologic pre-conditions hypothesised to be precursors for DHF (i.e. the evidence of previous infection by DEN virus types 1, 2 and 4), local children did not develop severe illness [ 29 ]. The authors concluded that this finding provides further evidence of a dengue-resistant genotype in black populations. In 1998 the Los Angeles County vital registration system reported DF/DHF incidence, but only among Hispanic and white ethnic groups (0.1 and 0.07/100,000) [ 30 ]. Genetic polymorphism in cytokine profiles and coagulation proteins has been proposed as a factor protecting persons of African origin [ 31 ]. Evidence for this hypothesis has been found in meningococcal disease, in which a genetic polymorphism in the gene encoding an essential protein involved in coagulation is a predictor for developing severe disease with lethal outcome. In Asia, two studies report racial differences in disease incidence. A 15-year study of the epidemiology of dengue reports a significantly higher incidence of DHF among Chinese compared to Malaysian males [ 32 ]. This finding was supported by a six-year surveillance data study in Singapore, which found the race-specific morbidity rate among the Chinese to be three times that of the Malays and 1.7 times that of Indians [ 23 ]. Although none of the above constitutes convincing evidence for the hypotheses, they highlight a useful area for better understanding of dengue pathogenesis and health service planning. Sex differences Understanding male-female differences in infection rates and severity of disease is important for public health control programmes. A few hospital-based studies and surveillance data show a male-female difference in infection rates and in severity of disease. Three independent studies from epidemics in India and Singapore found nearly twice the number of male patients compared to females (Lucknow and Singapore both report male to female ratios of 1.9:1 and Delhi 1:0.57) [ 33 - 35 ]. In his hospital-based study during the 1996 epidemic in Delhi, Wali reported an even higher ratio of 2.5:1 [ 27 ]. Another study during the same epidemic found a male to female ratio of 1:0.25 cases for DSS. However, of the three deaths in this sample, two were female [ 35 ]. Surveillance data from Malaysia revealed a male preponderance among Indian and Malay patients (1.5:1), but the ratio was almost equal for those of Chinese origin [ 32 ]. The Ministry of Health, Bangladesh reported a hospital patient DF/DHF male to female ratio of 1.5:1 during an outbreak in Chittagong in 1997 [ 36 ], although a later study of DHF only during the 2000 outbreak found no differences between sexes [ 37 ]. With the exception of the study by Shekhar, all the others were hospital-based and may represent those who sought care rather than the infected population [ 27 ]. Studies in South America generally report that both sexes are equally affected [ 26 , 38 ] although a male to female ratio of 0.65:1 was described as "typical" for dengue [ 9 ]. Kaplan, in a rare study testing for significance, found a higher proportion of women in all of his four Mexican samples (p < 0.001) [ 39 ]. Of significance are two studies in Asia by Kabra and Shekhar where severe illness and CFR were consistently higher among females despite higher incidence in males [ 32 , 40 ]. Halstead [ 41 ] had pointed out as early as 1970 that males predominate among those with milder disease but females account for more severe illness. He suggested that either immune responses in females are more competent than in males, resulting in greater production of cytokines, or the capillary bed of females is prone to increased permeability. Kaplan in Mexico suggests that an incidence bias in favour of females is related to the timing of the survey interviews, while Goh puts forward that low incidence among women occurs because they stay at home and are less exposed to infection [ 39 , 42 ]. No studies suggest gender bias in home care and male preferences in health care seeking, still prevalent in many Asian and other traditional societies. It is widely recognised that in many of the Asian communities, lower disease incidence in women may be a statistical artefact related to lower reporting and care-seeking for women from traditional practitioners who do not report to public surveillance systems. By the same token, women are less likely to be taken for care at a hospital when ill or are taken at late stages of disease, when no other options are available. Determining sex differences, both in infection and severity of disease, requires well-designed and targeted studies to capture both biological and social factors that drive disease patterns in a community. Rural spread Historically, DF/DHF has been reported as occurring predominantly among urban populations where density of dwellings and short flying distance of the vector create the right conditions for transmission. However, the literature shows that dengue transmission and, in some cases, outbreaks occur in rural settings in both Asia and Latin America. In the WHO Western Pacific region, WHO has confirmed that disease spread into rural areas from where it had not been reported previously [ 20 ]. Rural epidemics occurred as early as 1976 in Indonesia, and in 1994 the outbreak in Laos began in a remote, rural district of Nasaithong [ 43 , 44 ]. Today, Thailand has an incidence rate that is higher in rural (102.2 per 100,000) than urban areas (95.4 per 100,000) [ 28 ]. Similarly, in India, entomological investigation showed a widespread distribution of Aedes aegypti , both in rural and urban areas during an outbreak in Gujarat in 1988 and 1989 [ 45 ]. Increase in DF/DHF among rural populations is also observed in Central and South America and identical rates in both populations are reported [ 9 ]. Among jungle dwellers in Peru, antibody prevalence up to 67% compared to 66% among the urban population have been found [ 46 ]. In industrialised settings, the Centers for Disease Control and Prevention (CDC) reported an outbreak of DF among residents of the rural towns of Hana and Nahiku in Hawaii in 2001. The outbreak was historically unusual because infection occurred among residents who have no history of recent travel and the Aedes aegypti mosquito has not been seen in Hawaii since it was supposedly eradicated by pesticide spraying in 1943 [ 47 ]. Increased transport contact, mobility and spread of peri-urbanisation have been the most frequently cited reasons for spread of dengue to rural areas [ 48 ]. While some rural incidence linked to travel contact with urban areas is conceivable, outbreaks and infection rates equal to those in urban areas warrant further investigation. Improved reporting could also be a factor, but it would be less likely in areas such as Hawaii, USA. Standard epidemiological techniques such as spatial studies of cases and careful patient histories could shed further light into transmission patterns in rural populations. Health service structures and utilisation patterns differ substantially between urban and rural areas in many tropical countries and contextually appropriate strategies will be required for effective impact. Seasonality and climate variability The incidence and, in particular, epidemics of dengue have been commonly associated with the rainy season, and the El Niño phenomenon has been incriminated in the increases of certain vector-borne diseases, including dengue [ 49 , 50 ]. Despite the number of studies, convincing data or models supporting these hypotheses are scarce. The relationship between temperature, rainfall and vector-borne disease are increasingly seen as oversimplifications. A study modelling DF transmission and seasonal temperature on data from Puerto Rico from 1988 to 1992 revealed weak relationships between monthly mean temperature and incidence of DF [ 51 ]. The study concluded that factors related to history of herd immunity, introduction of new serotype or demographic transitions influence transmission. More recently, long-term meteorological trends were studied in four high-altitude sites in East Africa, where increases in malaria have been reported in the past two decades [ 52 ]. They did not observe any significant change in temperature, rainfall, vapour pressure and the number of months suitable for P. falciparum transmission in the past century or during the period of reported malaria resurgence. Others have questioned models linking global temperatures and disease incidence, stating that, historically, climate has rarely been the principal determinant of vector-borne disease prevalence. Neither does the literature provide an adequate evidence base establishing the impact of climate change on vector-borne disease [ 53 , 54 ]. The "bandwagon" of El Niño [ 55 ] and dengue incidence is now placed under scrutiny and further research will have to be done before climate variations can be nailed down as a culprit. Health systems issues Socio-economic context Social and economic factors play an essential role in the incidence and prevalence of DF and DHF. Air conditioning, screens and safe water supplies in wealthier countries help prevention and better health services reduce or eliminate mortality from DHF. Unplanned urbanisation and inadequate resources for vector control are factors that promote transmission and are characteristic of poor rather than richer countries. Reiter et al . (2003) studied dengue transmission on the Mexico-USA border and found higher rates in the Mexican city compared to the American one [ 56 ]. However, some anomalies persist despite the rich/poor divide in disease incidence. Despite energetic control programs in the wealthier endemic countries of southeast Asia such as Singapore, Malaysia and parts of China (eg. Hong Kong), dengue continues to be a problem. Malaysia reports some of the highest numbers of cases during epidemics compared to other countries in the region. In some of these cases, particular traditional practices, such as rainwater storage on roofs, expose them to higher risk. The major epidemic in Puerto Rico in 1977 serves as a reminder that advanced public health capacities and economic development may not guarantee protection against massive epidemics [ 9 ]. Despite high quality of health services and richer circumstances, complacency in endemic countries may lead to increased rates without continued vigilance. On an individual level, evidence points to greater susceptibility among well-nourished or middle-class communities rather than malnourished and poorer patients commonly associated with other tropical diseases. A case-control study of serologically-confirmed DHF patients, other infectious diseases patients and healthy children in the Children's Hospital in Bangkok showed that malnutrition amongst DHF patients was significantly lower [ 57 ]. In India, a hospital-based study found no association between nutritional status and severity of illness [ 40 ]. Middle classes have been specifically noted as the proportionally predominant group during the epidemic in Dhaka Bangladesh [ 25 ] and upper social classes had statistically higher sero-infection rates in Fortaleza and San Luis epidemics in Brazil [ 38 ]. Confounding factors for the preponderance of DF/DHF among the upper classes or well-nourished dengue patients were not discussed in any of these studies. Few studies specifically measure and test socio-economic determinants of exposure at community levels. Heukelbach in Fortaleza, Brazil did examine socio-economic variables but their study did not show an association with DF [ 58 ]. Since all 34 cases selected were from a shanty town (favela), a lack of heterogeneity may have been a factor for this result rather than a real absence of difference. In Taiwan, Ko, also in a case-control study, observed that patients who lived near markets and/or open sewers or ditches had a risk of contracting disease 1.8 times higher than those who did not [ 59 ]. Since housing near sewers and ditches is likely to comprise poorer families, the analysis should have tested for house site while controlling for use of screens, which were significantly associated with incidence. Costs On a macro level, the impact of dengue on the economy is likely to be increasingly similar to that of malaria. Prevalent in communities characterised by subsistence or daily wage labour, a week's illness can be catastrophic for poor families. As a primarily paediatric disease in the past, the active labour force or the family wage earners were less affected. Now, as the modal age of illness and incidence increases, losses in productivity and earning capacity may be expected. The economic lesson from malaria was learnt late and when the resurgence was already in full swing. Dengue fever risks the same fate. With regard to costs of care, few economic studies exist and most estimate economic loss to range in millions. Von Allmen et al . undertook a cost analysis of the epidemic of DF/DHF in Puerto Rico in 1977 using upper and lower limits of incidence [ 60 ]. They estimated the direct costs (medical care and epidemic control measures) to range between US$2.4 and $4.7 million and indirect costs (lost production of patients and parents of children) between US$6 and $15 million. Another economic study, still in Puerto Rico, assessed the loss in terms of DALYs due to dengue [ 61 ]. At 658 DALYs per year per million population, the study concluded that, in terms of its magnitude, DF ranks with TB, STDs (including HIV), childhood diseases or malaria. On a micro level, a detailed study on costs of care in 3 hospitals in Bangkok estimated direct adult patient costs at US$67. Including opportunity costs, this figure increased to US$161.49. The net hospital cost for each DHF patient was US$54.60 and the public sector cost of prevention and control of the outbreak was US$4.87 million. The total expenditure for DHF in 1994 was estimated to be at least US$12.56 million, of which 45% was borne by the patients [ 62 ]. These figures are reminders that most of the countries subject to DF/DHF cannot realistically afford a US$5 million prevention and control budget for a single disease and that the monthly income of many families in these countries is less than the direct cost of US$70 a month. Knowledge, attitude and practice (KAP) Much needs to be done in finding effective strategies for behaviour change. Since mothers are the first-line care-givers, this aspect is key, particularly for childhood diseases. KAP studies are rare and therefore little is known regarding knowledge and attitude of the exposed population towards dengue. However, the little that is known is encouraging. Straightforward community education to reduce breeding sites for mosquitoes performed better than chemical spraying in a controlled experiment in Mexico [ 63 ]. However, housewives, the unemployed and the elderly had significantly lower levels of knowledge of the disease compared to students and persons of younger ages (odds ratio (OR) = 0.44, 95% confidence interval (CI): 0.31–0.64). Other KAP studies have found that radio and television are very effective channels for knowledge dissemination. Nevertheless, these same studies found that while communities can score well in knowledge of the disease, they perform less well in attitude and practice, indicating that behaviour change is one area to target in social mobilisation programmes [ 64 - 67 ]. Treatment-seeking behaviour and lay symptom assessment is the first step in the chain to early diagnosis and was found to have an impact on duration of illness in Thailand [ 68 ]. In that context, it is discouraging to note that 45% of individuals in a population-based survey (23,970 households) in the urban municipality of Vientiane did not know what action to take when their children are diagnosed with dengue or what they should do for prevention [ 44 ]. Finally, reducing mortality from DHF and strengthening its control and prevention clearly cannot be done by the population alone. In most circumstances, these are poor populations with other pressing agendas. The programme requires public sector leadership with strong intersectoral collaboration. The WHO has made important progress to determine ways and mechanisms through which to achieve collaboration between sectors and state policy directions for control. Trends in case fatality rates Two aspects present themselves for useful discussion in this area. One relates to wide variations in CFRs between countries, sub-national units and hospitals under similar virological conditions. The other relates to differential risks of severe illness and mortality between children and adults. The global case-fatality rate (CFR) for DHF/DSS has been declining in most of the endemic countries according to government statistics. The overall CFR in the southeast Asia region is now less than 1% [ 20 ]. However, disaggregated data reveal a different picture. Rates vary significantly between countries, provinces and hospitals, pointing to a more complex situation. From 1995–2000, the CFR in the countries of WHO Western Pacific Region ranged from 0.06% in Singapore and 0.17% in Malaysia to 3.4% in Cambodia. Hong Kong reported no deaths [ 69 ]. In Vietnam, province-based 1998 data for DHF show CFR ranging from nearly 13% in Ha Tinh to 0.5% in Quang Tri [ 70 ]. Although the four provinces with the highest CFR were at some distance from Ho Chi Minh City or Hanoi, the four of the lowest were not particularly closer to these centres of tertiary care. In Laos, on the other hand, CFR for DHF during 1998 reached a high of 9.7% in Champassak province compared to 1.4% in Municipality of the capital city, Vientiane [ 44 ]. Wide variation in CFRs ranging from 0.1% to 5%, was also noted between the first administrative divisions in the Philippines [ 71 ]. During the 1998 epidemic in Cambodia the CFR in Kantha Bopha, a private, charitable hospital, was substantially lower (1.96%) than the national average (2.91%) [ 70 ]. Inter-district and inter-hospital variation is generally indicative of quality of care. Availability of medical supplies, equipment and economic status of patients can explain some differences but analyses to distinguish between the performances of provinces and countries in comparable settings would be useful for designing more effective disease control. Secondly, studies have postulated higher risk of DF/DHF morbidity and mortality among children compared to adults [ 15 ]. Recently, increasing reports of severe illness among adults and in some cases higher CFRs (e.g. age-specific CFRs from San Lazaro hospital over one year were 3.8% for 35–39 year olds, 8% for over 45s compared with 2% and 2.6% for 0–4 and 5–9 year age groups) merit closer looks at determinants of adult mortality [ 37 , 71 , 72 ]. Case management and early detection In addition to vector control, widely recognised as a preventive strategy of choice, key health sector response for reduction of mortality and morbidity lies primarily in two areas: early detection (including care-seeking behaviour change and better surveillance) and improved case management of patients. Mortality in excess of 1% may be considered the consequence of inadequate care, late diagnosis and delayed hospitalisation. A hospital-based study during the dengue outbreak in Delhi revealed that mortality could be very low in patients who came early to the hospital [ 73 ]. Late presentation was also strongly associated with increased mortality in children with DHF in the Philippines [ 74 ]. The short interval between onset of haemorrhage and death, especially in young children, makes rapid medical intervention for DHF/DSS a critical factor for survival. For most communities at highest risk of disease, intensive care facilities are only available at distant capitals requiring motorised transport, usually beyond the reach of many. Early diagnosis and leading indicators for DHF/DSS can ensure the availability of travel time to transfer the patient for effective treatment. Case-control studies have shown that low-normal hematocrit count at time of shock is a significant risk factor for haemorrhage [ 75 ] and potential predictors for clinical outcome, such as decrease in total plasma cholesterol, and high- and low-density lipoprotein, were associated with the severest cases [ 76 ]. However, research into predictive factors for severe illness is neither abundant nor conclusive. Moreover, as Van Gorp concludes, low capacity and lack of resources at secondary levels of health services limit the operational use of many of these findings [ 76 ]. At this time, the WHO classification of dengue diseases is often not feasible in many countries because of lack of trained health professionals, inadequate laboratories, and radiological support. Neither are facilities to detect DHF by using hematocrit and plasma leakage signs readily available in many tropical countries. As successful treatment of dengue depends on symptom recognition and careful fluid management, a simpler dengue disease classification scheme, realistic in poor, provincial conditions and better training of district-level personnel is needed. A few creative approaches to primary health care to improve quality of care and case management at primary health care levels have been reported in the literature. For example, encouraging results have been found in Vietnam where they reduced dengue mortality rates by 64% through innovative primary healthcare concepts, including paediatric priority training units for medical staff, health education for patient carers and promotion of outpatient treatment to avoid unnecessary admissions [ 77 ]. Reduction of CFRs from 10–15% (40% in some areas) in the early 1950s to less than 0.5% today in east Asian referral hospitals have been attributed to better training of the hospital staff [ 78 ]. The effect of strengthened health systems is recognised by public health authorities including WHO but is missing operational research and policies to put them into effect. Surveillance and reporting Unreliable statistics are an extremely serious weakness from many perspectives. Estimates of DHF/DSS CFR from surveillance data are consistently lower than those from single sample study data suggesting under-reporting or misclassification of deaths. Inadequate knowledge of case definitions among district health personnel compromise complete reporting even within the public health service system. Inappropriate denominators further add to the confusion in estimating prevalence and incidences. Reporting deviations can lead to seriously misleading CFRs in countries where reliable estimates are urgently needed for effective resource programming. In Laos, for instance, 8197 DHF cases and 24 deaths were registered by the WHO in 1996, compared to 2563 cases and 23 deaths registered by the Institute of Malariology, Parasitology and Entomology (IMPE) for a CFR of that is 3 times higher than WHO statistics [ 22 ], Most national surveillance data rely only on public sector institution reporting. An evaluation of the dengue reporting system in Bandung, Indonesia (covering private and public hospitals) found that only 31% of hospitalised DHF/DSS cases were reported to the Municipal Health Authorities [ 79 ]. In Puerto Rico, a hospital record review revealed a ratio of 3:1 total DHF cases compared to those detected by surveillance [80]. More alarmingly, in Texas, USA, an assessment of underdiagnosis of dengue was undertaken motivated by an outbreak in a town across the border in Mexico. A review of medical records between 23 July and 20 August 1999 found that 50% of suspected cases had undiagnosed dengue infection. [81]. Based on the above studies, a conservative estimate would be that a third of the total cases are captured by surveillance systems, indicating that the global incidence rate could be around 1.5 million cases of DHF on an average year rather than the 0.5 million estimated by WHO. While complete surveillance data may be an unrealistic option in many the affected countries, sentinel surveillance and sample surveys using reliable methodologies could be undertaken to provide more accurate estimates of the disease burden and fill in the gaps. Occasional sample surveys of the private sector could help better estimate the bias in disease burden. Conclusion On 18 May 2002, the WHO General Assembly confirmed dengue fever as a matter of international public health priority through a resolution to strengthen dengue control and research. Today, changing characteristics of the disease deserve serious research attention. Shifts in modal age, rural spread, social and biological determinants of race- and sex-related susceptibility have major implications for health service planning and control strategies. Behavioural risk factors, individual determinants of outcome and leading indicators of severe illness are poorly understood, compromising the effectiveness of control programmes. Early detection and case management practices have been noted as a critical factor for survival. Yet well-targeted operational research in these areas is rare. Population-based epidemiological studies with clear operational objectives should be launched as concerted efforts at regional levels. A major weakness is the inadequacy of sound statistical methods in some of the reviewed studies. Samples are exceedingly small in many cases, selection methods are often inadequately described or are self selecting, tests of significance are frequently not undertaken or not reported and denominators are not clearly described. Conclusions therefore do not have the full benefit of objective statistical analyses, reducing the scientific strength of the results. Furthermore, conclusions regarding case fatality or disease-specific mortality rates are hard to draw since they are frequently based on hospitalised patients who had actively sought care in tertiary centres. However, a systematic approach and a clear international research agenda can quickly bring forward the frontiers of knowledge. Better understanding of the above will not only feed into operational policy for dengue control, but also provide fertile terrain for vaccine application strategies in the future. Today, dengue control and prevention requires thinking outside the tropical disease box. Many of the affected countries are some of the poorest. Approaches that are realistic for their infrastructure need to be urgently developed. Competing interests The author(s) declare that they have no competing interests. Authors' contributions Debarati Guha-Sapir set out the plan of the paper, its focus areas and main messages. Barbara Schimmer carried out the literature search, summarised the studies and their results. She helped D. Guha-Sapir with the writing of the text.

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545599

Insertion bias and purifying selection of retrotransposons in the Arabidopsis thaliana genome

An analysis of the distribution and age of LTR retrotransposons in the Arabidopsis genome revealed that Pseudoviridae insert randomly along the chromosome and have been recently active whereas Metaviridae were more active in the past and preferentially target heterochromatin.

Background It has become increasingly clear that the activity of transposable elements (TEs) is a major cause of genome evolution. TEs are ubiquitous components of eukaryotic genomes. For example, 22% of the Drosophila melanogaster [ 1 ], 45% of the human [ 2 ], and up to 80% of the maize [ 3 ] genomes consist of TE fossils. TEs have influenced the evolution of cellular gene regulation and function, and have been responsible for chromosomal rearrangements [ 4 ]. Variation in genome size and the C-value paradox [ 5 ] can be attributed to a large extent to differences in the amount of TEs, particularly of retrotransposons, between the genomes of different species [ 6 ]. In plant genomes, large size and structural variation even among closely related species is mainly due to differences in their history of polyploidization [ 7 ] and/or amplification of long terminal repeat (LTR)-retrotransposons [ 3 , 8 - 10 ]. LTR-retrotransposons (LTR-RTs) are 'copy-and-paste' (class I) TEs that replicate via an RNA intermediate. Like retroviruses, their (intact) genome consists of two LTRs, which contain the signals for transcription initiation and termination, flanking an internal region (IR) that typically contains genes and other features necessary for autonomous retrotransposition. LTR-RTs are mainly classified into two major families, the Pseudoviridae (also known as Ty1/Copia elements) and Metaviridae ( Ty3/Gypsy ). The evolutionary forces that control copy number and shape the chromosomal distribution of different kinds of TEs in eukaryotic genomes are still poorly understood. Some large plant and animal genomes have expanded owing to an ability to tolerate massive amplification of retrotransposons, whereas in more compact genomes these elements are found in lower copy numbers, non-randomly distributed and mainly confined to heterochromatic regions [ 11 - 14 ]. TEs have mostly been regarded as parasitic DNA [ 15 , 16 ], and it has been suggested that important epigenetic mechanisms originally evolved to suppress the activity of TEs and other foreign genetic material [ 17 ]. Nevertheless, there are examples of individual elements that have been co-opted by, and entire TE families that have become mutualists to, their host genomes [ 13 ]. It is often hypothesized that the non-random genomic distribution of TEs in some species reflects the action of purifying selection on the host against the deleterious effects of TE insertions in certain regions. Models differ in the kind of deleterious effects they propose: chromosomal rearrangements due to 'ectopic' (unequal homologous) recombination [ 18 ]; disruption of gene regulation due to insertion near cellular genes [ 19 ]; or a burden on cell physiology as a result of the expression of TE-encoded products [ 20 ]. In compact genomes, clustering of TE insertions in silent heterochromatin, which has reduced rates of recombination, gene density and levels of transcription, is in principle consistent with a scenario of negative selection and of passive accumulation of TEs where their insertions would be less deleterious. As an alternative to purifying selection, another hypothesis to explain this clustering of TEs involves preferential insertion, or even positive selection for their retention, into heterochromatin [ 21 ]. To evaluate these hypotheses, I investigated the evolutionary history of different groups of LTR-RTs in the Arabidopsis thaliana genome. The total TE content of the compact genome of A. thaliana , with a haploid size of approximately 150 Mbp (million base-pairs), has been previously estimated as around 10%, and is known to cluster around the pericentromeric heterochromatin [ 14 ]. Despite the relatively low copy numbers, there is a high diversity of LTR-RTs in A. thaliana [ 22 , 23 ]. I have implemented an automated methodology for genome-wide sequence mining of LTR-RTs, and for estimating the age of insertion of different copies. This methodology is capable of identifying nested insertions, which are common in the pericentromeric regions. The technique for dating LTR-RTs has been previously used to reveal a massive amplification of these elements that doubled the size of the maize genome during the last 3 million years, by extrapolation of results found in a 240 kbp stretch of intergenic DNA [ 3 ]. Here I report genome-wide age profiles for different groups of LTR-RTs in A. thaliana . By comparing the age and chromosomal distributions of young and old insertions it is possible to distinguish between preferential targeting and passive accumulation of elements into heterochromatin. I show that members of the Pseudoviridae have recently been active, that they integrate randomly into the genome (relative to centromere location) and only passively accumulate in proximal regions, as purifying selection eliminates euchromatic insertions. In contrast, the Metaviridae (particularly members of the Athila group) preferentially insert into the pericentromeric heterochromatin, and their transpositional activity has declined in the last million years. Results Abundance and diversity Most of the retrieved elements are fragmented and truncated, and nested insertions are common particularly among pericentromeric elements belonging to the Athila superfamily, though the core centromere sequences themselves were not available. In fact, the size of the A. thaliana genome has been recently estimated as approximately 157 Mbp (around 20% larger than the estimate published with the genome sequence), and the additional size appears to be due to (unsequenced) heterochromatic repetitive DNA in the centromeres, telomeres and nucleolar-organizing regions [ 24 ]. Table 1 shows the relative abundance of each superfamily, and the numbers of complete and solo-LTR elements identified in the genome. Athila is the most abundant superfamily, followed by the Copia -like, Gypsy -like, and TRIM (terminal-repeat retrotransposons in miniature). The ratio of solo-LTRs to complete elements is around 2:1. In addition to solo-LTR formation, deletion and fragmentation of retrotransposon DNA in A. thaliana also occur via other mechanisms: 36% of the DNA in the Athila , 38% in the Gypsy -like, 32% in the Copia -like, and 21% in the TRIM superfamilies correspond to degraded insertions that are neither 'complete' elements nor solo-LTRs. Age distribution To obtain the genome-wide age distribution of each superfamily (except TRIM), 564 pairs of intra-element LTRs were (pairwise) aligned and their sequence divergence estimated. Many of the complete TRIM elements have highly divergent LTRs, and I suspect that extensive recombination between inter-element LTRs has occurred. In neighbor-joining trees of LTR sequences (of both complete and solo elements) from the TRIM families Katydid-At1 and Katydid-At2 , most intra-element LTR pairs did not cluster. In contrast, when trees were constructed for representatives of the Athila ( athila2 ), Gypsy -like ( atlantys2 ), and Copia -like ( meta1 , atcopia49 , atcopia78 ) superfamilies, intra-element LTR pairs always clustered (data not shown), providing evidence for the lack of inter-element recombination in those 'families'. The superfamilies differ significantly in their average age of insertions. Athila insertions are significantly older than the Gypsy -like (Wilcoxon rank-sum test, p < 0.0005), Gypsy -like older than Copia -like ( p < 0.0001). Age distributions are summarized in Figure 1 . Copia -like insertions are younger than host species Using the rate of 1.5 × 10 -8 substitutions per site per year [ 25 ], 97% of 215 complete Copia -like elements are younger than 3 million years (Myr), 90% younger than 2 Myr, and only two insertions estimated to be older than 4 Myr. This shows that complete insertions from the known Copia -like families in the A. thaliana genome are younger than the species itself, whose time of divergence from its closest relatives, such as A. lyrata has been estimated (with the same rate of evolution) to be 5.1-5.4 Myr ago [ 25 ]. The situation is less clear for Athila (and the Gypsy -like TEs), as 7% of 219 intra-element LTR pairs were estimated to be older than 5 Myr (3% of the Gypsy -like). Furthermore, the Athila and Gypsy -like superfamilies have an excess of degraded insertions relative to Copia -like (Table 1 ). Complete elements account for around 50% of the total amount of DNA in Athila and Gypsy -like, indicating that the majority of insertions remaining in the genome have been degraded or have become solo-LTRs. Some of these are likely to be older than the complete insertions. DNA loss (from LTR-RTs) has been shown to occur in A. thaliana [ 26 ], and the oldest insertions may have been degraded beyond detection. On the other hand, there is some evidence that synonymous sites in Arabidopsis are not evolving in a completely neutral fashion [ 27 ]. If this were the case for the chalcone synthase ( Chs ) and alcohol dehydrogenase ( Adh ) loci, their synonymous sites would be evolving more slowly than LTR-RT fossils, and the dating method described above would systematically overestimate the ages of their insertion events. Athila and Gypsy -like elements were more active in the past The age distribution of complete Copia -like elements appears to show a recent burst of activity (Figure 1 ), but I provide evidence (below) that the excess of very young elements is the result of the rapid (relative to Metaviridae insertions) elimination of these elements from the genome. In contrast, the age distributions of complete Athila and Gypsy -like insertions have peaks between 1 and 2 Myr ago (Figure 1 ). Moreover, whereas there are 34 Copia -like insertions with their intra-element LTRs identical in sequence, only four such Athila and three such Gypsy -like insertions are present. These results indicate that levels of transpositional activity of Athila and Gypsy -like elements have declined since their peak between 1 and 2 Myr ago. Physical distribution The chromosomal distribution of retrotransposons (and other TEs) in A. thaliana has been known to be non-random and dominated by a high concentration of elements in the heterochromatic pericentromeric regions [ 14 ]. However, this study has revealed significant differences in the chromosomal locations of the LTR-RT superfamilies. I have analyzed the distribution of complete elements and of solo-LTRs in each superfamily along all the chromosome arms combined, relative to the position of the centromeres (that is, the distribution of the distances between each insertion and the centromere, divided by the length of the respective arm), with results summarized in Figure 2 . Athila elements are almost exclusively inserted in the pericentromeric regions, and the other superfamilies in significantly and progressively less proximal regions of the chromosome arms (Wilcoxon rank sum tests: Athila more proximal than the Gypsy -like, p < 0.0001; Gypsy -like more proximal than Copia -like, p < 0.0001; complete Copia -like elements more proximal than complete TRIM elements, p < 0.05; there is no difference between Copia -like and TRIM solo-LTRs). Furthermore, except for TRIM, within each superfamily the solo-LTRs are significantly more distal than the complete elements (Wilcoxon rank sum tests, p < 0.001), suggesting that formation of solo-LTRs is more likely to occur in distal regions. The distribution of complete TRIM elements relative to the centromere is not significantly different from random (goodness-of-fit test, χ 2 = 4.22, df = 3, p > 0.2), although sample size is small, while their solo-LTRs are significantly clustered (goodness-of-fit test, χ 2 = 10.70, df = 3, p < 0.02). Accumulation in proximal regions by distinct evolutionary mechanisms: purifying selection and insertion bias The results above indicate that the older a superfamily is, the more its elements are concentrated in the proximal regions. This suggests that insertions into proximal (heterochromatic) regions are more likely to persist for longer periods of time. This interpretation assumes that the neutral mutation rate is the same for both the distal (euchromatic) and proximal (heterochromatic) portions of the genome. Intra-genomic variation in the per-replication mutation rate has been reported between the two sex chromosomes of a flowering plant [ 28 ] (although the difference could not be explained their different degree of DNA methylation, a feature often associated with heterochromatin). Given that the dating method used here is based on neutral sequence divergence (between intra-element LTRs), a higher mutation rate in heterochromatin in A. thaliana would affect age comparisons among different groups of elements, as they show different degrees of clustering into the pericentromeric heterochromatin. However, older estimates for the age of heterochromatic elements are consistent with the hypothesis that heterochromatin is a 'safe haven' where TE insertions persist for longer periods of time. Here I show that the mechanisms that led to the accumulation of LTR-RTs in proximal regions are distinct for different groups: elements of the youngest superfamily ( Copia -like) insert randomly into the genome (relative to the location of the pericentromeric heterochromatin), but there is negative selection (on the host genome) against their insertions in euchromatin; elements of the older superfamilies ( Athila , Gypsy -like) preferentially insert into the pericentromeric regions. These distinct mechanisms become apparent when temporal and spatial data are combined (Figure 3 ), and the chromosomal distribution of young elements compared with the distribution of older elements (within each superfamily). For complete Copia -like elements there is a highly significant negative correlation between relative distance from the centromere and age of the insertions (Spearman rank correlation, ρ = -0.39, p < 0.0001). Furthermore, the distribution along the chromosome arms of 34 Copia -like insertions with no divergence between their intra-element LTRs is not significantly different from random (goodness-of-fit test, χ 2 = 3.12, df = 3, p > 0.3). This is evidence that Copia- like elements integrate randomly relative to the location of the centromeres, but tend to get eliminated from distal, and passively accumulate in proximal regions. The average time to fixation ( t ) for a neutral allele is given by t = 4 N e , where N e is the effective population size. For A. thaliana t can be estimated using an average of estimates of nucleotide diversity ( θ ) for 8 different A. thaliana genes, θ = 9 × 10 -3 [ 29 ], and the synonymous rate of substitution per site per generation, μ = 1.5 × 10 -8 [ 25 ]. t = 2 θ / μ , yielding an estimate of t ≈ 1.2 Myr. This value for t is consistent with an independent estimate that placed the time since the divergence between A. thaliana and A. lyrata between 3.45 t and 5.6 t [ 30 ]. Given that 75% of all complete Copia -like insertions are younger than 1.2 Myr, most of them are likely to be polymorphic. Taken together with the highly significant negative correlation between age and distance from the pericentromeric regions, these results indicate that complete Copia -like insertions are less likely to get fixed in the distal, euchromatic portions of the chromosome arms than in the pericentromeric heterochromatin. In contrast, there is no correlation between age and relative distance from centromeres for complete Athila elements (Spearman rank correlation, ρ = 0.01, p = 0.9), as both young and old insertions are found only in proximal regions (Figure 3 ), compartmentalized into the pericentromeric heterochromatin. This strongly suggests that elements in the superfamily have evolved to preferentially target the pericentromeric heterochromatin, and their genomic distribution, unlike that of Copia -like elements, is not the result of passive accumulation therein. Only if Athila insertions were much more deleterious than Copia -like ones, so that they would be very rapidly removed by purifying selection, could passive accumulation be the case. Gypsy -like insertions display a similar pattern to Athila . Even though there is for complete elements a significant, negative correlation between relative distance from centromeres and age, this is due to an excess of recent insertions near the telomere of the short arm of chromosome II (data not shown). If the arm is excluded from the analysis there is no significant correlation (Spearman rank correlation, ρ = -0.09, p > 0.3). This suggests that for the Gypsy -like also there is an insertional bias towards proximal regions. This bias is not as strong as for Athila , as complete Gypsy -like insertions are not exclusively found around the centromeres, and they cluster (to a much lesser extent) in at least one other heterochromatic region (the telomere of the short arm of chromosome II). Included in the Gypsy -like 'superfamily' is a clade of elements, known as Tat , which is a sister group to Athila to the exclusion of the remaining Gypsy -like elements [ 31 ]. The age and physical distribution of Tat does not differ from those of the remaining Gypsy -like elements (Wilcoxon rank-sum tests, p > 0.4); Tat show insertion bias towards the pericentromeric regions, but again to a lesser degree than Athila . Half-life of complete Copia -like insertions Given that Copia -like elements have been active until recently but tend to be eliminated by purifying selection, their age distribution (Figure 1 , bottom) reflects the process of origin and loss of complete elements, when averaged over evolutionary time scales (and over all Pseudoviridae lineages). If this is assumed to be a steady-state process, it can be modeled by the survivorship function: N ( K ) = N o e - aK , where N(K) is the number of elements observed with intra-element LTR divergence K , and N o and a are constants to be fitted. The rate of elimination can then be estimated by linear regression of the log-transformed data (the half-life of insertions is given by ln2/ a ). Figure 4 shows the fit for all complete Copia -like insertions ( R 2 = 0.94), and for complete insertions outside the proximal regions (i.e. with relative distance from centromeres >0.2; R 2 = 0.95). Complete Copia- like elements are eliminated from the genome with a half-life of 648,000 years ( SE = 48,000 years). Insertions exclusively outside the proximal (heterochromatic) regions are lost more rapidly, with a half-life of 472,000 years ( SE = 46,000 years). Discussion The results above indicate that within a single genome, distinct evolutionary mechanisms can lead to the non-random distribution of retrotransposons, as in A. thaliana the accumulation of insertions in the pericentromeric heterochromatin is the result of both insertion bias (for Metaviridae elements) and a lower probability of fixation in euchromatin (Pseudoviridae). It has recently been shown that most TE lineages in A. thaliana were already present in its common ancestor with Brassica oleracea (the two species diverged around 15-20 Myr ago), and that copy numbers are generally higher in B. oleracea [ 32 ]. The authors suggested that differential amplification of TEs between A. thaliana and B. oleracea was responsible for the larger genome of the latter. Here I have shown that the major LTR-RT families have been active in A. thaliana since its divergence from its closest relatives, such as A. lyrata . The transpositional activity of Metaviridae elements has declined relative to its level between 1 and 2 Myr ago, perhaps suggesting that the host genome has more efficiently suppressed their transposition since. However, Pseudoviridae ( Copia -like) elements in A. thaliana have been subject to constant turnover. They have been recently active and show no insertion bias, and I estimate that the half-life of a complete element inserted in the euchromatic (non-coding) regions of the chromosome arms as around 470,000 years. Most of these Pseudoviridae insertions are lost before they reach fixation, and the half-life estimate provides a measure of the pace at which natural selection on the host constrains the genomic distribution and copy number of Pseudoviridae insertions. Turnover of Pseudoviridae insertions, in contrast to the longer persistence of Metaviridae elements that have declined in activity, is consistent with the higher sequence diversity among the Pseudoviridae than the Metaviridae in A. thaliana (107 Repbase update (RU) 'families' represented in 215 complete Pseudoviridaeelements, 25 RU 'families' in 349 complete Metaviridae elements, where 'families' were defined on the basis of sequence divergence); frequent reverse transcription during transposition would be likely to lead to faster evolution than that generated by the host genome DNA polymerase error rate on chromosomal insertions. The lower probability of fixation in euchromatin relative to heterochromatin implies that insertions into euchromatin are more deleterious to the host (and perhaps that purifying selection is less efficient in heterochromatin due to a much reduced rate of recombination). TE density in the A. thaliana genome does not correlate with local recombination rate [ 33 ], providing some evidence against the ectopic recombination model for the deleterious effects of insertions (if the occurrence of ectopic and meiotic recombination positively correlate). Consistent with my results, the same study supports a model of purifying selection against insertions in intergenic DNA, by inferring that they are less likely to be found near genes [ 33 ]. As an alternative to selection, a neutral mutational process that deletes (part of the) insertions could in principle be driving the distribution of Copia -like elements, if such a process occurred more often in the euchromatic than in the pericentromeric regions of the genome, and if it were frequent enough. One mechanism that removes LTR-RT DNA from the genome is solo-LTR formation via unequal homologous recombination between intra-element LTRs. However, this mechanism cannot be the driving force shaping the distribution of complete Copia -like elements because Copia -like solo-LTRs are also non-randomly distributed and clustered in proximal regions (goodness-of-fit test: χ 2 = 13.71, df = 3, p < 0.005). Copia -like solo-LTRs are either eliminated faster from distal than proximal regions, like complete elements, or solo-LTR formation on average occurs more slowly than extinction for euchromatic insertions. Despite clustering around the centromeres, Copia -like solo-LTRs are significantly more dispersed than complete elements. This suggests that solo-LTRs do form before extinction for distal insertions, but are probably less efficiently eliminated (possibly because they are less deleterious to the host genome) than complete elements. Another known mechanism of (general) DNA loss operates via small deletions due to illegitimate recombination (between short repeats); this has been shown to occur in the A. thaliana genome by an analysis of internal deletions in LTR-RTs [ 26 ]. In Drosophila , rates of spontaneous deletions in euchromatin and heterochromatin do not seem to differ [ 34 ]. In A. thaliana the relative rates between the two chromatin domains are unknown, but fragmented (that is, neither solo-LTR nor complete) Copia -like insertions are as clustered around the centromeres as complete ones (goodness-of-fit test: χ 2 = 80.36, df = 3, p < 0.0001). Therefore small, spontaneous deletions cannot account for the genomic distribution of complete elements. Larger deletions (that remove the entire LTR-RT sequence) occurring primarily in euchromatin would be necessary to explain the observed accumulation pattern; if such a mechanism existed it would be an important force for genome size contraction. As there is no evidence for such mechanism, and given that I estimate that the half-life of (complete) insertions to be less than half the average time to fixation for a neutral allele, a lower probability of fixation in euchromatin relative to the pericentromeric heterochromatin is more likely to be driving the genomic distribution of Pseudoviridae elements. It is interesting to note that the integrase proteins encoded by LTR-RTs differ between the Pseudoviridae and the Metaviridae in their carboxy-terminal domains, as they have different characteristic motifs [ 35 , 36 ]. This is the least conserved domain of integrase, and has been implicated in the insertion preferences of certain families of LTR-RTs in different organisms [ 37 ]. Examples of families of LTR-RTs whose integrase carboxy termini have been shown to interact with chromatin are known for both the Metaviridae [ 36 ] and the Pseudoviridae [ 38 ], and manipulation of this domain to engineer the targeting specificity of LTR-RTs has also been achieved [ 39 ]. Athila elements have been known to be present in the A. thaliana core centromeric arrays of the 180-bp satellite repeats and are abundant in pericentromeric heterochromatin [ 40 , 41 ]. In this study I have shown that in contrast with the passive accumulation of Copia -like elements, the striking compartmentalization of both recent and older Athila insertions in the pericentromeric heterochromatin indicates that these elements actively target those regions, and represents an example of a group of retrotransposons that have evolved to colonize a particular 'genomic niche'. Passive accumulation could not explain the distribution of Athila insertions unless they were generally much more deleterious to their host than Copia -like ones. Given the absence of complete Athila insertions from euchromatin, any one insertion would have to be so deleterious as to be almost immediately eliminated by purifying selection, even from intergenic DNA. Rather, it is likely that Athila elements preferentially insert into the pericentromeric heterochromatin and it is possible that this group of elements has been co-opted to play a part in centromere function. There is some evidence that such hypothetical role cannot be that of cis -acting sequences [ 42 ], but it could be a structural one. Studies on the appearance of neocentromeres [ 43 - 45 ] point to some degree of epigenetic regulation and function of centromeres via chromatin structuring. Although centromeric sequences are not conserved among plants [ 46 ], centromere-specific families of LTR-RTs seem to be common, as they have been found in cereals [ 47 - 51 ], chickpeas [ 52 ] and A. thaliana [ 40 ]. Both purifying selection (at the host level) against insertions (in euchromatin) and a decline in transpositional activity (of Metaviridae elements) appear to have limited the recent contribution of retrotransposon DNA to genome size expansion in A. thaliana . The rapid and recent genome evolution inferred for A. thaliana may be a feature common to other higher eukaryotes, in particular those with compact genomes. High turnover of TE insertions in euchromatin also occurs in Drosophila and pufferfish [ 53 ], for example, and accumulation of TEs into heterochromatin in those genomes may also, as in A. thaliana , be due to diverse evolutionary mechanisms. Materials and methods A methodology was developed for the automated mining of sequence data to retrieve the sequence and chromosomal location of genomic 'fossils' of LTR-RTs, identifying complete elements and solo-LTRs among the retrieved sequence fragments, and estimating the age of the insertion events that gave origin to these elements. This methodology was applied to the genome sequence of A. thaliana . Molecular paleontology of LTR-retrotransposons Sequences of the organellar and the five nuclear chromosomes (version 200303) were obtained from the Munich Information Center for Protein Sequences (MIPS) [ 54 ]. Computational mining for LTR-RT fragments in the A. thaliana genome (around 116 Mbp of available sequence) was performed using sequence-similarity search algorithms [ 55 ] against a library of representative sequences of LTR-RTs. This reference library was compiled by extracting from Repbase update [ 56 , 57 ] sequences of the LTRs and internal region (IR) of known A. thaliana 'families' of LTR-RTs. The programs RepeatMasker [ 58 ] and WU-BLAST [ 59 ] were used to search the whole genomic sequence (initially divided into 50 kbp chunks) and obtain the precise coordinates of chromosomal segments homologous to (a part of) the LTR or IR of library elements. The datasets of chromosomal coordinates of the complete LTR-RTs and solo LTRs identified are available as Additional data files 1 and 3. 'Families' of LTR-retrotransposons (as classified in Repbase update) are present in low copy numbers; therefore, for the purpose of this analysis they were grouped into three 'superfamilies': Athila , Gypsy -like (all 'families' belonging to the Metaviridae, excluding Athila ), and Copia -like (all 'families' belonging to the Pseudoviridae). The Metaviridae was split into two groups ( Athila and Gypsy -like), as initial mining of the A. thaliana genome revealed that Athila elements have been particularly successful in colonizing it. Their copy number is roughly double the number of all other members of the Metaviridae, and higher than the total of all Pseudoviridae elements. Athila form a clade and are retroviral-like elements that are likely to have an envelope ( env ) gene [ 60 ]. Most of the Copia - and Gypsy- like elements are typical LTR-RTs, although one of the Copia -like 'families' ( metaI ) comprises non-autonomous elements [ 22 ] and a few others ( endovir1 [ 61 ], atcopia41-43 [ 22 ]) are retroviral-like, featuring a putative env gene. A fourth 'superfamily' was used to include TRIMs. These are short, non-autonomous elements that feature LTRs but no coding genes and cannot currently be classified into either the Pseudoviridae or the Metaviridae; they are described in [ 62 ]. The four superfamilies comprise the following 'families'. Athila (10 families): athila2 - 5 , athila4A - D , athila6A , athila7 , athila8A and B ; Gypsy -like (15 families): atgagpol1 , atgp2 and 3 , atgp2N , atgp5 - 10 , atgp9B , atlantys1 - 3 , tat1 ; Copia -like (107 families): atcopia1 - 97 , atcopia8A and B , atcopia18A , atcopia32B , atcopia38A and B , atcopia65A , endovir1 , TA1-2 , meta1 ; TRIM (3 families): katydid-At1 , katydid-At2 , katydid-At3 . Identification of complete elements and solo-LTRs A Perl script, LTR_MINER (available on request), was written to parse all the chromosomal LTR-RT fragments reported by RepeatMasker (WU-BLAST hits of similarity to reference sequences) and identify complete elements and solo-LTRs. LTR_MINER performs the pattern-recognition function of assembling hits that originated from single LTR-RT insertion events. The algorithm involves: 'defragmentation' of LTR hits. If a chromosomal LTR fossil contains insertions/deletions (indels) relative to the most similar library sequence, it may be reported as multiple hits (fragments). Defragmentation is the identification of multiple hits that correspond to the same LTR. Parameters were set so that LTR hits were defragmented only when they were separated by no more than 550 bp, belonged to the same family, had the same orientation on the chromosome, and their combined length did not exceed the length of the corresponding family reference sequence by more than 20 bp. Identification of 'complete' elements An intact LTR-RT insertion consists of at least three hits: LTR-IR-LTR (an IR from a single element insertion may also yield multiple hits). After LTR defragmentation, LTR_MINER searches for contiguous patterns of LTR , IR , LTR . In order to check whether the pattern could be straddling a nested insertion of the same family, the search is then recursively extended from each end of the pattern for further contiguous hits to an IR and a LTR (of same family and orientation). The two LTRs of the innermost pattern are classified as a pair of intra-element LTRs. Identification of 'interrupted' elements: fossil elements containing insertions between the two LTRs LTR_MINER also identifies such elements provided an IR is present between the LTRs. A maximum pairing distance between LTRs was set at 30 kb. Identification of 'solo-LTRs' LTR_MINER was set to classify a LTR fragment as a solo-LTR if no other LTR or IR (of same family and orientation) is present within a 5 kbp radius from the fragment's ends. The aim was the identification of elements resulting from deletion (of the IR and one LTR) events via homologous recombination between intra-element LTRs, and not to classify as solo-LTRs sequences that are separated from IRs because of insertions. Dating of insertion events Nucleotide sequence divergence between pairs of intra-element LTRs was used as a molecular clock, as these pairs are identical at the time of insertion [ 63 ]. All mined pairs of intra-element LTR sequences were aligned using ClustalW [ 64 ] (with Pwgapopen = 5.0, Pwgapext = 1.0). To ensure correct alignment of any sequences with large indels, pairwise LTR alignments were position-anchored relative to reference sequences: if a chromosomal LTR fossil consisted of multiple hits (of similarity to segments of the reference sequence) then the intervening chromosomal sequence between such hits was replaced by a number of gaps, equal to the length of the region separating the corresponding segments in the reference. The number of nucleotide substitutions per site ( K ) between each intra-element LTR pair was then estimated using Kimura's two-parameter model [ 65 ]. To reduce sampling bias towards younger elements, elements with truncated LTRs were included in the analysis (provided both LTRs are present), as intact elements are likely to be younger than elements that have accumulated indels. Alignments with fewer than 80 nucleotides were discarded. As CLUSTAL-W alignments could be poor if LTR sequences were only partially overlapping, for all LTR pairs with K greater than 0.2 they were inspected by eye and manually edited if necessary (and K then recalculated). Estimates of the ages of insertion were obtained by using the equation t = K/2r , where t is the age, and r is nucleotide substitution rate for the host genome DNA polymerase. The value of 1.5 × 10 -8 substitutions per site per year was used for r (1.0 < r < 2.1 × 10 -8 95% confidence interval), estimated in [ 25 ] for the synonymous substitution rate in the Chs and Adh loci in Arabidopsis / Arabis species. Finally, if recombination between LTRs from different insertions had occurred frequently, the dating method above would be invalid for obtaining the age profiles of different families. To detect possible recombination events, multiple alignments of all LTRs (including solos) of certain families were generated using BLASTALIGN [ 66 ], a program that can handle datasets that may contain large indels. Neighbor-joining trees of the LTR sequences were then constructed using PAUP* 4.0b10 [ 67 ] with the HKY85 model, to check whether intra-element LTR pairs clustered. Additional data files The following additional data files are available with the online version of this article. Additional data file 1 contains the entire dataset of chromosomal coordinates and ages of complete LTR-retrotransposons in A. thaliana . Additional data file 2 describes the data fields in Additional data file 1 . Additional data file 3 contains the entire dataset of chromosomal coordinates of solo-LTRs in A. thaliana . Additional data file 4 describes the data fields in Additional data file 3 . Additional data file 5 contains the Perl script LTR_MINER, used to de-fragment sequence similarity hits to LTR-retrotransposons, and identify complete and solo-LTR elements. Additional data file 6 describes the utility and usage of the Perl script in Additional data file 5 . Additional data file 7 contains the Perl script used in conjunction with LTR_MINER, used to divide long sequences into smaller chunks labeled by their coordinate range. Additional file data 8 describes the usage of the Perl script in Additional data file 7 . Supplementary Material Additional data file 1 The entire dataset of chromosomal coordinates and ages of complete LTR-retrotransposons in A. thaliana Click here for additional data file Additional data file 2 A file describing the data fields in Additional data file 1 Click here for additional data file Additional data file 3 The entire dataset of chromosomal coordinates of solo-LTRs in A. thaliana Click here for additional data file Additional data file 4 A file describing the data fields in Additional data file 3 Click here for additional data file Additional data file 5 The Perl script LTR_MINER, used to de-fragment sequence similarity hits to LTR-retrotransposons, and identify complete and solo-LTR elements Click here for additional data file Additional data file 6 A file describing the utility and usage of the Perl script in Additional data file 5 Click here for additional data file Additional data file 7 The Perl script used in conjunction with LTR_MINER, used to divide long sequences into smaller chunks labeled by their coordinate range Click here for additional data file Additional data file 8 A file describing the utility and usage of the Perl script in Additional data file 7 Click here for additional data file

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519027

Predicting declines in physical function in persons with multiple chronic medical conditions: What we can learn from the medical problem list

Background Primary care physicians are caring for increasing numbers of persons with comorbid chronic illness. Longitudinal information on health outcomes associated with specific chronic conditions may be particularly relevant in caring for these populations. Our objective was to assess the effect of certain comorbid conditions on physical well being over time in a population of persons with chronic medical conditions; and to compare these effects to that of hypertension alone. Methods We conducted a secondary analysis of 4-year longitudinal data from the Medical Outcomes Study. A heterogeneous population of 1574 patients with either hypertension alone (referent) or one or more of the following conditions: diabetes, coronary artery disease, congestive heart failure, respiratory illness, musculoskeletal conditions and/or depression were recruited from primary and specialty (endocrinology, cardiology or mental health) practices within HMO and fee-for-service settings in three U.S. cities. We measured categorical change (worse vs. same/better) in the SF-36 ® Health Survey physical component summary score (PCS) over 4 years. We used logistic regression analysis to determine significant differences in longitudinal change in PCS between patients with hypertension alone and those with other comorbid conditions and linear regression analysis to assess the contribution of the explanatory variables. Results Specific diagnoses of CHF, diabetes and/or chronic respiratory disease; or 4 or more chronic conditions, were predictive of a clinically significant decline in PCS. Conclusions Clinical recognition of these specific chronic conditions or 4 or more of a list of chronic conditions may provide an opportunity for proactive clinical decision making to maximize physical functioning in these populations.

Background Comorbid chronic diseases are increasingly recognized as a significant factor in declining health. Of the 125 million Americans with chronic diseases, 48% are estimated to have at least one comorbidity, and 62% of persons over the age of 65 have two or more chronic illnesses.[ 1 , 2 ] Approximately 25% of persons with chronic illness have some limitation in activity and the percent of persons with disability increases with increasing numbers of coexisting conditions.[ 2 , 3 ] As the population in the United States ages, the number of persons with comorbid chronic disease will increase substantially. Primary care physicians will both provide and coordinate much of the care for this population [ 4 ]. Primary care for persons with chronic medical conditions differs from specialist care for these same conditions in the need to see both the forest and the trees: to address disease-specific issues and outcomes in the context of both coexisting medical conditions and the patient's psychosocial environment. To this end, practice guidelines developed from randomized controlled trials with strict inclusion criteria may not generalize well to the heterogeneity of primary care practice or the complex individual patient. In the environment of competing demands that characterizes medicine in general, and primary care in particular; information on health outcomes that can be inferred from the medical record problem list may be particularly relevant in clinical decision making for persons with multiple chronic conditions. We chose to investigate the health outcome of physical well being for several reasons: It is relevant to both clinical and quality-of-life decision making; and previous findings have demonstrated that different combinations of chronic medical conditions have been shown to be associated with type and/or severity of disability [ 5 , 6 ]. Longitudinal studies suggest that the cumulative effect of comorbid conditions is not simply additive: certain combinations of diseases may have a greater effect on outcomes than others. Combinations of diabetes plus obesity or heart disease [ 7 ], and arthritis plus diabetes, pulmonary disease or obesity [ 8 ] were significantly more detrimental to measures of health outcomes than either condition alone or in combination with other comorbidities. In a review of multiple longitudinal studies on comorbidity, Gijsen et al. found comorbidity to be a predictor of higher mortality, worse functional status, decreased quality of life, and increased health care utilization [ 9 ]. It is impractical for the primary care physician to maintain an awareness of specific combinations of chronic conditions that may characterize patients at risk for functional decline. However recognizing certain chronic medical conditions as potential 'red flags' for further investigation would be useful. We hypothesized that certain chronic diseases that often occur as comorbidities may have a greater impact than others on functional status outcomes over time. To explore this question, we analyzed data from the Medical Outcomes Study (MOS), a longitudinal study focusing on the care and medical outcomes of patients with specific common chronic conditions [ 10 ]. The MOS data have been previously used to study comparative effects of chronic conditions on physical well being over time [ 11 - 14 ]. These investigations have included analyses of the effects of anxiety disorder, varying levels of physical activity, and depression on health outcomes in the context of multiple chronic diseases.[ 12 , 15 , 16 ] We used this important data set to further explore the complexities of long-term health outcomes in a heterogeneous group of patients with comorbid chronic disease. Our study used MOS data to investigate the relative effect of six different common chronic conditions (diabetes, coronary artery disease (CAD), congestive heart failure (CHF), chronic respiratory disease, musculoskeletal disease and depression) on measures of physical well being over the course of four years in patients with comorbidities. This data base is comprised of information from respondents with certain chronic medical conditions. There is no information on 'healthy' respondents without any chronic conditions. Therefore we examined the relative effects of selected conditions relative to hypertension alone. We examined the presence of a specified level of decline in physical well being that had been verified to be clinically significant, rather than a change in the PCS score that might be statistically significant but of limited practical importance to patients. In addition, we analyzed the effect of specific diagnoses in the context of the total disease burden in an effort to identify possible 'sentinel' conditions that may specifically contribute to functional decline for patients with multiple comorbidities. Methods Study design The MOS was a four-year observational study that included assessment of health outcomes of chronically ill patients. Details of the study including design, sampling, site selection and clinician recruitment have been previously published[ 10 , 16 - 18 ] Study setting MOS study sites were selected from three cities (Boston, MA, Chicago, IL and Los Angeles, CA), from both primary care (family practice and internal medicine) and specialty (endocrinology, cardiology and mental health) practices, and from both managed care and fee-for-service payment plans. Sample and data collection The original study sample consisted of patients with one or more of five chronic "tracer" conditions (hypertension, adult onset diabetes, myocardial infarction within the past six months, congestive heart failure or depression) approached during a visit with an MOS clinician during a two-week period in 1986. Of the 28,257 patients originally approached, 20,232 agreed to participate. From this group, patients were selected for follow up on the basis of diagnosis and completion of baseline data collection, as described elsewhere [ 18 , 19 ]. Of the 3589 patients selected for follow-up, 2708 completed a baseline assessment, and 2235 were randomly selected for follow-up. Four-year follow-up data were obtained for 1574 of these 2235 (70%). For the current study, the chronic conditions of interest (diabetes, CAD, CHF, chronic respiratory disease, musculoskeletal conditions, and depression) were defined by combining the original tracer diagnoses with additional diagnoses determined by a structured medical history interview conducted by a trained clinician [ 17 ]. For example our category of CAD consists of persons with the original tracer condition of myocardial infarction within the past 6 months plus those with a history of angina, current symptoms of angina, and myocardial infarction more than one year ago. If information about a condition was missing, an independently derived probability of each diagnosis was substituted if the probability was at least 90%. The components of each of the main diagnoses are listed in Table 1 . We chose to analyze these conditions based on their high prevalence as well as frequent assessment in the literature on comorbidity and chronic disease management. Table 1 Components of main disease categories Main Disease Number (percent) a Diabetes 359 Type 2 diabetes mellitus 319 (88.9) Type 1 diabetes mellitus 40 (11.1) CAD 425 Myocardial infarction within past 6 months 76 (17.9) History of angina 104 (24.5) Current angina 233 (54.8) Myocardial infarction more than one year ago 135 (31.8) CHF 159 Respiratory disease 133 Asthma 42 (31.6) Chronic obstructive pulmonary disease 95 (71.4) Other lung disease 23 (17.3) Musculoskeletal disease 684 Back pain 446 (65.2) Musculoskeletal complaints 277 (40.5) Hip impairment 55 (8.0) Osteoarthritis 145 (21.2) Rheumatoid arthritis 30 (4.4) Depression 555 Diagnosed depression 260 (46.8) Symptoms of depression 295 (53.2) a) May sum to more than 100% due to coexisting conditions. The final sample included patients who had complete baseline and four-year follow-up information (including deaths), had completed a medical history questionnaire, and had definitive diagnostic information on their original tracer condition [ 17 ]. Patients from the longitudinal sample who were lost to follow-up did not have significant differences in initial health status from those who remained in the sample, however patients lost to follow-up tended to be younger and had lower income than those remaining [ 17 ]. Outcome measures We analyzed categorical change (worse versus same/better) in SF-36 ® Health Survey (SF-36) physical component summary (PCS) scores over four years. We chose this dichotomous outcome to emphasize the clinical importance of anticipating functional decline in patients with chronic medical conditions. Categorical change was defined as a decrease of 6.5 or more points in PCS. This was based on standards for PCS scores in which a change of 6.5 points is outside the 95% confidence interval for PCS scores. Longitudinal change norms for PCS classify patients with +/- two standard errors of measurement (SEM) as "better" or "worse" and those within two SEM as "staying the same" [ 17 , 20 ]. Changes of 6.5 points or more in PCS over time are clinically significant and correlate with changes in health and mortality [ 17 , 20 ]. We also assessed linear change in PCS scores over the four-year period. Statistical analysis Based on published sample sizes specifically calculated to detect differences in PCS between two groups using repeated measures over time, our sample size was adequate to detect a difference of five points in PCS with 80% power (alpha = 0.05, two tailed test with an intertemporal correlation of 0.70) [ 20 ]. As these published sample size calculations were designed to detect a slightly smaller difference in PCS than we chose to examine (5.0 versus 6.5 points), our sample sizes should be more than adequate. We used logistic regression to analyze the independent effect of each main chronic disease on categorical change (worse vs. same/better) in PCS relative to hypertension alone adjusting for the effect of the other main diseases. Logistic regression was again used to assess change in PCS over four years in patients with one, two, three or four or more of the main chronic conditions relative to those with hypertension alone. Due to the selection criteria for the original MOS study [ 10 ], the MOS data set does not include 'healthy' participants without any chronic conditions. Therefore we used persons with hypertension alone, but none of the other main chronic conditions, as the referent group for the analysis. In one analysis, hypertension alone had an effect on PCS that was comparable to the effect of aging in a 'healthy' population.[ 21 , 22 ] However another longitudinal analysis has shown slightly increased odds of a decline in health status over 2 years in patients with hypertension alone relative to those with no chronic conditions. This effect diminished with age [ 7 ]. We completed separate regressions to determine the effect on PCS due to each of the main chronic conditions of interest. In these models, the study population was divided into those with hypertension alone (referent group), those with the main chronic disease of interest (with and without other conditions), and those with any other of the main chronic conditions other than the condition of interest. Using similar modeling, linear regression analysis was used to assess the relative contributions of the explanatory variables. There were no significant interactions between each of the main conditions and the total number of conditions. As age has been shown to be associated with functional outcomes in persons with comorbidities [ 23 ], we assessed categorical change in PCS by number of comorbid conditions relative to hypertension alone for older and younger age groups (under 65 years vs. 65 and over). Four-year change in PCS relative to hypertension was comparable in both age groups, therefore the final analysis was not stratified by age. Analyses were additionally adjusted for age, a count from a list of 16 additional chronic conditions (in addition to adjustments for main diseases as mentioned above), poverty level, gender, race, educational level, employment status, and marital status. Subjects who died during the course of the study were assigned a four-year PCS score of zero and included in the 'worse' category. Assignment of a zero PCS sore to participants who died during the course of the study has been discussed by Diehr et al. as a reasonable approach for analyses in which a decline in health is the outcome of interest [ 17 , 24 ]. Failure to incorporate these subjects could substantially bias the results by limiting the assessment of outcomes to 'healthier' subjects. To partially account for level of physical well being at baseline, the analyses also adjusted for starting PCS score relative to age/gender norms. Results Of a total of 1574 subjects, 281 individuals carried a diagnosis of hypertension exclusive of any of the other major comorbid conditions, and were defined as the referent population for this analysis. (As participants in the original MOS study were selected on the basis of chronic medical diagnoses, the study population did not include a referent 'disease-free' population.) The remaining 1293 subjects had either one or more of the six comorbid conditions of diabetes, CAD, CHF, respiratory disease, musculoskeletal disease and depression with or without hypertension. In this heterogeneous population, subjects with the main conditions of interest had, on average, 1.8 of the main conditions and 0.8 from a list of 16 additional conditions. Referent subjects with hypertension and no other main conditions of interest had 0.3 additional conditions. The majority of all respondents had starting PCS scores within 1 standard deviation of age/gender norms, with an additional 10% above and 26% below age gender norms. Table 2 describes the characteristics of the study population. Table 2 Characteristics of study population N 1574 Age (mean) +/- SD 57.6 +/- 15.4 Male 41.3% Married 58.3% Employed 46.4% At or below 200% of poverty level 19.3% White (vs. non-white) 82.5% Education Education less than high school 14.6% High School graduate 28.5% Greater than high school 28.5% College graduate 12.1% Greater than college 16.3% Mean number of main diseases* 1.5 Hypertension alone (referent group) 0 Remaining subjects 1.8 Mean number of additional diseases** 0.7 Hypertension alone (referent group) 0.3 Remaining subjects 0.8 PCS Scores Baseline PCS score > 1 standard deviation above age/gender norms 10.0% Within 1 standard deviation of age/gender norms 63.7% >1 standard deviation below age/gender norms 17.9% >2 standard deviation below age/gender norms 8.5% * Diabetes, coronary artery disease, congestive heart failure, musculoskeletal disease, respiratory disease and depression. ** From a list of 16 additional conditions. Subjects with CHF, diabetes or chronic respiratory disease had increased odds of a clinically significant decline in PCS over 4 years. These odds ratios (confidence intervals) were 2.9 (1.7, 5.0), 2.1 (1.5, 2.9) and 1.7 (1.1, 2.8) respectively (Table 3 ). Subjects with diagnoses of CAD, musculoskeletal disease, or depression did not show a significant change in physical well being over 4 years relative to the referent population. The effects of these main conditions on physical well being over 4 years were confirmed by the linear model in which subjects with diagnoses of CHF, diabetes or respiratory disease had adjusted 4-year declines in PCS scores of -10.0, -3.2 and -3.1 points (p < = 0.05 for all). Table 3 Adjusted odds of a decline in PCS attributable to presence vs. absence of each main chronic disease a (Total N = 1574) Disease N b Adjusted odds ratio Hypertension 281 1.0 Diabetes 249 2.1 (1.5, 2.9) Coronary Artery Disease 364 1.1 (0.8, 1.5) Congestive Heart Failure 137 2.9 (1.7, 5.0) Respiratory Disease 125 1.7 (1.1, 2.8) Musculoskeletal Disease 514 0.9 (0.7, 1.2) Depression 319 1.3 (0.9, 1.8) a) Adjusted for number of main conditions, number of additional comorbid conditions, poverty level, gender, race, educational level, employment status, marital status. b) Sum to more than 100% due to coexisting conditions An absolute decrease in PCS of 6.5 points per subject was used as criteria for a clinically significant decline in PCS over time based on previous analyses of the MOS data and on validation of the SF-36 ® survey instrument [ 17 ]. The linear regression model presents changes in PCS for the population of subjects adjusted for characteristics of that population. Therefore the statistically significant changes in PCS scores over time resulting from the linear regression analysis may not necessarily be greater than 6.5 points Little increase in the odds of functional decline was evident in individuals with 1,2, or 3 of the main chronic conditions. However having 4 or more of these conditions predicted a decline in PCS (OR 2.8; CI 1.3, 5.9) (Table 4 and Figure 1 ). The effect on PCS of the number of chronic diseases was similar in older and younger age groups. Table 4 Number of main chronic conditions as predictors of a decline in physical well being over four years Number of main chronic conditions N Adjusted odds of a decine in PCS ab (N = 1574) Hypertension alone 281 1.0 One main chronic disease c 607 1.1 (0.8, 1.4) Two main chronic diseases c 423 1.2 (0.8, 1.7) Three main chronic diseases c 197 1.4 (0.9, 2.2) Four or more chronic diseases c 66 2.8 (1.3, 5.9) a) Adjusted for age, number of additional comorbid conditions, poverty level, gender, race, educational level, employment status, marital status. b) Number of main chronic diseases as a predictor of a decrease in PCS is statistically significant at p < = .05 c) Subjects with one, two, three or four or more of the following: DM, CAD, CHF, musculoskeletal disease, respiratory disease, or depression. May also include hypertension. Figure 1 Change in PCS relative to hypertension alone by number of main chronic diseases. Discussion Physical well being is particularly relevant for persons with chronic conditions and the clinicians who care for them. Declines in physical well being may have significant social, emotional and economic repercussions, as they correlate with job loss, high health care utilization and increased mortality [ 20 ]. Based on this analysis of a heterogeneous population from the MOS; persons with CHF, diabetes and/or chronic respiratory disease are at particular risk. Primary care providers are in an ideal position to help prevent, delay or proactively manage potential declines in physical functioning for these patients. In this analysis of change in physical well being over time in persons with a variety of chronic medical conditions, we found that specific diagnoses of CHF, diabetes and/or chronic respiratory disease; or the presence of 4 or more chronic conditions, were predictive of a clinically significant decline in PCS. We hypothesize that our findings reflect the different clinical courses of each of these conditions as well as the varying potential for therapeutic interventions in each case. The natural history of CHF, diabetes and some respiratory disease is progressive. Treatment of these conditions is aimed at optimizing long-term health outcomes. Ongoing management involves self-care that is replete with complex concepts and tasks. In contrast, management of CAD and musculoskeletal disease may include the potential for surgical intervention and protocol-driven rehabilitation programs. While some sub-populations of patients with these latter conditions may develop increasing disability, others may experience significant improvement in physical well being over time. For example, increases in PCS scores associated with hip replacement and therapy for low back pain can be in the range of 9.5 and 7.6 respectively [ 20 ]. We have no specific information on such interventions in our study population and therefore were unable to incorporate treatment interventions into our analysis. Disease management programs have been successful in improving health outcomes for persons with these and other chronic conditions [ 25 , 26 ]. However these programs are often disease specific [ 4 , 27 , 28 ]. As this study population illustrates, many chronic conditions do not occur in isolation. This may make disease-specific programs less beneficial for many patients. Some components of successful disease-management programs that are particularly relevant to persons managing multiple medical conditions include: guidance in problem solving, decision making, confidence building, self-management support, and systematic support of the disease management process [ 29 - 31 ]. In considering the issue of functional decline, any 'clinically significant change' over time is not only a function the starting and ending levels of functional status. It also is determined by the individual for whom the change has meaning, the instrument used to assess the change, and population norms that provide the context for the observed change [ 32 , 33 ]. Population-based studies (including ours) address parts of this equation, but do not address the most important dependent variable in the equation: the implications of a change in function to the individual. It is up to the provider and the patient to interpret the 'data' in the context of the individual and consider any subsequent recommendations in that same context. Our findings on depression deserve special mention. Depression in particular and mental health in general are well known to affect the management and outcomes of several chronic medical conditions [ 34 - 36 ]. Furthermore there is a high prevalence of coexisting depression in persons with chronic disease [ 37 ]. In this analysis, we found that depression did not predict a decline in physical functioning over time. We hypothesize that this is partly due to the natural history of the disease: Depression has a waxing and waning course, and disease symptoms that are subject to significant environmental and social effects. Therefore the effect of depression on physical functioning in this patient population may have varied significantly within the population over time. Our findings are also consistent with previous findings on depression in this data set: When MOS subjects with depression were followed over two years [ 12 ], they were noted to have similar or improved scores of physical functioning at the end of two years relative to those at baseline. It is possible that this trend continued in our sub-sample and accounted for the non-significant change in PCS score for subjects with depression as a comorbid condition. The study of comorbidity is, by definition, the study of inter-relationships: between different diseases and between diseases and age or other health-related sociodemographic variables. Caring for persons with chronic illness is, similarly, the care of heterogeneous populations of individuals with multidimensional medical, psychological and social issues. The heterogeneity of this study population may be relevant to the provider willing to sacrifice some internal validity in hopes of generalizing the findings to his or her patient population. Limitations Our sample size precluded stratification to investigate the relative contributions of different medical conditions to physical well being in sub-populations defined by specific combinations of conditions, smaller age groups, socioeconomic status or other demographic criteria that might further clarify the interactive nature of the comorbid chronic disease process. It is likely that the effect of CHF, diabetes, chronic respiratory disease or other conditions on measures of health related quality of life differs within different subpopulations. Specifically, vulnerable populations may be more at risk of poor health outcomes due to socioeconomic status or process of care factors than specific disease states [ 17 , 38 ]. In addition, there may be additional psychological and sociodemographic factors (e.g. levels of self-efficacy and social support systems) that we were unable to incorporate in our model, which affect outcomes in persons with comorbid conditions. In the original MOS cross-sectional analysis of functional status and well being, most of the variance in outcomes measured (including PCS scores) was not attributable to the diseases studied the same is true of our longitudinal analysis [ 16 ]. Due to the nature of the MOS data base, we compared the effect of selected chronic conditions on physical well being to a referent group with hypertension alone. While the effect of hypertension alone on physical well being over 4 years may be minimal ; it is possible that there is a synergistic effect between hypertension and certain conditions such as CAD and CHF on physical well being over time. If so, the effect of CAD or CHF on PCS scores for persons with these conditions may have been slightly magnified. It is unlikely that this bias would change the overall significance of the results of our analysis. We were unable to directly account for severity of illness for all main chronic conditions either at baseline or follow-up. However statistical adjustments for starting PCS score relative to age/gender norms and the inclusion of patients who died in the final analysis should indirectly account for some degree of severity of illness throughout the study population. As in all investigations, the conclusions reflect the population studied: primarily Caucasian, with a majority above 200% of the federal poverty level, relatively well educated and specifically selected for inclusion on the basis of certain medical diagnoses. Results from this study may not be generalizable to populations with different demographic characteristics and different constellations of comorbid conditions. Conclusions This analysis suggests that long term physical well being in persons with multiple chronic diseases is a function of both the number and type of medical conditions. Relative to persons with hypertension alone, those who carry diagnoses of CHF, diabetes and chronic respiratory disease have an increased risk of a decline in physical well being over 4 years. The presence of CAD, musculoskeletal disease or depression does not predict a similar decline. For the primary care physician for whom 'comorbidity' implies an exponentially increasing ratio of items on the problem list to available time during the office visit, the specific diagnoses of CHF, diabetes and chronic respiratory disease may serve as triggers for management decisions. Clinicians who care for patients with these common conditions should be alert to the possibility that a proactive approach incorporating generalizable principles of disease management may either attenuate this loss of function or help the patient and family anticipate future needs. Authors' contributions EB participated in the design of the investigation, performed analysis and drafted the manuscript. MB participated in the design of the investigation, performed additional analysis, data management, and manuscript revision. JW supervised the original data collection and MOS investigations and assisted in manuscript revision. JS participated in the design of the investigation, advised on the analysis and assisted in manuscript revision. All authors read and approved the final manuscript.

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519033

Simultaneous suppression of disturbing fields and localization of magnetic markers by means of multipole expansion

Background Magnetically marked capsules serve for the analysis of peristalsis and throughput times within the intestinal tract. Moreover, they can be used for the targeted disposal of drugs. The capsules get localized in time by field measurements with a superconducting quantum interference device (SQUID) magnetometer array. Here it is important to ensure an online localization with high speed and high suppression of disturbing fields. In this article we use multipole expansions for the simultaneous localization and suppression of disturbing fields. Methods We expand the measurement data in terms of inner and outer multipoles. Thereby we obtain directly a separation of marker field and outer disturbing fields. From the inner dipoles and quadrupoles we compute the magnetization and position of the capsule. The outer multipoles get eliminated. Results The localization goodness has been analyzed depending on the order of the multipoles used and depending on the systems noise level. We found upper limits of the noise level for the usage of certain multipole moments. Given a signal to noise ratio of 40 and utilizing inner dipoles and quadrupoles and outer dipoles, the method enables an accuracy of 5 mm with a speed of 10 localizations per second. Conclusion The multipole localization is an effective method and is capable of online-tracking magnetic markers.

Background The transport of capsules in the alimentary tract underlies complex influencing factors like the patients peristalsis, the hydration and the form and size of the capsules. A procedure which allows the instantaneous localization of the capsules supports a number of patient examinations as well as examinations of new drug forms [ 1 - 4 ]. Capsules can be marked radioactively (scintigraphy) or magnetically. The scintigraphy [ 5 ] has a lower time resolution compared to the magnetic localization, and due to radiation it is not appropriate for examinations with healthy probands. The localization of magnetically marked capsules (magnetic markers) must be spatially accurate and with high temporal resolution. For the spatial localization the marker field must be separated from the external magnetic disturbing fields. This separation can be achieved by splitting the magnetic field in multipole moments [ 6 ]. The method proposed utilizes the multipole moments directly for the determination of the position and the magnetic moment of the marker. Thus, the separation of disturbing fields and the localization are integrated numerically effective into one procedure. This allows a fast online-localization of the marker capsules. Multipole expansions are used also to model spatially distributed biological sources such as brain currents [ 7 , 8 ]. The application of multipoles for the localization of magnetic dipoles is described in [ 9 , 10 ], and is used in other technical areas without disturbing field suppression [ 11 ]. Marking of capsules and pills takes place by partially filling them with black iron oxide (Fe 3 O 4 ) which is subsequently magnetized up to saturation. The magnetic field measurement is performed within magnetically shielded rooms by the use of highly sensitive SQUID arrays. For the investigation at hand we conduct simulation runs to determine the performance of the multipole localization. Methods Algorithm The field of a magnetic marker located adjacent to the point of origin can be expressed by a multipole expansion in Cartesian coordinates ( x 1 , x 2 , x 3 ). If the distance between marker and origin is small compared to the distance between a magnetic sensor and the origin, the field of the marker at the sensor position is given by the first elements of the multipole expansion. With the notation follows with c m being the dipole, quadrupole and octopole moments of the field expansion. The form functions arise from a Taylor series expansion in the parameter with and . It holds With the Kronecker delta follows and Conversely, a Taylor series expansion of – compared to the sensor coordinates – far away located field sources in the parameter with yields a multipole expansion of external disturbing fields: We denote the multipole moments c ex of the expansion of fields of external sources as "outer moments" to distinguish them from the "inner moments" c m . To get the same normalization and symmetry properties for the outer and inner form functions, we define the outer form functions by It follows and The tensors of 3 rd order and of 4 th order own the following symmetry features which are identical for the inner and outer multipole expansion: We combine the resulting 3, 5 and 7 linearly independent components of the tensors of 2 nd , 3 rd and 4 th order to one vector for the marker field and one vector for the external disturbing field : The summation of equation (1) and equation (6) yields the field expansion for a magnetic marker with disturbing fields. We truncate this expansion after the octopole terms, and transcript it into a linear equation system for the determination of equivalent multipole moments c for a measurement B meas : The structure of the vectors B meas and c and the matrix F is given below in the formulas (15...24). The residuals 0(·) are sufficiently small, if the coordinates of the marker are small compared to the coordinates of the field sensors , and if the coordinates of the field sensors are small compared to the coordinates of the external disturbing field sources . B meas is a vector with the measurement values of the magnetometer sensor field in the positions with the directions : The matrix F is built from the linearly independent form functions for inner and outer field sources given in equation (13). Their scalar product with the sensor normal directions yields one row for every sensor: The number of columns of F is the sum of the numbers of inner and outer field functions used. Each column describes the field of one specific magnetic moment with unit strength measured by the sensor system. The Matrix F is called the forward matrix of all moments considered. The Matrix F is structured into submatrices for different moments: Matrix and are the forward matrices for inner and outer dipoles: Matrix and are the forward matrices for inner and outer quadrupoles. The size of is ( Nsen /5) with the rows belonging to quadrupole moments with indices (1,1; 3,3; 1,2; 2,3; 3,1). Matrix and are the forward matrices for inner and outer octopoles. The size of is ( Nsen /7) with the rows belonging to octopole moments with indices (1,2,2; 2,3,3; 3,1,1; 1,3,3; 2,1,1; 3,2,2; 1,2,3). The vector of multipole moments c is composed of inner and outer dipole moments c d , quadrupole moments c q and octopole moments c o : The inner dipole moments describe a dipole at the point of origin, the outer dipole moments describe a homogeneous disturbing field: The represent a quadrupole at the point of origin. The describe an external gradient field, whose field strength vanishes at the origin and which has no spatial derivations of 2 nd or higher order. This field can be measured by five ideal gradiometers at the origin and can be compared with the creation of software gradiometers. The represent an octopole at the origin. The describe an external gradient field of 2 nd order, which has no spatial derivations of 3 rd or higher order and whose field strength and spatial derivatives of 1 st order vanish at the origin. This field could be measured by 7 ideal second order gradiometers at the origin, it can be compared with the creation of software gradiometers of 2 nd order: Due to its small spatial extension, the magnetic marker can be described as a dipole of strength at position as a good approximation. The field of this dipole is With the Taylor series expansion follows in analogy to equation (1) A comparison of coefficients of (1) and (27) yields and The dipole strength can be determined by the dipole moment . An equation system for the adjacent calculation of the dipole position from the dipole strength and the quadrupole moment follows from (29) and (23): with This equation system is named shift equation in analogy to [ 10 ]. It is overdetermined, and can be solved by means of the pseudo inverse of m . We get the multipole moments c , which are required for the localization of the marker dipole, from solving the overdetermined equation system (14) by means of the pseudo inverse of F : c = ( F T · F ) -1 · F T · B meas .     (33) Here, the matrix of form functions F must contain columns at least for the moments and . Iterative dipole localization for a fixed dipole (e.g. one time point) is achieved by using the localization position as a new point of origin. The step length of the last localization step serves as a stop criterion for the iterative localization procedure. This is justified by considering the residuals of equation (14) within the convergence range of the procedure, and will practically be shown by the results of the following simulations. The tracking of a moved dipole based on measurements at consecutive time steps works by updating both the point of origin and the measurement data set after each localization step (Fig. 1 ). The localization step must be monitored, since it contains information about the marker speed and the noise dependent and speed dependent localization errors. Figure 1 Flow chart of the algorithm for online localization. This algorithm is meant for online localization, and therefore comprises only one iteration. A high signal to noise ratio and a high computing speed render 2–3 iterations per measurement cycle possible. Measurement system The simulations to determine the performance of the algorithm use the sensor geometry of the multi channel SQUID system Argos 200 from AtB (Advanced Technologies Biomagnetics, Pescara, Italy). The ARGOS 200 system contains fully integrated planar SQUID magnetometers produced using Nb technology with integrated pick-up loops. The sensing area is a square of 8 mm side length. The intrinsic noise level of the built in 195 SQUID sensors is below 5 fT Hz -1/2 at 10 Hz. Three sensors form one orthogonal triplet in each case. The measurement plane with a diameter of 23 cm consists of 56 of those triplets. The reference array consists of seven SQUID sensor triplets located in the second level in a plane which is positioned parallel to the measurement plane at a distance of 98 mm. The third (196 mm above the first plane) and fourth (254 mm above the first plane) levels contain one triplet each (Fig. 2 ). Figure 2 SQUID Array Argos 200. The ATB SQUID Array Argos 200 consists of 195 magnetometers which are arranged in orthogonal sensor triplets in four levels. The measurement area of each sensor is a square of 8 mm edge length. The measurement system is positioned within a magnetically shielded room, consisting of 3 highly permeable shieldings and one eddy current shielding. The shielding performance is 38 db at 1 Hz, 55 db at Hz and 80 db at 20 Hz. The sensor arrangement in orthogonal triplets facilitates the measurement of all 3 spatial components of the magnetic field. Thus, the required field coverage for the localization of a magnetic marker with unknown dipole strength is achieved. The subdivision into 168 measurement and 27 reference sensors is meant for the creation of software gradiometers. We can use all sensors simultaneously for the multipole method which integrates the suppression of disturbing fields. With the above described measurement system we performed simulations with different signal to noise ratios. Results We examined the localization characteristics of the multipole method by means of simulation runs at the sensor geometry of the measurement system Argos 200 (Fig. 2 ). All simulations performed are based on a dipole at position ( x , y , z ) = (0, 0, -300 mm), i.e. 30 cm below the measurement plane, with a dipole strength of 20 Amm 2 . This is a realistic dipole position for an examination within the digestive tract. The dipole field was superimposed by uncorrelated, Gauss distributed noise. The noise level in fT is also given as signal-to-noise-ratio (SNR), based on the channel with the strongest amplitude of the dipole field. The average localization accuracy over 100 simulations has been determined depending on the noise level and on the number of the multipole moments used in the vector c (21) (Fig. 3 ). Figure 3 Noise-dependent localization error. The mean squared localization error e rr ( ) over 100 simulations has been determined depending on the noise level and on the different number of multipole moments used. The curves are plotted up to the noise level, where all simulations still produced a stable localization result. We used the inner moments up to the 3 rd order , , plotted in curves 3χ and the inner moments up to the 4 th order , , , plotted in curves 4χ. The outer moments which were used to model the disturbing fields were none (curves χ0), 2 nd order moments (curves χ2: homogeneous fields), 2 nd and 3 rd order moments (curves χ3: homogeneous and gradient fields), and 2 nd to 4 th order moments (curves χ4: external fields up to 2 nd order). The simplest disturbing field to model is a homogeneous field having index χ2. The dipole field of a dipole with a strength of 20 Amm 2 at position ( x , y , z ) = (0, 0, -300 mm) is superimposed by white, Gaussian distributed noise, which is given in fT and as the signal to noise ratio (SNR). The localization was run up to a stable point. We define the localization error as the mean quadratic error of the 100 stable points based on the true dipole position. The localization error increases if we use higher order multipole moments. This holds true for the inner moments c m and for the outer moments c ex as well. As a good approximation the interrelationship between noise level and localization error is linear, with raising proportionality factor for higher mode numbers. This corresponds to parallel translation of the curves in double logarithmic plotting. We examined the localization speed depending on the distance of the starting point to the dipole position. For any tested distance the starting point has been moved from the dipole position into 100 random directions. The remaining mean distance to the dipole position after one localization step is depicted in Fig. 4 . The localization speed turns to be significantly higher when using inner octopoles. It gets higher with a shorter starting distance in both an absolute and a relative manner based on the starting distance. Both effects are to be expected directly from the residuals of equation (14). The influence of the outer multipoles on the localization speed is low. The convergence radius at which the dipole was found from all 100 directions decreases slightly with the raising number of outer multipoles used, and increases slightly if inner octopoles are used (unequal right ends of the respective curves in Fig. 4 ). The convergence radius ranges between 6 and 10 cm. The maximum number of iterations for a target accuracy of 1 mm can be estimated from Fig. 4 as 3. Figure 4 Localization error depending on the starting point distance for one iteration. For each distance d s the start position has been moved from the dipole into 100 random directions. The mean remaining distance d r after one localization step is shown. The curves are plotted until the starting distance d r where the localization was still stable from all 100 directions. To get the result after multiple iterative localization steps, the d r -value has to be taken as the starting distance d s of the following step. We used the inner moments up to the 3 rd order , , plotted in curves 3χ and the inner moments up to the 4 th order , , , plotted in curves 4χ. The outer moments which were used to model the disturbing fields were none (curves χ0), 2 nd order moments (curves χ2: homogeneous fields), 2 nd and 3 rd order moments (curves χ3: homogeneous and gradient fields), and 2 nd to 4 th order moments (curves χ4: external fields up to 2 nd order). In the following we examined the interrelationship between the convergence distance at y-direction and the noise level. The maximum y-distance of the starting position to the dipole, at which the dipole could be found with 100 random noise distributions, is depicted in Fig. 5 . It shows that the convergence distance remains unchanged almost up to the point of critical noise level (see Fig. 3 ) at which localization becomes impossible. The convergence distance, also compare the maximum convergence radius from the curve ends of plot (Fig. 4 ), depends only marginally on the choice of the inner ansatz functions. It decreases slightly when using outer multipoles. Having a convergence radius of at least 6 cm for the dipole position tested, the choice of the starting position can be regarded as noncritical. Figure 5 Convergence distance depending on noise level. The maximum y-distance dy between starting position and dipole position, at which for 100 random noise distributions the dipole could still get localized, is plotted. We used the inner moments up to the 3 rd order , , plotted in curves 3χ and the inner moments up to the 4 th order , , , plotted in curves 4χ. The outer moments which were used to model the disturbing fields were none (curves χ0), 2 nd order moments (curves χ2: homogeneous fields), 2 nd and 3 rd order moments (curves χ3: homogeneous and gradient fields), and 2 nd to 4 th order moments (curves χ4: external fields up to 2 nd order). The dipole field of a dipole with a strength of 20 Amm 2 at position ( x , y , z ) = (0, 0, -300 mm) is superimposed by white, Gaussian distributed noise given in fT and as the signal to noise ratio (SNR). The computing time used for one localization step is 5 ms with an implementation in Matlab at a standard Windows PC with a 2 GHz clock frequency. With maximum 3 iterations per localization step and additional computing time needed for data transfer and a basic visualization, 10 localizations per second are possible. This rate is normally sufficient for marker localizations. Discussion The localization speed rises when using inner octopoles , but this is associated with a higher localization error. At a signal to noise ratio lower than 10 3 inner octopoles cannot be used. An SNR of at least 10 2 is required for a source positioned 30 cm below the measurement plane. The outer moments c ex used enlarge the localization error depending on the uncorrelated sensor noise, as shown in Fig. 3 . Contrary, the localization error depending on the spatially correlated residual field within the measurement room lowers when using outer moments. Depending on the ratio between correlated and uncorrelated noise which has to be found with practical test series, noise suppression of homogeneous disturbing fields using and possibly noise suppression of gradient fields using are applicable. To ensure convergence, the starting point for the algorithm has to be within the convergence radius given in Fig. 4 . With typical measurement conditions, thus, a starting point 10 cm below the center of the measurement system will suffice. Conclusions The multipole localization is an effective algorithm because it unites a method for the suppression of disturbing fields with a localization method. It can be used iteratively and online for the tracking of magnetic marker timelines within the intestinal tract.

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Under-representation of developing countries in the research literature: ethical issues arising from a survey of five leading medical journals

Background It is widely acknowledged that there is a global divide on health care and health research known as the 10/90 divide. Methods A retrospective survey of articles published in the BMJ, Lancet, NEJM, Annals of Internal Medicine & JAMA in a calendar year to examine the contribution of the developing world to medical literature. We categorized countries into four regions: UK, USA, Other Euro-American countries (OEAC) and (RoW). OEAC were European countries other than the UK but including Australia, New Zealand and Canada. RoW comprised all other countries. Results The average contribution of the RoW to the research literature in the five journals was 6.5%. In the two British journals 7.6% of the articles were from the RoW; in the three American journals 4.8% of articles were from RoW. The highest proportion of papers from the RoW was in the Lancet (12%). An analysis of the authorship of 151 articles from RoW showed that 104 (68.9%) involved authorship with developed countries in Europe or North America. There were 15 original papers in these journals with data from RoW but without any authors from RoW. Conclusions There is a marked under-representation of countries in high-impact general medical journals. The ethical implications of this inequity and ways of reducing it are discussed.

Background National and international bodies concerned with research ethics need to confront the greatest ethical challenge-the enormous inequities in global health research [ 1 ]. Thus, less than 10% of the world's research resources are earmarked for 90% of the health problems [ 2 ]. Though 93% of the world's burden of preventable mortality occurs in developing countries, too little research funding is targeted to health problems in those countries [ 3 ]. This divide in research funding is mirrored by concerns regarding a divide in the proportion of publications arising from medical research in developing countries. A recent survey of six leading psychiatric journals revealed that only 6% of the articles originated from, or described data arising from, regions of the world that accounted for over 90% of the global population [ 4 ]. Following this paper, the issue of under-representation of developing countries was debated and discussed in many journals and by journal editors [ 5 - 7 ]. The aim of this study is to investigate the publication bias beyond the field of psychiatry to determine the overall contribution of different regions of the world to the medical literature published in high-impact journals and, in particular, to quantify the developing world's contribution. Methods The method used is the same as that which was used by two of the authors (AS and VP) in a recent survey of the international representation in the psychiatric literature [ 4 ]. A retrospective survey was conducted of all issues in one calendar year (the most recent, complete set available in the medical library in Colombo where data collection took place) of the following journals: BMJ, Lancet, NEJM, Annals of Internal Medicine & JAMA. These journals were selected because they have the highest impact amongst general medical journals [ 8 ]. These journals lay claim to their global legitimacy for many reasons: frequent publication, high impact, long history, credibility of the publisher, large numbers of full time editorial staff, membership of the International Committee of Medical Journal Editors, and influential joint statements [ 9 ]. The year for which data was collected was 2000 for all journals, except JAMA for which it was 1999 (all issues in year 2000 were not available). All articles, excluding the specified ones in each journal were reviewed. In the BMJ we excluded obituaries, multi-media, personal views, Minerva, news and soundings. In the Lancet we excluded Dissecting Room and news. In the Annals, we excluded on Being a doctor, Current clinical issues, Medical writings, Book notes, Ad Libitum and persona. In the NEJM, we excluded book reviews, This week in the journal and Abstracts. In JAMA, we excluded Medical news and Perspectives, Peace of my mind, JAMA hundred years ago, Abstracts, FDA, CDC, poetry and medicines, Books journals, New media, World in medicine and the section titled "from the JAMA websites". In the Lancet, editorials and commentaries were pooled together, because the commentaries in the Lancet resemble editorials in the other journals in terms of their contents. None of the other four journals had commentaries section. For the allocation of regions of the world, we categorized countries into four regions: UK, USA, OEAC (Other Euro-American countries) and RoW (Rest of the World). OEAC were European countries other than the UK but including Australia, New Zealand and Canada. RoW comprised all other countries including Eastern Europe, Central and South America, Asia and Africa. We scrutinized each article to examine the country of the authors' affiliation address with a view to categorizing them into relevant regions of the world. All authors were noted including multiple authors in large multi-center studies. We also scrutinized the methods section of all the original articles to ascertain the origin of data with special emphasis on whether the research was carried out in RoW countries. The affiliation of the first, last and corresponding authors was also noted. We worked with a negative bias against the USA and the UK from where the journals originated. Therefore articles, which included authors from the RoW, were considered as arising from the RoW even if the first author is from the UK, USA or OEAC. Similarly, articles from the OEAC, which involved collaborations from the UK or USA, were included in the OEAC category. In the event of a collaborative study between the USA and the UK, the allocation to region was based on the institutional affiliation of the first author. The RoW category included collaborative studies between any country in the RoW and developed nations. In some cases the origin of the author was difficult, particularly when there were two places named as their attached institutions. For an example when one was in USA while the other is in Kenya. We used our best guess in these instances. For an example the two authors AS and VP in this paper were based in RoW at the time we did this research but were employed in UK but with strong affiliations to the RoW countries where they were born and did their research. Attempts to analyse the nationality of the authors was therefore abandoned. Results The contribution of the RoW to the research literature surveyed in these five high-impact journals was 6.5%. In the two British journals, 7.6% of the articles were from the RoW, whilst the proportion in the three American journals was 4.8%. This averages hides the fact that there is considerable variation between journals; thus, around 3.5% of articles in the two journals of national medical associations of the UK (BMJ) and USA (JAMA) were from the RoW as compared to 12% in the Lancet articles (Table 1 ). Indeed, more than half the articles from the RoW were published in just one journal, the Lancet (Table 2 ). Table 1 The contribution of regions to the research literature in five leading journals BMJ Lancet NEJM JAMA ANNALS Total Editorials 261 350 4 124 102 39 876 UK 160 61.4% 167 47.7% 10 8.0% 02 1.9% 01 2.4% 340 38.8% USA 38 14.6% 87 24.9% 97 78.3% 97 95.0% 35 90.0% 354 40.4% OEAC 1 56 21.4% 89 25.4% 14 11.3% 02 1.9% 03 7.6% 164 18.7% RoW 2 07 2.6% 07 2.0 % 03 2.4% 01 0.9% 00 0.0% 18 2.6% Original papers 322 307 218 227 115 1189 UK 216 67.0% 66 21.5 % 06 2.7% 04 1.8% 03 2.6% 295 24.8% USA 22 7.0% 38 12.4% 107 49.1% 173 76.2% 81 70.4% 421 35.4% OEAC 76 23.6% 136 44.3 % 78 35.8% 42 18.5% 23 20.0% 355 29.9% RoW 08 2.4% 67 21.8% 27 12.4% 08 3.5% 08 7.0% 118 9.9% Correspondence 1118 1043 5 894 605 251 3911 UK 829 74% 374 35.8% 30 3.4% 13 2.1% 04 1.6% 1250 32% USA 69 6% 141 13.5% 601 67.2% 506 83.6% 183 72.9% 1500 38.4% OEAC 174 16% 400 38.3% 212 23.7% 71 11.7% 49 19.5% 906 23.2% RoW 46 4% 128 12.2% 51 5.7% 15 2.5% 15 6.0% 255 6.5% Review articles 286 50 65 36 66 503 UK 195 68.2% 17 34% 04 6.2% 01 2.7% 01 1.6% 218 43.3% USA 29 10.2% 15 30% 48 73.8% 28 78.0% 61 92.4% 181 36% OEAC 54 18.8% 14 28% 13 20.0% 04 11.1% 04 6.0% 89 17.7% RoW 08 2.8% 04 08% 00 0.0% 03 8.2% 00 0.0% 15 3% Others 3 47 208 149 145 27 576 UK 31 66.0% 68 32.8% 03 02.0% 00 00.0% 00 00.0% 102 17.7% USA 08 17.0% 34 16.3% 118 79.3% 120 82.7% 25 92.6% 305 53% OEAC 04 8.5% 78 37.5% 22 14.7 % 16 11.0% 01 3.7% 121 21% RoW 04 8.5% 28 13.4% 06 4.0% 09 6.2% 01 3.7% 48 8.3% Global total 2034 1958 1450 1115 498 7055 RoW total 73 3.6% 234 12% 87 6% 36 3.2% 24 4.8% 454 6.4 % 1. OEAC (Other Euro-American countries), OEAC were European countries other than the UK, Australia, New Zealand and Canada. 2. RoW comprised all other countries (i.e. all countries in Eastern Europe, Central and South America, Asia and Africa) 3. Others include: for The Lancet : case reports, view points, public letters, essays, public health, violence in health, department of medical history, the world, world ideas, health and human rights, development of ethics, eponymous, clinical picture, reportage, adverse drug reactions, personal papers, medicine and law, personal papers; for the JAMA : Patients relationships, grand rounds, clinical cross roads, rational clinical examinations, medicine and media, commentary, updates, letters from counters, public opinion and health, medical literature, health law and ethics, clinical cross roads; for the NEJM : images, clinical problem solving, case records from Massachusetts general hospital, clinical implications of basic research, sounding board, clinical problem solving, special articles, for the BMJ : drug point, practice and results, quality of life, history, practice point, lessons of every week; for the Annals : past present and future, time and medicine, social means of medicine, technology of time, personal time, media and publication, NIH conference, in the balance, abroad. 4. The Lancet had 52 editorials and 298 commentaries. Editorials were written in house. 5. The Lancet had 262 research letters. We did not include these either in the correspondence or with the Original papers category. Other journals did not have such a category to compare. Table 2 Proportion of articles contributed by different regions of the RoW category to the total number of editorials, original articles and reviews. BMJ Lancet NEJM JAMA Annals Total 1 (%) India 2 1 2 0 0 05 (3) China 2 5 6 1 1 15 (10) Other Asian 6 8 3 2 0 19 (12) Sub Saharan Africa 8 25 2 1 0 36 (24) Latin America 2 11 4 5 2 24 (16) Middle East/North Africa 0 6 1 0 0 07 (5) Japan 0 8 5 0 4 17 (11) Israel 3 6 4 1 0 14 (9) Multi national 0 8 3 2 1 15 (10) Total 2 (%) 23 (15) 78 (52) 30 (20) 12 (8) 8 (5) 151 1 The % figures in this column represent the contribution of the specific country or region to the RoW papers in the table 2 The % figures in this row represent the contribution of the specific journal to the total RoW papers in the table. Note: This analysis only includes Original Articles, Editorials and Review Papers Within journals, variations in regional contributions from other regions were also notable. Thus, the proportion of articles from the UK was highest in the two journals published in the UK (BMJ and Lancet) while the proportion of articles from the USA was highest in the three journals published in the USA. Table 2 shows the relative contribution of different regions of the RoW category to the total number of editorials, original articles and reviews. Two developed countries (Japan and Israel) contribute a fifth of the literature from RoW, while the two most populous countries in the world (India and China) contribute about 13% together. A detailed analysis of the authorship of the 151 RoW articles showed that 104 (68.9%) involved collaborative authorship with developed countries in Europe or North America; only 43 (31.1%) were entirely independent efforts from the RoW. There were 118 original papers with at least one author from the RoW. Forty-five (38%) of them had a first author from the RoW; 32 (27%) had a last author from the RoW; and 36 (30%) had a corresponding author from the RoW. Only 25 (21%) of them had first, last and corresponding authors all from the RoW. There were 15 original papers in these journals with data from the RoW but without any authors from the RoW; of these 15, nine were in Lancet, four in BMJ and one each in JAMA and NEJM. Thus, the majority of the original articles originating from the RoW had contribution from the developed world authors. Discussion This study presents findings of a survey of articles published in five high-impact general medical journals with the objective of describing the developing world's contribution to research literature over a single calendar year. Our way of classifying countries into developed (UK, USA, OEAC) and developing countries (RoW) has obvious inadequacies but we followed the same method adopted in an earlier paper [ 4 ]. Other euro-American countries (OEAC) shared many cultural and economic features. Rest of the world (RoW) included Eastern Europe, which although culturally related to Western Europe was not economically on the same level, and Japan, which was highly developed economically but did not share many cultural factors with OEAC. The key finding of our survey is that, only 6.5% of the publications in these journals have authors from countries where 90% of the world's population lives. There is a severe under representation of biomedical research from a vast section of the world in these five journals. In addition, there are a small but significant number of articles with data from the developing world without a single author from these countries. This is a troublesome finding that some would refer as 'safari research'. It is a separate ethical issue and journal editors need to look at it carefully. The reasons for under representation of researchers based in developing countries may include research barriers such as lack of funding, poor facilities, limited technical support and inadequate training. Many researchers from developing countries do not speak English as their first language. Fear of rejection by the journals, uncertainty about which journal would be best to publish research, a lack of the culture of publication, competing clinical commitments, different ministry and donor driven agendas for research, are some of the unseen barriers facing developing country researchers (10). The editors of leading medical journals may not have paid sufficient attention to these barriers, real and perceived, that clinical researcher in developing countries face [ 11 ]. We acknowledge that many developing country researchers choose to publish their work in national or regional journals that are not as high-impact as the journals we have reviewed. However, many researchers, irrespective of the country of their origin, also prefer to publish their work in journals with a high impact factor and circulation. This, inevitably, leads to a focus on leading journals published from the UK or USA. However, journals may be under some pressure to publish material, which is relevant to the majority of their readership. Thus, it is not surprising that journals from the UK have the majority of articles from UK institutions and similarly, journals from the USA tend to have most of their articles from US institutions. The BMJ admits that although the BMJ aspires to be more international, they cannot forget that they reach 80% doctors in Britain [ 12 ]. However all these journals are financially highly successful in global markets. Their international success brings responsibilities to the global community they serve and profits from [ 11 ]. Our findings are in agreement with other published findings in this area [ 13 ]. In a study of randomised controlled trials published in leading medical journals many of the diseases afflicting the south are understudied [ 14 ]. Even in the field of tropical medicine there were few contributions originating from countries with a low human development index (HDI) [ 15 ]. Another study, where the number of biomedical articles were normalized to the number of publications per million population, also shows an under representation of Asia, Africa and South America. The authors demonstrated that the number of biomedical publications increase according to the economic ranking of the country [ 16 ]. The same authors have shown in a similar study that publications per million population are more closely related to gross national product (GNP) and research and development expenditure [ 17 ]. The inescapable conclusion of this research is that USA, UK and other European countries dominate biomedical research, in part because of their wealth and investment in research. Irrespective of the method used to categorize countries in developing world, (by GNP, HDI or any other method), generation of new knowledge in biomedical research in these nations is insufficient. In a recent Nature paper on science publications Japan occupies 4th place, Israel 15th, China 19th, and India 22nd in the rank order of nations based on their share of top 1% of highly cited publications. Unsurprisingly the USA and UK occupies the two top positions [ 18 ]. There are many reasons to strive for a more just international distribution of biomedical research in leading journals. The burden of disease in the world falls heavily on developing countries and the pattern of this burden is likely to change in the next twenty years. Diseases like TB, HIV, Malaria, Dengue, and viral hemorrhagic fevers are now no longer tropical with increase in international travel, global warming, refugees, economic and military intervention and conflicts. Newly emerging infections from tropics such as SARS and bird flu can have devastating effects far away from tropics. In addition, diseases such as diabetes and obesity can no longer be considered as diseases in the developed world. As the impact and burden of these diseases are more in developing countries more research publications are needed from South. Clinicians and policy makers in many developing countries have limited access to international journals because medical libraries often need to make a choice from a number of journals due to financial constraints. Often, it is the high-impact journal, which is subscribed to. Thus, the proportion of international representation in these leading journals may be a crucial factor in influencing health policies in many countries. The importance of research goes well beyond its impact on health policies [ 11 ]. Thus, research on the epidemiology and management of diseases in different health systems raises the probability of identifying risk factors for diseases and identifying innovative approaches to their management. This fact was well reflected in the multi-center eclampsia trial [ 19 ]. Until this trial was carried out in Africa, South America and India, the subject of using magnesium sulfate in the management of eclampsia was controversial. The other good example is the only meningitis B vaccine in the world, which was developed by the Carlos J. Finlay Institute in Cuba and now saves lives all over the world [ 20 ]. Providing free journals electronically to the developing countries is a commendable step in correcting the thirst for new information [ 21 ]. However the one-way flow of information by making journals electronically free to developing world is unlikely to be sufficient [ 22 ]. By providing journals free to 'RoW', and propagating research which has been conducted in countries where only 10% of the disease burden is experienced is itself a moral and ethical issue. Ultimately, we believe that strengthening the health research capacity in developing world and providing reasonable opportunities for publications arising from the developing world are critical, not only for achieving biomedical research publication equity, but also advancing medical science. Competing interests We would like to state here that our conflict of interest if at all would be due to the fact that we come from the developing world. Authors' contributions All authors were involved in all the components of this paper, right from the start, in formulating and developing the idea, collecting the data, drafting the first version and revising it and approving the final version. Pre-publication history The pre-publication history for this paper can be accessed here:

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527877

Brain inflammation and oxidative stress in a transgenic mouse model of Alzheimer-like brain amyloidosis

Background An increasing body of evidence implicates both brain inflammation and oxidative stress in the pathogenesis of Alzheimer's disease (AD). The relevance of their interaction in vivo, however, is unknown. Previously, we have shown that separate pharmacological targeting of these two components results in amelioration of the amyloidogenic phenotype of a transgenic mouse model of AD-like brain amyloidosis (Tg2576). Methods In the present study, we investigated the therapeutic effects of a combination of an anti-inflammatory agent, indomethacin, and a natural anti-oxidant, vitamin E, in the Tg2576 mice. For this reason, animals were treated continuously from 8 (prior to Aβ deposition) through 15 (when Aβ deposits are abundant) months of age. Results At the end of the study, these therapeutic interventions suppressed brain inflammatory and oxidative stress responses in the mice. This effect was accompanied by significant reductions of soluble and insoluble Aβ1-40 and Aβ1-42 in neocortex and hippocampus, wherein the burden of Aβ deposits also was significantly decreased. Conclusions The results of the present study support the concept that brain oxidative stress and inflammation coexist in this animal model of AD-like brain amyloidosis, but they represent two distinct therapeutic targets in the disease pathogenesis. We propose that a combination of anti-inflammatory and anti-oxidant drugs may be a useful strategy for treating AD.

Introduction Alzheimer's disease (AD) is the most common, complex and challenging form of neurodegenerative disease associated with dementia in the elderly. Neuropathological examination of the AD brain shows extensive neuronal loss, accumulation of fibrillar proteins as extra-cellular amyloid β (Aβ) plaques, and as neurofibrillary tangles (NFTs) inside neurons [ 1 ]. However, besides these pathological hallmarks, AD brains exhibit clear evidence of chronic inflammation and oxidative damage [ 2 , 3 ]. Currently, data from human studies as well as animal models strongly support the concept that oxidative imbalance and subsequent oxidative stress are among the earliest events in the pathogenesis of AD [ 4 , 5 ]. Thus, an increase in lipid peroxidation, protein oxidation and DNA oxidation has been reported not only in AD patients, but also in subjects with mild cognitive impairment (MCI) [ 6 , 7 ]. Similarly, immunohistochemical and biochemical evidence for these signatures of oxidative stress have been shown in animal models of AD-like brain amyloidosis, i.e. the Tg2576 transgenic mouse model thereof [ 8 - 10 ]. Chronic neuroinflammation is another constant feature of AD, and this also is thought to play a significant role in the onset and progression of AD. Support for this hypothesis comes from epidemiological studies showing that prolonged use of nonsteroidal anti-inflammatory drugs (NSAIDs) decreases the risk of developing AD as well as delaying the onset of this disorder [ 11 ], while many mediators of inflammation have been detected in the AD brain [ 12 ]. Further, recent studies in AD mouse models have shown that chronic treatment with a subset of NSAIDs (e.g. ibuprofen, flurbiprofen, indomethacin) reduced brain inflammation and Aβ levels in addition to the deposition of Aβ in brain [ 13 , 14 ]. Despite this evidence, and the considerable theoretical and therapeutic interest, the relationship between brain inflammation responses and oxidative stress has not yet been clearly delineated in AD. For example, its is possible to consider these two events as elements of the same response mechanism, or they can be envisaged as two separate events. Alternatively, they also could work in concert to contribute synergistically to the pathogenesis of AD. In the present study, we examined whether the simultaneous administration of an anti-oxidant, vitamin E, with an anti-inflammatory drug, indomethacin, would exert an additive anti-amyloidogenic effect in the Tg2576 mouse model of AD-like Aβ brain amyloidosis, one of the most extensively studied mouse models of AD [ 15 ]. Significantly, we found for the first time that coincidental suppression of brain oxidative stress further augments the anti-amylodogenic effect of indomethacin. Materials and Methods Animals The genotype and phenotype features of the heterozygote Tg2576 mice that we studied here have been described in earlier reports on these mice from our group [ 9 ]. Mice were weaned at 4 weeks, kept on a chow diet, and males were always separated from females for the entire study. Eight-month-old Tg animals were divided in two groups (n = 10 each), and randomized to receive placebo, or simultaneously indomethacin (10 mg/liter) in their drinking water, and vitamin E (α-tocopherol) in their diet (2 I.U./mg diet) for seven months before being sacrificed. The detailed dosing of the animals receiving indomethacin or vitamin E alone (at the same concentration used in the present study) were described in two previously published studies which also included data on numerous non-transgenenic littermate controls of the Tg2576 mice [ 16 , 17 ]. Fresh drinking water and diet were always replaced every other day. Preliminary experiments demonstrated that the selected dose of indomethacin suppressed total cylcooxygenase-1 activity in vivo and significantly reduced brain inflammation [ 16 ]. The high dose of vitamin E was selected based on a previous study, which indicated that at this concentration it significantly reduced brain oxidative stress response [ 17 ]. During the study, all mice gained weight regularly, and no significant difference was detected between the two groups. Tissue preparation Animals were anesthetized and euthanized following procedures recommended by the Panel on Euthanasia of the American Veterinary Medical Association. They were always perfused intra-cardially for 30 min with ice-cold 0.9% phosphate buffer saline (PBS), containing EDTA (2 mM/L) and BHT (2 mM/L), pH7.4. Brains were removed and one hemisphere was fixed by immersion in 4% paraformaldehyde in 0.1 M PBS (pH7.4) at 4°C overnight, blocked in the coronal plane, and embedded in paraffin as previously described for immunohistochemistry [ 9 , 16 , 17 ], The other hemisphere was gently rinsed in cold 0.9% PBS, then immediately dissected in three anatomical regions (total cerebral cortex, hippocampus, and cerebellum) for biochemistry. Biochemical analysis Tissue samples were minced and homogenized, and total lipid extracted with ice-cold Folch solution (chloroform: methanol; 2:1, vol/vol). Lipids were subjected to base hydrolysis by adding aqueous 15% KOH and then incubated at 45°C for 1 hr for measurement of total iPF 2α -VI by ion chemical ionization gas chromatography/mass spectrometry assay, as previously described [ 9 , 16 , 17 ]. In brief, a known amount of the internal standard is added to each sample, after solid phase extraction samples are derivatized and purified by thin layer chromatography, and finally analyzed. An aliquot of these extracts was assayed for total levels of PGE 2 and TxB 2 by a standardized ELISA kit following the manufacturer's instructions (Cayman Chem. Com.). Briefly, extracts were diluted with acetate buffer and purified through an affinity column. The purified samples were evaporated, re-dissolved in the assay buffer and applied to 96-well plates pre-coated with goat anti-serum IgG and incubated with PGE 2 or TxB 2 monoclonal antibodies. The plates were rinsed with washing buffer and developed using Ellman's reagent for 60–90 min at room temperature with gentle shaking. Specific concentrations were determined spectrophotometrically and expressed as pg/mg tissue. IL-1β levels were measured by a standardized sandwich ELISA kit following the manufacturer's instructions (Endogen Pierce). Briefly, equal amounts of sample were loaded onto 96-well plate pre-coated with monoclonal antibody against mouse IL-1β overnight at 4°C. The plates were rinsed three times with washing buffer and developed with streptavidin-horseradish peroxidase (HRP) [ 13 ]. Specific concentrations were determined spectrophotometrically and expressed as pg/mg protein. Total protein carbonyls in tissue were determined by using the Zenith PC test kit according to the manufacturer's instructions (Zenith Tech.) [ 18 ]. Briefly, aliquots of the tissue homogenates were first reacted with dinitrophenylhydrazine (DNP), transferred to a multi-well plate, incubated with blocking reagent, washed and probed with anti-DNP-biotin solution. After washing, samples were incubated with streptavidin-HRP, washed again, and then developed. After 15 min, the reaction was stopped and absorbance immediately read at 450 nm. Oxidized protein standards, internal controls and blanks were always assayed at the same time and in the same way. All samples were always determined in triplicate and in a blind fashion. Immunoblot analysis An aliquot of brain homogenates was electrophoresed on a 10% acrylamide gel under reducing conditions. Protein were transferred to a polyvinylidene membrane before blocking in 10% nonfat dry milk for 2 hr. Blots were incubated with monoclonal antiboby against glial fibrillary acidic protein (GFAP) (2.2B10) (1:1,000), or an anti-beta actin (1:5,000) antibody overnight at 4°C. After three rinses, blots were incubated with HRP-conjugated goat anti-mouse for 45 min before development with chemiluminescent detection system using ECL (Amersham). Bands were quantified using densitometric software (Molecular Analyst). The anti-GFAP is a monoclonal antibody, and its characterization has previously been published [ 19 ] (Zymed Lab. Inc.). The anti-beta actin is from commercial sources (Novus Biological). Brain Aβ1-40 and Aβ1-42 levels Sequential extraction of brain samples was performed with high-salt buffer and formic acid, respectively to measure soluble and insoluble Aβ1-40 and Aβ1-42 levels, as previously described [ 9 , 16 , 17 ]. Briefly, cerebral cortex, hippocampus and cerebellum were serially extracted in high-salt Re-assembly buffer (0.1 M Tris, 1 mM EGTA, 0.5 nM Mgso4, 0.75 M NaCl, and 0.02 M NaF, pH 7.0) containing protease inhibitor mixture (pepstatin A, leupeptin, N-tosyl-L-phenylalanine chloromethyl ketone, soybean trypsin inhibitor, each at l μg/ml in 5 mM EDTA). Homogenates were centrifuged at 100,000 × g for 1 hr at 4°C. Supernatants were removed, pellets were re-suspended in 70% formic acid and sonicated and centrifuged at 100,000 × g for 1 hr at 4°C. Supernatants were diluted 1:20 with 1 M Tris base. Samples were mixed with buffer EC [0.02 M sodium Phosphate, 0.2 mM EDTA, 0.4 M NaCl, 0.2% BSA, 0.05% CHAPS, 0.4% Block-ace (Dainippon, Suita, Osaka, Japan), 0.05% sodium azide, pH7.0] and analyzed directly using Ban 50/BA27 for Aβ1-40 or Ban50/BC-05 for Aβ1-42/43 sandwich ELISA system as previously described [ 16 , 17 ]. Results were expressed as pmol/g tissue. The values were calculated by comparison with a standard curve of synthetic Aβ1-40 and Aβ1-42. Analyses were always performed in duplicates and in a coded fashion. Burden of brain Aβ deposits Serial 6-μm-thick paraffin sections were cut throughout each brain, and mounted on APES-coated slides. Sections were deparaffmized, hydrated, rinsed with PBS and pre-treated with formic acid (88%) for 10 min to antigen retrieval, and with 3% H 2 0 2 in methanol for 30 min to eliminate endogenous peroxidase activity in the tissue and with the blocking solution (5% normal horse serum in Tris buffer, pH 7.6). Subsequently, sections were incubated with a biotinylated antibody against Aβ (4G8) (1:10,000 dilution), at 4°C overnight [ 16 , 17 ]. Sections were then incubated with secondary antibody for 1 hr (dilution 1:1,000), then reacted with horse-peroxidase-avidin-biotin complex (Vector Lab.), and immuncomplexes visualized by using 3,3'-diaminobenzidine as the chromogen. Finally, they were dehydrated with ethanol, cleared with xylene and coversliped with Cytoseal. As control, sections from the same group of animals were treated in the same manner, except for the primary antibody. Light microscopic images from the somatosensory cortex, perihippocampal cortex, and hippocampus were captured from eight series of sections using a Nikon Microphot-FXA microscope with 4 × objective lens. The area occupied by Aβ-immunoreactive products in the region of interest were identified, and the total area occupied by the outlined structures was measured to calculate: 1) the total area with selected immunoreactive products, 2) the percentage of the area occupied by immunoreactive products over the outlined anatomical area in the image, as previously described [ 9 , 16 , 17 ]. Analyses were always performed in a coded fashion. Statistical analysis Data are expressed as mean ± standard error of mean (S.E.M.), analyzed by analysis of variance (ANOVA), and subsequently by student unpaired 2-tailed t test corrected for multiple comparisons. Significance was set at p < 0.05. Results Starting at eight months of age, Tg2576 mice were randomized to receive placebo or vitamin E (2 I.U./mg diet) added to their diet, plus indomethacin (l0 mg/liter) in their water, and they were treated until they were 15 months old. Notably, at 8 months of age, the Tg2576 mice show elevated brain levels of soluble and insoluble Aβ as well as isoprostanes, relative to their non-transgenic littermates, but they show no evidence of any brain Aβ deposits, while following the initial onset of mature plaque-like brain deposits at about 11–12 months of age, the Tg2576 mice show abundant Aβ deposits and higher levels of isoprostanes in neocortex and hippocampus a 15 months of age [ 9 , 15 , 20 ]. Assuming that each mouse eats 4–5 mg chow/day, the estimated average vitamin E intake for each animal was ~8–10 I.U./day. Assuming that each mouse drinks 3 to 4 mL water/day, the estimated daily intake of indomethacin was calculated around 30–40 ng. At the end of the study, body weight, total plasma cholesterol, triglycerides and peripheral blood cell count were not different between placebo and active treatment (not shown). Compared with placebo, Tg2576 mice receiving indomethacin plus vitamin E at the same time had a significant reduction in PGE 2 and suppression of TxB 2 levels in tissue homogenates from total cortex and hippocampus (Table 1 ). Further, the presence of vitamin E significantly reduced two independent markers of oxidative stress injury in both brain regions. Thus, neocortex and hippocampus levels of iPF 2α -VI (a reliable biomarker of lipid peroxidation), as well as protein carbonyls (known biomarkers of protein oxidation) were both significantly decreased (Figure 1 ). Compliance with the diet was evident from the rise in brain levels of vitamin E (+57%) in the mice receiving the supplemented chow. Table 1 Effects of indomethacin plus vitamin E on total brain cortex levels of PGE 2 , TxB 2 and IL-1β in Tg2576 mice. Mice were treated starting at 8-months of age until they were 15-month-old (n = 10 animals per group). Placebo Indomethacin Vitamin E P PGE 2 (pg/mg tissue) 92 ± 8 39 ± 5* <0.01 TxB 2 (pg/mg tissue) 148 ± 10 15 ± 4* <0.001 IL-1β (pg/mg protein) 75 ± 12 33 ± 8* <0.01 Values are expressed as means ± S.E.M. Figure 1 Effect of indomethacin plus vitamin E supplementation on markers of brain oxidative stress. Total cerebral cortex homogenates from Tg2576 receiving placebo (open bars) or the combination therapy (closed bars) were assayed for levels of iPF 2α -VI (upper panel) and protein carbonyls (lower panel) (*p < 0.01, n = 10 per group). Western blot analysis was used to determine the effect of the drug treatment on GFAP levels, a marker of astrocytosis [ 13 ]. These levels were significantly lower in the treated than in the placebo group (Figure 2 ). Another marker of brain inflammation was also assessed, i.e. IL-1β, which has been reported to be increased in these mice [ 13 ]. Compared with placebo, we found that IL-1β levels were significantly reduced by 55% in homogenates from neocortex (Table 1 ), and 61% in hippocampus (not shown) of the mice receiving the combination therapy. Figure 2 Effect of indomethacin plus vitamin E supplementation on GFAP levels. GFAP and actin levels were detected by immunoblots in homogenates from total cortex of Tg2576 administered with placebo (open bars) or indomethacin plus vitamin E (closed bars) (*p < 0.02, n = 8 per group). Next, we assessed the effect of indomethacin and vitamin E on brain levels of soluble and insoluble Aβ1-40 and Aβ1-42. As expected for their age, Tg2576 mice on placebo showed elevated levels of both forms of these peptides in their cortex and hippocampus (Figure 3 ), whereas cerebellum had much lower levels (not shown). Soluble Aβ1-40 and Aβ1-42 were reduced by ~65% in both neocortex and hippocampus homogenates from treated mice (Figure 3 ). Further, we found that the combination of vitamin E with indomethacin resulted in a significant reduction (55% and 59%) of the insoluble fraction of these peptides in both brain regions (Figure 4 ). The same treatment had no effect on both forms of Aβ in the cerebellum of Tg2576 compared with placebo (not shown). Amyloid deposits were widely present in the cerebral cortex and hippocampus of Tg2576 mice at 15 months of age, as previously reported [ 15 , 20 ]. To determine the effect of this treatment on amyloid deposition, the areas occupied by 4G8-immunopositive reactions were analyzed in three different brain regions: the somatosensory cortex (SSC), perihippocampal cortex (PHC), and hippocampus (HIP) areas. Comparison of the burden of Aβ positive deposits between placebo and combination therapy groups revealed a significant reduction for the amyloid burden in all three regions considered (Figure 5 , 6 ). Figure 3 Effect of indomethacin plus vitamin E supplementation on soluble Aβ levels. Levels of high salt soluble Aβ1-40 and Aβ1-42 in total cortex and hippocampus of Tg2576 on placebo (open bars), or indomethacin plus vitamin E (closed bars) (*p < 0.01, n = 8 per group). Figure 4 Effect of indomethacin plus vitamin E supplementation on insoluble Aβ levels. Levels of formic acid soluble Aβ1-40 and Aβ1-42 in total cortex and hippocampus homogenates from Tg2576 receiving placebo (open bars) or indomethacin plus vitamin E (closed bars) (*p < 0.001, n = 8 per group). Figure 5 Effect of indomethacin plus vitamin E supplementation on amyloid deposition. Percentage area of somatosensory cortex (SSC), hippocampus (HIP) and parahippocampal cortex (PHC) occupied by Aβ immunoreactive deposits in Tg2576 receiving placebo (open bars), or indomethacin plus vitamin E (closed bars) for seven months (*p < 0.001; n = 8 per group). Figure 6 Representative pictures of brain sections from mice on placebo or receiving indomethacin plus vitamin E. Discussion There is substantial evidence implicating both oxidative stress and inflammatory mechanisms in AD pathogenesis. Evidence for oxidative stress derives from both human (post-mortem and living patients) studies, and transgenic mouse models of the disease. There is a long list of surrogate markers of reactive oxygen species-mediated injury that have been found increased in the brain and cerebrospinal fluid of AD patients. It includes, just to mention a few, malondialdehyde, 4-hydroxynonenal, F 2 -isoprostanes (lipid peroxidation); protein carbonyls, nitrotyrosine (protein oxdidation); 8-hydroxy-2'-deoxyguanosine (DNA oxidation) [ 3 - 5 ]. Transgenic animals show the same type of oxidative damage that is found in AD, and it directly correlates with the presence of Aβ deposits [ 8 , 10 , 21 ]. Oxidative stress also precedes amyloid deposition in human AD, the Tg2576 and a transgenic Caernorhabditis elegans model, which over-expresses Aβ1-42 [ 9 , 22 , 23 ]. Furthermore, dietary or genetic perturbation of the anti-oxidant defense system causes exacerbation of the amyloid pathology characteristic of Tg models [ 24 , 25 ]. Taken together, the data accumulated so far clearly indicate that oxidative imbalance and subsequent chronic oxidative stress are not only early events, but they also play a functional role in AD pathogenesis. Based on this evidence we started the treatment at an early stage before the amyloid deposition occurs. Inflammatory mechanisms are also operative in the AD brain and significantly contribute to the pathophysiology of the disease. Although classical defined inflammation, including such features as edema and neutrophil invasion, is not seen in the AD brain, hallmark of innate immune response are constant elements of the neuropathology associated with brain degeneration in AD [ 12 ]. Further, evidence that inflammation contributes to the AD pathogenesis stems out from several retrospective epidemiological studies showing a significant reduction in the risk of AD associated with a prolonged usage of NSAIDs [ 11 ]. Tg2576 mice display age-related neocortical and hippocampal amyloid deposits, which correlate with microglia activation, reactive astrocytes with increased GFAP, IL-1β levels, and dystrophic neuritis [ 13 , 26 ]. Furthermore, plaque-associated reactive microglia in these animals show enhanced staining for TNFα and IL-1β [ 27 ]. Lim et al. first reported that chronic administration of the NSAID ibuprofen to Tg2576 reduces total Aβ levels, amyloid burden and brain inflammation [ 13 ]. More recently, we showed that a high dose of indomethacin, another NSAIDs member, which fully suppresses total cyclooxygenase (COX)-l activity, by modulating brain inflammation response reduces soluble Aβ1-40 and Aβ1-42, and insoluble Aβ1-42 but not Aβ1-40 levels in the same model. This effect was accompanied by a significant reduction of the amyloid burden in the hippocampus of these mice [ 16 ]. However, recent studies indicate that a subset of NSAIDs, including indomethacin, also possesses a direct, COX-independent Aβ-lowering capacity in cell cultures as well as transgenic models [ 28 ]. Further, we showed that vitamin E alone at the same high dose used in this study decreased soluble and insoluble Aβ1-40 and Aβ1-42 levels by ~28% and ~35%, respectively. This effect was associated with a significant reduction in amyloid deposition in the somatosensory cortex, but not in the hippocampus or parahippocampal areas [ 17 ]. In the present study, we extended these previous observations by examining whether administration of indomethacin in combination with vitamin E would result in a better anti-amyloidotic effect. Our findings show that soluble Aβ1-40 and Aβ1-42 levels were reduced by ~65%, while the insoluble fractions were decreased by ~55%. Consistently, we observed a better and more diffuse effect also on the amount of amyloid deposited in the brain at the end of the study. Finally, the two drugs together produced an additive affect on brain inflammation and oxidative stress [ 16 , 17 ]. Our results confirm previous observation where low-dose curcumin, a drug with reported both anti-oxidant and anti-inflammatory activities, reduced total Aβ and plaque burden [ 29 ]. However, several other mechanisms of action, unrelated to inflammation or oxidation, could underlie the effect of this compound in vivo, and the relative importance of each of them for the anti-amyloid effect observed is still unclear [ 30 ]. In our study, we used two different drugs with a more restricted therapeutic target to provide further evidence that both oxidative stress and inflammation are indeed functionally relevant in the development of the phenotype of these animals. However, we also provide new information on the critical issue of the in vivo relationship between these two events. Thus, our results suggest that brain inflammation and oxidative stress are two separate events, which work in concert to modulate the development of this AD-like brain Aβ amyloidosis model. Previously, we have shown that a full dose of indomethacin alone despite a significant reduction in brain inflammation had only a marginal effect on brain oxidative stress in the Tg2576 mice [ 16 ]. This finding suggests that lipid peroxidation products contribute minimally to brain inflammation in this model, and raise the possibility that vitamin E alone might have influenced amyloidosis by other mechanisms related to its anti-oxidant effect, such as inflammation. Thus, we observed that this antioxidant further suppressed both amyloidosis and brain inflammation when combined with indomethacin. In summary, our findings support the hypothesis that oxidative stress and inflammation represent important but distinct therapeutic targets in AD-like amyloidosis. We conclude that a combination of therapeutic agents targeting these different disease-modulating mechanisms might be rationally evaluated in the prevention or therapy of AD in humans. List of abbreviations AD: Alzheimer's disease Aβ: Amyloid β peptide Tg: Transgenic mouse model NSAIDs: Non-steroidal anti-inflammatory drugs PGE 2 : Prostaglandin E 2 TxB 2 : Thromboxane A 2 GFAP: Glial fibrillary acidic protein IL-1β: Interleukin 1-β IPF 2α -VI: Isoprostane F 2α -VI Competing interests The authors declare that they have no competing interests. Authors' contributions Yuemang Yao and Cinzia Chinnici have made substantial contribution to the acquisition of data and biochemical analyses. Hanguan Tang contributed to the immunohistochemical analyses. John Q. Trojanowski and Virginia M-Y Lee have been involved in the interpretation of data, and the critical revision of the manuscript for intellectual content. Domenico Praticò has been involved in the conception and design of the studies, interpretation of data, drafting and critical revision of the manuscript.

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548283

Etiology and prognosis of pregnancy-related pelvic girdle pain; design of a longitudinal study

Background Absence of knowledge of pregnancy-related pelvic girdle pain (PPGP) has prompted the start of a large cohort study in the Netherlands. The objective of this study was to investigate the prevalence and incidence of PPGP, to identify risk factors involved in the onset and to determine the prognosis of pregnancy-related pelvic girdle pain. Methods/design 7,526 pregnant women of the southeast of the Netherlands participated in a prospective cohort study. During a 2-year period, they were recruited by midwives and gynecologists at 14 weeks of pregnancy. Participants completed a questionnaire at baseline, at 30 weeks of pregnancy, at 2 weeks after delivery, at 6 months after delivery and at 1 year after delivery. The study uses extensive questionnaires with questions ranging from physical complaints, limitations in activities, restriction in participation, work situation, demographics, lifestyle, pregnancy-related factors and psychosocial factors. Discussion This large-scale prospective cohort study will provide reliable insights in incidence, prevalence and factors related to etiology and prognosis of pregnancy-related pelvic girdle pain.

Background In the Netherlands, little information is available about prevalence, incidence, etiology and prognosis of pregnancy-related pelvic girdle pain (PPGP). It is hypothesized that during pregnancy many women (about 80%) experience some degree of pain in the pelvic region and/or the low back and that in some of these patients pain becomes chronic or recurrent. Often, symptoms impact on activities of daily life, hobbies, participation in society, planning of next pregnancies and sometimes lead to a chronic disabling condition with considerable work absenteeism in the future [ 1 ]. Treatment and indirect costs of these chronic or recurrent patients constitute a considerable burden on health care services, health care insurers and other parties. The total health care expenditures incurred by patients with back pain in the United States in 1998 were approximately $91 billion, accounting for about 1 % of the Gross National Product [ 2 ]. Van Tulder et al. estimated the costs of back pain for Dutch society in 1991 at 1.7% of the Gross National Product [ 3 ]. These costs consist almost totally of indirect costs (97%) such as absenteeism and disablement; for that reason chronic back pain can be considered a major economical problem. It is therefore important that PPGP can be diagnosed and treated before PPGP becomes chronic. Consequently, tracking risk factors and characteristics influencing etiology and prognosis of pregnancy-related pelvic girdle pain is important. Absence of knowledge of risk and prognostic factors and the absence of evidence-based treatment strategies about PPGP has prompted the start of a large cohort study in the Netherlands. In January 2000, the Maastricht PPGP cohort study started. It was established (I) to examine the prevalence and incidence of pregnancy-related pelvic girdle pain (PPGP) during and after pregnancy, (II) to identify risk factors involved in the onset of PPGP and to identify which factors can play an important role in the early detection of PPGP and finally (III) to determine the prognosis of PPGP and to identify prognostic factors. Furthermore, a clinical trial is embedded in this cohort, aimed at studying the effectiveness of a tailor-made treatment program in PPGP after delivery [Bastiaenen et al., treatment, submitted]. Methods/design Design and study population In an observational prospective cohort study, etiology and prognosis of PPGP will be studied in 7526 pregnant women. The source population for the study comprises of pregnant women from the southeastern area of the Netherlands. Both midwives and gynecologists recruit women for the study when pregnant between 10 and 14 weeks. Women are considered eligible if they meet the following inclusion criteria: women are well versed in the Dutch language and at least 18 years. At inclusion, women receive a leaflet containing information about the research project, an informed consent form and a baseline questionnaire. After providing informed consent and filling out the first questionnaire, the women receive a second questionnaire at 30 weeks of pregnancy, a third one at 2 weeks after delivery and a fourth and fifth at 6 months and 12 months postpartum, respectively. In this study, extensive questionnaires are used with questions ranging from physical complaints (past and present), limitations in activities, restriction in participation, work situation, demographics, lifestyle, pregnancy-related factors and psychosocial factors. With the help of these questionnaires the prevalence and incidence of PPGP in the Netherlands will be described. In addition, possible risk factors and prognostic factors of PPGP will be examined. Exposure variables In the Maastricht PPGP cohort study several domains of exposure were measured, including individual characteristics, lifestyle, work situation, pregnancy-related factors and psychosocial factors. The majority of factors were assessed with existing, validated questionnaires. The Dutch translation of the Quebec Back Pain Disability Scale (QBPDS) [ 4 ] will measure low back functional status [ 5 ]. The QBPDS is a 20-item 6-point scale describing activities commonly affected by back pain. This questionnaire is not developed to study a pregnant population and some activities were unsuitable for women who were pregnant or just gave birth. We therefore added a 7 th option to the questionnaire, namely "not applicable". We also changed the phrase "because of my back " into "because of my back and/or pelvic pain" in questionnaires. Fear of movement is measured by means of the Dutch translation of the Tampa Scale for Kinesiophobia (TSK) [ 6 , 7 ]. The TSK consists of 17 items; each rated on a 4-point Likert scale. Pain catastrophizing is measured by the Pain Catastrophizing Scale (PCS) [ 8 ]. The PCS is a 13-item 5-point scale. A woman is said to catastrophize pain, when she views pain as extremely threatening. To measure the experience of negative affect and positive affect we used the 14-item Negative Emotionality Scale (NEM) and the 11-item Positive Emotionality Scale (PEM) [ 9 ]. Current mental health was measured by the General Health questionnaire (GHQ). The questionnaire was originally designed as a 60-item instrument, but we used the shortened version GHQ-12 [ 10 ]. The perceived stress scale (PSS) was used to measure to assess stress. The PSS is a 14-item instrument with a 5-point scale [ 11 ]. Outcome measurements Pregnancy-related pelvic girdle pain is currently not an entity that can be clearly diagnosed and described. Therefore, Bastiaenen et al. studied separate diagnostic strategies of four international authors in the field of PPGP [Bastiaenen et al., submitted]. They concluded that there was no similarity in the selection of patients with PPGP between the authors. Most of these classification-strategies of PPGP are based on expert-opinions. Therefore, a possible reason for the lack of similarity in the selection of patients can be that they all select different small parts of the same large patient-group. Because of the relatively unknown etiology of pregnancy-related pelvic girdle pain and the lack of an all-embracing definition, we will use an extensive description of PPGP. We expect that during pregnancy almost all women experience some form of pain in the lower back, the buttocks, the symphyses, the groins and/or radiation into the legs. This pain is probably caused by hormonal and physiological changes which are considered normal during pregnancy. However, some women experience pain in a very early stage of pregnancy while others only experience pain in the final stages of pregnancy. In addition, some women are more limited in their activities (due to pain) than others. This suggests that other factors might influence the hormonal or physiological changes during pregnancy [ 12 ]. Most women who had developed PPGP during pregnancy quickly recover after delivery [ 13 ]. In this study, pain during or after pregnancy is measured by using patients' self-reports. Women with pain can be identified by the question whether they experienced pain in the lower back, the buttocks, the symphyses, the groins or radiation into the legs during or after this pregnancy. To study etiology of PPGP, women who gave a positive answer to this question during this pregnancy were selected. For the prognosis of PPGP it is important that women have pain that started during pregnancy and persisted after delivery. At several moments during and after pregnancy, experienced pain in the lower back, the buttocks, symphyses, groins or radiation into the legs was measured. The answers to this question were assimilated into a flowchart (Fig. 1 ). Figure 1 Flowchart of pain in the lower back, the buttocks, the symphyses, groins and/or radiation into the legs: description of cases. Based on their self-reports, the women are stratified into a case group without a history of LBP/PPGP or a case group with a history of LBP/PPGP. Some women (N = 246) were not classified into groups for the following reasons; specific disorders of the spine, rheumatism, neurological disorders and cancer. The first group consists of women without a history of LBP/PPGP, but they experience pain during pregnancy and this pain does not disappear until (at least) 2 weeks after delivery. The second group experiences pain during and after pregnancy, but they also have a history of LBP/PPGP. Both groups of cases will be analysed to study the prognosis of PPGP. Women who experience recurrent pain episodes during pregnancy, that resolve within 2 weeks after delivery, will not be considered cases in the analyses for the prognosis of PPGP. They form a miscellaneous group. However, this miscellaneous group and women who experience no pain after delivery were not excluded from follow-up. Data analyses Participation rates and descriptions of baseline characteristics of the Maastricht PPGP cohort study will be presented. We calculated prevalence rates for PPGP by dividing the numbers of prevalent cases at several moments during pregnancy by the total number of subjects. Characteristics of the study population at baseline Recruitment of women into the study began in November 2000 and ended in November 2002. Approximately 10,850 women were asked to participate in the study by midwives and gynecologist in the southeast of the Netherlands. The locations of the participating midwives and gynecologists are shown in Fig. 2 . At the end of the recruitment period 7,526 pregnant women (73.4%) were willing to participate and were included in the cohort. Figure 2 The location of participating midwives and gynaecologists in the Netherlands. In Table 1 a number of selected characteristics of the study population at baseline are presented, including details of their age, education, BMI, smoking, work and reproductive history. Mean age of the study population is 31.5 years. The educational level of the participants is very high. Approximately 38 % of the participants have had higher vocational or academic education. The use of alcohol during pregnancy is limited. While 44.8% of the study population did not use alcohol before pregnancy, 91.2% did not use alcohol during pregnancy. With exception of the country of birth, the study population is heterogeneous with respect to demographics, work status and pregnancy-related factors. Table 1 Characteristics of the study population (N = 7526) at baseline (14 wks pregnancy) Aspect % Age (yrs) Mean 31.54 N = 7523 < = 20 0.4 21–25 5.3 26–30 32.4 31–35 47.8 36–40 12.8 >40 1.2 Country of origin Netherlands 96.5 N = 7516 Belgium, Germany, France, Austria, Switzerland, Luxemburg, Ireland and United Kingdom 1.9 Other countries 1.6 Education Primary school 0.6 N = 7498 Preparatory vocational education 7.2 Lower general secondary education 6.6 Vocational education 31.9 Higher general secondary education 8.5 Pre-university education 2.3 Higher vocational education 27.7 Academic education 10.3 Different 4.9 BMI before pregnancy < 18.5 Underweight 3,1 N = 7437 18.5–24.9 Normal weight 68,1 25.0–29.9 Overweight 20,5 > = 30 Extreme overweight 8,3 Smoking habits at 14 wks pregnancy Never 59.7 N = 7488 Ex 29.8 Current during pregnancy 10.5 Alcohol-usage (glasses/week) before pregnancy 0 44.8 N = 7482 1–10 53.5 11–20 1.6 >20 0.2 Alcohol-usage (glasses/week) during pregnancy 0 91.2 N = 7210 1–10 8.8 11–20 0.0 >20 0.0 Work (hours/week) No job 13.9 N = 7428 1–10 3.8 11–20 22.9 21–30 21.1 31–40 36.9 >40 1.5 Number of pregnancies 1 42.3 N = 7519 2 36.6 3 14.2 4 4.6 >4 2.3 To examine whether the response in our study affected the determinant distributions (e.g. did primarily women in their first pregnancy respond?), a comparison of response rates was carried out. We performed a pilot-study in which data was recorded from April 2001 until November 2001 of every pregnant woman who attended one of the cooperating practices. Information about parity, PPGP during pregnancy, delivery and PPGP after delivery (until 6 weeks) was collected of 283 women (170 participants and 113 non-participants). Results of this pilot-study showed that primipara compared to multiparous women were more willing to participate in the cohort study. Furthermore, data from the responders in the cohort was compared to available data on pregnant Women in The Netherlands from the "Centraal Bureau voor Statistiek". The results of this comparison are shown in table 2 . Table 2 A comparison between the total Dutch female population and the study population Total Dutch population Study population Period 2001 Period 2002 Period 2003 Period 2001 (N = 2892) Period 2002 (N = 3567) Period 2003 (N = 1061) BMI %* %* %* % % % - Underweight (<18.5) 5.3 4.9 3.0 2,9 3,2 3,5 - Normal weight (18.5–24.9) 70.3 71.9 71.2 66,8 68,5 70,4 - Overweight (25.0–29.9) 18.5 17.8 18.0 21,3 20,3 18,9 - Extreme overweight (> = 30) 5.9 5.4 7.8 9,1 8,1 7,2 Smoking %* %* %* % % % - Current/before pregnancy 35.7 34.0 33.3 29.7 29.3 31.7 Pregnancy-related factors Mean age mother - Total pregnancies 30.8 30.9 31.0 31.1 31.7 32.4 - First pregnancies 29.2 29.2 29.3 29.6 30.2 30.9 Child % % % % % % - Boy 51.2 51.3 51.4 51.0 52.2 50.5 - Girl 48.8 48.7 48.6 49.0 47.8 49.5 Number of pregnancies: % % % % % % - First 46.3 45.8 45.5 44,6 41,0 41.0 - Second 36.3 36.7 36.9 35,6 37,2 37.3 - Third 12.3 12.5 12.6 13,6 14,7 13.9 - Fourth or more 5.1 5.0 5.0 6,2 7,1 7.8 Multiple pregnancy % % % % % % - Twin 1.9 1.9 1.8 1.0 0.9 1.1 * Age-group comparable to study-population Data are presented in 3 separate years to show possible fluctuations. It is noticeable that the mean age of pregnant women is increasing during the 3-year period in the study population and in the total Dutch population. In the study population, pregnant women are slightly older and heavier compared to the total Dutch population. Approximately 34% of all Dutch women (age 18–45 years), compared to 30% in the study population, are smoking (see table 2 ). During pregnancy, 10.5% of the study population will continue smoking (see table 1 ). In general, data of the study population correspond with data of the total Dutch population. Prevalence of pregnancy-related pelvic girdle pain during pregnancy To determine the prevalence of PPGP during pregnancy, data at baseline (14 wks), 30 weeks of pregnancy and 2 weeks after delivery (information about 34–40 weeks of pregnancy) were used. Almost every woman develops pain in the lower back, the buttocks, the symphyses, the groins or radiation into the legs at some time in their pregnancy. Of the 7527 women, 84% reported pain in any or all of these areas during pregnancy. Women with a history of LBP/PPGP are more likely to develop PPGP during pregnancy then women without a history of LBP/PPGP (see fig. 3 ). Figure 3 The point prevalence of PPGP during pregnancy for women with (■) and without (▲) a history of LBP/PPGP. Discussion In this article, we describe the main characteristics of the Maastricht PPGP cohort study in terms of study design, study population, exposure variables and outcome measures. This design, in which both risk factors and outcomes are frequently measured between 14 weeks of pregnancy and 1 year after delivery, enables us to examine the etiology and prognosis of PPGP. Advantages and disadvantages of the study Although a cohort design (even incorporating a randomized controlled trial) has advantages over other epidemiological designs, it poses also a burden on the project in terms of logistics and recruitment. First of all, we are totally dependent on the cooperation and recruiting power of midwives and gynecologists. The number of pregnant women is about evenly divided between the two professions. Although professional workload for midwives in the Netherlands is extremely high, 62 % of the midwife practices took part in this study, recruiting about 90% of the patients. Whereas 58% of the hospitals in the recruitment area participate, they only recruit about 10% of the women. Coordination of the recruitment and frequent staff changes in the hospitals themselves seems to be a key problem. In etiological research there should be sufficient contrast in exposure. In this study, the study population at baseline is heterogeneous with respect to demographic variables, lifestyle characteristics and work-related factors. However, due to logistic constraints we have restricted the recruiting area to the southeast of the Netherlands, posing questions about the representation of our sample for the whole Dutch population. For instance the number of immigrants is significantly less in the southeast of the Netherlands. However, for future genetically oriented studies, this could be a major advantage. We have shown previously that our sample is slightly different from the national population of pregnant women with regards to several determinants (e.g. age, BMI). At baseline, 7526 pregnant women participated in our study. This is a response rate of 73.4%. Non-response and loss to follow-up might introduce selection bias in prospective studies. Loss to follow-up, especially in time series designs with repeated measurements, pose threats to representation of the sample. Especially when losses-to-follow-up are connected with negative pregnancy outcomes (miscarriage, birth defects), co-morbidity of the mother or factors predicting for PPGP. To evaluate whether differential loss to follow-up occurs, we will compare the profile of those lost to follow-up with other participants. In this study we were able to evaluate if there are significant differences between participants and non-participants. This evaluations shows that primiparous women are more willing to participate in the study compared to multiparous women. We expect that multiparity plays an important role in the etiology and prognosis of PPGP. Therefore, an underestimation of the prevalence of PPGP reported in this study cannot be ruled out. However, the collected sample should be able to provide reliable preliminary insights in incidence and prevalence of PPGP and factors related to etiology and prognosis of PPGP in the Netherlands. PPGP is a complex syndrome and for a greater understanding of pregnancy-related pelvic girdle pain, future studies should further disentangle the multifactorial etiology and prognosis of PPGP. Future research within the framework of the Maastricht PPGP cohort study will focus on disentanglement of the complex syndrome. Competing interests The author(s) declare that they have no competing interests. Authors' contributions All authors participated in the design of the study. JMB drafted the manuscript with input from the other authors. All authors read, revised and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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526214

Is cumulative frequency of mitochondrial DNA variants a biomarker for colorectal tumor progression?

To examine the relationship between mitochondrial DNA (mtDNA) alterations and colorectal tumorigenesis, we used high-resolution restriction endonucleases and sequencing to assess the mitochondrial genome from three histologic subtypes of colorectal adenomas (tubular = 8; tubulovillous = 9; and villous = 8), colorectal cancer (CRC) tissues = 27, and their matched surrounding normal tissue (MSNT) = 52. The mitochondrial genomes were amplified using 9 pairs of overlapping primers and systematically analyzed by means of high-resolution analysis. DNA fragments showing a shift in banding patterns between the three adenomas, CRC, in comparison to the MSNT were sequenced to identify the mtDNA alterations. A total of thirty-eight germ-line mtDNA variants were observed in this study. Twenty-two of the thirty-eight were identified as mutations and 59% (13 of 22) were silent mutations and one was a 1-bp insertion. Sixteen of thirty-eight were distinct SNPs in flanking regions of the restriction sites and, 6 of the 16 (37%) SNPs were not previously reported. Most of these mutations/SNPs were homoplasmic and distributed in various regions of mitochondrial genes including the 16S and 12S rRNA. Based on our results, mtDNA germline variants increased in prevalence with adenoma CRC progression. To the best of our knowledge, this is the first report to show an increased prevalence of mitochondrial gene variants in CRC tumorigenesis.

Findings Colorectal polyps are a frequent occurrence in the general population and adenomatous changes in these polyps are associated with the overwhelming majority of CRC. These adenomas are the precursor lesions of colon cancer [ 1 ]. Currently, clinical management of individuals with colorectal polyps is guided by the histology of the lesion [ 2 ]. However, the accuracy of an appropriate staging of colorectal polyps progression to cancer continues to confound clinical pathologists as well as surgeons. Alternative strategies, which augment pathological findings such as the identification of molecular markers that are associated with the colorectal tumor progression, may prove to be a useful as a prognostic tool and a preoperative stage-specific evaluation. Recently, there have been reports of the potential use of mtDNA mutations as a biomarker in the solid tumor of other cancers [ 3 - 6 ] due its repaired less efficiently compared with that of nuclear DNA. Also, Mitochondria have been implicated in cancer given their role in apoptosis [ 7 , 8 ] and its vulnerability to mutation due to the close proximity to a major source of reactive oxygen species (ROS). Nevertheless, definitive mtDNA mutations associated with the progression of tumors such as CRC have yet to be established. Given the connection between mitochondria, ROS, and neoplasia, mtDNA from CRC adenoma, cancer tissues, and pathologically determined matched surrounding non-cancerous tissue were screened for variants, which may be used as biomarkers for colorectal cancer progression. We speculated that there is an association between one or more mtDNA mutations in the adenomatous polyps and CRC progression and the cumulative frequencies of such mtDNA mutations may eventually be demonstrated to be an important marker in adenoma colorectal progression. This study was approved by the Institutional Review Board of Morehouse School of Medicine and the Research Oversite Committee of the Grady Memorial Hospital, Atlanta, Georgia and the University of Alabama at Birmingham School of Medicine, Birmingham, Alabama. Primary fresh frozen and paraffin embedded tissues from 3 histologic subtypes of adenomatous polyps (8 tubular; 9 tubulovillous; and 8 villous), 27 CRC tissues, and histologically MSNT, n = 52 were obtained from the Tissue Procurement Network at the University of Alabama at Birmingham and the Department of Surgery of Morehouse School of Medicine/Grady Hospital. Adenomas were defined by histologic type, degree of dysplasia, and presence of infiltrating adenocarcinoma in adenoma and classified as tubular, tubulovillus, and villous. CRC diagnosis was confirmed by histological examinations of biospied specimens for all patients and pathological tumor staging for these was based on American Joint Committee on Cancer [ 9 ]. The mean age of the study subjects was 66.3 ± 5.7 years. Genomic DNA was extracted from both frozen and paraffin embedded remnant tissues using Tri-Reagent (Molecular, Research Center Inc., Cincinnati, Ohio) and Purgene DNA purification kits (Gentra Systems, Minneapolis, Minnesota) according to the manufacture's protocol. The mtDNA from each tissue sample was amplified by PCR using nine overlapping mtDNA primer pairs as previously described [ 10 , 11 ], which resulted in large PCR products that exclude the possibility that nuclear pseudogenes were amplified. Each PCR product was digested with 14 restriction endonucleases (AluI, AvaII, BamHI, Dde I, HaeII, HaeIII, HhaI, HincII, HinfI, HpaI, MspI, MboI, RsaI, and TaqI) and then subjected to direct sequencing of both sense and anti-sense strands with ABI 3100 Genetic Analyzer to determine the exact nature of new length polymorphisms/mutations detected by restriction analysis. Sequences were compared (BLAST) to the human mitochondrial DNA sequence (Genbank Accession #J01415). The MITODAT database was used to identify mitochondrial genome sequence variants. The analysis of mtDNA samples by restriction digestion and sequencing of the DNA fragments showed non-predicted banding patterns either as homoplasmic single bandshifts or heteroplasmic multiple bands on the gel. The PCR product data point in overlapping regions were counted only once. However overlapping regions were used as internal controls for the identification of the variants. Results of the high-resolution restriction analyses and sequencing of the entire mitochondrial genome yielded 38 sequence variants (including 16 SNPs from the flanking restriction sites) for the precursor adenomatous polyps and cancer tissues. In no instance was a variant detected in the adenomatous tissue found in the histologically matched adjacent surrounding tissue, indicating that these were germ-line origin. Based upon our assessment of the mitochondrial DNA from non-cancer, precancerous (adenomatous polyps), and colorectal cancer tissues using a high-resolution restriction analysis, we have identified sequence variants. To our knowledge this is the first assessment of the mitochondrial genome using solely primary tissue rather than cell lines. This is important since tissues in culture can undergo clonal evolution which can distort frequency data. Although, the restriction analyses identified a number of band shifts indicating site gains or losses, these data only indicated that the predicted sequence had changed. They did not identify specific base pair changes. We therefore used the original primers and a series of nested primers to sequence the band shifts in order to identify these specific changes. A total of thirty-eight germ-line mtDNA variants were observed and all mutations/SNPs were considered as germ-line origin since the variants found in the colorectal adenoma polyps or cancerous tissues were also found in the MSNT tissues. Twenty-two of the thirty-eight were identified as mutations and 59% (13 of 22) were mostly silent mutations of T-to-C. or a G-to-A transition, which is consistent with the mutagenic spectra of oxidative, damage [ 12 , 13 ]. Among them, C3316T/A (ND1; Met-to Met/Ile), G2758A (16SrRNA), T2352C (16SrRNA), A4769G (ND2; Met-to-Met), A3759G (ND1;CUN), G5178T (ND2; CUN-to-Met) G7028A (COI; Ala-to-Ala) T7055C/G (COI; Gly-to-Gly) C7498T (S(UCN)), G6260A (COI; Glu-to-Gly), G8251A (COII; Gly-to-Gly) T8784C (ATPase 6; Gly-to-Gly) A8618G (ATPase6; Ile-to-Thr), G9055A (ATPase 6, Ala-to-Thr), A8860G (ATPase 6 Thr-to-Ala), T11641C (ND4; Met-to-Met), A10398G (ND3; Thr-to-Ala), C10400T (ND3; Thr-to-Ala), C12633A(ND5;S(UCN)), C16390T(D-loop), T16519C(tRNA Val ). However, one was a 9-bp Ins5892C in a non-coding region between MTTY and MTCOI. Some of these mutations are located in ATP synthase genes that are involved in mtDNA genome maintenance and integrity in yeast [ 14 ]. Seventy-five percent (39 of 52) of a G8860A (Thr-to-Ala) mutation in ATPase 6 gene was detected among CRC adenomas and cancer tissues, compared to 14% (7 of 52) in MSNT tissues. This mutational spectrum in ATPase gene could lead to a less efficient mtDNA replication and abnormalities as previously suggested by Maximo et al. [ 15 ]. Sixteen of thirty-eight were distinct SNPs in flanking regions of the restriction sites and 10 of the 16 (63%) have been reported, and 6 were not recorded in the MITODAT database [#J01415]. The identified SNPs are 3107delG (ND1; Frameshift), T2914G (16SrRNA), A2706G (16SrRNA), A2768G (16SrRNA), T2885C (16SrRNA), C7521T (tRNA Asp ), 7335insC (COI; Frameshift), G7256A (COI; Asn-to-Asn), T7146A (COI; Thr-to-S(UCN)), G8206A (COI; Met-to-Met), A16325G (D-loop), T16309G (D-loop), G16294A (D-loop), G16266A (D-loop), G16233A (D-loop), 8269-9bp del (noncoding). Worth mentioning is also the high frequency of 16S rRNA gene variants (> 65%) in the mtDNA among CRC tissue versus the < 25% in the precancerous tissues. Mutations in the 16S rRNA gene were commonly found in different cancers, except those of the thyroid [ 16 ]. We then sought to determine whether or not any of mtDNA variants observed in flanking regions of these restriction site variants may prove to be informative biomarkers. Although, a total of 38 of the mtDNA variants were found, none of the variants appeared to be a marker for a particular adenoma CRC tumor tissue type. Nevertheless, cumulative frequencies of mtDNA variants in the different tissue types resulted in a high prevalence of mtDNA sequence variants in CRC tissue and the trend was for the number of variants to be lowest in the precancerous (Fig. 1 ), suggesting that this may be a useful approach to distinguishing the progressive stages of CRC adenomas as previously observed in tumour progression in the thyroid [ 17 ]. Our sample of CRC adenomas is relatively small and these data must not be overinterpreted at this point. The data however are suggestive and the cumulative frequency approach is currently being followed-up. It is possible that the high frequency of variants in mtDNA in CRC cancer tissues may result from the high rate of mtDNA replication. Furthermore, most of the mtDNA mutations in this study could be a result of mtDNA aggression affected by reactive oxygen species [ 18 - 21 ] and could occur via a slipped replication mechanism [ 22 ]. Figure 1 Frequencies of mitochondrial genome variants based on high-restriction analysis in all of the colorectal tumors (tubular, tubulovillous, villous), cancer tissues in each of the 9 primer sets. Numbers on the abscissa represent PCR products obtained using overlapping primers sets 1–9, termed haploblocks (Rank methods; for all comparsion). These data should be interpreted cautiously as they are based on small number of sample size. Similar studies have looked at precancerous tissue types [ 20 ], however they have not assessed cumulative frequencies of mutations in these same tissues as we have done. Our study is also different in that we did not use cell lines for this work. Moreover, consistent with our findings were reports of cumulative mitochondrial DNA damage in the aging process as well as in cancer [ 23 ]. A similar mechanism may be involved in colorectal cancer progression, since age is a risk factor for CRC. Abbreviations MtDNA, mitochondrial DNA; ATP, adenosine triphosphate; CRC, colorectal cancer; MSNT, matched surrounding Normal tissue; SNPs single nucleotide polymorphisms; PCR polymerase chain reaction. Authors' contributions FOA performed few assays while MK performed almost all assays. DC provided the statistical support. JO provided some tissue samples and suggestions for finalization of the manuscript. RG provided some technical supervision to MK and suggestions for finalization of the manuscript. All authors read and approved the manuscript.

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524365

Improved gene delivery to human saphenous vein cells and tissue using a peptide-modified adenoviral vector

The establishment of efficient gene delivery to target human tissue is a major obstacle for transition of gene therapy from the pre-clinical phases to the clinic. The poor long-term patency rates for coronary artery bypass grafting (CABG) is a major clinical problem that lacks an effective and proven pharmacological intervention. Late vein graft failure occurs due to neointima formation and accelerated atherosclerosis. Since CABG allows a clinical window of opportunity to genetically modify vein ex vivo prior to grafting it represents an ideal opportunity to develop gene-based therapies. Adenoviral vectors have been frequently used for gene delivery to vein ex vivo and pre-clinical studies have shown effective blockade in neointima development by overexpression of candidate therapeutic genes. However, high titers of adenovirus are required to achieve sufficient gene delivery to provide therapeutic benefit. Improvement in the uptake of adenovirus into the vessel wall would therefore be of benefit. Here we determined the ability of an adenovirus serotype 5 vector genetically-engineered with the RGD-4C integrin targeting peptide inserted into the HI loop (Ad-RGD) to improve the transduction of human saphenous vein smooth muscle cells (HSVSMC), endothelial cells (HSVEC) and intact saphenous vein compared to a non-modified virus (Ad-CTL). We exposed each cell type to virus for 10, 30 or 60 mins and measured transgene at 24 h post infection. For both HSVSMC and HSVEC Ad-RGD mediated increased transduction, with the largest increases observed in HSVSMC. When the experiments were repeated with intact human saphenous vein (the ultimate clinical target for gene therapy), again Ad-RGD mediated higher levels of transduction, at all clinically relevant exposures times (10, 30 and 60 mins tissue:virus exposure). Our study demonstrates the ability of peptide-modified Ad vectors to improve transduction to human vein graft cells and tissue and has important implications for gene therapy for CABG.

Text Long term patency rates for CABG using autologous saphenous vein are poor, showing 1, 5 and 10 years post-CABG rates of 93%, 74% and 41%, respectively [ 1 ] and therefore represent a significant clinical problem. Long-term failures are due to neointima formation and superimposed atherosclerosis [ 2 - 4 ], a pathology that lacks a suitably efficient pharmacological therapy. Significant contributions of vascular smooth muscle cell (SMC) proliferation and migration have been documented [ 2 - 4 ]. Anti-proliferative strategies are in phase III clinical trial using decoy oligonucleotides to the transcription factor E2F, a strategy that has shown considerable promise pre-clinically [ 5 , 6 ] and also in early stage human trials [ 7 ]. We, and others have adopted the alternate strategy of gene therapy to prevent CABG failure [ 8 ]. CABG is an "ideal" clinical scenario for gene therapy since saphenous vein can be genetically modified ex vivo following leg harvesting and prior to coronary grafting. This unique "clinical window" has clear safety advantages over in vivo gene delivery since excess vector can be removed from the graft prior to coronary grafting. However, the clinical window is short (likely 10–60 minutes) and therefore necessitates the use of an efficient vector system for gene delivery. Adenoviral vectors have proven efficient for gene delivery in this context [ 9 ] although high titers are required to provide sufficient levels of gene delivery to achieve therapeutic gain using transgenes such as tissue inhibitor of metalloproteinases-3 [ 9 ], endothelial nitric oxide synthase [ 10 ] and p53 [ 11 ]. The latter study defined the rationale behind use of adenoviral vectors since long-term benefit on graft remodelling was shown at 3 months, even though the virus was only present for 2–4 weeks post grafting [ 11 ]. Any improvement in gene delivery above that mediated by adenoviral serotype 5 vectors would be very encouraging for clinical translation of pre-clinical therapies. To this end, a number of strategies have emerged including fiber switching (pseudotyping) and modification of adenovirus type 5 fibers with targeting peptides. Pseudotyping the fiber from adenovirus serotype 16, which binds CD46 [ 12 ], dramatically improves transduction to vascular cells including intact human saphenous vein allowing lower doses of vector to be used to achieve attractive levels of gene delivery to grafts ex vivo [ 13 ]. Likewise, gene delivery to vascular smooth muscle cells can be enhanced by incorporation of cell targeting peptides isolated by phage display into the HI loop of the adenovirus fiber [ 14 ], the preferred site for peptide insertion [ 15 ]. In the context of improved gene delivery mediated by the RGD-4C peptide, which was isolated by phage display and targets α v integrins [ 16 ], this has been shown for rabbit grafts [ 17 ] although the vast majority of data is based on gene delivery for cancer [ 18 ]. Since SMC show poor coxsackie and adenovirus receptor (CAR) availability [ 19 ], it is particularly relevant that the RGD-4C peptide may circumvent CAR deficiency on target cells to improve levels of transduction. In this study, we assess the ability of RGD-4C-modified adenovirus serotype 5 vectors to enhance gene delivery to human saphenous vein SMC and EC as well as to intact human saphenous vein ex vivo , the ultimate clinical target. HSVEC were obtained by enzymatic collagenase digestion of human saphenous vein and maintained in endothelial cell complete media (TCS CellWorks, UK) supplemented with 20% (v/v) foetal calf serum (FCS; PAA laboratories, UK). HSVSMC were grown from medial explants from the same material and maintained in Dulbecco's modified Eagle's medium (DMEM) with 4500 mg/l glucose supplemented with 20% (v/v) FCS and 100 IU/ml penicillin, 100 μg/ml streptomycin and 2 mmol/l L-Glutamine. All cells were grown in a humidified atmosphere with 5% CO 2 at 37°C. Cells were plated to reach 80% confluence 24 hours later. HSVEC or HSVSMC were infected in 96 well plates with increasing doses [plaque forming units (pfu) / cell] of Ad vectors for 10, 30, 60 mins at 37°C. The cells were washed twice in PBS and the media changed. 24 hours post-infection, the cells were again washed in PBS, lysed in PBS/0.2% Triton-X-100 and transduction quantified using the Wallac 1420 (Victor2) Multilabel Counter with recombinant eGFP (Clontech, Basingstoke, UK) as a standard. Reporter gene expression was normalised for total protein using the bicinchoninic acid (BCA) protein assay (Perbio, UK) with bovine serum albumin as standard, measured using a VICTOR2 plate reader. Exposure of HSVEC to Ad-CTL or Ad-RGD [50 plaque forming units (pfu) / cell] resulted in a time-dependent increase in the level of transduction (Figure 1A ). At each time point studied (10, 30 or 60 mins), Ad-RGD mediated a significantly enhanced level of transgene expression compared to Ad-CTL (Figure 1A ). Fluorescent microscopy demonstrated that control levels of infection with Ad-CTL were relatively high in HSVEC but further enhanced using the RGD-modified Ad (Figure 1A ) at all time points tested. This is consistent with HSVEC expressing moderate CAR levels [ 14 , 19 ] allowing transduction of cells by Ad-CTL but the RGD-4C vector can further improve virus uptake. In HSVSMC, Ad-RGD again mediated a marked and significant enhancement in levels of transgene expression at all time points studied (Figure 1B ). HSVSMC were much less permissive to non-modified Ad-CTL infection (Figure 1B ), consistent with our previous observations [ 14 ], but enhanced with Ad-RGD to near 100% transduction in HSVSMC by fluorescence microscopy (Figure 1B ). Again, this effect was evident at all virus:cell exposure time points – 10, 30 and 60 mins. For both HSVEC and SMC similar RGD-4C-mediated increases were observed with different viral doses (10 and 100 pfu/cell; not shown) thereby showing both time- and dose-dependence. Figure 1 Transduction of saphenous vein cells and intact tissue. Ad-CTL and Ad-RGD expressing eGFP were incubated with (A) HSVEC and (B) HSVSMC for different times and gene expression quantified and normalised to protein. Representative fluorescent images are shown. (C) Intact human saphenous vein was incubated with luciferase-expressing vectors and expression quantified. *Indicates p < 0.05 vs Ad-CTL. Based on the above we therefore assessed transduction in intact human saphenous vein. In order to quantify transgene expression accurately in tissue extracts we used luciferase-expressing viruses. Intact human saphenous veins were cleaned of surrounding connective tissue and cut into rings 3–4 mm in length. During preparation and infection, veins were maintained in wash medium (RPMI supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin and 2 mmol/l L-glutamine). Individual vein rings were incubated with Ad vectors for 10, 30 or 60 minutes (1 × 10 9 pfu / ring) before being washed twice in PBS and maintained in organ culture for 5 days. Rings were maintained in wash medium supplemented with 30% (v/v) FCS. Vein rings were snap frozen in liquid nitrogen and homogenised using a mortar and pestle for determination of reporter gene expression 5 days post-infection. Ex vivo homogenates were suspended in 100 μL reporter lysis buffer (RLB) and kept on ice for 1 hour before supernatants were analysed for luciferase expression using the Luciferase Assay System (Promega). 96 well plates were prepared using 10 μL/well of homogenate suspension diluted to a total volume of 100 μL with RLB. 100 μL of Luciferase Assay Reagent was added to each well and the plate immediately read for 10 seconds per well. Detection was achieved using a Wallac 1420 (VICTOR2) Multilabel Counter with recombinant luciferase (Promega) as a standard and normalised for total protein. Ad-RGDLuc mediated a time-dependent increase in the level of transgene expression that was evident at all exposure times studied – 10, 30 and 60 minutes (Figure 1C ). This demonstrates that the RGD-4C-modification of Ad vectors can increase transduction to human saphenous vein, especially at short exposure times. The kinetics of virus binding in relation to time is therefore improved through the RGD-4C peptide and has direct implications for the design of gene therapy vectors for use in human CABG gene therapy procedures in the future. Although we have previously shown that non-modified Ad vectors transduce both endothelial and smooth muscle cells during graft gene delivery [ 9 ], and here show increased transduction of both cell types in vitro with RGD-modification, it will be important to fully define the uptake of the RGD-modified virus in the intact vein at the cellular level by immunotechniques. In broader terms, the design and tailoring of viruses for individual cardiovascular gene therapy applications is an important aspect of translation from pre-clinical to clinical gene therapy. Competing interests The authors declare that they have no competing interests. Authors' contributions LMW performed all isolated cell culture and vein transduction experiments. PNR produced the viruses and AHB supervised all work as principle investigator The authors thank Nicola Britton and Margaret Cunningham for technical assistance. This work was supported by the Biotechnology & Biological Sciences Research Council (E17190 to A.H.B) and the British Heart Foundation (PG03/031 to A.H.B.)

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545957

SS-Wrapper: a package of wrapper applications for similarity searches on Linux clusters

Background Large-scale sequence comparison is a powerful tool for biological inference in modern molecular biology. Comparing new sequences to those in annotated databases is a useful source of functional and structural information about these sequences. Using software such as the basic local alignment search tool (BLAST) or HMMPFAM to identify statistically significant matches between newly sequenced segments of genetic material and those in databases is an important task for most molecular biologists. Searching algorithms are intrinsically slow and data-intensive, especially in light of the rapid growth of biological sequence databases due to the emergence of high throughput DNA sequencing techniques. Thus, traditional bioinformatics tools are impractical on PCs and even on dedicated UNIX servers. To take advantage of larger databases and more reliable methods, high performance computation becomes necessary. Results We describe the implementation of SS-Wrapper (Similarity Search Wrapper), a package of wrapper applications that can parallelize similarity search applications on a Linux cluster. Our wrapper utilizes a query segmentation-search (QS-search) approach to parallelize sequence database search applications. It takes into consideration load balancing between each node on the cluster to maximize resource usage. QS-search is designed to wrap many different search tools, such as BLAST and HMMPFAM using the same interface. This implementation does not alter the original program, so newly obtained programs and program updates should be accommodated easily. Benchmark experiments using QS-search to optimize BLAST and HMMPFAM showed that QS-search accelerated the performance of these programs almost linearly in proportion to the number of CPUs used. We have also implemented a wrapper that utilizes a database segmentation approach (DS-BLAST) that provides a complementary solution for BLAST searches when the database is too large to fit into the memory of a single node. Conclusions Used together, QS-search and DS-BLAST provide a flexible solution to adapt sequential similarity searching applications in high performance computing environments. Their ease of use and their ability to wrap a variety of database search programs provide an analytical architecture to assist both the seasoned bioinformaticist and the wet-bench biologist.

Background Evolution can be measured and studied on a number of different scales, one of which is through the determination and comparison of genetic sequence information. Current-day gene sequences in living organisms have arisen through modifications of an array of ancestral sequences. Duplication with modification is the central paradigm of protein evolution, wherein new proteins and/or new biological functions are fashioned from earlier ones. [ 1 ]. To detect these functionally (and structurally) related proteins based upon similarity between their primary nucleic acid or amino acid sequences, a variety of sequence comparison algorithms have been developed. When a new gene is cloned and sequenced, it is now standard practice to use these algorithms to search for similarities between the translated nucleic acid sequence and a protein sequence database such as the NCBI non-redundant protein database (nr) [ 2 ]. Sequence similarity that implies similar structure and therefore similar protein or enzymatic function is not definitive proof of such function; however, the results of database sequence similarity searches provide a starting point for researchers attempting to ascertain the function of an unknown gene by supporting the intelligent design of subsequent laboratory experiments. The basic local alignment search tool (BLAST) [ 3 , 4 ] is by far the most widely used pairwise-based sequence similarity comparison tool. It completes searches more swiftly than other tools, including FASTA, SSEARCH [ 5 ], and SCANPS [ 6 ]. BLAST uses an efficient, rapid algorithm to look for short segments or words of sequence similarity between two sequences that meet some predefined scoring threshold. After initially locating at least two of these words within a short distance of one another on a common diagonal, the algorithm uses them as "seeds" from which to extend the alignment to encompass longer regions of similarity, resulting in high scoring pairs (HSPs). The heuristic algorithm used by BLAST decreases search time dramatically in comparison to that of other search programs. However, the emergence of high-throughput DNA sequencing techniques has increased the size of sequence databases tremendously; thus conventional large-scale BLAST searching against the most commonly used databases has become infeasible on a PC or even a dedicated UNIX server. For this reason, new search strategies are needed. While BLAST provides a balance between search sensitivity and speed, in many cases a researcher would like to detect more distant sequence similarities by employing search strategies that maximize the sensitivity of the search. A profile-based comparison which, for example, compares a sequence to a hidden Markov model (HMM) representing an empirically derived estimate of all possible evolutionary changes for a protein of a particular function, generally permits identification of a much higher proportion of distantly related sequences [ 7 ]. There are two major profile-based comparison tools. PSI-BLAST [ 3 ] compares sequences with a profile model constructed dynamically during the initial search phase of a traditional BLAST search, while HMMPFAM in the HMMer package from Sean Eddy at Washington University compares sequences with a well-curated database of HMM profiles as well as models constructed by users. Well-curated profile databases such as Pfam [ 8 ] are being developed through the combined efforts of bioinformaticists and molecular biologists. Profile-based comparison has become a reliable way to gather information for predicting structure and function of unknown genes, and tools from the HMMer package are becoming a key centerpiece in many bioinformatics pipelines. The tradeoff of using HMM-based searches for increased sensitivity is the intrinsically slow nature of the Viterbi [ 9 ] or forward algorithm used in the application. Taking advantage of larger databases and more sensitive searching methods necessitates the use of high performance computing (HPC) platforms. Traditionally, HPC has been synonymous with high-priced vector or parallel supercomputers, but rapid advances in microprocessor and network bandwidth technologies are changing the definition [ 10 ]. Clusters of connected workstations utilizing commodity microprocessor systems provide enormous benefits in terms of cost and performance. Thus, cluster computing can meet the increased computational needs of resource- and data-intensive bioinformatics applications. HPC environments using workstations connected via high-speed networks are becoming more and more popular in the bioinformatics community [ 11 - 14 ]. Here we describe the SS-Wrapper package, which provides tools to adapt currently available similarity search applications onto HPC environments implemented through Linux clusters. Implementation The objective of this study was to implement a generic wrapper application that could deploy similarity searching applications on a Linux cluster. Design criteria for the wrapper included the ability to deploy applications without the need to alter the original application and the ability to increase the speed of the underlying application in a linear manner dependent on the number of cluster nodes available. To meet these objectives, tools in the SS-Wrapper package were written in C/C++ using the message passing interface (MPI) [ 15 ]. Tools in the SS-Wrapper should work with few if any changes on any Linux cluster running MPI utilizing any hardware platform. In addition to the SS-Wrapper executables (which are compiled using the MPI C++ compiler), executables for the underlying similarity search applications (blastall, formatdb, and hmmpfam) appropriate for the hardware platform underlying the cluster will be needed. These can be obtained from NCBI and Washington University in St. Louis. SS-Wrapper is available without charge under the Artistic License described in the Open Source Initiative [ 16 ]. The source code can be downloaded via ftp [ 17 ]. Using multiple processors is practical only when the computational task is too large to complete on a single processor in a reasonable amount of time. In the case of database searching, the database to be searched may be too large or the set of query sequences used in the search may be too large to accomplish the search on a workstation with a single or even double or quad processor architecture. Since the search must compare every query sequence to every database sequence, parallelizing the process can be accomplished by three different methods. The first method is to split the query sequence file into smaller subsets and apply each subset to one particular node of the cluster in a search against the entire database. The second method calls for the database to be split into a series of smaller files, one of which is distributed to each node of the cluster; then the entire file of query sequences is searched against each of the database segments. Finally, it is possible to use a combination of query sequence splitting and database splitting to accomplish the search. Using the query splitting approach does not require any modification to the output generated by search programs such as BLAST. When utilizing the database splitting approach, however, a correction must be made to the reported E-value for any particular hit. Most similarity comparison tools provide a statistical measure (e.g., the E-value reported for BLAST and HMMPFAM) that gives an indication of the statistical significance of a match between the query sequence and a particular database hit. This statistical measure is generally influenced by the size of the search space (which includes the total length of the database) and therefore the E-value needs to be recalculated when only a portion of the database is being searched. An added complication is that some search tools require that the database be intact. For example, PSI-BLAST [ 3 ] is a variant of BLAST that constructs a sequence profile model based on hits from an initial round of BLAST searching. This profile is then used and refined in subsequent rounds of searching to increase the sensitivity of the overall search. Because PSI-BLAST depends on the integrity of the database to guarantee that the resulting profiles are representative of the entire search space, it is best to perform parallelization using the query splitting approach. In contrast to database splitting, query splitting offers greater flexibility in that the query file segments can more easily be adaptively distributed to the nodes of the cluster according to the load on each particular node during the search process. As any one node becomes available, another segment of the query file can be distributed to that node during the search process, which improves performance. It is difficult to predict the workload on any one node before the search begins, and the time required to complete a program running on a cluster depends on the processor that finishes last. Adaptive distribution of the workload maximizes resource utilization. Optimizing resource utilization is dependent on finding a balance between having larger numbers of smaller tasks versus increased startup and communication overhead due to distribution of the required query and database files to each node. For this reason, for our query splitting wrapper (QS-search), we adopted a hybrid strategy wherein approximately 90% of the total workload is evenly divided and distributed to each node at the beginning of the search using a modified bucket algorithm (described below). Then the remaining 10% of the workload is divided and distributed to each node as it becomes available after completing its previous task. This strategy is accomplished using a master-slave model, where one node is set aside to act as the master, which is responsible for distributing the workload and supervising the slave nodes, which perform the computations. In general, the query splitting approach seems to be superior to the database splitting approach due to higher performance and fewer post-search processing tasks. Database splitting does provide a distinct advantage when the database is too large to fit into the physical memory of a single node [ 11 , 14 ]. NCBI BLAST uses memory mapped file I/O for database access. BLAST runs fastest when it can cache the database in memory. When the database size exceeds that of the available memory, however, the database splitting approach can reduce the possibility of swapping the database from physical memory to disk swap space, which could significantly slow the search process. Therefore, we have also developed a wrapper to support database splitting (DS-BLAST). DS-BLAST is specific for BLAST because it is necessary to include code to recalculate E-values following the search. As indicated above, a modified bucket algorithm is used to split up the query sequences for QS-search, and a similar algorithm is used to split up the database sequences for DS-BLAST. The modified bucket algorithm works as follows: First, the sequences are sorted according to length. In the first cycle, sequences are placed one at a time into each bucket in descending order of length. In the next two cycles, individual sequences continue to be placed into each bucket after first reversing the order of the buckets. Bucket order is reversed every two cycles and the process continues until all sequences have been placed into a bucket. At the end of this process, each bucket contains nearly the same number of sequences, and the total length of all sequences in any one bucket is also approximately the same as the total sequence length of any other bucket. In the end, therefore, the file of query sequences or the file of database sequences is evenly divided in both length and number. The BLAST E-value is a function of the size of the effective search space, which is dependent on three factors: the number of sequences in the database, the total combined length of all sequences in the database, and the length of the query sequence [ 13 , 18 ]. Figure 1 shows that when splitting the database into N fragments of equal sequence number and length, the effective search space of each database fragment is approximately 1/N that of the intact entire database. Therefore, the E-value calculated for any particular hit of a query sequence to a database sequence will approximate a linear function dependent on the value of N. For that reason, at the beginning of the search, DS-BLAST lowers the user-provided E-value cut-off to account for the number of nodes used in the search. Following the search, DS-BLAST recalculates the effective search space and each resulting E-value by multiplying by the value of N. Figure 1 Effective search space vs. database size and query sequence length. Relationship between effective search space and database size with different query sequence lengths. The GenBank non-redundant protein database (nr) was split evenly according to the modified bucket algorithm in order to construct databases of a size 1/64, 1/32, 1/16, 1/8, 1/4, 1/2, or 1 of the entire nr database. Query sequences of varying lengths were randomly assembled using a Perl script. A BLAST search was then carried out for each query sequence against each database. The effective search space and database size was extracted from the BLAST results and plotted for each query sequence. The length of each query sequence is indicated next to the line which plots the relationship between effective search space and database size for that query. Results and discussion Usage The QS-search executable (qssearch) provides the same interface for all search tools. The command line is as follows: qssearch -c <command> -q <query> -d <database> -o <output> -l <local scratch> -x <database files> DS-BLAST uses two executables: dsblast and dsformatdb. dsformatdb is responsible for splitting the database into fragments according to the modified bucket algorithm and then formats these fragments using the NCBI formatdb executable. The command line for dsformatdb is as follows: dsformatdb -n <number> -c <command> -d <database> -p <path> The command line for dsblast is as follows: dsblast -o <output> -c <command> -l <local scratch> -d <database> -q <query> The command-line variables are as follows: • -c command: normal command line used for the underlying application including all desired options • -q query: query filename in fasta format • -d database: database filename • -o output: output filename • -l local scratch: temporary directory on each node • -x database files: a space-delimited list of the database file names generated by the search program's formatting utility (formatdb for BLAST) • -n number: desired number of database fragments • -p path: directory to store database fragments. Benchmarking All benchmark experiments were performed on a Linux cluster in the Department of Engineering at the University of Alabama at Birmingham [ 19 ]. The cluster consists of one compile node and 64 compute nodes (IBM × 335s), as well as 2 storage servers (IBM × 345s). All machines have 2 × 2.4 GHz Xeon processors, 2 GB of RAM, an 18 GB SCSI hard drive, and are connected via Gigabit Ethernet to a Cisco 4006 switch. The NCBI non-redundant protein database (nr, 733 MB), downloaded from GenBank [ 2 ] in August, 2003, was used for testing both DS-BLAST and QS-search; it contained 1,508,485 sequences composed of 492,678,715 amino acids. Release 10.0 of the Pfam [ 8 ] database from Washington University (549 MB) was used in benchmarking HMMPFAM under QS-search; it contained 6190 profile models that, when combined, were 1,463,477 residues long. The same set of query sequences was used for all experiments. The query sequences represented all open reading frames of more than 30 amino acids from the genome of monkeypox virus strain WRAIR 7–61 (manuscript in preparation), and totaled 2068 sequences comprising 151,173 amino acids. Figure 2 demonstrates that QS-search provided a >40-fold acceleration for NCBI BLAST when using 64 processors, compared to the speed of 1 processor. Ideally, QS-search should provide an N-fold acceleration when using N processors, but this optimal result is rarely achievable. The major factor limiting the performance gain of QS-search is the time necessary to deliver the database to each node. As more nodes are employed, the portion of time spent searching decreases, but the communication overhead increases. When using QS-search with HMMPFAM, the search resulted in a 58-fold increase in processing speed when using 64 processors compared to a single processor (figure 3 ). QS-search therefore proved to be more efficient when running HMMPFAM in comparison to BLAST. Since the overall time required for the HMMPFAM search is much longer than that for BLAST, the portion of the search time devoted to communication overhead decreases thus increasing overall efficiency. Figure 2 BLAST performance of QS-search. Performance of QS-search with NCBI BLAST when searching all reading frames (>30 amino acids) from monkeypox virus against the GenBank non-redundant protein database. Vertical bars represent total time used while the line indicates increase in speed corresponding to the number of processors used. Figure 3 HMMPFAM performance of QS-search. Performance of QS-search for HMMPFAM when searching all reading frames (>30 amino acids) from monkeypox virus genome against Release 10.0 of the Pfam database. Vertical bars represent total time used while the line indicates increase in speed corresponding to the number of processors used. Figure 4 illustrates the performance of DS-BLAST. Since database preprocessing occurs before the search process, the times provided in figure 4 do not include this preprocessing time. As the same query, database and underlying application (NCBI-BLAST) were used in benchmarking DS-BLAST and QS-search, the results presented in figures 2 and 4 are directly comparable and indicate that QS-search appears to be more efficient than DS-BLAST. Two factors cause a reduction in performance of DS-BLAST. The first is the imbalance of the workload between nodes, and the second is the time necessary for the final merge phase of the output results from each node. When using QS-search, the workload is distributed dynamically during execution and therefore is well balanced between nodes. In contrast, when using DS-BLAST, the database is split into segments before the search, so the distribution of the workload between nodes is not as well balanced as for QS-search. The percent load imbalance for DS-BLAST (the time difference between completion of the first and last processors) has been as much as 5% of the total search time; for QS-search, on the other hand, the percent load imbalance is generally much less than 2%, and approached 3% only when 64 processors were used (data not shown). The merge phase of QS-search consists entirely of concatenating the results provided by each node into a single file. In contrast, the merge phase of DS-BLAST must parse the output from each node and combine the results for each single query sequence. As the number of nodes employed increases, the time required for the merge also increases. The advantage of the database splitting approach under limited memory conditions was not apparent in these benchmarks, since the memory available for each node in the cluster used in these experiments was large enough to accommodate the entire 733 MB database. Figure 4 Performance of DS-BLAST. Performance of DS-BLAST when searching all reading frames (>30 amino acids) from monkeypox virus genome against the GenBank non-redundant protein database. Vertical bars represent total time used while the line indicates increase in speed corresponding to the number of processors used. We also compared the performance of DS-BLAST to that of mpiBLAST [ 11 ] version 1.1.0. We found that DS-BLAST was almost twice as fast as mpiBLAST when both utilized 4 processors and more than twice as fast when both utilized 32 processors (data not shown). Both mpiBLAST and DS-BLAST required a substantial part of the total run time to merge and format the final BLAST output. Conclusions To increase the speed and efficiency of sequence similarity search programs, we have developed the SS-Wrapper package, a series of wrapper applications that supports the deployment of sequence similarity searches on high-performance computing clusters. QS-search implements a query sequence splitting approach for the deployment of NCBI BLAST and HMMPFAM. It also will support other similarity search programs, including all variants of NCBI BLAST (blastn, blastp, blastx, tblastn, and tblastx) as well as all options provided by the blastall executable. Because this implementation does not alter the original program, program updates and new programs should be easily accommodated. The output from QS-search is effectively identical to that produced by the underlying program. QS-search is designed to provide optimal load balancing and maximize resource usage when using computer clusters. The performance gain approaches linearity in proportion to the number of processors employed. When the database is too large to fit into the physical memory of a single node in the cluster, a database splitting approach should outperform the query splitting approach used by QS-search [ 11 , 14 ]. Therefore as a complementary application, the SS-Wrapper package also includes DS-BLAST, which implements a database splitting approach for BLAST searches and provides an effective solution to recalculate the E-value during the post-search phase of processing. SS-Wrapper provides a suite of tools that makes large sequence similarity searches feasible by deploying the search on a Linux cluster. These tools permit the bioinformatics community to take advantage of the power of high-performance cluster computing. Other tools such as Disperse [ 20 ] and TurboBLAST [ 21 ] are designed to deploy bioinformatics applications onto loosely connected machines. A more general approach to deployment uses grid computing as an increasingly popular alternative to cluster computing [ 22 ]. Grid computing organizes widespread, diverse collections of CPU resources (including desktop workstations, servers, and clusters) into a virtual supercomputer, where these collections of hardware, software, and data resources are organized into a more uniform, manageable, visual whole. In contrast, the CPUs in a Linux cluster are more tightly coupled and specialized. Grid computing has the advantage of utilizing large numbers of CPUs as they become available to the grid. The disadvantage of a grid is in managing the complexity of the disparate architectures of the available CPUs, minimizing overhead, and making maximum usage of network bandwidth. For maximal performance, tools in the SS-Wrapper package have been developed under the assumption of a homogenous Linux cluster in which every CPU is similar. We are currently exploring methods to extend our current work to take advantage of grid computing technologies. To accomplish this, the complexities involved will require significant modification and extension of the applications that are a part of the SS-Wrapper package. Availability and requirements The SS-Wrapper package is freely available under the Artistic License described in the Open Source Initiative. The source code can be downloaded via ftp [ 17 ]. Contact elliotl@uab.edu for information on obtaining the software. All tools have been tested on an IBM Intel ® processor-based Linux cluster with LAM/MPI [ 23 ] and should be compatible with other implementations of MPI. Authors' contributions CW was responsible for the conception, design, implementation, and testing of the SS-Wrapper package. EJL contributed to its conception and testing and provided overall project coordination. Both authors have read and approved the final manuscript.

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548297

Extragonadal aromatization increases with time after ovariectomy in rats

Background The circulating estrogen concentration elevated gradually along with time after ovariectomy in rats. To explore the source of the increased circulation estrogen, the extragonadal aromatization as well as the synthesis of androgen in the adrenal cortex of the ovariectomized rats was evaluated. Methods Female rats were divided into twelve groups: 1 month after ovariectomy (OVX1M), OVX2M, OVX3M, OVX4M, OVX5M, OVX6M; intact 1 month (INT1M), INT2M, INT3M, INT4M, INT5M, INT6M. The blood concentration of testosterone (T) was measured by radioimmunoassay. The mRNA expressions of P450 aromatase in the liver and subcutaneous abdominal (SA) adipose as well as the adrenal cytochrome P450 17 alpha hydroxylase/lyase (P450c17) were semiquantified by RT-PCR. The P450 aromatase protein expressions in the liver and SA adipose were detected by Western blot. Results The blood E2 concentrations increased gradually along with time after ovariectomy in the rats. The 58-kDa aromatase protein and mRNA expressions normalized to β-actin in the OVX6M rats' SA adipose tissues showed higher levels than those from corresponding tissues in the INT6M (p < 0.05). And the ratios of aromatase mRNA and protein to β-actin in the OVX6M rats' liver tissues increased significantly compared with those in the OVX1M rats (p < 0.05). The ratio of adrenal P450c17 to beta-actin in the OVX6M increased markedly, and was higher than OVX1M (p < 0.05), though the blood concentration of T decreased significantly in all the ovariectomized rats (p < 0.05). Conclusion Both the subcutaneous abdominal adipose tissues and the liver tissues contributed to the extragonadal aromatisation to promote the circulating E2 levels in the rats along with time after ovariectomy; the adrenal compensation might also be activated naturally.

Background Ovaries are the primary source of estrogen. In ovariectomized rats, the production of estrogen is shifted from ovary to a number of extragonadal sites [ 1 , 2 ]. Simpson developed the intriguing concept of extragonadal aromatization, i.e. androgens, particularly androstenedione produced primarily in the adrenal glands, can be converted (aromatized) into estrogens at extraglandular sites, including the mesenchymal cells in adipose tissue and skin, osteoblasts and chondrocytes in bone, vascular endothelial, aortic smooth muscle cells as well as numerous sites in brain [ 2 , 3 ]. Estrogen synthesised within these sites is biologically active in a paracrine or intracrine fashion, although it may escape the local metabolism and enter the circulation [ 2 , 3 ]. Estrogen production in extragonadal sites is dependent on an external source of C19 androgenic precursors [ 4 ]. Circulating levels of testosterone (T) and androstenedione as well as dehydroepiandrosterone (DHEA) and DHEA sulphate (DHEAS) become extremely important in terms of providing adequate substrate for estrogen biosynthesis in extragonadal sites. It is now recognized that much of the physiology of androgens is consistent with the concept that T functions as a circulating pro-hormone. In the postmenopausal woman, circulating T levels are an order of magnitude greater than circulating estrogen levels, which by itself suggests the importance of T for maintaining local estrogen levels in extragonadal sites [ 2 ]. 65–75% of the circulating T is formed peripherally from androstenedione, DHEA and DHEAS secreted by the adrenals, which form a large precursor reservoir that is available for conversion to T and finally to estrogen [ 2 ]. Thus in the species of rat, it is still unknown that whether it is the same situation of T in extragonadal aromatization. We have observed that the release of corticotrophin-releasing hormone (CRH) in the hypothalamic paraventricular nucleus of the ovariectomized rats increased significantly compared with the intact rats [ 5 ]. And more interestingly, the hypothalamic CRH expressions showed a gradual elevation along with time after ovariectomy (submitted data), which suggested that the activity of hypothalamus-pituitary-adrenal axis (HPAA) might promote. These observations lead us to hypothesize that more androgens from adrenal cortex might gradually be converted into estrogens by extragonadal aromatase in the rats along with time after ovariectomy. To test the hypothesis, we measured the blood concentrations of estradiol (E 2 ) and T as well as the aromatase expressions in adipose and liver tissues in the ovariectomized rats. Methods Animals Female Sprague-Dawley rats (180–200 g), with regular 4-day estrus cycles were purchased from Medical Experimental Animals Centre of Fudan University (Shanghai, China). The animals were housed under laminar flow in an isolated room with controlled temperature and at a 12 /12 (light /dark) schedule. Forty-eight of them underwent ovariectomy with ether anaesthesia, which were then divided randomly into six groups: 1 month after ovariectomy (OVX1M), OVX2M, OVX3M, OVX4M, OVX5M, OVX6M. The corresponding control groups were: intact 1 month (INT1M), INT2M, INT3M, INT4M, INT5M, INT6M. All experimental procedures involving the use of animals were conducted in accordance with NIH Guidelines and were reviewed and approved by the Animal Use and Care Committee for the Fudan University. Tissue collection and preparation At the time of sacrifice, the vaginal cytology of each rat was first examined. The tissues of the rats were collected respectively, and those of the intact control animals, during the period of diestrous. All the operations were carried out at 4°C. The liver tissues, subcutaneous abdominal (SA) adipose tissues and the adrenals were excised, and then snap-frozen in liquid nitrogen, and stored at -80°C. The preparation of the microsomal pellet was accordance with the report by Hiroshi [ 6 ]. Total tissue RNA was extracted using 'TRIzol Regent' (Biobasic Inc, Canada), and the purity and integrity of the RNA were checked spectroscopically and by gel electrophoresis before analytical procedures. Semiquantitative RNA analysis To compare the level of adrenal cytochrome P450 17α hydroxylase/lyase (P450c17), liver and SA adipose P450arom expressions after different treatments, PCR methologies were adapted to provide a semiquantitative measure of mRNA levels. Primers were synthesized based on published reports [ 7 , 8 ]. Table 1 summarizes the anticipated size of PCR products, sense and antisense sequences, and locations of primers. Total RNA (2 μg) was transcribed in reverse, in a final volume of 20 μl, using 200IU M-MLV reverse transcriptase in the presence of 25 pmol downstream primer (Sangon Inc), 0.5 mM deoxy-NTP and 20IU Rnasin (from Promega) for 60 min at 42°C before heat denatured for 5 min at 95°C. The cDNAs obtained were further amplified by PCR using 25 pmol of upstream primer (Sangon Inc). We first determined the linear range of amplification of cDNA using each of the primer sets, and then chose an appropriate amplification cycle within this range for each cDNA species. For P450arom and P450c17, we used 35 PCR amplification cycles, and 20 cycles for β-actin gene expression. Each PCR reaction underwent an amplification regimen characterized by (P450arom: 1 min at 94°C, 1 min at 60°C, 2 min at 72°C; P450c17: 1.5 min at 72°C, 2 min at 56°C, 4 min at 72°C) with Taq DNA polymerase (3U per tube) and 2.2 mM magnesium chloride (from Promega) in a final volume of 50 μl. To check the presence of DNA contamination, RT-PCR was performed on 2 μg of total RNA without M-MLV reverse transcriptase (negative control). An internal control (water instead of RNA) for each RT-PCR was performed to investigate RNA contamination of the mixture. For each sample 5 μl of the PCR amplification products were analysed on 2% agarose gels and stained with ethidium bromide. The intensities of the bands were evaluated using the Image Master Software (SYDR-1990, SYNGENE, U.S.A.). The RT-PCR products were extracted and purified from agarose gel by Golden Beads Gel Extraction kit (Sangon Inc., China) and sequenced using radioactive dideoxychain terminating method (Sangon Inc., China). Table 1 Properties of oligonucleotide primers used for PCR. Target mRNA Size of PCR products (bp) Sense (S) or antisense (AS) Primer sequence (location) P450c17 299 S AS 5'-TCATCAAGAAGGGAAAAGAA-3' 5'-TGAAGCAGATAGCACAGATG-3' P450arom 289 S AS 5'-GCTTCTCATCGCAGAGTATCCGG-3' 5'-CAAGGGTAAATTCATTGGGCTTGG-3' β-actin 550 S AS 5'-AAGCAGGAGTATGACGAGTCCG-3' 5'-GCCTTCATACATCTCAAG TTGG-3' Western blotting A 50 μg sample of the microsomal protein was loaded into each lane along with a prestained protein size marker (Bio-Rad Laboratories, Inc., Hercules, CA), electrophoresed on a 10% SDS-polyacrylamide gel at 18 V/cm, and electroblotted onto a polyvinylidene difluoride membrane (Micron Separations, Westboro, MA) using a wet electroblotter. After blocking in fat-free milk, incubation was conducted with the antiaromatase antibody (1:200; Boster Biological Technology LTD., China) and β-actin antibody (1:3000) at room temperature in 18°C for 4 h in TBS-T solution (20 mM Tris, 137 mM NaCl, and 0.1% Tween-20, pH 7.6). After extensive washing, blots were incubated with AP-labelled goat antirabbit antiserum (Sino-American Biotechnology Co., China) for 60 min at room temperature in 18°C and developed using NBT/BCIP detection system (Amersham Pharmacia Biotech). The intensities of the bands were evaluated using the Image Master Software (SYDR-1990, SYNGENE, U.S.A.), and values were normalized to β-actin immunoreactivity in each sample and expressed as percent of the control. Specificity of the aromatase immuno-staining was determined by preincubation of antiserum for 24 h at 4°C with varying concentrations of aromatase, with the primary antibody omitted to identify non-specific staining as well. RIA of blood estrogen and testosterone concentrations At the time of sacrifice, the blood samples (0.8 ml) of the rats were collected from tail veins respectively, and the corresponding intact controlling animals, during the period of diestrous. The plasma was separated by centrifugation and stored at -70°C until assayed. Concentration of blood hormones were determined by double-antibody RIA kits purchased from the National Atomic Energy Research Institute (Beijing, China.). The samples were assayed in duplicate, and all the subjects' samples were assayed together. The sensitivity of the kit was 0.8 pg/ml (testosterone) and 1.4 pg/ml (estrogen), the intra- and interassay coefficients of variation, 3.7–8.0% and 4.74–7.7%. Statistical analysis All data are presented as means ± S.E.M. Statistical analysis was performed on raw data using two-way analysis of variance (ANOVA), with the significance set at p < 0.05 and p < 0.01 in two-tailed testing chosen. Results Vaginal cytology of the animals The epithelial cells were stained by haematoxylin-eosin (HE). The intact rats (INT1M, 2M, 3M, 4M, 5M, 6M) showed regular 4-day estrus cycle change. The cyclic change disappeared in the ovariectomized (OVX1M, 2M, 3M, 4M, 5M, 6M) rats. A few of mature vaginal epithelia were observed in the smears of the OVX5M and OVX6M rats, and the percent of mature epithelia increased significantly in the OVX6M rats (p < 0.05) (Table 2 ). Table 2 The percent of mature vaginal epithelia and the blood concentrations of E 2 and T of the rats Groups The percent of mature vaginal epithelia Blood E 2 level (pg/ml) Blood T level (pg/ml) 1M INT (15.6 ± 0.88)% 56.60 ± 14.13 27.92 ± 1.74 OVX (0.19 ± 0.019)%* 4.06 ± 1.36* 18.91 ± 2.53* 2M INT (15.8 ± 0.91)% 59.12 ± 11.23 26.39 ± 2.04 OVX (0.17 ± 0.015)%* 8.94 ± 1.78* 18.03 ± 2.20* 3M INT (14.8 ± 0.90)% 58.25 ± 13.93 28.86 ± 2.19 OVX (0.20 ± 0.025)%* 16.72 ± 3.71* 20.21 ± 1.96* 4M INT (15.1 ± 0.86)% 59.35 ± 10.35 26.99 ± 2.14 OVX (0.19 ± 0.020)%* 28.10 ± 8.88* # 20.89 ± 2.69* 5M INT (16.4 ± 1.01)% 63.34 ± 15.85 27.03 ± 1.93 OVX (0.26 ± 0.034)%* # 28.97 ± 6.61* # 21.35 ± 2.33* 6M INT (16.8 ± 1.06)% 59.15 ± 13.34 29.36 ± 2.56 OVX (0.29 ± 0.027)%* # 31.05 ± 6.61* # 21.54 ± 2.09* *p < 0.05 vs corresponding intact; # p < 0.05 vs OVX1M Blood concentrations of estrogen and testosterone The blood E 2 concentrations decreased significantly in the OVX1M, 2M and 3M (p < 0.01) compared with those in the INT1M, 2M and 3M. The concentrations in OVX4M, 5M and 6M groups increased significantly (p < 0.05) compared with OVX1M, though still lower than INT4M, 5M and 6M. There were no disparities between the INT1M, 2M, 3M, 4M, 5M and 6M groups (Table 2 ). The blood T concentrations decreased significantly in the OVX groups compared with corresponding intact controls (p < 0.05). Though there were slightly increases in the OVX3M, 4M, 5M and 6M compared with OVX1M, no statistical significances were detected (Table 2 ). RT-PCR analysis: Effects of ovariectomy on adrenal P450c17, liver and SA adipose P450arom mRNA levels Comparison of the amplified PCR fragments with rat P450c17 and ovary aromatase cDNA sequences revealed 100% homology (data not shown). Densitometric analysis of the mRNA concentration using target product/β-actin was expressed as the mean with SEM. The ratios of liver P450arom to β-actin in the OVX1M, 2M, 3M, 4M and 5M groups were lower than the corresponding intact controls (p < 0.05). The ratio increased significantly in the OVX6M compared with OVX1M (p < 0.05), and no difference was detected between OVX6M and INT6M (Fig 1 ). The ratios of SA adipose P450arom to β-actin in the OVX1M, 2M, 3M, 4M and 5M groups appeared no significant changes compared with those in the corresponding intact control, and the ratio in the OVX6M was higher than that in all other groups (p < 0.05) (Fig 2 ). The ratio of adrenal P450c17 to β-actin in the OVX1M decreased significantly compared with that in the INT1M (p < 0.05), and those in the OVX4M and OVX5M increased slightly, with statistical disparities between OVX4M and INT4M, or OVX5M and INT5M. In the OVX6M, the ratio increased markedly, and was higher than OVX1M (p < 0.05) (Fig 3 ). However, no changes occurred on the ratios of adrenal P450c, liver and SA P450arom among INT1M, 2M, 3M, 4M, 5M and 6M(Fig 1 , 2 , 3 ). Figure 1 RT-PCR analysis of liver P450arom mRNA expressions of the rats. The upper picture shows the gel electrophoresis of the RT-PCR products for the liver P450arom. Densitometric analysis of the mRNA concentration using PCR product/β-actin expressed as the mean with SEM bar in each column indicated in the lower panel. * p < 0.05 vs corresponding intact controls, # p < 0.05 vs OVX1M. Figure 2 RT-PCR analysis of SA adipose P450arom mRNA expressions of the rats. The upper picture shows the gel electrophoresis of the RT-PCR products for the SA adipose P450arom. Densitometric analysis of the mRNA concentration using PCR product/β-actin expressed as the mean with SEM bar in each column indicated in the lower panel. * p < 0.05 vs corresponding intact controls, # p < 0.05 vs OVX1M. Figure 3 RT-PCR analysis of adrenal P450c17 mRNA expressions of the rats. The upper picture shows the gel electrophoresis of the RT-PCR products for the adrenal P450c17. Densitometric analysis of the mRNA concentration using PCR product/β-actin expressed as the mean with SEM bar in each column indicated in the lower panel. * p < 0.05 vs corresponding intact controls, # p < 0.05 vs OVX1M. Western blot analysis: Effects of ovariectomy on liver and SA adipose P450arom protein levels Densitometric analysis of the protein concentration using aromatase/β-actin was expressed as the mean with SEM. The ratios of liver P450arom to β-actin in the OVX groups were lower than the corresponding intact controls (p < 0.05). But the ratio increased significantly in the OVX6M compared with OVX1M (p < 0.05) (Fig 4 ). The ratios of SA adipose P450arom to β-actin in the OVX1M and OVX6M increased significantly compared with those in the INT1M and INT6M (p < 0.05). And the ratios in the OVX2M, 3M, 4M and 5M produced no disparities compared with corresponding intact controls (Fig 5 ). No disparities were detected among INT1M, 2M, 3M, 4M, 5M and 6M (Fig 4 , 5 ). No immunoreactive bands detected in the samples when using antiserum after preabsorption with excessive antigens and omission of the primary antibody. Figure 4 Western blot analysis of liver P450arom expressions of the rats. The upper picture shows the Western blot analysis of the liver aromatase P450. Densitometric analysis of the protein concentration using aromatase/β-actin expressed as the mean with SEM bar in each column indicated in the lower panel. * p < 0.05 vs corresponding intact controls, # p < 0.05 vs OVX1M. Figure 5 Western blot analysis of SA adipose P450arom expressions of the rats. The upper picture shows the Western blot analysis of the SA adipose aromatase P450. Densitometric analysis of the protein concentration using aromatase/β-actin expressed as the mean with SEM bar in each column indicated in the lower panel. * p < 0.05 vs corresponding intact controls, # p < 0.05 vs OVX1M. Discussion The most interesting findings in the present study were that the circulating E 2 levels increased gradually along with time after ovariectomy in the rats. If ovary is not the source of estrogen in ovariectomized rats, then the question arises as to the origin of the estrogens found in the circulation. Extragonadal aromatization has been in a general sense recognized, although its significance is only becoming to be appreciated, as will be explained further. It has been reported that aromatization in the adipose tissue is not negligible under normal and pathological conditions [ 9 ]. Due to the highest conversion of C19 precursor such as androstenedione to estrogen was observed in adipose tissue obtained from the subcutaneous abdominal wall but not from intraperitoneal cavity [ 10 ], our research focused on the aromatase activity of the SA adipose tissues. In the present results, increased aromatase protein and mRNA expressions were observed in the OVX6M rats' SA adipose tissue. Hemsell and co-workers [ 11 ] first addressed the significance of adipose tissue as a major source of estrogen production, i.e. there is a progressive increase in the conversion efficiency with advancing age, and the increase of estrogen production as a function of obesity [ 12 - 14 ]. The body fat of ovariectomized rats increases significantly [ 15 ], and the mesenchymal cells from the SA adipose tissue could be active, which might be closely related to the higher aromatase expression in the OVX6M. Yet, only in the OVX6M was there an associated significant increase of the aromatase expression in the SA adipose tissue, but not in other OVX groups, which by itself suggested that the elevated expression of aromatase in the OVX6M was not closely implicated in the obesity of the rats. We have also observed that the content of hypothalamic CRH increased along with time after ovariectomy in the rats, with a significant elevation in the OVX6M (submitted data). It has been reported that CRH may regulate the expressions of extragonadal aromatase via CRH receptor-II [ 16 ], which may help us understand the involved mechanism of the enhanced SA adipose aromatization and the eventual increased E 2 in the OVX6M rats, but further studies are earnestly needed. However, in the present results, it's confusing that the protein but not mRNA expression of SA adipose aromatase in the OVX1M showed an elevation compared with that in the INT1M. This might provide evidence indicating the inconsistency on the levels between transcription and translation of the aromatase gene. The factors possibly involved in the regulation of aromatase expression are still poorly understood [ 17 ]. Many studies have been performed to assess the possible dependence of the enzyme on androgens, though the data available are conflicting [ 17 , 18 ]. The situation is complicated by the fact that, in postmenopausal women, only about 25% of circulating T is derived by direct secretion from the ovaries [ 2 ]. The rest is formed largely from circulating precursors derived from the adrenal cortex. In the present study, the blood concentrations of T in the rats were detected. In the OVX groups there were decreases of the T level compared with corresponding intact ones, and no significant changes occurred along with the time after ovariectomy. These might be implicated in the less importance of T in the extragonadal aromatisation in rats than in women. Nevertheless, it's the first time to present the dynamic changes of T levels in the rats after ovariectomy. But further explorations are still needed on the way by which the aromatase enzyme expression is activated in ovariectomized rats. The microsomal enzyme cytochrome P450c17 is an important regulator of steroidogenesis. The enzyme has two functions: 17alpha-hydroxylase and 17,20-lyase activities. These functions determine the ability of adrenal glands and gonads to synthesize sex steroids (17,20-lyase activity) [ 19 ]. Its activity is abundant in testis, and lesser in ovary, and low levels of P450c17 activity in adrenals [ 20 ]. It is well known that adrenal is the principle organ to secrete sexual hormones except ovarian in females [ 21 ]. In the present study, due to the T levels did not show significant change along with the time after ovariectomy, the mRNA expressions of P450c17 were semi-quantified by RT-PCR to indirectly report the androgen synthesis in the adrenals. Excitedly, the ratio of adrenal P450c17 to β-actin in the OVX4M and OVX5M increased slightly, and in the OVX6M, the ratio increased markedly, which was higher than OVX1M. These suggested that the androgen synthesis activity of adrenal might be enhanced, and the more androgens might secrete from adrenal cortex. Though it has been reported that the splanchnic tissue is a minor site for extragonadal aromatization of androgens [ 22 ], there is a significant conversion of androstenedione to estrone by liver tissues [ 15 ]. In adult liver homogenates, C19 norsteroid (19-nortestosterone; NT) is readily aromatized to estrogens [ 23 , 24 ]. The present results showed that the aromatase expressions (mRNA and protein) of the liver tissue in the OVX1M, 2M, 3M, 4M and 5M groups were lower than the corresponding intact controls, and in the OVX6M the ratio increased significantly compared with that in the OVX1M. On one hand, after ovariectomy, the diminution of C19 precursor from ovaries for aromatization may induce the decreased expressions of aromatase in the OVX rats compared with the gonad-intact rats in our results. On the other hand, the results suggested that aromatization of liver tissue enhanced six months after ovariectomy. However, in the present study, the aromatase expressions in the OVX rats did not show consistent changes between the SA adipose and liver tissues, which may suggest that the role of SA adipose and splanchnic tissues in the extraglandular aromatisation might be different. Nonetheless, in the final analysis, our results suggested that both the SA adipose and liver tissues contributed to the extragonadal aromatisation to promote the circulation estrogen concentrations. Conclusions Both the subcutaneous abdominal adipose tissues and the liver tissues contributed to the extragonadal aromatization to promote the circulating E 2 levels in the rats along with time after ovariectomy; the adrenal compensation might also be activated naturally. Authors' contributions Hong Zhao and Zhanzhuang Tian designed the study, performed the studies and the statistical analysis, and drafted the manuscript. Junwei Hao performed the animal experiment. Boying Chen conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.

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549189

Influence of language and ancestry on genetic structure of contiguous populations: A microsatellite based study on populations of Orissa

Background We have examined genetic diversity at fifteen autosomal microsatellite loci in seven predominant populations of Orissa to decipher whether populations inhabiting the same geographic region can be differentiated on the basis of language or ancestry. The studied populations have diverse historical accounts of their origin, belong to two major ethnic groups and different linguistic families. Caucasoid caste populations are speakers of Indo-European language and comprise Brahmins, Khandayat, Karan and Gope, while the three Australoid tribal populations include two Austric speakers: Juang and Saora and a Dravidian speaking population, Paroja. These divergent groups provide a varied substratum for understanding variation of genetic patterns in a geographical area resulting from differential admixture between migrants groups and aboriginals, and the influence of this admixture on population stratification. Results The allele distribution pattern showed uniformity in the studied groups with approximately 81% genetic variability within populations. The coefficient of gene differentiation was found to be significantly higher in tribes (0.014) than caste groups (0.004). Genetic variance between the groups was 0.34% in both ethnic and linguistic clusters and statistically significant only in the ethnic apportionment. Although the populations were genetically close (F ST = 0.010), the contemporary caste and tribal groups formed distinct clusters in both Principal-Component plot and Neighbor-Joining tree. In the phylogenetic tree, the Orissa Brahmins showed close affinity to populations of North India, while Khandayat and Gope clustered with the tribal groups, suggesting a possibility of their origin from indigenous people. Conclusions The extent of genetic differentiation in the contemporary caste and tribal groups of Orissa is highly significant and constitutes two distinct genetic clusters. Based on our observations, we suggest that since genetic distances and coefficient of gene differentiation were fairly small, the studied populations are indeed genetically similar and that the genetic structure of populations in a geographical region is primarily influenced by their ancestry and not by socio-cultural hierarchy or language. The scenario of genetic structure, however, might be different for other regions of the subcontinent where populations have more similar ethnic and linguistic backgrounds and there might be variations in the patterns of genomic and socio-cultural affinities in different geographical regions.

Background Human society in a geographic area develops when colonizing populations bring along with them different languages, cultures and technological advancements over a period of time. As more populations migrate to settle in the same area, they are either eliminated, subjugated or absorbed [ 1 ]. In India, majority of incoming populations have been absorbed, forming heterogeneous and complex human societies. A few have subjugated the subservient cultures to establish a hierarchical caste system or have totally isolated some groups such as tribes, which still remain outside the social boundaries. This practice has enriched India with populations having varied socio-cultural and linguistic diversities that have flourished independently, nurtured by the vast geographical and ecological regime [ 2 ]. Studies based on various DNA markers on diverse populations occupying different geographical areas of the Indian subcontinent have revealed much about the presence of large extent of human genetic variation [ 3 - 10 ] and the distinct genetic difference between castes and tribal populations of India [ 11 - 13 ]. These studies, however fail to characterize the structure of populations in geographic contiguity, where populations with different language and social hierarchies cohabit together. Although distinct social demarcation between castes and tribes is well established, the origin of a few populations of India still remains controversial. Though many castes are known to have tribal origins [ 14 ], nevertheless their assessment with polymorphic DNA markers still remains incomplete. This study aims to understand the genetic diversity of populations of Orissa and examines the role of language and genetic origin on structure of populations inhabiting the same geographic region and evaluates some of the suggested population histories from a molecular perspective. Orissa is a coastal state in the southeast region of India, which is occupied by population groups having varied ethnicity, belonging to different strata of the hierarchical caste system and speaking languages belonging to different linguistic families. Its strategic geographic location between Northern plains and peninsular Southern India and cultures assimilated during the 4 th – 5 th century B.C. from southeast Asian countries of Java, Sumatra, Brunei and Indonesia [ 15 ] have enriched the socio-cultural diversity of contemporary populations of Orissa. The extant populations of the region can be broadly classified into two major social groups; castes and tribes. Brahmin, Khandayat, Karan and Gope comprise a large section of Indo-European speaking caste populations of Orissa, whose position in hierarchical caste system is governed by occupation and where ancestry is patrilineal. Brahmins form the priestly class who occupy uppermost strata in the caste hierarchy, with historical accounts that trace their migration from upper Gangetic regions of north India. Next in hierarchy is the Kshatriyā – a warrior group comprising the Khandayats; followed by Karans (Kayasthā), record keepers and Gope are cattle-breeders who occupy the subsequent strata in caste system [ 16 ]. Other than caste groups, tribes constitute a large number of aboriginal Australoid populations of Orissa who are predominantly forest dwellers, most of them having their own dialects. Linguistically, the tribal groups of the region can be categorized into three of the four major language families spoken in India: Indo-European, Austro-asiatic and Dravidian. Kharia, Juang, Gadaba, Ho, Munda and Saora are among few of the most ancient tribes whose dialects belong to the Austro-asiatic linguistic family, while those of Paroja, Oraon and Kondh belong to the Dravidian linguistic group [ 16 ]. Of these populations, only a few (Paroja, Agharia, Gaud, Tanti) have been included in studies using DNA markers to get a perspective of the overall genetic diversity present in the country [ 8 , 11 , 12 ]. Hence, to understand the genetic constitution of these ethnically and linguistically diverse populations, we have used autosomal microsatellites, genetic markers with proven precision in deciphering genomic diversity and affinities of human populations [ 17 ]. Microsatellites or short tandem repeats (STRs) are most extensively used for elucidating the genetic diversity and evolution of human populations because of their abundance and prevalence in the genome, high level of polymorphism and amenability to automation [ 18 - 23 ]. High mutation rates of STR loci facilitate inferences to be drawn about population substructure and short-term evolutions and to make a more reliable and precise estimation of phylogenetic relationships among populations both at racial and continental levels [ 24 - 29 ]. Also, most questions of anthropological interest involve processes occurring over relatively short time periods, during which substantial genetic drift and migration may occur but fewer mutations get accumulated. These minor changes are easily detected using STR markers rather than bi-allelic markers, where mutations accumulate slowly through evolutionary time. STR markers are therefore markers of choice for this study, which involves closely related populations that share similar ethnicity, language, culture or history of origin. In this study, we have examined variation at 15 autosomal STR loci in a sample of 404 individuals from Orissa (Table 1 , Figure 1 ) and compared the results with previously published data from other regions of the Indian subcontinent. Our aim was (i) to assess the genetic diversity and relationship of populations of Orissa with other Indian populations, and (ii) to find out the role of language and ancestry, if any, on genetic structure of populations living in geographic contiguity. This study also allows a finer resolution of population history of the region than has hitherto been possible. Table 1 Demographic characterization of the seven studied populations of Orissa POPULATION CODE n SOCIAL HIERARCHY ETHNICITY LANGUAGE GROUP 1 ORIYABRAHMIN OB 57 Upper Caste Indo-Caucasoid Indo-European 2 KARAN KR 62 Middle Caste Indo-Caucasoid Indo-European 3 KHANDAYAT KY 62 Middle Caste Indo-Caucasoid Indo-European 4 GOPE GP 60 Lower Caste Indo-Caucasoid Indo-European 5 JUANG JU 50 Tribe Proto-Australoid Austro-Asiatic 6 SAORA SA 35 Tribe Proto-Australoid Austro-Asiatic 7 PAROJA PR 78 Tribe Proto-Australoid Dravidian Figure 1 Geographical map of Orissa showing the location of sample collection Results Nature and extent of allelic diversity The distribution of allele frequencies and tests of Hardy-Weinberg Equilibrium (HWE) on the seven populations of Orissa have been previously reported [ 30 , 31 ]. Except for Saora, all other studied populations were found to be in HWE. Saora showed significant departures from HWE at three analysed parameters (p < 0.05 for exact test and homozygosity test; p < 0.1 for log-likelihood ratio test) and a lower heterozygosity value (0.571) compared to the expected estimates of allele frequencies at D3S1358 locus. Number of alleles and most common alleles at the fifteen STR loci along with gene diversity of each of the studied seven populations are shown in Table 2a , 2b and 3 . The most common alleles at each of the 15 STR loci were shared between 2–4 populations. These results agree with the analysis of 4 STR loci (CSF1Po, TPOX, THO1 and vWA) reported by Mukerjee et al, 1999 on three populations of Orissa (Agharia, Gaud and Tanti). The number of alleles observed in the studied population and heterozygosity values (0.615–0.967) indicate that the selected STR markers are highly polymorphic in all populations and that genetic variability within populations is significantly high across populations with mean gene diversity of 81%. Table 2a Allelic Diversity at 8 of 15 STR loci describing the extent of variation within the populations of Orissa D3S1358 THO1 D21S11 D18S51 D5S818 D13S317 D7S820 D16S539 Alleles MCA Alleles MCA Alleles MCA Alleles MCA Alleles MCA Alleles MCA Alleles MCA Alleles MCA Oriya Brahmin 6 15 6 6 11 29 11 15 6 12 8 11 7 11 6 11 Khandayat 6 15 6 9 11 29 10 14 7 11 8 12 8 11 8 11 Karan 7 15 5 6 10 32 11 14 6 11,12 8 12 7 11 8 11 Gope 8 15 6 9 10 32 12 14 8 12 8 11 8 11 7 11 Juang 6 15 6 9 8 29 10 15 6 11 7 11 7 8 7 11 Paroja 6 15 6 9 9 32 11 15 6 11 8 8 6 11 7 12 Saora 6 16,17 6 9 8 30 11 9 6 11 6 8 7 11 6 12 * Alleles = No. of Alleles, MCA = Most Common Allele Table 2b Allelic Diversity at 7 of 15 STR loci describing the extent of variation within the populations of Orissa CSF1Po vWA D8S1179 TPOX FGA Penta E Penta D Alleles MCA Alleles MCA Alleles MCA Alleles MCA Alleles MCA Alleles MCA Alleles MCA Oriya Brahmin 7 12 7 17 11 14 6 8 13 24 14 11 6 11 Khandayat 7 12 8 17 9 10,15 7 11 16 24 14 12 8 11 Karan 7 12 7 16 9 10 5 11 13 22 15 11 9 11 Gope 8 12 7 17 8 14 5 11 11 24 17 11,12 9 9 Juang 6 12 6 17,18 8 15 5 11 11 19,22 13 12 8 10 Paroja 5 12 8 14 8 10 5 11 14 23 18 11 9 10 Saora 5 11 5 16 8 13 5 8 10 21 15 11 8 9 * Alleles = No. of Alleles, MCA = Most Common Allele Table 3 Gene diversity estimated from 15 autosomal STR loci describing the total variation within the seven studied populations of Orissa Population Gene Diversity Oriya Brahmin 0.816 ± 0.017 Khandayat 0.811 ± 0.015 Karan 0.817 ± 0.018 Gope 0.815 ± 0.014 Juang 0.804 ± 0.015 Paroja 0.811 ± 0.013 Saora 0.810 ± 0.016 Extent of differentiation between populations To quantify the amount of genetic diversity that exists among populations, F ST was calculated separately for caste groups and tribes. The coefficient of gene differentiation was found to be significantly higher in tribes (0.014) than caste groups (0.004). Combining all seven populations yielded an F ST of 1%, demonstrating low level of population differentiation within Orissa. All values of F ST were significantly different from zero (p < 0.05). Analysis of molecular variance (AMOVA) presented in Table 4 , revealed that as a single group, a large extent of genetic variation (98.98%) was present within the populations of the region. To determine how the residual genetic variance was compartmentalized, we grouped the populations into (i) caste and tribes, (ii) linguistic groups; Indo-European speaking caste populations (Oriya Brahmins, Karan, Khandayat, Gope), Austro-Asiatic speakers Juang and Saora and Dravidian speaking, Paroja and (iii) according to their origins as suggested by historical accounts. The genetic variance between the groups varied from 0.25% to 0.34% and was equally distributed in both ethnic and linguistic clusters, but statistically significant only in the ethnic apportionment. Table 4 Variance in populations of Orissa due to ethnicity, language and history of origin at three different levels of hierarchy analysed with 15 autosomal STR loci Basis Grouping Populations in group % of total variance (p values) Within population Between population within group Between groups Ethnicity Castes vs Tribes (OB, KY, KR, GP) vs (JU, PR, SA) 98.84 (0.00) 0.83 (0.00) 0.34 (0.03) Language Indo-European vs Austro-Asiatic vs Dravidian (OB, KY, KR, GP) vs (JU, SA) vs (PR) 98.86 (0.00) 0.79 (0.00) 0.34 (0.05) History of Origin European vs Austro-Asiatic vs Admixed Gene Pool (OB, KR) vs (JU, SA) vs (KY, GP, PR) 98.91 (0.00) 0.84 (0.00) 0.25 (0.06) Because the amount of genetic variance between groups was found to be low, we also used clustering algorithm implemented in STRUCTURE analysis (Figure 2 ) to explore the population structure and relationship among these geographically contiguous but socially and linguistically disparate populations. When the populations were analysed assuming no admixture model and K varying from 1 to 7, only a single distinct genetic cluster could be found with the highest log likelihood value at K = 3. Most of the individuals of the seven populations clustered in cluster 1 and did not split into distinct clusters corresponding to their population affinities. A few of the individuals of Paroja and Khandayat were found in cluster 2 and 3 respectively. Figure 2 Assignment of samples from seven populations of Orissa to genetic clusters inferred from the STRUCTURE analysis for K = 3. Genetic relationship among populations The inter-population genetic relationship among Brahmins, Khandayat, Karan, Gope, Juang, Saora and Paroja was determined using principal component analysis. The plot (Figure 3 ) of principal component (PC) depicts population configurations in accordance with their ethnic affiliations. Together, the first two principal coordinates described almost 99.9% of the variance in the distance matrix. The caste populations (Brahmins, Khandayat and Karan) and the three tribal populations of Juang, Saora and Paroja were distinctly separated by the first component of the distance matrix. All the caste populations were found to cluster in the upper right quadrant while the tribes distinctly occupied the lower right quadrant. The only discordance was position of Gope, where this population was genetically separate from the other studied caste populations in the PC plot. Figure 3 PC plot for the seven populations of Orissa from centroid based on fifteen microsatellite loci The Neighbour-Joining (NJ) tree (Figure 4 ) gives a graphical representation of genetic distance of Orissa populations from populations of Bihar [ 32 , 33 ], Uttar Pradesh [ 34 ], Maharastra [ 35 , 36 ] and Tamil Nadu [ 37 ], belonging to similar rank and occupational affiliation in the caste hierarchy. The genetic closeness exhibited by Brahmins of Orissa to those of North India (NJ tree; Figure 4 ) was clearly discernible, supported by moderately high bootstrap values. While Karan belonging to the next level of hierarchy in the caste system showed similarity to Maratha, a warrior group of Maharastra; Khandayats and Gope depicted affinity to the tribal populations (Figure 4 ). Paroja, a Dravidian linguistic group, demonstrated affinity with Gonds, and the two Austro-Asiatic speakers Juang and Saora distinctly branched out in the phylogenetic tree. Figure 4 Neighbor-joining tree of genetic distances (D A ) based on fifteen microsatellite loci among studied populations of India Discussion India is a remarkable representation of a large segmented society that harbours rich genetic diversity within its human populations and offers myriads of attributes to study the various factors influencing demographics of human populations. It is of particular interest to study patterns of genetic affinities among endogamous groups inhabiting small geographical regions within the subcontinent because of their diverse origins and interethnic admixtures. We have typed a set of fifteen polymorphic autosomal microsatellite markers in linguistically and socially divergent populations with different histories of origin to elucidate the genetic diversities and affinities among them and to understand the role of genetic origin and language on the genetic structure of populations living in geographic contiguity. The most distinctive feature of our study was the clear delineation between castes and tribes, as was evident from both multivariate and phylogenetic analyses (Figure 3 and 4 respectively). The tribes seem to be the most unique and genetically isolated populations within Orissa. The two Austro-Asiatic tribes Juang and Saora were not significantly different from each other and both showed least number of alleles even at the most polymorphic STR loci such as, D21S11, D18S51, Penta E and FGA (Table 2a , 2b ) and lowest heterozygosity values in as many as six loci as compared to the caste groups [ 30 , 31 ]. The tribal groups show relatively high between group differentiation that probably can be attributed to reproductive isolation and drift. This finding is consistent with similar studies carried on tribal populations of Central India [ 6 ]. The low heterozygosity estimate of tribes suggests that they have probably undergone some stochastic processes that have resulted from limitations in mating practices and socio-cultural differences in them. The significantly low coefficient of differentiation among the seven populations (Fst: 0.010, p < 0.05), along with the number of alleles shared between them, confirms admixture and suggests an increased genetic affinity among populations residing in geographic proximity irrespective of their socio-cultural affiliation [ 3 , 38 , 39 ]. This is also substantiated with the AMOVA and Structure results, which showed that all the individuals of the studied populations cluster in one group and could not be subdivided further. The inability of STRUCTURE analysis to subdivide populations may be due to gene flow among groups or may be that more number of samples and loci are required to identify such close genetic subgroups. Among the caste groups, Orissa Brahmin showed close affinity to the other upper caste populations of North India rather than to its geographic neighbors. The affinity between Bihar Brahmin and Orissa Brahmin was supported with moderate bootstrap values in the phylogenetic tree (Figure 4 ), which could be attributed to gene flow between them because of sharing same hierarchical status in the Hindu caste system [ 9 ]. This observation corroborates prevalent historical accounts, which suggests that the Brahmin populations of different parts of the subcontinent were natives of upper Gangetic region, who later dispersed to different parts of the country to propagate their cultural and religious ideologies and to explore better economic opportunities [ 15 ]. The phylogenetic tree (Figure 4 ) also clearly depicted that Khandayat and Gope are genetically more related to each other than to other occupationally similar populations (Rajput, Thakur, Maratha and Yadav) of adjoining regions. These results are in congruence with the observations of Majumder et al 1998, where populations studied from widely separated geographic areas were found to exhibit closer genomic affinities with their geographic neighbors than with those sharing similar social ranks. It also substantiates the suggested origin of Khandayat from skilled individuals drawn from peasantry and aboriginals of the region [ 14 ]. Because the natives were assimilated into the caste system, they adopted the language and culture of the expanding and dominant upper caste population as a consequence of 'elite-dominance'. Their gene pool, however, still remains closer to aboriginals of the region. Therefore, except Brahmins, other groups were probably pooled from the local people to serve the needs of upper castes in the brahminical society. Thus, two castes bearing similar names simply represent affiliation to the same profession, but have probably different genetic constitution in different geographical regions. When populations of diverse geographic regions were included, the genetic difference among populations of the Indian subcontinent increased. This can probably be ascribed to drift caused by limitations imposed on social mobility between groups due to differences in culture and language. Juang and Saora speak Austro-Asiatic languages while Paroja follow the Dravidian language, both of which are unrelated to Oriya and by itself is a branch of Indo-European linguistic family spoken largely by the caste groups. PC analysis (Figure 3 ) revealed distinct isolation of the tribes from the Oriya speaking caste populations. The position of Juang and Saora in the NJ tree suggests that they are genetically still separate from other populations and extent of admixture in them from neighboring caste groups is negligible. It is also discernible that genetic distance among tribes is more strongly correlated with their genetic origin, with Paroja forming a close cluster with Madia Gond, a Dravidian tribe of India. This also substantiates the historical account describing Paroja to be an offshoot of the Gonds, one of the largest tribal populations of India. The NJ tree clearly shows that ethnic affiliation (caste/ tribe) and genetic ancestry are the key factors in shaping the genetic variation and sub-structuring among populations in geographic contiguity. Conclusions Our study on linguistically distinct but geographically contiguous populations of Orissa using autosomal microsatellite markers reveals a significant amount genetic homogeneity in them. AMOVA results suggest that linguistic differences probably play a negligible role in the present day scenario in restricting gene flow between these populations. The middle-order caste groups shared genetic affinity with the local people of the area, while the Brahmins were similar to those from northern regions. Tribal populations, on the other hand, because of their long-term isolation and mating patterns, were well differentiated from the upper caste groups. This paper provides evidence that for populations living in geographic contiguity, ancestry is the governing factor in fine-tuning of genetic differentiation. Methods Population samples analyzed Blood samples were collected from randomly chosen consenting volunteers, distributed across 17° -48' and 22° -34' North latitude and 81° -24' and 87° -29' East longitude of Orissa (Figure 1 ). A total of 404 individuals from seven populations, Brahmins (n = 57), Khandayat (n = 62), Karan (n = 62), Gope (n = 60), Juang (n = 50), Saora (n = 35), and Paroja (n = 78) were analyzed for the fifteen autosomal microsatellite loci. These populations were categorized based on ethnic and linguistic criteria (Table 1 ). The populations used for comparison in the study were selected on the basis of ethnicity, language and occupational similarity: Kanyakubj Brahmins (95), Bihar Brahmins (59), Kayastha (53), Yadav (44), Bhumihar (65), Rajput (58), Thakur (48), Irular (54), Maratha (65), Madia Gond (45), Katkari (72) and Pawara (51). DNA typing DNA was extracted from blood samples using standard phenol-chloroform procedure and amplified for fifteen autosomal microsatellite loci using primers multiplexed in the Powerplex 16 System (Promega Corp., Madison, Wisconsin). STR loci analyzed in the study included thirteen tetranucleotides D3S1358, THO1, D21S11, D18S51, D5S818, D13S317, D7S820, D16S539, CSF1PO, vWA, D8S1179, TPOX, FGA and two pentanuleotides, PentaD and PentaE. Analysis of data The genetic structure of the populations was analyzed at two hierarchical levels – within populations and among populations. The intrapopulation variability was estimated by analyzing the number of alleles and most common allele at individual loci and by estimating the average gene diversity [ 40 ] across the fifteen microsatellite loci. To understand the genetic variation among populations; F ST estimates, genetic distance and the analysis of molecular variance [ 41 ] were calculated. Genetic relationships among populations were analyzed using the Principal Component Analysis [ 42 ]. Genetic distances were estimated by using the D A distance measure [ 43 ], and were used to construct neighbor-joining tree [ 44 ]. The degree of support for the branches was evaluated by bootstrap analysis. To test the correspondence of genetic clusters with linguistically labeled groups, we used STRUCTURE program [ 45 ] assuming that each individual had ancestry in all clusters, so that fractions of ancestry in various clusters could be estimated. Authors' contributions SS carried out laboratory experiments, statistical analysis and drafted the manuscript and VKK conceptualized the paper, provided important intellectual inputs in intrepretation of data and preparation of the manuscript. Both authors read and approved the final manuscript.

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529457

Neoadjuvant or adjuvant therapy for resectable esophageal cancer: a systematic review and meta-analysis

Background Carcinoma of the esophagus is an aggressive malignancy with an increasing incidence. Its virulence, in terms of symptoms and mortality, justifies a continued search for optimal therapy. The large and growing number of patients affected, the high mortality rates, the worldwide geographic variation in practice, and the large body of good quality research warrants a systematic review with meta-analysis. Methods A systematic review and meta-analysis investigating the impact of neoadjuvant or adjuvant therapy on resectable thoracic esophageal cancer to inform evidence-based practice was produced. MEDLINE, CANCERLIT, Cochrane Library, EMBASE, and abstracts from the American Society of Clinical Oncology and the American Society for Therapeutic Radiology and Oncology were searched for trial reports. Included were randomized trials or meta-analyses of neoadjuvant or adjuvant treatments compared with surgery alone or other treatments in patients with resectable thoracic esophageal cancer. Outcomes of interest were survival, adverse effects, and quality of life. Either one- or three-year mortality data were pooled and reported as relative risk ratios. Results Thirty-four randomized controlled trials and six meta-analyses were obtained and grouped into 13 basic treatment approaches. Single randomized controlled trials detected no differences in mortality between treatments for the following comparisons: - Preoperative radiotherapy versus postoperative radiotherapy. - Preoperative and postoperative radiotherapy versus postoperative radiotherapy. Preoperative and postoperative radiotherapy was associated with a significantly higher mortality rate. - Postoperative chemotherapy versus postoperative radiotherapy. - Postoperative radiotherapy versus postoperative radiotherapy plus protein-bound polysaccharide versus chemoradiation versus chemoradiation plus protein-bound polysaccharide. Pooling one-year mortality detected no statistically significant differences in mortality between treatments for the following comparisons: - Preoperative radiotherapy compared with surgery alone (five randomized trials). - Postoperative radiotherapy compared with surgery alone (five randomized trials). - Preoperative chemotherapy versus surgery alone (six randomized trials). - Preoperative and postoperative chemotherapy versus surgery alone (two randomized trials). - Preoperative chemoradiation therapy versus surgery alone (six randomized trials). Single randomized controlled trials detected differences in mortality between treatments for the following comparison: - Preoperative hyperthermia and chemoradiotherapy versus preoperative chemoradiotherapy in favour of hyperthermia. Pooling three-year mortality detected no statistically significant difference in mortality between treatments for the following comparison: - Postoperative chemotherapy compared with surgery alone (two randomized trials). Pooling three-year mortality detected statistically significant differences between treatments for the following comparisons: - Preoperative chemoradiation therapy versus surgery alone (six randomized trials) in favour of preoperative chemoradiation with surgery. - Preoperative chemotherapy compared with preoperative radiotherapy (one randomized trial) in favour of preoperative radiotherapy. Conclusion For adult patients with resectable thoracic esophageal cancer for whom surgery is considered appropriate, surgery alone (i.e., without neoadjuvant or adjuvant therapy) is recommended as the standard practice.

Background Carcinoma of the esophagus is an aggressive malignancy that continues to kill more than 90% of people with the disease within five years [ 1 ]. The incidence of adenocarcinoma of the esophagus is rising faster than any other malignancy [ 2 ]. In 2001, there were at least 1,450 deaths due to esophageal cancer in Canada and many more people suffered because of the disease [ 3 ]. Its virulence, in terms of symptoms and mortality, justifies a continued search for optimal therapy. Surgical esophagectomy remains the preferred treatment for clinically localized thoracic esophageal carcinoma [ 1 , 4 - 6 ]. Two randomized trials comparing surgery alone to radiation alone found surgery to be the better treatment for resectable cancer [ 5 , 6 ]. Fok et al randomly assigned 39 patients to surgery and 35 patients to 45 to 53 Gy radiation over four to five weeks [ 5 ]. The median survival time and five-year survival rate for surgery were 21.6 months and 16%, respectively, compared with 8.2 months and 7% for radiation (p < 0.05). Badwe et al compared 47 surgical patients to 52 patients undergoing 50 Gy radiation in 28 fractions plus 15 Gy boost in 8 fractions or 15 Gy brachytherapy [ 6 ]. Overall survival was better with surgery (odds ratio [OR], 2.74; 95% confidence interval [CI], 1.51 to 4.98; log-rank p = 0.002). The swallowing status was better in the surgery arm at six months after treatment (p = 0.03). Survival data from these two trials were pooled. The pooled results favoured surgery alone. There was no statistical heterogeneity (X 2 = 0.02, p = 0.9) and a 52% relative increase in the risk of death at three years with radiotherapy compared with surgery alone (relative risk ratio [RR], 1.52; 95% CI, 1.23 to 1.86; p = 0.0007). The failure of surgery alone is attributed to the systemic nature of the disease at the time of presentation [ 7 , 8 ]. Early and effective systemic chemotherapy and local radiotherapy, directed at micro-metastases and added to surgical resection, could lead to increased survival. Many clinical trials have evaluated the role of adjuvant therapy, both preoperatively and postoperatively, with conflicting results. Patients with cervical esophageal cancer are generally treated with chemoradiation, either preoperatively or postoperatively, in an attempt to avoid a laryngoesophagectomy and preserve the larynx. Although the majority of studies have been performed in squamous cell carcinomas, adenocarcinomas were included in some studies, but a distinction between the two histological subtypes was not made in this guideline report because previous studies have not consistently found that they respond differently to chemotherapy or radiation [ 9 - 17 ]. The large and growing number of patients affected, the high mortality rates, the geographic variation in practice, and the large body of good quality research evidence warrants a systematic review with meta-analysis. Methods This systematic review was developed by the Practice Guidelines Initiative (PGI) of Cancer Care Ontario's Program in Evidence-based Care (PEBC). Evidence was selected and reviewed by two members of the PGI's Gastrointestinal Cancer Disease Site Group (DSG) and two methodologists. Members of the Gastrointestinal Cancer DSG disclosed potential conflict of interest information. This systematic review is a convenient and up-to-date source of the best available evidence on neoadjuvant or adjuvant therapy for resectable esophageal cancer. The body of evidence in this systematic review is primarily comprised of mature randomized controlled trial data; it forms the basis of a clinical practice guideline developed by the Gastrointestinal Cancer DSG published elsewhere (18). This systematic review and companion practice guideline are intended to promote evidence-based practice in Ontario, Canada. The PGI is editorially independent of Cancer Care Ontario and the Ontario Ministry of Health and Long-Term Care. Literature search strategy The MEDLINE (1966 through October (week 2) 2003), CANCERLIT (1983 to October 2001), Cochrane Library (2003, Issue 3), and EMBASE (to week 40, 2003) databases were searched with no language restrictions. "Esophageal neoplasms" (Medical subject heading (MeSH)) was combined with "chemotherapy, adjuvant" (MeSH), "radiotherapy, adjuvant" (MeSH), "immunotherapy, adjuvant" (MeSH), and each of the following phrases used as text words: "preoperative", "neoadjuvant", "chemotherapy", "radiotherapy", "radiation therapy", "irradiation", "immunotherapy", "chemoradiotherapy", "chemoradiation", and "hyperthermia". These terms were then combined with the search terms for the following study designs or publication types: practice guidelines, meta-analyses, and randomized controlled trials. Additionally, the conference proceedings of the 1997 to 2003 annual meetings of the American Society of Clinical Oncology (ASCO) and the 1999 to 2002 annual meetings of the American Society for Therapeutic Radiology and Oncology (ASTRO) were searched for reports of new or ongoing trials. Relevant articles and abstracts were reviewed, and the reference lists from these sources were searched for additional trials. This formal search was supplemented with published abstracts from thoracic surgery and oncology conferences, conversations with colleagues and experts in the field, and a review of textbooks related to esophageal oncology. Study selection criteria Articles were included in this systematic review if they were fully published reports, abstracts, or meta-analyses of randomized controlled trials (RCT) of neoadjuvant or adjuvant treatments compared with surgery alone or surgery plus another treatment in patients with resectable and operable thoracic esophageal cancer. Data on survival had to be reported. Other outcomes of interest were adverse effects and quality of life. Reports of carcinomas located in the cervical esophagus were excluded. Synthesizing the evidence Because diverse treatment strategies were evaluated, the eligible studies were grouped into 13 basic treatment approaches (Table 1 ), and each group was examined separately. Pooling was conducted using one-year mortality data for all meta-analyses except for the comparison of post-operative chemotherapy versus surgery alone, which was pooled at three years. Any time point selected for meta-analyses must be clinically credible and relevant but not so far along the survival curve that wide confidence intervals result from fewer patients contributing to the estimate. Since time points prior to the median will generally ensure that there are sufficient data to be credible, median survival times, weighted by the size of the treatment arms, were calculated to determine the time point for each meta-analysis as recommended in the literature [ 19 ]. Studies that did not provide values for survival at the time of pooling were not included in each meta-analysis, although they were included in calculating the weighted median survival time, if values for median survival were provided. All pooling was performed with Review Manager 4.2.1, available through the Cochrane Collaboration [Review Manager (RevMan) [Computer program]. Version 4.2 for Windows. Oxford, England: The Cochrane Collaboration, 2003]. Pooled results were expressed as mortality RR with 95% CI using the random effects model. An RR less than 1.0 favours neoadjuvant or adjuvant treatment, and an RR greater than 1.0 favours surgery alone. All analyses were made based on the intent-to-treat principle, except where only evaluable patient data were available. Potential sources of heterogeneity and sensitivity analysis Heterogeneity of study results was assessed using a visual plot of the outcomes and by calculating the X 2 (Chi-square) statistic using a planned cut-off for significance of p < 0.05. Potential sources of heterogeneity were postulated a priori and included study quality assessed with the Jadad scale [ 20 ] (>2 versus ≤2), full article publication versus abstract publication, squamous cell versus adenocarcinoma, type of chemotherapy (cisplatin-containing versus others), type of surgery (transthoracic versus transhiatal), and radiotherapy dose (BED>48 versus BED<48). To facilitate comparison across trials, radiotherapy dose was converted to biological equivalent dose (same as biological effective dose) using the equation BED = nd (1+d/α/β), where n = number of fractions, d = dose per fraction, and it is assumed that α/β = 10 for tumour effect. Due to limitations inherent with this model, no allowances can be made for any time gaps in split-course treatments. These factors were used to explore any significant heterogeneity of results across the trials. The robustness of our conclusions was examined through subsequent sensitivity analyses using these factors. The sensitivity analysis results are not detailed, as they would not change the conclusions. Results Literature search results Thirty-four randomized controlled trials were obtained. Of these, 30 were fully published reports [ 5 , 21 - 25 , 27 - 34 , 36 , 37 , 39 , 40 , 42 - 48 , 52 , 56 - 60 ], and four were available in abstract form only [ 35 , 41 , 51 , 53 ]. The four-arm trials by Fok et al [ 5 ] and Nygaard et al [ 24 ] contributed to multiple comparisons. Additionally, six meta-analyses were obtained, five fully published [ 26 , 38 , 49 , 50 , 55 ] and one abstract [ 54 ]. Literature search results appear in Table 1 . Outcomes Preoperative radiotherapy and surgery versus surgery alone Six randomized trials of preoperative radiotherapy and surgery versus surgery alone are presented in Table 2 [ 5 , 21 - 25 ]. The radiotherapy regimens varied, using low to moderate doses ranging from 20 Gy in 10 fractions to 53 Gy in 20 fractions. Treatment was delivered between one to four weeks prior to surgery. Quality-of-life assessments were not conducted in any of the six trials. The Gastrointestinal Cancer DSG pooled the five trials that reported one-year mortality data [ 5 , 21 , 22 , 24 , 25 ] (Figure 1 ). No statistically significant difference in the risk of mortality with preoperative radiotherapy at one year compared with surgery alone was detected (RR, 1.01; 95% CI, 0.88 to 1.16; p = 0.87). No statistical heterogeneity was detected (X 2 = 3.61, p = 0.46). A published meta-analysis [ 26 ] using updated individual patient data on 1147 patients from five trials [ 21 - 25 ] detected a hazard ratio for death of 0.89 (95% CI, 0.78 to 1.01; p = 0.062) for preoperative radiotherapy compared with surgery alone. This meta-analysis included additional patients from the study by Wang et al. [ 23 ] with no description of why these patients were excluded from the published report of the trial (a total of 418 patients from this study were included in the meta-analysis versus 206 included in the trial report). The trial by Fok et al. [ 5 ] was not included in the published meta-analysis. Postoperative radiotherapy and surgery versus surgery alone Five randomized trials of surgery and postoperative radiotherapy compared with surgery alone are presented in Table 3 [ 5 , 27 - 29 , 47 ]. Although all studies specified the absence of distant metastases as an inclusion criterion, Zieren et al. [ 29 ] and Teniere et al. [ 28 ] included patients with celiac node involvement (M1 disease). Fok et al. [ 27 ] included patients with positive margins and "a high chance of residual tumour". In the trials by Fok et al., Zieren et al. and Xiao et al., radiotherapy was delivered within six weeks postoperatively, while the trial by Teniere et al. specified within three months. The radiotherapy doses were higher than in the preoperative series. Of note, Fok et al. employed hypofractionation schedules using three fractions per week and 3.5 Gy per fraction to total doses of 49 Gy for patients with negative margins and 52.5 Gy for those with positive margins. Only Zieren et al. assessed quality of life. The results indicated more rapid recovery of quality of life with surgery alone compared with postoperative radiotherapy. Three trials [ 28 , 29 , 47 ] demonstrated no significant difference in survival while another [ 27 ] found significantly shorter survival with postoperative radiotherapy and surgery compared with surgery alone. The Gastrointestinal Cancer DSG pooled the five trials that reported one-year mortality data [ 5 , 27 - 29 , 47 ] (Figure 2 ). No significant difference in the risk of mortality with postoperative radiotherapy and surgery at one year compared with surgery alone was detected (RR, 1.23; 95% CI, 0.95 to 1.59; p = 0.11). No significant statistical heterogeneity was detected (X 2 = 7.53, p = 0.11). The rate of local recurrence with radiotherapy was lower in three of the trials [ 27 , 28 , 47 ], but two trials [ 27 , 28 ] noted this benefit was achieved at the expense of increased morbidity. Preoperative radiotherapy versus postoperative radiotherapy One randomized trial evaluated preoperative radiotherapy versus postoperative radiotherapy with curative esophagectomy as part of a four-arm study [ 5 ]. Patients in this trial, performed between 1968 and 1981, received from 24 to 53 Gy preoperatively (n = 40) or 45 to 53 Gy postoperatively (n = 42). The median survival was 11 months for both groups. No difference in the survival rate was detected, but there was increased morbidity with preoperative radiotherapy. Quality of life was not assessed in this trial. Preoperative radiotherapy and postoperative radiotherapy versus postoperative radiotherapy alone Iizuka et al. [ 30 ] reported a randomized trial of preoperative and postoperative radiotherapy versus postoperative radiotherapy alone in 364 Japanese patients. In an analysis of 207 eligible patients (157 patients were excluded because of the extent of disease or operative complications), preoperative and postoperative radiotherapy was associated with a significantly higher mortality rate compared with postoperative radiotherapy alone (median survival was 394 days versus 648 days; p = 0.0069). The major postoperative complications were pneumonia (13.5% versus 9.7%) and leakage (11.5% versus 9.7%). Preoperative chemotherapy and surgery versus surgery alone Seven randomized trials of preoperative chemotherapy and surgery versus surgery alone are presented in Table 4 [ 24 , 32 - 35 , 37 , 48 ]. Of these seven RCTs, six were available as fully published reports, and one was available as an abstract only [ 35 ]. Quality of life was not assessed in any of the trials. Additionally, three meta-analyses were obtained [ 38 , 49 , 50 ]. The Gastrointestinal Cancer DSG pooled the available data on preoperative chemotherapy with surgery versus surgery alone [ 24 , 32 - 34 , 37 , 48 ] (Figure 3 ). No significant difference in the risk of mortality at one year was detected (RR, 1.00; 95% CI, 0.83 to 1.19; p = 0.98). No statistical heterogeneity was detected (X 2 = 8.26, p = 0.14). The first meta-analysis, by Bhansali et al. [ 38 ], pooled data from 12 randomized trials of chemotherapy in a variety of combinations with radiotherapy with and without surgery, and no benefit for cisplatin-based chemotherapy was detected (OR, 0.96; 95% CI, 0.75 to 1.22; p > 0.10). This published meta-analysis included only four of the eight trials of preoperative chemotherapy versus surgery alone [ 24 , 31 - 33 ]. Trials that did not meet the inclusion criteria for this systematic review, such as trials involving patients with inoperable esophageal cancer and trials of combined chemoradiotherapy versus radiotherapy alone in the non-surgical management of esophageal cancer, were included in the Bhansali et al. meta-analysis. The second meta-analysis, by Urschel et al. [ 49 ], pooled data from 11 RCTs (a total of 1,976 patients). These 11 RCTs were graded for quality using the Jadad scale [ 20 ]. Pooling detected no statistically significant difference between combination preoperative chemotherapy with surgery over surgery alone for survival at either one year (OR 1.00; 95% CI 0.76–1.30; p = 0.98), two years (OR 0.88; 95% CI 0.62–1.24; p = 0.45), or three years (OR 0.77; 95% CI 0.37–1.59; p = 0.48). The third meta-analysis [ 50 ] was a Cochrane Review which pooled 11 RCTs (a total of 2,051 patients). Survival RRs were calculated at one, two, three, four, and five years, but a statistically significant difference in survival favouring preoperative chemotherapy was detected only at five years (RR = 1.44, 95% CI; 1.05–1.97; p = 0.02). Preoperative and postoperative chemotherapy and surgery versus surgery alone Two randomized trials of preoperative and postoperative chemotherapy and surgery versus surgery alone [ 31 , 36 ] (Table 5 ) were examined. Neither Roth et al. [ 31 ] (using a now out-dated combination of cisplatin, vindesine, and bleomycin) nor the largest North American trial as reported by Kelsen et al. [ 36 ] (using cisplatin and 5-FU) detected a statistically significant difference in overall survival. The Gastrointestinal Cancer DSG pooled these two trials (Figure 4 ). No significant difference in the risk of mortality with preoperative and postoperative chemotherapy and surgery compared with surgery alone was detected (RR, 0.99; 95% CI, 0.81 to 1.21; p = 0.93). No statistical heterogeneity was detected (X 2 = 0.65, p = 0.42). Postoperative chemotherapy and surgery versus surgery alone Three randomized trials of postoperative chemotherapy and surgery compared with surgery alone are presented in Table 6 [ 39 - 41 ]. All three trials used cisplatin-based regimens. Pouliquen et al. [ 39 ] found no improvement in the survival rate with postoperative chemotherapy. The patients were stratified into two groups: complete resections with or without nodal involvement and, palliative resections for positive margins or metastatic disease. Only the completely resected group was included in our analysis. Ando et al. [ 40 ] resected early (T1b) carcinomas and did not find any improvement in survival. In another study, reported in abstract form, Ando et al. [ 41 ] also found no survival benefit for postoperative chemotherapy in localized squamous cell carcinoma of the thoracic esophagus. Pouliquen et al. assessed quality of life and found that the duration of improved dysphagia was similar for both groups. The Gastrointestinal Cancer DSG pooled the three-year mortality data for two trials [ 39 , 40 ] (Figure 5 ). The trial by Ando et al. [ 41 ] could not be included in the pooled analysis because the abstract did not report three-year survival. No significant difference in the risk of mortality at three years for postoperative chemotherapy compared with surgery alone was detected (RR, 0.94; 95% CI, 0.74 to 1.18; p = 0.59). There was no significant statistical heterogeneity (X 2 = 0.08, p = 0.77). Preoperative chemotherapy and radiotherapy and surgery versus surgery alone Eight randomized trials of combined modality neoadjuvant chemotherapy and radiotherapy are presented in Table 7 [ 24 , 42 - 46 , 51 , 53 ]. A ninth trial obtained [ 52 ] provided updated five-year data for another report [ 44 ]. None of the trials reported data on quality of life. In contrast to the other trials, the study by Walsh et al. [ 44 , 52 ] reported a significant overall increase in three-year survival with combined preoperative chemoradiation but was closed prematurely following an interim analysis. This study was criticized for the lack of preoperative staging using CT scans, premature closure, and an unusually poor survival rate in the surgery-alone arm. The Gastrointestinal Cancer DSG pooled the one-year mortality data for the six trials with data available at one year [ 24 , 42 - 46 ] (Figure 6 ). No significant difference in the risk of mortality at one year for preoperative chemoradiation and surgery compared to surgery alone was detected (RR, 0.89; 95% CI, 0.76 to 1.03; p = 0.12). No significant statistical heterogeneity was detected (X 2 = 1.67, p = 0.89). The first meta-analysis, an abstract report by Fiorica et al. [ 54 ], pooled six RCTs comparing preoperative chemoradiation and surgery versus surgery alone. A systematic review, restricted to trials that included only patients with resectable esophageal carcinoma with no metastatic disease, obtained six RCTs. A significant difference in three-year mortality favouring neoadjuvant therapy with surgery versus surgery alone was detected (OR 0.53; 95% CI 0.31–0.92; p = 0.025). A conclusion was made that neoadjuvant chemoradiation and surgery significantly improved three-year survival compared to surgery alone in patients with resectable esophageal cancer but acknowledged that the magnitude of the benefit was relatively small. The authors recommend that research to determine the criteria that would identify patients likely to benefit from neoadjuvant chemoradiation be undertaken. The second meta-analysis, by Urschel et al. [ 55 ], pooled nine RCTs, eight of which were included in this practice guideline [ 24 , 42 - 46 , 51 , 52 ]. The RCTs were graded for quality using the Jadad scale. This meta-analysis did not find a statistically significant difference in mortality at one year (OR 0.79; 95% CI 0.59–1.06; p = 0.12) or at two years (OR 0.77; 95% CI 0.59–1.05; p = 0.10). However, as in the meta-analysis by Fiorica et al. [ 54 ], a statistically significant difference was found at three years in favour of preoperative chemoradiation (OR 0.66; 95% CI 0.47–0.92; p = 0.016). The authors noted that the three-year survival benefit was most pronounced when chemoradiation was given concurrently (OR 0.45; 95% CI 0.26–0.79; p = 0.005) as opposed to sequentially (OR 0.82; 95% CI 0.54–1.25; p = 0.36). To compare the results between the two published meta-analyses [ 54 , 55 ] with the trials included in this systematic review, the Gastrointestinal Cancer DSG pooled the data comparing neoadjuvant chemoradiation with surgery versus surgery alone at three years and obtained similar results. A significant difference in the risk of mortality at three years favouring neoadjuvant chemoradiation with surgery versus surgery alone was detected (RR = 0.87; 95% CI 0.80–0.96; p = 0.004). No statistically significant heterogeneity was detected (X 2 = 6.59, p = 0.25). Postoperative chemotherapy and radiotherapy versus surgery alone No randomized trials have evaluated postoperative chemotherapy combined with radiation versus surgery alone. Postoperative chemotherapy versus postoperative radiotherapy One randomized trial evaluated postoperative chemotherapy versus postoperative radiotherapy following curative esophagectomy [ 56 ]. Patients in this Japanese trial received cisplatin and vindesine (n = 126) or radiotherapy at a dose of 50 Gy (n = 127). The median survival was 38 months for both groups. No difference in survival was detected (52% for chemotherapy versus 51% for radiotherapy at three years; log-rank p = 0.806). There were significantly more cases of decreased white blood cell counts (12 versus 3 for grade 3–4; p = 0.026), elevated blood urea nitrogen (26 versus 11 for grade 1–2; p = 0.018) and elevated creatinine concentrations (27 versus 9 for grade 1–3; p = 0.006) among patients randomized to chemotherapy compared with radiotherapy. Quality of life was not assessed in this trial. Preoperative chemotherapy versus preoperative radiotherapy Two randomized trials evaluating preoperative chemotherapy compared with preoperative radiotherapy were reviewed [ 24 , 57 ]. Kelsen et al. [ 57 ] randomly assigned 96 patients to preoperative radiotherapy or chemotherapy. Postoperative crossover therapy (i.e., postoperative radiotherapy for those who received preoperative chemotherapy and vice versa) was given to patients who were found to have unresectable or locally advanced disease. Only 11 of 48 chemotherapy patients and 9 of 48 radiotherapy patients did not receive additional postoperative treatment. Overall median survival was similar in both groups (10.4 months for chemotherapy versus 12.4 months for radiotherapy; p = 0.61), but the crossover design precluded proper analysis. In the four-arm trial by Nygaard et al. [ 24 ], preoperative chemotherapy was compared with preoperative radiotherapy, and the results demonstrated a significant difference in survival favouring preoperative radiotherapy (21% versus 3% at three years; p = 0.01). However, when compared to surgery alone, there was no benefit to either preoperative radiation or chemotherapy. Neither trial report included data on quality of life. Preoperative chemoradiation versus preoperative radiotherapy One randomized trial evaluated the role of preoperative bleomycin in addition to radiotherapy [ 58 ]. Seventy patients received preoperative chemoradiation with bleomycin and 63 patients received preoperative radiotherapy alone. The results demonstrated no significant difference in survival between the two groups (median survival was 25 weeks versus 26 weeks; survival rate was 25% versus 19% at two years; p = 0.56). There was also no benefit for bleomycin in the palliation of dysphagia. Quality of life was not assessed in this trial. Postoperative immunotherapy in combination with radiotherapy or chemoradiation One Japanese trial evaluated protein-bound polysaccharide (PSK) as an adjunct to postoperative radiotherapy or chemoradiation in resected esophageal cancer [ 59 ]. This trial involved 174 patients who were randomly assigned to four treatment groups. The three-year survival rates for radiotherapy, radiotherapy + PSK, chemoradiotherapy, and chemoradiotherapy + PSK were 43.3%, 45.5%, 33.5%, and 44.3%, respectively. There was no significant difference in survival when radiotherapy and radiotherapy + PSK were compared, or when chemoradiotherapy and chemoradiotherapy + PSK were compared (log-rank p = 0.19 for chemoradiotherapy versus chemoradiotherapy + PSK). Some patients randomized to PSK experienced adverse effects, including mild nausea, erythema, liver dysfunction and leukopenia, but there were no reports of toxicity that were definitely attributed to PSK. There was no assessment of quality of life. Preoperative hyperthermia in combination with chemoradiation One Japanese randomized trial, reported by Kitamura et al. [ 60 ], evaluated preoperative hyperthermia and chemoradiotherapy and surgery (n = 32) versus preoperative chemoradiotherapy and surgery (n = 34). Median survival was 36 months and 20 months, respectively. The results showed a significant improvement in the survival rate (50.4% versus 24.2% at three years; p-value not reported) and local tumour control with hyperthermia compared with control. It was reported that both adjuvant treatments were well tolerated and resulted in no postoperative complications. Quality of life was not assessed. Adverse effects Adverse effects were inconsistently reported (Tables 2 , 3 , 4 , 5 , 6 , 7 ). Most patients experienced treatment-related adverse effects associated with radiotherapy or chemotherapy. Discussion Most trials excluded patients with cancers located in the cervical esophagus, and therefore the interpretation of this review is limited to tumours in the more distal two thirds. The options for neoadjuvant or adjuvant therapy for resectable thoracic esophageal cancer are many. On reviewing the results of randomized trials and meta-analyses, the Gastrointestinal Cancer DSG did not recommend neoadjuvant or adjuvant therapy based on the following: Preoperative radiotherapy does not improve survival compared with surgery alone. Postoperative radiotherapy may, in fact, be harmful [ 27 ]. Preoperative chemoradiation does not appear to improve survival compared to surgery alone. Although the pooled analysis shows that all six studies are in the direction favouring preoperative chemoradiation at one year, the pooled estimate did not achieve statistical significance. When examining the individual trial results, only the trial by Walsh et al. [ 44 ] detected a statistically significant survival benefit, but this trial has been criticized for its methodology. The most recent trials, conducted by Bosset et al. [ 45 ] and Urba et al. [ 46 ], have five-year data available, and neither detected a statistically significant difference in survival between preoperative chemoradiation and surgery alone. Preoperative cisplatin-based chemotherapy does not appear to improve survival. Four of the seven trials [ 24 , 32 - 34 ] detected a significant survival advantage favouring preoperative cisplatin-based chemotherapy. Kok et al. [ 35 ] reported a survival advantage for chemotherapy but only reported median survival results in abstract form. The two largest trials produced conflicting results [ 36 , 37 ]. Kelsen et al. [ 36 ] detected no survival advantage, while the MRC OE02 trial [ 37 ] detected a significant survival advantage for preoperative chemotherapy at two years. Although all chemotherapy protocols were cisplatin-based, the varying dosages, the number of cycles completed, and the other agents used contributed to clinical heterogeneity. The available evidence from three randomized trials does not support the use of postoperative chemotherapy over surgery alone [ 39 - 41 ]. Two novel approaches, immunotherapy and hyperthermia, were studied by two groups in Japan [ 59 , 60 ]. Ogoshi et al. [ 59 ] detected no significant survival benefit for patients treated with PSK versus without PSK. Although Kitamura et al. [ 60 ] found a significant improvement in survival and local control favouring preoperative chemoradiotherapy with hyperthermia versus without hyperthermia, the Gastrointestinal Cancer DSG felt the results should be interpreted with caution until further confirmatory trials are conducted. Examination of the results of randomized trials, including the pooled analyses, fails to support the use of preoperative or postoperative adjuvant treatment of any type at this time for patients with resectable carcinoma of the thoracic esophagus. Overall, the evidence does not support the use of neoadjuvant or adjuvant therapy for patients with resectable cancer of the lower two-thirds of the esophagus. Surgical resection alone should remain the standard for localized thoracic esophageal cancer. Patient staging information can be found in Appendix 1 (Additional file 1 ). Future trials should continue to assess multi-modality treatments for this patient population. For clinicians seeking guidance on treatments for patients with non-resectable esophageal cancer, the role of radiotherapy alone and chemoradiation alone without surgery is addressed in another clinical practice guideline by the Gastrointestinal Cancer DSG, Combined modality radiotherapy and chemotherapy in the non-surgical management of localized carcinoma of the esophagus [ 61 ]. List of abbreviations used In order of appearance: Gy, Gray; OR, odds ratio; DSG, Disease Site Group; RR, (relative) risk ratio; PGI, Practice Guidelines Initiative; PEBC, Program in Evidence-based Care; MeSH, Medical subject heading; ASCO, American Society of Clinical Oncologists; ASTRO, American Society for Therapeutic Radiology and Oncology; RCT, randomized controlled trial; CI, confidence interval; X 2 , Chi-square; BED, biological equivalent (or effective) dose; PSK, protein-bound polysaccharide; MRC, Medical Research Council; PGCC, Practice Guidelines Coordinating Committee. Competing interests The author(s) declare that they have no competing interests. Authors' contributions RM, RW and LZ performed the initial literature search and created the initial drafts of the systematic review with input from other members of the Gastrointestinal Cancer DSG. RM, RW and BR created the final draft of this systematic review. All statistical analysis was performed by BR in consultation with RM and RW. Creation of the submitted manuscript was performed by BR and RM. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional file 1 Click here for file

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545943

Vascular consequences of passive Aβ immunization for Alzheimer's disease. Is avoidance of "malactivation" of microglia enough?

The role of inflammation in Alzheimer's disease (AD) has been controversial since its first consideration. As with most instances of neuroinflammation, the possibility must be considered that activation of glia and cytokine networks in AD arises merely as a reaction to neurodegeneration. Active, healthy neurons produce signals that suppress inflammatory events, and dying neurons activate phagocytic responses in microglia at the very least. But simultaneous with the arrival of a more complex view of microglia, evidence that inflammation plays a causal or exacerbating role in AD etiology has been boosted by genetic, physiological, and epidemiological studies. In the end, it may be that the semantics of "inflammation" and glial "activation" must be regarded as too simplistic for the advancement of our understanding in this regard. It is clear that elaboration of the entire repertoire of activated microglia – a phenomenon that may be termed "malactivation" – must be prevented for healthy brain structure and function. Nevertheless, recent studies have suggested that phagocytosis of Aβ by microglia plays an important role in clearance of amyloid plaques, a process boosted by immunization paradigms. To the extent that this clearance might produce clinical improvements (still an open question), this relationship thus obligates a more nuanced consideration of the factors that indicate and control the various activities of microglia and other components of neuroinflammation.

Introduction Alzheimer's disease (AD) is a progressive degeneration of neural structure and function that arises in the cerebral cortex. Behaviorally, affected individuals usually present with semantic difficulty, followed by a deficiency in episodic memory, spatial disorientation, sleep disturbances, depression, agitation, loss of longer memories, general difficulty with the activities of daily living, and eventually, death. Neuropathological findings include a relatively high number of extracellular deposits of the amyloid β-peptide (Aβ), argyrophillic cytoskeletal aggregates in neurons, accumulation of α-synuclein, loss of synapses, loss of cholinergic and adrenergic fibers, loss of pyramidal neurons, and cerebral amyloid angiopathy (CAA) – deposition of Aβ around blood vessels. Most of the AD correlates above have been connected in some way to inflammation. For instance, the plaques – comprised primarily of aggregated amyloid β-peptide (Aβ) – are inundated with microglia that show profiles of morphology and gene expression consistent with inflammation. Indeed, if one characterizes any activity by microglia as a sign of "neuroinflammation," it can be said that inflammatory responses have been evident in AD for at least 40 years [ 1 ]. But, it was not until the late 1980s that investigators were willing to express the hypothesis that inflammatory events were causal or otherwise contributing to the progression of the disease. Recognition of the powerful impact of a cytokine like interleukin-1 (IL-1), elevated in AD microglia, permitted such speculation [ 2 ]. Similarly, research accrued showing that primary inflammation could lead to many of the aberrations found in AD, fueling the consideration that inflammatory events were seminal [ 3 - 5 ]. Many of the individual molecules produced by activated microglia and astrocytes are conditional neurotoxins: hydrogen peroxide, glutamate and other agonists of glutamate receptors, complement components, prostanoids. (Nitric oxide from inducible nitric oxide synthase, produced abundantly in rodent glia, may be less important in human tissues.) Retrospective epidemiological studies showed protection against AD – either in age of onset or rate of progression – by nonsteroidal antiinflammatory drugs (NSAIDs); such correlations have now been born out in a prospective study [ 6 ]. Perhaps most compelling, polymorphisms in the genes for proinflammatory cytokines are indicative of risk for AD [ 7 ]. Despite these indications, there are reasons to believe that the changes observed in glia and inflammatory cytokines constitute a compensatory response in AD. Indeed, some investigators have been reluctant to apply the term "inflammation" to the constellation of events related to AD pathology. Some of the cytokines and other gene products expressed in peripheral sites of inflammation are present in the AD brain, but there is no apparent vasodilation or extravasation of neutrophils. In general, there seems to be less of the molecular and cellular behavior that is responsible for bystander tissue damage in peripheral inflammation. This journal was founded partially out of recognition that "neuroinflammation" is distinct. In essence, the concept reflects a compromise befitting the difficult line that must be maintained between effective cell-mediated immune responses and damage to the precious components of the CNS. Microglia elevate their expression of neurotrophic factors under many of the same conditions in which they show inflammation-related responses such as phagocytosis, retraction of processes, release of excitotoxins, and production of IL-1β and IL-6 and tumor necrosis factor [ 8 ]; in fact, the latter cytokines can have neurotrophic effects themselves [ 9 , 10 ]. Astrocytes deposit proteoglycans around the Aβ deposits destined to become plaques [ 11 ], perhaps sequestering this neurotoxic peptide from doing its harm. Even the apparent benefits of NSAIDs can be parsed from their presumed mechanism of inhibiting cyclooxygenase-2 [ 12 , 13 ](and references therein). Discussion Recent experiments with anti-Aβ immunization have highlighted another beneficial effect of "activated" microglia: removal of Aβ. It has long been recognized that microglia can efficiently phagocytose and at least partially degrade Aβ both in vitro and in vivo. But the persistence of amyloid plaques suggests that microglia are stymied in this regard during the development of AD or in the deposition of Aβ in mice transgenically engineered to produce large amounts of the peptide. Introduction of antibodies recognizing Aβ, either by active vaccination or by passive immunization (injection of antibodies, typically monoclonal), results in removal of some Aβ deposits and/or prevention of their formation. Although the phenomenon has been studied most rigorously in the transgenic mouse models, similar clearance of parenchymal plaques seems to have occurred in two human subjects that participated in an Aβ-vaccine trial [ 14 , 15 ]. And microglia appear to contribute; Aβ can be readily detected in microglia of immunized mice [ 16 ] and was also abundant in some microglia and related syncitia in the AD trial subjects [ 14 , 15 ]. However, the only reason we are privy to the effects of the vaccination paradigm in humans is because these two individuals died after complications of meningeal encephalitis – rampant cranial inflammation brought on by the immunization. This iatrogenic event occurred in about five percent of the human subjects vaccinated against Aβ, prompting discontinuation. One interesting finding from both autopsies is that while parenchymal Aβ deposits were substantially lower than to be expected in AD victims, both individuals had relatively high levels of vascular deposition. This CAA was accompanied by microhemorrhage in at least one of the subjects [ 15 ], consistent with the majority of advanced cases of CAA [ 17 ]. Wilcock et al . [ 18 ] have now produced evidence that the appearance of CAA after immunization may represent an actual increase in this parameter triggered by anti-Aβ antibodies. Furthermore, the investigators also found that the CAA was accompanied by an increase in hemorrhages – similar to a previous report [ 19 ] – and a vascular accumulation of CD45 + cells presumed to be microglia. The experimental paradigm was one of passive immunization of transgenic mice at nearly two years of age, old enough to have accumulated substantial Aβ deposits. Consistent with expectations, injection of anti-Aβ antibody diminished deposits in the parenchyma, even those that were mature enough to stain with Congo red. However, vascular deposition of Congo-red staining was elevated by approximately four-fold in the anti-Aβ-treated animals. Pfeiffer et al . found similar results in another transgenic line [ 19 ]. Further, Wilcock et al . now show that the regional accumulation of vascular amyloid was accompanied by an elevated index of hemorrhages and a congregation of CD45 + cells, presumed to be microglia [ 18 ]. Given that stromal microglia show increased signs of activity and contain Aβ after passive Aβ immunization [ 20 ], one interpretation is that the immunization-induced shift in amyloid from the parenchyma to the vasculature is mediated by phagocytic microglia attempting to discard the Aβ into the bloodstream. Such a phenomenon is tenuously supported by the analogous transport of pyknotic neuronal nuclei to the vasculature by microglia, observed in 3-D time-lapse videos by Dailey and coworkers [ 21 ]. In those images, microglia are occasionally seen to transfer the nuclei to another cell, conceivably a perivascular macrophage or dendritic cell. Thus, it is not clear whether the CD45 + cells observed by Wilcock and coworkers are microglia or another cell type. It is also unclear whether the accumulation of amyloid and inflammatory cells at the blood vessels represents an arrested state in Aβ clearance or simply a bottleneck in the transport, one that would eventually yield to complete removal of the peptide. However, the appearance of CAA in the human subjects that suffered from acute encephalitis suggests that the vascular accumulation is an untoward event, created or facilitated by inflammation. Another vascular irregularity caused by Aβ has been linked to inflammatory events in both transgenic mice and isolated human blood vessels [ 22 ]. The apparent contributions of inflammatory mechanisms to both Aβ clearance and vascular pathology illustrate a somewhat unique example of microglial ambivalence. While many had argued that microglial "activation" by Aβ was at least partially responsible for AD-associated degeneration, others had pointed to microglial phagocytosis as a desirable consequence of activation. For the purposes of discussion, the term " mal activation" will be applied here to microglial activation which produces neurodegeneration. One obvious question is whether there might be a mode of "activation" that permits phagocytosis while limiting malactivation. In fact, stimulation of Fc receptors – the antibody receptors that initiate a good deal of antibody-triggered phagocytosis – can inhibit cytotoxicity in macrophages [ 23 ]. Similarly, phagocytosis of apoptotic cells inhibits macrophage expression of proinflammatory cytokines like IL-1, IL-8, tumor necrosis factor, and several prostanoids through stimulation of a phosphatidylserine receptor [ 24 ]. Evidence indicates that malactivation involves the production of reactive oxygen species like superoxide and peroxide, nitric oxide, and excitotoxins (glutamate, quinolinate, and D-serine). If these criteria are germane, malactivation certainly can be suppressed by specific cytokines, such as transforming growth factor β (TGFβ) [ 25 ]. Although TGFβ has often been characterized broadly as "anti-inflammatory," it does not inhibit the phagocytic activity of microglia in a setting where another "anti-inflammatory" cytokine (IL-4) does [ 26 ]. Interestingly, TGFβ1 transgenesis promotes the same apparent shift of Aβ from parenchyma to vessel that is observed after Aβ immunization [ 27 ]. Conclusions While some have argued that CAA is of little consequence in AD [ 28 ], the elaboration of the deposition that appears to occur under conditions of "beneficial inflammation" is on par with that seen in hereditary cerebral hemorrhage with angiopathy-Dutch type and is certainly a risk factor for devastating levels of hemorrhage. If such a response reflects a broad-acting realignment of cytokine profiles contingent upon immunization, it behooves careful consideration (and extensive animal testing) for any strategy for antibody-mediated reduction of Aβ in the AD brain. List of abbreviations AD: Alzheimer's disease Aβ: amyloid β-peptide CAA: cerebral amyloid angiopathy IL-1, -6, -8: interleukin-1, -6, -8 NSAID: nonsteroidal antiinflammatory drug TGFβ: transforming growth factor β Competing interests The author(s) declare that they have no competing interests.

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515180

Effects of evening light conditions on salivary melatonin of Japanese junior high school students

Background In a previous study, when adult subjects were exposed to a level of 400 lux light for more than 30 min or a level of 300 lux light for more than 2 hours, salivary melatonin concentration during the night dropped lower than when the subjects were exposed to dim illumination. It was suggested that such light exposure in adolescents or children during the first half of subjective night in normal life might decrease the melatonin level and prevent the falling into sleep. However, there has been no actual study on the effects of light exposure in adolescents. Methods Effects of exposure to the bright light (2000 lux) from fluorescent light bulbs during a period of three hours from 19:30 to 22:30 in one evening were examined on evening salivary melatonin concentrations from 19:45 to 23:40. The control group was exposed to dim light (60 lux) during these three hours. Both the dim light control group [DLCG] and the bright light experimental group [BLEG] consisted of two female and three male adolescent participants aged 14–15 y. Results The salivary melatonin level increased rapidly from 3.00 pg/ml at 21:45 to 9.18 pg/ml at 23:40 in DLCG, whereas it remained at less than 1.3 pg/ml for the three hours in BLEG. Melatonin concentration by BLEG at 22:30 of the experimental day was lower than that at the same time on the day before the experimental day, whereas it was significantly higher in the experimental day than on the day before the experimental day in DLCG. Conclusions Bright lights of 2000 lux and even moderate lights of 200–300 lux from fluorescent light bulbs can inhibit nocturnal melatonin concentration in adolescents. Ancient Japanese lighting from a traditional Japanese hearth, oil lamp or candle (20–30 lux) could be healthier for children and adolescents because rapid and clear increase in melatonin concentration in blood seems to occur at night under such dim light, thus facilitating a smooth falling into night sleep.

Background Night sleep duration of Japanese children aged 10–18 y has become shorter by one hour during the last 30 years in Japan [ 1 ]. The so called "24-hour society", which is currently in progress in Japan, seems to change environmental conditions surrounding children. For example, mobile phones are used by more than 90 % of university students and more than 30% of junior high school students living in the urban area of Kochi city (33°N) [ 2 ]. Students can communicate with their colleagues even in the middle of the night with mobile phones. Frequent or long-time (more than 30 min) usage of the mobile phone makes university and junior high school students more evening-typed [ 2 ]. "Convenience stores" are open for 24 hours and provide several kinds of food and other goods for general civilian life. Convenience stores are now common all over Japan even in suburban areas. Illumination inside the convenience stores is very bright (2000 lux or more at the level of the eyes). Bright lighting in retail stores seems to be a merchandising technique which has been in use worldwide for at least 60 years. Unconscious use of bright light in the evening or at night inside the convenience store may promote a circadian phase delay in students exposed to the bright light during the first half of subjective night. This hypothesis is based on a "light-pulse" experiment in the laboratory [ 3 ] as follows. Adult subjects were exposed to light pulse of 4000–6000 lux for 30 min at one of several phase points of their circadian rhythm, and the direction (advance or delay) and the extent of phase shift caused by the pulse were measured at each phase point. The light pulse delayed the phase of sleep-wake cycle by the subjects when they were exposed to the light pulse in the first half (about 19:00–24:00) of subjective night. However, it advanced the phase effectively when the subjects were exposed to it within three or four hours after the minimum point of inner body temperature (about 5:00–9:00). An epidemiological study about the effect of the convenience store usage was conducted on sleep habits and diurnal rhythm by about 500 students attending junior high school aged 12–15 y in Kochi Japan [ 4 ]. This latter paper reported the following three points: (1) Students going to convenience stores after sunset were more evening-typed and showed shorter night sleep of 7.0 hours on average than those going to convenience stores during the daytime, who showed night sleep of 7.3 hours on average, (2) Students who went to convenience stores every day slept only 6.4 hours on average and the sleep hours were significantly shorter than the 7.5 hours shown by students who went to convenience stores only 0–1 time per week, (3) Students who stayed more than 30 min in convenience stores took shorter night sleep of 6.6 hours on average than those who stayed there less than 15 min. (7.3 sleep hours). Younger children attending kinder garden and students attending elementary school were more sensitive to "light conditions" in normal life than university students, according to an epidemiological study [ 5 ]. However, no experimental field studies on the effects of "light conditions" during normal life have been conducted on sleep-wake cycles of healthy children younger than 15 y. Melatonin, which is synthesized in the pineal organ and secreted to the blood, is well known as a key substance which may be effective in promoting the falling into night sleep by humans [ 6 ]. Blood melatonin concentration by adult human subjects is extremely low during daytime and increases rapidly at 22:00–23:00 up to as much as ten or twenty times daytime values. The high level is maintained till the early morning and then decreases again rapidly to the extremely low concentration typical of the daytime [ 7 , 8 ]. The increase in melatonin concentration might occur in late evening and trigger the falling into sleep also for healthy children, although there have been no studies on melatonin concentration in salivary or blood under their normal life. In adult subjects, a single administration of 5 mg of melatonin at 13:00 was reported to induce higher subjective sleepiness during the following 2 hours and also higher EEG power density in the range of relatively low frequency of 5.25–9.0 Hz rather than that of placebo [ 9 ]. When adult subjects were exposed to 400 lux lights for more than 30 min or exposed to 300 lux lights for more than 2 hours, melatonin level during the night became lower than that when they stayed under dimmer lights [ 10 , 11 ]. In the case of adolescents and children, the exposure to lights of 300 lux or more during the first half of subjective night in the normal life might decrease their melatonin level and prevent the falling into sleep. Currently, more than 80% of junior high school students of the third grade aged 14–15 y in Kochi go to private school in the evening. If they take a short stop at the convenience store to get some fast food and enjoy talking with their colleagues in front of the store before or after going to the private school in the evening, they suffer the double exposure to bright lights at the school and at the convenience store. Such bright lights are from fluorescent light bulbs and include blue or blue-green lights with 470–500 nm wave lengths which were reported to be powerful to suppress melatonin concentration [ 12 ]. Based on the epidemiological studies made in 2001–2003 on junior high school students in Kochi Prefecture (33°N), 38.8% of the students who frequented convenience stores went there after sunset, and 30.2% and 6.5% of junior high school students who used convenience stores went there and stayed there for 15–30 min and longer than 30 min, respectively. Moreover, this epidemiological study showed that 62.4% and 18% of the students who went to the evening private school studied there for 2 and 3 hours until 9 or 10 o'clock in the evening, respectively. In total, junior high school students were estimated to be exposed to bright lights of more than 2000 lux inside private school and/or convenience store for 2–3.5 hours on average in the evening. Such exposures are expected to suppress the increase in blood melatonin level as a direct effect and also delay the phase of their circadian systems driving melatonin secretion rhythm and sleep-wake cycle. In this study, two light conditions were investigated. One was bright and high color-temperature light of more than 2000 lux, which is used in the evening and at night inside the convenience stores, at preliminary and private school for entrance examination, and at rental video shops in Japan. The other condition was a dark and low color-temperature light with less than 60 lux which is usual in the evening for traditional Japanese settings (a fireplace, candle, or a naked light bulb). Methods Participants Experimental participants were ten Japanese junior high school adolescent students (4 females and 6 males) aged 14–15 y who were attending Motoyama junior high school located in the mountain area of Reihoku district (33.5°N) in Kochi Prefecture. They had enjoyed New Year holidays for 7 days before the experiment. They were instructed to keep usual diurnal rhythm (for example bed time and wake-up time) during the holidays. Before the experiment, participants were divided into the two groups of "bright light experimental group (BLEG)" and "dim light control group (DLCG)". Participants in BLEG were selected to show similar circadian typology to those in DLCG based on the scores in the morningness-eveningness (M-E) questionnaire of Torsvall and Åkerstedt [ 13 ] (mean ± SD: 15.00 ± 4.30 by BLEG and 14.80 ± 4.09 by DLCG). Bed time, wake-up time and sleep hours shown by BLEG for the four days just before the experiment were 23.0 ± 4.2 hours, 8.4 ± 1.9 hours and 9.1 ± 1.4 hours, respectively; corresponding values for DLCG were 23.8 ± 1.3 hours, 8.9 ± 1.3 hours, and 9.5 ± 1.5 hours. Each group consisted of two females and three males. All the ten participants sampled their own saliva using "Salivette" collecting tubes (SARSTEDT Aktiengesellschaft & Co., Numbrecht, Germany) at 22:30–23:00 under the 200–300 lux light from fluorescent light bulbs in their home on the day before the experimental day. Japanese civilians seem to enjoy evening time during the first half of subjective night (after sunset till bedtime) under fluorescent light bulbs based on our unpublished questionnaire study on 950 families having small children aged 0–6 yrs in Kochi. More than 85% of the 950 families enjoyed evening life under fluorescent light bulbs. We measured the illumination at the level of 1 m above floor just under a usual type of round-shaped fluorescent light bulb in a typical one-room apartment for students and it was 340 lux. Procedure On the experimental day of the 5 th January 2003, all the ten participants got together in front of Motoyama junior high school at 8:00 in the morning. A wagon car took them to the experimental place which was a Japanese style hotel located at a mountain area, Yusuhara town in Kochi Prefecture, 126 km west from Motoyama town. During the driving, illumination inside the car was 350–500 lux. The car arrived at the hotel around noon. It was snowing through the day. Behavior of all the participants was controlled during the stay in the hotel till the next morning of the experimental day. All the participants played outside exposed to the sun light with 6000–7500 lux at the eye level during 12:30–13:30 and 14:00–14:50. They were allowed to have a rest in a living room in which the floor was filled with 12 tatami mats and the illumination at the eye level was 250 lux from fluorescent light bulbs during the rest of the time till 16:30. Participants took bath one by one between 16:30–18:00 and had supper all together between 18:15 and 19:20 in the living room. At 19:25, the participants of BLEG moved to a Japanese style room with 8 tatami mats where they were exposed to the light with 2000 lux at the eye level from fluorescent light bulbs, whereas DLCG group members moved to another Japanese style room with 8 tatami mats where they were exposed to the light of 60 lux and relatively low color-temperature from a electronic light bulb. All the participants included in both groups were home-working or making a small wooden folk craft object that is typical in the Yusuhara district, under each light condition till 22:30. Room temperature was controlled at 15 ± 2°C with an oil heater in both groups. Then they came back to the former living room (12 tatami mats) and stayed there under the light of 250 lux till 23:40. Then female and male participants moved to separate rooms and went to bed just before 24:00. Salivary samples were collected in collection tubes at 21:45, 22:30, and 23:40, and these salivary samplings were preserved in a refrigerator at less than -20°C. Melatonin concentration in the samples was analyzed by a professional analyzing company (MSL Co. Ltd.) which was a specialist for several chemical and microbiological analyses. All the participants from both groups were called out to get up at 7:00 in the next morning. All the participants got up between 7:00–7:15 responding to the calling out. After taking breakfast, they left the experimental place at 9:00 back for Motoyama junior high school. Throughout the study, light exposure was measured on the eye level with a digital illumination meter. Detailed explanation of the objectives and methods of the experiment was provided before the experimental performance to the participants and their parents. The research project received full and complete agreement from all of them. Results and Discussion The results are shown in Fig. 1 . Salivary melatonin concentration rose from 3.00 ± 3.34 (mean ± SD) pg/ml at 21:45 to 9.18 ± 7.66 pg/ml at 23:30 of the experimental day in the DLCG (t-test between values at 21:45 and 23:30: t = 3.60, df = 4, p < 0.05), whereas it remained at less than 1.3 pg/ml till 23:30 in BLEG (t = 2.07, df = 4, p < 0.2). There was no significant difference in the melatonin concentration between BLEG and DLCG in the day before the experimental day (Wilcoxon test: z = -1.163, p = 0.31). At 22:30 of the experimental day, melatonin concentration by BLEG tended to be lower than that on the day before the experimental day (Wilcoxon test: z = -1.604, p = 0.109), while the concentration became significantly higher in DLCG (z = -2.023, p = 0.043). On the day before the experimental day, all the participants were under Japanese standardized light condition with 200–400 lux from a fluorescent light bulb with relatively high color-temperature. On the experimental day, the bright light of 2000 lux in BLEG suppressed the expected night increase of melatonin concentration, whereas the relatively low color- temperature light with 60 lux did not. Figure 1 Effects of light condition on salivary melatonin concentration. Values shown are means (n = 5 per group) and SEM. In Japan, bright and high color-temperature light of more than 2000 lux is available in the evening or night inside convenience stores which are open for 24 hours and private schools for the preparation to go through the entrance examination to upper schools. Also in usual life, such exposures to bright lights in the evening private school and convenience store can suppress the night increase in blood melatonin level as a direct effect and possibly delay the circadian system that drives the melatonin secretion rhythm and sleep-wake cycle. The results of this study suggest that ancient Japanese lighting in the evening and at night, which could be supplied by a traditional Japanese hearth fire or a oil lamp or candle (20–30 lux), might be healthy for adolescents and children, because the ancient lights could allow rapid and clear increase in melatonin level leading to a smooth falling into night sleep [ 14 ]. Conclusions Bright lights of 2000 lux and even moderate lights of 200–300 lux can inhibit, as a direct effect, nocturnal melatonin concentration in children. Ancient Japanese light conditions which could be supplied by a traditional Japanese hearth fire or a small oil lamp or candle might be healthy for children, because the ancient lights could allow rapid and clear increase in melatonin level in the evening, leading to a smooth falling into night sleep. Competing interests None declared.

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555588

Nanopores: maltoporin channel as a sensor for maltodextrin and lambda-phage

Background To harvest nutrition from the outside bacteria e.g. E. coli developed in the outer cell wall a number of sophisticated channels called porins. One of them, maltoporin, is a passive specific channel for the maltodextrin uptake. This channel was also named LamB as the bacterial virus phage Lambda mis-uses this channel to recognise the bacteria. The first step is a reversible binding followed after a lag phase by DNA injection. To date little is known about the binding capacity and less on the DNA injection mechanism. To elucidate the mechanism and to show the sensitivity of our method we reconstituted maltoporin in planar lipid membranes. Application of an external transmembrane electric field causes an ion current across the channel. Maltoporin channel diameter is around a few Angstroem. At this size the ion current is extremely sensitive to any modification of the channels surface. Protein conformational changes, substrate binding etc will cause fluctuations reflecting the molecular interactions with the channel wall. The recent improvement in ion current fluctuation analysis allows now studying the interaction of solutes with the channel on a single molecular level. Results We could demonstrate the asymmetry of the bacterial phage Lambda binding to its natural receptor maltoporin. Conclusion We suggest that this type of measurement can be used as a new type of biosensors.

Nature created and optimized proteins for specific tasks which makes them often interesting in material science. For example, membrane transporters could control the permeability of artificial nanometer sized container. A typical application could be to control the enzymatic activity in a liposome [ 1 ]. Another possible application is to reconstitute channels into planar lipid bilayer and use time dependent conductance as a signal [ 2 , 3 ]. Application of an external electric field drives the ions through the nano (and subnano) meter sized channel. Any larger molecule that diffuses into and temporarily sticks to the channel interior will cause typical fluctuations of the ion current which allow to conclude on its mode of translocation. Such studies were used to follow sugar translocation through maltoporin [ 4 ]. Similar types of measurements were done to investigate the translocation of antibiotics like ampicillin [ 5 ]. Subtle changes in the channel size or small conformational changes can be recorded and this technique could be developed towards an instrument to probe very soft forces. Porins are attractive candidates for applications because they are very stable. Moreover, recombinant technology permits production of porins in E. coli with high yields [ 6 ]. A third advantage is the availability of the high resolution 3-D crystal structure showing details of substrate binding sites which facilitates enormously a rational engineering of modified proteins. The outer cell wall of Gram-negative bacteria from E. coli is fairly permeable to smaller solutes below a molecular weight of about 400 Da [ 6 ]. Such substances can freely permeate under a concentration gradient through general diffusion porins in the outer cell wall. Under stress, e.g. in case of lack of nutrition, the pure diffusion process is too slow and the bacteria need to improve the efficiency of the translocation. For those cases, nature has created a series of rather specific and highly sophisticated membrane channels. The most extensively studied examples of specific porins are the maltooligosaccharide-specific channel Maltoporin of E. coli [ 4 , 7 , 8 ]. Maltoporin forms ion-conducting channels when reconstituted into lipid bilayers [ 9 , 10 ]. The 3D structure of Maltoporin revealed that the monomer of Maltoporin of E. coli consists of an 18 stranded β-barrel with short turns at the periplasmic side and large irregular loops at the outside of the cell [ 11 ]. The bacteriophage Lambda is a virus recognizing Maltoporin at the outer cell surface [ 12 ]. In absence of this membrane channel, phage Lambda does not recognize the bacteria. Or, even minor mutations allow the bacteria to defend themselves against virus attacks. The virus itself can, in turn mutate to restore binding ability. According to the high resolution X-ray structure the water filled channel is far too small to permit the translocation of the double strain DNA (about 20 Å) [ 11 ]. The infection mechanism thus must involve one of the following processes: Phage binding will cause a strong conformational change within the Maltoporin or, after binding the phage releases a DNA translocation machinery to bring its DNA across the hydrophobic membrane. To date none of these intermediate steps has been observed so far and the underlying process remains unclear. Recently, gpJ, a protein in the phage terminal was identified to be involved in the Maltoporin recognition process [ 13 ]. A typical set-up for conductance measurements is shown in figure 1 . The measurement cell consists of two chambers separated by a hole (less than 0.1 mm diameter) in a thin poly(tetrafluorethylene) film sandwiched between two half-cells made of Teflon (Goodfellow, Cambridge, UK). Prior to each measurement this hole has to be pretreated to render it lipophilic by coating it with a hexadecane/hexane (1:100 v:v) droplet. After allowing for hexane evaporation, each chamber is filled with 1.5 ml buffer (for example, 1 M KCl, unbuffered, about pH 6). Black lipid bilayers were formed according to the classical Montal-Mueller technique by spreading lipids in hexane/chloroform (9:1) across the aqueous buffer [ 14 ]. For sake of stability we used diphytanoyl-phosphocholine (DphPC, Avanti Polar Lipids). After 20 min allowing for evaporation, the buffer level is lowered below the hole level and rose again. Typically after the first or second trial a stable unilamellar membrane is formed. In order to insert single porin trimers within reasonable time, but to avoid insertion of their multiples, a careful balance between the concentration of the protein solution, detergent concentration and buffer volume has to be found. One single porin trimer has to find the membrane and to insert while all others must be inactivated, e.g. by precipitation. Maltoporin from the stock (1 mg/ml in 1% OPOE) was diluted 10 2 -10 5 times in the buffer containing 1% OPOE. From our own experience in our laboratory the insertion was optimal if smallest amounts (less than 1 μl) were injected. In a second measurement we used painted membranes as described previously [ 15 ]. Here the Teflon chamber consists of a larger hole (diameter 800 μm and larger). Membranes were formed by painting 1 μl of a 1% solution of DphPC in n-decane across the hole. This type of membrane facilitates multichannel insertion. Figure 1 Schematic representation of a typical planar bilayer set-up for ion current recording. 1.a) Two half cells made of Delrine separated by a 25 μm Teflon foil with a hole in the center. Both parts are clamped together. 1.b) Below a microscope picture of the Teflon septum containing a hole. 1.c) Schema of a lipid bilayer with a reconstituted trimeric porin. The Cl - ions are attracted to the positive electrode and K + to the negative one. Ions are permeating the channel in the MHz range which is beyond the current time resolution. 1.d) The insertion of a single channel will give raise to a jump in conductance. Any objects diffusing in the channel may reduce the permeation time of ions and may be detected either in conductance fluctuations or an averaged reduced conductance. Membrane current was measured via homemade Ag/AgCl electrodes. One electrode was used as ground and the other connected to the headstage of an Axopatch 200B amplifier (Axon Instruments, USA), allowing the application of adjustable potentials (typically, 100 mV) across the membrane. A similar set-up was used in the second measurement. We recently investigated the sugar penetration on a single molecular level [ 4 ]. We were able to reconstitute a single Maltoporin trimer into the lipid bilayer. Addition of sugar into the bulk phase resulted in a blocking of the channel in a concentration dependent manner. At low sugar concentration individual closure of the channel could be observed. Maltohexaose induces higher frequencies of closure and longer closing times than a smaller sugar like maltose. The analysis of the time-resolved conductance as a function of sugar concentration yielded the binding constant as well as the "on" and "off" rates for the sugar binding. Here we used a modified sugar through covalent binding of an ANDS (3-amino-naphtalene-2,7-disulfonic acid) molecule to the reducing end of a Maltoheptaose as schematically shown in fig. 2A (for details, see [ 16 ]). The crystal structure suggests that the maltose molecule enter the channel only with the nonreducing end from the outside (or reducing end from the periplasmic side). Subsequently this molecule can only enter from the cis-side in our setup. In fig. 2B we see that addition on the periplasmic side (trans side) inhibit the entry whereas addition to the outer side (cis side) caused blocking. A good control experiment in order to test the activity is to add unmodified sugar molecules to the previous experiment. In fig. 2C we clearly observe the ability to translocate unmodified sugars. Addition of small amounts of unmodified sugar to the trans-side caused the expected number of events. Further addition of unmodified sugar to the opposite site enhances the sugar induced blocking. These data can be used for a fundamental analysis to probe e.g. the individual energy barrier and it seems that nature has optimized this channel to have the best turnover number. On the other hand these channels can potentially serve to discriminate sucrose from maltose. Figure 2 Typical recordings of ion current through a single Maltoporin trimer in presence of modified maltohexaose (see [16] for details). ( A ) Shows the unmodified maltohexaose and on the right hand side the modified sugar molecule. We designed this molecule according the crystal structure to guarantee the low penetration ability from one side. ( B ) M6-ANDS was added to trans (left) and then to cis (right). Sugar analogue modulates ion current only to the cis-side, the side of Maltoporin addition. The average residence time is 5.0 ms. ( C ) First, M6-ANDS was injected to the trans-side and no variation in the ion current occurs. As control, maltohexaose was added to the same side (left). The natural substrate is translocated demonstrating that it enters the channel from trans with the reducing end first. Then, M6-ANDS was added additionally to the cis-side (right) generating long current interruptions superimposed to maltohexaose blockade events seen in the figure of the left side. The dashed lines corresponding to zero current. Membrane bathing solution was 1 M KCl, 10 mM Tris, 1 mM CaCl 2 , pH 7.4, the applied voltage was + 150 mV. In a second series of experiments we were interested to probe for Lambda phage binding. In principle this should be possible despite the enormous size (about 100 nm size in comparison to 4 nm sized channels). However in a preliminary step we have produced larger quantities of the phage endterminal protein gpJ fused to Maltose Binding Protein (MBP). We reconstituted a larger number of maltoporin in solvent containing membranes and titrated small quantities of the fusion construct MBP-gpJ. We know from the experiments described above that most of the channels are oriented the same direction during the reconstitution. In fig. 3 we show a first result that titration of gpJ to the opposite side of protein addition had no effect. In contrast, addition of gpJ to the side of porin addition caused rapid blocking of the channel. This observation suggest that the porin inserts with the short turns first and that the protein part exposed to the extracellular side is naturally accessible to Lambda phages. These first results are promising and we currently work on improving the resolution. Here we have to note that this observation is in clear contrast by a report on phage lambda binding in a multichannel preparation [ 17 ]. The origin of this discrepancy might be simultaneous multiple insertion. Our observation here is in agreement with other reports showing the same orientation [ 4 , 5 , 18 ]. However, reason why porins inserts in artificial membranes differently than in natural ones remains unclear. One may speculate that the strong asymmetry of natural membranes or unknown chaperons will facilitate the entry with the long loops first. Figure 3 Here we show the ability to recognize bacterial phage Lambda by blocking the ion conductance through the natural receptor Maltoporin. We first reconstituted about 300 Maltoporin channel in a solvent containing planar lipid bilayer. This leads to a stable conductance after about 30 min with no further protein insertion. Titration of 7 and 42 nM of the fusion protein MBP-gpJ from the bacterial virus Lambda to the compartment corresponding the intracellular side of the channel showed no effect. However, titration to the opposite side corresponding to the extracellular side caused a significant reduction of the ion conductance. Membrane bathing solution was unbuffered 1 M KCl giving a pH of about 6. The applied voltage was + 20 mV. Sensing with membrane channel is a new way in screening for solute molecules and several promising examples are already shown [ 2 , 3 , 16 , 19 , 20 ]. The actual bottleneck is the complexity in membrane channel assembly. However, the current development in automatized patch-clamping will open a wide range of possibilities [ 21 , 22 ]. We plan to reduce the volume on each side of the membrane and the size of the lipid patch. We currently work with pore diameters of about 1 μm with less background capacitance and thus a better time resolution and to simplify the channel assembly.

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539324

Ancient DNA Provides New Insights into the Evolutionary History of New Zealand's Extinct Giant Eagle

Prior to human settlement 700 years ago New Zealand had no terrestrial mammals—apart from three species of bats—instead, approximately 250 avian species dominated the ecosystem. At the top of the food chain was the extinct Haast's eagle, Harpagornis moorei. H. moorei (10–15 kg; 2–3 m wingspan) was 30%–40% heavier than the largest extant eagle (the harpy eagle, Harpia harpyja ), and hunted moa up to 15 times its weight. In a dramatic example of morphological plasticity and rapid size increase, we show that the H. moorei was very closely related to one of the world's smallest extant eagles, which is one-tenth its mass. This spectacular evolutionary change illustrates the potential speed of size alteration within lineages of vertebrates, especially in island ecosystems.

Introduction Since the discovery of the first fossil in 1872 the sheer size of Harpagornis moorei ( Figure 1 A) has fuelled speculation about its evolutionary history, ecology, and extinction, which like many other New Zealand bird species, is linked ultimately to human arrival in the 13th century. Even though morphology indicates that H. moorei was approaching the upper limit of body mass for powered flight [ 1 ], it was still an efficient predator. Consistent with other members of the family Accipitridae, it killed by piercing and crushing its prey with its large talons ( Figure 1 B). Rock art, Maori oral history, and bone artefacts prove early Polynesians co-existed with the eagle; however, there is no evidence that humans were targets for this aerial predator. An exploratory skeletal analysis, using representative genera within the Accipitridae (but lacking Australasian representatives of the genera Hieraaetus ), placed H. moorei as a sister species to Aquila audax, the Australian wedge-tailed eagle (circa 4.5 kg; 2 m wingspan) [ 2 ]. However, shifts in body size are common in island ecosystems [ 3 ] and may distort skeletal characters used in phylogenetic reconstructions. Figure 1 Images and Phylogenetic Analysis of New Zealand's Extinct Giant Eagle, H. moorei (A) An artist's impression of H. moorei attacking the extinct New Zealand moa. Evidence of eagle strikes are preserved on skeletons of moa weighing up to 200 kg. These skeletons show the eagle struck and gripped the moa's pelvic area, and then killed with a single strike by the other foot to the head or neck. (Artwork: John Megahan.) (B) Comparison of the huge claws of H. moorei with those of its close relative the Hieraaetus morphnoides , the “little” eagle. The massive claws of H. moorei could pierce and crush bone up to 6 mm thick under 50 mm of skin and flesh. (C) Maximum-likelihood tree based on cyt b data (circa 1 kb), depicting phylogenetic relationships within the “booted eagle” group. Extraction numbers or GenBank accession numbers are shown along with taxa name. Harpagornis moorei (red) groups exclusively with the small Hieraaetus eagles, and genetic distances suggest a recent common ancestor about 0.7–1.8 million years ago (early to mid Pleistocene). The tree uses an HKY + Γ 4 + I likelihood model enforcing a molecular clock; maximum-likelihood bootstrap consensus values greater than 60% are shown. Results/Discussion To further investigate the evolutionary history of this raptor we performed an ancient DNA study on the fossil remains of two extinct H. moorei specimens together with 16 extant eagles ( Table S1 ). Short, overlapping segments of the mitochondrial cytochrome b (cyt b ) and ND2 genes were PCR-amplified, sequenced and analysed with data available on GenBank to build a maximum-likelihood tree ( Figures 1 C and S1). Appropriate ancient DNA controls were undertaken [ 4 ], including multiple, overlapping amplifications and independent replication (see Materials and Methods ). Surprisingly, the resulting phylogeny firmly placed the H. moorei in a “clade” with a group of small eagles of the genus Hieraaetus, H. morphnoides, the little eagle, and H. pennatus, the booted eagle (both circa 1kg; 1.2m wingspan), and not with A. audax . Moreover, the genetic distance separating H. moorei and the most recent common ancestor of the related Hieraaetus eagles is relatively small (1.25%). The lack of fossil calibration points for accipitrids precludes direct estimates of divergence times; however, when a molecular rate of 0.7%–1.7% per million years, as previously estimated for avian cyt b [ 5 ], is applied to the tree, a divergence estimate of approximately 0.7–1.8 million years ago is obtained. Although such indirect molecular dating estimates are error-prone, we believe that this range is the best available approximation of the “true” date when the lineages diverged (early to mid Pleistocene); however, additional molecular data and control region sequences may further clarify the topology and timing of the splits. The arrival of H. moorei into New Zealand's South Island appears to have been a recent event, probably involving a small bird-eating Asian/Australian Hieraaetus eagle that thereafter increased rapidly in size. An analysis of mean body mass using independent contrasts clearly demonstrated that the size of H. moorei is an anomaly in the context of the eagle phylogeny shown in Figure 1 C (see Materials and Methods ). Factors that may have influenced such rapid morphological evolution include the size of potential prey, competition with smaller harriers ( Circus spp.), and a complete lack of terrestrial predatory mammals in New Zealand. The speed and magnitude of the increase in body mass seems unique within vertebrate lineages, and is more significant because it occurred in a species still capable of flight. The avian faunas of Islands such as Hawaii (Hawaiian goose), the Galápagos (Galápagos finches), Mauritius (dodo), and New Zealand (moa) are often cited as examples of rapid evolution of flightlessness, shifts in body size, and other specialisations in birds [ 1 , 6 , 7 ]. Isolated island faunas derived from vagile colonists often have vacant niches for large herbivores and predators. In New Zealand the large herbivore niches were occupied for millions of years by the moa. Clearly, in the absence of mammalian predators, selection for large body size in an avian predator with a relatively generalised body form was not limited by competition. Other large predatory birds have evolved on islands in the absence of mammalian competitors, notably giant eagles and owls on Cuba [ 8 ], but the magnitude of the size increase in H. moorei over its sister taxa is unrivalled. H. moorei therefore represents an extreme example of how freedom from competition on island ecosystems can rapidly influence morphological adaptation and speciation. The phylogeny in Figure 1 C also reveals considerable problems with the current classification of the “booted eagle” group (eagles with feathered tarsi), especially the genera Aquila, Hieraaetus, and Spizaetus, which are clearly paraphyletic. Assignment of species within these genera has traditionally been problematic (see [ 9 ]), so this observation was not unexpected. However, it is apparent the name for the extinct New Zealand eagle should be amended to Hieraaetus moorei (Haast, 1872) (see Materials and Methods ). The inclusion of H. moorei with the small Hieraaetus eagles implies the body size of the New Zealand species has changed by almost an order of magnitude since these lineages diverged. This spectacular evolutionary change illustrates the potential speed of size alteration within lineages of vertebrates and represents yet another example of the remarkable evolutionary processes that occur within island ecosystems. Materials and Methods DNA extraction and amplification DNA was extracted and amplified from two H. moorei bones ( Table S1 ) as previously described [ 10 ] using appropriate ancient DNA techniques. “Modern” toepad tissue (museum specimens) was extracted using Qiagen (Valencia, California, United States) DNeasy tissue extraction kits. Multiple negative extraction and amplification controls were included, to detect contamination. All PCR reactions were conducted as described in [ 10 ] using Platinum Taq HiFi (Invitrogen, Carlsbad, California, United States) together with the cyt b and ND2 primers listed in Table S2 . Thermal cycling conditions were typically 40 cycles of 95 °C/55–60 °C/68 °C (30–45 s each). Sequences were determined using ABI Big Dye (v.3.1) on an ABI 3100 or 3730 (Applied Biosystems, Foster City, California, United States), according to manufacturer's instructions. Modern samples, and ancient samples subsequent to PCR amplification, were analysed in the Zoology Department, Oxford University. A single Harpagornis bone was sent to an ancient DNA facility at University College London (I. Barnes) for independent replication, where identical sequences were obtained for two cyt b amplifications. Similar cyt b and ND2 tree topologies, in addition to multiple overlapping sequences, make it unlikely that we are detecting a nuclear pseudogene. Phylogenetic methods Maximum-likelihood trees for cyt b and ND2 were selected using a heuristic search as implemented in PAUP*4.0b10 [ 11 ] under the HKY + Γ 4 + I substitution model. The assumption of a molecular clock was tested using a likelihood ratio test in which the χ 2 test statistic was two times the log likelihood difference between clock and non-clock models. For the cyt b tree the assumption of rate constancy was not rejected. Node support was evaluated for 1,000 bootstrap replicates. Bayesian Markov Chain Monte Carlo phylogenies were also generated on the cyt b dataset using BEAST [ 12 ] and MrBayes [ 13 ] under similar substitution models—the topology of these trees was consistent with Figure 1 C and generated posterior support values higher than the bootstrap values. Using the maximum-likelihood tree in Figure 1 C, an independent-contrasts analysis was employed to determine whether correlations existed between phylogenetic position and body mass. Mean live weight estimates were obtained from the literature, and the average mass of H. moorei was estimated from femur length [ 2 ]. A test to measure the index of phylogenetic dependence was conducted; this measures the degree to which traits vary across taxa (in a phylogeny) in accordance with predictions of a neutral Brownian model according to [ 14 ]. The results (not shown) clearly demonstrate that the mass of H. moorei is clearly an “outlier” in the context of the phylogeny presented here. Hieraaetus systematics The type species for the genus Hieraaetus is H. pennatus (Gmelin, 1788); therefore, the taxa grouping strongly with H. pennatus must remain in that genus. The close genetic relationship of H. morphnoides with H. pennatus firmly embeds this species in Hieraaetus . However, the New Guinea subspecies presently recognised as H. morphnoides weiskei is genetically, geographically, and morphologically distinct and warrants species status, which necessitates the new combination Hieraaetus weiskei (Reichenow, 1900). Harpagornis moorei is included in the clade with H. pennatus and H. morphnoides, and hence its generic assignment must reflect that. The name for the extinct Harpagornis moorei of New Zealand should therefore be amended to Hieraaetus moorei (Haast, 1872). Supporting Information Figure S1 Maximum-likelihood tree generated using 434 bp of ND2 data from a subset of eagle taxa. The tree topology seen here is identical to that seen in Figure 1 C and is an independent verification of the tree topology (89 KB PDF). Click here for additional data file. Table S1 List of Eagle Taxa Used in This Study along with Museum Accession Numbers and Sample Provenance (64 KB PDF). Click here for additional data file. Table S2 List of Avian cyt b and ND2 Primers Used in This Study (54 KB PDF). Click here for additional data file. Accession Numbers Sequences have been deposited in GenBank ( http://www.ncbi.nlm.nih.gov/Genbank/index.html ) under accession numbers AY754044 to AY754056.

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524371

Few amino acid positions in rpoB are associated with most of the rifampin resistance in Mycobacterium tuberculosis

Background Mutations in rpoB , the gene encoding the β subunit of DNA-dependent RNA polymerase, are associated with rifampin resistance in Mycobacterium tuberculosis . Several studies have been conducted where minimum inhibitory concentration (MIC, which is defined as the minimum concentration of the antibiotic in a given culture medium below which bacterial growth is not inhibited) of rifampin has been measured and partial DNA sequences have been determined for rpoB in different isolates of M. tuberculosis . However, no model has been constructed to predict rifampin resistance based on sequence information alone. Such a model might provide the basis for quantifying rifampin resistance status based exclusively on DNA sequence data and thus eliminate the requirements for time consuming culturing and antibiotic testing of clinical isolates. Results Sequence data for amino acid positions 511–533 of rpoB and associated MIC of rifampin for different isolates of M. tuberculosis were taken from studies examining rifampin resistance in clinical samples from New York City and throughout Japan. We used tree-based statistical methods and random forests to generate models of the relationships between rpoB amino acid sequence and rifampin resistance. The proportion of variance explained by a relatively simple tree-based cross-validated regression model involving two amino acid positions (526 and 531) is 0.679. The first partition in the data, based on position 531, results in groups that differ one hundredfold in mean MIC (1.596 μg/ml and 159.676 μg/ml ). The subsequent partition based on position 526, the most variable in this region, results in a > 354-fold difference in MIC. When considered as a classification problem (susceptible or resistant), a cross-validated tree-based model correctly classified most (0.884) of the observations and was very similar to the regression model. Random forest analysis of the MIC data as a continuous variable, a regression problem, produced a model that explained 0.861 of the variance. The random forest analysis of the MIC data as discrete classes produced a model that correctly classified 0.942 of the observations with sensitivity of 0.958 and specificity of 0.885. Conclusions Highly accurate regression and classification models of rifampin resistance can be made based on this short sequence region. Models may be better with improved (and consistent) measurements of MIC and more sequence data.

Background Rifampin, one of the principal drugs used in tuberculosis treatment, is a semi-synthetic antibiotic that inhibits transcription by preventing RNA synthesis. Isolates of Mycobacterium tuberculosis resistant to rifampin occur at low to moderate frequencies in many regions of the world [ 1 ]. Mutations in rpoB , the gene encoding the β subunit of DNA-dependent RNA polymerase, are associated with rifampin resistance. In the laboratory, drug resistance is quantified in terms of minimum inhibitory concentration (MIC), which is defined as the minimum concentration of the antibiotic in a given culture medium below which bacterial growth is not inhibited. Several studies have been conducted where MIC of rifampin has been measured and partial DNA sequences have been determined for rpoB in different isolates of M. tuberculosis [ 2 - 6 ]. However, no model has been constructed to predict rifampin resistance based on sequence information alone. Such a model might provide the basis for quantifying rifampin resistance status based exclusively on DNA sequence data and thus eliminate the requirements for time consuming culturing and antibiotic testing of clinical isolates. Tree-based statistical methods (see Methods) have generated very accurate models relating amino acid sequence of short (8-mer) peptides to their binding by major histocompatibility complex (MHC) class I molecules with higher accuracy than artificial neural networks [ 7 ]. Both tree-based models and aggregation of such models through random forests (see Methods) have proven to be quite successful in other problems involving sequence data as covariates such as HIV-1 replication capacity [ 8 ] and cytidine to uridine RNA editing in plant mitochondria [ 9 ]. The success of tree-based statistical models and random forests in these problems involving covariates derived from sequence data motivated our application of these models to the problem of rifampin resistance in M. tuberculosis . The response variable is a set of continuously distributed values for MIC, which makes the problem one of regression. These data are used to answer the following questions: What proportion of the variance in MIC is attributable to sequence differences in positions 511–533 of the β subunit of RNA polymerase of M. tuberculosis ? What particular positions, and what distribution of amino acids at those positions, are associated with most of the variance in MIC? Alternatively, the response variable could be cast in discrete terms: resistant or susceptible. This is possible by assuming a threshold value for MIC above which an isolate is considered resistant to rifampin. Among the specific questions we can answer with such a model are the following: What particular positions, and what distribution of amino acids at those positions, allow for distinguishing rifampin-susceptible and rifampin-resistant isolates of M. tuberculosis ? What is the misclassification error rate associated with susceptibility prediction for these data? We address these questions and evaluate the ability to predict MIC from protein sequence data (inferred from DNA sequence data) using tree-based regression and classification methods. We find that these methods generate highly accurate models of rifampin resistance. Results The data set used in the study consists of 173 observations with 60 distinct genotype-phenotype combinations (Table 1 ). The most frequent combination has 47 occurrences, and there are 40 unique (singleton) combinations. MIC for rifampin varies from 0.0625 μg/ml to > 512 μg/ml . The 173 sequences are distributed among 24 genotypes, 11 of which occur uniquely in the data set. The plurality genotype is represented by 69 samples; 98 samples differ from the plurality by one amino acid; and the remaining 6 samples differ from the plurality by two amino acids. Some genotypes defined by the partial sequence of rpoB are associated with several different phenotypes (MIC values). Also, some genotypic states are associated with large effects, while some have little or no effect on MIC phenotype. Finally, some changes in MIC are not associated with changes in the sequence region examined. These genotype data are short (69 bp) partial sequences of a single gene, and thus they may not contain all phenotypically relevant genetic information. Indeed, there is evidence that amino acid changes outside of the examined region are associated with changes in MIC for rifampin [ 3 ]. Additionally, the sample size is small (also typical of most genotype-phenotype datasets), which will decrease power. Nonetheless these data are typical of studies surveying the genetic variation associated with antibiotic resistance and of genotype-phenotype data in general. Thus they make an appropriate subject of investigation. Regression tree analysis The regression tree for the relationship of rpoB amino acid sequence and MIC has two splits defining three terminal nodes (Fig. 1 ). At each node in the tree, the MIC prediction given ( μg/ml ) is the mean of all isolates at that node. The first split of the topmost node (root node) consists of the entire sample and is based on the amino acid at position 531, with those sequences having serine (S) going to the left child node, and those having leucine (L) or tryptophan (W) going to the right child node. The best split for each node is that which gives the largest decrease in the error. Here error is measured as the deviance, which for a continuous variable is a constant multiple of the residual sums-of-squares. Reported values were determined using 10-fold cross-validation. Moving down the tree the error decreases, as the sum of the deviance for each pair of child nodes is less than the deviance of the parent node. Given the hierarchical nature of trees and the criterion used to choose splits, the first split, that based on position 531, explains the highest proportion of the overall phenotypic variance. This bisection of the data results in groups that differ one hundredfold in mean MIC (1.596 μg/ml and 159.676 μg/ml ). The subsequent partition based on position 526, the most variable in this region, results in a > 354-fold difference in MIC. The proportion of the variance in MIC explained across all splits involving the two amino acid positions (526 and 531) is 0.679. All proportions of variances explained by the model as reported here are those estimated through cross-validation and are not based on re-substitution, and thus represent appropriately conservative estimates. Classification tree analysis From a clinical perspective it may be most relevant to consider the level of drug resistance as a two-state categorical variable (susceptible or resistant) rather than as a continuously distributed variable. In clinical practice, if an isolate of M. tuberculosis is determined to be rifampin resistant then rifampin is replaced with another antibiotic. Although blood serum concentration of rifampin reaches levels of 6 – 7 μg/ml about 1.5 – 2 hours after ingestion [ 10 ], a clinically relevant MIC value for dichotomizing the MIC values would be lower than this peak. We conservatively define MIC values ≤ 1 μg/ml as susceptible and values > 1 μg/ml as resistant, a definition consistent with conventional standards [ 11 ]. With this dichotomization we can explore the use of tree-based statistical classification to predict rifampin resistance in a way that is more relevant to clinical practice. The predictor variables are again the unordered categorical designations of amino acids at polymorphic positions. The classification tree for these data (Fig. 2 ) has two splits based on two of the 11 variable amino acid positions. At each node in the tree, the prediction of rifampin susceptibility status (susceptible or resistant) is given for all isolates at that node. The first split is based on position 531; those isolates with serine (S) are predicted to be susceptible, and those with leucine (L) or tryptophan (W) are predicted to be resistant. The class counts for the full data set are given at each node. For example, the root node (top most node in the figure) contains all 173 cases of which 103 are resistant are resistant to rifampin, and the remaining 70 isolates are susceptible to rifampin. The proportion of correctly classified observations across all splits as determined by re-substitution of the observations on the cross-validation pruned subtree is 0.884. Comparing this tree to the pruned regression subtree (Fig. 1 ) reveals that the two split definitions in each tree are identical. Both the regression and classification tree models are significant ( P < 0.0001) based on permutation tests. Random forest analyses The random forest analysis, which aggregates results over many tree models, each constructed on subsamples of the data, produced markedly better models as compared to the single tree-based models. The random forest analysis of the MIC data as a continuous variable, a regression problem, produced a model that explained 0.861 of the variance. The random forest analysis of the MIC data as discrete classes (susceptible and resistant), a classification problem, produced a model that correctly classified 0.942 of the observations with corresponding sensitivity of 0.958 and specificity of 0.885. Although both the regression and classification random forest results are markedly better than the single tree-based models, they do lack the ease of interpretation of a tree model. However, variable importance can be assessed in random forests by measuring the increase in group purity based individual models containing the variable. As might be expected, the results for both regression and classification are similar and identify the same amino acid positions as being most important in determining response to rifampin as did the single tree models: primarily 531 and 526, and much less so for 513 and 516 (Figure 3 ). Discussion Analysis of genotype and phenotype data poses several significant challenges. Data characteristics such as mixture of variable types, high dimensionality, interactions between variables, and preponderance of unordered categorical variables render many candidate analytical methods inappropriate or ineffective. Tree-based statistical models adeptly deal with these all these challenges and do so in a way that produces readily interpretable results. Through the analyses described above, we have learned several things that were not previously apparent. We have distinguished phenotypically relevant from phenotypically irrelevant changes in genotype by establishing the relative importance of the polymorphic sequence positions, and amino acids at those positions, as they affect susceptibility to rifampin. For example, although they are polymorphic, changes at positions 511, 512, 515, 521 and 529 did not significantly affect MIC for rifampin. The hierarchical importance of changes, and their contextual/conditional relationships, are depicted in the resulting tree diagrams in a readily interpretable manner. Inherent in the tree structure is the fact that earlier splits explain more variation in phenotype then subsequent splits. For example, the first split, at position 531, explains more variation then does the split based on position 526. The models can be used to predict MIC for rifampin where genotype is known, as well as provide the basis for hypothesis testing involving future empirical work. Furthermore models can be refined to yield improved predictions by incorporating additional data as they become available. Improved models may be possible with additional data: full length sequence of rpoB may include sequence features that are responsible for some variation in MIC values for rifampin, and sequence data from additional strains might lead to even more general models. As demonstrated above, the relationship of genotype to phenotype can be quantified using tree-based statistical models and aggregations thereof. Our approach has been to use types of models in the analysis of genotype-phenotype relationships because they offer distinct advantages compared to other methods and allow for rigorous and ready interpretation of results. Tree-based and random forest analyses are readily applicable to other forms of genotypic information including data that take the general form of visualized fragments (bands on gels) such as microsatellites, restriction fragment length polymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs), and similar data. Tree-based and random forest analyses can also be applied directly to DNA sequence data including single nucleotide polymorphisms (SNPs). In general, tree-based statistical and random forest models are applicable to all cases where the goal is to examine the relationship between genotype and phenotype. Conclusions Relatively simple models provided accurate predictions of rifampin resistance in M. tuberculosis . These models demonstrated that only a few variable positions in the β subunit of DNA-dependent RNA polymerase were responsible for most of the variation in rifampin resistance. Such models might provide the basis for quantifying rifampin resistance status based exclusively on DNA sequence data and thus eliminate the requirements for time consuming culturing and antibiotic testing of clinical isolates. More generally, the results of this study demonstrate the usefulness of tree-based statistical models and random forests in genetic analysis. Methods Data sources Sequence data for amino acid positions 511–533 of rpoB and associated MIC of rifampin for different isolates of M. tuberculosis were taken from studies examining rifampin resistance in clinical samples from New York City and throughout Japan [ 2 - 4 ]. Minimum inhibitory concentration (MIC) is defined as the minimum concentration of the antibiotic in a given culture medium below which bacterial growth is not inhibited. Variables The predictor variables are unordered categorical designations of amino acid at polymorphic positions, and the response variable are continuous values for MIC represented by their log 2 transforms. Values given in the original sources as < x were set to log 2 ( x - 0.5), and those values given as > x were set to log 2 ( x + 0.5). The MIC values are converted back to μg/ml in figures to be consistent with Table 1 and to facilitate interpretation. Tree-based statistical analyses Analysis of the relationships between rpoB amino acid sequence and rifampin susceptibility was done through the use of tree-based statistical models [ 12 ], also known as classification and regression trees (CART) [ 13 ]. Analyses were done with rpart ( r ecursive part itioning) [ 14 ] using the rpart library [ 15 ] for the open source statistical package R [ 16 ]. Tree-based models operate by recursively partitioning a data set in two (binary split) based on the value of a single predictor variable to best achieve homogeneous collections of a nominal or ordinal response variable (classification) or to best separate low and high values of a continuous response variable (regression). The split definition can be considered as a question, which has the following general form: Is the observation x i ∈ A ? Here A is a region of the variable space. Thus answering the question for all observations produces two groups of observations; those for which the answer is yes (those in region A ) and those for which the answer is no ( x i ∉ A , those in the complement of A ). The specific criteria for choosing among the possible partitions (questions) is based on the change in deviance, which for regression problems is equivalent to least squares. Subsequent binary partitioning continues until stopping criteria (variously defined) are met. The result is a classification or regression tree: a hierarchical series of data bifurcations, which depicts the partition definitions and describes the resulting data subsets defined by each partition. For unordered categorical covariates, such as amino acid designation, the search through possible splits is exhaustive. For each variable amino acid position there are 2 n -1 -1 possible partitions, where n is the number of different amino acids observed. For example, in the case of amino acid position 526 of rpoB analyzed here there are 9 observed amino acids resulting in 255 possible partitions to be evaluated. The preferred way to construct an appropriately sized tree is to first build a large tree and subsequently prune it [ 12 , 13 ]. Pruning is the process of removing branches from a tree to produce a subtree. To objectively choose the appropriate size for a pruned tree, it is useful to employ the concept of cost-complexity [ 13 ]. Embodied within the cost-complexity measure is a reward for tree (model) fit and a penalty for tree size (number of parameters). A tree can be pruned by using the cost-complexity measure to identify subtrees to be eliminated. A more formal definition and discussion of cost-complexity is given elsewhere [ 13 ]. Performance of tree-based models can be assessed in a number of ways depending on the goals of the analysis. One way is to evaluate the fit of the data used to generate the model, which is known as the re-substitution error. The use of re-substitution error may be justified when the principal goal of the analysis is to explain the observations in hand. However, the re-substitution error provides an underestimate of the error if the goal is to produce a model for future prediction. Another scheme to assess performance is to partition observations into a subset for model building, the training set, and a subset to evaluate the model, the test set. To remove biases this general scheme can be expanded in the form of cross-validation. Typically 10-fold cross-validation is used, where the data are randomly divided into 10 equal or near equal portions. Nine of these portions are used to generate the model and the remaining portion, the test set, is used to evaluate the model. This step is repeated until all test sets have been used in model evaluation. We assessed the significance of our tree-based statistical models through permutation where the predictor variables are randomized with respect to the response variable [ 17 ]. The frequency of observing a result value equal to or better than the observed value in 1 × 10 4 permutations is the estimate of the probability associated with the observed result. Random forest analyses In a series of recent papers [ 18 - 21 ], Breiman has demonstrated that consequential gains in classification or prediction accuracy can be achieved by using an ensemble of trees, where each tree in the ensemble is grown in accordance with the realization of a random vector. Final predictions are obtained by aggregating (voting) over the ensemble, typically using equal weights. Bagging [ 18 ] represents an early example whereby each tree is constructed from a bootstrap [ 22 ] sample drawn with replacement from the training data. The simple mechanism whereby bagging reduces prediction error for unstable predictors, such as trees, is well understood in terms of variance reduction resulting from averaging [ 18 , 23 ]. Such variance gains can be enhanced by reducing the correlation between the quantities being averaged. It is this principle that motivates random forests. Random forests seek to effect such correlation reduction by a further injection of randomness. Instead of determining the optimal split of a given node of a (constituent) tree by evaluating all allowable splits on all covariates, as is done with single tree methods or bagging, a subset of the covariates drawn at random is employed. Breiman [ 20 , 21 ] argues that random forests (a) enjoy exceptional prediction accuracy, (b) that this accuracy is attained for a wide range of settings of the single tuning parameter employed, and (c) that over-fitting does not arise due to the independent generation of ensemble members. Here, our random forests comprised 1 × 10 4 individual trees constructed by sub-sampling eight predictor variables (regression) or two predictor variables (classification) at each node. Variable importance was assessed by measuring the increase in group purity when partitioning data based on a variable. We used the R package randomForest [ 24 ]. Authors' contributions MPC conceived of the study, and participated in its coordination. Both authors participated in study design, carried out the statistical analyses, wrote and approved the final manuscript.

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Harnessing genomics to improve health in the Eastern Mediterranean Region – an executive course in genomics policy

Background While innovations in medicine, science and technology have resulted in improved health and quality of life for many people, the benefits of modern medicine continue to elude millions of people in many parts of the world. To assess the potential of genomics to address health needs in EMR, the World Health Organization's Eastern Mediterranean Regional Office and the University of Toronto Joint Centre for Bioethics jointly organized a Genomics and Public Health Policy Executive Course, held September 20 th –23 rd , 2003, in Muscat, Oman. The 4-day course was sponsored by WHO-EMRO with additional support from the Canadian Program in Genomics and Global Health. The overall objective of the course was to collectively explore how to best harness genomics to improve health in the region. This article presents the course findings and recommendations for genomics policy in EMR. Methods The course brought together senior representatives from academia, biotechnology companies, regulatory bodies, media, voluntary, and legal organizations to engage in discussion. Topics covered included scientific advances in genomics, followed by innovations in business models, public sector perspectives, ethics, legal issues and national innovation systems. Results A set of recommendations, summarized below, was formulated for the Regional Office, the Member States and for individuals. • Advocacy for genomics and biotechnology for political leadership; • Networking between member states to share information, expertise, training, and regional cooperation in biotechnology; coordination of national surveys for assessment of health biotechnology innovation systems, science capacity, government policies, legislation and regulations, intellectual property policies, private sector activity; • Creation in each member country of an effective National Body on genomics, biotechnology and health to: - formulate national biotechnology strategies - raise biotechnology awareness - encourage teaching and training of biotechnology - devise integration of biotechnology within national health systems. Conclusion The recommendations provide the basis for a road map for EMR to take steps to harness biotechnology for better and more equitable health. As a result of these recommendations, health ministers from the region, at the 50th Regional Committee Meeting held in October 2003, have urged Member States to establish national bodies of biotechnology to formulate a strategic vision for developing biotechnology in the service of the region's health. These efforts promise to raise the profile of genomics in EMR and increase regional cooperation in this exciting new field.

Background In a recent study, University of Toronto researchers identified the "Top 10 Biotechnologies to Improve Health in Developing Countries" [ 1 ]. The study underscores the importance of harnessing new technologies to improve global health and development, a belief that is gaining widespread acceptance. For instance, the overall goal of the United Nations Millennium Project's Science and Technology Task Force is to address how science and technology can be leveraged to help countries achieve the Millennium Development Goals (MDGs) [ 2 ]. Its mission is guided by the understanding that most of the MDGs cannot be reached without a strong contribution from science and technology. The potential contribution of genomics and biotechnology to these goals has also been demonstrated [ 3 ]. However, these technologies are most beneficial to countries that have the scientific capacity to absorb and use them. The aim of the Inter-Academy Council on Science and Technology Capacity (IAC) was to develop a global strategy for promoting capacities in science and technology and the first report of the Council was recently presented to UN Secretary General Kofi Annan [ 4 ]. There is a wide range of scientific capacity and health system development across the Eastern Mediterranean Region (EMR) [ 5 ], which consists of 21 countries. Some countries in the region have taken the initiative in biotechnology by establishing regulations and encouraging private sector involvement. In other countries of the region, there is a serious lack of scientific capacity not only to conduct research and development in biotechnology but even to absorb the benefits of biotechnology and apply them to help meet the health and socio-economic needs of the population [ 6 ]. According to UNDP's Arab Human Development Report 2003, research and development in the Arab world represents less than 0.2% of Gross National Product (GNP). Fewer than one in 20 Arab university students were pursuing scientific disciplines, compared, for instance, to one in five in South Korea [ 7 ]. There is a risk that, as the genomics revolution gathers momentum, the imminent genomics divide between developed and developing countries will increase unless urgent action is taken to reverse the trend [ 8 ]. In order to assess the potential of genomics to address health needs in the Region, the World Health Organization-Eastern Mediterranean Regional Office (WHO-EMRO) and the University of Toronto Joint Centre for Bioethics jointly organized a Genomics and Public Health Policy Executive Course, held September 20 th –23 rd , 2003, in Muscat, Oman. This 4-day course/workshop was sponsored by WHO-EMRO with additional support from the Canadian Program in Genomics and Global Health. The overall objective of the Genomics Policy Executive Course was to familiarize participants with the potential of genomics and related biotechnologies to address health needs and to collectively address the question of how best to harness genomics to improve health in the region (table 1 ). Table 1 Objectives of the Genomics Policy Executive Course To familiarize participants with the current status and implications of health genomics/biotechnology, and to provide information relevant to public policy on health genomics/biotechnology To provide frameworks for analyzing and debating the policy issues and related ethical questions in health genomics/biotechnology, and to help understand, anticipate and possibly influence the legal and regulatory frameworks under which health biotechnology industries will operate, both nationally and internationally To begin developing an opinion-leaders network across different sectors (industry, academic, government, NGOs) by sharing perspectives and building relationship To formulate recommendations for future policy and strategic directions at the regional, national and individual levels. Methods We based the program and designed the sessions and lecture topics of this course on prior courses held in Nairobi, Kenya in March 2002; in Toronto, Canada in May 2002; and in Kumarakom, India in January 2003. The sessions and presenters of the course are shown in table 2 . The course participants and facilitators for each session were identified through a combination of recommendations from field experts in the region and from WHO's network of experts. People identified to participate in the course included scientists from academic institutions and industry, industry executives, regulatory officials, representatives from the legal sector, and media. The participants were carefully chosen in an attempt to represent a wide range of interests relevant to the emerging area of genomics and to have geographical, discipline and gender balance. They included scientists from academic institutions and industry, industry executives, legal and regulatory officials, WHO representatives and the media. In total there were 51 participants, from 13 countries of EMR. We drew as many of the faculty as possible from the region. Table 2 Program Saturday, 20 September 2003 08:00 – 08:30 Registration 08:30 – 10:30 Session I: Opening Address H.E. Dr Ali Bin Moosa, Minister of Health, Oman Dr H. A. Gezairy, Regional Director, EMRO Introduction and Course Overview Team from University of Toronto Session Chair: Dr Ali Jaffer Suleiman, Ministry of Health, Oman 10:30 – 11:00 Coffee break 11:00 – 12:30 Genomics: Scientific Developments Professor Riad Bayoumi, Sultan Qaboos University 12:30 – 14:00 Lunch Break 14:00 – 15:30 Session III: WHO Report on Genomics and World Health Professor Alexander Capron, Director, Department of Ethics, Trade and Human Rights, WHO/HQ 15:30 – 16:00 Coffee break 15:45 – 16:30 Session IV: Top 10 Biotechnologies for Improving Health in Developing Countries Professor Abdallah S. Daar, University of Toronto Sunday, 21 September 2003 08:30 – 09:00 Golden Nuggets (previous day's summary) Dr Peter A. Singer, University of Toronto 09:00 – 10:30 Islamic Perspective on Stem Cells, Cloning, Genetic Engineering, ..etc Dr Mohammed Al Bar, King Fahd Medical Research Centre, Saudi Arabia 10:30 – 11:00 Coffee break 01:00 – 12:30 Intellectual Property Rights Professor Richard Gold, McGill University, Centre for Intellectual Property Policy 12:30: – 14:00 Lunch Break 14:00 – 15:30 Business Models Mr Khalil Ahmed, Managing Director, Shantha Biotech, India Dr Peter A. Singer, University of Toronto 15:30 – 16:00 Coffee break 16:00 – 17:30 Group Work Monday, 22 September 2003 08:30 – 09:00 Golden Nuggets (previous day's summary) Dr Peter A. Singer, University of Toronto 09:00 – 10:30 Innovation Systems Dr Peter A. Singer, University of Toronto 10:30 – 11:00 Coffee break 11:00 – 12:30 Regulatory Systems and Related Issues Dr D.C. Jayasuriya, Director, UNAIDS, Pakistan Dr Anwar Nasim, Chairman, National Council of Biotechnology, Pakistan 12:30 – 13:00 TRIPS and Pharmaceutical Issues in Public Health. Dr. Abdel Aziz Saleh Special Advisor to the Regional Director for Medicine, WHO/EMRO 13:00 – 14:00 Lunch Break 14:30 – 15:30 Public Engagement Mr Ehsan Masood, Scidev.net, United Kingdom 15:30 – 16:00 Coffee Break 16:00 – 17:30 Group Work Tuesday, 23 September 2003 08:30 – 09:00 Golden Nuggets (previous day's summary) Dr Peter A. Singer, University of Toronto 09:00 – 10:00 Opinion Leaders Network Dr Peter A. Singer, University of Toronto, Dr Tara Acharya, University of Toronto 10:00 – 10:30 Break 10:30 – 12:30 Group Work 12:30 – 14:00 Lunch break 14:00 – 15:30 Group Presentations and Discussion 15:30 – 16:00 Coffee Break 16:00 – 17:30 Recommendations, Concluding Remarks and Closure of the Workshop Dr Peter Singer, University of Toronto The sessions dealt with a wide range of relevant topics, starting with recent scientific advances in genomics and stem cell research, followed by discussions on business models in genomics and biotechnology, intellectual property rights and regulatory frameworks, public engagement and an internet-based opinion leaders' network. The presentations were designed to be interactive and foster active discussion from, and among, the participants, and each presentation was followed by a moderated discussion period. Early in the course, the attendees were placed into one of five study groups – these groups were carefully designed to capitalize on the diverse backgrounds of the participants. Each group was assigned the task to on the deliberate the key question "How best to harness genomics and biotechnology to improve the health of the people in the Eastern Mediterranean Region?" The groups met frequently to discuss the presentations, and each participant was also provided a course reader with additional literature on the lecture topics (table 3). The overall task of these study groups was to draw upon the course material and their own experiences and propose a set of recommendations for genomics and biotechnology policy in the region. On the last day of the meeting each group presented these recommendations. Table 3 Course Readings Bhutta ZA (2002) Ethics in international health research: a perspective from the developing world. Bull World Health Organ .;80(2):114–20. Review. Bloom BR & Trach D (2001) Genetics and Developing Countries. BMJ ;322:1006–7. Capron AM (2001) Stem Cells: Ethics, Law and Politics. Biotech Law Report;5:678–699. Collins FS, Green ED, Guttmacher AE, Guyer MS; US National Human Genome Research Institute. (2003) A vision for the future of genomics research. Nature.24;422(6934):835–47. Daar AS, Thorsteinsdóttir H, Martin DK, Smith AC, Nast S, Singer PA. (2002) Top ten biotechnologies for improving health in developing countries. Nat Genet .;32(2):229–32. Gold ER (2003) SARS genome patent: symptom or disease? Lancet .;361(9374):2002–3. Juma C & Konde V (2002). The New Bioeconomy: Industrial and Environmental Biotechnology in Developing Countries. Geneva, Switzerland: United Nations, July 2002. Lundvall B, Johnson B, Andersen EA, Dalum B (2002) National systems of production, innovation and competence building. Research Policy ;31:213–231 Nasim A (2000) Ethical Issues of the Human Genome Project: An Islamic Perspective in Bioethics in Asia. Eubios Ethics Institute Eds: N Fujiki and DRJ Macer p. 209–214 Pang T & Weatherall D (2002) Genomics and Global Health. BMJ ;324:p.1051–52 Singer PA, Daar AS (2001) Harnessing genomics and biotechnology to improve global health equity. Science ;294(5540):87–9. Sulston J (2003) Beyond release: the equitable use of genomic information. Lancet .;362(9381):400–2. Thorsteinsdóttir H, Daar AS, Smith RD, Singer PA (2003) Genomics – a global public good? Lancet .;361(9361):891–2. Time to Unite Islam and Science. Nature . 2003 Mar 13;422(6928):99. In order to assess the level of interest in the formation of an email-based opinion leaders' network to continue discussion among the participants following the course, a brief survey was conducted on the participants' internet access and their expectations of the network. Results Dr Peter A. Singer, Director of the University of Toronto Joint Centre for Bioethics, described the overall aim of the course "How to best harness genomics to improve health in the region", as well as the 4-day program. Dr Ali Jaffer Suleiman of the Ministry of Health in Oman acted as chairperson of the meeting. Professor Riad Bayoumi of Sultan Qaboos University highlighted new scientific developments that have resulted from the genomics revolution, such as proteomics; mapping of single nucleotide polymorphisms (SNPs) to understand human genetic variation and its relationship with disease; gene expression chips to monitor differential gene expression and identify drug targets; and bioinformatics as a new field that combines biology, mathematics, statistics and computer programming to mine large-scale biological data. Professor Alexander Capron, Director, Department of Ethics, Trade and Human Rights at WHO, described the 2002 report of the World Health Organization "Genomics and World Health" [ 9 ]. He called attention to the recommendations from the report – these include improving technical cooperation between WHO and its member states (e.g. assessing the health impacts of genomics research to support informed priority setting; capacity building for genomics research and biotechnology in developing countries; development of ethical review structures and bioethics capacity). Professor Abdallah Daar described a recent study, conducted by the University of Toronto Joint Centre for Bioethics' Canadian Program on Genomics and Global Health, to identify the ten most promising biotechnologies for improving health in developing countries in the next five to ten years. These technologies offer guidance to those who can influence the direction of R&D in developing countries and challenge common assumptions about the relevance of biotechnology for these countries, as shown by the mapping of the Top 10 Biotechnologies onto the UN Millennium Development Goals [ 10 ]. However, to foster biotechnology in developing countries it is essential to build capacity (among researchers, politicians, legislators, entrepreneurs, etc). Professor Al Bar of King Fahad University, Saudi Arabia, illustrated Islamic perspectives on genetic testing, cloning, recombinant DNA technology and other genomics-related technologies. He showed that while Islam and science have always been aligned through history, today's religious leaders in the region must take the initiative to develop and formulate recommendations for genomics. Dr. Peter Singer described the concept of innovation systems and presented some observations of other countries' innovation systems and their successes. One definition of a national system of innovation (NSI) is the "network of institutions in the public and private sector whose activities initiate, import, modify and diffuse new technologies" [ 11 - 13 ]. The application of NSI to developing countries is a fairly recent concept. The Canadian Program on Genomics and Global Health is currently conducting studies of the NSI of 7 developing countries: Brazil, China, Cuba, Egypt, India, South Africa and South Korea [ 14 ]. Despite the identification of factors that foster innovation systems, there is no one model that guarantees success – each country follows its own unique path. Mr. Khalil Ahmed, Managing Director of Shantha Biotechnics, described the successes of this biotechnology company based in Hyderabad, India. Shantha was established in 1993, in part with seed money from the Government of Oman. Today, it is the first Indian company to receive WHO certification for a recombinant hepatitis-B vaccine Shanvac-B™, paving the way for UNICEF to buy 8.5 million doses for distribution globally. Hepatitis B vaccine is currently priced internationally as high as $8–10 per dose, while Shantha is selling it at $2 per dose [ 15 , 16 ]. Professor Richard Gold of McGill University's Centre for Intellectual Property Policy described the basics of intellectual property rights, patents and copyright issues, as well as policy options for developing countries in the international context. He outlined the characteristics of international agreements, which offer considerable flexibility to countries on how to apply patent laws to genomics and biotechnology. Developing countries have a number of options, such as compulsory licensing and research exemptions, to deal with property rights. Dr Jayasuriya, Director of UNAIDS in Pakistan described ways in which legal systems can facilitate best use of biotechnology. It is essential that legal reform keep up with the rapidly evolving science of genomics. Health law can facilitate best use of biotechnology – by providing for fast-track approval of biotechnology products; reducing import duties on health interventions; and allowing multiple channels of procurement and distribution to improve access and optimize prices. Dr Anwar Nasim, Chairman of Pakistan's National Council of Biotechnology, described the role of regulations both to promote useful biotechnologies and limit their risks for human health and the environment. A national-level regulatory body could provide guidelines for the use and release of biotechnology products, conduct biosafety reviews and risk assessments and formulate feedback mechanisms to improve the system through experience. The National Commission of Biotechnology of Pakistan, established in November 2001, focuses on biotechnology regulatory issues in health, agriculture, environment and industry. Similar commissions could be set up in other countries of the region and their interaction could further regional cooperation in biotechnology. Mr. Ehsan Masood of Scidev.net pointed out that active public engagement based upon knowledge can stimulate action to improve public health. Public engagement is far greater in today's world than it has ever been in the past. This is because of several factors including; a) the growing implications of research on public health, b) the increased awareness in civil societies to invest in health care and research, and to influence policy change and action, c) development and access of information technologies. On the last day of the course the five participant groups, who had been assigned the task to on the deliberate the key question "How best to harness genomics and biotechnology to improve the health of the people in the Eastern Mediterranean Region?" , were invited to present their findings. The main points of these group presentations are summarized in the recommendations that emerged from the course. Discussion The session discussions and group presentations underscored the urgent need for action to create the enabling environments (at regional, national and individual levels) for research and development in genomics and biotechnology. The issue of awareness of biotechnology among political leaders to garner support at the highest level was raised early on in the course, and was reinforced throughout the course discussions. The participants felt strongly that political commitment is crucial to the advancement of biotechnology in the region. Political leadership is a critical factor in raising the profile of science in developing countries. A prominent example in the Eastern-Mediterranean Region is that of the Sultan Bin Mohammed Al-Qassimi, Shaikh of the Arab emirate of Sharjah. He has made a dedicated effort to change the face of science in the Gulf [ 17 ]. For example, in an attempt to attract Arab scientists working abroad to return to work in the Gulf, and more specifically to Sharjah, he has built two universities, six museums and established a science foundation in just a decade. He is also actively engaged in creating an environment of regional cooperation. The course attendees recommended that WHO-EMRO should take the responsibility to engage political leaders in the region's national governments. The participants shared experiences of the importance of government support for biotechnology – in Egypt there appears to be relatively strong support for biotechnology by the government, with biotechnology activity in academic and other research institutions as well as in the private sector. In Iran, considerable advances have been made in private and academic research centres, leading to a number of biotechnology products that are soon to reach the market, as well as well-respected scientific journals such as the Iranian Journal of Biotechnology [ 18 ]. Other countries such as Pakistan, Tunisia, Lebanon, and Morocco are making steady gains in biotechnology, some in conjunction with their powerful public health sectors and others as a result of their strong scientific bases. It will also be crucial to involve, inform and engage religious leaders in the region in order to promote genomics and biotechnology for improving public health. In doing so, it is worthwhile to note that Islam and science have always been aligned through history and it is well-established that Islam has historically contributed tremendous achievements to the advancement of science. [ 19 ] A recent article argues that the Muslim world has neglected to pay attention to contemporary ethical issues in science and technology [ 20 ]. The author calls for the establishment of an independent Islamic bioethics panel to advise Islamic governments and communities. Other measures, such as training Muslim bioethicists, incorporating biomedical issues into school curricula and educating the community about such issues are also recommended. The active participation and involvement of organizations like COMSTECH was recommended to give support and guidance to WHO-EMRO's efforts. COMSTECH was established by the Islamic Summit in 1981 and includes among its objectives the building of indigenous capabilities in the fields of science and technology, promotion and continuing cooperation and coordination in scientific and technological areas of is member states and creation of effective institutional structure for planning research, development and monitoring of scientific and technological activities. COMSTECH has already launched networks across the region for exchange of information, and has valuable experience in the promotion of cooperation and coordination amongst the member states in science and technology activities in high technology areas [ 21 ]. A major discussion point at the workshop was the effective assessment of existing capacity in the region. Several participants shared disappointment over the relative lack of participation of the region in global science and technology advances, citing, for instance, poor representation of the region at international scientific conferences. Many felt the need to evaluate and assess the level of scientific activity and capacity in individual countries in the region, in order to identify, among other things, strengths and weaknesses, entry points for countries, opportunities for regional collaboration, and areas for improvement. The workshop attendees felt it important to carry out this type of survey of each country's innovation system as a prerequisite to revising and revamping national and regional genomics policy. The factors identified by the participants as important for assessment in this survey – scientific capacity within public and private sector, private enterprise, religious and political leadership and intellectual property rights – can be considered to be part of the National System of Innovation (NSI). This proposed survey of NSI in the region can help to identify factors for successful growth of biotechnology sectors. Similarly, the recently completed study by the Canadian Program on Genomics and Global Health of the health biotechnology innovation systems of Brazil, China, Cuba, Egypt, India, South Africa and South Korea may also help to identify some of these factors [ 22 ]. Workshop participants from Egypt, Iran, Lebanon, Pakistan, and Tunisia contributed to the discussion of NSI with descriptions of components of their countries' biotechnology innovation systems. In Egypt, there is increased recognition of the importance of intellectual property rights in fostering innovation. The Minister of Health has taken a keen interest in improving linkages between sectors to enhance inter-sectoral communication. The president himself was instrumental in the establishment of the Mubarak City for Scientific Research and Technology (MCSRT), which is currently engaging in collaborative projects with the United States and with China. Private sector development is of high priority, as indicated by government support of companies such as Vacsera, which is now making recombinant insulin in Egypt [ 23 ]. One successful story from Egypt is the development by scientists at the Agricultural Genetic Engineering Research Institute in Giza of a powerful bio-pesticide based on a new strain of Bacillus thuringiensis [ 24 ]. In Iran, the trade embargo has led to a shortage of funds, but has in some ways helped to strengthen the innovation system through the need for self-reliance. Iran and Cuba have reached an agreement for cooperation and transfer of technology to produce hepatitis-B vaccine, interferon-α, streptokinase, and erythropoietin. Iran has developed several products based on recombinant DNA technology, and the country is also actively building research collaboration networks. Pakistan has had some successes in agricultural biotechnology and made some advances in bioethics. Although there are powerful institutes in place, the country needs to continue to strengthen facilities, resources and human capital. There is a need to bring products to the public. Lebanon has a well-developed healthcare industry, and has developed expertise in genetic testing, bioethics as well as intellectual property rights. Tunisia has strong research infrastructure and has made advances in genetic counseling, cytogenetics, and diagnosis of genetic diseases, along with regulation and legislation. However the pharmaceutical industry is not able to optimize the potential of public sector research, for which regional cooperation would be valuable. The participants identified strengthening the linkages between academia and the private sector as key to strengthening capacity in genomics. One example of a concerted effort to improve academic-private sector links is Jeddah BioCity, a research facility with close ties to the King Faisal Specialist Hospital and Research Center. Founded by Sultan Bahabri, head of King Faisal Specialist Hospital and Research Center in Jeddah and other Saudi scientists, this private venture plans the construction of state of the art biotechnology laboratories and companies and is intended to make Saudi Arabia a world leader in biotechnology. As highlighted by the 2004 report of the UN Commission on Private Sector and Development report [ 25 ], the process of commercialization for development involves the dissemination and facilitation of knowledge flows between public and private sectors of both developed and developing markets. The report recommends action in both the public and private spheres, and emphasizes the linkages between these spheres, recognizing the importance of cooperation and partnerships to achieve goals. With stronger public-private linkages, the private sector in developing countries will be able to help provide new genomics-based technologies at affordable prices. Given that over the last few decades, market forces have driven the R&D agenda of pharmaceutical companies based in the North to de-emphasize the health concerns of developing countries, it is becoming increasingly important to develop indigenous capacity in private enterprise. The successes of biotechnology firms in developing countries such as India and Egypt were seen as a source of inspiration by the course attendees. The attendees observed that the key to effective participation in the genomics revolution is building scientific capacity, and expressed serious concern over the region's limited capacity to absorb and utilize genomics and biotechnology. Critical to capacity-building is access not just to technology but, more importantly, to scientific knowledge. One point-of-entry that was greeted with enthusiasm by the participants was bioinformatics, which is typically less-resource intensive compared with other genomics-related sciences, such as sequencing and proteomics. According to the report on the "Top 10 technologies to improve health in developing countries" countries of EMR can take advantage of genomic data and apply the power of bioinformatics to local health problems without having to invest heavily in the technologies used to produce them, A dedicated Genomics and Health Research Fund for the region may help to break down financial barriers to encourage scientific research and development in the region. The participants expressed enthusiasm for setting up National Biotechnology Commissions to address genomics policies at the national level and to contribute to the development of the above-mentioned national biotechnology strategy. A National Commission on Biotechnology (NCB) has been set up in Pakistan. The overall goal would be to help devise regulations to facilitate innovation in biotechnology. The NCB could help coordinate national biotechnology activities and policy, with activities including evaluation of biotechnology capacity (see above) to priority setting and public engagement. Accordingly, the NCB should have broad cross-sectoral representation in order to minimize the negative effects of inter-institutional rivalry. One of the main goals of the NCB (or equivalent national agency), following the above-mentioned national biotechnology survey, will be to help develop and adopt a national biotechnology strategy, perhaps as part of a long-term science and technology policy. Other important objectives are discussed below. At the outset of the conference, participants voiced their concern about the low level of awareness (which goes beyond just public awareness) of biotechnology and genomics in the region, and that this lack of awareness may be the biggest barrier to advances in genomics. Public awareness and engagement can help change the pace of research, leading to increased opportunities for greater societal involvement for improved health care. Media can be used as a catalyst to raise community awareness for social beneficence, equity and justice in health care. Recently, in May 2004, science journalists from across the region met in Cairo to discuss the hurdles they face in science reporting. The hurdles identified at this meeting included bureaucracy and poor access to scientific research taking place in the region [ 26 ]. The meeting concluded with the creation of a provisional network of Arab science journalists that will aim to provide its members with training, skills and contacts, as well as promote the coverage of scientific issues from a development perspective. Similarly, public health specialists and scientists should also be encouraged to engage actively disseminate evidence based and correct information in disease prevention and control through media. While it was agreed that science should be permitted to march forward, the participants emphasized that ethics, regulations and laws must keep up with the science [ 27 ]. For example, an important focus area of the NCB is that of intellectual property rights. The participants agreed that developing countries must build capacity and knowledge to choose the best policy options to benefit from the international patent regime. Compulsory licensing under specific circumstances may be a good option for developing countries and national laws should permit compulsory licensing [ 28 ]. The forces of globalization must balance forces of protectionism, especially by the developed world, and both national and regional regimes should respect international patent law to take advantage of globalization. If developing countries are involved in international research collaborations, they should ensure that they obtain patents. In view of this, it is essential for developing countries to build capacity and training in patent law and learn how to formulate effective patents, and the NCB could play a key role in this effort. Regional cooperation in science was given a boost this year, with the establishment of a network of science academies of the Organisation of Islamic Conference [ 29 ] at a meeting organized by the Third World Academy of Sciences. This formal network aims to provide the partner states with mutual support and supports discussion of the scientific aspects of common problems. It could help to build a unified approach to capacity building in science and technology within member states. Health advances in developing countries have lagged behind those in the developed world. The rapid advance in genomics research in developed countries compared with the relatively slow progress of genomics R&D in developing countries threatens to create a North-South genomics divide in the coming years, which may enhance existing health inequities. With the appropriate emphasis on its health needs, incentives for public-private R&D partnerships, and a sound set of regulatory policies, the Eastern Mediterranean Region may well reap the benefits of genomics and biotechnology. The overall goal of the Genome Policy Executive Course, a WHO-University of Toronto initiative, was to help provide the impetus for cross-sectoral dialogue on genomics and health policy in the region. The internet-based opinion leaders' network is expected to foster dialogue to help achieve the objectives outlined by the participants of the course. The participants and the organizers of the course felt strongly that the recommendations formulated at the course must be shepherded by individuals. People felt that progress could be achieved if individuals make a significant and concerted effort to ensure that these recommendations are fulfilled. One way to spur action and maintain the momentum generated by this course is by coordinating the participants into a network within which they can continue to interact and share information and experiences. This internet-based network will be moderated in order to streamline discussions. A number of short-term projects are envisioned that could be coordinated by various expert members of the network. The results of the survey (table 4 ) administered to the participants during the course suggested that 80% of them had reliable access to internet and would be willing to spend 1–2 hours a week taking part in the discussion. The main objectives of the network, as identified by the participants in the survey, would be dissemination of information, exchange of ideas, maintaining inter-connectivity, consensus building through wide participation, and influencing policy and media. The network is now established and is being used by the participants to exchange information. Table 4 Opinion leaders' network survey results in brief Goals of network • Dissemination of information • Exchange of ideas • Maintaining inter-connectivity • Consensus building through wide participation • Influencing policy and media Access • 41 (79%) no access issues – reliable connectivity from work • 3 needed some assistance with email access (internet connection at work; compensation for access; help to post responses) Obstacles to participation • 47 (92%) identified lack of time due to professional responsibilities • 98% of those with connectivity willing to dedicate 1 hour a week to the network • 3 people identified lack of connectivity as a barrier Conclusions The meeting concluded with a set of recommendations for the EMRO and Member States (table 5 ) [ 30 ]. The recommendations were developed through consensus among the participants. The process of consensus development involved the following steps: (i) the recommendations were drafted based on the group presentations, (ii) any recommendations which the participants did not support were deleted (iii) recommendations that were missing but deemed to be important were added (iv) the final list was scrutinized to sharpen language and consolidate points where possible. Table 5 Recommendations Recommendations for the Eastern Mediterranean Regional Office The workshop recommends that the Regional Director EMRO may be requested to address the governments at the highest level for actively considering the proposals of this workshop and for giving priority attention to genomics for health and health biotechnology. The political leadership may be provided effective advocacy material, with special reference to its link with poverty alleviation, public health objectives, and need for transfer (and internalization) of technology. EMRO and Organization of the Islamic Conference Standing Committee for Science and Technology (COMSTECH), and possibly other groups should provide coordination and networking among national biotechnology bodies (see below) and coordinators to exchange information, expertise, training, and Regional cooperation in production and utilization of health biotechnology. EMRO, in collaboration with member states and their national biotechnology bodies, should coordinate a national survey/inventory/situation analysis/needs assessment of health biotechnology innovation systems, including scientific and management capacity, government policies, legislation and regulations, intellectual property policies, private sector activity, and strengths/weaknesses, opportunities and threats. EMRO, in collaboration with COMSTECH and member states, should develop a proposal/feasibility study for a Regional Genomics and Health Research Fund emphasizing both peer-reviewed research and capacity strengthening. Recommendations for Member States Each member state should create an effective National Commission on Genomics, Biotechnology and Health, if this function has not otherwise been established, including a coordinator who will serve as the focal point for this activity. The membership should be multisectoral and include youth, women, and civil society. The focus should include ethical issues. Based on evidence from the national survey described above, governments of member states should develop and adopt, at the highest level, a national biotechnology strategy. The National Commission on Biotechnology should develop programs of public awareness and engagement. Important "publics" here include media and religious leaders as well as the public at large. The discussion should include ethical issues. The National Commission on Biotechnology should encourage academic institutions including schools and universities, to include health biotechnology topics within their curricula and create specialized programs and degrees where appropriate. There should be particular emphasis on ICT and bioinformatics. The National Commission on Biotechnology, in collaboration with the relevant ministries, should develop a plan to integrate genetic and genomics products (including diagnostics, vaccines, therapies, and other genomic priorities), within the health system and public health programs. The emphasis should be on accessibility and equity to improve the health of the poor. Recommendations for Individuals There is a need for strong personal commitment to strengthen the initiative on genomics and biotechnology to improve health and well-being of people in the EMRO Region. Workshop participants, as well as other concerned individuals, should are therefore encouraged to actively engage in the implementation of these recommendations. The participants felt the need for EMRO to: a) Request regional governments and policy makers at highest level to give priority to genomics for health and health biotechnology b) Develop linkages with Organization of Islamic Conference Standing Committee for Science and Technology (COMSTECH) and other international partners to build Regional networking and cooperation for developing and utilizing health biotechnology. c) In collaboration with Member States undertake a national survey/situation analysis/needs assessment of health biotechnology innovation systems including resource capacities, government policies (legislation, regulations intellectual property policies and private sector activity. d) In collaboration with COMSTECH initiate a research grant for applied (health) genomics and biotechnology The participants agreed that each member state would benefit from creating effective National Commissions on Biotechnology (NCB) to develop national biotechnology strategies. NCB should; a) develop national priorities, programmes and guidelines aimed at raising public education and awareness and b) collaborate with civil sectors to develop plans to integrate genetic and genomic products (including diagnostics, vaccines, therapies and other genomic priorities within the health systems and public health programmes and c) build capacities for utilization and access of health biotechnology to the needy and e) ensure ethical safeguards against unwanted harm and exploitation and improve equity to improve health of the poor. The participants also felt that there was a need for strong personal commitment at individual level by experts and key actors to engage actively in the implementation of the recommendations to strengthen the initiative on genomics and biotechnology to improve the health and well-being of people in EMR. One concrete outcome of the workshop that may contribute to strengthening capacity in the region is the joint agreement between WHO/EMRO and the University of Toronto Joint Centre for Bioethics to provide scholarships to the Master of Health Sciences Program at the JCB. Furthermore, there seems to be growing need to establish a Regional Health Biotechnology Network for EMR, and WHO-EMRO is now planning to hold a Regional meeting to discuss this in Iran in July 2004. WHO-EMRO has initiated follow-up of these recommendations. Ministers of Health from the region, at the 50 th Regional Committee Meeting held in October 2003, urged Member States to establish national bodies for genomics and biotechnology to formulate strategic vision for creating public awareness and for developing biotechnology for equitable health care in the region. Recently, a paper on harnessing genomics and biotechnology for public health was presented at the EM 28th Regional Consultative Committee Meeting (RCC) held in Cairo in April of this year. The recommendations in the paper were derived from those developed at the workshop. The paper will be presented at this year's Regional Committee Meeting to be held in October 2004. The methods and recommendations outlined in this paper demonstrate progress in bringing genomics and biotechnology to the forefront of science policy in developing countries. Our procedure has now led to the formation of three regional networks – the two previous ones being the African Genome Policy Forum, which encompasses participants from 10 African nations, the Indian Genome Policy Forum. We have also now held a course for Latin America and the Caribbean in association with PAHO and the UN University in Venezuela May 23–26 2004, and a similar network is being created of participants of that course. The next one will be held in the Southeast Asian region, most likely based in Hong Kong. These regional genome policy networks will provide models to establish a Global Genome Policy Forum. Competing interests The author(s) declare that they have no competing interests. Authors' contributions TA drafted the manuscript; PAS and ASD conceived of the course, and all authors participated in its design and coordination. All authors revised the manuscript for critical content and approved the final draft.

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535536

The role of d-dimer as first marker of thrombophilia in women affected by sterility: implications in pathophysiology and diagnosis of thrombophilia induced sterility

Background D-dimer is considered a marker of hypercoagulable state and of endogenous fibrinolysis, so increased d-dimer is detectable in patients affected by thrombosis. Yet, several studies showed that also infertility, in particular secondary infertility due to recurrent fetal losses, has been often related to thrombotic events, in particular in women carrying thrombotic risk factors such as inherited thrombophilia (MTHFR C677T , PTHR A20210G , Factor V Leiden polimorphisms and/or inhAfter this screening we selected 39erited protein C, protein S, AT III deficiency) or acquired thrombophilia (primary antiphospholipid syndrome, acquired protein C, protein S, AT III deficiency, drugs induced thrombophilia). However, because its high predictive negative value in case of suspected thrombosis, increased d-dimer has been often associated to subclinical thrombophilia. The aim of this study is to investigate the role of d-dimer as first marker of thrombophilia in women affected by unexplained infertility and subsequently to search the cause of increased d-dimer, such as inherited and/or acquired thrombophilia. Patients and Methods We selected 79 patients with unexplained primary or secondary infertility. We excluded 40 patients affected by hydrosalpinx, uterine fibroids, uterine malformations, endocrinological and immunological diseases, luteal insufficiency, cytogenetical alterations. All remaining 39 patients were tested for d-dimer and divided in two groups: the patients of group A (25 patients) showed increased plasma d-dimer, in group B were included 14 patients with normal plasma level of d-dimer. After this step all 39 patients were screened for MTHFR C677T , PTHR A20210G , Factor V Leiden polimorphisms, protein C, protein S, AT III, anticardiolipin IgM and IgG, lupus anticoagulant. In the control group were included 15 age matched women without sterility problems referred to our outpatient's section of vascular medicine for suspected deep venous thrombosis. Statistical analysis was based on χ 2 test, differences were considered to be significant if p < 0.05. Results D-dimer was increased in 25/39 and 20/25 showed inherited/acquired thrombophilia while patients with normal d-dimer showed inherited/acquired thrombophilia in 7/14 (p: < 0.05, s). Discussion D-dimer is a well known marker of hypercoagulable state, in particular its high predictive negative value in case of suspected thrombosis has been recognised by several reports. Yet, increased d-dimer has been identified also for subclinical thrombophilia besides for vascular thrombosis. Our data, in fact, for the first time suggest an interesting role of d-dimer to identify women affected by unexplained primary or secondary infertility and thrombophilia. So, probably there is a role for d-dimer in these subjects for its predictive positive value. Of course, further data on large based population are needed to confirm our results, because these findings may speed up a diagnostic screening in these patients also for a good cost/effectiveness of this test.

Introduction D-dimer is considered a marker of hypercoagulable state besides of endogenous fibrinolysis, so increased d-dimer is detectable in patients affected by arterial and/or venous thrombosis [ 1 ]. Yet, several studies showed increased d-dimer also in patients affected by subclinical thrombophilia without ongoing thrombosis [ 2 ]. Moreover, also in other clinical conditions, such as chronic inflammation as infectious disease (also as marker of disseminated intravascular coagulation if sepsis is associated) as cancer as necrosis as eldership and pregnancy we may observe an increase of plasma d-dimer [ 3 - 8 ]. So, for this reason d-dimer test is usually used in clinical management for its high predictive negative value in suspected thrombosis, particularly in deep vein thrombosis (DVT) [ 9 - 11 ]. However, several studies showed that frequently women affected by sterility, in particular secondary sterility for recurrent foetal losses, may be affected by an underlying inherited and/or acquired thrombophilia [ 12 - 20 ]. Besides, common thrombotic risk factors which include also a bad lifestyle (e.g. obesity, non regard to Mediterranean diet, sedentary life), a lot of molecular thrombotic risk factors such as inherited or acquired clotting inhibitor deficiency (i.e. protein C, protein S, antithrombin III), inherited thrombophilia (factor V Leiden, prothrombin A20210G mutation), primary or secondary hyperhomcysteinemia, primary or secondary antiphospholipid syndrome and increased plasma factor VIII levels have been identified [ 21 ]. Furthermore, these molecular alterations may be also associated in some subjects so inducing gene-gene interactions and/or gene-enviromental interactions [ 22 - 24 ]. So, because the high incidence of clotting abnormalities in these patients, according to the data of Brenner et al. [ 24 , 25 ], we investigated the role of d-dimer as first marker of thrombophilia in women affected by sterility in order to identify causes of increased d-dimer and probably of the induced sterility. Patients and Methods We selected 79 women affected by primary or secondary sterility (due to three or more fetal losses) referred to our sterility center. We excluded 40 patients affected by hydrosalpinx, uterine fibroids, uterine malformations, luteal insufficiency, anovulation, cytogenetical alterations, infectious diseases, endocrinological diseases (ie diabetes, subpituitarism), and by immunological diseases (inherited and/or acquired immunodeficiency, rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, vasculitis). After this screening we selected 39 patients (12 affected by primary sterility and 27 by secondary sterility due to recurrent foetal loss). These 39 patients were tested for d-dimer. D-dimer was measured by several methods [ 26 ]; d-dimer were tested randomly in various periods of the ovarian menstrual cycle in 31 patients, in one patient during menstrual bleeding and in seven patients during hormonal therapy in order to obtain controlled ovarian hyperstimulation (COH). Following d-dimer examination patients were divided in two different groups: group A including 25 patients with increased d-dimer levels and group B including 14 patients with normal d-dimer levels. As control group we selected 15 age-matched women, without sterility problem in their anamnesis, referred to our outpatient's section of vascular medicine for suspected deep venous thrombosis. Subsequently d-dimer evaluation, in order to identify a possible inherited and/or acquired thrombophilia, all patients were screened for methilene-tetra-hydro-folate-reductase C677T gene polimorphism (MTHFR C677T ), Factor V Leiden gene polimorphism (FVL), prothrombin A20210G gene polimorphism (PTHR A20210G ), protein S deficiency, protein C deficiency, antithrombin III deficiency (AT III), lupus anticoagulant, IgM and/or IgG anticardiolipin autoantibodies [ 22 , 27 ]. Moreover, all patients showing increased d-dimer were tested also for β-human-corionic-gonadotropin (β-HCG) to exclude early pregnancy, and lower limb ultrasound vascular examination associated to compression ultrasonography (CUS) to exclude a lower limb deep venous thrombosis (DVT); both conditions, in fact, are well known conditions associated to increased d-dimer [ 4 , 9 - 11 ]. Furthermore, patients with increased d-dimer (group A, 25 patients) and patients with normal d-dimer (group B, 14 patients) were compared also for possible differences in molecular markers of inherited and/or acquired thrombophilia. Statistical analysis was based on χ 2 test, differences were considered to be significant if p < 0.05. Results We found thrombophilia in group A, 80%, and in group B, 50%, so thrombophilia rate in all 39 selected was 65% if we consider together group A (i.e. women affected by sterility and showing increased d-dimer) and group B (i.e. women affected by sterility with normal d-dimer levels) (table 1, 'see additional file 1 '). Twenty patients of group A (80%) affected by sterility with increased d-dimer levels, showed inherited and/or acquired thrombophilia [(six MTHFR C677T homozygosity, four FVL heterozygosity, five PTHR A20210G heterozygosity, three inherited Protein S deficiency, two showing combined defects (one MTHFR C677T homozygosity associated to protein S deficiency and one MTHFR C677T homozygosity associated to FVL heterozygosity), none protein C deficiency or AT III deficiency, none positive for the presence of lupus anticoagulant, none with increased anticardiolipin autoantibodies IgM and/or increased anticardiolipin autoantibodies IgG)] (table 2, 'see additional file 2 '). Remaining five women of the group A did not show molecular thrombophilia, but in their anamnesis we found some possible correlation with an acquired thrombophilia: controlled ovarian hyperstimulation in one patient, monthlies in one patient, early pregnancy in one patient, miscarriage in one patient, none apparent cause in 1 patient; among them two of these five patients were heterozygous for MTHFR C677T . Furthermore, two patients of group A carrying inherited thrombophilia for the presence of heterozygous FVL and increased d-dimer revealed previous DVT with following pulmonary embolism in their anamnesis. Data of patients of group A are summarised in table 2 ('see additional file 2 '). Seven patients of group B (50%) showed inherited and/or acquired thrombophilia (one MTHFR C677T homozygosity, one FVL heterozygosity, five PTHR A20210G heterozygosity, none inherited Protein S deficiency, protein C deficiency, AT III deficiency, none presence of lupus anticoagulant, none with increased anticardiolipin autoantibodies IgM and/or IgG), as reported in table 2 ('see additional file 2 '). All remaining seven patients of group B showed all heterozygosity for MTHFR C677T . Moreover, none patients of group B revealed previous DVT and/or pulmonary embolism. Five patients of group C (i.e. control group) (33.3%) showed increased d-dimer as molecular markers of ongoing proximal DVT confirmed by ultrasound vascular examination associated to CUS; moreover, all five patients revealed an underlying inherited and/or acquired thrombophilia (three MTHFR C677T homozygosity, one protein S deficiency, one combined thrombophilia: FVL heterozygosity associated to protein S deficiency). Data of patients of group C are summarised in table 2 ('see additional file 2 '). In all groups positivity for anticardiolipin antibodies or lupus anticoagulant mimicking a primary antiphospholipid syndrome (APS) was not discovered. So, as showed in table 3 ('see additional file 3 '), increased d-dimer is frequently associated with thrombophilia in women affected by sterility, while this association is less present in patients with normal d-dimer, and this difference reaches statistical significance (p < 0.05); furthermore thrombophilia is more frequent in group A than in control group (i.e. group C) and also this difference reaches statistical significance (p < 0.05); finally, thrombophilia in group B is more frequent than in control group (i.e. group C), but this difference does not reach statistical significance (p: 0.08, ns). Discussion In this report for the first time the role of d-dimer was investigated in diagnostic screening of patients affected by sterility and this is a really innovative data available in this clinical setting. D-dimer is a fibrin degradation product which usually is extensively screened in patients with suspected thrombosis and/or pulmonary embolism [ 9 ]. An increased plasma d-dimer might have a predictive positive value for DVT and/or pulmonary embolism, but because increased d-dimer has been observed also in several conditions not associated with ongoing thrombosis (malignancy, chronic inflammation, infections, acute coronary syndromes, necrosis, eldership) [ 3 - 9 ] the really interesting role of d-dimer in this clinical setting is for its high negative predictive value as reported by Bounameaux et al. in a series of patients with suspected pulmonary embolism [ 9 ]. However, increased d-dimer has been observed also in subjects affected by thrombophilia (i.e. inherited thrombophilia and/or acquired thrombophilia) showing hypercoagulable state without ongoing thrombosis as reported by Arkel et al. and Humphries et al. [ 2 , 23 ]. So, our data showed that patients of group A, carrying increased d-dimer, has been extensively screened for inherited and/or acquired thrombophilia and 80% of them revealed a well known molecular condition associated to hypercoagulable state which may explain increased d-dimer levels (table 2, 'see additional file 2 '). Moreover, this our clinical and laboratory screening reaches statistical significance compared to group B and group C (table 3, 'see additional file 3 '). Furthermore, five patients with increased d-dimer did not reveal inherited and/or acquired thrombophilia, but a thorough anamnesis and a clinical evaluation permitted to identify other causes of increased d-dimer in four of these patients: one patient showed early pregnancy (confirmed by β-HCG measurements and following ultrasound scan), a known condition associated to hypercoagulability and increased d-dimer [ 4 , 28 , 29 ], one patient revealed an early abortion, confirmed by following decrease of β-HCG, and increased dimer levels might be related to uteroplacental thrombosis and/or necrosis [ 30 ], one patient was ongoing to controlled ovarian stimulation and this condition may be associated to alteration of haemostasis with a trend toward thrombophilia [ 31 , 32 ] and one patient showed ongoing monthlies, a condition associated to wound healing which involves also clotting factors and might explain increased d-dimer [ 33 ]; remaining one patient showed increased d-dimer for unknown causes probably related to not well studied thrombophilia [ 34 ] or idiopathic thrombophilia and/or other conditions although we excluded in our selection criteria several other diseases associated to increased d-dimer. So for the first time we showed an interesting and relevant role of d-dimer in the screening of sterility causes, particular an underlying thrombophilia may be suspected in pathophysiology of sterility if plasma d-dimer is increased. However, also an evaluation of other conditions associated to increased d-dimer (e.g. chronic inflammation, immunopathological diseases, infectious diseases, cancer, necrosis, eldership, pregnancy, controlled ovarian stimulation, monthlies) should be performed in order to avoid a misinterpretation. Also group B, with normal d-dimer levels, showed an increased rate of thrombophilia (50%, table 1, 'see additional file 1 '), so confirming one more time the clear relationship between thrombophilia and sterility, even if these data did not reach statistical significance compared to group C (table 3, 'see additional file 3 '). Yet, patients of group B, although affected by thrombophilia and sterility did not show increased d-dimer. This finding might be explained by several causes and a laboratory mistake cannot be excluded; furthermore, these patients of group B may show also transient and/or silent thrombophilia which may trigger a hypercoagulable state if associated to other causes (i.e. acquired conditions associated to thrombophilia) during their natural history and our evaluation of d-dimer might be done during a not-hypercoagulable transient state. An extensive screening of causes of increased d-dimer in our population was also performed. The association between thrombophilia and sterility due to recurrent foetal loss is well known as reported by several reports [ 12 - 20 ] and also by our data. However, recently an association between primary sterility and thrombophilia has been underlined such as also between thrombophilia and repeated in vitro fertilisation failures [ 35 , 36 ]. A clear relationship between thrombophilia and recurrent foetal loss has been reported for inherited deficiency of clotting inhibitors (i.e. protein C deficiency, protein S deficiency, AT III deficiency) [ 20 , 36 ], but we did not find in our population this strong association (only four cases of protein S deficiency, one of these associated to MTHFR C677T homozygosity, and none case of protein C deficiency and/or AT III deficiency). However, this aspect seems to be in agreement with other reports in which other thrombophilic conditions were more frequent than clotting inhibitor deficiencies (e.g. FVL, MTHFR C677T homozygosity, antiphospholipid syndrome and so on) [ 12 - 20 ]. FVL gene polymorphism has been frequently found in women affected by recurrent fetal loss, although the frequency of FVL differs in each study [ 15 , 24 ]. These differences could be related, besides to ethnic background, also to different inclusion criteria of investigated patients. However, FVL is associated to sterility also in our study (four cases in group A and one case in group B; table 2, 'see additional file 2 '). An increased MTHFR C677T homozygosity has been found in our study population (six cases in group A and one case in group B), so confirming a clear role of homocysteine metabolism and of the related hypercoagulable state in sterility pathophysiology [ 38 - 40 ]. Of course, MTHFR gene polymorphism and related homocysteine metabolism may influence sterility also through folic acid and vitamin B12 deficiency due to uncorrected diet and/or lifestyle [ 41 ]. We found also an increased frequency of PTHR A20210G in women affected by sterility (five cases in group A and five cases in group B), and these data seem to be different from data reported by Pickering et al [ 42 ] and Deitcher et al [ 43 ] and in agreement with data reported by Brenner et al [ 24 , 25 ]. As we previously underlined, these differences could be related to inclusion criteria established by Investigators of each study and also to an ethnic background; this gene polymorphism, in fact, is more frequent in Southern Europe than in Northern Europe [ 44 , 45 ]. A really interesting data is the absence of APS from our study population and this data differs from data of the Literature. A possible explanation could be offered by different selection criteria: we exclude, in fact, women with immunopathological diseases (e.g. rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, vasculitis), so excluding the most common causes of secondary APS and so searching only primary APS that is more rare than primary [ 46 ]. In conclusion, in this investigation both groups of women affected by sterility, group A and B, showed increased incidence of thrombophilia compared to control group (group A vs group C, p: < 0.05, s; group B vs group C, p: 0.08, ns; table 3, 'see additional file 3 '), so confirming, one more time the relevant role of thrombophilia in pathophysiology of sterility. So, the first relevant data we offer in this study is the role of d-dimer in the screening of sterility causes in order to early suspect an underlying thrombophilia; this screening, as also showed by our data, is in agreement with an elevated frequency of thrombophilia in women affected by sterility (80 % in group A, 50% in group B, 65% if we consider together group A and B). Of course, although several Authors already reported the association between thrombophilia and recurrent foetal loss we may testify that probably the role of thrombophilia is an underestimated problem if we consider all sterility conditions because usually thrombophilia is screened only for repeated foetal loss and not screened in any case of unexplained sterility as in this study. So, based on our data further studies on large population are needed not only to confirm our results but also to focus a possible different prognosis of these groups, in particular to sterility prognosis. Conclusion Our data demonstrated a clear role of thrombophilia in patients affected by sterility, but suggesting a clear diagnostic role of increased d-dimer in a lot of these patients. This diagnostic screening of thrombophilia in women affected by sterility, based on the d-dimer levels, may also represent a really speed method to suspect thrombophilia in these subject and has also a good cost/benefit ratio, although other causes of increased d-dimer should be always considered. In a second step, if increased d-dimer levels are present causes of hypercoagulable state may be investigated (i.e. inherited thrombophilia and/or acquired thrombophilia). This approach may play a role not only in differential diagnosis of sterility but also in the early diagnosis of sterility due to thrombophilia. After the first step in which d-dimer may be evaluated, causes of increased d-dimer should be subsequently identified in order to start a possible antithrombotic treatment soon. Nevertheless thrombophilia may be present in few cases also in subjects with normal d-dimer, it should be investigated always if other causes of sterility are not present. So, we strongly suggest to test d-dimer in patients affected by sterility as first step of a possible underlying thrombophilia in order to early identify the cause of thrombophilia and its prompt treatment but other data should be confirmed by further investigations on large based population. Supplementary Material Additional File 1 Thrombophilia frequency in studied groups Click here for file Additional File 2 Thrombophilia in studied groups Click here for file Additional File 3 statistical analysis according with χ 2 method Click here for file

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549199

Proteins with two SUMO-like domains in chromatin-associated complexes: The RENi (Rad60-Esc2-NIP45) family

Background Post-translational modification by Small Ubiquitin-like Modifiers (SUMO) has been implicated in protein targeting, in the maintenance of genomic integrity and in transcriptional control. But the specific molecular effects of SUMO modification on many target proteins remain to be elucidated. Recent findings point at the importance of SUMO-mediated histone NAD-dependent deacetylase (HDAC) recruitment in transcriptional regulation. Results We describe the RENi family of SUMO-like domain proteins (SDP) with the unique feature of typically containing two carboxy-terminal SUMO-like domains. Using sequence analytic evidence, we collect family members from animals, fungi and plants, most prominent being yeast R ad60, E sc2 and mouse NI P45 . Different proteins of the novel family are known to interact directly with histone NAD-dependent deacetylases (HDACs), structural maintenance of chromosomes (SMC) proteins, and transcription factors. In particular, the highly non-trivial designation of the first of the two successive SUMO-domains in non-plant RENi provides a rationale for previously published functionally impaired mutant variants. Conclusions Till now, SUMO-like proteins have been studied exclusively in the context of their covalent conjugation to target proteins. Here, we present the exciting possibility that SUMO domain proteins, similarly to ubiquitin modifiers, have also evolved in a second line – namely as multi-domain proteins that are non-covalently attached to their target proteins. We suggest that the SUMO stable fusion proteins of the RENi family, which we introduce in this work, might mimic SUMO and share its interaction motifs (in analogy to the way that ubiquitin-like domains mimic ubiquitin). This presumption is supported by parallels in the spectrum of modified or bound proteins e.g. transcription factors and chromatin-associated proteins and in the recruitment of HDAC-activity.

Background Among ubiquitin-related proteins, containing at least one domain with a ubiquitin-like fold, one can distinguish ubiquitin-like modifiers (UBLs) and ubiquitin domain proteins (UDPs) [ 1 ]. UBLs can be covalently attached to target proteins analogously to ubiquitin. Unlike ubiquitin, UBLs mostly do not directly target proteins for degradation [ 2 ], although functional links can exist. One of the most heavily researched single domain UBLs, the small ubiquitin-related modifier (SUMO), is known to act on transcription factors, chromatin associated proteins, nuclear body proteins and septins [ 2 ]. In contrast to UBLs, UDPs are not conjugated to other proteins and lack the C-terminal double glycine motif characteristic for ubiquitin and ubiquitin-like modifiers. They are a heterogeneous class of usually multi-domain proteins, which are unrelated outside of their ubiquitin-like domain [ 1 ]. In several cases, it has been demonstrated that the ubiquitin-domain within those proteins likely fulfills its cellular role by functionally mimicking ubiquitination [ 3 , 4 ]. The biological relevance of non-conjugatable multi-domain proteins having a domain with clear relationship to UBLs like SUMO, rather than ubiquitin, is yet unknown. Here, we present a detailed sequence analysis of a family of SUMO-like domain proteins (SDPs) containing one or two SUMO-like domains. Members of the proposed RENi family act as factors in transcriptional regulation, chromatin silencing and genomic stability. Results Sequence architecture of Drosophila melanogaster CG4449 During the study of the predicted nuclear subset of the Drosophila proteome, we encountered the unknown 424 amino acids long protein CG4449 (NP_651134). Initial analysis of its sequence complexity shows that the disordered N-terminal half of the protein is followed by a likely globular segment (predicted using Pdisorder by Softberry, Inc). Indeed, a compositionally biased, polar low-complexity region (LCR) spans almost the entire N-terminal 220 amino acids (AA) as reported by CAST (region 47–165, lysine-rich) [ 5 ] and SEG (regions 46–77 and 133–178, parameters 25/3.0/3.3) [ 6 ]. The C-terminal half of CG4449 turns out to contain an internally repeated segment identifiable with RADAR [ 7 ] (region 270–309 matching 368–407). In an attempt to confirm this repeat, we queried the protein against the conserved domain database using RPS-BLAST [ 8 ]. Thereby, we could define a similarity to SUMO-like domains overlapping with the second repeat-element (see Table 1 for details), while no significant hits emerged for the first of the repeat-constituents. Using profile-profile comparison, however, segment 220–325 is shown to possess a distant, yet significant similarity to SUMO sequences and, therefore, to share the SUMO fold (Table 1 ). In conclusion, we found that the Drosophila protein CG4449 (NP_651134) has a tripartite architecture: with a N-terminal LCR followed by two globular domains with a SUMO-like fold (termed SD1 and SD2). Whereas SD2's similarity to single domain SUMO-like sequences can be easily detected with BLAST tools, the identification of SD1 is non-trivial (Table 1 ). For both SD1 and SD2, a carboxy-terminal double-glycine motif, as it is known and necessary for the covalent attachment of SUMO proteins, is missing. This finding is remarkable as SUMO proteins are discussed in the scientific community solely as polypeptides that become covalently bonded to various targets [ 9 ]. Here, we present cases of non-conjugatable poly-SUMO fusion protein. Collecting animal NIP45-related proteins characterized by two SUMO-like domains A PSI-BLAST search started with the globular C-terminal half of CG4449 (220–424) including the two SUMO-like domains, collects a family of animal proteins with the same tripartite organization in C. briggsae (CAE71155.1, E = 0.001 round 2), H. sapiens (NP_116204.2, E = 0.003 round 2), M. musculus (NP_035030, E = 1e-39 round 3) and C. elegans (NP_497960, E = 2e-12 round 3). All these proteins contain a LCR at the N-terminus followed by two SUMO-like domains, the first of which has mostly diverged away beyond recognition thresholds using traditional sequence-profile searches (Table 1 ). The human and the mouse homologs correspond to the studied nuclear factor NIP45 (NF-AT interacting protein) [ 10 ]. All sequences and original database search results can be found at the RENi homepage [ 11 ]. Distant NIP45 homologs in fungi, other lower eukaryotes and plants Indications on the existence of NIP45 homologs in lower eukaryotes and plants A multiple sequence alignment of the globular C-terminal half of D. melanogaster CG4449 (220–424) and the corresponding sequences derived from A. gambie , X. laevis , C. elegans , C. briggsae , M. musculus and H. sapiens (Figure 1 ) was used to generate a Hidden Markov Model (HMM; in the global alignment mode). The protein family was enlarged using the HMMER2 tool [ 12 ] in searches against single model organism proteomes. These searches retrieved as best hits in the respective proteomes the likely homologs in D. discoideum (Sanger proteome identifier- JC3V1_0C0008_11033, 0.00059) A. thaliana (At1g68185.1, NP_564924.1, E = 0.00076), O. sativa (NP_917594.1, E = 0.0019), S. pombe (NP_595995.1, E = 0.00073), S. cerevisiae (NP_010650.1, E = 0.11), Y. lipolytica (CAG82446, E = 0.00087), C. glabrata (CAG57776, E = 0.52) and in other recently published fungi proteomes [ 13 ]. The S. cerevisiae and S. pombe homologs correspond to the studied Rad60 and Esc2 proteins, respectively [ 14 , 15 ]; the remaining proteins are uncharacterized. These HMM-search results suggest the most likely plant and lower eukaryote orthologues to the animal NIP45-like proteins. For establishing the orthology relationship, these initial results need to be confirmed by reciprocal searches independently performed for fungal and plant proteins. Further below, we present this evidence for the homology between the C-terminal part of the proteins found in the various taxonomic groups. Confirming fungal family members in reciprocal searches The set of fungal RENi proteins can be autonomously collected using a BLASTP search started with the C. glabrata representative (CAG57776) and retrieving the best and significant hits in the proteomes of S. cerevisiae , S. pombe , K. lactis , C. albicans , Y. lipolytica , D. hansenii , A. nidulans [ 11 ]. The domain architecture of these fungal homologs is likely also tripartite (Figure 1 , Table 1 ). It differs from the animal representatives by a longer sequence separating the two SUMO-like domains (many dozens of residues compared with ~10 in the case of animal proteins), which is typically of highly helical content (determined using NPL consensus secondary structure prediction [ 16 ]). A HMM was generated from a multiple sequence alignment of the SUMO-domain containing C-terminal half of the listed fungal homologs, where gap only columns replaced the compositional biased helical region between the two SUMO domains. A search with this HMM retrieved as best hits in the respective proteomes the RENi proteins in M. musculus (NP_035030, E = 0.005), A. thaliana (NP_564924, E = 0.016), C. elegans (NP_497960, E = 0.0012). Confirming plant family members in reciprocal searches Potential plant RENi homologs, derived in a full-length TBLASTN search with A. thaliana (At1g68185.1, NP_564924.1) against the TIGR Gene indices of barley, maize, rice, potato and soybean [ 17 ], show a length of 210–240 AA and are thus around 100 AA shorter than the shortest animal homolog from worm. The domain organization seems also to be distinct. A 100 AA N-terminal, very polar region (with two conserved motifs E [ED]LEPLFDY [SR]RVQP and DWLPPPP found with MEME [ 18 ]) is followed by ~40 AA with predicted strong helical preference (using NPL [ 16 ]) and a clear C-terminal SUMO-like domain (Table 1 ). There are no indications for another SUMO-like domain at the N-terminal side of the ~40 AA helical region. Further confirmation of the relationship between the listed RENi proteins of the Viridiplantae and Fungi/Metazoan group comes from the analysis of the reciprocal genomic best hits of A. thaliana in Y. lipolytica (At vs Yl 1e-07, Yl vs At 6e-04) and H. sapiens proteomes (At vs Hs 3e-05, Hs vs At 8e-05) [ 11 ]. Definition of the Rad60-Esc2-NIP45 (RENi) protein family We propose to name the collected group of protein sequences the RENi-family after its most studied members Rad60, Esc2 and NIP45. All representatives have a similar sequence architecture involving a N-terminal low complexity region with many polar and (positively) charged residues and a C-terminal globular part with one (plant proteins) or two (all others) SUMO-like domains. The use of a model representing the complete globular region of RENi proteins was essential for the successful collection of the family. A global HMM spanning the SD1 and SD2 domains tests for homology in the whole globular part and, correspondingly, directly collects the RENi family. In contrast, when using the C-terminal half of various RENi family members as query sequence in PSI-BLAST [ 19 ], the searches are invaded by SUMO proteins (hitting only the segment of SD2) before the RENi family can be collected. This means that the SD2 domain sequence segments of the RENi group and the family of single-domain SUMO proteins are not well separated in sequence space (Figure 3 ). For this reason, the similarity of NIP45, Esc2 and Rad60 could previously only been defined transitively via the similarity of their SD2 segment to SUMO proteins and their similar length [ 20 ]. Discussion The SUMO-like domains in proteins of the Rad60-Esc2-NIP45 (RENi) family While RENi proteins of the fungal, metazoan and mycetozoan taxa contain two C-terminal SUMO-like domains (SD1 and SD2), only the second one can be clearly defined in plant representatives (Table 1 , Figure 4 ). This very C-terminal SD2 domain, shares several features discriminating SUMO proteins from other ubiquitin-like modifiers, as for example the large negative charged cluster, seen in the alignment 5–15 residues from the very C-terminus (Figure 1 ). The negative surface patch formed by these residues has been suggested to shape a SUMO-typical interaction surface [ 21 ]. RENi proteins lack conservation of the carboxy-terminal double-glycine motif required for covalent attachment of SUMO to its substrates. Thus, they are likely linear non-cleavable SUMO fusions, which cannot be conjugated to target proteins, and have to be classified as UDPs. The SD1 SUMO-like domain contained in fungi, metazoa and mycetozoa, has sequentially diverged away from SUMO proteins, but structural prediction suggest its resemblance to SUMO (Table 1 ). The low sequence conservation of this domain does not understate a possible functional conservation in that region, as it has been shown that the structure rather than sequence is important for the function of ubiquitin-like domains (UD). For example, replacing the UD of the UDP Rad23 with ubiquitin renders a functional protein variant [ 1 ]. Indications on the functional importance of the first SUMO-like domain in RENi proteins come from the two fungal representatives of the family. The fission yeast rad60-1 (K263E) [ 15 ] and rad60-3 (F272V) [ 20 ] mutants, which are defective in the rad60 function of double strand break repair, contain a point-mutation within this first SUMO-like domain (SD1). The sequence alignment to human SUMO-1 (structure 1A5R, see Figure 1 ) indicates that both mutations most likely affect structurally important positions. They align to residues within human SUMO-1 (1A5R) (Gln55 and Phe66) that have been listed by Bayer et al. [ 21 ] among the contacts contributing with parts of their side chain to the formation of the hydrophobic core of the fold (Figure 1 and Figure 2 ). In the budding yeast Esc2p, the region containing the first SUMO-domain SD1 together with a 80 AA low-complex N-terminal segment can be defined as a sufficient fragment supplying its function in targeted silencing (residues 115–389 in Esc2p) [ 14 ]. A graph representation of the pair-wise similarity relationship for SD1 and SD2 sequences to other known ubiquitin-like domains (Figure 3 , created with the program Clans [ 22 ]) illustrates that both are most closely related to SUMO domains. From our analysis of sequence similarity, we suggest that, at least, the very C-terminal SUMO-like domain (SD2) in RENi proteins is able to mimic SUMO and potentially shares its interaction partners. On the other hand, the available experimental data confirms the functional importance of the SUMO-like domain SD1 preceding it. The N-terminal polar low complexity region in proteins of the Rad60-Esc2-NIP45 (RENi) family The occurrence of a N-terminal low complexity region with an excess of polar/charged residues is a characteristic element of the RENi protein architecture. Most likely, this is a conformationally flexible segment without inherent structural preference [ 23 , 24 ]. The molecular function of this region remains unknown. It should also be noted that homology considerations are not applicable to such compositionally biased regions for functional prediction. Boddy et al. [ 20 ] discussed the possible existence of coiled coils in the domain architecture of Rad60, Esc2 and NIP45. We found that the COILS [ 25 ] tool generates hits only in few representatives of the RENi family. They are positionally not conserved relative to the two SUMO-like domains. It is known that the COILS tool produces a considerable number of false-positive hits, especially in regions with many polar/charged residues, for example, as is likely the case for a glutamic-acid-rich part in Rad60. Experimentally verified functions of RENi proteins Functional information about RENi family members is restricted to the fungal Esc2, Rad60 and the metazoan NIP45 proteins. Here, unfortunately most of the existing data relates to the full-length sequences. Nevertheless, the quite divergent set of functions known for RENi proteins shows considerable overlap with the established cellular roles of SUMO proteins in genome replication and regulation of gene expression. The fission yeast Rad60 protein was shown to be essential in DNA double-strand break repair, and to be critical also for normal growth [ 15 ]. It physically and genetically interacts with the Smc5/6 complex, a complex with a housekeeping role in the genome [ 20 ]. Interestingly, the Smc5/6 complex [ 26 ] also includes Nse2, a protein containing a zf-MIZ domain commonly found in E3-like SUMO ligases (Pfam-search E = 0.0074). In addition, Rad60 is known to bind the replication checkpoint kinase Cds1 [ 20 ]. S. cerevisiae Esc2 (establishment of silent chromatin 2) is involved in chromatin silencing via the recruitment or stabilization of the Sir (silent information regulators) complex [ 27 , 28 ]. It is known to interact with Sir2, a histone NAD-dependent deacetylase (HDAC-class III) of the Sir complex, which is well conserved from bacteria to human [ 29 ] and, thus, might be an interaction partner of other RENi proteins as well. Similarly to other HDACs, Sir2 proteins are recruited to chromatin by DNA-bound factors [ 30 ] and act by deacetylating histones [ 31 ] as well as transcription factors such as p53 and forkhead transcription factor (shown for hSIR2) [ 29 , 32 ]. With respect to a possible intersection with known Rad60 functions, it is interesting that Sir2 is not only involved in heterochromatic gene silencing and euchromatic repression [ 30 ] but also in DNA double-strand break repair mediated by end-joining [ 33 ]. NIP45, the one studied RENi in metazoa, has been implied in gene regulation, where it needs its DNA-binding partner NFATp for this activity [ 34 ]. Strikingly, the NFAT family member NFAT1 that interacts with NIP45 [ 10 ] was independently shown to be sumoylated [ 35 ]. NFAT1 sumoylation acts in nuclear retention, regulation of transcriptional activity and recruitment to nuclear SUMO-1 bodies [ 35 ]. This analysis might suggest a potential role for SUMO-like NIP45 in its complex with NFAT proteins. Possible functional role of the SUMO-like domains in RENi proteins There is little experimental data on the importance of the predicted SUMO-like domains in RENi proteins. Nevertheless, all listed functions of RENi proteins conform with the known role of SUMO in transcriptional regulation and the control of genome integrity [ 36 ]. In the context of transcriptional repression, SUMO-modification has been suggested to recruit class I and II HDACs to promoter sites. Regarding genome stability, SUMOylation in DNA-repair proteins is thought to target these to DNA damage foci. The following parallels in RENi proteins become obvious: 1) HDAC recruitment has also been suggested for the fission yeast Esc2p [ 27 , 28 ]. 2) Mammalian NIP45 binds to transcription factors that can also be modified by SUMO [ 35 ]. 3) RENi and SUMO share a functional context in double-strand break repair and transcriptional regulation. On the basis of functional overlaps of SUMO and RENi proteins, we can speculate that RENi proteins act as SUMO stable fusion proteins "mimicking" SUMO and that they might have common interaction partners. Conclusions In this report, we use sequence-analytical methods to infer the homology relationships between RENi family members and determine their tripartite (bipartite for plant homologs) domain architecture. A N-terminal polar low-complexity segment and two consecutive SUMO-like domains in the C-terminal half characterize the functionally described fungal and metazoan RENi proteins. While the more C-terminal SD2 is easily detectable, it is the particularly divergent SD1 that was shown in fungi to be essential for the assayed molecular functions. Due to the likely limited sequential- (as opposed to structural-) requirements, this SUMO-like domain is difficult to detect and has been missed in previous analyses of individual family members. The identification of the more N-terminal SUMO-like domain SD1 helps rationalizing experimental findings for mutant fungal RENi family members. Methods RPS-BLAST [ 8 ] and FFAS [ 37 ] algorithms were used to search the COG [ 38 ], SCOP [ 39 ] and SMART databases [ 40 ]. SEG [ 6 ] and CAST [ 5 ] were applied in identifying low-complexity regions. Structural similarity was determined using the fold prediction methods FFAS [ 37 ] and BIOINBGU [ 41 ]. T-coffee was used for initial multiple sequence alignment [ 42 ]. CLANS [ 22 ] generated the pairwise similarity graph. VMD [ 43 ] was used for molecular visualization and POV-ray for the follow-up image rendering. Authors' contributions The sequence analytic work was executed by MN. All authors (MN, AB, BE, FE) contributed to evaluating the results and making the discoveries reported here. MN prepared all the figures and, together with FE, the manuscript text. All authors read and approved the final manuscript.

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524375

Isolation of suppressor genes that restore retrovirus susceptibility to a virus-resistant cell line

Background Genetic selections in mammalian cell lines have recently been developed for the isolation of mutant cells that are refractory to infection by retroviruses. These selections have been used to recover lines that block early postentry stages of infection, either before reverse transcription or before nuclear entry. The mechanisms of action of these blocks remain unknown. Results We have devised a method for the selection of genes from cDNA libraries that suppress the block to virus infection, and so restore virus susceptibility. The protocol involves the transformation of pools of resistant cells by cDNA expression libraries, followed by the selection for rare virus-sensitive cells, using multiple rounds of selection after infection by marked viral vector genomes. The suppressor genes were then recovered from these virus sensitive cells, and their ability to restore virus susceptibility was confirmed by reintroduction of these cDNAs into the resistant line. Conclusions The identities of these genes provide insights into the mechanism of virus resistance and will help to define new pathways used during retrovirus infection. The methods for gene isolation developed here will also permit the identification of similar suppressors that modify or override other recently identified virus resistance genes.

Background It is becoming increasingly apparent that mammalian cells harbor numerous genes that induce intracellular blocks to retrovirus infection [ 1 , 2 ]. These genes have presumably evolved and been maintained in the genome in response to the pathogenic and lethal consequences of infection, and are now thought to constitute an important part of the host defense against these viruses. Some of the genes and gene products responsible for this resistance have been recently identified, including the Fv1 locus in the mouse, which blocks infection after reverse transcription but before nuclear entry and establishment of the integrated provirus [ 3 ]; the APOBEC3G enzyme, which is incorporated into virion particles and catalyzes the destructive deamination of the viral cDNA during reverse transcription [ 4 ]; and the TRIM5a protein, which somehow blocks incoming virus soon after entry and prevents the activation of reverse transcription [ 5 ]. Others likely remain to be identified. We have been involved in the development of screens and selections for virus resistance genes, and have isolated mutant cell lines after chemical mutagenesis that are profoundly resistant to retrovirus infection. Two such lines isolated from a parental fibroblast cell line, Rat2 cells, have been characterized in some detail [ 6 ]. Mutant line R3-2 exhibited a nearly 1000-fold resistance to infection by genetically marked Moloney murine leukemia virus genomes, and was resistant to pseudotyped viruses utilizing the ecotropic envelope, the amphotropic envelope, or even the VSV G envelope protein. Infection of R3-2 resulted in the normal synthesis of the linear viral DNA by reverse transcription, but circular viral DNAs and integrated proviruses were not generated. The viral DNA was apparently trapped in the cytoplasm in a form that was not readily extracted by conditions that allowed DNA recovery from wild-type infected cells. Mutant line R4-7 exhibited about a 100-fold resistance to infection by M-MuLV, also independent of the envelope mediating entry. Infection of this line was blocked earlier, before the initiation of reverse transcription. Both lines R3-2 and R4-7 were also resistant to infection by pseudotyped HIV-1 viral vectors. To probe the nature of the blocks in these mutant cell lines, we have sought to identify and characterize suppressor genes that override the restriction exhibited by these cells. To identify such genes, we have developed methodologies that allow for the selection of rare virus-sensitive clones arising after transfer of gene libraries into populations of virus-resistant parents. We here report the isolation of two cDNA constructs that each restore virus sensitivity to the R4-7 mutant cell line. These DNAs constitute valuable tools in the characterization of this line's virus resistance. Results Selection for virus-sensitive clones from R4-7 mutant cells expressing cDNAs The R4-7 mutant cell line is approximately 100-fold resistant to transduction by MuLV-based vectors as compared to wild-type Rat2 cells [ 6 ]. To identify genes that could suppress this phenotype and restore virus sensitivity, a protocol involving multiple rounds of selection for virus sensitivity was devised (Fig. 1 ). First, R4-7 cells were transformed by a library of rat kidney cDNAs expressed from the constitutive CMV promoter. Recipient cells were selected by cotransformation with a DNA expressing puromycin resistance. Five pools of the puromycin-resistant cells were generated and maintained separately, each pool containing more than 1000 independent transformed clones. The expectation was that multiple rounds of selection for virus-sensitive clones would be required to recover such cells, with each round providing at most a 100-fold enrichment. Figure 1 Flowchart for isolation of cDNAs that suppress virus resistance and restore virus sensitivity to the R4-7 mutant cell line. See text for description. Four of the pools of transformed cells, with each clone in the pools overexpressing a small number of cDNAs, were sequentially exposed to a series of three genetically marked ecotropic MuLV-based vectors, and the rare successfully infected cells were recovered after each infection by selection for the marker carried by the vector (see Methods). The cells were first exposed to N2 virus, an MuLV vector carrying the neor marker, and infected cells were selected in medium containing G418. These cultures were then expanded and exposed to Eco-TK virus, an MuLV vector carrying the Herpes virus TK gene, and infected cells were selected with HAT medium. These cultures were expanded and finally exposed to Eco-His virus, and infected cells were selected with medium containing histidinol. In all cases, the selecting viral vectors were applied at low multiplicities of infection (MOI) so as not to override the resistance of the parental R4-7 cells, as can happen at high MOI [ 6 ]. Individual colonies were recovered after the triple selection. The number of colonies of infected cells recovered at each stage of the selection was determined for each of the four pools (Table 1 ). The number of colonies of wild-type Rat2 cells exposed to the virus in parallel was determined for comparison. In each of the first two rounds of selection, the pools of mutant cells yielded about 25-fold fewer transductants than the wild-type control, indicating retention of the resistance in the bulk of the population. In the third round, pools 3 and 4 yielded slightly higher numbers of colonies than the other pools, suggesting possible enrichment for virus sensitive clones, though still less than the wild-type cells. A total of 36 candidate colonies were isolated. Table 1 Numbers of colonies recovered after each round of infection and selection Initial Cell Population Pool 1 Pool 2 Pool 3 Pool 4 Rat2 Puro R colonies after transfection >1000 >1000 >1000 >1000 - Neo R colonies after N2 virus infection ~400 ~400 ~400 ~400 TMTC HAT R colonies after TK virus infection 100 60 40 30 2000 His R colonies after His virus infection 3 2 12 19 ~200 Virus S lines by GFP virus susceptibility 0 0 2 4 - To determine whether any of these candidate clones had become truly virus sensitive, all 36 colonies were individually picked and expanded into larger cultures. These cultures were then tested by infection with Eco-GFP, a virus vector expressing the green fluorescent protein, and the fraction of the cells expressing the marker was determined by inspection. While all the clones from pools 1 and 2 were as resistant as the parental R4-7 line, a total of 6 clones – 2 from pool 3 (dubbed A1, A2) and 4 from pool 4 (dubbed B1, B2, C1, C2) – were fully sensitive to infection. These cloned lines were thus candidates as potentially carrying cDNAs that could restore virus sensitivity to the R4-7 line. Recovery of cDNAs from virus-sensitive cell lines capable of suppressing virus resistance To recover the cDNAs present in the virus sensitive cell lines, total genomic DNA was isolated, and polymerase chain reactions were performed to amplify expression cassettes composed of the CMV promoter, the cDNA insert of the library and the poly(A) addition signal. The amplified DNA from each line was directly cloned into the TOPO plasmid DNA and used to transform bacteria. In this way cloned cDNAs were recovered from five of the six lines. Because the lines were expected to each carry a few different cDNAs, and because only one cDNA in each line would be expected to be responsible for the phenotype, a total of 50 bacterial colonies were isolated for each of the five lines. DNAs were prepared from these bacterial colonies and assigned to groups based on the pattern of restriction fragments after produced after digestion with MspI. The number of distinct cDNAs recovered from each of the five lines ranged from 1 to 11, and all together included 29 cDNAs (Table 2 ). Table 2 Numbers of cDNAs recovered from Virus S cell lines Origin of cell lines Total Pool 3 Pool 3 Pool 4 Pool 4 Pool 4 Pool 4 Virus S cell line A1 A2 B1 B2 C1 C2 Total cDNAs examined 50 50 50 50 50 - 250 Distinct cDNAs 10 2 5 1 11 0 29 Active cDNAs 0 0 1 0 1 0 2 The cDNAs isolated from the virus sensitive lines were then tested directly for their ability to suppress the virus resistance of R4-7 cells. Each cDNA (20 ug) was mixed with pGK-puro DNA (2 ug) and used to transform naive R4-7 cells, and recipients stably expressing the DNAs were selected by growth in puromycin. The resulting puromycin-resistant colonies derived from a given cDNA were pooled and grown into large cultures, and the resulting populations were tested for sensitivity to Eco-neo virus infection. Two of the cDNAs, one from cell line B1 (designated pB1-11) and one from cell line C1 (designated pC1-2), dramatically suppressed the virus resistance of R4-7 cells (Fig. 2 ). An inactive cDNA retained as a negative control did not suppress the resistance. The susceptibility to infection of the pooled R4-7 transfectants for the two active clones was similar to that of the wild-type Rat2 cells, and roughly 100-fold higher than that of the R4-7 parents. To further document the sensitivity of induced by pC1-2, two individual clones were isolated from the R4-7 populations expressing pC1-2 and a control cDNA, and these clones were similarly tested by infection with Eco-neo virus. Like the pooled populations, the clones expressing pC1-2 were virus-sensitive and the controls were not (Fig. 3 ). Thus, these cDNAs were sufficient to suppress the resistance, and were likely responsible for the virus sensitivity of the two lines in which they were recovered after the triple selection. The remaining lines had presumably become sensitive to virus independently of any of the cDNAs they carried, or as a result of a cDNA that was not recovered from the PCR amplified DNA products. Figure 2 Ability of the suppressor cDNAs to restore virus susceptibility to the R4-7 mutant cell line. R4-7 cells were cotransformed with the indicated cDNAs, and the transformants were pooled and grown into cell populations. These cultures were then exposed to equal amounts (approximately 10,000 cfu in NIH/3T3 cells) of an N2 virus preparation, and virus susceptibility was assessed by plating the infected cells in medium containing G418. While the mutant R4-7 control populations yielded only ~50 colonies, the populations expressing the active cDNAs produced nearly confluent lawns. Rat2: virus-sensitive subclone isolated after mutagenesis. R4-7: mutant line. No cDNA: pGKpuro marker DNA alone. Control cDNA: marker plus inactive cDNA. PB1-11, pC1-2: marker plus indicated cDNA. Figure 3 Restoration of virus susceptibility by pC1-2 DNA in clonal cell lines. Single-cell clones were derived from R4-7 cell populations cotransformed with either inactive control cDNA or pC1-2 DNA. The resulting lines were exposed to N2 virus (approximately 300 cfu in NIH/3T3 cells) and plated in medium containing G418. While the control yielded no colonies, the clonal lines containing pC1-2 showed 100–200 colonies. Characterization of biologically active suppressor cDNAs The pB1-11 and pC1-2 DNAs could function as general enhancers of retrovirus infection, or alternatively as specific suppressors of the block in the R4-7 mutant cell line. To distinguish between these possibilities, the DNAs were introduced into the wild-type Rat2 cells, the distinct R3-2 mutant line, and the R4-7 line by cotransformation, and stable transformants were selected and expanded. The resulting transformed lines were then tested for their sensitivity to infection by Eco-Neo virus. The Rat 2 lines expressing pB1-11 showed no change in virus susceptibility, and the Rat2 lines expressing pC1-2 showed at most a 2-fold increase in sensitivity (Fig. 4 ). The corresponding R3-2 lines gave similar results (data not shown). Thus, both cDNAs were highly specific in enhancing the virus susceptibility of the R4-7 line. Figure 4 Lack of effect of suppressor cDNAs in wild-type cells. Rat2 or a virus-sensitive subclone isolated after mutagenesis (RC-2) were cotransformed with the indicated DNAs, and the transformants were pooled and grown into cell populations. The resulting cultures were exposed to N2 virus (approximately 300 cfu in NIH/3T3 cells) and plated in medium containing G418. All the cultures yielded approximately equal numbers (~200) of colonies. The DNA sequences of the two cDNAs were determined and compared with the nucleic acid sequences of the NCBI databases. Clone pB1-11 contained an insert of 855 bp with close sequence similarity to the central portion of a transcript originally termed HCC1.3/1.4, identified as encoding a prominent autoantigen expressed in a human hepatocarcinoma [ 7 ]. The similar mouse gene product, dubbed CAPER, was subsequently shown to interact with c-Jun, a subunit of the AP-1 activator, and the estrogen receptors ERa and ERß, and to exhibit transcriptional coactivator activity when expressed in concert with these transcription factors [ 8 ]. The cDNA insert of pB1-11 aligned well with both the human sequences (92% identity match to bp 910–1767 of HCC1.4 (Genbank accession no. L10911)) and the mouse sequences (94% identity match to bp 1153–2006 of CAPER (accession no. AY061882)). Remarkably, the cDNA fragment was inserted in reverse orientation relative to the CMV promoter of the pcDNAI plasmid vector [ 9 ], and thus the active DNA would produce an antisense mRNA transcript. Clone pC1-2 proved to contain an insert of 1407 bp, with close sequence similarity to a central portion of the VL30 elements, a family of endogenous retrovirus-like elements widely expressed in many mouse [ 10 - 14 ] and Rat cell lines [ 15 , 16 ]. The pC1-2 sequences aligned best with particular Rat elements expressed in tumor cells (~88% identity to bp 5025–6151 of a 7.4-kb element [ 17 ]; Genbank accession no. D90005) and in the ovary (~90% identity to bp 3341–4677 of a 5.5-kb element [ 18 ]; Genbank accession no. U48828). There was weaker similarity to related retroviruses, such as the gibbon ape leukemia virus [ 19 ]. The insert was in the sense orientation relative to the CMV promoter, and if transcribed would result in formation of a plus strand RNA, corresponding to the central portion of the VL30 transcripts. Like most rat VL30 elements, the insert did not include any significant open reading frames, but rather contained numerous mutations that introduced frameshifts and stop codons that would preclude synthesis of any long protein products. These results suggest that both of the pB1-11 and pC1-2 DNAs might function by virtue of their RNA products rather than any encoded proteins. The sequences of the two inserts have been submitted to the NCBI database (pB1-11 accession number is AY769432; pC 1-2 accession number is AY769433; see figure 5 ). Expression of CAPER and VL30 RNAs in R4-7 mutant line The biological activity of the pB1-11 and pC1-2 DNAs in restoring virus susceptibility could be mediated through effects on their corresponding endogenous gene products expressed in the R4-7 mutant cell line, or could be indirect. If their activity was direct, then either one of the corresponding endogenous genes – the CAPER gene or a VL30 element – might be the locus that was originally mutated to give rise to the resistance of the R4-7 line. To examine this possibility, RNAs were prepared from R4-7 and wild-type cells, and analyzed by Northern blot. Hybridizing with the pB1-11 probe showed a single major RNA about 3 kb in length in both lines, with no significant change in level detected (Fig. 6 ). Hybridization with the pC1-2 probe showed an intense smear of RNAs in both lines as typically seen for VL30 RNAs (data not shown). No differences between the lines was apparent. Figure 6 Northern blot analysis of mRNAs in parental Rat2 and mutant R4-7 cell lines. RNA preparations from the indicated cells were separated, blotted, and hybridized with a 32P-labeled pB1-11 probe. The major mRNA at ~3.0 kb is indicated. The position of the 28S and 18S rRNA markers are indicated on the left. Although the levels of the CAPER mRNA was not detectably altered in the R4-7 line, it remained possible that the gene and its transcripts harbored point mutations that were responsible for the virus resistance. To test this possibility, CAPER cDNAs were isolated from the R4-7 and Rat2 cells by RT-PCR, and the amplified sequences were cloned into the TOPO vector. Ten cDNA clones from each line were recovered and sequenced. Clones of three distinct structures were recovered from each line, likely arising by alternative splicing, but the sequences of the corresponding clones from the two lines were identical (data not shown). These results suggest that the CAPER gene is likely not mutated in the R4-7 line. Nevertheless, to test whether any of these cDNAs could alter virus susceptibility, the cDNA inserts from both R4-7 and Rat2 cells were transferred into the expression vector pcDNA3.1/zeo (see Methods). Overexpression of the various CAPER cDNAs from the Rat2 cells in R4-7 cells did not restore virus susceptibility, and overexpression of the corresponding cDNAs from the R4-7 cells did not induce virus resistance. Thus, of the various CAPER expression constructs, only the original pB1-11 antisense DNA had biological activity. Discussion The results here document the development of an effective procedure for the isolation of cDNAs that allow virus infection of virus-resistant cells. The key feature is the repeated infection of the parental resistant line with viral vectors carrying distinct selectable markers, and has become possible only with the development of a multitude of such markers. In this way, rare susceptible cells in the R4-7 population are enriched by as much as 10 to 100 fold in each round of selection. The protocol should allow the recovery of DNAs that confer susceptibility from any large library, if present at an abundance of perhaps at least one in 106 clones. The system can only work if a single DNA is sufficient to enhance virus susceptibility. Once cell lines with restored virus sensitivity were isolated, the recovery of the cDNAs from genomic DNA and the screening for active clones were relatively straightforward. The identities of the two sequences in the active cDNAs isolated here were surprising and the mechanisms of action of the two distinct clones remains mysterious. Both are highly potent, restoring virus susceptibility essentially to wild-type levels (figure 2 ). The time of the block to replication in the R4-7 line that is overcome by these two DNAs is very early after virus entry, before the initiation of reverse transcription by the incoming virus [ 6 ]. One possibility is that the mutant line fails to uncoat the virions sufficiently to allow deoxyribonucleotides into the core. In this scenario the cDNAs would somehow facilitate the uncoating process or inhibit a block to uncoating. HCC1.3 and HCC1.4 are two closely related cDNAs first recovered from a patient with hepatocellular carcinoma [ 7 ]. The encoded protein was a prominent nuclear autoantigen. The deduced amino acid sequences contain an arginine/serine rich domain and three ribonucleoprotein consensus sequence domains, often found in RNA splicing factors; they show weak homology to S. pombe GAR2, a nuclear protein. A later report demonstrated that the gene product interacted with the transcriptional activators AP-1 and the estrogen receptors ERa and ERß, and had potent cotransactivation activity; the gene was renamed CAPER, for coactivator of AP-1 and ER [ 8 ]. The antisense orientation of the pB1-11 cDNA suggests that its mechanism of action might be to lower the level of the endogenous sense mRNAs and the encoded proteins produced from the CAPER gene. We were unable to directly assess the level of the mRNAs in the presence of the antisense cDNA by Northern blots because the level of expression of the antisense RNA was so much higher than the endogenous mRNA that these transcripts were obscured. However, it is possible that the reduction in levels of a protein factor involved in regulation of transcription could elicit profound changes in the patterns of gene expression in the cell. We cannot rule out the remote possibility that a cryptic promoter results in some production of sense mRNA, and a protein fragment with biological activity, from the pB1-11 cDNA. Whatever the mechanism of action of the pB1-11 cDNA, it is unlikely that the endogenous CAPER gene is the locus of the original virus resistance mutation in the R4-7 line. The levels of the major mRNA are similar in the mutant and the wild-type parent (Fig. 6 ), and sequence analysis of a variety of cDNAs from parent and mutant lines did not uncover any mutations. Further, the overexpression of the CAPER cDNAs from R4-7 did not cause resistance, and the overexpression of the wild-type CAPER cDNA did not suppress the resistance. Rather, the antisense cDNA must correct the phenotype indirectly, likely through effects on gene expression. The localization of the HCC1.3/1.4 or CAPER protein in the nucleus [ 7 ] rather than at the site of virus arrest also suggests that its mechanism is indirect. Possibly CAPER acts to maintain a program of cytoplasmic protein expression that blocks virus infection in the R4-7 mutant line. Figure 5 Sequence alignment of pB1-11 and pC1-2 DNA inserts with similar sequences from NCBI database. The insert of pB1-11 (855 bp) is an antisense sequence match to the central portion of the CAPER mRNA [8], and that of pC1-2 (1407 bp) is a sense sequence match to a portion of the VL30 endogenous retrovirus-like element [17]. The VL30 elements are a very large family of endogenous virus-like genes found in both mouse and rat genomes [ 10 - 14 ]. The various elements are dispersed and have significantly divergent sequences. Though gag - and pol -related sequences are often recognizable, nearly all the elements are grossly defective, with multiple frameshift and premature termination mutations interrupting the open reading frames. In addition, the majority of the elements have suffered deletions of various regions relative to the longer family members. Thus, while many of the elements are highly transcribed in rodent cell lines, very few of the transcripts code for protein products of significant length. However, the VL30 RNAs often contain recognition elements for packaging into virion particles encoded by murine leukemia viruses, signals for initiation of DNA synthesis and strong stop DNA translocation, and termini recognized by viral integrase proteins, and thus are competent for transfer by replication-competent viruses acting as helpers. The sequence of the insert in pC1-2 corresponds to a portion of the retroviral pol gene, specifically the integrase coding region, but is typical of the VL30s in containing no long ORF; furthermore, known cis -acting regions needed for replication are absent. We suppose that the RNA itself may be responsible for the activity, perhaps by binding some cellular protein. The region of the VL30 genome present in pC1-2 – the 3' portion of the pol gene – is not known to contain a binding site for any particular protein. The corresponding region of replication-competent viruses, however, would normally contain the splice acceptor site for the envelope mRNA. Although many VL30 elements do not contain env genes, and although the splice acceptor sites are not readily apparent in the pC1-2 sequence, the transcript might be hypothesized to bind splicing machinery. In this scenario, a possible mechanism of action of the clone is for the overexpressed RNA to bind up a splicing factor or other RNA binding protein, titering out the free protein and removing it from solution. If this factor were responsible for the viral resistance of the R4-7, either directly or indirectly, the binding might relieve that block. The mechanism would be surprising only because the endogenous VL30 RNAs are already so abundant in Rat2 cells, and they are not able to suppress the block to infection. However, the pC1-2 sequence must be an unusual element, in that some distinctive aspect of its sequence must be responsible for its peculiar biological activity. Perhaps identifying proteins that bind to the pC1-2 transcript would be informative. The mechanism of resistance exhibited by the R4-7 line remains uncertain. The block is early but likely not at virus entry: it occurs whether ecotropic envelope, amphotropic envelope, or even the VSV G protein is used for entry [ 6 ]. Further, the block is unlikely to involve VSV G function, since the cells are susceptible to infection by VSV itself (J.-W. Carroll and M. MacDonald, Rockefeller University, unpublished observation). The early block occurs at a similar stage of infection – before reverse transcription – as the dominant block induced by TRIM5a, a gene responsible for retrovirus resistance in primates [ 5 ]. There are no other indications, however, that the two blocks are related. We have observed that the R4-7 cells exhibit a slightly different morphology than the parental Rat2 cells, being somewhat more rounded and more easily detached from the substrate during trypsinization. This phenotype could in principle be unrelated to the virus resistance, since the cells were subjected to heavy chemical mutagenesis before their isolation [ 6 ]. However, the R4-7 cells expressing both pB1-11 and pC1-2 were restored to a flatter morphology, much closer to that of the parental line, suggesting that the two phenotypes may be causally linked. If this notion is correct, changes in the cytoskeleton may be involved in the resistance. Further analysis of the R4-7 cells by gene expression profiling may help reveal the basis for its behaviors. Conclusions The power of genetic selections in mammalian cells for alterations in virus susceptibility is increasing rapidly. We believe that selections like the one devised here will be applicable to the isolation of suppressors of other blocks to infection, including the prototypical Fv1 gene [ 3 ], the APOBEC3G cytosine deaminase [ 4 ], and the TRIM5a gene [ 5 ]. The identity of such suppressors may provide important clues into the mechanism of their action and regulation. Methods Cell lines, cell culture The Rat2 cell line is a TK-negative fibroblast line that is highly sensitive to MuLV infection. RC-2 is a subclone isolated after mutagen exposure but also sensitive to virus, and was used in many experiments as a wild-type control line. Lines R3-2 and R4-7 are virus-resistant mutants of Rat2 isolated after exposure to ICR-191 [ 6 ]. 293T cells are human embryonic kidney cells transformed by adenovirus E1 and also expressing SV40 T antigen. All these lines were maintained in DMEM with 10% fetal calf serum. DNA transformations A rat kidney cDNA library in the pcDNAI vector [ 9 ] was purchased from Invitrogen (Carlsbad, CA). To increase the library transfection efficiency and maximize the integrity of the cDNAs, the library was digested in the vector sequence with the restriction enzyme SfiI and then religated. R4-7 cells in ten 10-cm dishes were cotransformed with 20 ug of the religated library DNA and 2 ug of pGK-puro plasmid by calcium phosphate-mediated transformation. Cells expressing transformed DNAs were selected by growth in culture medium containing 5 ug/ml puromycin. Each transformed cell was expected to receive about 2–10 different cDNAs. The cultures were expanded before the selections for virus susceptibility such that a pool of 105 cells contained about 2000 distinct puromycin resistant clones. Thus, there were about 50 sibling cells of each transformant in the pools at the time of selection for virus susceptibility. Retrovirus preparations Eco-neo or MuLV-N2 virus [ 20 ]; Eco-TK virus, and Eco-GFP virus [ 21 ] were as previously described [ 6 ]. To generate Eco-His virus, the Neo resistance gene in the N2 vector was replaced with His resistance gene, and GP+E86 packaging cells [ 22 ] were stably transformed with the resulting vector DNA. Recipients were selected with histidinol and the resistant cells were pooled to generate Eco-His producer cells. Typical titers of the virus preparations on Rat2 cells were 107 cfu/ml for N2 virus; 2 × 104 for TK virus; 2 × 106 for Eco-His virus; and 105 cfu/ml for Eco-GFP virus. Viral transduction and selection Selections for virus sensitive cells were performed by infecting approximately 105 R4-7 cells per 10-cm dish in each round, with virus titers determined by infection of Rat2 cells. In the first round approximately 104 cfu of N2 virus were applied, and transductants were selected with 800 ug/ml G418. In the second round, approximately 2 × 103 cfu of Eco-TK virus were used, and transductants were selected with HAT medium (Gibco). In the third round, approximately 200 cfu of Eco-His virus were applied, and transductants were selected with medium containing 1 mg/ml histidinol. The multiplicities of all these infections with the selecting viruses were kept low, at less than 0.1 These low MOIs were required because infection of R4-7 cells at high MOI can override the block, perhaps by saturation of a titratable factor. Even at this low MOI, the presence of many siblings of each transformant implied that most cDNAs in the pool were tested for inducing virus susceptibility. Polymerase chain reactions cDNA inserts from the expression library were recovered from cell lines by PCR as follows. Genomic DNA was extracted (DNAeasy kit, Qiagen) and subjected to PCR with primers hybridizing upstream from the CMV promoter (sequence 5'-GGGCCAGATATACGCGTT-3') and downstream from the poly(A) addition region (sequence 5'-AATTTGTGATGCTAT-3') of the pcDNAI vector. Conditions for the PCR were: ten cycles of 94°C for 10 sec, 55°C for 30 sec, and 68°C for 3 min, followed by 20 cycles of the same conditions but with an increase in the polymerase reaction time of 5 sec in each cycle. The amplified DNAs were cloned directly into the TOPO vector (Invitrogen) and used to transform DH10b bacteria to ampicillin resistance. DNAs were isolated from approximately fifty bacterial colonies for each original cell line. CAPER cDNAs were prepared from RC-2 mRNA preparations by standard RT-PCR methods using primers spanning the entire ORF (sequences: 5'-ATATAGCTTAAGGCCACCATGGCAGACGATATTGATAT-3' and 5'-ATATAGGCGGCCGCTCATCGTCTACTTGGAAC-3'), and cloned into the pcDNA3.1/zeo expression plasmid using AflII and NotI restriction sites. Authors' contributions GG carried out all the experiments and participated in their design. SPG participated in the experimental design and drafted the manuscript. Both authors read and approved the final manuscript.

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536007

A Gradual Process of Recombination Restriction in the Evolutionary History of the Sex Chromosomes in Dioecious Plants

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 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.

Introduction Newly evolved sex chromosome systems, such as those in plants [ 1 ] and fish [ 2 ] allow study of the evolutionary processes causing degeneration of Y chromosomes. The genetic theory of sex chromosome evolution [ 3 ] predicts that initially one part of a chromosome pair containing the sex-determining genes evolves reduced recombination. Two questions are then particularly interesting. First, how is recombination suppressed throughout most of the initially homologous X and Y chromosomes, as in mammalian and Drosophila sex chromosomes and some plants [ 1 ], but not others [ 4 ]? Second, why does recombination suppression lead to genetic degeneration? Processes leading to degeneration in large nonrecombining genome regions have been well studied theoretically [ 5 ], and empirical data on the first stages of degeneration are starting to be obtained from the plant genus Silene [ 6 , 7 ] and from the neo-sex chromosomes of Drosophila miranda [ 8 ]. Recent neo-sex chromosome systems in Drosophila are excellent for studying the rate and causes of degeneration, but cannot shed light on question (i). 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” [ 9 , 10 ]. Part of the reason for different sequence divergence is that mammalian sex chromosomes are ancient neo-sex chromosomes [ 11 ]. In addition, the “strata” suggest a series of Y inversions disrupting X-Y recombination [ 9 ]. Strata have also been found in the chicken Z chromosome, which, like the Y, is present only in one sex (females in birds) and does not recombine with its homolog [ 12 ]. To further understand the evolution of suppressed recombination between X and Y chromosomes, we describe results from the plant genus Silene . This genus is a model for the study of plant sex chromosome evolution, since the sex chromosomes evolved recently [ 7 , 13 ]. One group of closely related dioecious 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 [ 1 , 14 ], while many Silene species are hermaphroditic or gynodioecious (i.e., some plants bear hermaphrodite flowers and others female flowers). Dioecy and sex chromosomes thus probably evolved within this genus [ 13 ]. All diploid Silene species have n = 12 chromosomes [ 15 ], so there is no evidence for neo-sex chromosome formation, although an autosomal region of unknown size has been duplicated on the Y [ 16 ]. Several sex-linked genes from S. latifolia have recently been identified and sequenced ( Table 1 ), allowing progress in understanding the evolution of these sex chromosomes. Four genes have functional X- and Y-linked homologues. Very different X-Y divergence of two gene pairs suggested that different Y chromosome regions probably ceased recombining at different times in these species' evolutionary history [ 17 ]; testing this hypothesis requires knowing the genes' locations on the sex chromosomes. We here describe a new gene pair in S. latifolia, SlX3 and SlY3 (together termed locus 3; Table 1 ), and present the first genetic map for the X chromosomes in three dioecious species. Divergence between the X and Y chromosomal copies of the different genes indeed correlates with increased distance from the PAR, but the time scale is very different from that in mammals. Three genes (locus 3, the SlX4-SlY4 pair [termed locus 4], and DD44 ) 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. Table 1 Description of the Four X-Y Gene Pairs and the PAR Marker Used in the Analyses a For the four genes, the alignments include coding sequences of both X and Y copies in S. latifolia , S. dioica, and S. diclinis , and the orthologous sequence from a close outgroup ( S. vulgaris or S. noctiflora ). The values correspond to the number of sites with no gaps or ambiguous bases). Values in parentheses indicate the numbers of diverged sites b DD44 is also single-copy in S. latifolia [ 20 ], but at least two copies are found in other Silene species, including S. dioica (V. Laporte, unpublished data) and other species (J. Ironside, Univ. of Birmingham, UK, unpublished data).In our S. dioica material, there are three tightly linked X-linked copies (B. Janousek, unpublished data). Thus this duplication does not affect our mapping conclusions Results Characterization of Gene 3 Locus 3 was identified from S. 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 [ 18 ]. Thus, locus 3 is probably a sex-linked housekeeping gene, like the previously characterized X-Y-linked genes in S. latifolia [ 17 , 19 ]. Phylogenetic Analysis of the Four Sex-Linked Genes Figure 1 shows the estimated phylogenetic relationships based on single X and Y copies of the four loci from each species in which sex linkage has been confirmed. Except for locus 1 (discussed below), each gene falls into distinct X and Y clades, showing that these genes ceased recombining well before the split of the present dioecious species, consistent with large X-Y divergence in both S. latifolia and S. dioica [ 17 , 20 ]. Not surprisingly for such closely related species [ 13 ], the phylogenies of the three dioecious species are inconsistent for these genes. For example, one Y-linked gene supports each of the possible clades latifolia-dioica, latifolia-diclinis, and dioica-diclinis ( Figure 1 ). Figure 1 Phylogenetic Trees for DD44 and Loci 1, 3, and 4 All trees were estimated from coding sequence alignments (using all sites except gaps) under the BIONJ method with Kimura-two-parameters corrected distances, using Phylo_Win software [ 43 ]. Other methods (maximum parsimony and ML) give very similar results. Branch lengths correspond to total sequence divergence under the model assumed (see scale bars). Values indicated at the nodes are bootstrap values exceeding 50% (based on 500 replicates). S. vulgaris was used as an outgroup (except for locus 1, for which a closer outgroup, S. noctiflora , was used). Dic = S. diclinis , Dio = S. dioica , Lat = S. latifolia . The numbers of sites analyzed are in Table 1 . Gene 1 X-Y divergence is much less than that of the other genes studied [ 17 ]. We therefore tested whether divergence between the X and Y copies started before or after the speciation event. The grouping of this gene by species in Figure 1 suggests independent X1-Y1 divergence in the three dioecious lineages. For such closely related sequences, however, analysis using single X and Y sequences from each species confounds fixed differences between species with within-species polymorphisms, and can be misleading, given that S. latifolia is a highly variable species [ 21 ]. Ancestral polymorphisms persisting through the speciation event also obscure close phylogenetic relationships, particularly inferences using X-linked genes, which have large within-species polymorphism [ 7 , 22 ]. Finally, the well-documented introgression between S. latifolia and S. dioica [ 23 ] may contribute to the phylogenetic discrepancies. We therefore analyzed the X1-Y1 gene pair separately, using multiple sequences from two species. If X1-Y1 divergence started sufficiently long before the species split, some sites should share the same fixed differences between X and Y sequences in both S. latifolia and S. dioica . The number of such sites depends on the amount of time after recombination ceased; for the genes other than gene 1, this number is large (see above), 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 [ 24 , 25 ]. However, because the dioecious Silene species are very closely related [ 13 ], there are few fixed differences, and, using global gap removal to be conservative, none between the 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 (some may actually be polymorphic in this species). 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 ( Figure 2 ), implying suppression of X-Y recombination within these species. This suggests the possibility of independent cessation of recombination after speciation. However, we cannot exclude the possibility that recombination stopped shortly before the dioecious species split. Under this alternative, if the Y1 genes retained some polymorphism, variants in the Y1 genes would become fixed differences when Y chromosome diversity was lost within each species; according to this hypothesis, however, each species must, by chance, have retained Y1 variants closest to its own X sequences. Figure 2 Phylogenetic Tree for Gene 1, including Within-Species Diversity The tree was estimated using PHYML software [ 52 ] from a DNA alignment including coding sequences and introns of 12 X and 11 Y S. dioica alleles, and 26 X and 22 Y from S. latifolia [ 22 ]. There were 973 sites, excluding gap regions, among which 154 variable sites were used. The estimation used the BIONJ algorithm with global gap removal. The percentage of invariant sites, the transition-transversion ratio, and the α parameter of a γ distribution of substitution rates, were estimated by the program, and we assumed four categories of evolutionary rates, to take into account the different evolutionary dynamics of coding and intron sites. The HKY substitution model was used. Bootstrap values exceeding 50% (based on 100 replicates) are indicated at the nodes, but some bootstrap values exceeding 50% for terminal nodes are omitted because of lack of space. Correlation between X-Y Divergence and Position on the X Chromosome The gene order is the same in S. latifolia and in the S. diclinis × S. latifolia hybrid ( Figure 3 A). Locus 1 is closest to the PAR. If the S. diclinis and S. latifolia maps differed by an inversion or other rearrangement, the map using hybrid parents should contain a non-recombining region; this was not observed. Thus, the gene order determined in the S. diclinis x S. latifolia hybrid must also apply in S. diclinis . In S. dioica , however, the map order of locus 1 and DD44 is reversed relative to the other species ( Figure 3 A). Figure 3 X Genetic Maps in the Three Dioecious Species versus Plot of Synonymous Divergence (A) Gene orders and the map positions of the genes. The PAR is not drawn to scale, as there is only one marker, and the map shows only the portion of the X chromosome containing our marker genes. Vertical lines connect the homologous genes in the three species, and the chromosomal rearrangement in S. dioica , are shown by crossed lines for locus 1 and DD44 . (B) Plot of synonymous divergence between X and Y pairs ( dS X-Y ), estimated using PAML, against the map position using the gene order in S. latifolia and S. diclinis (see text). Synonymous divergences are statistically significantly distinguished for the following three groups of genes: locus 1, the DD44 gene, and loci 3 and 4 (see text). The figure also indicates minimum and maximum X-Y divergence time estimates for the genes, assuming a molecular clock and 1.8 × 10 –8 synonymous substitutions per synonymous site per year (the mean of the values 1.4 × 10 –8 and 2.2 × 10 –8 discussed in the text). Synonymous divergence (dS) between the X and Y sequences of S. latifolia and S. diclinis ( dS X-Y ) correlates with the gene's distance from the PAR in the X chromosome genetic map ( Figure 3 B). X-Y synonymous divergence in S. latifolia does not differ significantly between genes 3 and 4, but these genes' synonymous divergence values differ significantly from that for genes 1 or DD44 (with p < 0.01). 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 [ 26 ]. Comparing Sequence Divergence of X and Y Copies Analysis of the coding sequences shows that all four Y-linked genes appear to encode functional sequences; in each case, the nonsynonymous divergence (dN) was less than dS for divergence between X and Y sequences ( dN / dS values in Table 2 ); although dN is high for the DD44 gene pair, it is considerably below dS . These results are consistent with cDNA representation of all sequences except the Y-linked copy of gene 3; despite repeated attempts, this copy never amplified from leaf cDNA, whereas the X chromosome copy amplified consistently (see Materials and Methods ). Table 2 Comparison of Evolutionary Rates in the X and Y Clades LR tests between models with X = Y and X ≠ Y were performed using the ML-based software HyPhy. R YX is the X/Y ratio of dS or dN values. For the dN/dS analyses, the values in the table are the dN/dS values for X and Y lineages Nonsignificant differences are indicated as “X = Y.” a Significance of the LR test is p < 5 × 10 −2 b Significance of the LR test is p < 5 × 10 −3 The Y copies of all genes have higher dS, dN, and dN / dS values than the X-linked copies, except for DD44 ( Table 2 ). However, the differences are significant only for dN . The differences in the numbers of synonymous differences are also nonsignificant, taking into account diversity within species. Synonymous site evolution is significantly faster in DD44 -X than in DD44 -Y, in contrast to the other genes, where the Y tends to evolve faster than X copies (although the differences are nonsignificant; Table 2 ). Exon 1 of DD44 is particularly divergent [ 20 ], but our results for this gene are similar if we exclude this exon (unpublished data). The results for gene 1 presented in Table 2 cannot be interpreted reliably because of polymorphisms within the species (see above), which would cause overestimation of numbers of substitutions. Overall, therefore, dN is clearly higher in the Y copies of genes 3 and 4, but its mutation rate is not higher, since X-Y differences in dS are nonsignificant; combining the probabilities from the likelihood ratio (LR) tests for these two genes, the dN / dS difference between Y and X is highly significant (χ 2 = 11.7, with 4 degrees of freedom). Our observation of similar dS values contrasts with previous analyses [ 27 ], probably because we used only synonymous sites, rather than synonymous plus noncoding sites. The S. diclinis Y3 gene also seems to evolve faster than the other Y3 genes (see Figure 1 ); for this gene, the difference is seen for both synonymous sites (6-fold increase) and nonsynonymous ones (3.6-fold increase), but it is significant only for synonymous sites. Discussion Progressive Differentiation of the X and Y Chromosomes The correlation of dS X-Y of these dioecious plants with distances from the PAR in the X chromosome genetic map suggests that suppression of recombination between X and Y genes progressed, starting from an “ancient” sex chromosomal region (presumably containing the primary sex determining loci) and moving toward the current PAR. This pattern resembles the “evolutionary strata” for mammalian X-Y gene pairs based on K s values, a measure of divergence per site similar to dS [ 9 , 10 ]. However, the time scale is much shorter for the plant sex chromosomes. The largest dS X-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 (mean K s values 8% and 30%, respectively); 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 [ 9 , 11 ]. The 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 [ 16 ]; all these genes appear to be functional. This divergence is also similar to that for MROS3 -X/Y, whose Y-linked copy is degenerated [ 6 ]. Unless genes with higher divergence are discovered in the male-determining region of Y chromosomes of dioecious Silene species, the Silene sex chromosomes must have evolved much more recently than mammalian sex chromosomes. There are few reliable absolute molecular clock calibrations in plants [ 28 ], and none for 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 [ 29 ], and a similar value was estimated for Ipomoea [ 30 ]. Using synonymous site divergence values suggests an age estimate of 5–10 MYA for the sex chromosomes of the 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 [ 31 ]. Thus, a greater age cannot be excluded. It is nevertheless clear that the X and Y copies of genes3, 4, and 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 ( Figure 1 ). If this event occurred shortly before the dioecious species split, our results show that it must have happened in such a way that the Y-linked copy of gene 1 retained some diversity, in other words, by some mechanism other than an inversion (see below). Suppression of X-Y recombination (diminution of the PAR) has occurred quickly, and probably independently, in different mammalian and bird lineages [ 24 , 25 , 32 ]. 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 [ 9 ]. Inversions exist between human X and Y chromosomes [ 10 ], but have not yet been explicitly related to the strata, so they may not be the sole cause of the divergence differences. Moreover, new pairs of X-Y linked genes recently analyzed do not suggest clear-cut boundaries between strata; divergence values for strata 3 and 4 genes are not discontinuous [ 10 ]. Finally, the amelogenin gene, at the strata 3–4 boundary, is not disrupted by an inversion [ 25 ]. Thus, gradual modification of recombination rates may have played a part in reducing recombination in some regions of the X-Y pair, in both Silene and mammals. Testing this for the dioecious Silene species requires a Y-chromosome map. The present map, based on deletion mutants in Silene [ 20 , 33 ], requires further markers and deletions for detailed comparison with the genetic map of the X chromosome. S. latifolia Y deletion mutants with altered meiotic X-Y pairing (unpublished data) 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 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 Silene . 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 [ 34 ], although it seems unlikely that all three dioecious species studied here could recently have acquired advantageous Y-linked genes. Involvement of selectively favored inversions causing the formerly pseudoautosomal gene 1 to become Y-linked might be detectable from sequence data, since a selective sweep would be expected. This would contribute to low diversity for all the Y-linked genes, consistent with the long branches in Y lineages ( Figures 1 and 2 ). However, although Y-linked diversity is low, there is no evidence of such events in the frequency spectra of the genes [ 7 , 22 ]. Degeneration of the Y Chromosome Our analyses suggest that both reduction of recombination and Y degeneration may be in progress for Silene sex chromosomes. Degeneration is likely, since genotypes with a Y but no X chromosome are inviable [ 1 , 35 ], but so far, only one degenerated plant Y-linked gene has been found, MROS3 -Y in S. latifolia [ 6 ]. The extent of genetic degeneration and gene loss in the 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 [ 4 ]. Some degeneration of Y-linked genes in 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 ( Table 2 ). Differences in dS are systematically lower than in dN (the ratio of dS values for X and Y lineages is close to 1, but dN is roughly 3-fold larger overall for Y lineages). 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. These observations, plus those for gene 1 ( see Results ), suggest that Y copies of genes loci 1, 3, and 4 evolve faster than X copies, due either to a higher rate of fixation of advantageous mutations in the Y copies or to accumulation of slightly deleterious amino acid variants in the Y copies (Y degeneration). To discriminate between these hypotheses, McDonald-Kreitman tests can be done to compare fixed differences (divergence) and polymorphisms and test for an excess of selectively advantageous nonsynonymous substitutions [ 36 ]. At present, this is possible only for genes 1 and 4; there were no nonsynonymous polymorphisms for DD44, 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 [ 27 ]. 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 [ 7 , 22 ]. This difference is predicted in a degenerating Y chromosome, because various hitchhiking processes leading to degeneration, including selective sweeps, background selection, and weak Hill-Robertson effects [ 5 ] reduce diversity, even at loci that are not themselves degenerating. Why is degeneration so slight for our Silene 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 [ 8 ], which are much younger than the 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 [ 4 ]. More likely, the Y-linked genes we have studied (which are all housekeeping genes) are under selective constraints. The lower effective population size of Y-linked genes, and thus the expected reduced efficacy of selection ([ 5 ]) may thus be too slight to allow the Y copies of these genes to lose function, but merely allows higher amino acid substitution. Our findings parallel those for most loci on the D. miranda neo-Y chromosome [ 37 ], the bird W chromosome [ 38 ], and in other situations in which effective population size in reduced, such as protein-coding genes of the endosymbiont Buchnera [ 39 ]. In all these cases, genes evolving without recombination retain homology with their ancestral copies, but undergo faster amino acid replacement (including several frameshift and deletion mutations in the D. miranda neo-Y [ 37 ]), suggesting that the common factor is weakened ability of natural selection to preserve adaptation. Materials and Methods Plants used and nucleic acid extraction. S. latifolia plants were from Edinburgh (D. Charlesworth personal collection) 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 [ 19 ]. For RT-PCR from total leaf RNA, first-strand cDNA was reverse transcribed using RevertAid M-MuLV RT (Fermentas, Vinius, Lithuania) and the oligo-dT primer T11VN (5′- TTTTTTTTTTTVN-3′). Isolation of SlX3 / SlY3 Locus 3 was identified in S. latifolia by the approach that yielded loci 1 ( SlX1 / SlY1 [ 19 ]) and 4 ( SlX4 / SlY4 [ 17 ]). From an initial partial cDNA sequence of a clone that hybridized to a probe containing Y-linked sequences, both 3′ and 5′ RACE-PCR were performed [ 19 ], and the final coding sequence was obtained by sequencing the RT-PCR product obtained using primers 11S10 (5′- ATCACCATCATCATTTCCACC-3′) and 11AS11 (5′- CAGTGAAATCTTTCATTTACCACG-3′). Segregation analysis (see below) showed that this sequence corresponds to SlX3 . The SlY3 sequence was obtained from genomic DNA by PCR genomic walking [ 40 ], using the specific primers 11AS15 (5′- TCAGTGTCTCCTTGAGTTTCTTGCAC-3′) and 11AS15C (5′- TGCACAAGATGGACTGGCTACAATACG-3′) for the first and second PCR, respectively, and Ex Taq polymerase (Takara Bio, Otsu, Shiga, Japan) for both PCR reactions. Similarly to gene 4 [ 17 ], Southern blot analysis showed that gene 3 is present as a single copy in the S. latifolia genome. Amplification and sequencing of orthologous sequences. The orthologs of each of the four gene pairs in Table 1 were amplified in S. dioica, S. diclinis, and S. noctiflora or S. vulgaris using primers designed from S. latifolia sequences ( Table 3 , which also provides GenBank accession numbers). All sequences were amplified from cDNA, except for 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 ( Table 3 ); and a final extension at 72 °C for 5 min. Table 3 Primers and Annealing Temperatures Used for RT-PCR Amplification of the Genes Listed in Table 1 All genes, except gene 1, are single-copy (see Table 1 ). In S. latifolia, three or four copies of gene 1 are detected in Southern blots [ 19 ], and the A. thaliana genome has five copies, AtMSI4 (GenBank accession number AF028711) being the probable ortholog of SlY / X1 . In S. latifolia, some of the copies have been shown not to be sex-linked (F. Monèger, personal communication). 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 [ 20 ]. For locus 3, the 3′ two-thirds of the coding sequences of 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 (Applied Biosystems, Foster City, California, United States). Sex linkage and genetic mapping. Sex linkage of three gene pairs studied here was previously demonstrated in either S. dioica or S. latifolia . We have now confirmed sex linkage of all four loci by segregation analysis in all three dioecious species ( Figure 4 ), and, for genes 1, 4, and DD44, by population studies using allele-specific PCR reactions to show that the putative Y-linked alleles are consistently present only in males, while the X-linked ones amplify in both sexes [ 17 , 22 ] and unpublished data). Figure 4 Segregation Analysis of Locus 3 in S. dioica To test for sex linkage, the female and male parents of the family and six progeny of each sex were genotyped. Genomic DNA preparations from these plants were used in PCR amplifications. The PCR products were separated by electrophoresis and visualized using ethidium bromide. (A) With primers specific for the X3 allele, the restriction enzyme RsaI reveals an allelic polymorphism. The maternal plant is heterozygous and has both cut and uncut alleles while the male parent has only the uncut allele. Female progeny always have the uncut allele (like the father), and male progeny have one of the maternal alleles, but never both, corresponding to the expected segregation of the X chromosome. (B) Primers specific for the Y gametolog. A product amplifes only with male DNA, corresponding to the expected segregation of the Y chromosome. Only X-linked genes can be mapped genetically, because the Y chromosome recombines with the X only in the PAR. For each locus, gene-specific primers were used to amplify X alleles from genomic DNA of potential seed parents. The PCR product was directly sequenced and the chromatograms inspected for polymorphisms scorable by restriction enzyme digestion ( Table 4 ). Progeny of heterozygous mapping family females were sexed and genotyped for the maternal alleles. In S. diclinis, no suitable polymorphisms were found, so the loci were ordered in a S. diclinis × S. latifolia hybrid plant pollinated by a S. latifolia male. Table 4 Primers Used for Mapping Included here are primers used for mapping the four X-Y gene pairs and the PAR marker; nature of the polymorphisms used as markers (i.e., DFLP indicates length polymorphism of amplified fragments; restriction enzymes used for CAPS analysis are included in parentheses); and accession numbers of either the partial cDNA sequences obtained by RT-PCR (listed as X sequence, Y sequence) or the genomic sequence of the pseudoautosomal sequence. Details of PCR primer annealing temperatures for the different species are available from the authors on request. ND, no data a The annealing temperature for ScOPA09 was 55 °C, and elongation time was 1 min 30 s 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 S. dioica family in which this marker was originally developed [ 41 ]. The sequence encodes a protein with similarity to an Oryza sativa putative non-long terminal repeat reverse transcriptase (E value = 2.5 × 10 –10 ; accession number Q9FW98 in the UniProt/TrEMBL database, but contains stop codons. Primers (ScOPA09F1: 5′- GCAATTCACCATCCTCTGCT-3′ and ScOPA09R1: (5′- ATGGTCTTTGGGCCCTTATC-3′) 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 [ 42 ]. Thus the gene orders are well established; Table S1 gives estimated genetic distances between markers and their standard errors. Phylogenetic analysis. 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 ( http://pbil.univ-lyon1.fr/ ) [ 43 ]. Alignment lengths are given in Table 1 . Phylogenetic trees were estimated including all sites except those with gaps by neighbor joining (NJ), maximum parsimony, and maximum likelihood (ML), using Phylo_Win ( http://pbil.univ-lyon1.fr/ ) [ 43 ]. For NJ trees, we used Kimura two-parameter corrected distances; results with other distances corrected for multiple hits were similar, as the sequences are not highly diverged and have similar GC content (unpublished data). Branches were tested by bootstrapping (500 replicates). Trees were edited with NJplot ( http://pbil.univ-lyon1.fr/ ) [ 44 ] and TreeView ( http://taxonomy.zoology.gla.ac.uk/rod/treeview.html ) [ 45 ]. Divergence analysis. Both dS and dN site divergences were estimated using PAML 3.13 ( http://abacus.gene.ucl.ac.uk/software/paml.html ) [ 46 ] and JaDis ( http://pbil.univ-lyon1.fr/ ) [ 47 ]. Estimates of dS and dN are similar under various substitution models (namely, Goldman and Yang 1994 [ 46 ], Yang and Neilson 2000 [ 48 ], and Nei and Gojobori 1986 [ 49 ], implemented in PAML; and Li 1993 [ 50 ] using JaDis). We report values from the ML approach based on the Goldman and Yang 1994 codon-based model [ 46 ]. Values for dS or dN of X and Y sequences were compared using HyPhy 0.99 (Kosakovsky Pond, personal communication; http://www.hyphy.org , using the alignment and NJ tree for each gene, including the outgroup species ( Figure 4 ) to polarize the synonymous and nonsynonymous substitutions between X and Y genes into X-specific and Y-specific lineages, using ML. To build the likelihood function, we used the MG94xHKY85 codon-based substitution model with different transversion and transition rates. We compared dS values under two models for each gene. Model 1 (“relative synonymous rates”) expresses dS values for Y lineages as multiples of values for X lineages: R syn = dS Y / dS X . In model 2 (“equal synonymous rates”), R syn was constrained to be equal to 1 ( dS Y = dS X ). 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 R syn ). 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 (“shared and equal dN / dS ”) assumed ( dN / dS ) X = ( dN / dS ) Y . To test whether S. diclinis Y3 evolves faster than other Y3 sequences, we assumed a common R syn for S. dioica and S. latifolia, as in model 1 above, but added a further parameter, the dS Y / dS X 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. McDonald-Kreitman tests were done using DNAsp software, version 3.95 [ 51 ]. The divergence and polymorphism data used are from previous work and were available only for genes 1 and 4 [ 17 ]; there were no nonsynonymous polymorphisms for DD44, and no diversity data are yet available for gene 3. 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 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. Analysis of gene 1. 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 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. As this dataset includes the first approximately 2,000 sites of gene 1, including coding and intron sequences [ 22 ], a more sophisticated model for sequence evolution is required for phylogenetic analysis than for the coding sequences analyzed above. We estimated the percentage of invariant sites, and the transition to transversion ratio, and fitted a GAMMA distribution, estimating its ALPHA parameter with four categories of sites evolving at different rates, using the HKY (Hasegawa, Kishino, Yano [ 52 ]) model as the global substitution model. The tree was estimated using NJ (BIONJ) with global gap removal, using a fast ML-based program, PHYML ( http://www.lirmm.fr/guindon/phyml.html ) [ 53 ], excluding fixed differences from the multiple alignment (to avoid conflicting phylogenetic signals between fixed and polymorphic differences). The statistical support for the tree was estimated by bootstrapping (100 replicates), using SEQBOOT, followed by CONSENSE to make a consensus tree with the resulting 100 PHYML trees. Supporting Information Table S1 Recombination Fractions (Rf) between the Loci, and Standard Errors of Rf Values (36 KB DOC). Click here for additional data file. Accession Numbers The GenBank ( 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.

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Effect of the G-308A polymorphism of the tumor necrosis factor (TNF)-α gene promoter site on plasma levels of TNF-α and C-reactive protein in smokers: a cross-sectional study

Background Plasma levels of tumor necrosis factor (TNF)-α and of C-reactive protein (CRP) are elevated in smokers. Previous studies failed to show an association between the G-308A polymorphism in the promoter region of the TNF-α gene and coronary artery disease (CAD). We investigated whether smoking would interact with the TNF-α G-308A polymorphism in determining plasma levels of TNF-α and CRP. Methods Study participants with a complete data set in terms of smoking and the TNF-α G-308A polymorphism were 300 middle-aged male and female industrial employees. After excluding 24 irregular smokers, analyses were performed on 198 "non-smokers" (life-long non-smokers or subjects who quit smoking >6 months ago) as compared to 78 "regular smokers" (subjects currently smoking >3 cigarettes/day). All subjects had a fasting morning blood draw to measure plasma levels of TNF-α and CRP by high-sensitive enzyme-linked immunosorbent assays. Results The cardiovascular risk factor adjusted analysis regressing log-transformed CRP levels against smoking status, genotype, and smoking-status-genotype interaction revealed a significant main effect for smoking status (F 1,250 = 5.67, p = .018) but not for genotype (F 1,250 = 0.33, p = .57). The interaction-term between genotype and smoking status was not significant (F 1,250 = 0.09, p = .76). The fully adjusted model with plasma TNF-α failed to show significant main effects for smoking and genotype, as well as for the smoking-status-genotype interaction. Conclusions The findings suggest that the TNF-α G-308A polymorphism does not mediate the effect of smoking on plasma CRP levels. It remains to be seen whether other genetic polymorphisms along the inflammatory pathway may modulate vascular risk in smokers.

Background Coronary artery disease (CAD) is associated with chronic inflammatory processes in which blood-derived macrophages play a key role [ 1 ]. Tumor necrosis factor (TNF)-α is essentially produced by monocytes and macrophages, and, in turn, it is the strongest known paracrine activator of monocytes and macrophages [ 2 ]. Upon stimulation, these cells secrete a variety of products including interleukin (IL)-6 stimulating the liver to produce the acute phase reactant C-reactive protein (CRP) [ 3 ]. CRP is an acknowledged indicator of increased systemic inflammation across a wide range of diseases [ 4 ]. TNF-α and CRP both are found in considerable quantities in atherosclerotic lesions [ 5 , 6 ], and they have also been associated with increased cardiovascular risk in numerous large population-based studies [[ 7 , 8 ]; for review]. Smoking is one of the strongest cardiovascular risk factors for atherosclerotic diseases [ 9 ]. Several studies have revealed increased plasma levels of TNF-α and of CRP in smokers as compared to non-smokers [ 10 , 11 ], suggesting that part of the coronary risk associated with smoking may relate to increased inflammatory activity. However, the prevalence of cardiovascular disease varies substantially among smoking individuals [ 12 ]. This could indicate that genetic factors are important determinants of the biological pathways linking smoking with cardiovascular disease risk [ 13 ]. In fact, it has been recently shown that the CC polymorphism in the promoter region of the CD14 gene (CD14 -159C/T) was associated with common carotid artery intima-media thickness in smokers, but not in non-smokers [ 14 ]. Similarly, in young and healthy individuals carrying the C allele of the interleukin-6 promoter polymorphism -174 genotype, those who smoked had higher leukocytes, lymphocytes, and monocytes than those who did not smoke [ 15 ]. Another candidate polymorphism that might mediate the cardiovascular risk with smoking, and that is at the initiation of the inflammatory cascade, is the G-308A polymorphism of the TNF-α gene promoter site. The TNF-α G-308A polymorphism is a single base pair polymorphism located at position 308 in the TNF-α gene that maps to human chromosome 6 (p21.1-p21.3) resulting in substitution of the nucleotide adenine (A) for guanine (G) [ 16 ]. In the present study, we investigated the possible impact of this polymorphism on the association between smoking severity and plasma levels of TNF-α and CRP in vivo . While the TNF-α G-308A polymorphism has been associated with increased production of TNF-α in vitro [ 17 , 18 ], previous studies failed to show an association of this polymorphism with CAD [ 19 - 25 ]. However, there is a dearth of data regarding possible interactions between environmental risk factors for cardiovascular disease (e.g., smoking) and the G-308A polymorphism on plasma levels of proinflammatory markers. It is conceivable that the TNF-α G-308A polymorphism alone is of negligible importance in CAD, but that the presence of certain environmental conditions (i.e., exposure to tobacco smoke) and specific alleles may influence CAD risk [ 26 - 28 ]. If alleles are randomly distributed between smokers and non-smokers, case-control studies not explicitly investigating the possible environmental-genetic interaction might fail to unravel the role of genetic polymorphisms in CAD. Therefore, we speculated that smoking would affect plasma TNF-α and CRP levels depending on the TNF-α genotype. If so, the genotype might affect the rate of disease progression rather than the existence of atherosclerotic lesions. Methods Study participants The study was conducted as part of a larger project in an airplane manufacturing plant in southern Germany. From a total of 1,760 employees, participation was offered to a representative sample of 647 men and women. Of those, 537 (accrual rate 83%) volunteered to participate. The Institutional Review Board approved the study protocol. All 537 subjects completed questionnaires on medical and psychosocial health status. A subsample of 332 subjects agreed to have a variety of biological measures assessed. Three hundred of these individuals had complete data on smoking status, smoking history and the TNF-α G-308A gene polymorphism. Based on self-reported smoking history, we categorized life-long non-smokers or those, who had quit smoking for at least 6 months as „non-smokers" (n = 198), smokers who were currently smoking >3 cigarettes per day as "regular smokers" (n = 78), and smokers reporting to consume up to 3 cigarettes/day or who had stopped smoking for less than 6 months as „irregular smokers" (n = 24). Because of the resulting small numbers of irregular smokers, the latter 24 individuals were excluded from further analyses. Of the remaining 276 individuals, we excluded those with a history suggestive of symptomatic atherosclerotic disease, individuals reporting intake of drugs or conditions that might affect CRP levels (including chronic inflammatory diseases such as active rheumatoid arthritis), and subjects for whom CRP data were missing because of occasional assay problems. Subjects reporting a positive history of elevated blood glucose were not excluded. This selection procedure resulted in a final dataset of 261 subjects. Experimental protocol Data were collected on two occasions: First, subjects completed a medical questionnaire and examination. The medical assessment consisted of a 96-item questionnaire assessing the medical history and smoking behavior. The questionnaire was based on the Nurses Health Study [ 29 ] with questions asking for smoking behavior adapted from the MONICA study [ 30 ]. After completion of questionnaires, subjects had a 15-min rest period while sitting. Thereafter, systolic and diastolic blood pressure (BP) were measured twice within 5 min by sphygmomanometry, and the average of the two readings was computed. The waist-to-hip ratio was calculated based on waist circumference (as measured at its narrowest point between the ribs and iliac crest) and hip circumference (as measured at the maximal buttocks). Blood samples after overnight fasting were collected within one day to two weeks after having obtained medical data. Blood sampling was scheduled two hours after awakening to minimize circadian variation in variables of interest. Biochemical analyses Venous blood was obtained using cooled (4°C) citrate tubes for the TNF-α assay. Plasma was snap-frozen after centrifugation until further processing. We chose high-sensitive enzyme-linked immunosorbent assays (ELISA) to measure plasma concentrations of TNF-α (Quantikine HS, R&D Systems Europe, Abington, United Kingdom) and of CRP (detection limit 0.1 mg/l; Immunolite, DPC Biermann GmbH, Germany). In contrast to standard CRP assays, the high-sensitivity assay for CRP allows stratification of subjects with CRP levels below the range used for infectious disease workup [ 31 ]. Moreover, it has been argued that the high-sensitive CRP assay is required for risk assessment of cardiovascular disease [ 32 ]. Low-density lipoprotein cholesterol (LDL-C) cholesterol and high-density lipoprotein cholesterol (HDL-C) as well as hemoglobin A1c (HbA1c) were determined by a commercial laboratory (Synlab, Augsburg, Germany) applying standard procedures. Gene analysis To determine the TNF-α -308 G/A gene polymorphism, we extracted genomic DNA from the leukocyte-containing pellets remaining after centrifugation of coagulated blood using the QIAmp DNA Blood Mini Kit (Qiagen, Hilden, Germany). The TNF-α G-308A polymorphism was assessed by fluorescent real-time polymerase chain reaction with melting curve analysis on a LightCycler (Roche Diagnostics, Rotkreuz, Switzerland) using the TNF-α G-308A ToolSet for LightCycler (Genes-4U, Neftenbach, Switzerland) containing specific primers and fluorescent mutation detection oligonucleotide probes, in conjunction with the Roche Light Cycler Hyb Probe Master Mix (Roche Diagnostics, Rotkreuz, Switzerland) according to the manufacturer's protocols. For statistical analyses, we used the following groups: a) the GG variant, and b) the rarer AA and GA genotypes combined. Statistical analyses Descriptive data are presented as means ± SD or as median and interquartile range for severely skewed data. To approximate a normal distribution, we log transformed TNF-α and CRP values. General linear models were employed to elucidate the proportion of variance explained of log-transformed plasma TNF-α and CRP values (dependent variables). Independent variables were smoking status (regular smokers vs. non-smokers), gene variant, and an interaction term between smoking status and gene variant. Following this crude analysis, we entered possible covariates (age, gender, self-reported physical exercise, self-reported alcohol intake, gender, BP, and lipoproteins) into the equation. Results were considered statistically significant at the p ≤ .05 level; all tests were 2-tailed. To minimize possible type II errors when assessing interaction terms, we considered interaction terms when the F-statistic on the interaction term had a p-value < 0.2 [ 33 ]. All regression and variance analyses were performed using generalized linear models (PROC MIXED) to account for the unbalanced nature of the data (SAS version 8.2, SAS Inc, Cary, NC). Analyses were repeated including the small group (n = 24) of irregular smokers either with the non-smokers or with the regular smokers. None of these additional analyses changed our main results to a relevant degree. Therefore, only the results comparing non-smokers with regular smokers are reported. Results Study population and smoking status Table 1 compares health variables between non-smokers (n = 198), irregular smokers (n = 24) and regular smokers (n = 78). Regular smokers tended to be younger (p = 0.09), and they had significantly lower HDL-C levels (p = 0.01) than non-smokers. Regular smokers also had higher plasma levels of CRP than non-smokers (median 1.75 mg/l vs. 1.0 mg/l, p = 0.006). In our study sample, irregular smokers had plasma levels of CRP comparable to non-smokers (median 0.81 vs. 1.0 mg/l, p = 0.94). TNF-α was not significantly different between regular smokers and non-smokers. Table 1 Subject characteristics in relation to smoking status Smoking status Variable Non-smokers for >6 months n = 198 Irregular smokers n = 24 Regular smokers n = 78 P-value a Gender [% male] 86 80 88 0.52 Age [years] 42.1 ± 9.0 40.6 ± 9.2 40.0 ± 9.6 0.09 LDL [mg/dl] 122.3 ± 28.5 117.4 ± 30.4 115.9 ± 28.3 0.10 HDL [mg/dl] 45.3 ± 10.8 47.1 ± 8.3 41.4 ± 9.2 0.01 Systolic blood pressure [mmHg] 132.9 ± 15.2 128.7 ± 9.9 131.2 ± 13.6 0.43 Diastolic blood pressure [mmHg] 82.9 ± 9.7 79.2 ± 8.0 81.6 ± 8.3 0.33 Glycosylated hemoglobin [%] 5.15 ± 0.5 5.1 ± 0.4 5.25 ± 0.4 0.12 Tumor necrosis factor-α [ng/l] 1.8 ± 0.5 1.8 ± 1.0 2.2 ± 2.5 0.61 C-reactive protein [mg/l] 1.0 (0.47–1.9) 0.80 (0.5–2.0) 1.75 (0.7–3.0) 0.006 a P-values for comparisons between non-smokers and regular smokers. Tumor necrosis factor-α and C-reactive protein (CRP) levels were compared using the Wilcoxon test; for all other continuous variables the Student t -test was employed. Values are means ± SD except for CRP where the median and interquartile range is provided. LDL = Low-density lipoprotein cholesterol, HDL = High-density lipoprotein cholesterol Gene distribution After excluding irregular smokers, we found the GG wild type polymorphism in 203 subjects (74%); GA heterozygote were 70 participants (25%) and AA homozygote were 3 participants (1%). Calculated allele frequencies amounted to 0.86 for the G allele and to 0.14 for the A allele. Regular smokers were slightly though not significantly more frequent amongst subjects with the GG genotype than amongst individuals carrying the GA or AA genotype (odds ratio 1.41; 95% CI 0.77–2.6, p = 0.26). Table 2 compares individuals with the GG wild type with participants having the GA or the AA genotype. The table reveals that genotype was not associated with any of the examined health variables in crude bivariate comparisons, including plasma levels of TNF-α and CRP. Table 2 Subject characteristics in relation to the TNF-α G-308 polymorphism GG n = 203 GA/AA n = 73 P-value Gender [% male] 87 86 0.73 Age [years] 41.0 ± 9.1 42.9 ± 9.4 0.37 Regular smokers [%] 28.3 21.8 0.26 Low-density lipoprotein cholesterol [mg/dl] 119.5 ± 32.6 124.8 ± 29.1 0.22 High-density lipoprotein cholesterol [mg/dl] 44.1 ± 11.9 44.62 ± 10.9 0.76 Systolic blood pressure [mmHg] 131.7 ± 11.7 134.6 ± 17.4 0.18 Diastolic blood pressure [mmHg] 82.5 ± 11.1 82.7 ± 8.1 0.87 Glycosylated hemoglobin A1c [%] 5.1 ± 0.5 5.2 ± 0.6 0.18 Tumor necrosis factor-α [ng/l] 1.7 (1.3–2.2) 1.9 (1.4–2.2) 0.39 C-reactive protein [mg/l] 1.04 (0.53–2.36) 1.05 (0.49–3.14) 0.35 Tumor necrosis factor (TNF)-α and C-reactive protein (CRP) levels were compared by the Wilcoxon test; for all other continuous variables the Student t -test was employed. Values are means ± SD except for TNF-α and CRP where the median and interquartile range is provided. Genotype, smoking status, TNF-α and CRP levels The adjusted analysis regressing log-transformed CRP levels against smoking status, genotype, and smoking-status-genotype interaction revealed a main effect for smoking status (F 1,250 = 5.67, p = .018), but not for genotype (F 1,250 = 0.33, p = .57). The interaction-term between genotype and smoking status failed to gain significance (F 1,250 = 0.09, p = .76), indicating that the effect of smoking on plasma levels of CRP is not affected by the TNF-α -308G/A polymorphism (Figure). Of the considered covariates, gender (F 1,250 = 12.5, p = .0005), age (F 1,250 = 4.55, p = .03), and HDL-C (F 1,250 = 3.4, p = .065) were associated with CRP levels, but not BP or LDL-C. Men, older individuals, and those with lower HDL-C had higher plasma levels of CRP. Figure 1 The figure depicts plasma levels of C-reactive protein (CRP) in relation to smoking status and the TNF-α G-308 polymorphism in the final sample of 261 subjects. CRP levels are anti-log transformed least square means estimates from the fully adjusted model controlling for age, gender, blood pressure and blood lipids. Error bars denote the standard error of the mean estimate. Simple effects comparing genotypes across smokers were not significant (p = .14). The fully adjusted model with plasma levels of TNF-α also failed to show significant main effects for genotype (F 1,246 = 1.08, p = .30) and for smoking (F 1,246 = 0.33, p = .56), as well as for the smoking-status-genotype interaction. However, the adjusted model revealed a significant main effect for HDL-C (F 1,246 = 20.7, p < .0001), suggesting that individuals with higher plasma levels of TNF-α had lower HDL-C. Post-hoc analyses revealed no interaction between smoking status and HDL-C or genotype and HDL-C. Discussion Genes, health-behavior and the psychosocial environment interact to determine whether or not, and, if at all, how rapidly silent atherosclerosis will progress to the clinical manifestation of CAD [ 34 , 35 ]. The development of coronary artery sclerosis is a life-long process that probably has its onset in childhood [ 36 ]. Particularly, as suggested by post mortem studies, inflammation-related endothelial damage plays an important role in atherosclerosis onset and progression early in life [ 37 ]. The understanding of atherosclerosis as an inflammatory disease [ 1 ] has kindled much interest in a number of genetic polymorphisms coding for inflammatory molecules potentially related to CAD [ 38 ]. While plasma levels of the proinflammatory cytokine TNF-α are regulated by several polymorphisms of the TNF-α gene [ 39 ], it is the TNF-α G-308A gene polymorphism which has been most intensely scrutinized as one candidate polymorphism underlying CAD [ 8 ]. Plasma TNF-α levels predicted second myocardial infarction [ 40 ], and have been associated with common carotid intima-media thickness [ 41 ]. TNF-α also stimulates the liver to produce CRP [ 8 ], which, itself, has been shown to predict coronary risk in numerous population based studies [ 7 ]. Interestingly, blood cells from individuals who carry the A allele of the TNF-α G-308A gene polymorphism express more TNF-α in vitro upon stimulation with lipopolysaccharide than cells from individuals being homozygous for the G allele [ 18 ]. Despite this association, several studies did not find a significant association between the TNF-α G-308A gene polymorphism and incident CAD [ 19 - 25 ]. There are, however, no studies examining whether established cardiovascular risk factors might interact with the TNF-α G-308A gene polymorphism in determining plasma levels of TNF-α and eventually CRP downstream in the inflammatory cascade. We thus investigated the effect of an interaction between smoking severity and the G-308A polymorphism of the TNF-α gene on plasma levels of these two proinflammatory markers. Our specific hypothesis was that there was a cumulative increase of TNF-α and CRP related to the TNF-α G-308A polymorphism in subjects who regularly smoke as compared to non-smokers. In spite of two recent studies, which found an interaction between smoking and polymorphisms of molecules participating in the inflammatory response [ 14 , 15 ], the results from the present study fail to support our hypothesis. More precisely, we found that the interaction between smoking status and the TNF-α G-308A polymorphism did not significantly affect plasma levels of TNF-α and CRP in both unadjusted and adjusted analyses. Also, there was no main effect for the polymorphism investigated in terms of plasma levels of TNF-α and CRP. On the other hand, although not an aim of our study, we confirmed previous findings of increased plasma CRP in regular smokers as compared to non-smokers [ 11 ], while, rather unexpectedly, plasma levels of TNF-α were not different between smokers and non-smokers. It must be noted that our findings are preliminary, and, they do not allow us to reject the overall hypothesis of a smoking-gene interaction modifying inflammatory processes contributing to atherosclerosis initiation and progression [ 14 , 15 ]. For instance, because the number of homozygous carriers of the A allele in our study population was low reflecting low frequency of the AA genotype in the general population, we were unable to analyze whether there might be a "dose-response" relationship between the A allele dosage and CRP levels. Larger sample sizes are clearly needed to detect a potential difference in regulation of proinflammatory markers in plasma across the GG, AG, and AA polymorphism and with respect to their interaction with different cardiovascular risk factors. This reasoning becomes even more obvious with respect to the higher absolute difference in the mean estimates of plasma CRP levels between the GG and the GA/AA genotypes in smokers as compared to non-smokers (Figure). A highly powered study might raise the odds of this absolute difference to become statistically significant. Moreover, the biological plausibility of our hypothesis was straightforward given the important role of smoking, inflammation and their link in CAD [ 9 - 11 ]. However, we do not know in how far interactions between smoking, the TNF-α G-308A polymorphism, and other polymorphisms of molecules involved in the inflammatory pathways not investigated in our study [ 14 , 15 ] might affect plasma TNF-α and CRP levels in an unexpected way. The lack of a difference in plasma TNF-α levels between subjects with the A allele as compared to those homozygous for the G allele stands in contrast to previous in vitro studies [ 17 , 18 ]. However, aside from a power issue, our measured values of TNF-α levels only slightly exceeded the assay's sensitivity limit, incurring a larger chance of measurement error. We may speculate that a relation between gene variant, smoking, and circulating TNF-α levels might have been uncovered among patients with atherothrombotic disorders or other inflammatory conditions. Moreover, we measured systemic TNF-α; circulating TNF-α may not necessarily reflect TNF-α secretion at sites of confined subendothelial atherosclerotic lesions, where regulatory polymorphisms are most likely to affect reactions of immune cells. Finally, interactions also involving IL-6 polymorphisms [ 15 ] may play a role in the association between smoking, elevated CRP, and increased CAD risk. Future studies thus may want to investigate whether IL-6 polymorphisms might be associated with plasma CRP levels and whether they interact with TNF-α and CRP in smokers. Conclusions Our study suggests that both plasma TNF-α and CRP levels are not regulated by an interaction between smoking and the G-308A polymorphisms of the TNF-α gene promoter site. We thus remain far from adopting a clinical practice that would counsel smokers to quit smoking based on a particular gene polymorphism. Nonetheless, our results do not refute the overall hypothesis that genetic polymorphisms along the inflammatory pathway may account for the differential effect of tobacco consumption on the cardiovascular risk in individuals who smoke. Competing interests The authors declare that they have no competing interests. Authors' contributions MG participated in the design of the study and drafted the first version of the manuscript. JF participated in the design of the study, in data acquiring, performed the statistical analyses, and critically revised the manuscript. FM carried out and supervised the molecular genetic studies. RvK participated in the design of the study and wrote the final version of the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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Using item response theory to explore the psychometric properties of extended matching questions examination in undergraduate medical education

Background As assessment has been shown to direct learning, it is critical that the examinations developed to test clinical competence in medical undergraduates are valid and reliable. The use of extended matching questions (EMQ) has been advocated to overcome some of the criticisms of using multiple-choice questions to test factual and applied knowledge. Methods We analysed the results from the Extended Matching Questions Examination taken by 4 th year undergraduate medical students in the academic year 2001 to 2002. Rasch analysis was used to examine whether the set of questions used in the examination mapped on to a unidimensional scale, the degree of difficulty of questions within and between the various medical and surgical specialties and the pattern of responses within individual questions to assess the impact of the distractor options. Results Analysis of a subset of items and of the full examination demonstrated internal construct validity and the absence of bias on the majority of questions. Three main patterns of response selection were identified. Conclusion Modern psychometric methods based upon the work of Rasch provide a useful approach to the calibration and analysis of EMQ undergraduate medical assessments. The approach allows for a formal test of the unidimensionality of the questions and thus the validity of the summed score. Given the metric calibration which follows fit to the model, it also allows for the establishment of items banks to facilitate continuity and equity in exam standards.

Background It is acknowledged from medical student learning behaviour that assessment often drives learning [ 1 ]. Therefore, if students are learning what is being assessed then it is vital that the content of the assessment reflects the learning objectives. This process, known as blueprinting, maps the content of assessments against the clinical competencies (knowledge, skills and attitudes) that the student is expected to acquire [ 2 ]. The pyramid of competence developed by Miller provides a conceptual framework for ensuring that student assessments are valid and cover core aspects of factual knowledge and problem solving (e.g. Extended Matching Questions – EMQ), performance assessment in "vitro" (e.g. Objective Structured Clinical Examinations – OSCE) and performance in "vivo" (e.g. case presentations, log books) [ 3 ]. At the University of Leeds the undergraduate medical course includes an integrated medical and surgical specialities program (Rheumatology, Orthopaedics, Rehabilitation, Anaesthetics, Dermatology, Infectious diseases, Oncology and Palliative Medicine, Genitourinary Medicine and Accident and Emergency medicine). Acknowledging that no single assessment format can adequately assess all the learning objectives within the course blueprint, a combination of assessments (including OSCE, EMQ, slides with problem solving, reflective learning log books and case presentations) are currently used to assess the student's competence. Although a combined score is used to assess the competence of the students, test scores within the individual assessments reflect a richer profile of the individual, allowing an understanding of strengths and weaknesses that can result in improvement in the individual and the educational programme. Analysis of the quality of individual assessments is essential to this process. The focus of this paper is the use of item response theory to examine the validity of the typical use of a single score obtained from the summative EMQ examination, to characterise each student and their individual differences, in short the investigation of the relative unidimensionality of the EMQ examination The EMQ is a form of multiple-choice type question [ 4 ] designed to test the student's knowledge. EMQs are written by experts from each of the medical specialties. EMQs have four components; a theme (e.g. leg or cancer pain), the lead-in for the questions that gives the students instructions on what to do (e.g. "for each patient select the most likely diagnosis"); the questions in the form of vignettes giving the pertinent information based on which the student is to select the correct answer; and finally the potential answers (e.g. a list of potential diagnoses relevant to the theme) (Figure 1 ). The response option includes one correct answer for each question, and other possible responses as distractors, a reasonably plausible response if the student does not know the correct response for whatever reason. Figure 1 Example of Extended Matching Question (EMQ) Format. The use of EMQs has been advocated to overcome some of the criticisms levelled at the use of multiple-choice questions to test factual and applied knowledge. There are advantages to using EMQs [ 4 ]: • The format of themes aid the organisation of the examination, and the use of blueprinting is a natural aid to the process of writing EMQs • As questions are written in themes or general topic it allows the teacher to write many questions for that theme and then share these questions out randomly to create more than one examination paper • Good questions provide a structure designed to assess application of knowledge rather than purely recall of isolated facts • The approach to writing these questions is systematic, which is very important when several people are contributing questions to one exam • The extended list of options allows the inclusion of all relevant options, and reduces the opportunity for students to 'guess' the correct answer as in MCQs • EMQs were found to be more discriminating than two and five option versions of the same questions resulting in a greater spread of scores, and reliability was higher as a consequence of this [ 5 , 6 ]. The importance of ensuring validity and reliability of the EMQ procedure is crucial. Current evidence for this is limited but does support good reliability and validity [ 1 , 6 , 7 ]. This study considers EMQs as used in the medical education at two levels. Within the individual EMQ it explores the operation of the distractors, and across EMQs it explores whether (a) summary scores are justified; (b) how EMQs vary in difficulty across specialities and (c) whether the EMQ scores taken together discriminate students of different abilities. Traditionally, methods of analysis based on classical test theory have been used to evaluate such tests. The focus of the analysis is on the total test score; frequency of correct responses (to indicate question difficulty); frequency of responses (to examine distractors); reliability of the test and item-total correlation (to evaluate discrimination at the item level) [ 8 - 11 ]. Although these statistics have been widely used, one limitation is that they relate to the sample under scrutiny and thus all the statistics that describe items and questions are sample dependent [ 12 ]. This critique may not be particularly relevant where successive samples are reasonably representative and do not vary across time, but this will need to be confirmed and complex strategies have been proposed to overcome this limitation. Developments of the Classical Test Theory can be found in modern test theory and, in particular, the Rasch model [ 13 ] of Item Response Theory (IRT). This too uses the total test score, but in this instance from a theoretical basis. It is also concerned with reliability (in the form of a person separation statistic which is similar to Cronbach's alpha). However, in addition it provides a mechanism for testing the invariance of items which allows the construction of a bank of calibrated questions that facilitates a direct comparison over different administrations of the test [ 14 ]. From such an item bank, different combinations of questions can be incorporated into an examination to ensure that the difficulty of the exam remains consistent for successive cohorts of students. The use of the Rasch model entails a different perspective, or paradigm, from IRT approaches in general [ 15 ]. Where data do not conform to the expectations of the Rasch model, the main challenge is not to find a model that better accounts for the data, but to understand statistical misfit as substantive anomalies that need to be understood, and by being understood, to lead to the construction of more valid and reliable tests. This is the approach taken in this study. That is, analysis of data based on existing items will be considered closely both statistically and substantively with a view to guiding better question construction. Thus the aim of this paper is to explore the use of Rasch analysis to determine the validity of the EMQ examination currently taken by 4 th year medical undergraduates. Methods Data collection Responses to the EMQ examination taken by one hundred and ninety three 4 th year medical students were used as the source data. The examination is designed to test factual and applied knowledge taught in the Medical and Surgical Specialties course and is taken by the students at the end of the 16 weeks course. The course is run three times per year and rotates with two other courses (Paediatrics / Obstetrics / Gynaecology and Primary Care / Public Health / Psychiatry). All questions were devised by the lead educational supervisor within each specialty. Training in EMQ writing was provided to the medical specialty supervisors. The examination consisted of 98 EMQs distributed across eight specialties and 27 themes, each with two to four EMQs. Each themed group of EMQs had eight to 15 possible response options (e.g. see example in Figure 1 ). There were 12 Oncology, 14 Anaesthetics, 12 Dermatology, 12 A&E, 12 Infectious Diseases, 16 Orthopaedics, 8 Rheumatology and 12 Rehabilitation EMQs. The final exam mark is the sum of correct answers to all themes, summed across specialties, giving an indication of the applied knowledge across the range of medical and surgical specialties which comprised the Medical and Surgical Specialities module. No other information was collected about the students other than which term they had sat the EMQ examination. The students take this examination at the end of the course and the medical and surgical specialties course is repeated three times a year. Differential Item Functioning (see below) was used to determine the impact of the term in which the examination was taken on student performance. Parameter estimation The Rasch model is a probabilistic unidimensional model which asserts that (1) the easier the question the more likely the student will respond correctly to it, and (2) the more able the student, the more likely he/she will pass the question compared to a less able student. The model assumes that the probability that a student will correctly answer a question is a logistic function of the difference between the student's ability [ θ ] and the difficulty of the question [ β ] (i.e. the ability required to answer the question correctly), and only a function of that difference From this, the expected pattern of responses to questions can be determined given the estimated θ and β . Even though each response to each question must depend upon the students' ability and the questions' difficulty, in the data analysis, it is possible to condition out or eliminate the student's abilities (by taking all students at the same score level) in order to estimate the relative question difficulties [ 14 , 16 ]. Thus, when data fit the model, the relative difficulties of the questions are independent of the relative abilities of the students, and vice versa [ 17 ]. The further consequence of this invariance is that it justifies the use of the total score [ 18 , 19 ]. In the current analysis this estimation is done through a pair-wise conditional maximum likelihood algorithm, which underlies the RUMM2020 Rasch measurement software [ 20 , 21 ] If the above assumptions hold true then the relationship between the performance of students on an individual question and the underlying trait (applied knowledge within the medical and surgical specialties course) can be described by an S shaped curve (item response function). Thus the probability of answering the question correctly consistently increases as the location on the trait (knowledge) increases (Figure 2 ). The steepness of the curve indicates the rapidity with which the probability that a student responding to the question correctly, changes as a function of this location (ability). The location of the curve along the horizontal axis (defined by the point at which the 0.5 probability level bisects the horizontal scale) indicates the difficulty of the question. The location of the student on the same axis indicates their level (of knowledge, ability etc.) on the trait. Figure 2 An Item Response Function (Item Characteristic Curve). When the observed response pattern does not deviate significantly from the expected response pattern then the questions constitute a true measurement or Rasch scale [ 22 ]. Taken with confirmation of local independence of questions, that is, no residual associations in the data after the person ability (first factor) has been removed, this supports the unidimensionality of the scale [ 23 , 24 ]. General tests of fit In this analysis, responses to the EMQ are analysed as dichotomous options, that is, one correct answer and all of the other options are analysed together as one incorrect response. To determine how well each question fits the model, and so contributes to a single trait, a set of 'fit' statistics are used which test how far the observed data match those expected by the model. The trait refers to the required knowledge base that the student must acquire within the medical and surgical specialties course. The Item – Trait Interaction Statistic (denoted by the chi-square value), reflects the degree of invariance across the trait. A significant chi-square value indicates that the relative location of the question difficulty is not constant across the trait. In addition, question fit statistics are examined as residuals (a summation of the deviations of individual students responses from the expected response for the question). An estimate of the internal consistency reliability of the examination is based on the Person Separation Index where the estimates on the logit scale for each person are used to calculate reliability. Misfit of a question indicates a lack of the expected probabilistic relationship between the question and other questions in the examination. This may indicate that the question does not contribute to the trait under consideration. In the current study students are divided into three ability groups (upper third, middle third and lower third) denoting each Class interval with approximately 65 students in each. Furthermore, significance levels of fit to the model are adjusted to take account of multiple testing (e.g. for 24 items the level would be 0.002 and for 98 the level would be 0.0005) [ 25 ]. As well as invariance across the trait, questions should display the same relative difficulty, irrespective of which externally defined group is being assessed. Thus, the probability of correctly answering a question should be the same between groups given the same ability level of the student. For example, given the same ability, the students should not be more likely to answer a question correctly simply because they sat the exam in the third term instead of the first or second term. This type of analysis is called Differential Item Functioning (DIF) [ 26 ]. The basis of DIF analysis lies in the item response function, and the proportion of students at the same ability level who correctly answer the question. If the question measures the same ability across groups of students then, except for random variations, the same response curve is found irrespective of the group for whom the function is plotted [ 26 ]. Thus DIF refers to questions that do not yield the same response function for two or more groups (e.g. gender or the cohort of students). DIF is identified by two way analysis of variance (ANOVA) of the residuals with the term in which the examination was taken by the student as one factor and the class interval as the other [ 27 ]. Two types of DIF are identified: (a) uniform DIF demonstrating that the effect of the term in which the exam was taken are the same across all class intervals (main effect), and (b) non-uniform DIF which demonstrates that the effect of which term the student sat the exam in is different across class intervals (interaction effect). Where there are more than two levels of a factor, Tukey's post hoc test is used to indicate which groups are contributing to the significant difference. Although EMQ are analysed as though they have dichotomous response categories (correct or incorrect), it is possible to examine how the separate incorrect options within an individual EMQ are contributing to the student's response. This procedure is very similar to the technique of Graphical Item Analysis(GIA) [ 28 ], though in this case the RUMM 2020 programme [ 21 ] produces the analysis with no extra user effort. The proportions of students in each class interval who have selected the various response categories, including the correct option, are plotted on a graph of the item response function. This visually illustrates how often the various response options are being selected by the students in relation to one and other, and can be compared across themes given that different options are likely to have different response patterns for different questions within a theme. This is particularly useful in improving the quality of the distractor responses. In view of our limited sample size (and particularly the ratio of students to items) we elected in the first instance to examine in detail the psychometric properties of the musculoskeletal component of the EMQ examination, acknowledging the limitations associated with the accuracy of the person estimate based upon 24 items (29). Subsequent analysis of the whole examination is reported to demonstrate the potential benefits of using Rasch analysis, but again acknowledging the limited conclusions that can be drawn on student ability and question difficulty estimates for the whole examination as a result of looking at 98 items with 193 students. Results Data were collected from 193 students (Term 1 = 61, Term 2 = 64, and Term 3 = 68). Total scores ranged from 36 to 78 out of a maximum mark of 98 (mean = 60.3, median = 61). Initially, analysis of data was undertaken from the combined specialties of rheumatology and orthopaedic questions, which consisted of 24 EMQs'. Analysis of the musculoskeletal component of the EMQ examination To estimate individual question difficulty using the Rasch model, all the incorrect response options were treated together as one incorrect option. The fit of the 24 questions to the Rasch model was acceptable, both in terms of individual item fit (Table 1 ) and over all Item-Trait Interaction (χ 2 = 79.73, p = 0.003). This suggests that the musculoskeletal questions mapped on to a single dimension of applied knowledge in this case and within the power of the test of fit. This was further supported by a principal components analysis of the residuals identifying a first residual factor accounting for just 8% of the variation. However, the Person Separation Index was low, 0.50, indicating a low reliability. This, however, can be ascribed to the intrinsic homogeneity of the students who are selected under rigorous criteria and who are all studying for the same exam. Table 1 Individual Item difficulty (location) and Tests of Fit (residuals and chi-square and its probability) for the 25 musculoskeletal EMQs. Question Location SE Residual ChiSq DF Prob OR63 -2.68 0.47 -0.60 0.06 1 0.81 OR64 -2.32 0.40 -0.67 0.43 1 0.51 OR65 -2.03 0.35 -1.02 0.96 1 0.33 OR66 1.03 0.15 2.48 1.85 1 0.17 OR67 -2.27 0.39 -1.12 2.32 1 0.13 OR68 1.74 0.16 0.20 0.02 1 0.88 OR69 -0.29 0.19 -0.79 0.52 1 0.47 OR70 -3.49 0.68 -0.25 0.59 1 0.44 OR71 0.81 0.16 1.62 1.19 1 0.28 OR72 -1.66 0.30 -0.52 0.44 1 0.51 OR73 -0.48 0.20 0.21 0.47 1 0.49 OR74 1.79 0.16 1.64 7.80 1 0.01 OR75 0.12 0.17 -0.64 0.33 1 0.57 OR76 1.70 0.16 -0.71 3.90 1 0.05 OR77 1.12 0.15 -0.52 2.95 1 0.09 OR78 1.20 0.15 1.76 0.08 1 0.78 RH79 -1.47 0.28 -0.61 1.00 1 0.32 RH80 1.57 0.15 -1.00 2.01 1 0.16 RH81 0.22 0.17 1.52 4.22 1 0.04 RH82 1.11 0.15 1.01 0.00 1 0.96 RH83 -1.08 0.24 -0.58 0.43 1 0.51 RH84 2.08 0.16 -0.18 0.09 1 0.76 RH85 1.50 0.15 -0.12 0.00 1 0.99 RH86 1.78 0.16 -0.88 3.28 1 0.07 OR** representing Orthopaedic EMQ RH** representing Rheumatology EMQ ChiSq Chi – squared statistic SE Standard error Location Value identifies question difficulty on logit scale Residual Fit of question to underlying trait DF Degrees of freedom None of the items displayed DIF by the term in which the examination was taken. The location (a logit scale – Table 1 ) measures the item difficulty of the musculoskeletal EMQs and shows the range of difficulties from easy to hard (negative values indicating easier questions and positive values indicating harder questions). Analysis of the whole EMQ examination (all eight specialties) An initial exploration of the overall fit of the 98 questions to the Rasch model was poor with a significant question – trait interaction (χ 2 = 291.4, p < 0.0001). Three out of the 98 EMQs showed significant misfit to the model at the individual level; one Infectious Diseases question, one Oncology question and one Rehabilitation Medicine question. Once these misfitting questions were omitted from the analysis, the overall fit of the remaining 95 EMQs to the Rasch model improved, and showed no significant Item – Trait Interaction. The questions from each of the component specialties within the course had reasonable spread of difficulty across the logit scale (Figure 3 ). Overall the students were more likely to answer the A&E EMQs correctly than other themes. Five questions (Oncology2, Oncology3, A&E41, Rheumatology84 and Rehabilitation93) displayed DIF by term in which the examination was taken (p < 0.01) indicating that the student's responses to these questions were influenced by the term in which the students sat the examination. Note that when the overall trait from all specialties is considered, Rheumatology84 shows DIF, but not when just the musculoskeletal-related trait was considered. Figure 3 Map of question difficulty and student ability on Rasch transformed logit scale. Right hand side shows questions in order of difficulty and the left hand side shows the distribution of the students abilities based on their total examination score. Response option analysis The analysis of response options is descriptive. The proportions of students who have selected the various response categories, including the correct option, are plotted on a graph of the item response function. This visually illustrates student behaviour in their response to questions. Three main patterns of response to the questions were identified. a) the incorrect response option selected more frequently than the correct answer The majority of the students are selecting the same wrong answer, irrespective of their ability level (Figure 4 ). In contrast, a more typical pattern of responses to a hard question would be students choosing a range of incorrect response options randomly, rather a single option. Figure 4 A question where the vast majority of the students are selecting the same incorrect response option (0 – blue). b) the distractor response option is selected more frequently than right answer in some ability groups Responses to this EMQ show that students with lower ability are likely to select the incorrect answer, while the more able students select the correct response (Figure 5 ). Response option 0 is considered by most to be wrong, but options 1 and 2 clearly attract students of lesser ability. Figure 5 Distractor response option (2 – green) selected more often than the correct answer (3**) by lower ability students, but not higher ability students. c) the correct answer too obvious The majority of the students in all ability groups select the correct answer (Figure 6 ). An "easy" question is not in itself undesirable as it may test the student on a critical piece of knowledge. Figure 6 Correct answer is too obvious (2** – green). Discussion In this study, Rasch methodology was used to analyse the Extended Matching Questions examination taken by our fourth year medical students in 2001. Analysis of the musculoskeletal subset of questions showed that they mapped on to the trait under investigation (assessment of the musculoskeletal applied knowledge content of the course) and thus supported the notion of a valid unidimensional scale. Exploratory analysis of the whole examination showed that only three out of the 98 EMQs displayed significant misfit to the measurement model. These EMQs should be reviewed for ambiguities in question format and relationship to the course blueprint. DIF was identified in five of the 98 questions (these were different to the items displaying misfit) suggesting that the pattern of student responses to these EMQs were dictated by the term in which the exam was undertaken. The reason for this apparent variation needs to be understood before such questions are included into the bank of EMQs used for future examinations. In the present analysis only 'term' was considered for DIF, but DIF by gender also needs to be considered as gender can influence the response students make to the distractors [ 30 ]. Rasch analysis revealed the variety of response patterns made by the students. In some questions the incorrect response is selected more frequently than correct response , suggesting that the question appears extremely hard in that the majority of students are consistently selecting the same wrong option. This may relate to the stem being ambiguous, poor delivery of the teaching with students having experienced difficulty in understanding the topic such that the chosen response seems plausible to everyone. In those questions where the incorrect response is selected more frequently than the correct response but in only some ability groups (distractor), this suggests that the distractor option creates a strongly discriminating question. In this case less able students are more likely to select a distractor response while the more able students are likely to select the correct option. Where most students select the correct answer, regardless of their overall ability level , the question provides little information to distinguish the ability of the students. Kehoe [ 31 ] has suggested that "questions that virtually everyone gets right are useless for discriminating between students and should be replaced by more difficult items", particularly in the case where the pass mark of the exam is criterion referenced. However as the question might test an essential component of the course that is important for all students to know it may be reasonable for such questions to be included in the examination even though they may be poor discriminators of low and high ability students. The content of the examination needs to have a mixture of questions that are discriminatory as well as those that define the pass standard (which would include questions that appear to be too easy but test an essential component of the course). If a question is too easy and does not test an essential component of the course then it needs to be revised. The data presented in this paper on the analysis of response pattern within individual questions is purely descriptive. Although visual inspection of the response patterns is informative, Wang [ 32 ] is currently developing a quantitative Rasch model based on the analytical approach traditionally used in Classical Test Theory. This alternative would appear to provide a more statistical, as opposed to descriptive analysis about how the response options are working within the context of the Rasch measurement model. The use of the extended matching format with up to 14 response options reduces the impact of guessing on the student's overall mark compared with standard multiple choice questions. However, it could also be argued that setting questions within themes puts the test at risk of a breach of the local independence assumption, in that responses to questions on the same theme may be locally dependent. The PCA of the residuals reject this and support the assumption of local independence. Rasch analysis also allows for the development of a bank of questions that have been calibrated with one another in terms of their difficulty. This allows different examinations to be constructed (from combinations of the calibrated questions) while retaining the same level of overall difficulty. This will reduce the likelihood of students in consecutive years taking harder or easier exams when the standard they have to attain is unchanged. Classical test theories (including Generalisability theory), widely used to investigate the quality of student assessment, make few assumptions about the characteristics of the questions such as whether they form a unidimensional construct. Therefore this approach can be used in a variety of measurement situations. A comparison between the classical approach and the Rasch approach with regard to discrimination is given in Figure 7 . However the statistics obtained from classical analysis only apply to the specific group of students who took the test (i.e. the analysis is sample dependent). This analysis cannot separate the attributes of the questions from the attributes of student (e.g. ability) making it difficult to compare the performance of different sets of students who take the same format examinations with year on year content variations. Figure 7 Graph of a misfitting EMQ (classic test theory and Rasch); EMQ 3. In contrast, the Rasch measurement model checks two important assumptions: (a) the probability of answering one question correctly does not increase the probability of answering another question correctly within the examination (local independence) and (b) all questions in the examination map on to one construct (unidimensionality). With respect to a) above, questions that incorporate sections whose answers influence the response to other sections within the same question cannot be analysed using this approach and, to b), unidimensionality is a requirement for the summation of any set of items (33). In addition, arguments have been made to use the two parameter and three parameter logistic models (the latter which adds a guessing parameter) [ 34 ] as these much better reflect the type of curve derived from educational data. Unfortunately, apart from sample size requirements which are very high for these type of model, it is known that almost 100% of the time their parameters violate interval scaling [ 35 ]. Thus these models do not provide the invariance or quality of measurement which is required for summative unidimensional scales. The property of the invariance of the ratio of difficulty across items (that is this ratio between any two items is constant, irrespective of the ability of the students) is again an essential requirement for measurement. Finally, a consistent problem with criterion related tests is the apparent low reliability, as expressed by a low person separation index. This is to be expected, as traditional tests of reliability are not appropriate for criterion-referenced tests [ 36 ] where the score distribution is likely to be very narrow. Conclusion Case and Swanson [ 4 , 5 ] have set out clear guidelines on how to write an EMQ. The Rasch measurement model, and the associated analysis used in this study, will ideally be the next stage in the process of EMQ writing. It can be used to give feedback to the question writers on how to revise the problem questions. The analysis clearly shows how students use options, and this information coupled with expert knowledge and understanding of the questions will help questions writers to create and improve the quality of EMQs. It allows for a formal test of the unidimensionality of the questions and thus the validity of the summed score. Given the metric calibration which follows fit to the model, it also allows for the establishment of items banks to facilitate continuity and equity in exam standards. Thus modern psychometric methods based upon the work of Rasch provide a useful approach to the calibration and analysis of EMQ undergraduate medical assessments. Competing interests The author(s) declare that they have no competing interests. Authors' contributions All authors have contributed to the analysis and writing of this paper and its revisions, and all have read approved the final version. Pre-publication history The pre-publication history for this paper can be accessed here:

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555567

Ethmoidal osteoid osteoma with orbital and intracranial extension – a case report

Background Osteoid osteoma is a benign bone neoplasm which is seen in the long bones of appendicular skeleton. It is rarely seen in the cranium. Skull base osteoid osteoma is extremely rare and has been anecdotally reported. Case presentation The authors report a case of a large osteoid osteoma of the ethmoid with intraorbital and intracranial extension in a 33 year old male patient. He presented with loss of vision in the left eye. The intra-cranial extension was excised through a single burr-hole fronto-orbitotomy. The ethmoid and orbital portions were approached and excised through a Weber-Ferguson incision and inferior orbitotomy. Radical excision of the tumor could thus be achieved through a craniofacial approach. Conclusion Although benign and rare, skull base osteoid osteoma can present with neurological deficit due to its mass effect and involvement of vital structures. A multispeciality team approach is advisable in such cases if radical excision is to be achieved. A craniofacial approach made radical single stage excision of this multicompartmental osteoid osteoma possible with an uneventful postoperative period.

Background Osteoid osteoma is a rare benign osteoblastic lesion usually involving the long bones of the lower limbs. Cranial involvement has been mainly localised to the skull vault. Osteoid osteoma of the skull base is a rare entity [ 1 ]. Surgical management of skull base osteoid osteoma may be challenging due to its proximity to vital structures, access and hard consistency. This report deals with a case of an ethmoidal osteoid osteoma invading the adjacent orbit and anterior cranial fossa and the team approach employed to achieve radical excision. The radiological findings and the surgical procedure employed are presented. Case presentation Clinical presentation A 33 year old male presented with decreased vision in the left eye and left sided headache of 3 months duration. Examination revealed left eye blindness with primary optic atrophy and no other neurological deficit. Imaging revealed a bony tumor in the left ethmoid sinus invading the left orbit and compressing the left optic nerve. Intracranial extension into the anterior cranial fossa on the left side was noted (Figure 1 ). Core biopsy of the mass showed an osteoid osteoma. Surgical management A multispeciality team approach was devised to achieve radical excision of the tumor. Bicoronal scalp flap and pericranial flaps were raised separately. A single burr hole left fronto orbital bone flap was raised including the orbital roof and left zygomaticofrontal process (Figure 2 ). Dura was retracted and the bony hard whitish tumor visualised. This was excised using the high speed drill (Figure 3 ). Weber Ferguson incision was used to access the orbital portion of the tumor. Medially the tumor could be felt in the orbit but retraction of the globe was difficult. Hence inferior orbitotomy was done by removing the lower and lateral orbital margins. The intraorbital contents could now be retracted laterally and the tumor visualized (Figure 4 ). The tumor was then detached from the ethmoid sinus and the intraorbital extension excised. The ethmoidal portion was drilled and radical excision achieved (Figure 5 ). Dural tears were covered with temporalis fascia and glue. The ethmoidal sinus was packed with free temporalis muscle graft. Vascularised pericranial graft was used to cover the anterior skull base. The frontal sinus was exenterated and packed with gelfoam. The bone flap and orbital margins were replaced. Postoperative period and follow up Post-operatively patient had a frozen left eye (possibly due to retraction of the orbital contents) with no improvement in the left eye vision in the immediate postoperative period. His postoperative period was otherwise uneventful. Histopathological examination revealed an osteoid osteoma (Figure 6 ). On follow up after 12 months patient was disease free (Figure 7 ). Discussion Osteoid osteoma is a benign osteoblastic lesion and constitutes 1% of all bone tumors and 11% of benign bone lesions [ 1 ]. It is usually seen in the second and third decades and a male preponderance has been noted. It can occur throughout the skeleton but the long bones of the lower extremities and the vertebrae are most commonly affected. They are usually metaphyseal but may be epiphyseal occasionally. It is frequently localized to the cortex (85%) but may also occur in spongiosa (13%) and subperiosteal region (2%) [ 2 ]. Cranial cases are seen to generally arise from the skull vault. Skull base osteoid osteomas are extremely rare and occur in the frontal or ethmoidal sinuses [ 1 , 3 ]. It usually presents with sharply localized pain and tenderness especially at night. In our case the osteoid osteoma was seen to originate from the ethmoid sinus and pain was not a presenting feature. The radiological diagnosis rests on Computerised Tomography and isotope bone scan [ 1 ]. Radiographically osteoid osteoma appears as a radio opaque lesion with a nidus which has a radiolucent centre surrounded by dense sclerosis [ 2 ]. This may at times be mistaken for Garre's osteomyelitis. Occasionally the nidus may have a radio opaque centre with a surrounding radiolucent area. In our patient no definite nidus could be visualized probably due to the large size and unusual location. The treatment generally consists of en bloc resection or curettage of the tumor. Recurrence rate after incomplete resection may be upto 10% [ 1 ]. If asymptomatic and small, the lesion may be left alone and observed. However a rare complication of a large pneumocephalus has been reported by Ferlito et al from a frontoethmoidal osteoid osteoma [ 4 ]. Our patient presented with loss of vision due to a large osteoid osteoma of the ethmoid invading the left orbit. Anterior skull base lesions have been approached through a frontoorbitotomy which is usually removed as two separate parts. We have found that a single burr hole frontoorbitotomy flap gives excellent exposure to the anterior skull base without excessive retraction on the brain and is also cosmetically superior. Combining the craniotomy with a Weber Ferguson incision and orbitotomy made a single stage radical excision possible. Histologically the nidus is sharply delineated from the surrounding variably thick layer of dense bone. The nidus is composed of more or less calcified osteoid lined by plump osteoblasts within a highly vascularised connective tissue stroma [ 2 ] (Figure 6 ). It does not invade the adjacent tissue. No malignant transformation has been reported [ 1 ]. A differential diagnosis of benign osteoblastoma may be entertained. However, in the case of the latter, active osteoblasts are more numerous and the stroma is richly vascularized and extravasated blood with large number of multinucleated giant cell macrophages are noted [ 5 ]. Several authors have stressed the fact that the two are identical histologically and the differentiation between them if any can only be on the basis of size [ 5 ]. In our patient although the tumor was large, none of the above mentioned histological features of a benign osteoblastoma could be noted. Conclusion Osteoid osteoma of the skull base is rare and anecdotally reported. Radical excision is difficult especially if the tumor involves major blood vessels and cranial nerves. The surgical team constituted the neurosurgeon, surgical oncologist and plastic surgeon. A craniofacial approach made radical single stage excision of this multicompartmental osteoid osteoma possible with an uneventful postoperative period. Competing interests All the authors of the article "ETHMOIDAL OSTEOID OSTEOMA WITH ORBITAL AND INTRACRANIAL EXTENSION -CASE REPORT" hereby declare that there are no competing interests – financial and non financial. Authors' contributions SBP performed the craniotomy, excised the intracranial extension of the tumor, assisted in excising the orbital portion, drilled the ethmoidal portion and drafted the manuscript. KH excised the orbital portion and helped draft the manuscript. MSV and US performed the orbitotomy, helped in tumor excision and closure. DJ did the histopathological examination and helped in drafting the manuscript. All authors have read and approved of the manuscript . Pre-publication history The pre-publication history for this paper can be accessed here:

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526210

Community nursing needs more silver surfers: a questionnaire survey of primary care nurses' use of information technology

Background In the UK the health service is investing more than ever before in information technology (IT) and primary care nurses will have to work with computers. Information about patients will be almost exclusively held in electronic patient records; and much of the information about best practice is most readily accessible via computer terminals. Objective To examine the influence of age and nursing profession on the level of computer use. Methods A questionnaire was developed to examine: access, training received, confidence and use of IT. The survey was carried out in a Sussex Primary Care Trust, in the UK. Results The questionnaire was sent to 109 nurses with a 64% response rate. Most primary care nurses (89%) use their computer regularly at work: 100% of practice nurses daily, compared with 60% of district nurses and 59% of health visitors (p < 0.01). Access to IT was not significantly different between different age groups; but 91% of practice nurses had their own computer while many district nurses and health visitors had to share (p < 0.01). Nurses over 50 had received more training that their younger colleagues (p < 0.01); yet despite this, they lacked confidence and used computers less (p < 0.001). 96% of practice nurses were confident at in using computerised medical records, compared with 53% of district nurses and 44% of health visitors (p < 0.01.) One-to-one training and workshops were the preferred formats for training, with Internet based learning and printed manuals the least popular (p < 0.001). Conclusions Using computers in the surgery has become the norm for primary care nurses. However, nurses over 50, working out in the community, lack the confidence and skill of their younger and practice based colleagues.

Background In the UK its National Health Service (NHS) is investing in computerisation. Large contracts have been set that will run until 2013 to provide electronic patient records [ 1 ]. Over this timeframe, with the exception of patient-held records, written medical records will largely be phased out, and replaced by the computerised. Instead of each hospital, or other NHS service that a patient attends creating its own record in isolation separate records will be linked via an "electronic spine." To help integration of records the whole NHS is due to move to a single coding system. Currently most of general practice uses Read codes to record structured data in computers [ 2 , 3 ]. There are many other systems currently in use in the other parts of the health service, but this is all due to come to an end with the whole NHS moving to use a classification system called SNOMED CT (Systematized Nomenclature of Medicine – Clinical Terms [ 4 , 5 ]) In addition, computer use will be become more and more necessary, as ever increasing amounts of information to support learning and the delivery of high quality care, are placed on-line [ 6 , 7 ]. Every general practice premises has a connection to NHSnet and via this the Internet, to facilitate access [ 8 ]. In this environment primary care professionals will need the skills to read and enter data in computerised medical records, access guidelines and other information on-line, receive alerts about changes in practice via email and use computer as a learning resource. Nurses: practice nurse, district nurses, health visitors, and others, make up a substantial proportion of the primary care workforce. Previous studies have suggested that they have limited access and low levels of confidence and competence in using IT (Information Technology) [ 9 - 11 ]. However, the data reported in these publications is at least two years old. We therefore conducted this study to discover the extent to which primary care nurses had access to, and were using IT; and to examine whether any particular groups were being left behind. Methods A literature review was carried out of standard bibliographic data bases: CINAHL, British Nursing Index and Medline to identify any recent publications about use of IT or computerised medical record by practice or district nurses and health visitors. In addition the primary care electronic library (PCEL) [ 12 ], and information departments in the Community Practitioners and Health Visitors Association (CPHVA) [ 13 ] and Royal College of Nursing (RCN) [ 14 ] were contacted by email. We did not find any contemporary reports of UK primary care nurse use of IT. The study took place in a single Primary Care Trust in Sussex, a mixed urban and rural county in southeast England. A Primary Care Trust is a geographically defined locality of approximately 1 – 200,000 people that commissions hospitals, general practitioners and other health providers to deliver comprehensive local NHS healthcare. A PCT can also provide care itself. Most community nurses are employed directly by their PCT, and this PCT is no exception. All the practices in this PCT are computerised with computers linked to the patient database located in all consulting and treatment rooms; and most offices. In common with nearly all UK general practice the practice is networked and the network is connected to the internet via a fixed ISDN-2 (Integrated Services Digital Network) link. The ISDN (64 K link) actually connects the practices to the NHS own intranet (NHSnet) and via secure gateways to the internet [ 8 ]. The NHSnet connection also provides primary care professionals email, as part of this service. In theory there should not be problems with access to IT in NHS primary care. This PCT has a predominantly female (97%) workforce with one-and-a-half times as many nurses in the over 40 age group compared with the national average for nursing [ 15 ]. The Nursing and Midwifery Council does not keep separate age-sex details for community nurses, however some information was available from the RCN. The RCN provided information about the age range of community nurses in southeast England. These, although collected using different age bands, show a similar population distribution as that found in this sample. The department of health produces details of community nurses by age band [ 16 ]. Although, these data do not include practice nurses, and are a national sample, they also show a similar distribution. The proportions by age group are shown in Figure 1 . Figure 1 Comparison of the age distribution of the study population with the Royal College of Nursing community nurse members in the southeast of England. A questionnaire was considered the most appropriate way to gather the information required. Two previous studies indicated that primary care nurses appeared willing to complete the questionnaires and provide rich data [ 10 , 11 ]. The design consisted of questions which required "tick box" answers, or rating choices on a simple four point scale. It had 12 questions spread over three sides of A4 with a fourth side available for comments; it could be completed in less than five minutes. A copy of the questionnaire is attached as an appendix to this paper [see Additional file 1 ], or can be downloaded from: . The questions were based on those used in two earlier studies; with the clarity of questions was improved. Especially distinguishing between a clinical computer system (i.e. one that is used to enter data into an electronic patient record) from other "work" computer systems; and, between home and work use. The questionnaire was tested by a pilot group of nurses located in another PCT in a neighbouring county. The pilot group of six nurses were asked to complete the questionnaire on their own. They were then invited to join a discussion group – where the purpose of the study was explained. Then the questions were gone through one by one to identify any ambiguity. The nurses were also invited to suggested alternative or additional questions. They were given SB's contact details should anything come to mind later. Ethical approval was obtained from the local research ethics committee to conduct the study. Primary care nurses were identified using a list provided by the PCT. The questionnaire was sent out with a covering letter explaining the nature of the study. A courier addressed envelope was provided so the questionnaire could be returned without cost. A reminder and second questionnaire was sent out to non-responders. The questionnaire responses were coded and entered into SPSS (Statistical Package for Social Sciences) for analysis. The results were analysed by age bands and professional grouping as we wished to test whether either of these factors predetermined the level of computer use. Additional comments were entered into Microsoft Word, and the SPSS data from the questionnaire was placed alongside the relevant comments – they were flagged with the professional group and age group of their originator. Microsoft Excel was used to allow them to be sorted by age group and profession. To simplify the analysis only three age-groups were considered: age under 30, 30 to 39, 40 to 49, and 50 and over. There were no nurses under 30, and only one over 60. The nursing professions were divided into: District nurses, health visitors and midwives, practice nurses, and others. In this study qualified nurses who work in the community primarily supporting district nurses or health visitors were analysed as part of that nursing specialist group. The health visitors and community midwives were combined into the "health visitors" group. The term "primary care nurse" is used to embrace all qualified nurses included in this survey; "community nurse" refers to nurses who still do a proportion of their work in patients' homes, Figure 2 . Figure 2 Typology of UK primary care nurses Results There was a 67% (67/105) response rate to the questionnaires. There was some variation between nursing groups: the response from practice nurses was 73% (n = 22/30), for district nurses 51% (n = 17/33), for health visitors and midwives 67% (n = 19/28), and other nurses, community psychiatric nurses and managers 64% (9/14). Computers were readily available to primary care nurses. Over 90% of nurses had access to a personal computer (PC) at work, though for nearly half (45%) this was shared access; home access was even higher – 96%, with very few (12%) describing their access as shared. The remainder had access elsewhere: library, college etc. Not all the computers were connected to the Internet: 11% of those provided a PC at work and 6% at home could not go on line. After taking into account "other" access to the Internet only 3% of primary care nurses lacked access. The pattern of access to computers, the Internet, and receiving training changed with age. The younger nurses had more access to PCs and the Internet at work, while older nurses had higher levels of access at home, and had received more training. These trends are shown in Table 1 ; only the receipt of more training in nurses 50 and over was statistically significant (Chi-squared test.) There were more striking differences between the different nursing professions. 90.1% of practice nurses had their own computer at work, with the balance of 9.1% having shared access – three-quarters (76.2%) had Internet access via this computer. By way of contrast their community based colleagues had significantly less access – only 56% of district nurses and 17% of health visitors had their own computer. Most had shared access and a few, 6.3% and 8.7% respectively, no access at all (p < 0.001). Table 1 Trends in access to computers, the Internet and receipt of training with age Age 30–49 Age 50 & over All Pearson X 2 p = Has access to own PC when working 59.1% 37.0% 50.7% 0.074 Has own access to Internet when working 47.7% 29.6% 40.8% 0.225 Has Access to PC at home 81.8% 85.2% 83.1% 0.656 Has Access to Internet at home 77.3% 77.8% 77.5% 0.999 Have received computer training 50% 81.5% 62.0 0.008 (n=) 44 27 71 62% of nurses had received some training, although it was often superficial and run in house to provide basic knowledge of how to use the clinical computer system. Some learned from family members, colleagues or were self-taught, in the use of Microsoft Word and Excel. A few respondents received more formal training on the use of Internet for literature searching from a library, evening classes or as part of high education. Open question data shows that the training received are very diverse both in subjects and level of skills: "...two fifteen minute sessions in 1991 when we first got computers, a second when system was updated" "...only one day. All other courses since have been at difficult times or locations" "...very basic" "...not sufficient though" "...no formal courses, only shown by colleague when I was new in post" "Protected time for PC training would be most valuable – so often this is hurriedly given during the odd 5-minute break rather than in a training session." "...adequate training is really necessary and protected time; 10–15 minutes between patients is not adequate." Respondents were asked to rate their preferred format for ICT training on a 4-point scale: from 0 for least desirable to 3 for highly desirable. The result showed that 'one-to-one' training and workshops were the preferred formats for training, printed manual the least (Table 2 .) Table 2 Preferred format for training Training format Mean rank Printed manual 1.73 Tutorial on the Internet 2.68 Lectures 2.46 Workshops 3.75 One to one 4.38 (Friedman Test: n = 42; X 2 = 87.01; df = 4; p < 0.001). The preferred location for the training reflected the format of training requested (Table 3 .) Most wanted training to take place one-to-one in their workplace with those wanting workshops looking to see them held in an education or teaching centre. No age or professional group differences were found for the format or venue of ICT training required. Table 3 Preferred location of IT training. % of responses % of cases At work 63.7 84.1 In education/teaching centre 29.7 39.1 Library 2.2 2.9 Home (self directed learning) 4.4 5.8 100% 131.9% (Multi-response analysis: 3 missing cases; 69 valid cases) Confidence with computers appeared to be age related, with younger nurses having higher levels of confidence across all the areas of competence than their more senior colleagues. However, only two of these trends, use of spread sheets and electronic patient records (EPR) were found to be statistically significant, see Table 4 ; though the latter is critically important for patient care. Practice nurses had significantly higher levels of confidence in working with the EPR. 95.5% of practice nurses felt confident compared with 53% of district nurses and 44% of health visitors (p < 0.01). Table 4 Confidence in the use of IT among primary care nurses Age 30–39 Age 39–49 Age 50 & over X 2 Mouse 97.7% 96.3% 97.2% 0.724 Key board 93.3% 81.5% 88.7% 0.291 Word processor 70.5% 51.9% 63.4% 0.128 Spread sheet 43.2% 25.9% 36.6% 0.095 e-mail 79.5% 66.7% 74.6% 0.243 Internet 81.8% 70.4% 77.5% 0.457 Electronic library 52.3% 33.3% 45.1% 0.292 Electronic medical records 79.5% 37.0% 63.4% P = 0.001 n= 44 27 71 The raw data also suggested that there was a discernable trend in computer usage with age. 90% of nurses age 30 to 39 years used their workplace computer daily, 70% of those 40 to 49, and only 59.3% of those over 50. This trend was not significant, see Table 5 . Table 5 Use of computers at work for different age groups of community nurses Age 30–49 Age 50 & over Total At Least daily 75.0 59.3 69 (n = 49) At least weekly 13.6 25.9 18.3 (n = 13) At least monthly or never 11.4 14.8 12.7 (n = 9) n= 44 27 71 Colleagues were the most used source of information across all ages and nursing professions. Older nurses tended to use books and journals from their personal collections, and to use libraries. However, the use of libraries, books and journals is quite low. Younger nurses seem to use journals at work or electronic resources more. Neither of these trends, shown in Table 6 , were statistically significant. A larger proportion of practice nurses were more confident about using electronic libraries (63%), compared with health visitors (35%), and district nurses (24%); this trend was also not statistically significant. Table 6 Sources of information and knowledge for primary care nurses Age 30–49 Age 50 & over All Fisher 1 tail p = Books personal collection 75.0% 85.2% 78.9% 0.238 Journals personal collection 43.2% 51.9% 46.5% 0.320 Libraries 36.4% 29.6% 33.8% 0.376 Journals at work 75.0% 77.8% 76.1% 0.513 Electronic resources 29.5% 33.3% 31.0% 0.469 Books at work 68.2% 74.1% 70.4% 0.401 n= 44 27 71 Nurses summarised their experiences in access information in a number of free text comments: "don't know where to look", "don't know website address", "vast amounts of irrelevant articles under same headings", "I am not as good as I should be with CIHNAL, Medline", "information not found or not relevant some difficulty sometimes refining the search for specific information" "lack of training/unable to access information I am looking for quickly enough – quicker to look in a book" "like trying to get a glass of water from Niagara Falls" Discussion This study demonstrated that primary care nurses have high levels of access to IT and only a small minority have no access at all. Practice nurses, working within the surgery, all use the computerised medical record and nearly all feel confident to use it; over three-quarters have access to the Internet. However, community bases nurses in the second half of their careers despite receiving more training; have less access and are less confident about computers, especially use of the EPR, and prefer to use paper-based information resources. Across all ages and professions using libraries, reading journals and accessing on-line resources are minority activities. One to one learning in the workplace, and workshops were believed to be the best way to learn, with printed manuals and on-line tutorials of least value. The implication of this study is that community nursing needs more silver surfers. Silver surfers are those over 50 years who enjoy using the Internet. The primary care nurses over 50, despite more training, are still looking for information on paper; using their computers less at work; and less confident in computer use. Market strategies are making the over 50 s the fastest expanding group of Internet users this decade [ 17 , 18 ]; but the benefits of this growth in the market have not as yet had sufficient impact on primary care nurses. The successful aspects of these strategies need to be replicated for community over 50 years if they are to become effective users of the EPR and access the ever increasing number of online information resources. Nurses have clear ideas about the sort of training they want; however their chosen options is the most labour intensive and potentially expensive for the health service to provide. The small sample size and single locality of this study are its principal weaknesses. The former prevented any more detailed sub-group analysis, as the groups became too small. A further reminder may have improved the response rate. This study shows that nurses have made enormous strides in acquiring IT skills and access to computers since earlier surveys [ 10 - 12 ]; although these surveys included a smaller proportion of practice nurses – the group that appears to have embraced information technology the most. However, the use of Internet sources of information has changed relatively little. The change strategies proposed by the nurses fit with much of the literature, even though most of this was written in the context of implementing evidence based guidelines. It is likely that lessons about changing one type of behaviour in medicine are likely to apply to others [ 19 - 21 ]. Further research is needed with a larger sample to see if some of the non-statistically significant trends seen in this data become significant in a larger sample drawn from a national sample. It is important for the NHS to understand what input is needed to raise the level of use of the EPR and online information; and how quickly this change can be achieved. What nurses believe to be most effective ways of changing behaviour, and promoting computer use needs to be tested. Conclusions There is a consensus among primary care nurses of the sort of training they require to improve their computer use. A significant proportion of community nurses in the second half of their careers need this additional support if they are to achieve silver surfer status; and, have the skills needed to work in the new computerised NHS. Studies need to test whether providing the training wanted overcomes the barriers reported. Although primary care nurses have acquired more computer skills than previously reported there is no room for complacency – community nurses need the opportunity to develop into silver surfers. Abbreviations CPHVA Community Practitioners and Health Visitors Association, a representative body of community nurses. EPR Electronic patient record, synonymous with Computerised Medical Record. IT Information Technology NHS National Health Service. The UK's state funded health service. PCEL Primary Care electronic Library, an on-line information resource for primary care. PC personal computer PCT Primary Care Trust – organises and commissions care for a locality of about 100,000 population. RCN Royal College of Nursing SNOMED CT Systematized Nomenclature of Medicine – Clinical Terms SPSS Statistical Package for Social Sciences – a computerised statistical package. Competing interests The author(s) declare that they have no competing interests. Authors' contributions All authors conceived the study, and contributed to all aspects of the paper. The major contributions of each author is as follows: TC Analysed the data for the study, SB Recruited the nurses, sent out and collated the questionnaire, SdeL helped develop the questionnaire used in a earlier study and wrote the initial draft of the paper. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 Community nurses access to and use of computers: questionnaire Click here for file

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549600

Injections and HIV in Rural Zimbabwe

Of the 40 million people worldwide with HIV, 30 million live in the developing world. By far the worst hit region is sub-Saharan Africa, where nearly four million children have lost one or both parents to HIV/AIDS since 2000. Is heterosexual transmission the driving force behind the HIV epidemic in sub-Saharan Africa? In a controversial debate, some researchers have suggested that other factors such as unsafe medical injection practices may also be to blame, and that by overlooking, and even suppressing, analysis of this possible route of transmission, the current focus on preventing sexual transmission may be misguided. In this month&apos;s PLoS Medicine , Ben Lopman and colleagues argue that although it is right to criticize the lack of evidence on unsafe medical injection, field data are hard to collect. They note that in the only published study addressing this issue, Kiwanuka and colleagues found no link between unsafe injections and HIV spread in rural Uganda. In an effort to “inform the debate” further, Lopman and colleagues looked at the association between HIV and unsafe injection practices in rural Zimbabwe. Are medical injections an important cause of HIV in rural Africa? The team analyzed data from adults in Manicaland, a rural part of Zimbabwe, who were taking part in the Manicaland HIV/STD Prevention Study. In 1999 and 2000, eligible patients were tested for HIV and surveyed (86.7% were HIV negative at the start of the study), and were followed up three years later. The team collected survey data on injections in the patients, who were male and female adults aged 15 to 54 years old, and tested for an association between injection exposure and HIV infection. In 2002 and 2003, 505 of the men and 1,342 of the women, representing a 69.7% follow-up, were again interviewed and tested for HIV infection. Of these, 40% reported having had an injection or needle prick during the study period. A total of 67 patients developed HIV during the study; of these 13 (19%) said they had not had sex during the study period and 40 (60%) said they had not had an injection. The statistical analysis found no significant association between injections and HIV infection in men or women. Patients who had HIV when the study began did not have higher rates of injections. Instead, injections were highly associated with childbirth and pregnancy. But since HIV–positive women have reduced fertility, a reduction in the use of maternal services may partially explain why injections were not more common in these HIV-positive patients. In this study, the strongest predictor of HIV infection was symptoms of sexually transmitted disease. Despite problems of recall bias and under-reporting of sexual activity—a particularly difficult problem in studies in Africa—sexual behavior is consistently linked with HIV incidence. Where does this leave the debate over injections in Africa? Certainly, for this community, they do not seem to be a major source of HIV infection, and local policy-makers would therefore do best to concentrate on the prevention of sexually transmitted infections.

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548287

Patient-focused measures of functional health status and health-related quality of life in pediatric orthopedics: A case study in measurement selection

The objectives of this report are to review the assessment of patient-focused outcomes in pediatric orthopedic surgery, to describe a framework for identifying appropriate sets of measures, and to illustrate an application of the framework to a challenging orthopedic problem. A detailed framework of study design and measurement factors is described. The factors are important for selecting appropriate instruments to measure health status and health-related quality of life (HRQL) in a particular context. A study to evaluate treatment alternatives for patients with neurofibromatosis type 1 and congenital tibial dysplasia (NF1-CTD) provides a rich illustration of the application of the framework. The application involves great variability in the instrument selection factors. Furthermore, these patients and their supportive caregivers face numerous complex health challenges with long-term implications for HRQL. Detailed summaries of important generic preference-based multi-attribute measurement systems, pediatric health profile instruments, and pediatric orthopedic-specific instruments are presented. Age-appropriate generic and specific measures are identified for study of NF1-CTD patients. Selected measures include the Activities Scale for Children, Gillette Functional Assessment Questionnaire Walking Scale, Health Utilities Index, and Pediatric Inventory of Quality of Life. Reliable and valid measures for application to pediatric orthopedics are available. There are important differences among measures. The selected measures complement each other. The framework in this report provides a guide for selecting appropriate measures. Application of appropriate sets of measures will enhance the ability to describe the morbidity of pediatric orthopedic patients and to assess the effectiveness of alternative clinical interventions. The framework for measurement of health status and HRQL from a patient perspective has relevance to many other areas of orthopedic practice.

Review Assessments of patient-focused health status and health-related quality of life (HRQL) are being recognized increasingly by clinicians, patient advocates, regulatory authorities, administrators and policy makers as primary measures of the need, efficacy, effectiveness and efficiency associated with health care services. These types of measures are commonly reported in the published literature of many health care disciplines although there are few published reports concerning orthopedic surgery. However, the orthopedic community is becoming interested in using evidence reported by patients and patients' family members to determine what is best for patients. Patient-focused evidence (including patient-reported outcomes, PROs) refers to results from functional health status (FHS) and HRQL studies of orthopedic outcomes with measurements obtained from the perspective of patients or their non-clinician caregivers (e.g., parents of patients too young to self-report). The field of orthopedics is concerned with all musculo-skeletal problems from the top to the bottom of the human body. This broad anatomical range is associated with a wide range of functional problems. Some orthopedic problems are highly focused in regards to anatomy and associated with highly effective therapies. However, other orthopedic problems are of a diverse nature, the etiology is often poorly understood, and effective treatment strategies are sometimes elusive. The breadth and depth of some particularly challenging orthopedic problems has stimulated an interest in patient-based perspectives of their own FHS and HRQL. These types of measures will be referred to as "health measures" for the purposes of this paper. Health measures can be used to assess the burden of morbidity associated with orthopedic diagnoses, and to assess the effectiveness and efficiency of therapies. The diversity of issues also led the American Academy of Orthopedic Surgeons and the Pediatric Orthopedic Society of North America to develop the Pediatric Outcomes Data Collection Instrument (PODCI), and led other researchers to develop function-specific instruments such as the Activities Scale for Kids (ASK). However, there are a large variety of potentially appropriate health measures and there are limits to the number of these measures that can be used in any given study. The most appropriate set of health measures should be selected on the basis of underlying study objectives and design criteria [ 1 ]. Objectives and designs vary greatly across orthopedic studies. In general, orthopedic procedures and programs are undertaken to improve the health of patients. The health of patients can be measured in different ways. Conventional clinical or physiological measures are generally very useful for diagnostic and therapeutic purposes. Conventional clinical measures may, however, provide an incomplete assessment of health. For example, clinical examination and gait measures have been used to describe the quality and quantity of limitations in walking ability associated with hip flexion contracture but these types of assessments provide little information about the importance of this contracture relative to that of other serious health problems. In addition, various measures may provide conflicting results: some indicating improvement while others suggest decline in patients' health. Therefore, in the interest of scientific rigor, protocols to assess the overall effectiveness of therapy should include a priori specification of the comprehensive measure of health and a credible assessment viewpoint for the purposes of primary analysis of study results. Secondary study objectives may involve use of data collected from other health measures or from other assessment perspectives. FHS and HRQL measures are important for a variety of reasons that complement conventional clinical measures [ 2 ]. FHS measures provide descriptive information and HRQL measures add some type of valuation about the desirability of overall health status. Valuation may be based on preference-based scores or by including only items identified as important to patients. HRQL is a more comprehensive concept than FHS as noted in this leading definition. "Health-related quality of life is the value assigned to duration of life as modified by the impairments, functional states, perceptions, and social opportunities that are influenced by disease, injury, treatment or policy" [ 3 ]. It is generally accepted that a goal of therapy is to make patients feel better [ 4 ] and this is supported by statements of leading policy analysts such as "The goal of health care is to protect, promote, and maintain the health status of people" [ 5 ] and "Since the ultimate goal of all health care is to improve, restore, and preserve HRQL" [ 6 ]. However, physiological measures may change without people feeling better and people may feel better without measurable change in physiological function. Furthermore there is often a need for trade-offs between treatment-related benefits and adverse side effects. There is an increasing awareness also that various stakeholders in clinical decisions often have differing opinions about disability and that the opinions of patients should count. Numerous valid and reliable questionnaires are now available to collect FHS and HRQL measurements from patients and their representatives. The measures may be used for discriminative (comparing groups at a point in time such as assessing the burden of illness), evaluative (assessing within-person change over time as in clinical trials), and predictive purposes (providing prognostic information) [ 7 ]. A series of studies from a randomized controlled clinical trial (RCT) and prospective cohort study of elective total hip arthroplasty are illustrative of these purposes. These examples, while not from a pediatric setting, are orthopedic and demonstrate how data can be used for a variety of purposes. In the RCT, Rorabeck and colleagues [ 8 ] undertook a discriminative analysis of a set of FHS and HRQL measures, and reported there were no differences in outcomes between cemented and non-cemented protheses. Evaluative analyses of data from the same trial have documented improvements according to six HRQL measures and the six-minute-walk test [ 9 ], and have shown important differences among results from four major generic HRQL measures [ 10 ]. In the prospective cohort study by Mahon et al [ 11 ], waiting time for surgery was inversely related to baseline WOMAC (Western Ontario and McMaster University Osteoarthritis Index) scores for patients at time of referral to an orthopedic surgeon. There are many factors to consider when designing studies using patient-focused health measures for orthopedic patients. Among the most important factors are the study objectives, types of patients, the dimensions and FHS constructs associated with the health problem under study, the appropriateness of the questionnaire for the age range of the patients, the viewpoint of the assessors answering the questionnaire, the method of collecting questionnaire information (self-completion or interviewer-administration), the period of time respondents are asked to consider when answering the questionnaire (i.e., assessment recall period), and measurement properties of assessment tools. Measurement properties include content and construct validity, test-retest and inter-rater reliability, responsiveness, and practical limitations of data collection. It takes many years, and often decades, for credible and relevant evidence to be accumulated about the measurement properties of instruments. Instruments with well-established measurement properties should be used whenever possible. Two prime issues for assessments of children are the developmental stage of the subjects and the most appropriate respondent for questionnaires. The methods section of the paper presents a framework of important factors for consideration in designing studies to assess FHS and HRQL for orthopedic patients. The results were generated by applying the framework in the context of developing a proposal for a comprehensive international study of patients with neurofibromatosis type 1 (NF1) and congenital tibial dysplasia (CTD). The NF1-CTD protocol provides a rich illustration because it encompasses great variability in the instrument selection factors, and these patients with their supportive caregivers face numerous complex health status challenges with long-term implications for HRQL. Furthermore, there is little information in the published literature about the comprehensive health status and HRQL of patients with NF1-CTD. The rationale and approach to studying FHS and HRQL from a patient perspective have relevance to many other areas of orthopedics. Methods: a framework for orthopedic applications This section identifies specific study design and measurement instrument factors that should be considered in selecting measures for use in studies, and presents some background information for illustrating the application of these factors. Study design factors Study design factors are specified in detailed protocols that clearly define the study objectives and conditions. Well-defined study objectives identify the study subjects, and type and number of measurements required. Study conditions describe practical issues including data collection sites, and budgets for time and resources. Variability in study objectives A highly focused, single objective study may require relatively few instruments while a broad-based, multi-faceted study will require numerous measures. For example, a RCT of efficacy for an experimental technique to achieve union of bone compared to a conventional technique might specify patients' reports from a single well-established walking ability scale as the primary outcome measure. However, an economic evaluation of the cost-effectiveness and cost-utility of the experimental technique would require at least two instruments: the single well-established walking ability scale to estimate effectiveness for the numerator in the cost-effectiveness ratio; and a utility-based scale of overall HRQL to estimate quality-adjusted life years for the numerator in the cost-utility ratio. Age range of subjects The age of study subjects affects the choice of measurement instruments and the method of collecting data. Each instrument is valid for a specific age range and the age range is quite small for many pediatric instruments. For example, developmental changes during childhood make it difficult for single instruments to be valid from infancy through adolescence. Many instruments are valid for one or more of the following age categories: infants, pre-school children, primary-school children, adolescents, young / middle-age adults, seniors and elderly. Inability to read well (e.g., young children or populations with high illiteracy rates), or see well (e.g., many elderly populations), or concentrate well (e.g., people on certain strong medications) inhibits use of self-complete questions. Well-designed interviewer-administered questionnaires pose clear, short, well-focused questions with readily understood and easily remembered response options (e.g., yes or no response options). Assessment perspective It is becoming widely accepted in many disciplines that studies of health care programs and technologies should include measures of patients' perceptions of their FHS and HRQL [ 12 ]. Measurements of these types have been shown to vary by assessment viewpoint and, although many different viewpoints are valid, the patient perspective is considered to be one of the most important. It has been shown that children as young as 7 years can reliably complete interviewer-administered disease-specific and generic questionnaires about their own health [ 13 ]. Type of data collection sites The locations and cultural characteristics of the study population will determine the language and sometimes the choice of assessors. Relatively few instruments have been carefully translated and culturally-adapted to facilitate use in a large variety of communities. Some cultures accept a variety of assessment viewpoints (e.g., patient, family, nurse, physician, and other allied health professionals) while other cultures recognize only a physician viewpoint for health assessments. Mode of data collection The literature suggests that there may be important effects due to mode of data collection [ 14 ] and, therefore, mode of administration should be standardized across subjects, assessors, and assessment points. In general, self-complete questionnaires tend to elicit reports of greater morbidity than interviewer-administered questionnaires. It is hypothesized that subjects may be less inhibited to report disabilities on self-complete questionnaires than to report disabilities to interviewers. The mode of data collection for a study may be determined by numerous factors other than the characteristics of study subjects mentioned above. A low budget study may rely on a mail survey, or distribution of questionnaires in a busy clinic setting, and use self-complete questionnaires. Alternatively, a study involving serial assessments may require follow-up data to be collected by telephone and therefore require use of questionnaires designed for interviewer-administration. Assessment recall period FHS questionnaires should have well-specified assessment periods to help ensure that the subjects and researchers know the period of time covered by the responses. Assessment recall period refers to the period of time the assessor is asked to consider when answering the questionnaire. The period should match the objectives of the study. For instance, if a new surgical technique is thought to reduce the peri-operative burden of morbidity then frequent assessments with a brief recall period (24 hours) might be suitable. Standard assessment periods include the past 24 hours, the past 1 week, the past 2 weeks and the past 4 weeks. Short assessment periods should be used in studies of patients whose FHS varies over time and in studies involving serial assessments to ensure that there is no overlap in assessment times. Relatively long recall assessment durations may be used when it can be assumed patients' health status is fairly stable. For example, a four-week recall assessment period was used to measure the HRQL of patients in a randomized clinical trial and economic evaluation of two alternative treatment strategies for patients with knee osteoarthritis [ 15 , 16 ]. Other factors There are limits to the number and type of measures that respondents can be expected to complete without getting tired or frustrated, and/or that the study budget can afford in regards to both data collection and analysis. The evidence about the limits of respondent burden is sparse. All else being equal, studies involving serial assessments should expect to collect fewer measurements per assessment than single-assessment cross-sectional surveys. To maximize efficiency, instruments should be selected to provide complementary rather than overlapping information. Instrument factors Types of measures There are numerous definitions of FHS and HRQL. For the purposes of the paper, a FHS measure is defined as being descriptive in terms of functional ability and HRQL is defined as involving some form of valuation of that health status. One published taxonomy [ 7 ] suggests that measures may be classified as specific or generic. Specific measures are focused on a specified health problem, disease, or age group of subjects. An example is the Western Ontario McMaster Osteoarthritis Index [ 17 ]. Specific measures designed for evaluative purposes are often able to detect small but clinically important differences among subjects and be responsive to small but clinically important intra-subject changes over time. Generic measures are applicable to a broad range of subjects, including a wide variety of clinical groups and general populations. There are 2 types of generic measures: generic health profile instruments such as the Rand-36 [ 18 ] and SF-36 [ 19 ]; and generic preference-based instruments. There are 2 types of generic preference-based instruments: direct measurement instruments, such as standard gamble [ 20 ]; and multi-attribute classification systems with preference-based scoring functions [ 21 ], such as the Health Utilities Index [ 22 , 23 ] and the Quality of Well-Being Scale [ 24 , 25 ]. A detailed description of direct measurement techniques is beyond the scope of this paper but provided in a recent review paper by Torrance and colleagues [ 20 ]. Direct preference measurement is generally not practical for application in most clinical studies, especially those involving very young children. Customized instrumentation, usually relying on administration by highly skilled interviewers, must be developed for each study. Further, measurement questions are cognitively demanding. Direct preference measurement will, therefore, not be considered further in this paper. Each multi-attribute system includes a descriptive classification scheme to describe and assess health status, and a preference-based valuation system. HRQL scores for health states defined by multi-attribute systems are calculated from models fitted from directly measured preference measurements (see below). Multi-attribute systems provide descriptive information about comprehensive health status, and interval-scale preference scores of overall HRQL from a community perspective on a scale where 0.00 is the score for being dead and 1.00 is the score for being in perfect health. Several multi-attribute systems define negative scores of overall HRQL to represent preferences for states considered worse than being dead. A few systems include single-attribute preference-based scales of morbidity. Single-attribute morbidity scales are defined such that the least desirable level within an attribute (dimension of health status such as vision) has a score of 0.00 (blind) and the most desirable level has a score of 1.00. The community perspective is most widely recommended for technology assessment and reference case economic evaluation analyses [ 26 - 29 ]. Interval-scale properties, and a score of 0.00 for being dead, are important features of HRQL scales for integrating the effects of morbidity and mortality in descriptive studies and in cost-utility economic evaluations. Interval-scale preference scores of HRQL may be either utilities (e.g., Health Utilities Index Mark 3) or values (e.g., EQ-5D). Utility preference measures are based on von Neumann-Morgenstern utility theory, include an element of risk attitude, and are therefore appropriate for decision problems with uncertainty. Value scores are preferences measured under certainty. Details about differences between utilities and values, and about direct preference measurement, appear in recent papers by Torrance et al [ 20 , 30 ]. Uncertainty is an important factor in many orthopedic procedures and therefore utility scores are more appropriate than value scores in this context. Evidence of measurement properties: validity, reliability, and responsiveness A valid measure is "sound and sufficient" [ 31 ]. There are many ways to assess validity of measures. Assessments of FHS and HRQL measures should consider at least six types of validity: face validity, content validity, construct validity, convergent validity, discriminative validity, and predictive validity [ 32 ]. Face validity requires that a measure appear on the surface to make sense in regards to being relevant and useful. Content validity requires that the measure include all important and relevant domains or dimensions of health status. Construct validity describes the extent to which a measure corresponds to theoretical concepts and convergent validity describes the association between related variables. Discriminant validity is a lack of correlation between dissimilar variables or groups. Predictive validity, one type of criterion validity, describes the relationship between current and future measurements. A measure is reliable if it is sound and dependable [ 31 ]. Reliability is assessed by tests of repeatability or reproducibility. Reliability is often assessed in terms of agreement between intra-subject test-retest measurements and inter-assessor measurements [ 33 ]. Responsiveness is also referred to as sensitivity to change. It is an important feature for determining a measure's ability to detect effects of treatments or natural changes over time (e.g., due to the aging process). Husted and colleagues reviewed the literature and defined two major types of responsiveness: internal and external responsiveness [ 34 ]. Internal responsiveness describes the ability of a measure (instrument) to change and has been assessed using a variety of techniques including the magnitude of statistical significance tests (e.g., p < 0.05 versus p < 0.001), the mean change score divided by the standard deviation of scores at baseline (effect size), and a sensitivity coefficient calculated as the proportion of the variance in change scores due to treatment [ 32 ]. It has also been assessed as the ratio of the mean change in patients' scores and the pooled standard deviation of the mean change scores [ 35 ], and as the mean change score among those who changed divided by the standard deviation of change scores among stable patients [ 33 ]. External responsiveness is concerned with the relationship between change in a measurement and change in a reference measurement of health status. External responsiveness has been assessed using the receiver operating characteristic method, correlations (e.g., Pearson product moment correlation), and regression models. The minimum important difference (MID) is the smallest size of difference that is important from patients' or clinicians' perspectives. The MID between two measurements is a concept closely related to responsiveness when assessing change over time [ 36 ]. Ceiling and floor effects are undesirable properties that reduce the validity, reliability, and responsiveness of measures. A ceiling effect may occur when a large proportion of measurement observations are close to the upper bound of the measurement scale. A ceiling effect results in a positively skewed distribution of measurements, limited ability of the measure to discriminate among subjects at the upper end of the scale, and attenuated responsiveness to improvements in health in longitudinal studies. A floor effect may occur when a large proportion of measurement observations are close to the lower bound of the measurement scale. Floor effects create a negatively skewed distribution of measurements, limited ability of the measure to discriminate among subjects at the lower end of the scale, and decreased responsiveness to decrements in health in longitudinal studies. Many generic and specific measures of HRQL may be subject to ceiling effect problems in that they may not be able to describe patients or subjects with above average (supra-normal) health. Some measures are subject to floor effect problems. Some are subject to both. Typically floor effect problems are more serious in clinical studies (which often involve patients with disabilities) and ceiling effect problems may be more problematic in general population studies. Limits of respondent burden The limits of respondent burden depend upon many factors including the number of questions presented, how the questions are presented, the complexity of questions, the sophistication of respondents, and the respondents' interest in the questions. In general, the allowable length of questionnaire is shorter for mail and phone administration than for face-to-face interviewer administration [ 37 ]. One set of guidelines specifies the following maximum lengths: 20 questions for phone surveys; 60 questions for mailed surveys; and 80 questions for face-to-face interview surveys [ 38 ]. Another guideline recommends that telephone interviews not exceed 5 to 10 minutes [ 39 ]. These guidelines are in general agreement with maximum recommended number of pages for self-administered questionnaires: 2 to 4 page upper limit for topics not especially salient [ 40 ]; 12 page upper limit for self-administered questionnaires [ 41 ]; and 4 to 6 page upper limit for mailed surveys [ 42 ]. For mail-out surveys, the evidence suggests no effect of length on response rates for questionnaires varying from 3 to 9 pages [ 43 , 44 ] but reduced response rates with questionnaires greater than 12 pages [ 41 ]. Availability of support services Applications of FHS and HRQL measures are greatly facilitated by expert advice, detailed instructions and other services designed to support users of a measure. Supporting documentation is usually protected by copyright and should not be used without written permission of the original developers. Documentation obtained from third-party sources should be considered suspect because it is frequently invalid. Licensing fees are used to fund high quality, readily accessible service centers. Permission to use copyright materials is typically granted one study at a time. Support services may also include consultation about the most appropriate versions of questionnaires for use in a specific study. Application packages may include data collection instruments such as questionnaires, procedure manuals, coding algorithms and scoring systems, as well as background information about the conceptual and measurement properties of the instrument. NF1-CTD: A case study Recently there has been interest in using measures of FHS and HRQL to evaluate treatment alternatives for NF1-CTD. NF1 is one of the most common genetic disorders in childhood [ 45 ]. It is estimated that at least 1 million people throughout the world have NF1 [ 46 ]. NF1 has a wide range of clinical manifestations including abnormalities of the skin, nervous system, bones and soft tissues [ 46 ]. Other conditions experienced by children with NF1 include short stature and neurologic problems such as learning disabilities or unspecified school performance problems (36%), frequent headaches (28%), mental retardation (6%), and reduced reproductive potential [ 46 - 49 ]. CTD is rare in the general population, approximately 1 per 140,000 [ 46 ]. It has been estimated that approximately 1% of people with NF1 have CTD [ 46 ]. CTD is diagnosed usually during the first year of life and fractures often occur before 3 years of age. Frequently, initial presentation is tibial bowing followed by subsequent fracture and pseudoarthrosis [ 45 ]. There is no generally accepted standard for management of CTD although most surgeons would suggest initial treatment of either intramedullary fixation with bone grafting or resection and bone transplant. Surgical procedures for the treatment of CTD are fraught with complications and failure of union. For the treatment of CTD, pre-fracture bracing until skeletal maturity may be a better alternative than surgery. CTD is associated with severe complications due to nonunion or pseudoarthrosis after osteotomy and amputation may be required. Conventional clinical measures of CTD include the Crawford classification system [ 46 ]. These measures provide clinicians with important information used in diagnosis and management of well-established symptoms. A list of important concerns could be prepared by interviewing patients and members of their families. Standardized comprehensive tools that integrate multi-dimensional effects would also be useful in quantifying the number and extent of problems experienced by NF1-CTD patients, and other pediatric orthopedic patients with complex issues. The published literature on NF1 and NF1-CTD contains virtually no information based on FHS or HRQL measurements. The exception is a recent paper by Wolkenstein and colleagues [ 50 ] who reported results from 128 adult patients in France using the generic health profile SF-36 and a skin-disease-specific measure, Skindex-France. Surveys of the published literature, experts in the fields, web sites and other sources of information were conducted to determine the dimensions of health that are affected by NF1-CTD, the types of FHS and HRQL measures that have been used, which measures should be considered as potentially useful for studies of NF1-CTD, the measurement properties of potentially useful measures, and the relative merits of various measures. A review of the on-line Quality of Life Instruments Database (QOLID) developed by Dr. Marcello Tamburini and the MAPI Research Institute [ 51 ], and correspondence with instrument developers, identified a short list of potentially useful measurement tools in each of the following categories: generic preference-based HRQL systems, major pediatric and other generic health profiles, and disease or function specific measures. Selected measures should have demonstrated properties in accordance with currently accepted criteria [ 12 , 52 , 53 ] and should provide commensurate measurements for patients across a wide age range. Problems with mobility, cognition, pain, emotion (including impacts of problems with self-image), self-care, vision, and fertility are aspects of health reported in the published literature to be compromised in NF1 patients. Illustrative study design criteria There are five important research objectives of an NF1-CTD study that provide a context for applying the framework described in the Methods section: 1) to document long-term health outcomes associated with the disease and its treatment; 2) to measure the burden of disease and treatment during active therapy; 3) to investigate the hypothesis that improved HRQL is associated with initial amputation compared with multiple limb-saving procedures; 4) to determine relationships of FHS and HRQL with conventional clinical variables used in diagnosis and management; and 5) to assess the measurement properties (e.g., construct validity, patient versus parent inter-rater reliability, and responsiveness to change) of selected FHS and HRQL measures in NF1-CTD patients. These detailed objectives require the identification and assessment of leading FHS and HRQL measures for use in both cross-sectional and prospective longitudinal surveys. The prevalence of NF1-CTD is relatively low. Patients will need to be recruited from numerous clinical centers in North America to generate precise estimates of FHS and HRQL. Questionnaires should be available in at least 3 major languages: English, French and Spanish. The survey population ranges in age from newborn into adulthood and linking results across the study objectives requires that at least some of the assessment tools be in common across the age range of study patients. To avoid potential confounding effects, data collection techniques should be consistent across subjects and measures. The patient-focus will be represented by collecting measurements from all patients old enough to provide self-assessments, and from parents acting as proxy assessors for all children and adolescents. Self-complete questionnaires requiring minimal supervision should be used to eliminate the need for interviewers at each clinical center, to facilitate use of mail-out surveys, and to avoid potential "interviewer" effects. The number and type of measures per assessment, and the number of serial assessments per patient, should be sufficient to address all the study objectives within the limits of study resources and assessor burden. Measures of morbidity associated with NF1-CTD should be comparable with data on norms from surveys of general populations and other patient groups, and be useful for assessing the effectiveness and efficiency of health care services. Existing patient-focused health measures The HRQL measure should be comprehensive and preference-based, to facilitate a broad variety of comparisons. A pediatric health profile measure and other specific measures will be selected to complement the selected preference-based HRQL measure. FHS measures may be focused on one or more of the following: the population of interest (e.g., pediatrics); the major underlying disease (e.g., NF1); the major human function of most interest (e.g., walking ability); the medically-defined health problem of most interest (e.g., tibial dysplasia); the medical speciality most involved with treatment of the health problem (e.g., pediatric orthopedics). There are six major generic preference-based HRQL utility systems [ 21 ], presented here in chronological order of development: QWB [ 25 ], 15D [ 54 ], HUI [ 23 ], EQ-5D [ 55 ], AQOL [ 56 ] and SF-6D [ 57 , 58 ]. HRQL scores from these systems represent mean community preference scores. The 15D and AQOL have not been widely used outside of Finland and Australia respectively and, therefore, will not be described further in this paper. The SF-6D has been developed only recently so there is as yet little evidence to report. The major features of QWB, HUI, EQ-5D, and SF-6D systems are summarized in Table 1 [ 21 , 25 , 57 , 59 , 60 ]. The major characteristics vary greatly among the systems. For example, linear additive scoring models do not include effects of preference interactions among attributes or domains but multiplicative scoring functions include these effects. The QWB is available in both self-complete and interviewer-administered formats [ 61 ]. The symptoms attribute is a dominant feature of the QWB health status classification system. This emphasis is reflected in the population-derived preference weights. HUI health status classification systems cover more than 10 attributes. There is evidence that HUI scores agree well with mean directly measured standard gamble utility scores from a representative sample of the general population [ 59 , 60 , 62 , 63 ]. Numerous versions of HUI questionnaires are available and HUI has a service center [ 64 - 66 ]. It is available in numerous languages. A closely-related comprehensive health status classification system for pre-school children (CHSCS-PS) has been developed recently [ 67 - 69 ] for children age 2 through 5 years of age. EQ-5D is very simple and concise. It consists of 5 attributes with 3 levels per attribute, assesses "current" health status, has been used in a large number of studies, and is available in numerous languages. Information, including a long list of references, about EQ-5D is available on the EuroQol Group web site [ 70 ]. SF-6D is a multi-attribute health status classification system based on the SF-36 [ 19 , 71 , 72 ]. The SF-36 was not designed to be commensurate with the fitting of a multi-attribute utility function. The SF-6D health status classification system is a sub-set of the attributes defined in the SF-36 health status classification system [ 57 ]. SF-6D utility scores may be useful in retrospective studies analyzing previously collected SF-36 data. Table 1 Major Characteristics of Five Generic Preference-Based Multi-Attribute Systems Instrument QWB HUI EQ-5D SF-6D HUI2 HUI3 Developed 1970s 1980s 1990s 1990s 1990s # Attributes (# levels per attribute) 4 (3 to 27) 7 (3 to 5) 8 (5 or 6) 5 (3) 6* (4 to 6) # of unique health states 1215 24000 972000 243 18000 Attributes Mobility, Physical activity, Social activity, Symptoms / problems. Sensation, Mobility, Emotion, Cognition, Self-care, Pain, Fertility. Vision, Hearing, Speech, Ambulation, Dexterity, Emotion, Cognition, Pain. Mobility, Self-care, Usual activities, Pain / discomfort, Anxiety / depression. Physical functioning, Role limitations, Social functioning, Pain, Mental health, Vitality. Types of HRQL scores and measurement Values; VAS Utilities; VAS and SG combination Utilities; VAS and SG combination Values; TTO Utilities; SG HRQL scoring model form Linear additive Multiplicative Multiplicative Modified linear additive Modified linear additive HRQL scale interval 0.00 to 1.00 -0.03 to 1.00 -0.36 to 1.00 -0.59 to 1.00 0.00 to 1.00 Questionnaire formats (languages) 2 (numerous) 16 (numerous) 1 (numerous) 2 SF-36 versions (numerous) SG – standard gamble; TTO – time trade-off; VAS – visual analog scale. * – 6 attributes but role limitations attribute above includes emotional and physical so encompasses 7 of 8 SF-36 domains. The major population-specific health profiles include the Child Health Questionnaire (CHQ), Pediatric Inventory of Quality of Life (PedsQL), Pediatric Evaluation of Disability Inventory (PEI) and TNO-AZL Pre-School Children Quality of Life questionnaire (TAPQOL). The PEI is limited to children age 0.5 – 7 years of age and requires a structured parent interview or clinician observation [ 73 ]. TAPQOL [ 74 , 51 ] is limited to children 0.5 to 5 years of age. Therefore, PEI and TAPQOL will not be discussed further. The major pediatric disease-/function-/specialty-specific instruments include the PODCI (also referred to as the POSNA or Pediatric Orthopedic Society of North America instrument), ASK, Gillette Functional Assessment Questionnaire Walking Scale (FAQ walking scale), and Wee-FIM. Wee-FIM [ 75 ], a popular measure of functional independence, is not being considered because it involves clinician assessments rather than assessments from a patient or parent perspective. In general, disease-specific scales in orthopedics focus on pain and physical function because these factors are major areas of concern for orthopedic patients and no generic health measures have been developed specifically for orthopedic application [ 73 ]. No relevant disease-specific measures or disease-specific preference-based tools were identified. A summary of the major pediatric generic health profiles appears in Tables 2 and 3 . The CHQ [ 76 ] covers relevant physical domains and provides detail on emotion/psychological health. The PedsQL [ 77 ] assesses physical, emotional, social and school functioning. It has demonstrated a return to health 3 months after acute limb fractures [ 78 ] and has been used in large general population surveys [ 79 ]. Table 2 Major Characteristics of Two Pediatric Health Profile Systems Instrument CHQ PedsQL 4.0 Full Name Child Health Questionnaire Pediatric Inventory of Quality of Life Origin 1996 1998 Version Parent Forms (PF) Parent Form for Infants & Toddlers Youth- Completion Form Child Self-Report Forms 1) ages 5–7 years 2) ages 8–12 years 3) ages 13–18 years Parent Report Forms 1) ages 2–4 years 2) ages 5–7 years 3) ages 8–12 years 4) ages 13–18 years Measurement Constructs Measures of physical and psycho-social health concepts Measures of core dimensions delineated by WHO Age Bounds (years) 5 to 17 2 months to 5 years 11 to 17 5 to 18 2 to 18 # Items 98 (PF98) or 50 (PF50) or 28 (PF28) short form 102 87 (working on shorter form) 23 # Domains 10 child & 4 parent concepts 8 infant & 5 parent concepts 10 child concepts 4 "Generic Core" Overall Score 2 Global Physical & Global Psycho-Social 1 overall, for primary analyses Sub-scale Scores 14 13 10 4 sub-scales, for secondary and descriptive analyses Completion Time (minutes) 10 to 20 for PF50; 7 to 15 for PF28 20 to 30 15 to 30 n/a < 4 Spanish Language Yes Yes Assessment Perspective Parents' Parents' Patients' Patients' Parents' Legend: WHO – World Health Organization. Table 3 Domains and Constructs of Forms for Two Pediatric Health Profile Systems Instrument CHQ: Child Health Questionnaire PedsQL 4.0 Version Parent Forms (PF) Youth-Completion Form Parent Form for Infants & Toddlers Child Forms (3 versions: 5–7, 8–12, 13–18 yrs) Parent Forms (4 versions: 2–4, 5–7, 8–12, 13–18 yrs) Domain Names 1. Physical functioning – 6 items. - e.g., Child is greatly limited in performing all physical activities, including self-care, due to physical health. 2. Role (school and activities) limitations due to emotional.* 3. Role limitations due to behavioral problems* – 3 items. - e.g., Child is limited a lot in school work or activities with friends due to behavioral problems. 4. Role limitations due to physical problems – 2 items. - e.g., Child is greatly limited in school work or activities with friends due to physical health. 5. Bodily pain – 2 items. - e.g., Child has extremely severe, frequent and limiting body pain. 6. General behavior – 6 items. - e.g., Child very often exhibits aggressive, immature, delinquent behavior. 7. Mental health – 5 items. - e.g., Child has feelings of anxiety and depression all the time. 8. Self-esteem – 6 items. - e.g., Child is very dissatisfied with abilities, looks, family/peer relationships and life overall. 9. General health perceptions – 6 items. - e.g., Parent believes child's health is poor and likely to get worse. 10.Change in health – 1 item. - e.g., Child's health is much worse now than 1 year ago. 1. Physical Functioning. 2. Behavior (perception). 3. Growth & Development. 4. Pain. 5. Behavior (getting along). 6. Temperament & Mood. 7. General Health. 8. Change in Health. 1.. Physical Functioning – 8 items. - hard to walk more than one block, hard to run, hard to do sports or exercises, hard to lift something heavy, hard to take a bath, hard to do chores around the house, hurt or ache, low energy. 2. Emotional Functioning – 5 items. - feel afraid or scared, feel sad or blue, feel angry, trouble sleeping, worry about what will happen. 3. Social Functioning – 5 items. - trouble getting around, other kids not wanting to be friends, teased, doing things other peers do, hard to keep up when playing with others. 4. School Functioning – 5 items. - hard to concentrate, forget things, trouble keeping up with schoolwork, miss school because not well, miss school for doctor appointments. *Role Emotional and Role Behavioral are separate scales in PF98 but combined into one in the PF50 and PF28. Table 4 summarizes the major characteristics of four orthopedic-specific measures. The PODCI [ 80 ] was designed specifically as a very comprehensive measure of musclo-skeletal outcomes associated with pediatric orthopedic problems. ASK was designed to measure children's activities in terms of both capacity and performance [ 81 ], and it assesses domains not covered in detail by other instruments [ 82 ]. The FAQ walking scale provides the most complete measure of walking abilities. Table 4 Major Characteristics of Orthopedic-Specific Systems Instrument PODCI ASK FAQ walking scale Full Name Pediatric Outcomes Data Collection Instrument Activities Scale for Kids Gillette Functional Assessment Questionnaire: Functional Walking Scale Origin 1997 1996 1993 Measurement constructs Musculoskeletal outcomes Physical disability in terms of capacity or performance dimensions Complete range of functional walking abilities Age range (years) 2 – 18 2 – 18 2 + # Items 108 30 1 # Domains; and Domains 8 Comorbidity, Upper Extremity & Physical Function, Transfers & Basic Mobility, Sports & Physical Function, Pain, Treatment Expectations, Happiness, Satisfaction with Symptoms. 9 Personal Care, Dressing, Eating & Drinking, Miscellaneous, Play, Locomotion, Standing Skills, Stairs, Transfers. 1 Walking. Overall Score Yes, Global Function, mean of sub-scales for domains 1 to 4 above Yes, additive and not weighted Yes Sub-scale Scores Yes, additive No No Completion Time (minutes) 10 – 20 5 – 12 < 2 Spanish Language version No No No Assessment Perspective Patient &/or Parent (Diagnostic, complications and aims section by clinician) Patient Parent The choice of existing measures is based on a process of elimination considering the relative strengths of each instrument and the complementarities among measures. Neither the SF-36 nor the EQ-5D is valid for use in adolescent patients with orthopedic problems [ 83 ]. A review of measurement of HRQL in children by Eiser & Morse [[ 84 ]; see also [ 85 , 86 ]] identified HUI and CHQ and PedsQL as the only 3 generic measures that fulfill all specified review criteria: established reliability and validity; suitable for self- and proxy-report; and brief (<30 items). PODCI outperformed CHQ physical functioning scale for orthopedic patients [ 87 ]. However, PODCI has considerable problems with missing data, especially in upper extremity function and physical function and sports scales for children ages 2 to 5 years, associated with the use of "too young" response options [ 88 ]. ASK is reported to be more sensitive to change in disability levels than HUI [Young N, personal email communication to W Furlong 2002-02-18]. The FAQ walking scale provides the most complete assessment of functional walking abilities, especially at the upper end of the scale [ 89 ]. In summary, there are few measures available for assessing subjects less than 5 years of age and even fewer for subjects less than 2 years of age. Most relevant measures are available in self-complete format only. Preliminary recommendations for the NF1-CTD study were that PedsQL be used as the generic health profile, HUI be the multi-attribute preference-based measure of HRQL utility scores for children age 5 years and older and that CHSCS-PS be the measure for children age 2 through 4 years, ASK be the measure of activity limitation, FAQ walking scale be the measure of walking ability, and that a small feasibility study of these instruments be completed with a convenience sample. Feasibility study A pilot feasibility study surveyed 8 NF1 patients using HUI and FAQ walking scale measures. Questionnaires were completed by 6 NF1 patients and 3 parents. The combined HUI and FAQ walking scale questions took respondents an average of 13 minutes (range, 9–20 minutes) to complete. The patients were 11 to 50+ years old and had health problems ranging from mild to severe. One patient with tibial dysplasia and 2 patients with scoliosis were included. HUI data were collected from 5 patients, 2 parents and both the patient and parents in one case. Health problems were reported in 7 of the 8 HUI3 attributes (vision, speech, ambulation, dexterity, emotion, cognition, and pain; no problems with hearing were reported). The attributes associated with the most morbidity, as assessed using HUI3 single-attribute utility scores [ 60 ], were pain (mean score = 0.81), speech (0.94), cognition (0.94), and emotion (0.94). For the 7 patients having complete data, 5 had two or more HUI2 and HUI3 attributes at less than full function. On the conventional utility scale in which being dead = 0.00 and in perfect health = 1.00, the HUI3 scores ranged from 0.45 to 1.00. The mean HUI3 score, 0.73, is similar to the mean score of 0.77 for adults with arthritis [ 90 ]. FAQ walking scale data were collected from 4 patients (1 of the 5 survey patients did not answer the question), 2 parents and both the patient and parents in one case. Five patients were reported to be at Level 10 (walks, runs, and climbs on level and uneven terrain and does stairs without difficulty or assistance), one patient to be at Level 8 (walks outside the home for community distances, is able to get around on curbs and uneven terrain in addition to level surfaces, but usually requires minimal assistance or supervision for safety), and one patient at Level 6 (walks more than 15–50 ft. outside the home but usually uses a wheelchair or stroller for community distances or in congested areas). In summary, the feasibility study showed that the HUI and FAQ walking scale questions were acceptable to patients' families and that results, especially for HUI, reflected the large variability in HRQL of the sample of patients. Choice of measures for illustrative study No single measure will provide sufficient data to address all the important study objectives. A set of measures is required. The set of measures should provide complementary data of health status and preference-based scores of HRQL. Redundancy in measurement is reduced, and efficiency of measurement is increased, by selecting the most comprehensive generic measures and then supplementing these measures with the most appropriate set of specific measures. It is recommended that HUI be selected as the comprehensive generic measure for ten reasons: a) it includes both generic health profile and preference-based scoring systems; b) the preference-based scoring systems are well-validated; c) it is the most comprehensive, compact and efficient of these types of systems; d) it includes many of the most important domains in the context of NF1-CTD; e) it is applicable for all people age 5 years and older; f) well-developed data collection questionnaires are available to match the study design criteria; g) HUI results facilitate integrating effects of morbidity and mortality, and cost-utility economic evaluations; h) it has been used successfully in a variety of studies of musculoskeletal problems; i) population norm data are available; and j) a closely-related health status system, the CHSCS-PS, is available to assess children 2 through 4 years of age. The HUI will provide a broad set of measures for comparisons with other populations and for estimating HRQL on a general scale such that dead = 0.00 and perfect health = 1.00. As a generic measure, HUI also has the ability to capture side effects and the effects of co-morbidities. However, these broad measures may not be responsive to small but important changes in health status. Therefore, HUI should be complemented by a set of instruments focused on pediatric, orthopedic and walking issues. The PedsQL 4.0, a pediatric generic health profile, should also be included in the set of measures because: a) it includes domains, social and school function, which complement HUI and CHSCS-PS domains; b) it is appropriate for children ages 2 through 18 years; c) it is not overly burdensome in terms of data collection; d) patient and parent assessment questionnaires are available; and e) it can be interviewer-administered to facilitate data collection by telephone, if necessary. Two specific measures should also be part of the set of instrumentation: the ASK and the FAQ walking scale. ASK is an orthopedic-specific instrument which has been shown to cover the most important domains in the context of musculoskeletal disorders, including the impact of limb lengthening surgery experienced by many children with tibial dysplasia. ASK is also attractive because it provides overall summary scores for both performance and capability measures, and is only moderately burdensome to complete. Walking ability is one of the most important aspects of health that is frequently compromised in NF1-CTD patients, and the FAQ walking scale is the most complete scale of functional walking ability currently available and it only requires asking one question. CHQ is not recommended because it does not add much to the set of recommended measures and it is burdensome to complete. PODCI is not recommended because it is very burdensome to complete, it has been reported to have major problems with "missing data", and the system for collapsing questionnaire responses into summary scores is not well validated. Children ages 2 to 5 years of age should be assessed by their parents using three questionnaires: the CHSCS-PS (12 questions), the PedsQL (23 questions); and the FAQ walking scale (1 question). It is expected that all three of these questionnaires can be completed in an average of 15 minutes. Children and adolescents ages 5 to 17 years of age should be assessed by their parents using four questionnaires: the HUI (15 questions), the PedsQL (23 questions), the FAQ walking scale (1 question), and the ASK (30 questions). These four questionnaires are expected to be completed in an average of 20 to 30 minutes. Children and adolescents older than 11 years should provide self-assessments using four questionnaires: the HUI (15 questions), the PedsQL (23 questions), the FAQ walking scale (1 question), and the ASK (30 questions). On average, it is expected that respondents will complete all four questionnaires in 20 to 30 minutes. Conclusions This paper highlights reasons why patient-focused measures of FHS and HRQL should be considered important tools in the field of orthopedic surgery. It has also noted that there is increasing competition for scarce health-care resources, that allocation decisions about these resources are being informed by evidence based on patient-focused health measures, and that these measures are being under-utilized by the orthopedic surgery community. The orthopedic community faces numerous obstacles in utilizing FHS and HRQL measures. One major obstacle is that the multitude of existing measures makes it difficult to decide which measures may be appropriate for a specific application. A second obstacle is that most of the information about FHS and HRQL measures is not reported in the orthopedic literature. A third obstacle is that usually no one measure can capture all the important aspects associated with a specific orthopedic issue. The framework outlined in the paper provides guidance for selecting appropriate FHS and HRQL measures. The framework guides orthopedic investigators to combine their basic study criteria, including objectives and clinical context, with key criteria for FHS and HRQL measures from the published literature. The results in this paper identify some major sources of information about health measures, identify some of the most widely used measures of FHS and HRQL, and provide summaries of key characteristics for selected measures in three major taxonomical classes: generic preference-based multi-attribute systems; generic pediatric health profile systems; and orthopedic-specific systems. It is clear that there are many important differences among measures both within and across taxonomical classes. All measures are not equal. There are sound factors for making judgements about which measures are most appropriate for a given application. A process of appraisal and elimination was used to select one measure from each taxonomical class for inclusion in the NF1-CTD study illustrative example, and a pilot study of the most readily available selected measures confirmed the feasibility of their use in a small sample of NF1-CTD patients. The paper shows that a set of relevant, valid, reliable, responsive and practical patient-focused health measures for use in an orthopedic study can be readily identified and selected from the published literature and information available on the worldwide web. We encourage orthopedic researchers to use the framework to identify and select appropriate patient-focused health measures in their future studies. Conflict of Interest W. Furlong and D. Feeny have a proprietary interest in Health Utilities Inc. which distributes copyright Health Utilities Index (HUI ® ) instrumentation and provides methodological advice on the use of HUI. Authors' contributions All authors were involved in critical review of drafts for intellectual content and have given final approval to the version submitted for publication. W Furlong was responsible for much of the overall design, acquisition of data, and initial drafting. R Barr and D Feeny made important contributions to the analysis and interpretation of results. S Yandow conceived the idea of presenting the information in a published manuscript.

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Young age: an independent risk factor for disease-free survival in women with operable breast cancer

Background The incidence of breast cancer in young women (age < 35) is low. The biology of the disease in this age group is poorly understood, and there are conflicting data regarding the prognosis for these women compared to older patients. Methods We retrospectively analyzed 2040 consecutive primary invasive breast cancer patients who underwent surgical procedures at our institution between 1990 and 1999. The younger age group was defined as patients aged <35 years at the time of diagnosis. The clinicopathological characteristics and treatment outcomes were compared between younger and older age groups. Results A total of 256 (12.5%) patients were aged <35. There was a significantly higher incidence of nuclear grade 3 and medullary histological-type tumors in younger patients compared to older patients. Axillary lymph node status, T stage, histological grade, c-erbB2 expression and estrogen receptor status did not differ significantly between the two age groups. Younger patients had a greater probability of recurrence and death at all time periods. Although there was no significant difference in disease-free survival between the two age groups in lymph node-negative patients, the younger group showed worse prognosis among lymph node-positive patients (p < 0.001). In multivariate analysis, young age remained a significant predictor of recurrence (p = 0.010). Conclusion Young age (<35) is an independent risk factor for relapse in operable breast cancer patients.

Background Breast cancer is relatively rare in women less than 35 years of age, with this group accounting for less than 4% of the total number of breast cancer cases diagnosed in Western countries [ 1 , 2 ]. Despite the disease being relatively uncommon, it has a severe negative effect on the patients and their families. It remains controversial whether young age at diagnosis is an adverse prognostic factor in primary breast cancer. While some studies have found that younger patients have worse clinical outcomes than older patients [ 3 - 7 ], others report younger patients have a more favorable prognosis, or that there is no relationship between outcome and age [ 8 - 10 ]. Various explanations have been given for these conflicting results, including small numbers of patients comprising the study population, differences in patient selection criteria and differences in the age groupings used in the analyses. Moreover, it has long been debated whether breast cancer diagnosed at a young age is a clinically and etiologically distinct disease from breast cancer diagnosed later in life. Some researchers reported that tumors in younger women were of higher grade, higher proliferation fraction, had more vascular invasion, and expressed fewer estrogen and progesterone receptors compared to tumors in older women [ 11 - 14 ]. It is important for clinicians to clarify the existing controversy as to whether aggressive treatment for young women with breast cancer is justified. Breast cancer is the most frequent cancer in Korean women and its incidence is increasing [ 15 ]. Breast cancer in young Korean women is a serious problem, with the proportion of young age-onset breast cancer much higher than in western countries. According to the 2002 annual report of the Korean central cancer registry, breast cancers that developed before the age of 35 comprised 9.5% of all breast cancers [ 16 ]. The aim of the present study was to retrospectively investigate clinicopathological characteristics and prognosis in a large, ethnically homogeneous group of young breast cancer patients (less than 35 years old) treated with the same strategy at a single institution. Methods A retrospective review was performed of the medical records of all consecutive primary invasive breast cancer patients (not including phyllodes tumor) undergoing curative surgery in the Department of Surgery, Seoul National University Hospital between January 1990 and December 1999. Patients with distant metastasis detected at the time of surgery or within 4 months of surgery were excluded. Those patients whose surgical margins were positive for malignancy were also excluded. Patients' records were reviewed for the following: age of onset, family history of breast cancer in 1st or 2nd degree relatives, histological type of cancer, tumor size in pathology reviews, axillary lymph node status, histological grade (HG: Scarff-Bloom-Richardson classification), nuclear grade (NG: Black's nuclear grade), type of surgical procedure and adjuvant therapy administered. Disease was staged according to the American Joint Committee of Cancer (AJCC) system [ 17 ]. The 'younger' group was defined as patients less than 35 years old at the time of breast cancer diagnosis. Expression of immunohistochemical tumor markers such as estrogen receptor (ER), progesterone receptor (PR) and c-erbB2 were determined in over 70% of cases. The expression was determined in assays performed immediately after surgery for each case. The primary antibodies for ER (DAKO, Glostrup, Denmark), PR (DAKO, Glostrup, Denmark) and c-erbB2 (Novocastra, Newcastle, UK) have been previously characterized. A cut-off value of 10% or more positively stained cells out of total cells in ten high-power fields was used in the classification of ER, PR and c-erbB2 expression levels. Statistical analysis The χ 2 test (Pearson statistic) was used to determine the differences in clinicopathological features between the two groups of patients. The follow-up duration was calculated from the date of diagnosis until the date of death or last contact. The disease-free survival was the time between diagnosis and confirmation of disease recurrence. The overall survival was the time between diagnosis and death as a result of any cause, regardless of recurrence events. Survival estimates were computed using the Kaplan-Meier method [ 18 ] and the differences between survival times were assessed by means of the log rank test [ 19 ]. Multivariate analyses were carried out using Cox's proportional hazards model [ 20 ]. All statistical analyses were carried out using the SPSS (version 10.0) software package (Chicago, IL, USA). Results A total of 2040 patients were eligible for this study, of which 256 (12.5%) were aged <35 at the time of diagnosis. The median follow-up was 74 months. Histology showed the incidence of medullary carcinoma was significantly higher than ductal carcinoma in the younger group (p = 0.018). There was a significantly higher incidence of nuclear grade 3 in the younger group than in the older group (p = 0.015). Axillary lymph node status, the most prominent prognostic factor in breast cancer, was not significantly different between the two age groups. Also, neither the family history of breast cancer in 1 st or 2 nd degree relatives, T stage, histological grade, c-erbB2 expression, nor ER or PR status were different between the two groups (Table 1 ). Frequencies of ER and PR positivity were low, and frequency of c-erbB2 positivity was high, in both age groups compared to frequencies reported in western populations and other Asian studies [ 21 - 23 ]. The proportion of breast-conserving surgery compared to mastectomy was similar in both groups. Axillary lymph node dissection, at least to the first Berg level [ 24 ], was performed in 250 (97.7%) younger patients and 1735 (97.3%) older patients. No sentinel lymph node procedure was performed. Adjuvant radiation therapy was administered to patients who underwent breast-conserving surgery and after mastectomy in patients who had four or more positive lymph nodes or a tumor >5 cm in diameter. Adjuvant chemotherapy was administered to 68.0% of younger and 58.7% of older patients. The most common chemotherapy regimen was a combination of cyclophosphamide + methotrexate + 5-FU (CMF) for 6 cycles or anthracycline containing regimen (AC). In terms of hormone therapy, tamoxifen was used for as long as 5 years after completion of surgery and adjuvant therapy. We classified a patient as tamoxifen treatment group if she got tamoxifen through more than a year before recurrence. The proportion of tamoxifen treated patients was significantly lower in young age group. Neither the type of surgery nor the postoperative adjuvant choemotherapy was significantly different between the two age groups (Table 2 ). Younger patients had a worse disease free survival (greater probability of recurrence) at all time periods (Fig 1A ; p < 0.001). At 5 years, the actuarial recurrence rate for patients <35 years old was 30.4% as compared with 18.7% for older patients. This difference persisted at 10 years, at which time the actuarial recurrence rates were 40.1% and 28.6%, respectively. Overall survival among younger patients was significantly worse than for older patients (Fig 1B ; p = 0.002). The 5-year survival rate was 80.0% for patients aged <35 years as compared with 88.5% for older patients. Stratified analysis according to axillary lymph node status was performed for disease-free survival. In lymph node-negative patients there was no significant difference in disease-free survival between the two age groups (Fig 2A ; p = 0.223). However, in lymph node-positive patients, disease-free survival was significantly worse in younger patients (Fig 2B ; p < 0.001). In multivariate analysis, young age (<35 years) remained a significant predictor of recurrence when entered into a model containing all potential demographic, pathologic and immunohistochemical variables (Table 3 . Hazard Ratio (HR), 1.7; 95% confidence interval, 1.1–2.6; p = 0.010). However, young age was not a significant independent predictor of overall survival in the same Cox model (table not shown. HR, 1.4; p = 0.242). Because hormone therapy was done more frequently in older patients than young age group (Table 2 .), we made another multivariate model involving hormone therapy in patients with ER positive and/or PR positive cancer to address the effect of hormone therapy on the prognostic significance of young age. In this analysis, young age was still an independent significant prognostic factor while hormone therapy showed borderline significance (Table 4 .). Discussion Our results showed that operable young breast cancer patients (<35 years old) have a worse prognosis than older patients in terms of both overall survival and recurrence. The difference in disease-free survival was clear in patients with axillary lymph node metastasis, but was not observed in lymph node-negative patients. Even after controlling for differences in distribution of potential prognostic factors, young age remained a significant predictor of recurrence. The present findings support previous reports showing that women diagnosed with breast cancer at a younger age have a poorer prognosis compared with their older counterparts [ 3 - 7 ]. However, those reports suffered from limitations including a small younger patient sample size, a study period spanning too many years during which treatments changed, lack of information about pathological and protein markers, and a heterogeneous case population in terms of ethnicity and treatment strategy. To our knowledge, the present study is the largest to directly compare the prognosis of younger (<35) breast cancer patients with that of their older counterparts. Moreover, the data in this study were generated from patients of the same ethnicity undergoing treatment at a single institution under the same contemporary strategy of surgery and adjuvant therapy over a relatively short time period (10 years). In addition, this study included a multivariate analysis of the difference in distribution of potential prognostic markers between the two age groups. The biomarker results in the present study are different to those reported for other populations, including ethnically-related Asian patients. In a study of 1052 Chinese breast cancer patients in Hong Kong, 53% and 61.6% of pre- and postmenopausal women were ER-positive, respectively, and 51.5% and 46.2% were PR-positive, respectively [ 21 ]. Those figures are higher than the figures reported in the present study. A recent study of Japanese patients showed 62.2% were ER-positive and only 17.2% were c-erbB2-positive [ 22 ]. Merchant et al. found c-erbB2 expression in 30% and 24% of British and Japanese breast cancer patients, respectively [ 23 ]. In contrast, Choi et al. reported significant differences in c-erbB2 expression between Korean and white patients (47.5 vs. 15.8%, respectively) using immunohistochemistry and fluorescence in situ hybridization (FISH) techniques. These data suggest c-erbB2 expression may be related to race [ 25 ]. Although not a major focus of this study, we found PR and c-erbB2 expression were significant independent predictors of disease recurrence. Currently, the role of PR status as a prognostic factor is not clear, with some evidence to suggest it is useful [ 26 , 27 ] and other evidence to the contrary [ 28 ]. As for c-erbB2, its prognostic importance is also controversial. A large number of studies have been published, some reporting positive results and others reporting negative results [ 29 - 31 ]. The prognostic significance of PR and c-erbB2 in this data set can be investigated further as an independent analysis later. The St. Gallen Consensus Conferences in 1998 and 2001 concluded that age under 35 was a high risk factor for relapse in node-negative breast cancer patients [ 32 , 33 ]. Kroman et al. [ 34 ] reported that young women with low-risk breast carcinoma who did not receive adjuvant treatment had a significantly increased risk of death from the disease. Furthermore, Fowble et al. [ 4 ] reported that young women with early stage breast cancer, especially those with lymph node-negative disease, had a relatively worse prognosis than older counterpart. In the present study, although no significant difference was observed between the two age groups in lymph node-negative patients, the pattern of survival curves implied younger patients may have a worse prognosis. It may be that a study with a larger case size and a longer follow-up duration would provide enough statistical power to show a significant difference in prognosis for node-negative patients. It has been suggested that younger women with breast cancer have a poorer prognosis because they present with later stage disease due to either physician or patient delay in diagnosis. However, in this study, no significant difference was found between the two age groups in terms of tumor size or lymph node status. Moreover, multivariate analysis indicated that young age is an independent negative prognostic factor. This issue of delayed diagnosis is not conclusive now and should be elucidated further in subsequent studies. One possible limitation of this study is that the control group was heterogeneous and contained a mixture of premenopausal and postmenopausal patients. Adami et al. showed complex pattern of survival as a function of age at diagnosis of breast cancer [ 12 ]. However, as shown in Table 4 , young age remained an independent prognostic factor in multivariate analysis even after patients aged over 50 years were excluded from the control group ( p = 0.008). Although we did not have full information on each patient's menopausal status at the time of diagnosis, this result suggests that patients under 35 years have even worse prognosis than relatively "less young age" premenopausal patients. Up to 15–30% of women aged less than 35 years diagnosed with breast cancer are likely to have germ-line BRCA1 or BRCA2 mutations [ 35 , 36 ]. Although we did not investigate BRCA gene mutations in all patients in the present study, we performed BRCA1 and BRCA2 gene mutation scanning in 22 patients who had two or more breast cancer patients in their 1 st degree relatives. We found four BRCA2 and one BRCA1 mutations that are thought to be disease-causing (data not shown). Only one of these 5 patients was less than 35 years old at the time of cancer development. It is known that young breast cancer patients are more likely to have an inherited form of the disease [ 37 ]. However, the current study showed there was no significant difference in the family history of breast cancer between the two age groups. In the recent report by Choi et al. the prevalence of BRCA1 and BRCA2 mutations in Korean women with breast cancer at a young age (<40) was as high as western population. However, most of the BRCA-associated patients had no family history of breast and/or ovarian cancer. That is, the penetrance appears to be low. They suggested that there may be different genetic and etiologic factors affecting transmission and penetrance of the BRCA genes in Korean patients with breast cancer diagnosed at a young age [ 38 ]. Although most current breast cancer investigators agree that young age is an adverse prognostic factor for breast cancer, there have been few studies designed to elucidate the molecular or genetic differences associated with young age breast cancer. Recent CGH (Comparative Genomic Hybridization) analysis suggested that alterations in specific regions on chromosomes might be responsible for the poor outcome of early onset breast cancer [ 39 ]. Future research must be focused on this area in order to confirm the characteristics of young age-onset breast cancer at the molecular level. Conclusions These results show that operable young breast cancer patients (<35 years old) have a worse prognosis than older patients in terms of both overall survival and recurrence. Even after controlling for differences in distribution of potential prognostic factors, young age is an independent predictor of recurrence. The underlying biology of young age breast cancer needs to be elucidated and development of tailored treatment for this patient population is crucial. Competing interests The author(s) declare that they have no competing interests. Authors' contributions WH selected cases, reviewed medical records, analyze data, and drafted the manuscript. SWK participated in the data collection and input. IAP carried out the pathological diagnosis and immunohistochemistry. DK performed the statistical analysis. SWK, YKY, SKO, KJC, and DYN conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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548278

CASCAD: a database of annotated candidate single nucleotide polymorphisms associated with expressed sequences

Background With the recent progress made in large-scale genome sequencing projects a vast amount of novel data is becoming available. A comparative sequence analysis, exploiting sequence information from various resources, can be used to uncover hidden information, such as genetic variation. Although there are enormous amounts of SNPs for a wide variety of organisms submitted to NCBI dbSNP and annotated in most genome assembly viewers like Ensembl and the UCSC Genome Browser, these platforms do not easily allow for extensive annotation and incorporation of experimental data supporting the polymorphism. However, such information is very important for selecting the most promising and useful candidate polymorphisms for use in experimental setups. Description The CASCAD database is designed for presentation and query of candidate SNPs that are retrieved by in silico mining of high-throughput sequencing data. Currently, the database provides collections of laboratory rat ( Rattus norvegicus ) and zebrafish ( Danio rerio ) candidate SNPs. The database stores detailed information about raw data supporting the candidate, extensive annotation and links to external databases (e.g. GenBank, Ensembl, UniGene, and LocusLink), verification information, and predictions of a potential effect for non-synonymous polymorphisms in coding regions. The CASCAD website allows search based on an arbitrary combination of 27 different parameters related to characteristics like candidate SNP quality, genomic localization, and sequence data source or strain. In addition, the database can be queried with any custom nucleotide sequences of interest. The interface is crosslinked to other public databases and tightly coupled with primer design and local genome assembly interfaces in order to facilitate experimental verification of candidates. Conclusions The CASCAD database discloses detailed information on rat and zebrafish candidate SNPs, including the raw data underlying its discovery. An advanced web-based search interface allows universal access to the database content and allows various queries supporting many types of research utilizing single nucleotide polymorphisms.

Background Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation within species. As a result, SNPs are now becoming the most popular type of marker in genetic association and mapping studies. SNPs are also most likely to be the molecular basis for the majority of phenotypic variation in (outbred) populations. In particular, SNPs in regulatory and protein-coding regions can have an effect on gene expression levels and protein activity, respectively. The phenotypic differences observed between selected (sub) strains in model organisms may be the result of specific (combinations of) natural occurring polymorphisms. Hence, a comprehensive inventory of SNPs, including extensive annotation will be extremely valuable in the search for functional polymorphisms. There is often a vast unexplored potential in large sequence datasets that have been collected for other purposes, for example, EST and whole genome sequencing (WGS) projects. In an effort to address these two issues, we have developed an in silico candidate SNP mining pipeline that uses all publicly available sequence data for a specific organism, and designed a database, CASCAD (CAscad SNP CAndidates Database), that allows storage of a wide variety of primary source data, cross-annotation to other databases, and analysis parameters for SNPs associated with expressed sequences. Construction and content We applied the SNP discovery pipeline to both rat [ 1 ] and zebrafish (unpublished results) and identified about 33,000 and 52,000 high-quality candidates, respectively, that were extensively annotated and stored in the CASCAD database (Table 1 ). The database includes detailed primary information on which the discovery of the candidate SNPs was based. This information, including sequence quality information (Phred score), the number of supporting reads for every nucleotide observed at the SNP position, and expected alignment lengths, was found to be very valuable for filtering for predicted variants that have the highest likelihood to be experimentally confirmed. Two verification experiments for the rat resulted in confirmation rate estimates of 59% (68 candidate SNPs in 10 different laboratory rat strains) and 50.3 % (340 candidates in 5 laboratory and 2 wild rat isolates) [ 1 ]. A set of 139 CASCAD entries tested in 7 zebrafish isolates confirmed 67.6% of them as true polymorphisms (unpublished data). The success rate values obtained are likely to be underestimates since only limited number of isolates/samples were used and we were unable to include exactly the same isolates that were used for generating the primary data (e.g. EST sequencing). Table 1 Input data (number of sequence reads) for the CASCAD pipeline and number of predicted candidate SNPs. Rattus norvegicus Danio rerio Input data mRNA 25, 634 3, 366 EST 244, 518 283, 572 WGS 19, 813, 313 11, 588, 394 Candidate SNPs predicted 33, 305 51, 769 synonymous 3, 842 9, 111 non-synonymous 3, 708 6, 217 nonsense 162 158 We designed a web-based interface with Perl scripts communicating to a MySQL database, and displaying HTML pages through Apache server running on SuSE Linux. The interface provides simple, advanced (Figure 1 ), and sequence-based search forms. Parameters that can be used in a search include strain information, different formats of sequence identifiers (e.g. GenBank, UniGene, LocusLink accessions or gene symbol), map positions (genetic and physical), and a wide variety of SNP characteristics, such as experimental evidence, a likelihood score for verification as deduced from extensive verification experiments [ 1 ], and information regarding restriction sites that have been affected, facilitating the design of RFLP based assays. To this end CASCAD is tightly linked to primer design [ 2 ] and local genome assembly [ 3 ] interfaces, enabling a fast, reliable, and universal primer design for a chosen SNP candidate even when there is no assembled genome sequence available. To facilitate the retrieval of candidate SNPs with higher verification rates, represented by more reliable and common SNP candidates, we implemented the possibility to restrict searches to entries characterized by location at hypervariable CpG site, by minimal basecalling quality (Phred score), sequence match size, and/or minimal number of supporting reads for either allele. Figure 1 CASCAD advanced search form. In addition to primary sequence data analysis, the effect of all SNPs on protein coding capacity was evaluated and non-synonymous SNPs were categorized in classes reflecting the severity of the polymorphism using a BLOSUM-based score. The predicted missense SNPs were analyzed by SIFT [ 4 ] and Polyphen [ 5 ] programs that utilize not only substitution information but also phylogenetic conservation and structural protein information to predict a potential effect of the polymorphism on protein function. Query results are summarized on the SNP details page (Figure 2 ), listing the SNP characteristics and including active links to other databases and resources, such as dbSNP, Ensembl, UniGene, and LocusLink. More detailed information regarding raw data underlying the candidate SNP (links to the original sequence files and a full nucleotide and protein alignment) can be obtained by clicking on the observed nearly exact hits between nucleotide sequences. Moreover, statistics on the data that support the SNP (number of occurrences for every nucleotide at SNP position, range of Phred basecalling quality scores) are provided. Figure 2 SNP details page Utility and discussion For many applications, it is important to be able to distinguish between SNP candidates by their characteristics, as they may be predictive for verification success rate or carry biologically relevant information. Non-confirmed candidate polymorphisms may represent variants uncommon for a given population, but also sequencing errors (all types of sequences), RNA editing events and reverse transcriptase errors (EST reads). In order to minimize the contribution of false positives, one can exclude polymorphisms based on a single read for either allele, as is common for many in silico discovery pipelines [ 6 ]. Although this is a valid approach when selecting SNPs for population or association genetics, one could inadvertently discard many rare variants that may be associated with phenotypic variation, for example by affecting protein structure or function. Information on such polymorphisms can be very useful when mapping disease or QTL alleles. We have developed our database to fulfill the needs of any particular SNP application by providing control over every parameter we used in the polymorphism discovery step. Applications of the CASCAD database include queries for potentially deleterious SNPs in a specific genomic region of interest, for example a QTL interval, design of SNP-based mapping panels using either RFLP or any other technology, and identification of informative SNPs for fine-mapping. Custom sequences can be provided to search for known SNPs in any sequence of interest. In addition, the CASCAD pipeline [ 1 ] can be used to build a candidate SNP database for any model organism of interest for which sufficient sequencing data is available. Conclusions The main purpose of CASCAD database is to provide flexible access to candidate single nucleotide polymorphisms, which were predicted using a computational approach from publicly available sequence data of the rat and zebrafish. The resulting database is crosslinked to most common public databases and can be queried for SNPs using accession numbers, sequence context, SNP characteristics, but also using parameters specific to the SNP discovery process, allowing stringent or relaxed conditions suitable for different types of applications. Availability and requirements The database is freely accessible through the website . Programs, scripts, MySQL database dumps, and instructions for setting up a species-specific SNP database can be obtained from the authors upon request. Authors' contributions VG designed and implemented the CASCAD database. EB tested database and interface. EC provided supervision and guidance for the project.

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524361

Generation of competent bone marrow-derived antigen presenting cells from the deer mouse (Peromyscus maniculatus)

Background Human infections with Sin Nombre virus (SNV) and related New World hantaviruses often lead to hantavirus cardiopulmonary syndrome (HCPS), a sometimes fatal illness. Lungs of patients who die from HCPS exhibit cytokine-producing mononuclear infiltrates and pronounced pulmonary inflammation. Deer mice ( Peromyscus maniculatus ) are the principal natural hosts of SNV, in which the virus establishes life-long persistence without conspicuous pathology. Little is known about the mechanisms SNV employs to evade the immune response of deer mice, and experimental examination of this question has been difficult because of a lack of methodologies for examining such responses during infection. One such deficiency is our inability to characterize T cell responses because susceptible syngeneic deer mice are not available. Results To solve this problem, we have developed an in vitro method of expanding and generating competent antigen presenting cells (APC) from deer mouse bone marrow using commercially-available house mouse ( Mus musculus ) granulocyte-macrophage colony stimulating factor. These cells are capable of processing and presenting soluble protein to antigen-specific autologous helper T cells in vitro. Inclusion of antigen-specific deer mouse antibody augments T cell stimulation, presumably through Fc receptor-mediated endocytosis. Conclusions The use of these APC has allowed us to dramatically expand deer mouse helper T cells in culture and should permit extensive characterization of T cell epitopes. Considering the evolutionary divergence between deer mice and house mice, it is probable that this method will be useful to other investigators using unconventional models of rodent-borne diseases.

Background Hantaviruses (family Bunyaviridae ) are rodent-borne and can cause hemorrhagic fever with renal syndrome (HFRS 4 ) or hantavirus cardiopulmonary syndrome (HCPS) [ 1 ]. While HFRS is usually associated with Eurasian hantaviruses, HCPS is caused by any of several recently described New World hantaviruses [ 2 - 4 ]. In North America, the great majority of HCPS cases have occurred in the western United States and Canada and were caused by Sin Nombre virus (SNV). Patients afflicted with HCPS exhibit pronounced pulmonary inflammation due to capillary leak syndrome, with the consequent hypotension often leading to rapid decline and death [ 5 ]. Virus is found in the lungs of infected humans, but without discernible cytopathology, and mononuclear infiltrates are observed that produce proinflammatory cytokines, including IL-2, IL-4, IFN-γ, TNF and lymphotoxin (LT) [ 6 ], suggesting that HCPS is an immunopathologic response to the virus. To date, more than 370 infections with hantavirus have been documented in the United States, with a 36% fatality rate. Deer mice ( Peromyscus maniculatus ) are the principal reservoir host of SNV [ 4 , 7 ]. As is usual with some natural hosts, SNV infection of deer mice does not result in discernible pathology [ 8 ]. Infection parallels that of humans, with virus infecting capillary endothelial cells in many tissues, including the lungs, but without conspicuous cytopathology. However, in contrast to human HCPS, no pulmonary inflammation, capillary leakage, or mononuclear infiltrates are observed, and most, if not all, deer mice remain persistently infected for the remainder of their lives [ 9 ]. Deer mice are among the most common mammals in North America, found from the subarctic to central Mexico, except for the Atlantic seaboard and the southeast United States where other peromyscine species predominate [ 10 , 11 ]. Serosurveys of natural rodent populations suggest that hantavirus infections occur throughout the range of deer mice [ 7 , 12 - 15 ], which thus poses a potential threat to individuals who are in contact with these rodents. In addition, deer mice and other peromyscine rodents have been shown to harbor other human pathogens [ 16 - 28 ]. Very little is known about the mechanism by which the deer mouse immune system engages SNV because few reagents and methodologies have been developed and no susceptible inbred deer mice are available. The only useful immunological data that can be acquired is by use of serology; infected deer mice produce a neutralizing IgG response that is inadequate to clear the virus [ 8 , 9 , 29 ]. We previously cloned several deer mouse cytokine genes [ 30 - 32 ], but quantitative assays for the detection of the expression of these genes have not been developed. These limitations have made it difficult to determine what immunological events occur that impair an effective immune response without pathology. In some viral infections, persistence has been shown to occur because of impairment of helper and cytotoxic T cell responses, antigen presenting cell (APC) function, and development of APC from bone marrow progenitors [ 33 - 37 ]. Currently, none of these functions can be evaluated in deer mice. Recent advances in hematopoietic stem cell research have identified an important role for granulocyte-macrophage colony stimulating factor (GM-CSF) in the expansion and maturation of bone marrow cells into competent APC [ 38 - 40 ]. We previously cloned a partial cDNA representing deer mouse GM-CSF and found that one of its receptor-binding domains is nearly identical to that of the common laboratory house mouse ( Mus musculus ) [ 30 ]. This led us to hypothesize that house mouse GM-CSF, which is commercially-available, might be useful in expanding and differentiating deer mouse bone marrow cells into competent APC. If so, then it should be possible to generate large pools of APC from individual deer mice that could be aliquotted and frozen for use in long-term T cell cultures, which would preclude the necessity for inbred deer mice. We present evidence that such cells can be propagated in vitro and that they are capable of processing antigen and stimulating antigen-sensitized autologous T cells. This technique could provide sufficient APC, such that conventional T cell cloning and peptide-mapping experiments could be performed. In addition, because deer mice (New World rodents) and house mice (Old World rodents) are divergent by 25 to 50 million years [ 41 ], it is possible that this approach may be useful to investigators using other unconventional rodent models of infectious diseases. Results Cloning of the 5' end of deer mouse GM-CSF We used RACE to obtain the complete 5' end of GM-CSF. This sequence was translated using the default translation table within MacVector. The polypeptide is predicted to have a 25 residue signal peptide based upon orthologous sequences from other species [ 42 - 45 ] (Figure 1 ). The receptor-binding domains of deer mouse and house mouse GM-CSF share 13/15 identical residues. This region forms the α-helix (helix A) that binds with high affinity to β chain subunit of the GM-CSF receptor complex that is shared with the IL-3 and IL-5 receptors [ 46 - 49 ]. Figure 1 Amino acid alignment of deer mouse (DM), cotton rat (CR), house mouse (HM) and human (HU) GM-CSF. Polypeptides were aligned with the clustal algorithm in Macvector. Conserved (light shading) and identical (dark shading) amino acids are enclosed in boxes. The 25-residue signal peptide is enclosed in box A. Helix A, which binds to the β chain subunit of the GM-CSF receptor, is enclosed in box B. Deer mouse and house mouse GM-CSF share 13 of 15 identical residues in this domain. Morphologic characteristics of bone marrow-derived APC Deer mouse bone marrow cultures contained mostly cells that appeared dead or dying after 24 hours in culture with GM-CSF. However, at 48 hours clusters of cells were apparent, while control wells without GM-CSF had fewer live cells than at 24 hours. By day 3, adherent stromal cell foci were conspicuous, while semiadherent and nonadherent cells were more evident and these became the prominent cells for the duration of culture. Day 12 bone marrow cells incubated for an additional 48 hours were large, ranging from 12 to 18 μm in diameter, and possessed macropinocytic vesicles and processes (Figure 2A ). Although the method that was employed selects for DC in the house mouse [ 39 ], the deer mouse cells appeared to resemble macrophages, with abundant cytoplasmic vesicles, rather than dendritic cells, with lamellipodia or characteristic long processes extending from the cell. (S. K. Chapes, pers. comm.). However, the cells' exact definition will not be complete until better phenotypic characterization is possible. Treatment of the cells with recombinant TNF diminished the macropinocytic vesicles (Figure 2B ). Figure 2 Morphologic characteristics of deer mouse bone marrow-derived APC. Day 14 bone marrow cells cultured in GM-CSF were processed by cytospin and stained with Wright's stain. The cells exhibited conspicuous cytoplasmic vesicles and small processes (A). Cells collected on day 12 and incubated for 48 hours with 20 ng/ml of hmTNF displayed less conspicuous cytoplasmic vesicles (B). Proliferation of deer mouse cells to house mouse GM-CSF and human IL-2 Day 8 deer mouse bone marrow cells were cultured with various concentrations of house mouse GM-CSF. Two days later, proliferation was assessed (Figure 3A ) and maximal proliferation was observed at about 0.5 ng/ml of GM-CSF. Deer mouse splenocytes proliferated in response to human IL-2 (Figure 3B ). In this experiment, deer mouse splenocytes were cultured with a suboptimal concentration of PHA and various concentrations of recombinant human IL-2. Maximal proliferation occurred at 20 U/ml of IL-2. In another proliferation assay, in vitro deer mouse T cells that were collected 8 days after stimulation with APC and antigen exhibited slightly greater proliferation to IL-2 (data not shown). Figure 3 Proliferation of deer mouse cells to recombinant cytokines. (A) After 8 days of incubation with GM-CSF, deer mouse bone marrow cells were washed and then cultured with dilutions of GM-CSF in duplicate for 48 hours, then proliferation assessed by MTS assay. The data are representative of four deer mice. (B) To assess proliferative capacity of deer mouse T cells to human IL-2, splenocytes were cultured with a suboptimal dose of PHA (2 μg/ml) and dilutions of recombinant human IL-2 in duplicate for 48 hours, and proliferation assessed by MTS assay. The data are representative of two deer mice. Expression of MHC class II I-Eβ and TCRβC by deer mouse cells propagated in vitro BM-APC and T cells were examined for the expression of orthologous I-Eβ and TCRβC, respectively, by RT-PCR (Figure 4 ). For I-Eβ, primers were designed from previously published deer mouse sequences [ 50 ], while primers for TCRβC were those that are described in this work. In each instance, products of the expected sizes were amplified. The amplified BM-APC product was cloned, sequenced, and verified to be I-Eβ. Figure 4 RT-PCR of TCRβC and MHC class II I-Eβ in deer mouse cells. Total RNA was extracted from T cells and bone marrow-derived APC. Expression of the constant β chain of the TCR by the T cells and class II I-Eβ chain by the APC were detected by RT-PCR. β-Actin primers were used as controls for each sample. BM-APC induce antigen-specific proliferation of autologous T cells Deer mice were immunized with keyhole limpet hemacyanin (KLH), and 10 days later the lymph nodes, spleens and bone marrow were processed for in vitro expansion of polyclonal T cells (lymph node cells), while the bone marrow cells and splenocytes were frozen. Sera were tested for antibodies to KLH by ELISA and in each deer mouse tested the titer was greater than or equal to 8,000 (data not shown). For recall proliferation, KLH, in vitro-propagated T cells (14 days with IL-2) and BM-APC (14 days with GM-CSF) or freshly thawed splenocytes were cultured together for 72 hours, and proliferation was assessed by MTS assay (Figure 5 ). For each deer mouse, the BM-APC were between 10× to 20× more efficient at stimulating antigen-specific helper T cell proliferative responses. While each T cell line exhibited a 50% maximal stimulation in the presence of about 10 to 20 μg/ml antigen with splenocytes, 50% maximal stimulation was usually near 1 to 2 μg/ml antigen with BM-APC. In parallel experiments, cultures incubated with 20 ng/ml house mouse TNF did not exhibit noticeably different proliferative responses compared to control cultures (data not shown), despite morphological evidence suggesting an effect on macropinocytosis (Figure 2 ). Figure 5 BM-APC stimulate helper T cell proliferation. Deer mice were immunized with 20 μg of KLH subcutaneously and 10 days later the lymph nodes, bone marrow and splenocytes were retrieved for expansion of helper T cells and BM-APC. After expansion of these cells in culture, proliferation assays were performed comparing mitomycin-C-treated autologous splenocytes (SC) and BM-APC (BMC) for their capacity to stimulate T cells. In each instance, the BM-APC were more effective at stimulating T cell responses. The data are of two deer mice (DM212 and DM213). Antibody augments BM-APC stimulation of T cells Deer mouse antiserum raised against KLH and incubated with antigen for one hour prior to addition of cells increased the sensitivity of T cell proliferation (Figure 6 ), suggesting that Fc receptors are present on the surface of the BM-APC. The presence of antibody increased the 50% maximal T cell proliferation from one deer mouse (DM223) about 10-fold, from about 500 ng/ml without antibody to 50 ng/ml with antibody. Similarly, for another deer mouse (DM224) the presence of antibody was substantially more effective at inducing T cell proliferation, increasing 50% max 50-fold (from 50 ng/ml to about 1 ng/ml). Notably, the T cell response in DM223 was also less vigorous (50% max 500 ng/ml) without antibody compared to DM224 (50% max 50 ng/ml). The proliferative responses of six other deer mouse T cell lines were similar to those of these deer mice (data not shown). Figure 6 Antigen-specific antibody augments BM-APC-induced T cell proliferation. T cell proliferation responses from deer mice 223 and 224 were assessed as described in Figure 5 using BM-APC. KLH-specific antiserum or normal deer mouse serum were diluted 1:2,000 in DMM-5 and incubated with dilutions of KLH for 1 hour in 96-well plates at room temperature. BM-APC and T cells were added to the wells and incubated 72 hours, and proliferation was assessed by MTS assay. Discussion To our knowledge, no previous efforts have been made to develop long-term cultures of T cells from unconventional laboratory rodents. The principal reason for this is that highly inbred strains, required for conventional long-term T cell work, are not available from rodents not routinely used in laboratory work. At least for deer mice, we have developed a method of fulfilling this need by using commercially-available house mouse GM-CSF. This cytokine apparently binds to the GM-CSF receptor on deer mouse cells such that it generates competent APC from the bone marrow. These cells are capable of processing and presenting soluble antigen to autologous antigen-specific helper T cells. Our initial suspicions that house mouse GM-CSF might bind to deer mouse GM-CSF receptor was the result of previous work [ 30 ] in which we cloned a partial cDNA of deer mouse GM-CSF, including most of its A helix that is involved in binding to the β chain subunit of the receptor. We used 5' RACE to obtain the complete N-terminus and found that all but two of the residues from helix A are identical between the two species. Subsequent experiments demonstrated that GM-CSF induces proliferation of deer mouse bone marrow cells, and since GM-CSF is routinely used to generate APC from the bone marrow we hypothesized that it would do so with deer mouse bone marrow. We used a method that has been shown to generate dendritic cells in house mice; however, the cells obtained from deer mouse bone marrow more closely resembled macrophages rather than DC. These cells contained many large macropinocytic vesicles, but conspicuous dendrites typical of DC were not observed. Microscopically, these cells also appeared sensitive to TNF, which decreased macropinocytosis, but it had no effect on the capacity of these cells to present antigen to T cells as has been reported for human DC derived from blood mononuclear cells [ 51 ]. TNF can induce a physiologic change in the APC from an active pinocytotic cell into one that becomes highly efficient at MHC class II antigen presentation, thus facilitating transition from the innate phase to the adaptive phase of the immune response. It is unclear why TNF treatment does not augment T cell proliferative responses with deer mouse APC, but it may be that species-specific differences in GM-CSF and TNF signaling occur that account for these disparities in APC development and behavior. It is also possible that TNF does not induce complete maturation of the cells into highly efficient APC, as has been reported for some DC [ 52 ]. It is currently impossible to phenotype these cells because no antibodies specific to deer mouse APC subpopulations, such as CD markers, are available, nor have genes for these markers been cloned, despite many attempts (unpublished observations), that might facilitate identification of these cells. Regardless, the cells are highly efficient at processing and presenting antigen, and inclusion of antigen-specific antibodies augments these functions. Since the deer mice are outbred, this method requires the immunization and collection of cells from individual animals (Figure 7 ). These cells are derived from lymph nodes (T cell source), splenocytes (APC source) and bone marrow (APC source). Most of the recovered cells can be propagated in vitro and/or aliquotted and stored frozen so that viable cells can be used as necessary to propagate and characterize helper T cell lines. We routinely recover 10 7 bone marrow cells from a deer mouse, which is sufficient for freezing 5 vials at 2 × 10 6 cells each. Each vial is used to seed a 100 mm bacterial Petri dish, which produces about 10 7 BM-APC at 14 days of culture. For deer mice, the most significant limitation for cells is from the spleen. Although deer mice are slightly smaller than BALB/c mice, their spleens are disproportionately small (unpublished observations). We routinely recover 7 × 10 6 splenocytes from a deer mouse, while BALB/c house mice usually provide 10-fold more. Because of this limitation, we have begun to use BM-APC to propagate T cells. This method involves culturing of bone marrow cells with GM-CSF for 10 days, then freezing aliquots of 10 6 cells. Three days before T cell restimulation, the 10-day BM-APC are thawed and cultured with GM-CSF, then used for restimulation with fresh antigen in one well of a 24-well plate. The T cells are fed fresh IL-2 DMM-5 at two-day intervals for expansion. Figure 7 Schematic overview of the process for culturing autologous deer mouse BM-APC and T cells. Ten days post immunization, lymph nodes, spleens and bone marrow are harvested from euthanized deer mice. The lymph node cells are cultured with antigen for four days, while the splenocytes and bone marrow cells are frozen at -70°C. On day 4, the blasting lymph node T cells are recovered and cultured with fresh antigen and thawed splenocytes (without mitomycin-C treatment). Simultaneously, bone marrow cells are thawed and cultured with GM-CSF. Expansion of the T cells is performed with huIL-2 and the BM-APC with GM-CSF for 14 days. These cells are then used for proliferation experiments. We have used this method to establish nine T cell lines, six specific for KLH and three specific for SNV nucleocapsid antigen (data not shown). Based upon typical cell yields, it should be possible to assay several thousand wells on 96-well plates, which we estimate to be sufficient for many T cell activities, including cloning, peptide epitope mapping, TCR variable gene segment usage, and cytokine profiling. We believe the methods described in this work will allow the characterization of antigen presentation and T cell responses in infected deer mice. Many viruses impair pathways involved in APC and T cell functions so that they can evade a sterilizing immune response. With hantaviruses and their rodent hosts, millions of years of evolution have presumably allowed a coadaptation of the viruses and host immune responses such that pathology does not occur and the virus is not eliminated. It is possible that hantaviruses possess some as yet unidentified mechanism for suppressing an aggressive inflammatory immune response in rodent hosts, which is ineffective in human infections and often leads to inflammatory immunopathology. Because of the substantial evolutionary divergence of deer mice and house mice (about 25–50 million years) [ 41 ], it is likely that this method could be used with many divergent rodent species, and thus useful for examining APC and T cell responses in a variety of rodent systems, including natural hosts and animal models of disease. A limitation of this approach is that BM-APC lose their ability to proliferate in the presence of GM-CSF after four to six weeks, similar to what has been observed with house mouse bone marrow-derived cells [ 53 , 54 ]. Since a finite number of bone marrow cells can be harvested, this limitation prevents indefinite propagation of deer mouse T cells. It is possible that competent BM-APC might be propagated indefinitely by the introduction of oncogenes, such as transforming retroviruses [ 55 ]. Alternatively, other house mouse or human hematopoietic cytokines that are commonly used to propagate bone marrow progenitor cells may bind to deer mouse receptors. For example, human or house mouse Flt3 ligand is active on both human and house mouse cells, suggesting that one or both would have an effect on deer mouse cells as well. In this manner, large numbers of progenitor cells might be produced in vitro for storage and then thawed, as needed, for culturing in GM-CSF to produce functional APC. Conclusions We have developed a method for generating large numbers of competent antigen presenting cells from deer mouse bone marrow using house mouse GM-CSF. This method resulted in the production of antigen-specific T cell lines from outbred deer mice. Inclusion of antigen-specific antibody in cultures augments T cell proliferation, suggesting the APC express Fc receptors. This method will allow characterization of APC and T cells in deer mice and may be extended to other rodent species that are important in infectious disease research. Methods Deer mice The deer mice used in these experiments were from a colony of animals established with deer mice trapped in western Colorado [ 30 ]. All procedures were approved by the Mesa State College Institutional Animal Care and Use Committee and in accordance with the Animal Welfare Act. Cloning of the deer mouse GM-CSF 5' cDNA The 5' end of the deer mouse GM-CSF cDNA was obtained by using RACE. Briefly, a primer (Table 1 ) was designed from a partial cDNA of deer mouse GM-CSF [ 30 ] and used to amplify the 5' end according to manufacturer's directions (SMART RACE, BD CLONTech, Palo Alto, CA) using con A-activated spleen cell culture cDNA. The fragment was cloned into pGEM-T Easy (Promega, Madison, WI) and sequenced using the Big Dye Terminator sequencing kit (Applied Biosystems, Foster City, CA) and an ABI 310 DNA Analyzer, and the sequence was deposited into Genbank (AY247762). The signal peptide and receptor-binding domain was identified by comparison to house mouse GM-CSF [ 49 ]. GM-CSF polypeptides from the house mouse (X02333), cotton rat ( Sigmodon hispidus , AAL55394) and human (NP_000749) were aligned using MacVector's (Accelrys, San Diego, CA) clustal algorithm. Table 1 Primers used in this work 1 Gene Forward Reverse Size (bp) I-Eβ GTC ATT TCT ACA ACG GGA CG TCT CCG CTG CAC AAT GAA GC 242 TCRβC AGG ACC TGA GCA AGG TGA GC GCA CAG CAT ACA GGG TGG CC 474 β-Actin ATG TAC GTA GCC ATC CAG GC TCT TGC TCG AAG TCT AGG GC 283 GM-CSF 5' RACE N/A GTT GCC CCG TAG GCC CTT CTC ATA TAA CT 273 1 All sequences are listed 5' to 3'. Cloning of the deer mouse T cell receptor β constant domain cDNA Total RNA from activated splenocytes was reverse-transcribed using an oligo-dT primer and Superscript II (Invitrogen, Carlsbad, CA). TCRβC cDNA sequences from house mouse, rat and human were aligned with MacVector. PCR primers (Table 1 ) were designed from highly conserved regions within the alignment. PCR was performed on activated splenocytes with 95°C for 30 sec, 58°C for 30 sec, and 72°C for 1 min for 35 cycles. The amplified fragment was cloned and sequenced as described above, and deposited into Genbank (AY307417). Immunization of deer mice Deer mice were bilaterally immunized subcutaneously at the base of the tail with 20 μg of KLH (Sigma Chemical Co, St. Louis, MO) emulsified in CFA (Sigma). Ten days later, draining lymph nodes, spleens and bone marrow were recovered for in vitro experiments. For production of high-titer KLH antiserum, deer mice were immunized i.p. with 20 μg of KLH emulsified in CFA and boosted with 20 μg in IFA one month later. Sera were collected 7 days after boosting. Processing of tissues from immunized deer mice Immunized deer mice were euthanized by cervical dislocation and the draining lymph nodes, spleens and bone marrow from individual animals were separately collected in Hank's balanced salt solution for processing. The lymph nodes served as a source of antigen-specific T cells, while the splenocytes were treated with ammonium chloride (Cambrex Bioproducts, Walkersville, MD) to lyse RBCs, then frozen in 10% DMSO/5% FBS deer mouse medium (DMM-5: 5% FBS, RPMI-1640 supplemented to 315 mOsm [with 2.5 ml of 5 M NaCl/L], 2.5 μg/ml Fungizone, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml gentamicin, 50 μM β-ME, 10 mM HEPES, 2 mM L-glutamine) in aliquots for use as autologous APC for additional rounds of in vitro T cell stimulation. The bone marrow cells were collected from tibiae and femurs, washed twice in DMM-5, and aliquotted at 2 × 10 6 cells per vial in 1 ml of 10% DMSO/DMM-5 and stored at -70°C. Bone marrow culture Bone marrow-derived APC (BM-APC) were generated with modification of a previously described method for dendritic cells (DC) [ 39 ]. One vial of bone marrow cells (2 × 10 6 ) was quick-thawed in a 37°C water bath and cultured without washing in 100 mm bacterial Petri dishes in DMM-10 containing 20 ng/ml recombinant house mouse GM-CSF (R&D Systems, Minneapolis, MN) at 37°C under 7% CO 2 . Fresh GM-CSF/DMM-10 was provided on days 3, 6, 8, 10, and 12 for the generation of APC. Cells were collected with a scraper for use in experiments. Cells were processed by cytospin and stained with Wright's stain for morphological examination. Assessment of T cell sensitivity to human IL-2 Deer mouse splenocytes depleted of RBC by ammonium chloride treatment were incubated with a suboptimal dose of PHA (2 μg/ml) (Sigma) in DMM-5 and recombinant human IL-2 (R&D Systems) for 48 hours. Proliferation was determined by MTS assay (Cell Titer-96 AQ, Promega). The means and standard deviations of duplicate samples were calculated, with the mean of cells without IL-2 subtracted from sample means. Assessment of T cell receptor and MHC class II expression Total RNA was extracted from 14 day T cell and BM-APC cultures (Versagene RNA, Gentra Systems, Minneapolis, MN) and converted into cDNA. Class II expression of the bone marrow cells was assessed by PCR using a forward primer from exon 2 and a reverse primer that overlaps the boundaries of exons 2 and 3 of deer mouse I-Eβ (Table 1 [ 50 ]). The amplified fragment was cloned and sequenced as described above. T cells were defined by PCR amplification of the TCRβC chain using the primers listed above (forward, exon 1; reverse, exon 2). β-Actin expression was assessed for each population. ELISA serology Sera were collected at euthanasia by cardiac puncture. Plates were coated with 5 μg/ml KLH in PBS overnight and washed 5× with wash buffer (PBS-0.1% TWEEN-20). Plates were then blocked with blocking buffer (5% nonfat powdered milk in wash buffer) for 1 hour at room temperature. The sera and remaining reagents were diluted in blocking buffer. Sera were incubated in duplicate for 2 hours at room temperature, followed by goat anti- P. leucopus IgG (H&L) (KPL, Gaithersburg, MD) for 1 hour, then horse anti-goat IgG-HRP conjugate for 1 hour (Vector, Burlingame, CA). ABTS substrate (Sigma) was incubated for 15 min, and plates were read at 414 nm. Means were calculated with the background (1:100 normal deer mouse serum) subtracted. In vitro helper T cell expansion In vitro stimulation of helper T cells was performed essentially as described elsewhere [ 56 , 57 ]. Lymph nodes from immunized deer mice were made into single-cell suspensions by gently disrupting the capsule between the ends of sterile frosted microscope slides. The cells were washed twice in HBSS and plated at 5 × 10 6 cells per well (24 well plate) with 20 μg/ml KLH in DMM-5. After a 4 day incubation, the lymph node cells were collected and washed twice in DMM-5. The number of recovered lymph node cells varied between animals, but between 2 × 10 5 and 5 × 10 5 cells were recovered and plated with fresh antigen and 3 × 10 6 thawed autologous splenocytes in DMM-5 in 24 well plates in 1 ml of DMM-5. At 2 day intervals, cultures were fed by removing 750 μl of media and replacing it with DMM-5 containing 20 U/ml of recombinant human IL-2. When cultures were greater than 80% confluent, cells were passaged 1:2 into additional wells or into T25 tissue culture flasks. This process was continued for 14 days to expand T cells. Cultures were also restimulated at two week intervals with fresh mitomycin-C treated autologous splenocytes as above, or 2 × 10 6 BM-APC to continue propagation of T cell lines. Functional assessment of bone marrow-derived APC BM-APC were examined for functional capacity to process and present KLH to sensitized autologous deer mouse T cells expanded in culture. For these experiments, mitomycin-C treated BM-APC (10 4 ) or splenocytes (2 × 10 5 ) were used to stimulate 10 5 T cells in the presence of KLH in 96-well plates. In other experiments, the effects of house mouse tumor necrosis factor (20 ng/ml) were evaluated on APC morphology and capacity to stimulate T cell proliferation. Lastly, antisera to KLH were produced in deer mice and used to assess the capacity of the BM-APC to capture and process antigen for presentation to T cells. In these experiments, antiserum or normal deer mouse serum were diluted to 1:2,000, a saturating dilution in ELISA as described above, with KLH and incubated for 1 hour at room temperature. BM-APC and T cells were then added and the cultures incubated for 72 hours prior to determining proliferative responses by MTS. Means and standard deviations were calculated from duplicate samples. List of abbreviations HFRS, hemorrhagic fever with renal syndrome; HCPS, hantavirus cardiopulmonary syndrome; APC, antigen presenting cell; SNV, Sin Nombre virus; GM-CSF, house mouse GM-CSF; DC, dendritic cell; CFA, complete Freund's adjuvant; RT-PCR, reverse transcription polymerase chain reaction; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium Authors' contributions BD conducted bone marrow cell culture work. DGW and TAC performed RT-PCR experiments. JP cloned and sequenced the TCRβ cDNA. RMF cloned and sequenced the MHC class II cDNA. TS immunized deer mice and generated T cell lines.

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423156

Taking the Stem Cell Debate to the Public

In response to the Blackburn and Rowley essay on the President's Council on Bioethics, several thought-provoking opinions on ethical challenges in biomedical research are expressed by prominent stakeholders

In their essay in the April 2004 issue of PLoS Biology , Elizabeth Blackburn and Janet Rowley (2004) , two distinguished cellular biologists and members of the President's Council on Bioethics, strongly question the scientific foundation of two reports from the Council ( President's Council on Bioethics 2003 , 2004 ). The Council on Bioethics was formed by executive order “to advise the President on bioethical issues that may emerge as a consequence of advances in biomedical science and technology.” An open discussion between ethicists and scientists is critical to the advisory system. The recent administrative dismissal of Dr. Blackburn from the Council is very alarming. By stacking the deck with conservative opinions, and not accurately discussing the scientific issues, the Bioethics Council has become irrelevant to the scientific community and presents a jaundiced view to the public. Stem cell research and its applications have the potential to revolutionize human health care. Recent polls show support for embryonic stem cell research, even with conservative voters. The public, as the major benefactor of biomedical research and the target population of beneficial clinical advances, has the right to a fact-based discussion of the science regarding stem cells. It is therefore time that the debate on stem cell research, with its risks and benefits, be taken to the public. A debate on stem cell research restricted to the President's Council on Bioethics is a disservice to the public. Nearly three decades ago, the advent of recombinant DNA technology and in vitro fertilization (IVF) techniques, raised similar concerns regarding research. Contrary to apprehensive expectations, recombinant DNA technology has boosted enormous advances in the health care and pharmaceutical industry. IVF evolved to be a widely accepted, safe medical procedure, with over one million healthy babies born by IVF and related treatments. Similarly, once stem cells are successfully used in the clinic, most of today's political and ethical issues will evaporate. The International Society for Stem Cell Research (ISSCR), a society whose membership encompasses the bulk of the stem cell research brain trust, holds the position that research on both adult and embryonic stem cells will guarantee the fastest progress in scientific discovery and clinical advances. The ISSCR also strongly opposes reproductive cloning and supports the National Academy of Science's proposal to develop voluntary guidelines to encourage responsible practices in human embryonic stem cell research. One of the original recommendations of the President's Council on Bioethics was a four-year moratorium on stem cell research. The purpose of this moratorium was theoretically to open a large, national discourse on the topic of stem cell research, a debate intended to bring all sides into thoughtful reflection on the issue. To that end, the ISSCR has repeatedly and consistently offered an open forum for all sides in the debate at our conferences, and has carefully offered invitations to join our society and to speak at our annual meeting to members of the President's Council, including colleagues whose opposition to stem cell research has been clear. None have accepted. Dr. Kass, in particular, has received several direct appeals but has turned down every such opportunity to make his case to the researchers who arguably are his discourse partners, from whom he could learn much, and whom he should be actively engaged in teaching. It is tragic that voices of dissent and debate are stilled, for it is this very quality of open debate that is at the heart of both the scientific method and an ethically directed American democracy—surely a goal that we all share.

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527873

Gastric T-cell lymphoma associated with hemophagocytic syndrome

Background Lymphoma-associated hemophagocytic syndrome (LAHS) occurs in mostly extra nodal non-Hodgkin's lymphoma. LAHS arising from gastrointestinal lymphoma has never been reported. Here we report a case of gastric T-cell lymphoma-associated hemophagocytic syndrome. Case presentation A 51-year-old woman presented with pain, redness of breasts, fever and hematemesis. Hematological examination revealed anemia. Gastroscopy revealed small bleeding ulcers in the stomach and the computed tomography scan showed liver tumor. She underwent total gastrectomy for gastrointestinal bleeding and the histopathology revealed gastric T-cell lymphoma. She continued to bleed from the anastomosis and died on the 8th postoperative day. Autopsy revealed it to be a LAHS. Conclusions If Hemophagocytic syndrome (HPS) occurs in lymphoma of the gastrointestinal tract, bleeding from the primary lesion might be uncontrollable. Early diagnosis and appropriate treatment are needed for long-term survival.

Background Hemophagocytic syndrome (HPS) in adults is characterized by reactive and systemic proliferation of benign histiocytes that phagocytose blood cells [ 1 ]. It is often associated with infections, malignant neoplasms, autoimmune diseases and various immunodeficiencies. Lymphoma-associated hemophagocytic syndrome (LAHS) mostly occurs from extra nodal lymphoma and is known to have a poor prognosis. Here we report a case of LAHS arising from gastric lymphoma with a fulminant clinical course and difficult diagnosis until the time of autopsy. Case presentation A 51-year-old female was admitted on May 9, 1995, because of severe hematemesis. The patient had been treated elsewhere for one month for pain and redness of both breasts and fever (≥ 38°C). There was no generalized lymphadenopathy. On gastroscopic examination multiple small ulcers were observed in the stomach. An abdominal computed tomographic (CT) scan showed liver tumor and a normal spleen. Hematological and biochemical examination at admission showed the following results: RBC 352 × 10 4 /mm 3 , hemoglobin 10.3 g/dl (post transfusion), WBC 4,900/mm 3 , Platelets 51,000/mm 3 , serum albumin 1.5 g/dl, total bilirubin 0.6 mg/dl, AST 691 IU/l, ALT 187 IU/l, LDH 2976 IU/l, fibrinogen 134 mg/dl, FDP 10 μg/ml, and AT-III 40%. Bleeding from the stomach continued and did not stop with conservative treatment; therefore, two days later the patient underwent total gastrectomy and a partial liver resection. Histopathology of the resected specimen showed it to be a gastric lymphoma (pleomorphic medium-large cell type, non-Hodgkin's T-cell lymphoma) with liver metastasis (Fig. 1 ). From first postoperative day (POD), bleeding from the esophagojejunostomy continued; the patient developed disseminated intravascular coagulopathy and died on 8 th postoperative day. Figure 1 Photomicrograph showing medium-large sized atypical lymphoid cells with pleomorphic features in the stomach suggesting a gastric lymphoma (Hematoxylin and Eosin, ×170). On autopsy, malignant lymphoid cell infiltration and hemophagocytosis were observed in the liver, spleen, heart, small bowel, lung, both breasts, kidney, pancreas, uterus, and gastroduodenal lymph nodes (Fig. 2 ). The bone marrow presented hyperplasia and hemophagocytic macrophages but no infiltration by lymphoma cells. Immunohistochemically the neoplastic cells were positive for T-cell marker UCHL1 (CD45RO) and EBV by EBER in situ hybridization. The final diagnosis was EBV-related T-cell LAHS. Figure 2 Photomicrograph of the lymph node at autopsy illustrating histiocytes that show hemophagocytosis of normoblast in a lymph node (Hematoxylin and Eosin, ×200). Discussion HPS is a clinicopathological entity characterized by systemic proliferation of benign hemophagocytic histiocytes, fever, cytopenia, liver dysfunction, hepatosplenomegaly, and coagulopathy [ 1 ]. This syndrome has been observed during the clinical course of a wide variety of disorders, including viral infections and malignant neoplasms. Diagnostic guidelines of Henter et al , [ 2 ] are widely used for the diagnosis of HPS. However, these guidelines are not satisfactory in diagnosing HPS in adults; therefore, a number of studies on adult HPS have used their own criteria [ 1 , 3 , 4 ]. On the other hand for the diagnosis of LAHS, in addition to the clinical features, it is also important to confirm the presence of malignant lymphoid cells histopathologically. Takahashi et al , [ 5 ] has proposed a set of new diagnostic criteria for adult LAHS that has been detailed in Table 1 . Table 1 Diagnostic criteria for adult lymphoma associates hemophagocytic syndrome (LAHS) 1 High fever for more than a week (peak 38.5°C) 2 Anemia (Hb < 9 g/dl) or thrombocytopenia (platelet < 100,000 μ/l) 3 a) LDH ≥ 2 × upper limit b) Hyperferritinemia (≥ 1,000 ng/dl) c) Hepatosplenomegaly on CT, US or MRI d) FDP ≥ 10 μg/ml 4 Hemophagocytosis in bone marrow, spleen or liver 5 No evidence of infection 6 Histopathologically confirmed malignant lymphoma A diagnosis of LAHS requires that all of the above conditions are fulfilled. Of the item 3, at least two of the four sub-items (a~d) should be fulfilled. When item 1 to item 5 are present for 2 weeks and glucocorticoid or γ-globulin therapy is not effective, a diagnosis of probable LAHS can be made and chemotherapy against malignant lymphoma can be started. In Japan, T-cell LAHS accounts for 48.5% of all adult LAHS [ 5 ]. T-cell LAHS mostly occurs in extra nodal, especially nasal, cutaneous, or malignant lymphoma involving liver and spleen. There have been no reports on LAHS from gastric lymphoma. As the diagnosis in the present case was made at autopsy it is not clear as to when the HPS occurred initially. One possibility is the setting of disseminated T-cell lymphoma. This is supported by the patient's fever, which continued for one month, liver dysfunction, and coagulopathy, which existed from the initial stage of the disease, however the bone marrow did not show any lymphoma infiltration. It could also be considered that the hemophagocytic syndrome occurred as a result of the surgery as pancytopenia and hepatosplenomegaly were not observed before the operation and hemophagocytosis was not recognized on histopathological examination in the resected stomach. In T-cell lymphoma, the hemophagocytic syndrome is assumed to be caused by cytokines, especially, tumor necrosis factor-α, and interferon-γ released from neoplastic T-cells [ 4 , 6 ]. Uncontrolled secretion of cytokines may stimulate the proliferation and phagocytic activity of macrophages. It seems likely that hypercytokinemia due to surgical resection might have contribute to the development of HPS in the present case. In our opinion the former is more likely however based on the findings of this case the second hypothesis too cannot be rejected. The poor prognosis of LAHS, especially T-LAHS, is well known. The median survival time from the diagnosis is reported to be 143 and 69 days respectively in Japan [ 5 ]. For LAHS prompt initiation of treatment with multi agent chemotherapy is required to improve the symptoms and survival [ 7 ]. Bone marrow transplantation is considered to be a treatment for chemotherapy-resistant LAHS [ 8 ]. The median survival time of LAHS patients without chemotherapy is only 11 days [ 5 ]. In this case, the initial presentation was mastalgia and hence it took a considerable amount of time to reach a diagnosis. Furthermore, bleeding from the anastomosis continued leading to a rapidly progressive fatal clinical course. In HPS occurring in lymphoma of the gastrointestinal tract uncontrollable bleeding from the primary lesion might occur. Therefore, an earlier diagnosis of HPS should be made by bone marrow aspirates, and appropriate treatments should be started as soon as possible. Surgery if performed, must be performed with utmost caution. Conclusions LAHS could also occur from lymphoma of the gastrointestinal tract. For long-term survival; early diagnosis and appropriate treatment are needed. Surgery if performed without a proper diagnosis could prove fatal. Competing interests The author(s) declare that they have no competing interests. Authors' contributions RF, FH, TY, RD, KO and KH were gastrointestinal surgeons. MO referred this patient to us. KK and MS performed pathological examination and the autopsy. KS was a member of the intensive care team. TH, YY, HN gave us helpful comments about the manuscript

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544867

Progress, challenges, and responsibilities in retrovirology

In this editorial, Retrovirology's choice for best basic science "retrovirus paper of the year" and a perspective on challenges and responsibilities facing HIV-1 and HTLV-I research are presented.

Progress The beginning of a year provides an occasion to look back upon progress made over the past 52 weeks. With the end of 2004, Retrovirology concluded its first calendar year of publishing. In actual fact, Retrovirology launched as an Open Access journal the final week of February 2004 and has been publishing continuously for a little more than 10 months. Over that period, with the wonderful efforts from my 6 very capable Associate Editors (Monsef Benkirane, Ben Berkhout, Masa Fujii, Mike Lairmore, Andrew Lever, and Mark Wainberg), the journal has thrived. The goal that we set for Retrovirology is to provide a visible forum for retrovirologists so that their works can be read by all in a free and openly accessible manner. What this means is that if you are a human immunodeficiency virus (HIV)-researcher and you had published a paper in Retrovirology , a graduate student in Sri Lanka updating his/her research protocol, an AIDS activist in South Africa looking for the latest information, and even your long-lost high school sweetheart wondering what you have been doing all these years, can all find your work (i.e. through a simple Google or PubMed search) and read your most recent findings. Perhaps more relevant to the enterprise of scientific communication is that numerous academic peers in Eastern Europe, Asia, South America, Africa and elsewhere do not have funds which would permit them to read Cell , Science or Nature . Hence, while some can read your research in Cell , Science , or Nature , all colleagues, rich or poor alike, can read your Retrovirology paper. Are they reading Retrovirology ? You bet! Our monitored statistics tell us that in 2004, the most highly accessed review article [ 1 ] published in Retrovirology was read by over 3,500 individuals while a comparably popular original research article [ 2 ] was read more than 2,400 times. Readers also read Retrovirology articles with great immediacy. Thus, a recent paper by Rana and colleagues [ 3 ] appeared in Retrovirology on December 27 th , 2004; and already by December 31 st , 2004, a short 4 days later, that study had been read 389 times. Just as readers are quick to read our papers, I am equally pleased by our unmatched speed in publishing authors'works. In 2004, based on all papers we published in Retrovirology , the time from submission to publication averaged 40 days. From my personal experience of publishing in other virological journals, this duration is 3 to 4 times faster than our best competitors. Different journals/magazines recognize "Molecule of the Year", "Breakthrough of the Year", or even "Person of the Year". With this editorial, Retrovirology will initiate the annual recognition of the best basic science "retrovirus paper of the year". The Associate Editors and I decided that in 2004, the best basic science retrovirus paper was the work from Joseph Sodroski and colleagues describing the HIV-1 restrictive property of the tripartite motif 5 (TRIM5) protein [ 4 ]. Thus, these researchers characterized in primates a restriction factor, similar to the Friend virus susceptibility factor-1 (Fv1) in mice, which counters the ability of infecting retrovirus to establish a proviral form in target cells. In coming years, I anticipate that Retrovirology Editors will find it fitting to recognize a Retrovirology paper as the "best basic science retrovirus paper" of the preceding year. Challenges and responsibilities I explain to my postdoctoral fellows that challenges are those issues which you think others should solve, while responsibilities are items that you think you should tackle. As a retrovirologist depending on how you regard yourself, pressing problems are either others' challenges or your responsibilities. I study two retroviruses, HIV-1 and human T-cell leukaemia virus type 1 (HTLV-I). The start of a new year offers me a chance to review briefly my personal bias on the important research question that confronts HIV-1 and HTLV-I, respectively. For HIV-1, the "holy grail" remains the development of an effective vaccine against the virus. As we enter 2005, mortality from AIDS is staggering. It is estimated that in 2004, 3.5 million individuals perished worldwide from AIDS; or nearly 10,000 AIDS deaths each day. We can view this number in another way. The recent tsunami in South Asia is estimated to have caused 150,000 fatalities. AIDS in 2004 is then the equivalent of 23 tsunamis. Imagine, unrelentingly tsunami-like casualties every fortnight from people dying from HIV-1! While it is laudable that the World Health Organization has a goal to treat three million HIV-1 positive individuals globally using anti-retroviral (ARV) medicine over the next five years, that approach will unlikely address the full magnitude of the AIDS problem, especially in developing nations. On the other hand, 100 million infants (even those in remote regions of the world) receive basic vaccinations each year. This fact suggests that when an AIDS vaccine does become available, that vaccine could be logistically and practically effective. Separately, statistics from the World Cancer Report for the year 2000 show that 5.3 million men and 4.7 million women developed malignancy, and altogether 6.2 million persons died from cancer worldwide. The American Cancer Society estimates that approximately 553,000 individuals succumb to cancer in the United States each year. I see HTLV-I, the etiological agent for adult T-cell leukaemia (ATL), as perhaps the best retroviral system for retrovirologists to study human cancer. Substantive progress has indeed been made in our understanding as to how HTLV-I transforms cells in tissue culture [ 5 ]. What remains needed is the development of a good non-human primate model to investigate the genesis of adult T-cell leukaemia by the virus in vivo . Opportunities and limitations at mid-career I first started studying viruses in the fall of 1977 at age 19 as a MD-PhD student at the Johns Hopkins school of medicine. For me personally, 2004 marked over a quarter-century of virus-research. At age 46, the unbridled youthful optimism of 19 is now tempered by realizations of physical and career limitations (i.e. some very interesting research problems are going to take longer to resolve than the remaining span of my scientific and physical endeavor). Nonetheless, I am optimistic and hopeful that, despite enormous odds, the opportunity to see a successful AIDS vaccine will come before I leave retrovirology research in 20 or some years. Regarding Retrovirology , I am also optimistic that I started this project at an age that provides ample time to develop this journal into a premier research forum. Let me conclude this writing by thanking all authors, reviewers, Editorial board members, and our wonderful staff at Biomed Central who have contributed to Retrovirology's progress in our first year.

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423142

Information Transport across a Membrane

From a biochemical perspective, a living cell is a collection of molecules jampacked into a confined space by a flexible barrier, called the plasma membrane. A diverse array of proteins embedded in the plasma membrane act as conduits between the cell interior and its external environment, conveying nutrients, metabolites, and information. The life of a cell—as well as that of any multicellular organism—depends on a cell's ability to communicate with its neighbors, both near and far. One way cells do this is with transmembrane receptors outfitted with both extracellular and intracellular domains that mediate information flow between the cell's external and internal environment. One class of transmembrane receptors, called integrin receptors, specializes in interacting with and binding to other cells and the extracellular matrix, a complex of molecules surrounding cells that provides structural support. By integrating various components of the extracellular matrix, integrins (also known as adhesion receptors), play an important role in such diverse processes as cell differentiation, programmed cell death, wound healing, and metastasis. Association between integrin α and β subunit transmembrane domains Integrins can be regulated by signals within the cell to bind to their ligands with either low or high affinity. While a multitude of integrin ligands have been identified and the general mechanics of both the extracellular and intracellular domains of these receptors are known, exactly how a signal crosses the receptor's transmembrane segment to regulate affinity has remained obscure. Now, Bing-Hao Luo, Timothy Springer, and Junichi Takagi have taken a mutational approach to shed light on the inner workings of the transmembrane segment and to explain how it transmits information. Much of what we know about the function of integrins has come from studying the crystal structures and models obtained from structural analysis. These analyses have generated information not only about the structure and composition of the extracellular and intracellular domains of integrins, but also about the conformational changes that accompany signaling events. Integrins contain a large extracellular domain, a transmembrane segment, and a relatively short intracellular “tail.” Integrins are heterodimers—molecules that contain two subunits composed of different amino acids—made up of an α chain and a β chain. Tight association of the two subunits is associated with an inactive, or low-affinity, state of the extracellular ligand-binding domain. Separation of the intracellular subunits is associated with a dramatic conformational change and activation of the extracellular domain, changing a bent structure with a downward-pointing ligand-binding site into an extended one with an outwardly stretched ligand-binding site. This mechanism differs from most transmembrane signaling molecules, which usually achieve activation through association with their target molecules. To investigate how the transmembrane segment mediates these changes, Luo, Springer, and Takagi systematically replaced amino acids in both the α and β transmembrane domains of the heterodimer with cysteines, creating the potential for binding interactions through a chemical reaction, disulfide bond formation, between the two subunits. By analyzing 120 possible cysteine pairs, the researchers not only confirmed the structure of the transmembrane region as helical but also mapped the proximal amino acid residues between the helices. To understand how the helical transmembrane domains transmit signals, the team introduced activating mutations in the amino acids of the α subunit cytoplasmic tail. Using this approach, they observed the loss of the contact between the subunits, indicating a separation of the transmembrane helices. Furthermore, when disulfide bond formation occurred, linking the transmembrane segments together, activation was suppressed. While previous models had proposed various modes of subunit movements, including hinge- and piston-like models, these results strongly support the notion that lateral separation of the subunits is the driving force behind the signal. As many diseases arise from defects in integrin adhesion, understanding the conformation and mechanism of integrin activation could suggest promising avenues for drug development aimed at correcting such defects.

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545953

Gene expression in the brain and kidney of rainbow trout in response to handling stress

Background Microarray technologies are rapidly becoming available for new species including teleost fishes. We constructed a rainbow trout cDNA microarray targeted at the identification of genes which are differentially expressed in response to environmental stressors. This platform included clones from normalized and subtracted libraries and genes selected through functional annotation. Present study focused on time-course comparisons of stress responses in the brain and kidney and the identification of a set of genes which are diagnostic for stress response. Results Fish were stressed with handling and samples were collected 1, 3 and 5 days after the first exposure. Gene expression profiles were analysed in terms of Gene Ontology categories. Stress affected different functional groups of genes in the tissues studied. Mitochondria, extracellular matrix and endopeptidases (especially collagenases) were the major targets in kidney. Stress response in brain was characterized with dramatic temporal alterations. Metal ion binding proteins, glycolytic enzymes and motor proteins were induced transiently, whereas expression of genes involved in stress and immune response, cell proliferation and growth, signal transduction and apoptosis, protein biosynthesis and folding changed in a reciprocal fashion. Despite dramatic difference between tissues and time-points, we were able to identify a group of 48 genes that showed strong correlation of expression profiles (Pearson r > |0.65|) in 35 microarray experiments being regulated by stress. We evaluated performance of the clone sets used for preparation of microarray. Overall, the number of differentially expressed genes was markedly higher in EST than in genes selected through Gene Ontology annotations, however 63% of stress-responsive genes were from this group. Conclusions 1. Stress responses in fish brain and kidney are different in function and time-course. 2. Identification of stress-regulated genes provides the possibility for measuring stress responses in various conditions and further search for the functionally related genes.

Background Until recently multiple gene expression profiling was applied almost exclusively to human and a few model organisms. At present cDNA microarrays are being constructed for new species including teleost fishes [ 1 - 6 ]. Since EST sequencing projects are carried out with a large number of species, continuous development of new platforms can be expected in the future. We designed a salmonid fish cDNA microarray primarily to characterize responses to stress, toxicity and pathogens. This paper focuses on time-course comparisons of stress responses in rainbow trout and the usage of functional annotation to conduct analyses of gene expression data. Functional annotation of genes, especially Gene Ontology [ 7 ] is increasingly being used for analyses and interpretation of microarray results [ 8 - 13 ]. We applied Gene Ontology in several modes to facilitate implementation of our research tasks. Furthermore, experimental results generated guidelines for the development of specialized microarrays. Well designed platforms are expected to ensure identification of differentially expressed genes while containing representative coverage from important functional groups. Custom made microarrays include clones from cDNA libraries and/or selected genes, which have advantages and drawbacks. Indiscriminant spotting of EST may result in under representation of many functional classes. On the other hand selection of genes fully relies on annotations and hypotheses, which can be misleading and limit possibilities for nontrivial findings. We used clones from normalized and subtracted cDNA libraries as well as genes selected by the functional categories of Gene Ontology for inclusion onto a microarray targeted at characterizing transcriptome responses to environmental stressors. Designing a new platform requires balancing a large number of genes versus multiple replications of spots, which enhances statistical analyses of data. The rainbow trout microarray was prepared by spotting of relatively small number of genes (1300) in 6 replicates. We show that multiple replications combined with the dye-swap design of hybridization [ 14 , 15 ] allows for accurate detection of relatively small alterations in expression levels, which is important for the functional interpretation of results. Stress is closely associated with many diverse issues in fish biology and environmental research (reviewed in [ 16 ]). Stress is generally defined as the reaction to external forces and abnormal conditions that tend to disturb an organism's homeostasis. To illustrate the major trends in the studies of stress in fish, we performed a computer-assisted analysis of Medline abstracts covering this area (Table 1 ). Salmonids have been studied more extensively than any other fish species. Research has focused on various biotic and abiotic factors including toxicity, environmental parameters (oxygen, temperature, salinity, acidosis), diseases, social interactions (crowding, aggressiveness) and farming manipulations. Analysis of Medline abstracts indicated physiological processes, cellular structure and selected proteins that have been the major foci of previous fish stress studies. This provided an outline for interpretation of our results. We analyzed the effects of stress on the transcriptome in the brain and kidney, which are considered important target tissues along with muscle, blood cells, liver and epithelia. We report a profound difference of stress response in these tissues and the identification of a diagnostic set of genes. Table 1 Thematic associations in studies of fish stress. Computer-assisted analysis of 11129 Medline abstracts was performed as described in Methods. Terms that were over-represented in the abstracts (exact Fisher's test, P < 0.05) are ranked by the numbers of occurrence. Category Terms (counts/1000 abstracts) Species Salmonids (126.4), carp (68.9), eels (67.0), catfish (37.7), tilapia (38.7) Stressors Toxicity (440.6), temperature (178.3), oxygen (91.5), confinement (52.8), salinity (46.2), hypoxia (54.7), diseases (20.8), crowding (23.6), acidosis (17.9), aggressiveness (11.3) Messengers Cortisol (208.5), catecholamines (159.4), steroids (92.5) Tissues Muscle (197.2), blood cells (152.9), pituitary (119.8), liver (123.6), epithelia (96.2), brain (90.6), kidney (89.6), heart (51.9), skin (42.5) Cellular structures Cytosol (42.5), collagen (17.0), cytoskeleton (15.1), microsome (15.1), microtubule (14.2), lysosomes (13.2), peroxisome (4.7) Oxidative stress Glutathion (167.9), oxidant (93.4), antioxidant (90.6), peroxide (66.0), radical (55.7), superoxide (40.6), catalase (35.8), redox (18.9) Other processes Immunity (91.5), secretion (80.2), metabolism (74), transport (56.6), defense (52.8), necrosis (28.3), apoptosis (18.9), phosphorylation (15.1), proteolysis (7.5) Metabolites Ion (987.7), iron (215.1), glucose (141.5), lactate (67.9), lipid (74.5), zinc (51.9), phospholipid (11.3), triglyceride (11.3), lipopolysaccharide (9.4) Proteins Enzymes (180.2), heat-shock proteins (84.0), hemoglobin (37.7), metallothionein (37.7), transferase (32.1), phosphatase (26.4), chaperones (21.7), glutathion-S-transferase (17.0), transaminase (17.0), Na/K-ATPase (17.0), aminotransferase (8.5), mitogen-activated kinases (4.7) Results 1 Design of cDNA microarray The rainbow trout cDNA microarray was composed of EST and selected genes. The cDNA libraries were prepared from tissues of stressed fish using suppression subtractive hybridization, SSH [ 17 ] and a modification of the cap-finder method [ 18 ] supplemented with enzymatic normalization [ 19 ]. We sequenced 2000 clones and redundancy of the subtracted libraries was markedly greater than that of the normalized (306% and 134% respectively). In addition to EST we selected rainbow trout transcripts from the normalized multi-tissue cDNA library [ 20 ] based on their assignment to functional categories of Gene Ontology (stress and defense response, regulation of cell cycle, signal transduction, chaperone activity and apoptosis). The selected genes substantially improved the coverage of many functional classes (Table 2 ), though the number of differentially expressed genes in this group was markedly inferior to EST (Figure 1 ). Subtraction cloning enriched genes that showed strong alteration of expression at response to stress (p < 0.01 or lower, Figure 1A ), however the SSH clone set did not provide any advantage when microarray was used for the related research tasks (Figure 1B ). Table 2 Presentation of the Gene Ontology functional categories in the microarray. Table shows the numbers and frequncies of genes in the clone sets that were used for spotting (SSH – subtracted libraries, EST – normalized libraries). Gene Ontology classes N on slide SSH EST Selected Response to external stimulus 147 11 (0.07) 48 (0.11) 88 (0.31) Response to stress 145 7 (0.04) 30 (0.07) 108 (0.38) Defense response 105 6 (0.04) 34 (0.08) 65 (0.23) Humoral immune response 42 3 (0.02) 13 (0.03) 26 (0.09) Apoptosis 79 6 (0.04) 10 (0.02) 63 (0.22) Cell communication 139 11 (0.07) 45 (0.11) 83 (0.29) Cell proliferation 82 8 (0.05) 23 (0.05) 51 (0.18) Cell cycle 62 2 (0.01) 17 (0.04) 43 (0.15) Signal transduction 114 5 (0.03) 32 (0.07) 77 (0.27) Receptor activity 49 3 (0.02) 18 (0.04) 28 (0.10) Intracellular signaling cascade 49 3 (0.02) 15 (0.04) 31 (0.11) DNA metabolism 47 5 (0.03) 15 (0.04) 27 (0.09) Transcription 67 9 (0.05) 21 (0.05) 37 (0.13) Chaperone activity 41 4 (0.02) 12 (0.03) 25 (0.09) Figure 1 Performance of the clone sets used for preparation of the microarray. Figure shows frequencies of genes that were differentially expressed in at least 5 samples at different p-values (Student's t-test). A: this study (stress response), B: related experiments (exposure to aquatic contaminants [34], response to stress, cortisol and combination of these treatments, challenge with bacterial antigens, M74 disease). SSH – subtracted cDNA libraries, EST – normalized libraries, Select – genes chosen by the Gene Ontology functional categories. 2 Stress response in the brain and kidney of rainbow trout 2.1 Differentially expressed genes Fish were stressed with netting and samples were collected 1, 3 and 5 days after the first exposure. We used plasma cortisol as a stress marker [ 21 ]. The hormone levels increased 7.6-fold after 1 day and did not change significantly to the end of experiment (Figure 2 ). Figure 2 Plasma cortisol levels. The data are mean ± SE (n = 4). Difference between the control and stressed fish is significant (Student's t-test, p < 0.05). Microarray results were submitted to GEO ( GSM22355 ). Two genes were up-regulated in both tissues at all time-points (Figure 3 ). One is a putative homolog to the mammalian N-myc regulated genes, which are induced with steroid hormones in the brain [ 22 ] and kidney [ 23 ]. Mitochondrial ADP, ATP carrier can be implicated to both normal functions and cell death [ 24 ]. Metallothionein-IL, a classical stress marker was induced to the end of experiment and a similar profile was seen in midkine precursor (growth factor), histone H1.0 and B-cell translocation protein 1. In kidney we observed consistent up-regulation of genes related to energy metabolism, such as mitochondrial proteins (cytochromes b and c, cytochrome oxidases), enzymes (glyceraldehyde 3-phosphate dehydrogenase, fructose-bisphosphate aldolase, serine-pyruvate aminotransferase) and similar profiles were seen in two heat shock proteins and two signal transducers (cytohesin binding protein and GRB2-adaptor). The repressed genes were related to actin binding (coronin and profilin) and immune response (meprin, immunoglobulin epsilon receptor, thymosin and lysozyme). Figure 3 Examples of differentially expressed genes . Pooled RNA from 4 fish was hybridized in dye-swap experiments to two microarrays on which each gene was printed 6 times (total of 12 replicates). Differential expression was analysed with Student's t-test (P < 0.01); the expression ratio is coded with color scale. Rapid alteration of gene expression was a remarkable feature of stress response in the brain. Only one gene, aquaporin, was up-regulated for the duration of the experiment. Water channel aquaporin plays a key role in water homeostasis being implicated in various physiological processes and pathological conditions [ 25 ]. A panel of genes which showed markedly increased expression after 1 day was also suppressed after 5 days. Surprisingly, this group included mainly genes that are predominantly expressed in skeletal or cardiac muscle (myosin light chain 1 and 2, skeletal and cardiac isoforms, myosin heavy chain, troponin I, T and C) or are involved in regulation of muscle contraction (parvalbumin alpha and sarcoplasmic reticulum calcium ATPase). An opposite tendency was shown by a large group of genes however the magnitude of expression changes was smaller. We analysed 5 differentially expressed genes with qPCR and the results were in close concordance with the microarray data (not shown). 2.2 Functional classes The search for enriched Gene Ontology functional categories in the lists of differentially expressed genes found almost no overlap between the tissues (Table 3 ). In the brain stress affected binding and transport of metal ions, especially calcium and manganese, chaperones and heat shock proteins, cytoskeleton and microtubules and a number of signaling pathways; whereas, mitochondrion, extracellular structures and peptidases appeared the primary targets in the kidney. Table 3 Enrichment of Gene Ontology categories in the lists of differentially expressed genes. Analysis with exact Fisher's test, (p < 0.05) was made using the composition of microarray as a reference. The numbers of differentially expressed genes and genes on the microarray are in parentheses. Brain Kidney Intracellular signaling cascade (19/47) Mitochondrion (19/71) RAS protein signal transduction (6/9) Electron transporters (13/43) GTPase mediated signal transduction (11/16) Extracellular (19/70) Chaperones (16/40) Endopeptidases (8/22) Heat shock proteins (8/16) Metallopeptidases (7/12) Metal ion binding (31/80) Zinc ion binding (8/24) Carriers (15/37) Potential-driven transporters (7/9) Calcium ion binding (20/41) Magnesium ion binding (8/14) Cytoskeleton (27/76) Myofibril (16/16) Microtubule-based process (6/6) Comparison of the differentially expressed genes by the Gene Ontology categories suggested coordinated regulation of various cellular functions in the brain. Early stress response was marked with transient induction of the cytoskeleton proteins and similar profiles were observed in the metal binding proteins and enzymes of carbohydrate metabolism (Figure 4 ). An opposite expression pattern was shown by a large group of genes involved in stress and immune response, regulation of growth and cell cycle, apoptosis, signal transduction and cell to cell signaling. This was in parallel with enhancement of transcription and translation, ubiquitin-dependent protein catabolism and protein folding. In the kidney the temporal alterations were much weaker. Expression of metal binding proteins increased slowly in parallel with peptidases. Strong induction of collagenases coincided with decrease of collagen expression. At the same time a number of metabolic functions were suppressed (oxidative phosphorylation and oxidoreductase activity, amine metabolism and RNA binding). Figure 4 Time-course of stress response in the brain and kidney . Differentially expressed genes were grouped by the Gene Ontology categories and mean log (expression ratios) were analysed with Student's t-test. Panel presents examples of categories that showed significant difference between the time points (p < 0.05). The values are coded with color scale. 3 Stress-responsive genes Microarray design included genes from functional categories which were expected to be affected by stress (Table 2 ). Overall observations of differences in gene expression from this group in response to handling stress were minimal; however, this could be accounted for by its heterogeneity. Therefore we searched for the subgroups of genes with correlated expression profiles within the functional classes using results of 35 microarray experiments conducted by our laboratory. Both factorial and cluster analyses revealed 9 defense response genes that showed tightly coordinated expression being induced with stress. We continued search using the consensus profile of this subgroup and found 47 positively and 1 negatively correlated genes (Pearson r > |0.65|). Of these 29 were identified by the protein products (Figure 5A ), 19 being from the set of selected clones. Expression of the stress-responsive genes changed significantly in several experiments including this study (Figure 5B ). They were up-regulated in kidney with stress and injection of cortisol, combination of these treatments showed an additive effect (Figure 5C ). These genes also responded to the model water contaminants, being induced with low and medium and repressed with high doses (Figure 5D ). Figure 5 Expression of stress-responsive genes . A : Experiments. 1–6 : response to handling stress, this study. Kidney, 1 day (1), 3 days (2) and 5 days (3); brain, 1 day (4), 3 days (5) and 5 days (6). 7–12 : response to handling stress and exogenous cortisol in kidney. Cortisol, 1 day (7) and 3 days (8); stress, 1 day (9) and 3 days (10), combination of stress and injection of cortisol, 1 day (11) and 3 days (12). 13–20 : exposure of yolk sac fry to model contaminants [34]. β-naphthoflavone, low (13) and high (14) dose; cadmium, low (15) and high (16) dose; carbon tetrachloride, low (17) and high (18) dose; pyrene, low (19) and high (20) dose. 21–22: response of yolk sac fry to transportation stress, rainbow trout (21) and Atlantic salmon (22). Ranks are coded with color scale; correlation coefficients (Pearson r) with the mean expression profile are indicated. B-C: the mean ranks ± SE of the stress-responsive genes in 3 experiments. A : this study; B – response to handling stress and injection of cortisol in kidney; C – exposure of yolk sac fry to β-naphthoflavone, cadmium and pyrene at low, medium and high doses. Discussion I Stress response in rainbow trout Our study aimed at comparison of time-course of stress response in rainbow trout brain and kidney and finding of a diagnostic set of genes. These tasks were implemented with an aid of Gene Ontology annotation, which was used in several modes. The most straightforward and commonly used approach is counting of Gene Ontology classes in the lists of differentially expressed genes. Statistical inference of enrichment and depletion is made with Z-score of hypergeometric distribution, exact Fisher's test or its modifications. Such analyses helped us to interpret differences of stress responses in the brain and kidney (Table 3 ). In the brain handling stress mainly affected expression of transcripts for structural proteins (especially cytoskeleton), signal transduction, and binding of metal ions, whereas mitochondria, extracellular structures and peptidases appeared the key targets in the kidney. Computer-assisted analysis of Medline abstracts suggested that most of these themes have not been addressed in the studies of fish stress (Table 1 ). Searches of the enriched Gene Ontology categories associated with differentially expressed transcripts is useful for rapid screens of microarray data; however, it presumes coordinated expression of functionally related genes. This assumption is not valid for many classes, especially large and heterogenous groups, such as stress and defense response. Because the gene composition of microarray is used as a reference, uneven presentation of functional categories can distort the results. Finally, this analysis does not take into account direction and magnitude of differential expression. To overcome these problems, enrichment of Gene Ontology classes is analysed in groups of genes with similar expression profiles revealed with cluster or factorial analyses. In this study we preferred straight comparison of Gene Ontology classes by the mean log expression ratios which helped in interpretation of the time-course of stress response. In the kidney temporal alterations were relatively weak though significant. Expression of peptidases (especially collagenases) increased steadily, which implied possible degradation of tissue with prolonged stress. We could expect abrupt fluctuations in the rainbow trout brain, since transient induction and up-regulation of gene expression was observed in response to cold in the brain of channel catfish [ 4 ]. In our study most differentially expressed genes fell into two groups with distinct temporal profiles which showed remarkable coherence of the functional classes. Early phase was associated with dramatic up-regulation of structural and metal binding proteins, which were repressed in later phases. Expression of genes involved in stress and defense response, apoptosis and signal transduction, cell cycle and growth changed in a reciprocal fashion. Activation of metal binding proteins could be accounted for the role of ions (particularly calcium) in multiple pathways of gene expression regulation in the brain [ 26 ]. Motor proteins of cytoskeleton play key roles in the transport of vesicles and the establishment and rearrangement of neuronal networks [ 27 - 29 ] which also could be implicated to the stress response in fish. However, in mammals these functions are associated with non-muscle isoforms and therefore differential expression of the sarcomeric proteins was unexpected. Additional experiments confirmed induction of these proteins at early phase of stress response. Previously we observed high activity of skeletal α-actin and myosin light chain 2 promoters in the neural tissues of rainbow trout embryos [ 30 ]. Sequencing of salmonid fish cDNA libraries provided evidence for the brain expression of sarcomeric proteins, but their role remains fully unknown. At present there is sparse evidence for differential expression of structural muscle proteins in the mammalian brain. For example regulation of troponin I with dextromethorphan (antagonist of excitatory amino acid receptors) was reported in the rat hyppocampus and cortex [ 31 ]. Grouping of individual differentially expressed genes by the functional classes reduced noise and enhanced cluster and factorial analyses. This helped to identify stress-responsive genes that showed correlated expression in 35 microarray experiments (22 experiments are shown in Figure 5A ). Association with stress is well established for most of these proteins and some are used as stress markers. The list of enriched Gene Ontology categories (stress, defense and humoral immune response, signal transduction and response to oxidative stress, p < 0.05) suggested biological relevance of this group. Computer analysis of Medline abstracts (Table 1 ) showed that immunity and metabolism of reactive oxidative species are prioritized in studies of fish stress and these functional categories were enriched in the list of stress-responsive genes. Thus Gene Ontology provided a useful starting point for search of functionally related genes and results of these analyses can be used further for the revision of annotations. 2 Construction of microarrays Results of our experiments helped to evaluate the strategy used in construction of the rainbow trout microarray. Researchers developing microarrays for new species are commonly choosing between specially selected genes and clones from normalized and subtracted cDNA libraries. We used SSH, which is at present probably the most popular method of subtraction. Though proven efficient in many studies, this method has a number of drawbacks. Subtraction requires re-association of tester and formation of double-stranded DNA, hence many rare transcripts are not cloned and variations in concentrations of cDNA and hybridization conditions may have strong impacts on library composition. High redundancy is a common feature of the SSH libraries. Apart from these problems, rapid alterations of gene expression observed in this study and many other microarray experiments make the advantages of subtractive cloning ambiguous. Subtraction achieves enrichment of the transcripts, which are over or under represented in the test sample. In many cases one sample will not provide coverage of differentially expressed genes for the whole series, whereas pooling of samples may reduce fluctuations. Furthermore, we observed relatively high ratio of differentially expressed genes among the clones from the unsubtracted cDNA libraries, which are easier for construction and much less redundant. The advantage of subtractive cloning becomes negligible when microarrays are used for different, though related research tasks (Fig. 1B ). At present selection of genes for microarrays is facilitated with advances of functional annotation. This helped us to improve presentation of many functional categories (Table 1 ) and enhanced interpretation of results. Most of the selected genes did not show differential expression in our studies, however 63% of stress-responsive genes were from this group. In our view, this finding is a strong argument for utilizing Gene Ontology in the development of specialized platforms. Given the limited number of spots on slides, microarray design requires a careful balance between the number of genes and replication of spots. Apparent advantages of genome-wide platforms are compromised with the problems associated with identifying significantly differentially expressed genes. We preferred combination of multiple spotting and dye-swap normalization, which ensured robust normalization and accurate detection of differential expression at low ratios. Coordinated expression of functionally related genes suggested biological relevance of relatively small alterations in the transcription levels. Selection of differentially expressed genes by the cutoff values would result in loss of valuable information in our experiments. For instance, most of the stress-responsive genes showed small or moderate expression changes, the identification of this group would not be likely without multiple replications. Conclusions 1. Combination of EST and selected genes appears a reasonable way for construction of cDNA microarrays. Multiple replications of spots and dye swap design of hybridization ensure robust normalization and high power of statistical analyses. Finding of differential expression at small ratios is essential for the functional interpretation of microarray data. 2. Stress response in fish brain and kidney is different both by the target functions and time-course. In brain slow progression of adaptive response was preceded with dramatic transient induction of motor and metal ion binding proteins. Prolonged stress was likely to result in slow degradation of extracellular matrix in kidney. 3. Finding of stress-responsive genes provides possibility for measurement of stress in various conditions and search for the functionally related genes. Methods 1. Computer-assisted analysis of Medline abstracts Search of Medline was made with queries: "fishes AND stress" (1060 abstracts) and "fishes NOT stress" (10069 abstracts). Abstracts were split into separate words and a list of non-redundant terms was composed. The numbers of abstracts including each term were estimated. The terms were ranked by the Z-scores of hypergeometric distribution and enrichment was analysed with exact Fisher's test (p < 0.05). 2. Experiments with fish, exposure and sampling One year old rainbow trout were stressed with netting for 2 min, this treatment was repeated once a day for a duration of 5 days. Fish were killed with over-dose of anaesthetic (MS-222) and blood was taken from the caudal vein. The kidneys and brains were snap-frozen in liquid nitrogen. Plasma cortisol was determined with RIA using Orion Spectra Cortisol kit. 3. Preparation of microarrays RNA was extracted with Trizol reagent (Invitrogen) and mRNA was purified with Dynabeads kit (Dynal). SSH cloning was performed as described [ 17 ]. For preparation of normalized libraries, synthesis of cDNA with PowerScript reverse transcriptase (Clontech) was primed with oligonucleotides including EcoRI and NotI sites: 5'-ACGAGGC GAATTC ACAGAGAGGAG(T)VN-3', 5'-GAGAGAGAGTGGT GCGGCCGC GGTGTATGGGG-3'). Double-stranded cDNA was generated using Advantage DNA polymerase mix (Clontech) and PCR primers: 5'-ACGAGGC GAATTC ACAGAGAGGAG-3' and 5'-GAGAGTGGT GCGGCCGC GGTGTA-3'. The PCR products were purified with QIAquick kit (Qiagen), precipitated with ethanol and dissolved to 1 μg/μl in hybridization buffer (1 M NaCl, 50 mM HEPES (pH 8.3), 1 mM EDTA). DNA was denaturated for 5 min at 94°C. Following re-association at 72°C for 16 hours, DNA was ethanol precipitated and digested with 150 U of exonuclease III (MBI Fermentas) for 15 min at 37°C. This treatment eliminates re-associated double-stranded DNA [ 19 ]. Single-stranded DNA was PCR amplified, size separated with agarose gel electrophoresis and cloned into pGEM ® -11Zf (+) (Promega). Normalized and subtracted cDNA libraries were prepared from the stressed fish (whole fry, brain, kidney and spleen of 1-year old fish). A number of clones were from the rainbow trout and Baltic salmon cDNA libraries constructed in University of Turku. The sequences were analysed with stand-alone blastn and blastx [ 32 ] Microarray incldued 315 genes selected by the Gene Ontology functional categories. Of these, 282 clones were from the normalized multi-tissue library [ 20 ] and the rest were produced with RT PCR. The cDNA inserts were amplified with PCR using universal primers and purified with Millipore Montage PCR96 Cleanup Kit. DNA was spotted onto poly-(L) lysine-coated slides and each clone was printed in 6 replicates. 4. Microarray analyses Total RNA was extracted with Trizol reagent (Invitrogen) and 4 individuals were pooled in each sample. Stressed fish was compared with time-matched control. Labeling with Cy3- and Cy5-dCTP (Amersham Pharmacia) was made using SuperScript III (Invitrogen) and oligo(dT) primer; cDNA was purified with Microcon YM30 (Millipore). We used a dye swap experimental design [ 14 , 15 ] and each sample was hybridized to two microarrays. For the first slide, test and control cDNA were labeled with Cy5 and Cy3 respectively, and for the second array dye assignments were reversed. The slides were pretreated with 1% BSA, fraction V, 5 x SSC, 0.1% SDS (30 min at 50°C) and washed with 2 x SSC (3 min) and 0.2 x SSC (3 min) and hybridized overnight in cocktail containing 1.3 x Denhardt's, 3 x SSC 0.3% SDS, 0.67 μg/μl polyadenylate and 1.4 μg/μl yeast tRNA. All chemicals were from Sigma-Aldrich. Scanning was performed with ScanArray 5000 and images were processed with QuantArray (GSI Luminomics). The measurements in spots were filtered by criteria I/B ≥ 3 and ( I - B )/( S I + S B ) ≥ 0.6 , where I and B are the mean signal and background intensities and S I , S B are the standard deviations. After subtraction of mean background, lowess normalization [ 33 ] was performed. Differential expression was analysed with Student's t-test (p < 0.01) and the genes were ranked by the log(p-level). 5. Quantitative RT PCR Primers (Table 4 ) were designed to amplify 194–305 b fragments. RNA was processed with Rnase-free Dnase (Promega). Synthesis of cDNA with Superscript III reverse transcriptase (Invitrogen) was primed with oligo(dT). Analyses were carried out using Dynamo SYBR Green kit (Finnzymes) and ABI Prism 7700 (Amersham-Pharmacia). Table 4 Primers used for qPCR. Gene Sequence GRB2-related adaptor protein 2 Forward 5'-GCCAGAGCACCCCAGGAGAT-3' Reverse 5'-GGCTGAGAGGATGGGGCTGA-3' Collagenase type IV Forward 5'-AACATCAGAAACGCCCTCAT-3' Reverse 5'-TGGTGGTAGTGGTAGTGGAC-3' Troponin T Forward 5'-TGGGAAGAAGGAAACTGAGA-3' Reverse 5'-CTCTTACGCAGGGTTGTGAC-3' 40S ribosomal protein S12 Forward 5'-AGACCGCACTCATCCACGAC-3' Reverse 5'-CCACTTTACGGGGTTTTCCT-3' EST1 Forward 5'-CGGAGAAGGAGAACCCACAG-3' Reverse 5'-CCCTCAAACAAGCAAAGTG-3' EST2 Forward 5'-GCAAATGACAGCCCTCTTAG-3' Reverse 5'-AGCAGGTTTTCATCAAGGA-3' Author's contributions AK designed microarray, carried out experiments with fish and data analyses and drafted the manuscript. HK conducted the microarray analyses. PP developed software for annotation of genes and analyses of Medline abstracts. CR constructed the multi-tissue cDNA library and provided the selected genes. SA developed software for management of microarray data and performed the statistical analyses. HM coordinated research. All authors read and approved the final manuscript.

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548522

Association between fetal growth restriction and polymorphisms at sites -1 and +3 of pituitary growth hormone: a case-control study

Background Fetal growth restriction is associated with significantly increased risks of neonatal death and morbidity and with susceptibility to hypertension, cardiovascular disease and NIDDM later in life. Human birth weight has a substantial genetic component, with at least a quarter of the variation attributable to additive genetic effects. Methods One hundred twenty-five subjects (83 control and 42 case) were selected using stringent inclusion/exclusion criteria. DNA sequencing was used to identify 26 single nucleotide polymorphisms in the pituitary growth hormone gene (GH1) at which all subjects were genotyped. Association with fetal growth restriction was tested by logistic regression for all sites with minor allele frequencies greater than 5%. Results Logistic regression identified significant association with fetal growth restriction of C alleles at sites -1 and +3 (relative to the start of transcription) that are in complete linkage disequilibrium. These alleles are present at higher frequency (6% vs. 0.4%) in fetal growth restricted subjects and are associated with an average reduction in birth weight of 152 g in normal birth weight and 97 g in low birth weight subjects. Conclusions There is suggestive association between fetal growth restriction and the presence of C alleles at sites -1 and +3 of the pituitary growth hormone gene.

Background Fetal growth restriction (FGR) is a major risk factor for illness in the perinatal period and throughout life, with the smallest 7.5 percent of infants accounting for two-thirds of infant deaths [ 1 ]. Term, low birth weight infants are at least five times more likely to die in the first year [ 2 , 3 ] and are second only to premature infants in their rates of morbidity and mortality [ 4 ]. FGR infants have an increased frequency of hypoglycemia, hypothermia, polycythemia, neurodevelopmental deficits, and cerebral palsy [ 5 ]. Later in life, individuals born FGR are at elevated risk of hypertension, cardiovascular disease, and non-insulin dependent diabetes [ 6 ]. For example, FGR increases the risk for adult onset of non-insulin dependent diabetes two- to three-fold [ 7 , 8 ]. The ability to both diagnose and treat FGR early in gestation has enormous potential to reduce childhood and adult illness. It is difficult to distinguish the genetic and environmental components of human birth weight variation, but recent studies support a major genetic component to birth weight variation. Clausson et al.'s [ 9 ] study of the offspring of female dizygotic and monozygotic twins (2,009 twin pairs) estimated heritability for human birth weight of 42%, although the confounding influence of shared environmental effects must be considered. In a study of 3,562 captive macaques that minimized environmental heterogeneity, Ha et al. [ 10 ] estimated a total heritability for birth weight of 51%, with an additive genetic component of 23%. These findings demonstrate that comparatively simple and readily identifiable genetic factors influence birth weight. In concert with this recent research, FGR tends both to cluster in families and to recur in successive generations [ 11 - 14 ]. The five genes of the human growth hormone locus reside within about 45 kb on chromosome 17 [ 15 ]. Pituitary growth hormone (GH1) is by far the most thoroughly studied of the genes and lies at the 5' end of the cluster. The remaining four genes, placental growth hormone (GH2) and three chorionic somatomammotropins (CS1, CS2, and pseudogene CS5 or CSHP1), are expressed only from the placenta. The promoter region of GH1 is unusually polymorphic, with 16 SNPs having been identified in a span of 535 bp [ 16 - 18 ]. Most of these SNPs occur at the comparatively small number of sites that exhibit sequence differences among the five genes of the GH locus, and this has been interpreted as evidence of gene conversion [ 16 , 19 ]. Here we use DNA sequencing to identify and to determine the frequencies of both 12 newly-identified single nucleotide polymorphisms (SNPs) in the promoter and coding region of the GH1 gene and 15 previously reported SNPs [ 16 , 18 ]. Using a case-control design, we identify two SNPs in complete linkage disequilibrium near the start of transcription of the GH1 gene that may predispose to reduced birth weight. Methods Human subjects DNA was extracted from placental tissue from 125 live births (83 normal birth weight, 42 low birth weight) at Baystate Medical Center (Springfield, MA) in a case-control study of the genetic predisposition to fetal growth restriction. All subjects were Caucasian, both Hispanic and non-Hispanic. Classification of newborns as fetal growth restricted (or IUGR, intrauterine growth retarded) followed the definition of the American College of Obstetricians and Gynecologists as those newborns below the 10 th percentile of size for gestational age [ 20 ]. Our cut-off of 2,500 g at term (>37 weeks gestation) corresponds to the lowest 7.5 percentile of all US Caucasian deliveries, or the lowest 10 th percentile of female and lowest 6.5 percentile of male deliveries [ 21 ]. Stringent inclusion/exclusion criteria (Table 1 ) were employed by a placental pathologist (TKB) to reduce nongenetic contributors to birth weight variation. Ethical approval to conduct this study was obtained from the Human Subjects Institutional Review Board of the University of Massachusetts. Table 1 Inclusion and exclusion criteria for the study of fetal growth restriction Inclusion Criteria ≥ 37 weeks gestation (full-term) Mother 17–35 years old Singleton pregnancy Case (fetal growth restricted): <2,500 g birth weight Control: 3,000 to 4,000 g birth weight Exclusion Criteria Karyotypic abnormalities, including confined placental mosaicism Placental abnormalities Birth defects or syndromic conditions (i.e., Silver-Russell Syndrome) Pregnancy complications Preeclampsia Type 1, Type 2 or gestational diabetes Meconium staining Uterine infection Maternal chronic illnesses (i. e., hypertension, AIDS, hepatitis, endocrinological) Known illicit drug use Rh disease Polymerase chain reaction (PCR) and sequencing The region from -624 (relative to the start of transcription; GenBank accession J03071) to +1,726 (197 nucleotides after the termination codon) of GH1 was amplified in two overlapping fragments: -624 to +541 (GHN-1F 5' AGGGACCTGGGGGAGCCCCAGCAA 3', GHN-1R 5' TCACCCCTTCCTGCCACCCCTGAT 3') and +450 to +1,726 (GHN-2F 5' CCATCGTCTGCACCAGCTGGCCTT 3'; GHN-2R 5' GCCCTACAGGTTGTCTTCCCAACT 3'). Approximately 50 ng of DNA was used as a template in a polymerase chain reaction with 30 cycles of 95°C (1 minute), 62°C (1 minute), and 72°C (2 minutes 30 seconds). PCR products were purified from agarose using a QIAquick PCR Purification kit (Qiagen) and sequenced directly with BigDye v2.0 chemistry (Applied Biosystems) and run on either an ABI Prism 377 or 3100 automated DNA sequencer. PCR products were sequenced with the PCR primers and additional internal primers: 5' AAGCACAGCCAATAGATTG 3', -459 to -441; 5' GCACAAGCCCGTCAGTGGCC 3', -108 to -89; 5' GGATTTTAGGGGCGCTTACC 3', +71 to +90; 5' CATCTCCCTGCTGCTCATC 3', +931 to+949; 5' GCGCTTGGGYACTGTTCCCT 3', +1280 to +1299. Single nucleotide polymorphism (SNP) genotyping Sequence traces were aligned and assembled into contigs by the program Polyphred [ 22 ]. Contigs were viewed in either the program Consed [ 23 ] or Sequencher (Gene Codes Corp.) and polymorphisms confirmed visually. Twenty-six polymorphic sites were identified (Table 2 ), including all of the sites identified by Horan et al. [ 18 ] in 154 British military recruits with the exception of site -339 which had a minor allele (deletion of G) frequency of 3.6% in their study. Table 2 Frequency of alleles at 26 single nucleotide polymorphisms in the promoter and coding region of pituitary growth hormone and the nucleotide present at the homologous site in other members of the human GH locus Frequency GH1 paralogs Position* Alleles This Study Horan et al. CS-5 CS-1 GH2 CS-2 Categorization Function** -580 A 0.985 A A A A Constant G 0.015 -476 A 0.012 0.013 A G A G Variant G 0.988 0.987 -360 A 0.972 G G G G Variant G 0.028 -352 G 0.012 T G G G Variant T 0.988 -308 G 0.732 0.753 T C T C Variant T 0.268 0.247 -301 G 0.732 0.753 T T T T Variant T 0.268 0.247 -278 G 0.628 0.601 T A T A Variant NF1 T 0.372 0.399 -168 C 0.024 0.019 T C T C Variant T 0.976 0.981 -75 A 0.900 0.886 G A G A Variant PIT-1 G 0.100 0.114 -57 G 0.687 0.633 G T A T Variant Vitamin D Receptor T 0.313 0.367 -31 G 0.882 0.867 G G - G Variant Vitamin D Receptor - 0.118 0.133 -6 A 0.565 0.588 A G A G Variant Transcription Start G 0.435 0.412 -1 A 0.847 0.932 C T A T Variant Transcription Start C 0.044 0.003 T 0.109 0.065 3 C 0.044 0.003 C G G G Variant Transcription Start G 0.956 0.997 16 A 0.976 0.981 G A A A Variant 5' UTR G 0.024 0.019 25 A 0.980 0.981 C A A A Variant 5' UTR C 0.020 0.019 59 G 0.072 0.049 G G G G Variant 5' UTR T 0.928 0.951 69 A 0.968 G C G G Variant Thr/Ala G 0.032 124 A 0.988 G A G A Variant Intron G 0.012 128 A 0.988 C C T C Variant Intron T 0.012 140 A 0.004 G G G G Constant Intron G 0.996 144 A 0.012 G G G G Constant Intron G 0.988 281 C 0.024 T C C C Variant Intron T 0.976 596 C 0.986 T T T T Variant Intron T 0.014 1070 A 0.004 G G G G Constant Synonymous G 0.996 1169 A 0.331 T T T T Constant Intron T 0.669 * Relative to the start of transcription ** Polymorphisms in known transcription factor binding sites are shown. Site +69 is part of the signal peptide. Statistical analyses The five genes of the human growth hormone locus exhibit high sequence similarity, and the paralogous regions corresponding to the portion of GH1 sequenced in this study (-624 to +1,726) were multiply aligned. Nucleotide positions (Table 2 ) were designated as invariant if all five genes had the same nucleotide or the four paralogs of GH1 were identical and matched the major allele at that site in GH1. This categorization explicitly assumes that only the minor alleles in GH1 are the product of gene conversion and that minor alleles not observed in paralogs of GH1 are the result of unique mutations. The proportion of GH1 polymorphisms at invariant versus variant sites was compared by Fisher's Exact Test to determine if there was an over-representation of polymorphic sites among variant sites. Logistic regression, using FGR status as the outcome, was performed on gestational age and genotypes at sites with a minor allele frequency above 5% (Table 3 ). Sites -301 and -308 (relative to the start of transcription) are in complete linkage disequilibrium and the minor C alleles at sites -1 and +3 are in complete linkage disequilibrium. Therefore, sites -301 and +3 were excluded from logistic regression to avoid multicollinearity. Based on the results from logistic regression, separate ANOVA (Table 4 ) was performed on gestational age and the AA versus AC genotypes at site -1 within low birth weight and within normal birth weight subjects. Empirical p values for the F statistic for the genotypic effect, corrected for multiple comparisons, were determined by 2,000 random permutations of the genotypic data [ 24 ]. All statistical analyses were performed using the Stata program (Stata Corp., College Station, TX). Table 3 Logistic regression on FGR status based on gestational age and SNP genotype for GH1 polymorphisms with minor allele frequency greater than 5% Variable Odds Ratio 95% CI Z Score P-value Gestational Age 0.42 0.26–0.66 -3.75 <0.001 -308 2.66 0.54–13.19 1.20 0.23 -278 1.62 0.27–9.80 0.52 0.60 -75 1.70 0.55–5.23 0.93 0.35 -57 3.34 0.62–18.12 1.40 0.16 -31 0.85 0.27–2.65 -0.29 0.78 -6 2.11 0.66–6.74 1.26 0.21 -1 A/T 0.76 0.23–2.44 -0.47 0.64 -1 A/C 0.10 0.01–0.77 -2.21 0.03 +59 1.48 0.32–6.95 0.50 0.62 +1169 1.05 0.38–2.95 0.10 0.92 Table 4 ANOVA on A/C genotypes at site -1 and gestational age in normal and low birth weight subjects Normal Birth Weight Low Birth Weight Site -1 Gestational Age Site -1 Gestational Age Mean AA 3382.8 2287.9 Mean AC 3230.2 2190.4 ANOVA F 3.75 4.55 3.12 2.25 P-value 0.056* 0.002 0.073* 0.099 * Empirical P value corrected for multiple testing determined by 2,000 random permutations of the genotypic data [24] Results Polymorphism in the GH1 gene Among the 125 subjects sequenced from -624 to +1,726 of the GH1 gene, 26 polymorphic sites were identified (Table 2 ). These included all but one of the 15 sites characterized by Horan et al. (2003). In the region of overlap between the two studies, we failed to detect variation at site -339, where there is a minor allele deletion of a single nucleotide with frequency 3.6% in British army recruits, and identified two additional variants at sites -360 and -352 with minor allele frequencies 2.8% and 1.2%, respectively. Therefore, the discrepancies between the two studies in the identification of SNPs can most likely be ascribed to sampling error. Outside the region surveyed by Horan et al. (2003), we detected 10 additional polymorphisms. All of these had minor allele frequencies ≤ 3.2%, except an intron four polymorphism at site +1169 with a minor allele frequency of 33.1%. In general, polymorphisms in the promoter of GH1 tend to be more densely clustered and exhibit higher minor allele frequencies than in the transcribed region. Evidence of gene conversion An alignment of the region of GH1 sequenced in this study with the paralogous sequences of placental growth hormone and chorionic somatomammotropins indicated that there are 1,979 invariant sites and 293 variant sites (excluding 78 sites that could not be unambiguously aligned), as defined in the methods. Of the invariant sites, 5 are polymorphic in GH1, while 21 of the variant sites are polymorphic. A comparison of the proportion of polymorphic sites at invariant and variant sites by Fisher's Exact Test is highly significant (P <<0.001). This result indicates that there is a strong bias for polymorphisms in GH1 to occur at the minority of sites that exhibit sequence divergence among the paralogous genes. The high correspondence between the sequence of minor alleles in GH1 and nucleotides present in paralogs of GH1 supports previous assertions that the unusually high polymorphism of the GH1 gene is driven by gene conversion [ 16 , 18 ]. A proportion of this bias may be explained by selective constraint, in that sites that are polymorphic within GH1 may be under less selective constraint and thus more free to exhibit sequence divergence among paralogs. However, to entirely explain the bias towards polymorphism at sites of divergence, one must assume that about 1,804 (91%) of the invariant sites are selectively constrained and not free to vary. Given that a substantial proportion (814 bp, ~36%) of the sequence surveyed in this study is composed of introns, this assumption seems unreasonable. Association with fetal growth restriction Logistic regression (Table 3 ) was performed on gestational age and SNPs with minor allele frequencies greater than 5%, excluding sites -301 and +3 because they are in complete linkage disequilibrium with other sites, to identify associations with fetal growth restriction. Gestational age was significant because the FGR subjects exhibit a slightly younger average estimate of gestational age (38.1 vs. 39.1 weeks, t = 4.9, p < 0.0001; gestational ages rounded to nearest week). However, even accounting for the effect of gestational age, the C allele at site -1 was significantly associated with FGR in the combined regression. However, this allele did not retain significance in a regression on only gestational age (p < 0.001) and the A/C polymorphism at site -1 (p = 0.242). Although the A/C polymorphism at site -1 was not significant in a reduced model, we decided to investigate this site for three reasons. First, the C allele at site -1 is in complete linkage disequilibrium with C at site +3. Second, both of these sites are located at the start of transcription, making them good candidates for affecting the level of transcription of GH1. Third, the only paralog that shares C at these sites is CS-5, a pseudogene, consistent with the possibility that C at sites -1 and +3 is disadvantageous. The C allele at both sites exhibits a much higher frequency in low birth weight (6%) versus normal birth weight (0.4%) subjects. Restricting examination only within the normal or low birth weight subjects, the AC genotype is associated with an average reduction of birth weight of 152 g and 97 g in normal and low birth weight subjects, respectively. However, this difference does not achieve significant in an ANOVA on the A/C polymorphism and gestational age (Table 4 ). Discussion Birth weight in humans and other primates exhibits substantial heritability [ 9 - 13 ]. Although a suite of environmental and genetic/karyological insults are known to cause fetal growth restriction, perhaps as many as 40% of FGR cases have no known etiology [ 25 ]. Therefore, identification of the underlying genetic variants that predispose to FGR could have significant medical significance if it allows us to identify early in gestation those pregnancies that are at increased risk of growth retardation. A logical place to begin such a search is among those genes that are known to be major regulators of fetal growth and to exhibit significant differences in circulating protein concentrations between normal and FGR pregnancies, such as the members of the growth hormone-prolactin and insulin-IGF families of hormones, receptors, and binding proteins. Here, we use a stringently selected set of subjects to report suggestive association of SNPs in GH1 with fetal growth restriction. Adjusted for gestational age, the C alleles at sites -1 and +3 of the GH1 gene appear to be associated with reduction in birth weight. The marginal significance of these results may be the result of several factors. First, we examined 125 total subjects, roughly one-third of them FGR, and this number may give inadequate statistical power to identify weak genetic effects. Second, the C allele is low frequency, providing a small number of heterozygotes for the allele and no homozygotes. It is worth noting that among normal birth weight subjects we observed a very similar frequency for the C allele as Horan et al. (2003) did among British army recruits (0.4% vs. 0.3%) but that among the low birth weight subjects the frequency of the C allele is substantially higher (6%). Comparing all the other allele frequencies (Table 2 ) between the two studies of Caucasian populations, no other allele shows such a large magnitude of difference, although the proportional difference in frequency of C at -1 and +3 may be somewhat distorted by sampling error at low allele frequencies. Third, FGR undoubtedly has a heterogeneous genetic etiology, and it may be unlikely to find any genetic variant that accounts for more than a small proportion of cases. If the C allele at -1 and +3 exhibits true association with FGR, it unfortunately may be very difficult to determine if the effect is due to one or both sites because they are in complete linkage disequilibrium. Nevertheless, the presence of both alleles at the start of GH1 transcription provides both variants with a biologically plausible possibility to affect the level of transcription of GH1. Future work should be devoted to examining the effect of these alleles on transcription levels both alone and in combination. Site -1 is somewhat unique among single nucleotide polymorphisms in that three alternate alleles exist at that site (the major allele A and the minor alleles C and T). There are two possible explanations for this observation. First, site -1 could be a hotspot for nucleotide mutation, with transversions from A to C/T occurring often. Second, GH1 may be the recipient of gene conversion events from more than one paralog within the GH locus. Among the four other genes of the GH locus, all three alleles observed at site -1 occur (Table 2 ). Given the predominant effect that gene conversion [ 16 , 18 ] appears to have on the patterns of nucleotide polymorphism in the GH1 gene, the latter explanation may be more plausible. It must be pointed out that the common wisdom is that GH1 plays no role in regulating fetal growth because the GH receptor is expressed fairly late in gestation and because anencephalic infants or those born without a pituitary achieve nearly normal length [ 26 ]. Importantly, we restricted our study to full term deliveries. It is possible that late in gestation, when fetal GH1 is expressed and GHR receptors are present in a wide variety of fetal tissues, pituitary growth hormone begins to have a growth stimulatory role sufficient to account for the 90–150 g difference in birth weight between genotypes for the A/C polymorphism at -1 and +3 that we observed. Conclusions In a stringently selected set of subjects, C alleles at sites -1 and +3 relative to the start of transcription of GH1 have a higher frequency (0.4% vs. 6%) in fetal growth restricted newborns. These two alleles are in complete linkage disequilibrium and their presence is associated with a reduction in birth weight of 152 g in term, normal birth weight subjects and 97 g in term, low birth weight (<2,500 g) subjects. In combination with environmental, behavioural and other genetic factors, these alleles may contribute to fetal growth restriction. Competing interests The author(s) declare that they have no competing interests. Authors' contributions RMA conceived the project and directed its design and execution. CC and RV performed the molecular genetic work and participated in preliminary analyses. TKB performed the subject selection. Pre-publication history The pre-publication history for this paper can be accessed here:

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Relaxed Molecular Clock Provides Evidence for Long-Distance Dispersal of Nothofagus (Southern Beech)

Nothofagus (southern beech), with an 80-million-year-old fossil record, has become iconic as a plant genus whose ancient Gondwanan relationships reach back into the Cretaceous era. Closely associated with Wegener's theory of “Kontinentaldrift”, Nothofagus has been regarded as the “key genus in plant biogeography”. This paradigm has the New Zealand species as passengers on a Moa's Ark that rafted away from other landmasses following the breakup of Gondwana. An alternative explanation for the current transoceanic distribution of species seems almost inconceivable given that Nothofagus seeds are generally thought to be poorly suited for dispersal across large distances or oceans. Here we test the Moa's Ark hypothesis using relaxed molecular clock methods in the analysis of a 7.2-kb fragment of the chloroplast genome. Our analyses provide the first unequivocal molecular clock evidence that, whilst some Nothofagus transoceanic distributions are consistent with vicariance, trans-Tasman Sea distributions can only be explained by long-distance dispersal. Thus, our analyses support the interpretation of an absence of Lophozonia and Fuscospora pollen types in the New Zealand Cretaceous fossil record as evidence for Tertiary dispersals of Nothofagus to New Zealand. Our findings contradict those from recent cladistic analyses of biogeographic data that have concluded transoceanic Nothofagus distributions can only be explained by vicariance events and subsequent extinction. They indicate that the biogeographic history of Nothofagus is more complex than envisaged under opposing polarised views expressed in the ongoing controversy over the relevance of dispersal and vicariance for explaining plant biodiversity. They provide motivation and justification for developing more complex hypotheses that seek to explain the origins of Southern Hemisphere biota.

Introduction An important principle of evolutionary inference is that explanations for the past require an understanding of mechanisms and processes applicable in the present [ 1 ]. It is perhaps this sticking point more than any other that has polarised views over the relative importance of vicariance and dispersal for explaining extant plant biodiversity. In 1915, Alfred Wegener put forward a testable hypothesis and mechanism that could explain the transoceanic distribution of animal and plant species. In the 21st century, with many DNA studies now implicating the importance of long-distance dispersal for explaining plant biodiversity [ 2 , 3 , 4 , 5 ], it is disconcerting that there is currently a very poor understanding of the mechanisms of transoceanic dispersal (but see [ 6 , 7 , 8 , 9 , 10 ]). Indeed, the inference that the seeds of extant Nothofagus species are not suited for dispersal across large distances has played a major role in motivating the hypothesis that transoceanic distributions of Nothofagus ( Figure 1 ) can only be explained by vicariance [ 11 , 12 , 13 , 14 , 15 ]. This hypothesis posits that following the Cretaceous breakup of Gondwana, Nothofagus rafted and evolved in situ upon different Southern Hemisphere lands. Whilst very attractive, this hypothesis fits somewhat uncomfortably with the findings from analyses of morphological and molecular data. In particular, whilst earlier molecular data have been insufficient for rigorous molecular clock analyses, their interpretation has favoured hypotheses of transoceanic dispersal [ 16 , 17 , 18 ]. Figure 1 Southern Hemisphere Maps and Present-Day Nothofagus Distribution (A) Transoceanic distribution of Nothofagus subspecies Lophozonia and Fuscospora and South American species N. nitida (subgenus Nothofagus ). Map adapted from Swenson et al. [ 43 ]. ASE, Australia; NCA, New Caledonia; NGU, New Guinea; NZE, New Zealand; SAM, South America; TAS, Tasmania. (B) Relationship of Australia, New Zealand, and South America 65 Myr and 35 Myr before present, reconstructed from http://www.odsn.de/ (link “Plate Tectonic Reconstructions”). Based on the sequence of Gondwana breakup, a hypothesis of vicariance most parsimoniously predicts that Australian Nothofagus species should be most closely related to South American species. This follows since South America and Australia were connected via Antarctica until approximately 35 million years (Myr) ago ( Figure 1 ). In contrast, New Zealand is thought to have separated from Australia 80 Myr ago [ 19 , 20 ]. Thus to explain the close relationship between Australian and New Zealand species by vicariance, it is necessary to argue that extinction of Australian and/or closely related South American species has occurred [ 12 ]. Whilst this explanation is ad hoc, the fossil record does provide evidence for numerous Nothofagus extinctions in Australia, South America, and New Zealand [ 21 , 22 , 23 ]. However, the fossil record has also been interpreted as indicating multiple events of transoceanic dispersal of Nothofagus from Australia to New Zealand. Whilst the extinct “ancestral” Nothofagus pollen type occurred in New Zealand prior to the breakup of Gondwana, Fuscospora pollen first appeared in New Zealand during the Palaeocene (65 Myr ago) and Lophozonia pollen first appeared during the late Eocene (50 Myr ago; [ 24 ]). Sixty-five Myr ago the Tasman Sea had already reached its present-day size [ 19 , 20 ]. Hence it is possible that extant New Zealand Nothofagus subgenera did not have the opportunity to reach New Zealand via overland migration. Hill [ 25 ] has also described the species Nothofagus cethanica, which first appeared in Oligocene macrofossils from Tasmania. This species shares unique features with extant N. fusca and N. truncata from New Zealand and may share a sister relationship with these species explained by trans-Tasman Sea dispersal [ 26 ]. A contribution to the debate over the relative importance of vicariance and dispersal can be made by estimating the divergence times of extant species. However, DNA sequences of insufficient length have prevented robust molecular clock analyses from being undertaken. For this reason, we report the sequencing of a 7.2-kb chloroplast genome fragment covering the gene regions ( trnL–trnF and atpB–psaI; see Table 1 for accession numbers) for 11 species of three Nothofagus subgenera ( Lophozonia, Fuscospora, and Nothofagus ). Our aim has been to date divergence of extant species in the subgenera Lophozonia and Fuscospora. We have carried out relaxed molecular clock analyses using the methods of Sanderson [ 27 , 28 ] and Thorne et al. [ 29 ]. Our findings are that, whilst vicariance is likely to explain some transoceanic relationships amongst Nothofagus species, phylogenetic relationships between trans-Tasman species in both Lophozonia and Fuscospora can only be explained by mid- to late-Tertiary transoceanic dispersal. Table 1 Origin of Nothofagus Samples and Sequence Accession Numbers Results Figure 2 shows an optimal maximum-likelihood reconstruction of phylogenetic relationships for chloroplast DNA sequences (7.2-kb comprising the atpB–psaI region and the trnL–trnF region; 7,269 nucleotide sites) for Nothofagus (subgenera or pollen groups (a) Lophozonia, (b) Fuscospora, and (c) Nothofagus ) and outgroup Castanea sativa (not shown). In a sensitivity analysis of 60 substitution models, the tree shown in Figure 2 was always recovered with very little difference in branch lengths regardless of the substitution model used. Of all substitution models evaluated, K81uf+G was identified as the best fitting one for the data based on hierarchical likelihood ratio tests and the Akaike Information Criterion. This substitution model and also the F84+ Γ 8 model were used for further analyses. The latter was included because the Bayesian relaxed molecular clock (BRMC) approach as implemented in the program MULTIDIVTIME (see Materials and Methods ) only allows the use of the JC and the F84 models. Thus analysis with the F84+ Γ 8 model allowed a comparison of date estimates to be obtained using different relaxed molecular clock methods. All nodes of the optimal ML tree recovered in the sensitivity analysis received nonparametric bootstrap support greater than 97%, with the only exception being the grouping of N. cunninghamii with N. moorei, which received 72% support. Figure 2 ML Tree Indicating Evolutionary Relationships for Nothofagus Species Based on the atpB–psaI and trnL–trnF Region of the Chloroplast Genome (7,269 bp) Divergence dates (in Myr) were obtained with an F84+ Γ 8 substitution model using the BRMC approach of Thorne et al. [ 29 ]. For the dates indicated, the age of the root node and that of F/N1 were constrained to 70–80 Myr; L2 was also constrained in accordance with fossil data [ 26 ] at 20 Myr. Violet numbers show bootstrap values. The pollen grains represent the first appearance of the respective pollen type in the New Zealand fossil record. Plio, Pliocene; Oligo, Oligocene; Palaeo, Palaeocene; Ma, Maastrichian; Campan, Campanian. L1–L4, Lophozonia 1–4; F1–F2, Fuscospora 1–2; F/N1, Fuscospora / Nothofagus 1. Divergence times for the nodes in this tree ( Figure 2 ) were estimated using the penalized likelihood (PL) method [ 27 ] and BRMC method [ 29 , 30 , 31 ]. For these analyses, a period of 70–80 Myr was used to calibrate the divergence between the three fossil pollen groups representing subgenera Lophozonia, Nothofagus, and Fuscospora. These four pollen groups all first appeared in the fossil record approximately 75 Myr ago [ 32 ]. A second constraint of a minimum of 20 Myr for the divergence of N. cunninghamii and N. moorei was also used. This constraint was based on observations reported by Hill [ 26 ] that 20-Myr-old fossils intermediate between N. moorei and N. cunninghamii were recorded from Tasmania and that fossils closely resembling N. moorei were also present at that time. The inferred ages for the remaining nodes of the tree, obtained under the F84+ Γ 8 model of substitution are given in Table 2 and graphically illustrated on Figure 2 . The variance on these estimates was low and the values were little influenced by the choice of substitution model ( Table 3 ). The robustness of the estimates to calibration error was tested by constraining the divergence of Australian and New Zealand sister taxa to 65 Myr (the time before present when the Tasman Sea reached its present position; thus this date provided us with a lower bound for divergence times of trans-Tasman Nothofagus disjunctions that might be explained by vicariance). Constraining these two nodes in this way produced unrealistic age estimates for all basal nodes. For example, using the BRMC method, which additionally required a prior expectation to be specified for the age of the root node (which we specified at 75 Myr—the time of appearance of all four extant pollen types), we estimated a more likely age for the root node at 191 Myr. For the PL approach, which does not require specification of a prior, we estimated the age of the root node at 634 Myr. Other basal nodes in both the Fuscospora and Lophozonia lineages were also much older than reasonably expected (see Table 2 ). Table 2 Estimated Divergence Dates and Standard Deviations (in Brackets) of Different Nothofagus Clades The numbers in bold are all the nodes that were estimated without constraints Dates are based on different calibration dates and estimation approaches and are given in Myr before present a Node fixed b Node constrained DOI: 10.1371/journal.pbio.0030014.t002 Table 3 Variation of Estimated Divergence Times (in Myr) under 60 Symmetrical Models of DNA Substitution Dates estimated using PL approach a Node constrained Discussion Our findings from molecular clock analyses using five independent calibrations (for four nodes), suggest that the sister relationships of the Australasian (Australia and New Zealand) species within both Lophozonia and Fuscospora lineages are too young to be explained by continental drift (as indicated by the inferred ages of nodes F1 and L3). Transoceanic dispersal appears the most likely explanation for the trans-Tasman sister relationships indicated in Figures 1 and 2 . In contrast, the age inferred for node F2, using both relaxed clock methods is compatible with a hypothesis of continental drift as an explanation for the sister relationship between South American and Australasian Fuscospora lineages. The age for node L4, which separates Australasian and South American Lophozonia, may also be consistent with vicariance. The BRMC method dates it at 34 Myr before present. However, the PL method estimates this node to be only 25 Myr old, an age too recent to be consistent with vicariance. Thus we regard our results for node L4 as equivocal. Nevertheless, southern beeches are likely to have been present in Antarctica 25 myr ago [ 33 ], and thus long-distance dispersal across the young southern ocean between South America and Australia via Antarctica may be conceivable. The robustness of our phylogenetic inferences has been investigated by varying the substitution model (60 symmetric models were used), estimating the variance of age estimates, and evaluating the influence of calibrations on divergence times. With the exception of the root node, the PL method consistently gave more recent age estimates than did the BRMC method. Both methods showed sensitivity to the number of calibration points used, a finding consistent with recent observations on the performance of relaxed molecular clock methods [ 34 ]. In general, the date estimates produced by the BRMC approach were more consistent with the fossil record [ 26 ]. A relevant question is whether or not additional calibration points could make date estimates older and thus change our conclusion of trans-Tasman dispersal. We suggest that this may be unlikely, given the observation that constraining a minimum age for trans-Tasman sister species to 65 Myr leads to greatly inflated and unrealistic age estimates for all basal nodes. Hence to explain this finding we would need to invoke a further hypothesis of a dramatic and independent slowing in the rate of evolution in Lophozonia, Fuscospora, and Nothofagus lineages. Thus the hypothesis that present-day distribution patterns of Nothofagus can be explained by continental drift following the breakup of Gondwana and subsequent extinction of some species [ 24 ] can be rejected on the basis of the divergence dates that we have estimated. These dates also indicate that present-day Nothofagus species in New Zealand are not the direct descendants of the Fuscospora and Lophozonia southern beeches that reached New Zealand in the Palaeocene and Eocene eras, respectively [ 24 ]. This finding highlights the caution that needs to be taken when interpreting fossil evidence for the apparent first appearance of extant taxa. Fossils that identify specific evolutionary lineages may not necessarily indicate the origins for extant taxa or suggest a continuous presence for these taxa. Similar concerns follow from the findings of molecular analyses for Ascarina and Laurelia in New Zealand [ 2 , 4 ]. The strength of our molecular analyses highlights the importance of future research into potential mechanisms of long-distance dispersal, and in particular reinvestigation of the transoceanic dispersal properties of Nothofagus seeds. For the reasons that we outline in our introduction, it seems likely that only once the mechanisms of long-distance dispersal are understood will hypotheses based on DNA divergence time estimates be truly convincing. DNA sequence analyses have also suggested that long-distance dispersal and continental drift are both important for explaining distributions of the conifer Agathis (Araucariaceae) in the South Pacific [ 35 ]. Although the molecular evidence for Agathis is not as strong as it is for Nothofagus, the findings from the molecular studies on these genera highlight the importance of considering more complex hypotheses of relationship in debates concerning the relative importance of dispersal and vicariance. Materials and Methods Sequence data Chloroplast DNA sequences (7.2 kb comprising the atpB–psaI region and the trnL–trnF region) were determined for each of 11 accessions of Nothofagus (subgenera or pollen groups Lophozonia, Fuscospora, and Nothofagus ) sampled in South America, Australia, and New Zealand (see Table 1 ). These genome regions were also determined for C. sativa (an outgroup taxon from Fagaceae) and aligned using progressive multiple-sequence alignment: ClustalX version 1.81 [ 36 ]. This resulted in an unambiguous alignment of 7,269 nucleotide sites. Data are missing for approximately 250 bp of the atpB gene and atpB – rbcL intergene region of Nothofagus. Tree building Phylogenetic analyses were conducted with PAUP* version 4.0b10 [ 37 ] under the ML criterion. A model sensitivity test was conducted, investigating a range of 60 symmetrical models of DNA substitution corresponding to the 56 implemented in MODELTEST version 3.06 [ 38 ] ( http://darwin.uvigo.es/software/modeltest.html ) plus F84, F84+I, F84+Γ 8 , and F84+I+Γ 8 . ML parameters of these models were estimated by PAUP* following the approach used in MODELTEST. These parameters were then used to conduct 60 individual ML heuristic searches in PAUP* with tree bisection-reconnection branch swapping and a neighbour-joining starting tree. ML bootstrap proportions were obtained after 100 replications, using the same search strategy and ML parameters as for the analysis of the original dataset. Molecular dating: The PL method Divergence dates were obtained using the PL method of Sanderson [ 27 ] as implemented in the program r8s, version 1.60 [ 28 ] ( http://ginger.ucdavis.edu/r8s/ ) with the TN algorithm. The outgroup was excluded using the “prune” command. The degree of autocorrelation within lineages was estimated using cross-validation as suggested by Sanderson [ 27 ], and the correcting smoothing parameter λ defined accordingly. Divergence dates were estimated on the 60 ML phylograms recovered in the phylogenetic model sensitivity analysis. Ages for each node across the 60-ML trees were summarized using the “profile” command. Confidence limits on dating estimates were computed by using nonparametric bootstrapping of the original dataset as suggested by Sanderson and Doyle [ 39 ]. The program SEQBOOT of the PHYLIP 3.6 package [ 40 ] was used to generate 100 bootstrap resampled datasets of 7,269 sites in length. ML branch lengths of the optimal topology were then estimated under the F84+ Γ 8 model for each of the bootstrap resampled datasets using PAUP*. Divergence estimates were then calculated for each of the 100 bootstrap replicates using r8s to obtain standard deviations on each node by the “profile” command and the settings described above. Molecular dating: The BRMC method The BRMC approach was applied using the program MULTIDIVTIME as implemented in the Thornian Time Traveller (T3) package [ 41 ]. First, the program BASEML of the PAML package version 3.13 [ 42 ] ( http://abacus.gene.ucl.ac.uk/software/paml.html ) was used to estimate the ML parameters of the F84+ Γ 8 substitution model, using the ML topology previously identified. Second, the program ESTBNEW ( ftp://abacus.gene.ucl.ac.uk/pub/T3/ ) was used to estimate branch lengths of the ML topology and the corresponding variance–covariance matrix. Finally, the program MULTIDIVTIME was used to run a Markov chain Monte Carlo for estimating mean posterior divergence times on nodes with associated standard deviations from the variance–covariance matrix produced by ESTBNEW. The Markov chain was sampled 10,000 times every 100 cycles after a burn-in stage of 100,000 cycles. We used a 75-Myr (SD = 37.5 Myr) prior [ 32 ] for the expected number of time units between tip and root and a prior of 200 Myr for the highest possible number of time units between tip and root. Other priors for gamma distribution of the rate at root node and the Brownian motion constant describing the rate variation (i.e., the degree of rate autocorrelation along the descending branches of the tree) were derived from the median branch length. As for the PL method, the outgroup was not included in this analysis. Supporting Information Accession Numbers The GenBank ( http://www.ncbi.nlm.nih.gov/ ) accession numbers for the sequences discussed in this paper are given in Table 1 .

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519023

Food and nutrient intake in relation to mental wellbeing

Background We studied food consumption and nutrient intake in subjects with depressed mood, anxiety and insomnia as indices of compromised mental wellbeing. Methods The study population consisted of 29,133 male smokers aged 50 to 69 years who entered the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study in 1985–1988. This was a placebo-controlled trial to test whether supplementation with alpha-tocopherol or beta-carotene prevents lung cancer. At baseline 27,111 men completed a diet history questionnaire from which food and alcohol consumption and nutrient intake were calculated. The questionnaire on background and medical history included three symptoms on mental wellbeing, anxiety, depression and insomnia experienced in the past four months. Results Energy intake was higher in men who reported anxiety or depressed mood, and those reporting any such symptoms consumed more alcohol. Subjects reporting anxiety or depressed mood had higher intake of omega-3 fatty acids and omega-6 fatty acids. Conclusions Our findings conflict with the previous reports of beneficial effects of omega-3 fatty acids on mood.

Background Diet has an effect on mood and cognitive function [ 1 ]. There is some evidence that deficiency or supplementation of nutrients can affect not only mood, but also behavioral patterns. A double-blind placebo-controlled trial with 30 patients showed that omega-3 essential fatty acid supplements alleviated symptoms in patients with bipolar disorder [ 2 ]. In a recent double-blind, placebo-controlled trial on 231 young adult prisoners, by comparing the number of their disciplinary offences before and during the supplementation, antisocial behavior was reduced by the supplementation of vitamins, minerals and essential fatty acids [ 3 ]. Vitamin D supplementation during winter was reported to improve mood in a double-blind, placebo-controlled trial on 44 healthy volunteers [ 4 ]. A number of studies have shown that acute tryptophan depletion produces depressive symptoms and results in worsening of mood [ 5 ]. Folic acid deficiency may also correlate with depression, and it has particular effects on mood, cognitive as well as social functioning [ 6 ]. Recently, it have been reported that low levels of dietary folic acid are associated with elevated depressive symptoms in middle-aged men [ 7 ]. In general, a low-fat diet may have negative effects on mood [ 8 ], and altered dietary fat intake can lead to acute behavioral effects such as drowsiness, independent of energy consumption [ 9 ]. A high intake of proteins also seems to increase alertness [ 1 ]. Increased dietary serine and lysine may be linked to the pathogenesis of major depressive disorder [ 10 ]. Apart from specific nutrients or vitamins, certain foods may have an effect on mental wellbeing. Warm milk, for instance, has been traditionally used as self-medication for insomnia. Individuals drinking regular coffee with caffeine have reported to have decreased total sleep time and sleep quality, and increased sleep latency [ 11 ]. It has been reported that people with a high consumption of fish appear to have a lower prevalence of major depressive disorder [ 12 , 13 ]. Recently, it has been also reported that increased fish intake in people without depressive symptoms had no substantial effect on mood [ 14 ]. Depressed subjects tend to consume more carbohydrates in their diets than non-depressed individuals [ 15 ], and they show heightened preference for sweet carbohydrate or fat rich foods during depressive episodes [ 16 ]. High carbohydrate intakes increase brain uptake of tryptophan, which in turn stimulates the synthesis of serotonin [ 1 ]. At present, there are some studies focusing on the use of dietary supplements in individuals with mental disorders, but there is a lack of consistent data concerning the impact of nutrition, diet and eating habits on mental health. Aims We set out to study whether food consumption and intake of nutrients in subjects with depressed mood, anxiety and insomnia differed from those in subjects without any such symptoms. Methods This study was based on the cohort of a randomized, double-blind, placebo-controlled primary prevention trial testing the hypothesis that daily supplementation with α-tocopherol or β-carotene reduces the incidence of lung and other cancers [ 17 ]. The study participants were recruited between 1985 and 1988 from the total male population 50–69 years of age, residing in southwestern Finland (n = 290,406). These men were sent a questionnaire on current smoking status and willingness to participate in the trial. Smokers of at least five cigarettes per day and who were willing to participate were then invited to visit their local study center for further evaluation of their eligibility. A previous cancer diagnosis, current severe angina with exertion, chronic renal insufficiency, cirrhosis of the liver, alcohol dependence, or a disorder limiting participation in the long-term trial, such as mental disorder or physical disability, were reasons for exclusion. A total of 29,133 men were randomly assigned to receive supplements of either α-tocopherol, β-carotene, both, or placebo, in a 2 × 2 factorial design. The ethics review boards of the participating institutions approved the study, and all subjects provided written informed consent prior to randomization. At baseline, subjects completed a questionnaire on their background and medical history, including three questions on mental wellbeing. These items concerned anxiety, depressed mood and insomnia experienced in the past four months. Height and weight were also measured, and a blood sample was drawn for determining total and high-density lipoprotein (HDL) cholesterol concentrations. Diet and alcohol consumption was assessed from a self-administered dietary history questionnaire [ 18 ], which asked the frequency of consumption and the usual portion size of 276 food items during the past year, using a color picture booklet as a guide for portion size. Complete dietary data were available for 27,111 participants. Dietary nutrient data were analyzed by linking the questionnaire data to the food composition database of the National Public Health Institute, Finland. For analysis, we considered three main groups: principal nutrients, specific nutrients selected on the basis of a priori hypotheses, and certain foods. The principal nutrients were energy, carbohydrates, proteins and fats. The hypothesis-based nutrients were omega-3 and omega-6 fatty acids, lysine, serine, tryptophan, and two vitamins, vitamin D and folic acid. Omega-3 fatty acids from fish consist of long-chain fatty acids, while the omega-3 fatty acids in vegetables are shorter-chain molecules. The food items included were fish, milk, meat, vegetables, margarine, coffee and alcohol. We also evaluated the total energy intake. The trial involved three follow-up visits annually. At each follow-up visit the participants were asked whether they had felt anxiety, depression, or insomnia since the preceding visit (Have you felt feelings of depression in last three months? Have you felt feelings of anxiety in last three months? Have you had insomnia in last three months?). To identify subjects who suffered chronically from these symptoms we took into account the symptoms reported throughout the first follow-up year, i.e. at baseline and the three follow-up visits (at baseline, 4 months, 8 months and 12 months). Men reporting anxiety, depression, insomnia, or all these symptoms at all four visits were included in these analyses. Statistics As potential risk factors, baseline age, body-mass index (BMI), energy intake, alcohol consumption, education level, marital status and smoking were entered into regression models as covariates. Dietary factors were adjusted for energy intake in the models [ 19 ]. Results At study entry, 4314 (16%) men reported depressed mood in the past four months, 6498 (24%) feelings of anxiety, and 5550 (21%) insomnia. The mean intake of energy was 1 to 3% greater and consumption of alcohol 30 to 33% greater in subjects with any such symptoms, compared with symptom-free individuals (Table 1 ). Men reporting all three symptoms consumed as much as 47% more alcohol than those without any symptoms. Subjects with insomnia consumed 7% less coffee than symptom-free individuals, whereas those with depressed mood or anxiety consumed only about 2% less coffee (Table 2 ). Table 1 Baseline characteristics of subjects with self-reported depressed mood, anxiety or insomnia, and subjects with all three or none of the symptoms. Depressed mood Anxiety Insomnia All three symptoms No symptoms (n = 4314) (n = 6498) (n = 5550) (n = 1670) (n = 19116) Mean SD Mean SD Mean SD Mean SD Mean SD Age (years) 57.2 4.9 57.0 4.8 57.8 5.1 56.9 4.8 57.8 5.1 Energy (kcal/day) 2877 813 2888 801 2828 818 2886 863 2793 777 Alcohol consumption (g/day) 21.7 26.2 21.5 25.1 22.0 25.4 24.3 28.5 16.5 19.8 BMI (kg/m 2 ) 26.3 3.9 26.2 3.9 26.1 3.9 26.1 3.8 26.3 3.7 Total serum cholesterol (mmol/l) 6.16 1.19 6.22 1.18 6.15 1.19 6.13 1.21 6.26 1.16 Serum HDL-cholesterol (mmol/l) 1.24 0.36 1.26 0.36 1.27 0.37 1.27 0.38 1.23 0.34 Table 2 Baseline daily food consumption and nutrient intake of subjects self-reporting depression, anxiety or insomnia, and all three or none of the symptoms. Depressed mood (n = 4314) Anxiety (n = 6498) Insomnia (n = 5550) All three symptoms (n = 1670) No symptoms (n = 19116) Mean SD Mean SD Mean SD Mean SD Mean SD Fish (g) 39.3 30.2 39.9 30.2 40.1 30.3 40.3 32.9 39.3 29.8 Milk (g) 212 315 203 316 226 321 219 325 220 322 Coffee (ml) 595 374 601 372 567 364 583 382 609 349 Meat (g) 78.0 38.4 80.2 38.8 77.6 38.6 78.0 37.8 78.6 37.2 Vegetables (g) 256 103 264 104 253 104 255 106 263 101 Margarine (g) 11.5 21.1 11.5 20.8 10.7 20.3 11.8 21.3 10.2 19.8 Carbohydrate (g) 308 97.7 309 96.8 300 96.7 304 97.6 303 94.0 Protein (g) 105 30.6 105 30.2 103 31.2 105 31.6 103 28.9 Fat (g) 125 41.8 125 41.8 123 42.2 125 43.4 122 40.6 Sugar (g) 38.5 27.5 38.3 28.0 36.9 26.7 37.7 27.6 38.1 26.5 Lysine (g) 6.42 1.97 6.44 1.95 6.37 2.01 6.44 2.04 6.30 1.86 Serine (g) 4.12 1.31 4.27 1.30 4.22 1.33 4.28 1.35 4.18 1.24 Tryptophan (g) 1.28 0.38 1.29 0.38 1.27 0.39 1.29 0.40 1.26 0.36 Omega-3 fatty acids (total) (g) 2.21 0.93 2.24 0.92 2.16 0.92 2.23 0.97 2.14 0.87 Omega-3 fatty acids (from fish) (g) 0.47 0.28 0.48 0.29 0.48 2.89 0.49 0.30 0.46 0.28 Omega-3 fatty acids (from vegetables) (g) 1.77 0.82 1.79 0.80 1.70 0.80 1.77 0.86 1.70 0.77 Omega-6 fatty acids (g) 10.12 6.82 10.14 6.70 9.70 6.65 10.17 7.05 9.44 6.31 Omega-6/omega-3 ratio 4.47 2.00 4.45 2.01 4.41 2.03 4.50 2.30 4.34 1.85 Folic acid (μg) 342 106 344 105 335 106 340 107 336 103 Vitamin D (μg) 5.59 3.21 5.65 3.18 5.60 3.18 5.72 3.23 5.45 3.08 In subjects with depressed mood, the mean intake of omega-6 fatty acids was 7% greater than in symptom-free subjects. In individuals with anxiety, the mean intake of omega-6 fatty acids was 7% greater and that of omega-3 fatty acids from vegetables 5% greater than in subjects with no symptoms. Intake of fish or omega-3 fatty acids from fish were not associated with anxiety or depressed mood. When the symptoms reported during the first trial follow-up year were taken into analysis, 782 men reported depressed mood, 1237 feelings of anxiety, 1234 insomnia, and 166 men all three symptoms on all four occasions. The mean intake of energy was 7% greater in subjects reporting all three symptoms repeatedly compared with symptom-free individuals. Subjects with insomnia consumed 11% less coffee but 10% more milk than those with no insomnia. Both in subjects with depressed mood and with anxiety, the mean intake of total omega-3 fatty acids was 9% greater and that of omega-3 fatty acids from vegetables 6% greater than in respective symptom-free subjects, whereas the mean intake of omega-6 fatty acids was 6% greater in subjects with depressed mood and 9% greater in subjects with anxiety. Discussion Our subjects reporting anxiety had higher intakes of omega-3 and omega-6 fatty acids, but omega-3 fatty acids from fish were not linked to anxiety. Margarine was the main source of both omega-3 fatty acids from vegetables and omega-6 fatty acids. Subjects with depressed mood also had a higher intake of omega-6 fatty acids. Because 3138 (73%) subjects with depressed mood also had feelings of anxiety, it may be that anxiety is the dominant symptom, and the greater intake of omega-3 and omega-6 fatty acids is primarily related to feelings of anxiety. Previously, it has been suggested that omega-3 fatty acids may alleviate the effects of depressive symptoms but not those of mania [ 20 ]. Recently, we have reported that the low dietary intake of omega-3 fatty acids is not associated with depression [ 21 ]. Our present results show now that individuals suffering from symptoms of depressed mood have higher intakes of omega-6 and omega-3 fatty acids. More investigation is needed to elucidate the specific effects of omega-3 fatty acids on mood. Subjects with any or all of the symptoms consumed more alcohol than the symptom-free subjects. Subjects with all three symptoms consumed most alcohol of all, and they received 6% of their total energy from alcohol, compared with 4% in subjects with no symptoms. Energy from alcohol, however, did not explain the differences in the mean intake of energy between groups. Body-mass index was lower, despite a higher caloric intake, in subjects with any of the symptoms compared with symptom-free subjects. Subjects reporting insomnia drank more milk than symptom-free subjects, but less coffee. Warm milk has long been taken as a self-medication for insomnia, and our finding among those with insomnia accords with this traditional habit. In addition, they avoided consuming large amounts of coffee, which is known to have impact of sleep. We also found that subjects reporting depressed mood consumed more carbohydrates than subjects with no symptoms. This finding is consistent with the attempt by depressed subjects to alleviate the carbohydrate craving associated with symptoms of depression. Tryptophan intake showed no association with mental wellbeing in our study population. Interestingly, a number of negative studies has been published recently, suggesting that the effects of tryptophan depletion on mood are inconsistent [ 22 - 24 ], and the rationale for augmentation has now been challenged [ 25 ]. The intakes of vitamin D and folic acid exceeded the daily recommendations and showed no association with mental wellbeing. Neither did the consumption of fish, milk, meat or vegetables. Limitations There are some limitations in our study. Our study was a cross-sectional study, and it cannot provide causal evidence on the association between the diet and symptoms of depression, anxiety or insomnia. The study participants included only men, aged 50 to 69 years, and all were smokers. Our exclusion criteria limit the generalization of our findings, but the study still provides valid and reliable data on a community-based, homogenous sample of older men. Dietary intake and alcohol consumption were assessed with a validated food use questionnaire to measure the habitual dietary intake over the previous year as completely as possible. For most nutrients, both the reproducibility and the validity of this method are 0.6 to 0.7 [ 18 ]. For example, they are 0.66 and 0.73 for energy intake, 0.88 and 0.85 for alcohol, 0.70 and 0.75 for carbohydrates, and 0.70 and 0.64 for vitamin D, respectively. The assessment of self-reported depression was based on a single item only that might have compromised the specificity, but not sensitivity. For example, two questions only may be as effective as more detailed screening instruments in detecting probable cases of major depression [ 26 ]. One of these questions ("During the past month, have you often been bothered by feeling down, depressed, or hopeless?") is rather similar to the item that we applied for being indicative of depressed mood. Conclusion The scientific examination of relationships between nutrition and mental wellbeing is a relatively new area of study. Most of the studies focus on the use of dietary supplements, which provide more concentrated amounts of specific nutrients than most food sources. There are few data evaluating food consumption and nutrient intake among subjects with compromised mental health. Our main finding was that we did not find any association between omega-3 fatty acids from fish and mental wellbeing. In general, more attention need to be paid to the intake of nutrients in patients suffering from symptoms of depression, anxiety or insomnia. Further studies are needed to clarify complex associations between the diet and mental wellbeing, and to elucidate their mechanisms of action.

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529321

Are Animals As Irrational As Humans?

Animals in the wild are constantly confronted with decisions: Where to nest? Who to mate? Where's the best forage? To explore the mechanisms underlying such decisions, animal behavior studies often incorporate concepts from economic theory. Mainstream models of choice in both economics and biology predict that preferences will be rational, or consistent across contexts, as a result of being motivated by self interest or, in the case of animals, reproductive success. Yet many studies report that when making decisions people often take shortcuts, using cognitive heuristics that may lead to economically irrational decisions, with similar claims now showing up in animal behavior studies. In a new study, Cynthia Schuck-Paim, Lorena Pompilio, and Alex Kacelnik ask whether studies applying economic rationality to animal behavior are controlling for potentially confounding effects inherent in such approaches. The authors suggest that observed “breaches of rationality” may stem from differences in the physiological state of animals “unwittingly imposed” by experimental design rather than from real irrational decisions. Choice studies typically offer subjects a range of choices that include clearly superior and inferior alternatives. While humans can simply hear about the various alternatives and their respective properties, animals must be trained to learn about the different choices. This difference is far from trivial, Schuck-Paim et al. argue, and could well require different interpretations of results in animal and human studies. In fact, economic theory states that optimal choices depend on both the properties of the option and the chooser's state. Training animals to learn of different choices typically involves giving them food rewards, which means that an animal's energetic state—that is, hungry versus sated—will change over a day of training. A bird that's eaten an ounce of birdseed is more likely to opt for an “irrational” option—say, a choice that dispenses little food—than one that's hungry. To examine this theoretical constraint under experimental conditions, Schuck-Paim et al. trained European starlings to choose between two rich food sources (called focal options) and one of two poorer “decoys” in different contexts. One of the focal options offered more food while the other offered a shorter delay between pecking a key and receiving the food, but their amount/delay ratios were equal. The decoys were considered less preferable because their ratio of amount to delay was lower than that of the focal options. But the decoys could potentially confound the results because repeated training to each decoy could sate a bird's appetite to different degrees: although amount/delay was equal among the decoys, their long term energetic consequences differed. European starlings make rational decisions The authors tested for preference between the focal options under three experimental conditions: altering the birds' food intake/energetic state with no decoys; changing the decoy and not controlling for its corresponding energetic contributions; and changing the decoy but controlling for its energetic consequence (by supplemental feeding). Schuck-Paim and collaborators show that the birds' preferences between the focal options differed significantly between treatments, in apparent breach of economic rationality; the preference for the larger reward option over the shorter delay option was much stronger when the trial involved lower accumulated intake than when the accumulated intake was high. Introducing the decoys resulted in an “irrational” preference only when the decoys were allowed to have an effect on food intake, suggesting that the choice resulted from the birds' energetic state rather than from cognitive mechanisms of choice similar to those used to explain irrationality in human subjects. The authors offer an evolutionary and mechanistic explanation for why animal preference might be governed by energetic state, including the possibility that animals are less motivated to focus exclusively on the richest option when they are well fed. But they are careful to disabuse the notion that “state-dependence accounts for all reported inconsistencies in animal choice” or that animals do not employ cognitive mechanisms of choice similar to those of humans. Altogether, Schuck-Paim and co-authors argue, these results warn that studies appropriating ideas from other disciplines can introduce confounding effects. And that researchers would do well to carefully examine the underlying causes of observed animal behaviors when testing ideas formulated in a nonbiological framework.

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529309

SIVdrl detection in captive mandrills: are mandrills infected with a third strain of simian immunodeficiency virus?

A pol-fragment of simian immunodeficiency virus (SIV) that is highly related to SIVdrl-pol from drill monkeys ( Mandrillus leucophaeus ) was detected in two mandrills ( Mandrillus sphinx ) from Amsterdam Zoo. These captivity-born mandrills had never been in contact with drill monkeys, and were unlikely to be hybrids. Their mitochondrial haplotype suggested that they descended from founder animals in Cameroon or northern Gabon, close to the habitat of the drill. SIVdrl has once before been found in a wild-caught mandrill from the same region, indicating that mandrills are naturally infected with a SIVdrl-like virus. This suggests that mandrills are the first primate species to be infected with three strains of SIV: SIVmnd1, SIVmnd2, and SIVdrl.

Findings To date over 30 strains of simian immunodeficiency virus (SIV) have been isolated from African primate species and sequenced [ 1 ]. Mandrills ( Mandrillus sphinx ) are quite exceptional among African monkeys in that they harbour two distinct SIV strains, designated SIVmnd1 and SIVmnd2, with a separate geographic distribution [ 2 , 3 ] (see also Figure 1A ). SIV infections are mostly non-pathogenic in their natural hosts. SIVmnd1, despite high virus levels in chronically infected mandrills, has only a small effect on the T-cell counts, and primary infection does not induce clinical symptoms [ 4 , 5 ]. However, two cases of immunodeficiency were reported in mandrills after long-term (>18 years) SIV infection [ 6 ]. In 2003, a 20-year old male captive mandrill (mandrill CAS) housed at Artis Zoo (Amsterdam, The Netherlands), suffering from heart failure and poor general condition, was found to be positive for serum SIV antibodies. Inspection of the other three mandrills of his group, a ten-year old female (mandrill REB) and their offspring (mandrills RAF, 3 years old and HAB, 2 months old), showed that the female and one of the offspring (HAB) were also SIV antibody-positive. Figure 1 A) Geographic distribution of the genus Mandrillus , based upon mitochondrial haplotypes (adapted from [10]), and the SIV strains they harbour. B) Phylogenetic tree generated with Kimura-2-parameter distances and the NJ option of the MEGA package , from 267 nt of the mitochondrial cytochrome b gene of the four captive mandrills and reference sequences from GenBank (accession numbers are indicated). C) Phylogenetic tree generated with the NJ option of MEGA, based upon Kimura-2-parameter distances of SIVpol nucleotide fragments from captive mandrills CAS and REB, and reference sequences for SIVdrl, SIVmnd1, and SIVmnd2, respectively. Numbers shown are bootstrap confidence levels (BCL). D) SIV-pol amino acid consensus sequence from captive mandrills compared with homologous sequences from SIVdrl and SIVmnd2, respectively. The translated SIV-Artis sequence is the consensus sequence of 14 PCR clones derived from two animals. The YMDD motif within the catalytic core of the RT enzyme is underlined. Both EDTA-plasma and PBMC (isolated with the OptiPrep system (Nycomed, Oslo, Norway)) was obtained from the animals for further analysis. Our goal was to investigate whether the monkeys were virus carriers, and which strain of SIV they harboured. To detect both SIVmnd1 and SIVmnd2, we designed two nested primers sets based on published pol gene sequences that amplify an RT fragment of 282 nucleotides (Table 1 ). Nucleic acids were isolated from PBMC by a procedure using silica and guanidium thiocyanate [ 7 ]. cDNA was synthesized with the 3'primer and AMV-RT (Roche Diagnostics, Penzberg, Germany). PCR amplifications were performed using the following protocol: denaturation for 5 min at 95°C and amplification for 35 cycles (first PCR) or 25 cycles (second PCR) of 1 min at 95°C, 1 min at 55°C, and 2 min at 72°C, followed by an extension of 10 min at 72°C. Products were cloned with the TOPO TA cloning kit (Invitrogen, San Diego, Calif.). Sequencing of at least four clones per sample was done with the Bigdye Terminator Cycle Sequencing kit and an ABI 377 automated sequencer (both from ABI, Foster City, Calif.), using M13 forward and M13 reverse primers. For species identification, fragments of the mitochondrial 12S and cytochrome B genes were amplified [ 8 ], and sequenced. Table 1 PCR primers used to amplify Mandrillus SIV-pol Primer Sequence ('5→'3) Description Fragment size SIVmnd1A AGATATAGGGGATGCCTATT 5' first primer A-B = 356 nt SIVmnd1B TCTTCCACTTATCTGGGTGT 3' first primer SIVmnd1C AGATTATAGACCCTATACTGC 5'second primer C-D* = 282 nt SIVmnd1D CATCCAATGAAAGGGAGGTTC 3' second primer SIVmnd2A GGACATAGGGGATGCCTATT 5' first primer A-B = 356 nt SIVmnd2B CTGTCCATTTCTTTGGGTGC 3' first primer SIVmnd2C GGACTTTAGAAAGTACACTGC 5'second primer C-D* = 282 nt SIVmnd2D CATCCACTCAAAGGGAGGTTC 3' second primer SIVdrlA GGATGTAGGTGATGCCTATT 5' first primer A-B = 356 nt SIVdrlB CTGTCCACTTCTTTGGATGC 3' first primer SIVdrlC = SIVmnd2C 5'second primer C-D* = 282 nt SIVdrlD CATCCATTCATAAGGAGGATTG 3' second primer * corresponding to nucleotides 2503–2784 of SIVdrl (acc. no. AY159321) Mitochondrial 12S and cytochrome B sequences were identical in all four animals and confirmed that the monkeys were M. sphinx [ 9 , 10 ]. In addition, the cytochrome B sequences were indistinguishable from the recently described northern mandrill haplotype (Figure 1B ) [ 10 ], suggesting that the captive animals descended from founders originating from a locale north of the Ogooué River (see Figure 1A ). SIV-pol fragments could be amplified from PBMC of both adult mandrills CAS and REB with the primer set specific for SIVmnd2, but not with SIVmnd1 specific primers. However, analysis of the cloned fragments showed that these were 96–97% identical to SIVdrl-1FAO (GenBank acc. no. AY159321) isolated from a drill monkey ( Mandrillus leucophaeus ), with a lower sequence identity to SIVmnd2 (± 85% to GenBank acc. no. AF367411), and to SIVmnd1 (<64% to GenBank acc. no. M27470). Although SIVmnd2 and SIVdrl are more closely related to each other than the two SIVmnd strains, SIVdrl has several mismatches with the PCR primers designed for detection of SIVmnd2. This could explain why one seropositive animal was tested as PCR-negative. Therefore, we designed a new primer set that amplifies the same gene fragment based on the SIVdrl sequence (Table 1 ). Reanalysis of the mandrill PBMC samples with this drill-specific primer set again resulted in only two positive samples from the two adult animals: mandrills CAS and REB. Sequence analysis confirmed the high similarity to SIVdrl-1FAO (97%), and a lower similarity to SIVmnd2 (87%). SIV pol fragments from both mandrills were 98–99% similar to each other. Clones obtained from a single animal with the two primer sets were not identical to each other (98–99% identity), suggesting that each PCR amplified a subset of the virus population (Figure 1C ). Mandrills presently inhabit Cameroon, Gabon, and the southwestern part of the Republic of Congo. Two mitochondrial haplotypes are described in this species, separated by the Ogooué River in Gabon [ 10 ]. Interestingly, the distribution of mandrill SIV strains follows approximately the same geographic distribution, with SIVmnd1 being present in the southern part of the mandrill range, and SIVmnd2 in the northern part. Drill monkeys are found in Nigeria and Cameroon separated from the mandrill territory by the Sanaga River (Figure 1A ). Mandrills and drills are currently believed to be non-sympatric, but it is not unlikely that the situation was different in the past. SIVmnd2 and SIVdrl are closely related, and both are equidistant from SIVmnd1. SIVmnd2 is found in northern mandrills, which are closest to the current drill habitat. A wild-caught mandrill from south Cameroon was found to harbour a SIVdrl virus strain [ 3 , 11 ], suggestive of cross-species transmission [ 11 ]. Multiple cross-species transmissions are now believed to obscure the evolution and distribution of SIV strains in African primate species [ 1 ]. SIV cross-species transmissions are ongoing, and African green monkey strains have recently been detected in patas monkeys and baboons [ 1 , 12 , 13 ], species that are found in close proximity to each other. All four mandrills examined here were born in captivity. Male CAS was born in 1983 at the now closed Wassenaar Zoo (The Netherlands), and moved to Artis Zoo in 1986. The female, REB, was born in Budapest Zoo (Hungary), and moved to Artis Zoo when she was 5 years old. Their offspring, RAF and HAB, were both born in Amsterdam. Exposure to drills during their lifetime is unlikely as none of the zoos kept drills ( M. leucophaeus ) at any time. Drills are rare in European zoos, and only the zoos of Nikolaev (Ukraine) and Saarbruecken (Germany) reported keeping both drills and mandrills in a 1992 survey [ 14 ]. So, it is improbable that the Artis mandrills acquired SIVdrl from a recent contact with captive drills. Another way of acquiring a drill SIV strain could be if one of the monkeys was actually a hybrid between a drill and a mandrill. Hybridisation between different species of Cercopithecinae is possible, and offspring is sometimes fertile depending upon the exact species. The genus Mandrillus cannot hybridise in the wild, as the habitats of the two species do not overlap, but it does so in captivity. The morphologic differences between female drills and mandrills are less obvious than those between males and are mainly noticeable in the colouration of the muzzle and the size of the animal. A single hybrid M. sphinx × M. leucophaeus has been reported from Vienna Zoo, Austria, in a 1992 survey [ 14 ], and two hybrid M. leucophaeus × M. sphinx were described from a Wildlife Rescue Centre in Cameroon [ 15 ]. Mitochondrial sequencing as performed in this study cannot alone be used to resolve hybridisation, as it only characterises the mother lineage. The male mandrill is, however, unlikely to be a hybrid as its description fits exactly that of a male mandrill. The female was also listed in the Artis Zoo database records as a non-hybrid. She was registered with the European studbook programme (ESB) for mandrills supervised by the Budapest Zoo, Hungary, and was born from registered mandrill parents. Because SIVdrl was also found in a wild-caught Cameroonian mandrill [ 3 , 11 ], it is plausible that a SIVdrl-like virus is naturally present in mandrills, making them the only primate species that is naturally infected with three strains of SIV. The presence of SIVdrl in one of the two adult captive mandrills occurred probably through transmission from a wild ancestor, and this animal probably infected the other mandrill once joined in Artis Zoo. Sexual and mother-to-child transmission of SIV in mandrills have been reported to be rare [ 16 , 17 ]. Here, in offspring born to a SIV-positive mother SIV could not be detected. The persistence of maternal antibodies could explain why the 2-month old young tested seropositive, although there is a possibility of the animal having a viral copy number below the detection limit of the PCR assay. Transmission of SIV between mandrills and drills could have taken place either by biting or sexual contacts. SIVmnd1 is mainly transmitted between males during aggressive contacts [ 17 ], and might also be transmitted when fighting off other receptive primates species. Sexual transmission could have occurred during interbreeding. Each of these possibilities requires an overlap of habitats, which could have existed in the past. If we assume that SIVdrl(Artis) left Africa at least ten years ago (when the female was born in captivity), its genome conservation is remarkable: only 2 conserved amino acid differences separate the consensus pol-sequence from the SIVdrl reference sequence (Figure 1D ). However, to gain a deeper insight into the characteristics and evolution of this virus strain, a full-length sequence, or at least additional sequences of the gag or env regions, would be required. Only then could it be determined whether the virus carried by the captive mandrills is really SIVdrl or a novel recombinant virus. Several recombinant SIVs have been described in naturally infected primate species (see: [ 1 ]). Unfortunately, further sequence analysis is difficult due to shortage of material as the monkeys were euthanized soon after the SIV antibody test results became known. Competing interests The author(s) declare that they have no competing interests. Authors'contributions ACvdK designed the study, analysed the sequences, and drafted the manuscript. RvdB carried out the PCR assays and performed the cloning and sequencing. MJH did the medical examinations and collected the blood samples. RAG carried out the SIV antibody assays. ADMEO and BB conceived of the study, and participated in its coordination.

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526760

Improving epidemic malaria planning, preparedness and response in Southern Africa

Malaria is a major public health problem for countries in the Southern Africa Development Community (SADC). While the endemicity of malaria varies enormously across this region, many of the countries have districts that are prone to periodic epidemics, which can be regional in their extent, and to resurgent outbreaks that are much more localized. These epidemics are frequently triggered by climate anomalies and often follow periods of drought. Many parts of Southern Africa have suffered rainfall deficit over the past three years and countries expect to see increased levels of malaria when the rains return to more 'normal' levels. Problems with drug and insecticide resistance are documented widely and the region contains countries with the highest rates of HIV prevalence to be found anywhere in the world. Consequently, many communities are vulnerable to severe disease outcomes should epidemics occur. The SADC countries have adopted the Abuja targets for Roll Back Malaria in Africa, which include improved epidemic detection and response, i.e., that 60% of epidemics will be detected within two weeks of onset, and 60% of epidemics will be responded to within two weeks of detection. The SADC countries recognize that to achieve these targets they need improved information on where and when to look for epidemics. The WHO integrated framework for improved early warning and early detection of malaria epidemics has been recognized as a potentially useful tool for epidemic preparedness and response planning. Following evidence of successful adoption and implementation of this approach in Botswana, the SADC countries, the WHO Southern Africa Inter-Country Programme on Malaria Control, and the SADC Drought Monitoring Centre decided to organize a regional meeting where countries could gather to assess their current control status and community vulnerability, consider changes in epidemic risk, and develop a detailed plan of action for the forthcoming 2004–2005 season. The following is a report on the 1 st Southern African Regional Epidemic Outlook Forum, which was held in Harare, Zimbabwe, 26 th –29 th September, 2004.

Introduction The Southern African region has a long and varied history of malaria control with periodic epidemics occurring [ 1 , 2 ]. These epidemics can be regional in scale, as in 1996 and 1997, or much more focal, affecting specific districts or sub-districts. The countries of the Southern African Development Community are committed to the Abuja Targets for Roll Back Malaria in Africa, and this includes improved detection and response to epidemics [ 1 ]. To meet these targets countries are expected to detect 60% of malaria epidemics within two weeks of onset, and respond to 60% of epidemics within two weeks of their detection. The countries recognize that to achieve these targets they need improved information on where epidemics are most likely to occur, and ideally some indication of when they are likely to occur. The WHO guidelines on the development of Malaria Early Warning Systems (MEWS) for Africa are seen as offering a useful framework for an integrated approach to epidemic preparedness and response planning [ 3 - 5 ]. Experience and evidence of the successful application of this approach within the National Malaria Control Programme in Botswana over the past few years was demonstrated by the national malaria programme manager at the Southern Africa Regional Malaria Planning and Consultation Meeting in Gaborone in July 2004. Other countries in the SADC region considered this approach to provide a useful framework for planning epidemic preparedness and response strategies and, in view of the perceived vulnerability of communities throughout much of the region, called for a regional meeting that could launch the scaling-up of this process to include other epidemic prone countries beyond Botswana. The WHO Southern Africa Inter-Country Programme for Malaria Control (SAMC) responded to this demand and together with SADC's Drought Monitoring Centre (DMC) organized the 1 st Southern African Regional Epidemic Outlook Forum, which was held in Harare, Zimbabwe, 26 th –29 th September, 2004 and hosted by Zimbabwe's Ministry of Health and Child Welfare. Representatives from malaria control services in nine Southern African countries participated in the meeting: Angola, Botswana, Madagascar, Mozambique, Namibia, Swaziland, Tanzania, Zambia, and Zimbabwe. The purpose of the meeting was: to enable malaria control services from epidemic prone countries to gather and review their control programme status and epidemiological trends for the past 3–5 years and identify and map districts they consider to be vulnerable to epidemics; to learn about advances in the science of seasonal climate forecasting and review the implications of the forecast for the forthcoming season; to learn about environmental variables pertinent to epidemic risk and readily available sources of monitoring information; to review methods of early detection using case surveillance data; and, using the WHO framework for MEWS, to develop plans of action for epidemic preparedness and response for the forthcoming season. Discussion The MEWS framework as set out by WHO consists of four components: 1) vulnerability monitoring; 2) seasonal climate forecasting; 3) environmental monitoring; and 4) sentinel case surveillance. This framework is illustrated in Figure 1 . Figure 1 MEWS gathering cumulative evidence for early and focused response (WHO 2004) Vulnerability monitoring There are many factors that increase the vulnerability of a population to malaria epidemics [ 6 , 7 ] and increase the severity of disease outcome should a malaria epidemic occur. Co-infection with other diseases such as HIV-AIDS is a major consideration for Southern African countries. Resistance to therapeutic drugs and insecticides has also been a recent problem throughout much of the region. Drought, food insecurity and associated population movements between areas of differing endemicity combine to make certain populations more vulnerable to epidemics. These factors and consideration of the where and how to get appropriate information were discussed and countries were encouraged to identify measurable indicators and key informants. Seasonal climate forecasting In recent years there have been significant scientific advances in our ability to predict climate on the seasonal timescale [ 8 ]. The skill associated with these predictions varies from region to region, but is generally higher within the tropics. Scientists from the SADC Drought Monitoring Centre and the International Research Institute for Climate Prediction (IRI) joined with meteorologists from Democratic Republic of Congo, Malawi, Namibia, Zimbabwe and the World Meteorological Organization (WMO) to deliver the climate forecast for the forthcoming 2004–2005 season. An overview of climate variability in the SADC region was presented. The inherent issues of probability and uncertainty in climate forecasting were discussed with participants from the malaria control services. A number of myths were exploded and the variables that could or could not be skilfully forecast were reviewed. The malaria control participants gave their views on how communication of the forecast should be improved and made more understandable to the non-climate-specialist. Following a subsequent working session by the climate and meteorological specialists, an outline of additional or alternative forecast indicators was provided. Environmental monitoring The availability of environmental variables pertinent to malaria transmission, such as rainfall, temperatures, humidity, and flooding, were discussed and information on where they could be obtained was provided. The two basic sources of such information are periodic summaries (usually satellite-derived and interpolated estimates) available through the internet from the SADC DMC, the Famine Early Warning Systems Network (FEWS-NET) or the International Research Institute for Climate Prediction, Columbia University (IRI) websites; or directly from national meteorological services' ground-based weather observations. Generally, summary products are available free of charge, whereas the meteorological services may need to charge for raw data. Countries were encouraged to begin dialogue with their national meteorological services and discuss the more specific information requirements and support they may need. Sentinel case surveillance The paramount importance of developing good health information and sentinel surveillance systems was acknowledged. The process of MEWS development is seen as offering opportunities for strengthening integrated health systems surveillance. It is in itself dependent on good epidemiological data for testing and validating the relationships between the component parts. Methods of using indicators for epidemic early detection were discussed. Various indicators such as the mean × 2 standard deviations, the 'normal channel', cumulative-sum and weekly case thresholds have been tried, tested and used in a number of Southern Africa countries, and countries are encouraged to develop and use what is most appropriate and effective for their purposes. However, a number of the countries acknowledged having a poor statistical basis on which to develop and test early warning and detection indicators. Following the formal presentations setting out the MEWS components and epidemiological trends, the discussions centred around the countries' perceived control needs over the coming season and the information requirements for developing appropriate plans of action for epidemic preparedness and response. The countries represented varied markedly in their current levels of endemicity/epidemicity, surveillance and control coverage. Tanzania is for the most part a highly endemic country with an estimated 16–19 million cases per year. Botswana and Swaziland, by contrast, are currently recording cases in the low thousands and hundreds respectively. Zimbabwe's economic situation has recently compromised its control programme, and two of the countries, Mozambique and Angola, are in process of reconstructing their control programmes after recently emerging from major disruption due to long-term conflict situations. However, all of the countries did acknowledge the integrated MEWS approach as offering a useful framework for improving their epidemic planning, preparedness and response capabilities. Based on the the climate forecast for October, November, December, and the extended forecast for January, February, March, which are posted on the DMC website . The participants discussed the difficulties in access to and interpretation of meteorological data. The representatives from the meteorological services expressed a willingness to engage in closer collaboration to address these issues. The participants voiced a clear need to improve the availability of the seasonal climate forecast to the epidemic prone districts. They also highlighted the need for better communication of the forecast to non-climate users. Requests were specifically made for forecasts that are more 'meaningful' to the health sector. In response, the meteorological sector pointed at the necessity to know more specifically what information the health sector requires in order to then meet this need. Forecasts could, for example, be expressed simply as the probability of the coming season being wetter or drier than the previous season, or two, or three, or n seasons; or compared to that of the last epidemic season; or as probabilities of exceeding a given threshold for the season. However, it was stressed that forecasts will always be probabilistic and not deterministic. Moreover, countries were encouraged to refer to forecast updates as the season progresses. The issues of how to communicate better the probabilities and uncertainties associated with seasonal climate forecasts were addressed more closely. While many activities in malaria control are based on probabilistic, uncertain premises (clinical diagnosis and presumptive treatment, for instance), public health professionals are well aware of the limitations of their own indicators. While recognizing the potential value of advance lead-times for planning, they are understandably cautious in basing critical decisions on uncertain information from others, and the health and meteorological sectors probably need to work this through in more local collaborative settings. One additional issue that came out strongly during the meeting was the need for broader cross-border collaboration on epidemic prevention and control as 'true epidemic' prone areas are often based on particular environmental zones rather than administrative boundaries. For example, high rainfall anomalies in Angola may ultimately find their way as increased stream-flow into Botswana and Namibia, and create extensive breeding sites for vectors. Drought, food security, or a range of other factors, may lead to migrations of people across borders from one level of endemicity to another and pose a significant increase in epidemic risk. Development of national epidemic risk maps therefore ought to reflect the situation in neighbouring countries. There are a few examples of cross-border initiatives in the region: Republic of South Africa, Swaziland and Mozambique; Republic of South Africa and Zimbabwe. Both are showing promising results. Conclusions The meeting ended with the presentation of the recommendations, to be followed up within the next twelve months. The majority of the recommendations highlighted the need for stronger collaboration a) within the health sector itself; b) among the health sector and the climate-meteorological sector, and other relevant sectors; and c) among the various countries in the region. The participants committed themselves, with their partners, to developing integrated early warning systems as a decision support tool for improving epidemic preparedness and response planning. They recognized that this will be best achieved by drawing on appropriate scientific and technological advancements (and challenging these where necessary), by conducting operational research, and with the help of technical development and support, strengthen the capacity for improved epidemic preparedness and response in the districts at risk. The successful implementation of MEWS will depend on close cooperation among several partners: National Malaria Control Programmes must work closely with National Meteorological Services, supported by the regional Drought Monitoring Center, WMO and WHO. Opening these channels of communication will allow public health professionals and climate-environmental scientists and practitioners to incorporate more meaningful variables into the seasonal forecasts. In addition, it is necessary to exchange information with institutions dealing with vulnerable populations such as food security and refugee agencies, to develop mutually beneficial mechanisms that ensure easy access and utilization of relevant information for planning or decision-making. It was recognized that by adopting the MEWS approach for malaria control planning the overall health information and surveillance system would be strengthened as other diseases have strong climate and environmental components to their distribution, and further dialogue with Integrated Disease Surveillance and Response services would be useful. Training and capacity building requirements were discussed with WHO-AFRO regarding implementation within the national IDSR activities in the Southern Africa sub-region, and sub-regions elsewhere in Africa. In the final evaluation of the meeting participants, from both health and climate communities, considered that the meeting had provided a very useful overview of the issues and offered a good starting point for them to develop more flexible Plans of Action for Epidemic Preparedness and Response in their countries. It was recommended that a similar meeting be held each year prior to the onset of the rainy season.

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544873

The Latin American Social Medicine database

Background Public health practitioners and researchers for many years have been attempting to understand more clearly the links between social conditions and the health of populations. Until recently, most public health professionals in English-speaking countries were unaware that their colleagues in Latin America had developed an entire field of inquiry and practice devoted to making these links more clearly understood. The Latin American Social Medicine (LASM) database finally bridges this previous gap. Description This public health informatics case study describes the key features of a unique information resource intended to improve access to LASM literature and to augment understanding about the social determinants of health. This case study includes both quantitative and qualitative evaluation data. Currently the LASM database at The University of New Mexico brings important information, originally known mostly within professional networks located in Latin American countries to public health professionals worldwide via the Internet. The LASM database uses Spanish, Portuguese, and English language trilingual, structured abstracts to summarize classic and contemporary works. Conclusion This database provides helpful information for public health professionals on the social determinants of health and expands access to LASM.

Background Public health practitioners have long recognized the connections between patients' socioeconomic conditions and their health [ 1 - 8 ]. Yet these practitioners and their empirically oriented researcher colleagues have faced difficulties in establishing the precise linkages between socioeconomic variables and sub-optimal health status. Social medicine is a diverse field that studies these relationships between society (and its socioeconomic conditions) and the health of populations. In Latin America, social medicine consists of a widely respected and influential field of research, teaching and professional practice [ 9 ]. Professionals working in this field seek to identify and to understand better the linkages between socioeconomic conditions and patients' health. Until recently, however, most of the knowledge base in this discipline has remained largely unknown outside Latin America. Language barriers and disincentives to distribute this information more widely are two major reasons for this lack of awareness. Some readers might have first learned about Latin American social medicine (LASM) through recent critical reviews [ 9 ] or through a special issue of the American Journal of Public Health that focused on LASM [ 10 , 11 ]. LASM traces its historic origins to European researchers such as Rudolf Virchow and the belief systems of indigenous cultures. Both the European and indigenous sources of current social medicine practices emphasized the importance of linking social conditions to health status. Contemporary social medicine in Latin America continues to emphasize these linkages between social conditions and the health of populations. Social medicine professionals participate in a wide array of settings in Latin America and represent diverse specialties. Their integration into healthcare systems has varied by era and country [ 12 ]. Construction and content Innovative approaches to disseminating work in LASM have become increasingly available due to Internet technology. The project "Enhanced Access for Latin American Social Medicine" at The University of New Mexico with funding from the U.S. National Library of Medicine seeks to make information on the connections between social conditions and health problems available to a wide audience. The project has sought to bridge the prior information gap primarily through delivering structured abstracts of social medicine publications in Spanish, English, and Portuguese via the Internet on the LASM database beginning in 2001. Other goals of this project include: publishing full text social medicine electronic journals on behalf of medical societies in Latin America; and, maintaining a repository for key classic and contemporary social medicine publications. Structured abstracts are posted in three languages in the LASM database at The University of New Mexico for both the classic and contemporary social medicine literatures. The first phase of this project involved preparing and posting the structured abstracts of 25 landmark books, 50 book chapters, and 100 journal articles from the classic social medicine literature in Spanish, Portuguese, and English. A peer selection committee identified and agreed upon the specific selections of classic books, book chapters, and journal articles to be abstracted for this project. Table 1 lists the members of this committee, representing institutions in Brazil, Chile, Colombia, Cuba, Ecuador, Mexico, the United States, and Venezuela [ 13 ]. Representative examples of some of the classic books [ 14 - 16 ], book chapters [ 17 - 19 ], and articles [ 20 - 22 ] can be found in the list of references following this article. Table 1 Members of the Peer Selection Committee Country Name Institution Brazil Emerson Elias Merhy University of Campinas, São Paulo Chile Alfredo Estrada L. Investigation and Training Group in Social Medicine, Santiago Colombia Saul Franco Agudelo National School of Public Health, Bogotá Cuba Francisco Rojas Ochoa National School of Public Health, Havana Ecuador Jaime Breilh Health & Research Advisory Center, Quito Mexico Ángeles Garduño Autonomous Metropolitan University-Xochimilco, Mexico City Mexico Asa Cristina Laurell Secretariat of Health, Mexico City Mexico Francisco Mercado Martínez University of Guadalajara, Guadalajara Peru Marcos Cueto Peruvian University "Cayetano Heredia," Lima United States Elizabeth Fee National Library of Medicine, Bethesda, MD United States Norman Frankel American Medical Association, Chicago, IL United States Allen Jones American Public Health Association, Washington, DC United States Antonio Ugalde University of Texas, Austin, TX Venezuela Oscar Feo Department of Public Health, Maracay Venezuela Maria Urbaneja Latin American Social Medicine Association and Foreign Ministry of the Venezuelan national government, Caracas The contemporary literature summarized in the LASM database has been drawn primarily from the 12 journals currently or previously published in Latin America. Table 2 lists the 12 journals, with their titles translated into English. These specific journals also have been identified and approved by the peer selection committee. The website that hosts the LASM database provides further information about this committee's members, including their institutional affiliations and areas of research. The peer selection committee consists of experts in social medicine and information technology. The LASM steering committee based at The University of New Mexico meets with the peer selection committee twice a year via online conferencing to decide on selection policies, actual lists of resources slated for inclusion in the LASM database, and administrative matters regarding the project [ 23 ]. Table 2 Current journal subscriptions monitored for noteworthy articles on social medicine Country Title Argentina Cuadernos Médico Sociales (Medico-Social Notebooks) 197?- Argentina Salud Problema y Debate (Health – Problem and Debate) + Brazil *Cadernos de Saúde Pública (Notebooks of Public Health) 1985- Brazil Ciência & Saúde Coletiva (Science and Collective Health) 1996- Brazil Interface (Interface) 1997- Brazil Revista Brasileira de Epidemiología (Brazilian Journal of Epidemiology) 1998- Brazil Saúde e Sociedade (Health and Society) 1992- Brazil Saúde em Debate (Health in Debate) 1976- Cuba Archivos del Ateneo Juan César García (Archives of the Juan César García Circle) 2000- Cuba *Revista Cubana de Medicina Tropical (Cuban Journal of Tropical Medicine) 1996- Cuba Revista Cubana de Salud Pública (Cuban Journal of Public Health) 1975- Mexico Salud Problema (Health – Problem) + Notes: * Indexed by the MEDLINE database from the National Library of Medicine + Start date occurred during 1900s but exact date not known As a pilot, the project has made two social medicine journals available in electronic full text format. Several issues of these journals, Saúde em Debate (Brazil) and SaluCo Bulletin (Cuba), are available from the host website. The host website also posts structured abstracts of important articles from these full text journals. The LASM database encompasses several broad themes within LASM. Subject areas include the history, theories, methodologies, and organizational dimensions of social medicine. Other subjects pertain to institutional analysis, social/critical epidemiology, and strategic planning. Table 3 summarizes the specific topics emanating from these broad subject areas. Readers will recognize that many of the subjects have direct bearing on the health of populations, such as health disparities and managed care. Table 3 Subjects covered by Latin American social medicine Health policy analysis Violence and health Medical education reform Mental health services and mental health policies Primary care research and preventive services Determinants of mental illness in race, ethnicity, social class, or gender Strategic planning Indigenous, complementary and herbal medicine Environmental health Social, environmental and nutritional causes of infant and perinatal mortality Labor and health Economic development, demographic change, and aging Ethnic/racial disparities and health Socioeconomic barriers to cancer prevention Social class disparities and health Social processes of alcohol and drug abuse Gender disparities and health Chronic illnesses Infant and perinatal mortality Urban health International health Managed care The LASM database is a web application developed using the Cold Fusion application server. The data are stored in a Microsoft SQLServer relational database. The data sources are the original Latin American publications, which are summarized in Spanish, Portuguese, and English languages in structured abstract format. The English-language structured abstracts are quality checked by the principal investigator, who reviews the abstracts for substantive content, and then by the librarian investigator who reviews the structured abstracts for final quality assurance purposes. Each record contains fields for author, title (book, book chapter, or article), place of publication, publisher and structured abstracts in each language. Records contain volume, number, and pagination when applicable. All records contain terms the from Medical Subject Headings (MeSH) system, a controlled vocabulary developed and maintained by the National Library of Medicine. Users can search the database on controlled fields of author, title, and MeSH terms in any of the three available languages. Full text abstract searching is also available. In addition to the abstract searching facilities, the LASM database can be browsed alphabetically by title. The browsing interface offers a convenient way to become familiar with the extent and diversity of the LASM literature. Utility and discussion From January 2002 through December 2003, a total of 17, 853 visits were made to the website hosting the LASM database. The largest numbers of visits in descending rank order originated from Brazil, Mexico, Argentina, Spain, and Colombia. A preliminary, qualitative evaluation has been favorable. Following completion of this project, we will conduct and publish a comprehensive summative evaluation. A total of 250 structured abstracts in Spanish, Portuguese, and English had been posted to the LASM database as of June 30, 2004. The host website presents more detailed information about this project, as well as the structured abstracts themselves. The LASM database comprises a dynamic and searchable web application containing structured abstracts in English, Portuguese, and Spanish. It is designed using industry standard web application design principles, but its content is unique to the LASM domain. A database searching design and utility problem unique to the LASM database, and other web applications like it, relates to the problem of web-based multilingual searching. Widely available search engines cannot preprocess a multilingual language translation of search terms (e.g. retrieving results containing the Spanish word "pública" for the English search term "public"). Additionally, search engines do not recognize that the unaccented "publica" (as it might be entered by an English speaker as a search string) might be the same word as the Spanish accented "pública" and therefore will not return the user's expected search result. Key combinations and modifiers that are used to create special and accented characters in word processing programs like Microsoft Word often do not work in browser based search and form fields. Our primary instruction to users for constructing search terms containing special and accented characters (i.e., diacritics) suggests that they use a separate text editor that accepts keyboard modifiers to construct special and accented characters to build the search term and then copy and paste the completed search string into the field. On our search pages we also list common special and accented characters in Spanish and Portuguese that users can copy and paste into browser based search fields. Although there are no other alternatives for entering special and accented characters into browser form fields, both methodologies are somewhat cumbersome. Therefore, to improve the searching utility of the LASM database we also preprocess entered search strings to allow the search engine to perform selected character substitution on the entered search string and attempt to solve the accented character problem from the search engine side. All search strings are first passed through a regular expression routine, which substitutes single character search engine wildcards for the following characters: A, E, I, O, U, a, e, i, o, u, N, C, n, c. This effectively removes potential special and accented character misspellings. To return to the previous example, a user might enter the search string "publica" ("public" in English) in a search field as an attempt to find article titles that contain the Spanish or Portuguese word "pública". Properly spelled, "Pública" uses the accented character "ú" rather than "u" (ASCII 163 rather than ASCII 117). Unfortunately, current search engines are not intelligent enough to infer that "pública" is a match for the search string "publica". Therefore, the literal search for publica (unaccented) will not return any search results that contain "pública" although there are hundreds of instances of the word "pública" in the LASM database. In the case of "publica", the preprocessed search string that is finally submitted to the search engine is "p*bl***". The search engine will return all seven-letter word matches that contain the letters p, b, and l in the first, third, and fourth positions respectively. The net effect of this technique is to under-specify the search result. That is, the search engine may possibly return records that contain other words that happen coincidentally to match the submitted search string. On the other hand, the returned result set can be guaranteed to contain the desired search result. Problems created from under-specifying the search are limited based on experience gained from using this technique. The positional constraints of submitted characters within the search string generally are restrictive enough to prevent most problems. Specifically, within a limited domain search surface like the LASM database, the likelihood of the occurrence of most alternative word matches is very low. The character substitution methodology described here is not perfect, and many other alternative strategies for addressing the multilingual search problem have been explored by LASM technical staff. Most of the alternative strategies considered, however, involved much higher costs in terms of acquiring or developing specialized search engine capabilities or much higher abstract preparation costs. Therefore, we chose the character substitution strategy as a compromise between implementation cost and search utility. Conclusions Internet technology via websites and web browsers has created numerous opportunities for public health colleagues to inform one another and to collaborate across wide geographic space. The LASM database clearly demonstrates the efficacy of the Internet for communicating its informative structured abstracts posted in the Spanish, Portuguese, and English languages. This database provides useful information that would otherwise be unavailable for public health professionals on the social determinants of health. Furthermore, it expands access to LASM through its inclusion of both the classic and contemporary literature. Availability and requirements Anyone with access to the World Wide Web and a web browser can access all structured abstracts in the LASM database . As the data reported above indicate, the LASM database already has been accessed steadily since its initial small-scale publicity began in 2002. We hope that public health readers will utilize the LASM database to improve their research, teaching, and practice. Competing interests Neither the authors nor any support personnel possess competing interests related to the LASM database or to this article's publication. Authors' contributions JE conceived of and wrote the initial version of this article, served as an early collaborator on and helped design the project, was responsible as an investigator for acquiring journals for the Latin American social medicine collection, provided quality assurance editing on structured abstracts, played a major role in the formative evaluation of the project, and coordinated all revisions to this article. HW initiated and designed the project, served as principal investigator, obtained funding, translated and edited abstracts in the English-language, and edited this manuscript. HSB served as co-principal Investigator, helped design this project, obtained funding, and led the effort for the formative evaluation of this project. JT helped administer the project and edited this manuscript. CI coordinated the project, wrote Spanish-language structured abstracts, and provided reference materials for this article. KW and JT developed all programming aspects of the website including the search strategies, managed the web interface and underlying database and contributed the text for portions of this article. Pre-publication history The pre-publication history for this paper can be accessed here:

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555956

Influence of immunomagnetic enrichment on gene expression of tumor cells

Background Metastasis is the leading cause of cancer-related death. Bone marrow (BM) is a frequent site for the settlement of disseminated tumor cells which occurs years before overt metastases signal incurability. Methods Here we describe a new method to assess the initial stage of metastasis development in cancer patients. By immunomagnetic selection with HER2/neu and EpCAM as catcher antigens single disseminated tumor cells can be enriched from BM samples. To examine whether the immunomagnetic enrichment technique may change gene expression in the selected tumor cells, we performed differential expression profiling with the breast cancer cell lines MCF-7 and BT474 as models. The profiles were performed using 1.2 Cancer Arrays (Clontech) containing 1176 cDNAs that can be grouped into different functional categories, such as signal transduction, cell cycle, adhesion, cytoskeleton plasticity, growth factors and others. Results The reproducibility of the gene expression profiling between duplicate cDNA-array experiments was assessed by two independent experiments with MCF-7 breast cancer cells. Scatter blot analysis revealed a good reproducibility of the cDNA array analysis (i.e. less than 10% difference in the gene expression between the arrays). Subsequent comparative cDNA-array analyses of immunobead-selected and unselected MCF-7 and BT474 cancer cells indicated that the antibody incubation during the immunomagnetic selection procedure did not considerably alter the gene expression profile. Conclusion The described method offers an excellent tool for the enrichment of micrometastatic tumor cells in BM without largely changing the gene expression pattern of these cells.

Introduction Solid tumors derived from epithelial organs are the main form of cancer in industrialized countries. The first phase of the metastatic development consists of local tumor cell invasion, followed by tumor cell circulation in the blood and homing to secondary distant organs [ 1 , 2 ]. As indicator organ for early systemic dissemination of epithelial tumor cells to distant sites, BM has played a prominent role [ 3 ]. BM can easily be aspirated from the iliac crest and single metastatic cells are already present in 20–40% of patients with epithelial tumors (e.g., breast, lung or colon carcinomas) years before overt distant metastases occur in the skeleton or other distant organs [ 3 - 5 ]. The molecular description of these cells has been, however, hampered by the low concentration of these cells (e.g., 10 -5 -10 -6 per BM cell). To predict and monitor therapeutic responses the assessment of the gene expression profile of disseminated tumor cells seems to be of utmost importance. However, it is uncertain to which extent incubation with antibodies used for immunomagnetic isolation of these cells might affect their expression profile. We have addressed this aspect, using monoclonal antibodies against two prominent antigens, EpCAM and HER2/neu that are frequently and independently expressed on micrometastatic tumor cells [ 3 ]. Materials and methods Ficoll density gradient centrifugation and immunocytochemistry The enrichment of tumor cells from BM by Ficoll density gradient centrifugation and the immunocytochemical detection of epithelial tumor cells in cytological BM preparations has been described elsewhere in detail [ 5 , 6 ]. Immunomagnetic cell separation and immunocytochemistry of BM samples Two ml of a BM sample usually containing 2 × 10 6 mononuclear cells were washed with Hank's Salt Solution (Biochrom KG, Germany). The pellet was resuspended in 2 ml Hanks and 3.2 × 10 7 (80 μl) CELLection™ and pan-mouse immunomagnetic beads (Dynal, Oslo, Norway) coated with anti-EpCAM (MAb 3B10) and anti-HER2/neu (MAb 7C1) antibodies (Micromet, Munich, Germany) were added. All solutions and cell preparations were kept at 4°C during the whole procedure to avoid nonspecific binding of immunomagnetic beads. After an incubation time of 30 min at 4°C and 20 min at room temperature on a rotating mixer the magnetically labeled cells were isolated in a magnetic particle concentrator and resuspended in 200 μl bead removing buffer (40 mM Tris, 10 mM MgSO 4 , and 1 mM CaCl 2 , pH7.4, prewarmed to room temperature). The immunomagnetic beads were removed by DNase treatment with 15 μl DNase (50 U/μl) at room temperature for 15 min. After separation in a magnetic particle concentrator the supernatant was collected and centrifuged onto glass slides. Tumor cells were identified by immunostaining with monoclonal anti-cytokeratin antibody A45-B/B3 according to the manufacturer's instruction (Micromet, Munich, Germany). Cytokeratins are specific constituents of the epithelial cytoskeleton and they have become the marker antigen of choice for the detection of disseminated epithelial tumor cells in mesenchymal organs such as BM [ 3 , 7 ]. To avoid unspecific binding of the antibody via Fc-receptors present on leukocytes, we used F ab fragments of A45-B/B3 that were directly conjugated to the marker enzyme alkaline phosphatase. Cell culture and antibody incubation MCF-7 cells and BT474 cells were maintained in RPMI (Invitrogen, Karlsruhe, Germany) supplemented with 5 % glutamine (Invitrogen) and 10 % FCS (C-C Pro, Neustadt, Germany). MCF-7 and BT474 cells (ATCC HTB-22 and HTB-20) were allowed to reach a logarithmic growth phase in culture. At 90 % confluency, the cells were incubated for 30 min at 4°C and 20 min at room temperature in Hanks containing 1 μg/ml of anti-EpCAM (MAb 3B10) as well as anti-HER2/neu (MAb 7C1) antibodies according to the immunomagnetic cell separation protocol. In a negative control experiment, cells were suspended in Hanks devoid of antibodies. cDNA probe preparation and hybridization Total RNA was isolated using the peqGold TriFast™ (Peqlab, Erlangen, Germany) according to the manufacturer's instruction. In order to remove genomic DNA contamination, a DNase step was included using the DNA-free™ kit (Ambion, Cambrigeshire, England) according to manufacturer's instructions. RNA was dissolved in RNase-free H 2 O with 1 U/μl RNase inhibitor (SUPERase. IN™, Ambion). 5 μg purified total RNA was used for [α- 33 P] dATP (3000 Ci/mmol, 10 μl; Amersham, Freiburg, Germany) labeled cDNA synthesis as previously described [ 8 ]. The cDNA probe was purified with nucleotide removal columns (Qiagen, Hilden, Germany). The Atlas Human 1.2 Cancer Arrays (Clontech, Heidelberg, Germany) were hybridized according to the manufacturer's protocol. cDNA- array data analysis The membranes were exposed to phosphoimager plates (Raytest Isotopen-Meβgeräte, Straubenhardt, Germany) for 3 days, and plates were scanned with the phosphoimager Fuji Bas (Raytest) at a 100 μm-resolution. The images were analyzed using the Imagene 5.5 software (Biodiscovery, CA, USA). The data of the arrays were normalized on the basis of the genes ubiquitin, HLAC and beta actin. The ratio between antibody-treated and non-treated cells was calculated for each gene. Ratios lower than 0.5 or higher than 2 were considered as differentially expressed if at least one sample showed an expression above 0.5. We performed duplicates of each experiment and created scatter blots with the SPSS software for windows. Results In a recent work [ 9 ] we demonstrated that our immunomagnetic separation works on clinical samples (Figure 1 ) and is superior to the standard Ficoll density centrifugation technique, used in most previous studies on cancer micrometastasis [ 4 , 5 , 7 , 10 ]. Figure 1 CK-positive cells detected after immunomagnetic enrichment of BM from breast cancer patients. One single cell and one 2-cell cluster is shown in a 400× magnification. To test whether the antibody incubation during the immunomagnetic enrichment approach affects gene expression in the selected cells, we applied cDNA-array analysis. We subsequently evaluated whether the immunomagnetic enrichment method affected gene expression in the selected cells. This aspect is of utmost importance for further molecular description of disseminated tumor cells and has not been addressed before. The profiles were performed using 1.2 Cancer Arrays (Clontech) containing 1176 cDNAs that can be grouped into different functional categories, such as signal transduction, cell cycle, adhesion, cytoskeleton plasticity, growth factors and others [ 11 ]. As models, we used the breast cancer cell lines MCF-7 and BT474 (ATCC HTB-22 and HTB-20), because they express heterogeneous levels of the target antigens HER2/neu and EpCAM comparable to micrometastatic breast cancer cells in vivo [ 3 ]. The reproducibility of the gene expression profiling between duplicate cDNA-array experiments was assessed by two independent experiments with MCF-7 breast cancer cells. As shown in Figure 2A , scatter blot analysis revealed a good reproducibility of the cDNA array analysis (i.e. less than 10% difference in the gene expression between the arrays). We plotted the data of the antibody-treated and untreated MCF-7 (B) or BT474 (C) cells two dimensionally in a scatter plot; y-axis represents the data of untreated cells and the x-axis represents the data of cells treated with anti-HER2/neu or anti-EpCAM. For both cell lines, the scatter plots show that expressed genes in antibody-treated versus untreated cells (Figure 2B, C ) was in principal within the range observed in the duplicate experiments with MCF-7 cells (Figure 2A ). However, subtle changes in the expression of individual genes after antibody incubation were observed in particular in BT474 cells. In this cell line 38 genes were strongly differentially expressed (ratio >3) in antibody-treated versus untreated cells (Table 1 ). Most of these genes play a role in extracellular matrix remodeling, signal transduction and replication, as well as repair and transcription. MCF-7 cells showed in this experimental approach 31 differentially expressed genes with a ratio of over 3 (data not shown). Although similar group of genes were affected, only 3 common genes (CDC7, SGI and KIR) were differentially expressed in both cell lines after antibody incubation. Figure 2 Representative scatter blots of breast cancer cells using the 1.2 Cancer Array for expression analysis. (A) untreated MCF-7 breast cancer cells (results of duplicate experiments), (B) antibody-treated versus untreated MCF-7 cells, and (C) antibody-treated versus untreated BT474 breast cancer cells. Table 1 Genes differentially expressed in antibody-treated versus untreated BT474 cell Genes GenBank Accession# Ratio * Extracellular matrix remodeling : COL11 J04177 - 6.2 MMP17 X89576 - 4.7 MMP16 D50477 -4.6 SPARC J03040 5.7 Adhesion: PKD1 U24497 - 7.3 NCAM AF002246 7.3 M-cadherin D83542 3.5 Cytoskeleton plasticity: SPTA1 M61877 7.4 Signal transduction: RGS4 U27768 - 10.1 GAS L13720 - 7.1 BMP1 U50330 - 7.0 FGFR4 L03840 - 6.5 PMEL17 M77348 - 6.5 ETS-1 - 5.9 ERBB2 M95667 - 5.8 TGF-beta X02812 - 5.2 N-ras X02751 - 4.8 HRS D84064 - 4.7 KIR U10550 9.0 BMP6 M60315 7.5 BIN1 U68485 7.4 SGI Y00064 4.1 CDC7 AF015592 3.8 CNTF S72921 3.4 SH3BP2 AF000936 3.1 Apoptosis: CD27BP U82938 6.0 DR5 F016268 4.8 Metabolism: PPAT U00238 6.3 HPRT P00492 5.4 Immune response: MHC class I U65416 8.4 Replication/repair/transcription: CHAF1A U20979 - 12.3 NEK3 Z29067 - 5.6 BTG U72649 8.9 HRC1 M91083 6.2 TOP1 J03250 4.9 CLK1 L29222 4.0 Functionally unclassified PIG7 AF010312 - 4.9 menin U93236 3.1 *Ratio of normalized data from antibody-treated versus untreated BT474 cells as described in the Materials and Methods section. Negative values indicate downregulated and positive values upregulated genes. Discussion Here, we investigated whether an immunomagnetic enrichment procedure for micrometastatic cancer cells present in BM aspirates leads to significant changes in the gene expression pattern of the enriched tumor cells. In order to mimic the biological conditions of a tumor type with frequent BM involvement, we used two breast cancer cell lines (MCF-7 and BT474). Both cell lines expressed the target antigens, EpCAM and HER2/neu, for immunomagnetic separation at different levels [ 12 ] and they were incubated with the anti-EpCAM and anti-HER2/neu antibodies according to the same immunomagnetic enrichment protocol used for the BM samples from cancer patients analyzed recently [ 9 ]. It has been shown by other groups that some of the mAb directed against HER2/neu (e.g., Herceptin R ) can specifically block cell proliferation and affect gene expression in HER2/neu-positive breast cancer cells [ 13 , 14 ]. Furthermore the incubation of human cells with anti-EpCAM-specific mAbs (e.g. KS1/4 mAb) can induce considerable changes in the expression of insulin and glucagons [ 15 ]. However, our present results suggest that the two antibodies against EpCAM and HER2/neu used for the immunomagnetic selection process did not considerably influence the gene expression pattern of the enriched cells, although the HER2/neu- positive cell line showed a slightly increased number of differentially expressed genes. These genes are involved in extracellular matrix remodeling, signal transduction and replication, repair and transcription, and they were either up or downregulated after antibody incubation. For example, HER2/neu gene expression was downregulated after antibody binding, as expected from reports in the literature [ 16 ]. Taken together, we cannot exclude subtle changes in the expression of individual genes after antibody incubation, but we observed no obvious shift in the expression pattern that exceeds the normal variability of duplicate experiment. Thus, we conclude that the immunomagnetic selection protocol described here might be useful for experimental approaches aimed to determine the gene expression profile and genome of disseminated CK-positive cells [ 17 , 18 ]. As we performed our study only on two breast cancer cell lines, larger series of similar experiments with further cancer cell lines as well as with enriched tumor cells from the blood or BM must be investigated to draw firm conclusions. The detection and characterization of micrometastatic cancer cells will provide new insights into the biology of the metastatic process in cancer patients. This will lead to an improved molecular staging of cancer patients and to the identification of new biological targets for adjuvant systemic therapies aimed to eradicate micrometastatic disease before the onset of overt metastasis signals incurability.

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340127

Wnt Signaling Relies on Nuclear Armadillo

A couple of years ago, a paper was published in a high-profile journal that challenged a long-established model of cell signaling. While researchers in the field mostly greeted the results with skepticism, some went into the lab to investigate the discrepancy. Many elements of this pathway, called the Wnt pathway, have been well characterized. The standard model of Wnt signaling holds that when the Wnt protein binds to its receptor, it initiates a labyrinthine signaling cascade that sends a protein called β-catenin into the cell's nucleus where, together with a protein complex, it initiates transcription. In the absence of this signal, β-catenin binds to an inactivating complex in the cytoplasm and is targeted for degradation. The paper that disputed this view suggested that β-catenin can effect gene expression without entering the nucleus and that it can activate the Wnt pathway while tethered to the cell membrane. Before that paper was published, Nicholas Tolwinski and Eric Wieschaus had shown that β-catenin, also known as Armadillo (Arm) in the fruitfly, is sent into the nucleus in response to Wnt signaling. Upon entering the nucleus, Arm interacts with a second protein complex to activate transcription. Now Tolwinski and Wieschaus have reexamined the function of Arm in the fruitfly and have demonstrated that the pathway “in fact does depend on the nuclear localization of β-catenin.” While their paper was in the final stages of acceptance, the dissenting paper was retracted, after it was learned that the results had been fabricated. Tolwinski and Wieschaus' findings confirm what had already been known about Arm's role in Wnt signaling and also fill in important details about how it works. Multicellular organisms rely on elaborate communication networks of signaling proteins and enzymes to exchange information between cells. The Wnt signaling pathway regulates the expression of a host of different genes during embryogenesis to control body patterning and cell differentiation in organisms from fruitflies to mammals. Miscommunications in this tightly regulated pathway contribute to a variety of developmental defects and cancers. In the developing fruitfly, Wnt signaling is normally restricted to the front of each larval segment, where it produces a smooth surface; the rear of the segments, where Wnt signaling is absent, is hairy. If Wnt signaling is overexpressed, it produces fruitfly larvae with only smooth segments; lack of Wnt signaling produces hairy segments. Using the smooth phenotype as a measure of Wnt signaling, Tolwinski and Wieschaus delved deeper into the role of Arm in this signaling process. These experiments are complicated because Arm functions not just in Wnt signaling, but also in cell adhesion. The trick is to make the endogenous Arm (the version encoded by the fly genome) defective for signaling, while leaving the cell adhesion functions fairly normal. Set against this “background,” an additional Arm protein is expressed that is tethered to the membrane; it still retains the protein domains required for signaling, but it's stuck on the inside of the membrane and can't move into the nucleus. In this background, any signaling in response to Wnt must mean that Arm can signal to the nucleus without actually having to get inside. Tolwinski and Wieschaus prove—again—that this is not the case. They do this by showing that the weak, medium, and strong endogenous Arm mutants—these classifications reflect the severity of the mutations' effects—have different effects on signaling in the presence of the membrane-tethered Arm. It's clear, they argue, that tethered Arm cannot signal on its own and must somehow be helping the weaker mutants signal. To further investigate how the tethered Arm activates the endogenous mutants, Tolwinski and Wieschaus developed two new and “cleaner” Arm mutants that impair Arm's signaling ability but have no effect on its cell adhesion function. The tethered Arm could not produce a completely smooth phenotype with these nonsignaling endogenous mutants. These experiments, the authors conclude, indicate that the tethered form of Arm produces its transcriptional effects by activating the endogenous Arm protein. Normal activation of the pathway liberates Arm proteins from the inactivating complex, which allows them to enter the nucleus and activate transcription. Tethered Arm appears to accomplish this by sequestering the inactivating complex at the cell membrane, preventing it from interfering with endogenous Arm. Even though Tolwinski and Wieschaus started these experiments based on what turned out to be fabricated results, their investigations produced valuable contributions. They not only reaffirm the standard model of Wnt signaling, but reveal important new insights into the workings of a major player in the pathway.

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545210

Predicting Tumor Responses to Gefitinib and Erlotinib

Tyrosine kinases regulate signaling pathways that control cell growth, proliferation, motility, and other critical cellular processes. Mutations in tyrosine kinase genes can lead to abnormal kinase activity, and some tumors become dependent upon this activity for growth and survival. Thus, kinases are attractive targets for anti-cancer drugs. Examples of new kinase inhibitors include gefitinib and erlotinib, which have recently shown promise in treating non-small-cell lung cancer. Unfortunately, gefitinib and erlotinib work only in a subset of patients, and they can have severe side effects, albeit infrequently. So researchers have been trying to find ways to predict who will benefit from therapy with these drugs and who won't. Assessing lung tumors for gene mutations could help guide therapy Following the work of Lynch et al. (N Engl J Med 350: 2129–2139) and Paez et al. (Science 304: 1497–1500), William Pao and colleagues have previously shown that the epidermal growth factor receptor (EGFR), a tyrosine kinase, is often mutated in non-small-cell lung cancers, and that tumors that harbor such mutations are sensitive to gefitinib and erlotinib. In this new study, they focused on a signaling protein called KRAS, which functions downstream of many tyrosine kinases, including EGFR. The KRAS gene is also often mutated in lung cancers, but very few cancers have mutations in both EGFR and the KRAS gene. To find out whether KRAS mutations could help to predict which patients would respond to gefitinib or erlotinib, the researchers looked for mutations in EGFR and KRAS genes in 60 tumors for which sensitivity to either drug was known. They extended their earlier findings that EGFR mutations (which were found in 17 of the tumors) were associated with sensitivity to the kinase inhibitors, and found that tumors that had mutations in KRAS (a total of nine) were refractory (i.e., did not respond) to either drug. These results need to be validated in larger and prospective trials that use standardized mutation detection techniques. If they are confirmed, knowing the mutation status of EGFR and KRAS in tumors could help physicians decide which patients should receive gefitinib and/or erlotinib. As Inoue and Nukiwa state in a Perspective that accompanies the article, “By combining all the factors that relate to response or resistance, patients who will benefit from treatment can hopefully be identified. Undoubtedly we have taken a great step forward in molecular therapy for lung cancer treatment.”

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524188

Extraabdominal fibromatosis in retroperitoneal space

Background Fibromatosis or desmoid tumor covers a broad spectrum of benign fibrous tissue proliferations. It is characterized by infiltrative growth and a tendency towards recurrence; however, unlike sarcoma, it never metastasizes. Case presentation We report on a case of extraabdominal fibromatosis originating from the retroperitoneal space in a 43-year-old woman. Seven years earlier she had undergone ureterolysis and ureteroureterostomy for ureteral obstruction. Computed tomography revealed a tumor between the iliocostalis and the psoas muscle. Histopathological evaluation revealed uniform proliferation of spindle cells, with a moderate amount of collagen fibers, suggesting extraabdominal fibromatosis (desmoid tumor). The tumor was surgically resected, and since then, the patient has remained asymptomatic without any restrictions of daily living activities and without any signs of tumor recurrence during the two-year follow-up. Conclusions Complete resection is the treatment of choice. Adjuvant therapy using non steroidal anti-inflammatory agents, tamoxifen, interferon, anti-neoplastic agents, and radiotherapy, either alone or in combination finds application for unresectable or recurrent cases.

Background The term "fibromatosis" covers a broad spectrum of benign fibrous tissue proliferations, the biological behavior of which is similar to both benign fibrous lesions and fibrosarcoma. Like fibrosarcoma, fibromatosis is characterized by infiltrative growth and a tendency towards recurrence; however, unlike sarcoma, it never develops metastasis [ 1 ]. Therefore, the most important strategy is to prevent direct invasion into adjacent tissues. Extraabdominal fibromatosis principally originates from the connective tissue of muscles and the overlying fascia or aponeurosis. It may occur in a variety of anatomical locations, including the muscles of the shoulder, the chest wall and back, thigh, and head and neck. However, solitary occurrence is rare in retroperitoneal space [ 1 , 2 ]. Here, we report on a case of extraabdominal fibromatosis in the retroperitoneum. Resection was successfully performed, and the patient has been tumor-free for two years after surgery. Case presentation A 43-year-old woman with a history of schizophrenia since 1982, and a history of hospitalization to help the patient to acquire social communication abilities, at the age of 23 presented with slight pain on her left flank and back. In 1995, she was treated with ureterolysis and ureteroureterostomy because of left-sided ureteral obstruction. Histological evaluation of the biopsy revealed benign fibrous tissue proliferations; however, no further evaluation and surgical excision was planned as her mental state was deteriorating. She was put on regular follow-up with computed tomography (CT) scans. In May 2002, she was referred from the psychiatric hospital to our Department of Urology, as the tumor tended to grow. CT scan with contrast enhancement revealed a tumor located between iliocostalis and psoas muscles in retroperitoneal space. The peripheral part of the tumor was enhanced, while the central part did not. The left paravertebral muscles around the tumor showed atrophy. The medial margin of the tumor was deformed by a left transverse process of the second lumber spine, suggesting invasive behavior (Figure 1 ). Coronal magnetic resonance imaging (MRI) demonstrated the tumor to be located beneath the left kidney. The central part of the tumor was found to be of iso/low intensity in the T1-weighted phase and of heterogeneously high intensity in the T2-weighted phase. The marginal part showed very low intensity in both phases (Figure 2 ). In May 2002, needle biopsy was performed and revealed that the tumor consisted of well-proliferated spindle cells rich in collagen fibers, an observation that was inconsistent with the histological pictures made in 1995. In June 2002, resection was performed using a paraspinal approach. Although the tumor strongly adhered to adjacent tissues, including the urinary tract and peritoneum, it was marginally resected, including paravertebral muscles and part of the spine. As the kidney was less affected by the tumor, ablation posed no problem. On gross examination, the cut surface appeared homogeneously gray and glossy (Figure 3 ). Histologically, a uniform proliferation of spindle cells with a moderate amount of collagen fibers led to a diagnosis of extraabdominal fibromatosis in the retroperitoneal space (Figure 4 ). No adjuvant treatment was given and during the two years of follow-up, the patient has remained asymptomatic, with no restrictions of daily living. There were no clues as to recurrence of the tumor in computed tomography. Figure 1 Contrast enhanced computed tomography showing the tumor location between iliocostalis and psoas muscles in retroperitoneal space. Figure 2 Coronal magnetic resonance imaging demonstrating the tumor beneath the left kidney. Figure 3 Gross appearance of the resceted tumor. The cut surface homogeneously appears gray and glossy. Figure 4 Photomicrograph showing the tumor composed of uniform spindled proliferation with moderate account of collagen fibers (hematoxylin and eosin ×400). Discussion Extraabdominal fibromatosis may occur in a variety of anatomic locations; the principle sites of the involvement are the shoulder, chest wall and back, thigh and head and neck. Origin of extraabdominal fibromatosis from any mesenchymal tissue is now well recognized [ 1 , 2 ]. Several authors have reported retroperitoneal fibromatosis in patients with familial adenomatous polyposis (Gardner syndrome) [ 2 , 4 - 6 ], however, solitary occurrence of fibromatosis is very rarely reported [ 1 , 2 , 7 - 9 ]. Our patient did not have a family history and upper gastrointestinal endoscopy, colonoscopy, or opthalmoscopy were normal suggesting that our patient may be negative for the syndrome. The exact histological origin of the tumor remains to be verified. The findings of CT suggested an origin from paravertebral muscles. Interestingly, this assumption was corroborated by a computed tomography performed in April 2000, which revealed that the previous tumor was located intramuscularly Principally, complete resection is the therapy of choice for this type of tumors [ 10 ]. Adjuvant therapy using non steroidal anti-inflammatory drugs (NSAIDs), tamoxifen, interferon, anti-neoplastic agents, radiation, and a combinations of these, have been reported for cases that are difficult to resect [ 1 ], the exact benefit offered by them is not known due to thin literature. Radiation therapy is accepted as an effective treatment after incomplete resection [ 11 , 12 ]. Recently, preoperative radiotherapy was reported to be useful for the local control [ 13 ]. In our case the tumor detection was delayed because the psychiatric status of our patient which has been unstable for several years. As wide resection of the tumor reduces the risk of recurrence, an early diagnosis is required for this type of tumor, which is difficult as most of these patients are asymptomatic. While the silent area contains several vital organs, extraabdominal fibromatosis should be considered for the differential diagnosis for such a lesion. Competing interests The authors declare that they have no competing interests. Authors' contributions AKik and AKid performed the operation, are responsible for the clinical work and helped with the preparation of the manuscript. TK is the orthopedic consultant and helped with the preparation and editing of the manuscript. TH coordinated and drafted the manuscript. All authors read and approved the final manuscript.

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529447

Validity of the Clock Drawing Test in predicting reports of driving problems in the elderly

Background This study examined the use of the Folstein Mini Mental Status Exam (MMSE) and the Clock Drawing Test (CDT) in predicting retrospective reports of driving problems among the elderly. The utility of existing scoring systems for the CDT was also examined. Methods Archival chart records of 325 patients of a geriatric outpatient clinic were reviewed, of which 162 had CDT results (including original clock drawings). T -test, correlation, and regression procedures were used to analyze the data. Results Both CDT and MMSE scores were significantly worse among non-drivers than individuals who were currently or recently driving. Among current or recent drivers, scores on both instruments correlated significantly with the total number of reported accidents or near misses, although the magnitude of the respective correlations was small. Only MMSE scores, however, significantly predicted whether or not any accidents or near misses were reported at all. Neither MMSE nor CDT scores predicted unique variance in the regressions. Conclusions The overall results suggest that both the MMSE and CDT have limited utility as potential indicators of driving problems in the elderly. The demonstrated predictive power for these instruments appears to be redundant, such that both appear to assess general cognitive function versus more specific abilities. Furthermore, the lack of robust prediction suggests that neither are sufficient to serve as stand-alone instruments on which to solely base decisions of driving capacity. Rather, individuals who evidence impairment should be provided a more thorough and comprehensive assessment than can be obtained through screening tools.

Background Assessment of cognitive function pertaining to capacity for safe and independent living among elderly patients is a central responsibility of many geriatric medical clinics and service agencies. Specific concerns pertaining to judgments of driving capacity are also befalling upon the medical profession in primary care settings [ 1 ]. To aid in this task, a number of brief assessment screens are often employed to identify cognitive problems that may be indicative of a range of pragmatic concerns, including driving capacity [ 2 ]. Specifically, results on assessment instruments purported to assess attention, reaction time, and visuospatial abilities are often used to inform clinical judgment about driving capacity in such settings. Two such screening instruments typically used to gauge general cognitive function, and inform questions pertaining to driving capacity specifically, are the Folstein Mini Mental Status Exam (MMSE) [ 3 ] and the Clock Drawing Test (CDT). The MMSE is a widely used cognitive screening tool, due to its brevity, ease of administration, and relative breadth [ 4 ]. Numerous studies over the past 40 years have supported its utility as a valid and reliable indicator of general cognitive function [ 5 ]. The MMSE consists of 30 items comprising subscales assessing orientation, word registration, attention (via a serial sevens or spelling task), word recall, and language. Additionally, a figure copy exercise is included to examine visuospatial abilities. The CDT is hypothesized to assess more specific aspects of planning, organization and visuospatial skill. Directions for completing the CDT involve asking a patient to draw the face of a clock, including the numbers, and then to place the hands to designate a certain time, such as "ten minutes after eleven." Although different scoring templates for the CDT exist, most often code for features such relative size, spacing and placement of numbers or hands, disorganization, perseveration, completeness, and other potential errors that are hypothesized to indicate cognitive impairment [ 6 - 8 ]. In addition to the MMSE, results on the CDT are often used in clinical settings to inform clinical impressions pertaining to whether or not patients are impaired to such an extent that they should not be driving [ 9 ]. Although empirical reviews note that performance on the CDT should only be examined in conjunction with other assessments in this regard, anecdotal evidence also suggests that the CDT is often used as a stand-alone instrument to inform judgments of driving capacity, in both medical and non-medical settings. Despite this apparent widespread use, there appears to be a dearth of research addressing the validity of the CDT for detecting driving impairment. Although a small number of studies exist that suggest CDT scores may relate to driving problems, the size of this literature base coupled with methodological concerns indicate a need for further research. For example, one study [ 10 ] examined the effectiveness of the CDT and MMSE, in addition to the Trail Making Test, Part A [ 11 ] and a visual acuity test, in predicting driving ability as judged by a driving instructor after participants completed a road test. A discriminant function analysis indicated that the set of test scores and participant age correctly identified 80% of drivers judged to be impaired, and 85% of drivers judged not to be impaired, according to driving instructor assessments. The authors reported that the discriminant model did not include the MMSE, however, because it did not add significant discriminatory power. The authors then suggested that the overall battery may be useful as a screening instrument in primary health care settings for detecting potential problems in driving that would warrant further examination. Separate univariate data on the predictive power for each of the separate instruments, however, was not provided. Additionally, the authors incorporated a 4-point scoring system for the CDT that was created for the study and differs from scoring systems used in other studies. Furthermore, given that only the component instruments are typically used in practice as opposed to the more extensive batteries advocated, the unique predictive power of the CDT warrants further investigation. Additional evidence for the potential utility of the CDT in predicting driving behaviors is provided in an examination of neurophysiologic phenomena related to caregiver reports of driving impairment in 79 individuals with Alzheimer's disease [ 12 ]. Single photon emission computerized tomography was incorporated to examine brain function. Additionally, scores on the MMSE, CDT, and caregiver ratings of driving ability were analyzed. CDT scoring was based upon a 5-point system that was constructed for the study. Results indicated that MMSE scores did not significantly differ between individuals based upon driving ability, but that CDT scores were predictive of driving impairment based upon level of impairment and whether participants were instructed to simply copy an existing clock, or construct their own according to specific directions. Furthermore, imaging also indicated that level of driving impairment related positively to changes in cortical function. These authors hypothesized that cognitive tests assessing visuospatial abilities and executive function may thus show greater discriminative power between driving impaired and non-impaired subjects than MMSE scores, which may be impacted to a greater extent by other non-relevant verbal tasks. The validity of the scoring system constructed for the CDT in comparison to other scoring systems, however, was not further explored. A pilot study examining the comparability of simulated driving tests in predicting actual driving problems also suggested that CDT scores may be significant predictors [ 3 ]. A small sample of nine older adults was incorporated, four of whom were classified as cognitively impaired based in part on abnormal CDT and MMSE scores. It was found that simulated driving tasks correlated moderately with actual driving problems across the groups. No data was provided, however, on the extent to which the CDT or MMSE uniquely predicted impairment. Given the typically low rate of follow-up for patients referred for more formal driving assessments, it would be beneficial to further investigate the relations between scores on the CDT and reports of actual driving problems. Furthermore, the predictive power of the CDT alone and in conjunction with other assessment tools in predicting reported driving problems has yet to be fully assessed. Additionally, previous studies examining CDT scores and driving behaviors have employed markedly small sample sizes, warranting future research with greater numbers of participants. Finally, previous studies differ in terms of what, if any, scoring systems were used to score the clock drawings. Thus, further investigation of the comparability of different scoring systems is needed. To address these concerns, this study explored the relations of patient scores on the CDT and MMSE to patient or family reports of driving problems. In so doing, the utility and comparability of three scoring systems for the CDT that are commonly used by researchers and practitioners, namely the Shulman et al. [ 6 ], Sunderland et al. [ 7 ], and Wolf-Klein et al. [ 8 ]systems, were also examined. Specifically, the Shulman system incorporates a 1–6 rating scale, where higher scores indicate higher levels of impairment. Conversely, scores on the Wolf-Klein and Sunderland systems range from 1–10, with lower scores indicating greater levels of impairment. Although specific criteria differ, each system codes for elements pertaining to spacing, organization, and comprehension of the task, among other criteria. Exploratory analyses also were conducted to examine the predictive utility of the CDT and MMSE in predicting whether driving problems, namely accidents or near misses, were reported. Further analyses examined whether linear relationships existed between CDT and MMSE scores and the reported number of such incidents. Finally, regression tests examined whether the CDT and MMSE uniquely predicted the number of reported incidents. Methods IRB approval was obtained for the study, and data was collected from archival records of patients seen over a 10-year period at a geriatric assessment center of a general teaching hospital in the Midwest. The center operated as a full-service outpatient clinic, where new patient assessments included a full medical and psychosocial history. This history included patients' and collateral others' reports of driving behaviors within the past year, including whether patients were currently driving or had recently stopped driving within the past year, and number of driving accidents or near misses. The data was often collected during the initial intake assessment, when both the patient and available family members or caregivers were interviewed by a geriatrician, social worker, and/or a nurse specialist. In addition, the MMSE and CDT were typically administered to patients to assess cognitive functioning. Chart records did not clarify whether the reported driving problems were acknowledged by the patient or caregiver, nor the extent to which any discrepancies existed, but rather only reported the number of incidents. The content of the incidents was also not always documented, but examples that were provided typically included crashes or minor accidents for which the patients were at fault. Nevertheless, despite the subjectivity inherent to such reports, it was the intent of these authors to remain true to the figures documented in the patient charts. Indeed, given that medical professionals typically must rely to some extent on subjective reports of patients or caregivers during intake evaluations to inform initial judgments about patient safety, it was decided that incorporation of such data in the present study would nonetheless be useful. Data was obtained from charts of 325 patients, including 162 original clock drawings that were scored according to the systems provided by Shulman et al. [ 6 ], Sunderland et al. [ 7 ], and Wolf-Klein et al. [ 8 ]. Two advanced students in psychology were trained in each of the three scoring systems, and subsequently scored the clocks independently of each other and blinded to information about driving. MMSE scores, driving status, and reports of driving problems were also coded for subsequent analyses. The initial sample consisted of 81 men and 242 women (gender data was unavailable for 2 individuals). Of these, 287 (88.3%) were Caucasian, 34 (10.5%) were African American, and one individual was Asian American. Ethnicity data was not available for the other three individuals. The mean patient age was 79.75 ( SD = 6.67), and ranging from 58 to 99 years of age. As is typical of many outpatient geriatric populations, there was a range in type and severity of presenting concerns, with some patients reporting relatively few problems and others evidencing diagnoses of vascular dementia, Alzheimer's disease, or depression in addition to other health concerns. Of these, concerns due to cognitive function predominated; approximately 60% of the patient sample was referred to the clinic for evaluation of memory loss, cognitive decline, or dementia. MMSE data was available for 311 patients; of these, 159 also had CDT data sufficient for analysis. Of the 162 charts that had CDT data, only 3 did not also have MMSE data. Results and discussion The raters' corresponding CDT scores for each scoring system correlated above 0.70, suggesting adequate inter-rater correspondence. The corresponding scores for each scoring system were then averaged to create three composite scores for each clock drawing, one for each scoring system. Descriptive data pertaining to scores for the overall sample on the MMSE and CDT is provided in Table 1 . Table 1 Descriptive statistics for overall sample scores on cognitive measures Test N Mean SD Range MMSE 311 21.51 6.15 0–30 CDT (Shulman Score) 162 3.87 1.25 1–6 CDT (Wolf-Klein Score) 162 6.58 2.10 1–10 CDT (Sunderland Score) 162 6.35 2.49 1–10 Initial exploratory t -tests were conducted to examine whether CDT scores and MMSE scores differed between individuals who had been currently or recently driving, versus those who had not been reported to be driving for a more extensive time period. In each case, current and recent drivers evidenced better scores on all of the cognitive measures than individuals who had not been driving. Results for these analyses are provided in Table 2 . Table 2 Mean differences in CDT and MMSE scores based on driving status Variable N Mean SD t df MMSE Score Currently or Recently Driving 114 24.32 4.87 6.41** 305 Not Currently driving 193 19.98 6.18 Shulman Score Currently or Recently Driving 61 3.39 1.28 -3.94** 157 Not Currently driving 98 4.16 1.15 Wolf-Klein Score Currently or Recently Driving 61 7.26 1.83 3.29** 157 Not Currently driving 98 6.18 2.13 Sunderland Score Currently or Recently Driving 61 7.18 2.28 3.28** 157 Not Currently driving 98 5.89 2.49 Note. **p < .01. Further analyses examined whether CDT or MMSE scores predicted the presence of reported driving problems among individuals who had been current or recent drivers. Patients who had not been driving for a longer period of time were excluded from the analyses, since no driving problems would have been reported as a function of not driving. Specifically, t -tests were incorporated to examine whether CDT and MMSE scores differed among individuals for whom driving problems had been reported, versus those with none. Drivers with reported problems evidenced significantly lower MMSE scores, but no significant differences were obtained for CDT scores. Nevertheless, the trends for the overall mean differences on CDT scores, although small, were in the same direction as the findings for the MMSE. Specifically, in every case the CDT scores for each scoring system were worse for drivers with reported problems than those with none. Overall, these results suggest that the presence of driving problems may have been reflective of greater levels of cognitive impairment, although the overall differences reflected in CDT scores were nonetheless very small in magnitude. These results are detailed in Table 3 . Table 3 Mean differences in CDT and MMSE scores based on presence of reported driving problems among current or recent crivers Variable N Mean SD t df MMSE Score Did Report Problems 51 23.08 6.03 -2.44* 112 Did Not Report Problems 63 25.32 3.41 Shulman Score Did Report Problems 27 3.57 1.35 1.03 59 Did Not Report Problems 34 3.23 1.22 Wolf-Klein Score Did Report Problems 27 7.09 2.25 -.64 59 Did Not Report Problems 34 7.40 1.43 Sunderland Score Did Report Problems 27 6.72 2.64 -1.41 59 Did Not Report Problems 34 7.54 1.91 Note. *p < .05. Next, the linear relations for both CDT and MMSE scores in predicted the number of reported problem incidents were examined. Specifically, correlation coefficients were calculated separately for number of reported accidents or near misses, and scores on the CDT and MMSE. Patients who had not been currently or recently driving were excluded from the analysis, since no problems would have been reported if they had not been driving. The number of reported incidents correlated significantly and positively with the level of cognitive impairment as measured by MMSE and CDT scores. Additionally, each of the CDT scoring systems appeared to evidence similar predictive utility, as they correlated highly (above 0.80). Means, standard deviations, and correlations for these variables are provided in Table 4 . Given that not all patient charts necessarily contained all of the requisite MMSE and CDT data, cases that were missing data were excluded from some of the cells. Thus, the n of the resultant cases is included for each cell. Table 4 Means, standard deviations, and correlations for cognitive measures and reported number of problems among current or recent drivers Variable N M SD 1 2 3 4 1. MMSE Score 114 24.32 4.87 2. Shulman Score 61 3.39 1.28 -.45** (59) 3. Wolf-Klein Score 61 7.26 1.83 .50** (59) -.80** (61) 4. Sunderland Score 61 7.15 2.30 .58** (59) -.82** (61) .83** (61) 5. Reported Number of Driving Problems 116 .62 .90 -.27** (110) .23* (57) -.24* (57) -.27* (57) Note. ** p < .01, *p < .05. The N for each cell is provided in parentheses. Total reported number of driving problems ranged from 0–4 for each patient. Finally, hierarchical regression analyses examined whether MMSE or CDT scores uniquely predicted number of reported accidents or near misses. The non-significant R-squared change term in the second step of each regression indicates that neither the MMSE nor set of CDT scores predicted significant incremental variance. Thus, it appears that the variance in reported accidents or near misses predicted by the MMSE and CDT was redundant. Regression results are provided in Table 5 . Table 5 Hierarchical regression analyses predicting number of reported driving problems from CDT and MMSE scores among current or recent drivers ( N = 54) Regression Criterion and Steps R R 2 F df R 2 change F change Reported Number of Accidents or Near Misses Step 1: CDT Scores .25 .06 1.17 3,51 .06 1.17 Step 2: MMSE Score .31 .10 1.37 1,50 .04 1.92 Step 1: MMSE Score .30 .09 5.19* 1,53 .09 5.19* Step 2: CDT Scores .31 .10 1.37 3,50 .01 .18 Note. *p < .05 Conclusions The results of this study suggest that both the MMSE and the CDT appear to have only limited utility in predicting retrospective reports of driving problems among elderly drivers. The finding that MMSE and CDT scores were worse among patients who had not been currently or recently driving may be due to a number of factors, including the possibility that some individuals may have never driven at all before. Nevertheless, it appears likely that many of these individuals probably had been driving in the past, but may have since stopped due to problems related to cognitive impairment. This assertion is supported by the finding that individuals who had been currently or recently driving at the time of the assessment, and who had lower MMSE scores, were more likely to have had reports of accidents or near misses. Although similar mean tests with the CDT were not significant, it is notable that the direction of the obtained differences for each scoring system of the CDT was consistent with the findings of the MMSE. Furthermore, the relatively modest n -sizes within each cell may have limited statistical power. More robust findings were obtained, however, for the correlations examining number of reported accidents or near misses to CDT and MMSE scores. Among individuals who had been currently or recently driving at the time of assessment, greater levels of cognitive impairment as evidenced by MMSE and CDT scores also predicted greater numbers of reported accidents or near misses. This finding held regardless of which CDT scoring system was incorporated, suggesting that each may have equal utility. Finally, the results of the regression analyses appear to indicate that the predictive power of the CDT and MMSE are somewhat redundant, since neither added significant incremental variance to prediction. Although it is possible that the regressions may have had limited power to detect significant incremental differences due to the relatively small sample sizes, in each case the increment to R-squared was small nonetheless. Thus, it appears that both the MMSE and CDT served as gross assessments of general cognitive function, versus more specific cognitive capacities, in predicting reported numbers of accidents or near misses. Although the current results appear to suggest limited predictive utility for the MMSE and CDT in predicting driving problems, an additional cautionary note is in order. The significant predictive power for each instrument as demonstrated by the magnitudes of the correlation coefficients was nevertheless small. Furthermore, significant predictive utility was not obtained for every test in the current research. Additionally, the use of a retrospective design does not necessarily allow for definitive conclusions about predicting instances of future driving problems. Thus, although poor CDT or MMSE scores appear to indicate greater potential for driving problems, the current data do not support the use of the CDT or MMSE alone in making definitive decisions pertaining to driving competence. Rather, the empirical findings of the current research appear to best support the use of the CDT and MMSE solely as their originally intended purpose as screening tools. Thus, scores evidencing impairment on either of these instruments may indicate a need for driver caution, followed by more comprehensive and extensive assessment of driving capacity on which to base decisions regarding safety. As such, the role and utility of these instruments in predicting driving problems may be more fully understood through future research that incorporates a prospective design, along with a more comprehensive assessment of specific and relevant cognitive skills (like psychomotor speed or executive function) and objective assessment of driving abilities (such as can be obtained through simulated or practice driving situations). Competing interests The author(s) declare that they have no competing interests. Authors' contributions ND, AG, and JM conceived of the study purpose and design. ND, EB, and MM collected, entered, and analyzed the data. ND conducted the literature review and critique, and drafted the manuscript. AG, JM, EB, and MM provided comments on the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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526774

Mental health first aid training of the public in a rural area: a cluster randomized trial [ISRCTN53887541]

Background A Mental Health First Aid course has been developed which trains members of the public in how to give initial help in mental health crisis situations and to support people developing mental health problems. This course has previously been evaluated in a randomized controlled trial in a workplace setting and found to produce a number of positive effects. However, this was an efficacy trial under relatively ideal conditions. Here we report the results of an effectiveness trial in which the course is given under more typical conditions. Methods The course was taught to members of the public in a large rural area in Australia by staff of an area health service. The 16 Local Government Areas that made up the area were grouped into pairs matched for size, geography and socio-economic level. One of each Local Government Area pair was randomised to receive immediate training while one served as a wait-list control. There were 753 participants in the trial: 416 in the 8 trained areas and 337 in the 8 control areas. Outcomes measured before the course started and 4 months after it ended were knowledge of mental disorders, confidence in providing help, actual help provided, and social distance towards people with mental disorders. The data were analysed taking account of the clustered design and using an intention-to-treat approach. Results Training was found to produce significantly greater recognition of the disorders, increased agreement with health professionals about which interventions are likely to be helpful, decreased social distance, increased confidence in providing help to others, and an increase in help actually provided. There was no change in the number of people with mental health problems that trainees had contact with nor in the percentage advising someone to seek professional help. Conclusions Mental Health First Aid training produces positive changes in knowledge, attitudes and behaviour when the course is given to members of the public by instructors from the local health service.

Background Community surveys have shown that the public in many countries have poor mental health literacy [ 1 ]. Many people cannot recognise mental disorders correctly, they differ from mental health professionals in their beliefs about causes and the most effective treatments, and they have stigmatizing attitudes which hinder recognition and appropriate help-seeking. This lack of mental health literacy limits the uptake of evidence-based treatments and leads to lack of support for people with mental disorders from others in the community. To help improve mental health literacy, a Mental Health First Aid training course has been developed. This course uses the first aid model that has been successfully applied to training members of the public to help in accidents and emergencies [ 2 ]. The Mental Health First Aid course is designed to give skills to provide initial help in mental health crisis situations and for on-going mental health problems. The course teaches a five-step approach to first aid: 1. Assess risk of suicide or harm, 2. Listen non-judgmentally, 3. Give reassurance and information, 4. Encourage person to get appropriate professional help, and 5. Encourage self-help strategies. These steps are applied to depression, anxiety disorders, psychosis and substance use disorders. In addition, participants are given specific instruction on how to help in the following mental health crisis situations: a suicidal person, a person having a panic attack, a person who has experienced a traumatic event, and a psychotic person threatening violence. An initial uncontrolled evaluation of the course involved comparing the first 210 participants at the beginning and end of the course, and at 6 months follow-up [ 3 ]. The course was found to produce improvement in ability to recognize a mental disorder in a case vignette, to change beliefs about treatment to be more like those of health professionals, to decrease social distance from people with mental disorders, to improve confidence in providing help to others, and to increase the amount of help actually provided. The next stage in the evaluation of Mental Health First Aid involved a randomized controlled trial with 301 employees of two large government departments [ 4 ]. Participants were assigned to either receive the course immediately or were placed on a wait-list for 5 months and received the training after the trial was completed. The trial found a number of benefits, including greater confidence in providing help to others, greater likelihood of advising people to seek professional help, improved concordance with health professionals about treatments, and decreased social distance from people with mental disorders. A surprising finding was that the course improved the mental health of the participants themselves, even though they were not recruited to have mental health problems and no therapeutic benefit was promised. The mental health benefits of the course had not been assessed in the earlier uncontrolled trial. This study involved an "efficacy" trial in that it was carried out under fairly ideal conditions which permitted rigorous experimental control. There was only one instructor who was the originator of the Mental Health First Aid course and very experienced, the trial was carried out in a workplace setting where employees were allowed time off to participate, the participants were a relatively well educated group of civil servants, and it was possible to randomly allocate participants relatively easily. In order to evaluate the course under more typical circumstances, we have now carried out a second trial. This "effectiveness" trial involved members of the public in a large rural area of Australia, who were taught by trained Mental Health First Aid instructors from the local health service. As in the previous trial, participants who received training were compared to a wait-list control group. Participants were randomized by Local Government Area clusters rather than individually because (1) there might have been contamination of information provided across allocated groups (2) the wait list group might have been difficult to maintain if others in the same locality were seen to be receiving training, and (3) individual randomization in some small communities may not have produced sufficient numbers to run a course. The reason for basing the trial in a rural area is that people living in rural Australia are less likely to receive general practitioner services for common mental disorders and also have more limited access to specialist mental health services [ 5 , 6 ]. There is therefore a greater need to develop community capacity to support those with mental disorders. Methods The details of this trial have been reported according to the CONSORT statement for cluster randomized trials [ 7 ]. Participants Eligible participants were residents of the catchment area of the New South Wales (Australia) Southern Area Health Service who were over 17 years of age, who volunteered for training in response to publicity, who were available over the period of the trial, and who were willing to receive interviews assessing trial outcomes. Participants had to volunteer as individuals rather than as a group (e.g. a whole workplace). Publicity took the form of talks to community groups, newspaper ads, a press release and radio interviews. Eligible clusters were the 16 Local Government Areas (cities or shires) in the catchment area of the Southern Area Health Service in 2003. This catchment is located in south-east New South Wales, runs approximately 370 km from north to south and approximately 160 km from east to west, and had a population of 194,435 in 2001. The Local Government Areas varied from popular coastal areas to farming communities to rural towns and ranged in population size from less than 5000 to over 50,000. Intervention Participants received a nine-hour Mental Health First Aid course, in three weekly sessions of three hours each. Training was administered in the local area in groups of up to 25 participants, with a minimum of 10 participants per course. As documentation of the intervention, there is a lesson plan for each session and a participants' manual containing material that was given to take away [ 2 ]. All instructors were given training and a teaching kit of lesson plans, videos, books, master copies of handouts and a set of transparencies. Educators received a one-week training program in how to conduct Mental Health First Aid courses and subsequent supervision in running a course. They were trained by Betty Kitchener who devised the Mental Health First Aid course. The course teaches how to help people in the crisis situations of being suicidal, having a panic attack, being exposed to a traumatic event, or in an acute psychotic state. The symptoms, risk factors and evidenced-based treatments (medical, psychological, alternative and self-help) for the mental disorders of anxiety, depressive and substance use and psychotic disorders are also taught. Figure 1 shows the five steps of providing mental health first aid taught in the course. Participants received training either immediately (experimental Local Government Areas) or after 6 months on a wait-list (control Local Government Areas). Figure 1 The five steps in providing mental health first aid. Training was administered by educators who were recruited from the staff of the Southern Area Health Service. Expressions of interest to become Mental Health First Aid instructors were sought from staff of the Area Health Service and associated community organisations. Five Mental Health first Aid instructors were recruited from a pool of 10 applicants for these positions. All the instructors had experience in mental health work and also a background in training, working with communities or health promotion work. A project coordinator with experience in mental health and health promotion (Ms Karen Peterson), who was employed to work on the project half time, also trained as an instructor. The same instructors taught courses in each paired Local Government Area, so that this factor did not differ between the immediate and wait-list Local Government Areas. The coordinator monitored a sample of courses taught during the trial to assess fidelity to the lesson plans. A fidelity checklist of topics that had to be covered was developed for each session. Four of the instructors had all three course sessions checked, while one of the instructors only had two sessions checked. The percentage of topics covered correctly was 100% for four of the instructors and 81% for one of the instructors. Objectives The hypotheses were that individuals trained in Mental Health First Aid, when compared to wait-list controls, would have increased knowledge of mental disorders and their treatments, decreased social distance, increased confidence in providing help, and that they would provide greater help to people experiencing mental health problems. Outcomes Outcomes were measured in January–February of 2003 (the pre-test assessment), the courses were run for the intervention group in March–April of 2003, and outcomes were measured again in July–August 2003 (the follow-up assessment). The wait-list control group received courses in September–October 2003, after the follow-up assessment was completed. All outcomes were measured at the individual level by telephone interview. The interview content was based on the questionnaire used in the uncontrolled trial of Mental Health First Aid [ 3 ]. The pre-test interview covered the following: whether the participant had ever experienced a mental health problem (yes/no), whether a family member had ever experienced a mental health problem (yes/no), the participant's confidence in helping someone (five-point scale from 1. not at all to 5. extremely ), contact in the last six months with anyone with a mental health problem (yes/no), how many people, whether any help offered (yes/no), what type of help (open-ended question), recognition of the problem in a case vignette (randomly assigned to be a case of depression or one of schizophrenia), what participant would do to help if they knew the person in the vignette (this "mental health first aid intention" involved the presence or absence of 8 elements, arrived at by a qualitative analysis of a sample of the responses, and added up to give a scorefrom 0–8), ratings of the likely helpfulness of a range of interventions for the person in the vignette (scored to give a scale of percentage agreement with mental health professionals about treatment [ 3 ]), a social distance scale relating to the person in the vignette [ 8 ], whether the participant had had a problem like the one in the vignette, whether a family member had had a problem like the one in the vignette, participant's reason for doing the course, and sociodemographic characteristics of the participant (age, gender, education, non-English speaking background, aboriginality). The follow-up questionnaire was the same as the pre-test questionnaire except that it omitted the sociodemographic questions. All outcomes were measured by a scripted telephone interview administered by professional interviewers. In order to reduce the length of the interview, participants were individually randomly assigned to receive either a depression vignette or a schizophrenia vignette, with the same questions asked in respect to each vignette. The interviewers were provided with an ID, name and phone number of each participant and knew whether they were giving the first or second interview to the participant. While they were not told whether the participant was in the experimental or control group, information about which group they were assigned to was given at the end of the interview script. As far as was practical given the very different sizes of the Local Government Area pairs, the same interviewers interviewed participants in each pair. Sample size determination For power calculations and sample size determination, a conservative assumption was made that the waitlist control group would show improvements, possibly due to increased awareness of mental health issues, of about 50% of that of the experimental group. This corresponds to effect sizes in the range 0.28–0.31 for changes on scales and in the range 0.02–0.04 for changes in identifying the correct diagnosis. Sample size estimates using nQuery Advisor software [ 9 ] indicated that a sample size of 200 participants in each of the two groups would be sufficient to detect differences with power of at least 80% in 2-sided tests at the 0.05 level. Clustering effects of individuals in 16 Local Government Areas involved design effects of unknown magnitude in the analysis. It was assumed that these would be of the order of 20%, so that a total achieved sample sizes of 250 in each group would be sufficient to detect differences with 80% power. Randomization: Sequence generation Randomization to immediate participation or wait-list was at the level of Local Government Area. The Local Government Areas were matched in pairs to have similar population and social characteristics. The variables used for matching were population size, interior vs coastal location, and an index of population education/occupation. The first listed LGA of each pair was assigned to the immediate or wait-list group at random, using the Random Integers option of Random.org [ 10 ] to generate a 1 or a 2 for each pair. For LGA pairs receiving a 1, the first member of the pair received immediate training, while for those receiving a 2 it was the second member of the pair. Each individual participant was randomly assigned a variable (values of 1 or 2) to determine which case vignette they received during their interviews. This was done using the Random Integers option of Random.org [ 10 ]. Those assigned a 1 received the interview based on a vignette of a person who is depressed and those assigned 2 received a vignette of a person with schizophrenia. Randomization: Allocation concealment Allocation was on the basis of cluster. In other words, the participant's Local Government Area determined whether they received immediate or wait-list training. Participants were not informed about their allocation to immediate or wait-list training until the end of their baseline interview. Randomization: implementation Local Government Areas were matched in pairs and Anthony Jorm assigned these randomly to immediate training or wait-list. Participants were not able to attend a class from outside their own Local Government Area. There was a recruitment period for all Local Government Areas which was organized by the coordinator Karen Peterson. The coordinator and the participants who were recruited were blind to the allocation of the Local Government Area during the recruitment period. Anthony Jorm revealed the allocation to Karen Peterson after the recruitment period ended. Karen Peterson then organized class times either immediately or after a waiting period, depending on the allocation of each Local Government Area in the pair. Randomization: Blinding (masking) At the time of the baseline interview, the participants did not know whether they were in an immediate or wait-list Local Government Area. However, interviewers had information at the end of the interview script telling whether the participant was assigned an immediate class or had to wait. Blinding of participants was not possible at subsequent interviews. Participants knew whether or not they had received training. While interviewers were not told the allocation of the participants in subsequent interviews, this might have become obvious during the interview if participants mentioned whether or not they had done the course. Interviewers were given a scripted interview to minimize any bias in the assessment due to knowledge of allocation. Ethics Ethical approval for the study was given by the Australian National University Human Research Ethics Committee and by the ethics committee of the South Western Sydney Area Health Service. Statistical methods For outcomes measured on a numeric scale, the change from pre-test to follow-up was analysed using linear regression. For binary outcomes, individuals scoring the same at pre-test and at follow-up were not used, and for those who changed, the direction of change was analysed as a binary outcome using logistic regression. Standard errors and p-values were adjusted for the cluster design using the Huber-White "sandwich" variance estimator, treating the 16 LGAs as the clusters. Analyses were corrected for differences between the LGA pairs by including this as an 8-level fixed-effect factor in the regression models. Missing data were imputed using best-subsets regression. All analysis was done using Stata version 8.2 [ 11 ]. Results Recruitment and Participant flow Recruitment of participants took place in October and November of 2002. Figure 2 shows the number of participants and clusters at each stage of the trial. Figure 2 Flow diagram of the number of participants and clusters at each stage of the trial. Baseline data Table 1 shows the characteristics of each group at the cluster and individual level. The two groups appear to be well matched in terms of sociodemographic characteristics and in history of mental health problems in self and family. However, there was a significant difference in reason for doing the course, with more people in the control group doing it for work reasons. Table 1 Baseline characteristics for each group given at the individual and cluster levels. Mental Health First Aid group Control group P-value Local Government Area characteristics at baseline Number 8 8 Population size: 1.0 <5,000 3 3 5,000–9,999 2 1 10,000–19,999 1 2 20,000–29,999 1 0 30,000–39,999 1 2 Number of participants in each area (smallest to largest) 9,17,18,29,30,48,100,165 8,9,12,16,28,50,53,161 Individual participant characteristics at baseline Number 416 337 Mean age (years) 47.14 47.97 0.42 Number (%) men 79 (19.0) 57 (16.9) 0.40 Number (%) with university degree 85 (20.6) 81 (24.1) 0.36 Number (%) aboriginal 11 (2.6) 10 (3.0) 0.40 Number (%) non-English speaking background 5 (1.2) 7 (2.1) 0.12 Reason for doing course: 0.011 Relating to workplace/voluntary work 180 (43.3) 188 (55.8) Relating to family/close friends 56 (13.5) 29 (8.6) Relating to own mental health status 20 (4.8) 10 (3.0) Duty as a citizen 49 (11.8) 44 (13.1) Just interested 111 (26.7) 66 (19.6) Note: P-values are adjusted for clustering by Local Government Area Numbers analyzed The data were analyzed by an intention-to-treat approach, with single imputation used for missing data. As shown in Figure 2 , the number of participants analyzed was the same as the number randomly allocated. Outcomes and estimation Tables 2 and 3 show the changes found for the dichotomous and continuous outcome measures respectively and the P-value of the comparison between the Mental Health First Aid and control group on these changes. From pre-test to follow-up a significantly larger percentage of the Mental Health First Aid group than the control group changed from not reporting experiencing a mental health problem to reporting experiencing one, from incorrectly to correctly diagnosing the case vignette and from reporting not offering help to a person with a mental health problem to reporting offering help. The Mental Health First Aid group changed significantly more than the control group in their agreement with health professional about treatment, in the degree of reduction in reported social distance from the person in the vignette and in their confidence in providing help. Table 2 Changes in dichotomous outcome measures. Outcome Mental Health First Aid group Control group OR (95% CI) P-value Mental health problems in self Pre-test 154 (37%) 118 (35%) Follow-up 172 (41%) 118 (35%) Change (95% CI) 4% (2 to 6) 0% (-3 to 3) 0.548 (0.304, 0.986), P = 0.045 Mental health problems in family Pre-test 233 (56%) 183 (54%) Follow-up 277 (67%) 205 (61%) Change (95% CI) 11% (4 to 17) 7% (2 to 11) 0.575 (0.318, 1.037), P = 0.066 Correct diagnosis of vignette Pre-test 282 (68%) 249 (74%) Follow-up 337 (81%) 255 (76%) Change (95% CI) 13% (8 to 19) 2% (0 to 4) 0.311 (0.250, 0.387), P < 0.001 Help offered to person with mental health problem Pre-test 305 (73%) 256 (76%) Follow-up 340 (82%) 270 (80%) Change (95% CI) 8% (4 to 13) 4% (-2 to 10) 0.602 (0.380, 0.953), P = 0.031 Professional help advised to person with mental health problem Pre-test 81 (19%) 71 (21%) Follow-up 104 (25%) 73 (22%) Change (95% CI) 6% (3 to 8) 1% (-4 to 5) 0.734 (0.452, 1.191), P = 0.21 Note: P-values and confidence intervals are adjusted for clustering by Local Government Area Table 3 Changes in continuous outcome measures. Outcome Mental Health First Aid group Control group Treatment effect (95% CI), P-value Agreement with health professionals about treatment Pre-test mean (SEM) 60.55 (3.89) 69.46 (2.18) Follow-up mean (SEM) 74.74 (1.91) 70.81 (2.27) Change (95% CI) 14.19 (9.53 to 18.85) 1.35 (-6.04 to 8.75) 11.77 (5.98, 17.56), P = 0.001 Social distance Pre-test mean (SEM) 8.13 (0.24) 8.06 (0.13) Follow-up mean (SEM) 7.59 (0.17) 7.90 (0.20) Change (95% CI) -0.53 (-0.99 to -0.08) -0.17 (-0.41 to 0.07) -0.26 (-0.49, -0.03), P = 0.032 Mental health first aid intention Pre-test mean (SEM) 1.81 (0.04) 1.88 (0.04) Follow-up mean (SEM) 1.83 (0.03) 1.85 (0.07) Change (95% CI) 0.02 (-0.11 to 0.15) -0.03 (-0.15 to 0.08) 0.06 (-0.00, 0.12), P = 0.066 Confidence in providing help Pre-test mean (SEM) 3.13 (0.08) 3.17 (0.07) Follow-up mean (SEM) 3.39 (0.05) 3.21 (0.07) Change (95% CI) 0.27 (0.11 to 0.42) 0.04 (-0.02 to 0.11) 0.21 (0.10, 0.33) P = 0.001 Number of people in contact with who had mental health problem Pre-test mean (SEM) 3.97 (0.31) 4.56 (0.20) Follow-up mean (SEM) 3.89 (0.30) 4.34 (0.29) Change (95% CI) -0.08 (-0.64 to 0.49) -0.22 (-0.83 to 0.40) 0.22 (-0.18, 0.63) P = 0.25 Note: Standard errors of the mean (SEM), confidence intervals and P-values are adjusted for clustering by Local Government Area The intraclass correlations for the continuous outcomes were: for agreement with health professionals about treatment, 0.15 (95% confidence interval 0.01, 0.29); for number of people in contact with that had a mental health problem, 0.02 (0, 0.06); for confidence in providing help, 0.03 (0, 0.07); for mental health first aid intention, 0.002 (0, 0.02); and for social distance, 0.04 (0, 0.08). Thus for all but one outcome, the correlation was small, justifying our assumption of a modest design effect. Adverse events Given that an educational intervention was evaluated with a non-clinical sample, there was no justification for a systematic inquiry into adverse events. Informally, no adverse events were reported. Discussion This study has found that the Mental Health First Aid training produced a number of significant changes in participants compared to a wait-list control group. A number of changes related to how people responded to a vignette of a person with either depression or schizophrenia. We found that there was greater recognition of the disorders in a vignettes, increased agreement with health professionals about which interventions are likely to be helpful, decreased social distance towards the people portrayed in the vignettes. These changes were seen equally with both vignettes. There was also a non-significant trend for those in the trained group to have more ideas for how to help the person in the vignette if it had been someone they knew. Other outcomes with significant changes related more directly to the provision of mental health first aid. There was increased confidence in providing help to others and an increase in help actually provided. There was no change in the number of people with mental health problems that trainees had contact with or in the percentage advising someone to seek professional help. One potential concern of Mental Health First Aid training is that it will lead to over-diagnosis of life problems as mental disorders. In previous trials we have found no evidence that the training affects the perception that the participant or their family have mental health problems [ 3 , 4 ]. By contrast, in the present study there was a significant increase in the percentage who perceived themselves as having a mental health problem and a non-significant trend for an increased perception of family members as having mental health problems. However, in absolute terms the changes were not so great as to be a concern and may, in fact, reflect accurate re-labelling. These findings are similar to those of the earlier efficacy trial. However, the courses were taught by instructors who were not the originators of the Mental Health First Aid program under conditions which more closely approximate those that are typical in practice. The findings are therefore more generalizable than those reported previously. While the more typical conditions of this trial are an advantage for generalizability, they produced greater practical difficulties in running the trial. An important weakness was that attendance data on participants were not collected by some of the instructors. We are therefore uncertain what proportion of the participants received the complete training course. A similar problem was determining the adherence of the instructors to the curriculum. We were able to carry out some formal observation of the instructors'adherence to a list of topics covered by the curriculum and found 100% adherence for most of the instructors, but one had only 81% adherence. Another limitation of this study is that we did not directly measure the mental health of participants. In the earlier trial, we unexpectedly found a mental health benefit and this requires replication. The reason that a mental health measure was not included was that we did not have the results of the earlier trial at the time we designed this one. Another factor was the limited time available in the telephone interviews used to assess outcomes. We used an intention-to-treat approach to the data. Whereas many trials use a last observation carried forward approach to handle missing post-test data, we used data imputation by best-subsets regression. This approach is likely to give better estimates than conventional approaches to missing data even when the missing-at-random assumption is not met [ 12 ]. Since this and the earlier trials were started, the Mental Health First Aid course has been extended from 9 to 12 hours on the basis of consistent requests from trainees for a longer course. The longer course does not add new content, but rather extends the time available to deal with each topic. We have yet to evaluate whether this extension adds to the effectiveness of the training. Conclusions A nine-hour Mental Health First Aid training produces positive changes in knowledge, attitudes and behavior when the course is given to members of the public by instructors from the local health service. This finding shows that the effects of the course are generalizable beyond its originators and when run under typical conditions. Competing interests BAK and AFJ were the developers of the Mental Health First Aid course. Authors' contributions AFJ was involved in securing funding for the study, had a major role in the design of the study, co-developed the evaluation questionnaire, contributed to the data analysis and had a major role in writing the manuscript. BAK was involved in securing funding for the study, developed and taught the Mental Health First Aid Instructor course, had a role in the design of the study, co-developed the evaluation questionnaire, organized the outcome assessment and had a minor role in writing the manuscript. ROK was involved in securing funding for the study, had a role in the design of the study, had a major role in planning and managing the trial's implementation in its initial stages, recruited and supervised the study staff, established and maintained organisational support in the Southern Area, and had a role in the writing of the manuscript. KBGD had a major role in the data analysis and a minor role in writing the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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Identification of hip fracture patients from radiographs using Fourier analysis of the trabecular structure: a cross-sectional study

Background This study presents an analysis of trabecular bone structure in standard radiographs using Fourier transforms and principal components analysis (PCA) to identify contributions to hip fracture risk. Methods Radiographs were obtained from 26 hip fracture patients and 24 controls. They were digitised and five regions of interest (ROI) were identified from the femoral head and neck for analysis. The power spectrum was obtained from the Fourier transform of each region and three profiles were produced; a circular profile and profiles parallel and perpendicular to the preferred orientation of the trabeculae. PCA was used to generate a score from each profile, which we hypothesised could be used to discriminate between the fracture and control groups. The fractal dimension was also calculated for comparison. The area under the receiver operating characteristic curve ( A z ) discriminating the hip fracture cases from controls was calculated for each analysis. Results Texture analysis of standard radiographs using the fast Fourier transform yielded variables that were significantly associated with fracture and not significantly correlated with age, body mass index or femoral neck bone mineral density. The anisotropy of the trabecular structure was important; both the perpendicular and circular profiles were significantly better than the parallel-profile ( P < 0.05). No significant differences resulted from using the various ROI within the proximal femur. For the best three groupings of profile (circular, parallel or perpendicular), method (PCA or fractal) and ROI ( A z = 0.84 – 0.93), there were no significant correlations with femoral neck bone mineral density, age, or body mass index. PCA analysis was found to perform better than fractal analysis ( P = 0.019). Conclusions Both PCA and fractal analysis of the FFT data could discriminate successfully between the fracture and control groups, although PCA was significantly stronger than fractal dimension. This method appears to provide a powerful tool for the assessment of bone structure in vivo with advantages over standard fractal methods.

Background The NIH Consensus Statement defines Osteoporosis as "a skeletal disorder characterised by compromised bone strength predisposing to an increased risk of fracture" [ 1 ]. Bone strength was defined as "the integration of two main features: bone density and bone quality". Currently, clinical diagnosis is based solely on bone mineral density (BMD) in accordance with the World Health Organisation guidelines [ 2 ]. Previous studies, however, have found that trabecular bone structure also plays a significant role in determining bone strength [ 3 - 5 ] with BMD explaining only 60 to 80 % of the variability in mechanical resistance [ 6 ]. Trabecular bone structure is visible on standard pelvic radiographs and many attempts have been made to quantify the quality of the structure and assess its relationship to osteoporosis and BMD. These range from visual scoring systems, such as the Singh index [ 7 ], through to sophisticated computerised methods based on fractals [ 8 - 10 ] and other image processing methods [ 11 - 13 ]. A review of the literature suggests that fractal analysis has been a method of choice in recent years for the analysis of trabecular bone structure in CT scans [ 14 , 15 ], MRI [ 16 ], histology [ 17 ] and radiographs [ 18 - 21 ], although it has not been established categorically that it is preferable to other methods of texture analysis [ 22 , 23 ]. By reducing all the information in the image to one descriptor, the fractal dimension [ 24 ], a large part of the information is lost. The Fourier transform of an image expresses the information in the image in terms of spatial frequencies rather than distances. Various methods can be applied to extract information from the Fourier transform [ 25 ], including the fractal dimension [ 24 ]. However such methods have not been fully exploited for analysing bone structure [ 8 , 26 - 30 ]. In this study we investigate the use of Fourier transforms and Principal Components Analysis to generate a mathematical model of the data which can be used to help classify individuals according to the presence or absence of a hip fracture. Principal component analysis (PCA) [ 31 ] is a data reduction technique that has been applied in many fields of study, including investigation of gene expression [ 32 ], development of an electronic nose [ 33 ] and tracing of the evolutionary changes in fish morphometry [ 34 ]. It describes data in terms of a small number of orthogonal, linearly independent components which contain the majority of the information. PCA has no preconditions, such as relying on the data to fit a normal or fractal distribution, but builds a mathematical model based on the correlations present in the data. An eigenanalysis of the correlation or covariance matrix is used to perform PCA. The resulting components are then selected in order of the amount of variance they account for, enabling an efficient mapping of the data. As the first few components account for the vast majority of the variance in the original data, they can be selected for analysis whilst the remainder are discarded as 'noise'. In this way, the number of variables can be greatly reduced whilst maintaining the information present in the original data. In this pilot study we used these methods to investigate the similarities and differences between trabecular bone structure in fracture and control groups using standard radiographs of the proximal femur. Methods Study data A set of digitised standard pelvic radiographs was available from a previous investigation into the morphology of the proximal femur [ 35 ]. These radiographs were taken from an earlier study [ 36 ], that had examined three groups (osteoporotic, osteoarthritic and control) of age matched, postmenopausal women (30 subjects per group). Subjects with osteoarthritis were excluded from the present study. All patients had undergone a scan of the unfractured hip by dual-energy x-ray absorptiometry (DXA) using a Norland XR-26 scanner (CooperSurgical Inc, Trumbull, CT). The controls had had their left hip scanned. All patients and controls had had a pelvic antero-posterior radiograph recorded within a year of the DXA scan. We used those radiographs and the femoral neck BMD (Neck-BMD) data in the current study. A data set of 50 digitised radiographs was available comprising 26 hip fracture patients (HIP) and 24 controls (CNT). The radiographs were digitised, using a Howtek MultiRAD 850 scanner (Howtek, Hudson, New Hampshire) at a resolution of 584 dpi (44 μm per pixel) and a depth of 12 bits. The age, height and weight of each subject were also recorded. Region selection Five regions of interest (ROIs) were selected relative to the principal trabecular systems in locations known to be related to hip fracture via the Singh index [ 7 ] and BMD analysis [ 37 ]. To ensure reproducibility, their locations were determined in relation to the centre and angle of the narrowest part of the femoral neck and the centre and radius of the femoral head on each image, as shown in Figure 1 . Figure 1 Regions of interest. Displays the five regions of interest, upper femoral head (UH), central femoral head (CH), upper femoral neck (UN), Ward's triangle area (WA) and the lower femoral neck (LN) used for analysis. Points A to G are determined by the femoral head and neck and used to locate the ROIs. Points A and E mark the femoral neck width. Points B, C and D lie at 1/4, 1/2 and 3/4 along this line. Point F is the centre point of the femoral head, point G at 1/2 the radius of the femoral head at an angle of 45 degrees to the neck width, 135 degrees to the neck shaft, shown as a dashed line through point C. Each ROI was 256 × 256 pixels (11.3 mm square), to enable use of the fast Fourier transform, and were selected as follows. The upper region of the head (UH) lies on the upper part of the principal compressive trabeculae, the central region of the head (CH) is at the intersection of the principal compressive and tensile trabeculae, the upper region of the neck (UN) lies on the principal tensile trabeculae, the lower region of the neck (LN) is at the base of the principal compressive trabeculae and finally Ward's triangle (WA) which lies between these structures. The points and regions were identified using a macro written for Image Pro Plus software (version 4.1.0.0, Media Cybernetics, Silver Spring, Maryland). The femoral head was described by a best-fit circle, calculated from a series of manually marked points around the outline of the femoral head. Between 15 and 20 evenly spaced points were used to describe the outline, depending on the size of the head. The radius and centre (marked as F in Figure 1 ) of the femoral head were then taken from this circle. The narrowest part of the neck (neck-width) was determined using two automatic edge traces, marking the upper and lower outlines of the femoral neck. The first point and the direction for each trace were marked manually; the edge of the neck could then be identified automatically by the software. The neck width (A – E in Figure 1 ) was calculated by finding the smallest Euclidean distance between the traces. The centre of the neck was located at the mid-point of this line (point C) and the axis of the femoral neck was taken to be a line perpendicular to this through the centre of the neck (dashed line). The top right corner of the WA region was located at the midpoint of the neck width (point C). Points B and D were placed 25% and 75% of the way along the neck width and used as the midpoints of the UN and LN regions respectively. Point F, the centre of the femoral head marked the centre of the CH region and point G, the centre of the base of the UH region. Point G was placed one half of the femoral head radius above point F, at a 45-degree angle to the neck width (A-E). Region analysis Analysis was performed using Matlab software (version 6.1.0, MathWorks Inc, Natick, Massachusetts). A fast Fourier transform was generated for each ROI and three profiles were generated using data from the power spectrum. Firstly a global or circular profile (CircP) was generated, composed of the magnitude at each spatial frequency averaged across all angles, resulting in a profile with 128 data points. To create this profile, each pixel in the Fourier transform was assigned to the integer spatial frequency that most closely matched its' distance from the zero'th component. The angle of preferred orientation was calculated by finding the angle of the maximum value in the power spectrum for the first 25 spatial frequencies [ 38 ]. The maximum value over this range relates to the dominant texture orientation within the image, the trabecular structure. As data in the frequency domain relate to features in the spatial domain rotated by 90°, the median of the values plus 90° was taken as the angle of preferred orientation for each image. Due to the symmetry of the Fourier power spectrum, angles were only calculated between 0° and 180°, rather than 0° and 360°. Two more profiles were then generated, parallel with (ParP) and perpendicular to (PerP) the angle of preferred orientation. In this case the average value was calculated at each spatial frequency from all points lying within ± 5° of the desired angle (Fig. 2 ). Figure 2 Profile generation. (A) Shows a typical region of interest (contrast enhanced for visualisation) showing the trabecular bone structure, in this case aligned approximately 22° to the vertical. (B) The central section of the FFT (128 × 128 pixels). The horizontal and vertical axes have been marked with a mid-grey tone to indicate that they have been excluded from the angle calculation. The bright strip at the centre (running from top left to bottom right) shows the preferred orientation of the trabeculae. Angles calculated from the Fourier power spectrum correspond to the same angles in the spatial domain, rotated by 90°. (C) The pixels with the maximum values are marked using white squares for the first 25 spatial frequency values of the Fourier power spectrum. The median angle, lying 21.8° from the horizontal is shown by a dashed white line. (D)_The regions used to generate the parallel (shaded black) and perpendicular (shaded white) profiles, based on the orientation of the trabecular structure. Principal component analysis Principal component analysis [ 31 ] was used to model statistically the shape of each set of profiles (parallel, perpendicular and circular). This was performed using an eigenanalysis of the correlation matrix. The eigenvectors then become the principal components and are selected in order, depending on their eigenvalue. The eigenvalues are associated with the components in decreasing order, the largest eigenvalue is associated with the first component and the smallest with the last. In order to choose the number of components for analysis, a scree plot [ 31 , 39 ] was generated by plotting the eigenvalues (representing the proportion of variance described by each component) against the component number (Figure 3 ). In each case, the first few principal components were selected for analysis using the scree test [ 39 ] to find an 'elbow' in the slope of the plot. This is used as a threshold between the components that contained useful information, which were then used as input variables for further analysis, and those that could be attributed to noise. Figure 3 Scree plot. Example of a scree plot from the perpendicular profile. The first component typically accounts for the largest amount of variance. The components are chosen to the left of an 'elbow' in the plot. Here components 1 to 5 are included in the analysis as they lie before the 'elbow' at point6 (eigenvalue = 1.63). Fractal analysis Fractal analysis was performed on each profile using a method similar to the Fourier transform technique described by Majumdar et al [ 40 ]. The average power spectrum of the circular profile was plotted on a log-log scale, three approximately linear regions were defined and the gradient ( slope ) of a straight line fitted to each region was found; slopeA , a 'coarse' slope, where the log of the spatial frequency is less than or equal to 1.0, slopeB a 'medium' slope, where the log of the spatial frequency lies between 1.0 and 1.75 and slopeC , a 'fine' slope where the log of the spatial frequency is above 1.75. The fractal dimension was calculated for each slope using the formula suggested by Majumdar et al [ 40 ] Statistical analysis Stepwise discriminant analysis was used to select principal components that could be combined to build a linear classifier. If the stepwise procedure failed to select any components, the most accurate of the individual components was chosen. The same procedure was used to discriminate between the groups using the fractal dimension. Measurement of the area under the ROC curve was used to compare the classifiers built using the discriminant analysis [ 41 ]. A three way ANOVA was applied in order to determine whether there were significant differences between the performance of classifiers depending on the type of analysis, the profile used or the region analysed. Pearson product moment correlation was applied to examine the relationship with age, BMI and Neck BMD for the strongest classifiers. A one-way ANOVA was used to test for significant differences in the performance of the slopes from each spatial frequency band used in the fractal analysis. T-tests, correlation and ANOVA were performed using SigmaStat (version 2.03, SPSS Science, Chicago). Principal component analysis, discriminant analysis, and measurement of the area under the ROC curve were calculated using SPSS (version 10 SPSS Science, Chicago). Results There were no significant differences between the age, height, weight or body mass index (BMI) of the fracture and control groups (Table 1 ). As expected femoral neck-BMD was significantly lower in the fracture group in comparison to the control group ( P = 0.001). Table 1 Summary of anthropometric variables for the fracture and controls groups. Mean and standard deviation (SD) of the age, height, weight, BMI and BMD of the fracture and control groups. P values were obtained from a two-tailed t-test. Variable Control Group (n = 24) Fracture Group (n = 26) Mean SD Mean SD P Age, years 69.1 6.5 69.2 6.3 0.97 Height, cm 158.6 7.1 157.1 0.4 0.38 Weight, kg 63.4 9.5 61.0 9.0 0.38 Body Mass Index, kg/m 2 25.2 3.2 24.8 4.1 0.72 Femoral neck BMD (g cm -2 ) 0.70 0.11 0.604 0.066 0.001 The Receiver Operating Characteristic (ROC) curve is a plot of True Positive Fraction v False Positive Fraction (or Sensitivity v 1 – Specificity). The area underneath the curve ( A z ) represents the performance of the classifier ranging from a value of 0.5 if it is no better than chance to 1.0 for a perfect discriminator. Table 2 shows A z for PCA analysis by region for the circular, perpendicular and parallel profiles respectively, discriminating fracture and control cases. A wide range of values was observed (overall mean 0.70, standard deviation 0.11). Some were little better than chance ( A z = 0.5) (mostly derived from the parallel profile) and the strongest ones were from the perpendicular profiles. The 5 largest areas under the ROC curve were obtained by PCA of the perpendicular profile of the lower neck, upper and central head regions (Table 3 ) ( A z = 0.93, 0.84 and 0.84 respectively), followed by PCA analysis of the circular profile in the upper head region ( A z = 0.76) and, finally, fractal analysis of the parallel profile in the upper neck region ( A z = 0.75). Femoral neck BMD lay between the third and fourth best texture measures ( A z = 0.79 95% CI = 0.66 – 0.91). Plots of the ROC curves for the strongest combinations of image analysis classifier are shown in Figure 4 . Table 2 Classification accuracy for each region-profile combination. Area under the ROC curve for principal component analysis of each profile by region of the femoral neck. Analysis using three-way ANOVA found that the area under the ROC curve was significantly higher in the perpendicular profile than in the parallel profile. ( P < 0.05) Region Circular (95% CI) Parallel (95% CI) Perpendicular (95% CI) Upper head 0.76 (0.63 – 0.89) 0.57 (0.41 – 0.73) 0.84 (0.73 – 0.95) Central head 0.59 (0.43 – 0.75) 0.56 (0.40 – 0.73) 0.84 (0.72 – 0.95) Upper neck 0.72 (0.58 – 0.86) 0.72 (0.57 – 0.86) 0.67 (0.52 – 0.82) Wards triangle 0.74 (0.61 – 0.88) 0.61 (0.45 – 0.76) 0.71 (0.56 – 0.86) Lower neck 0.71 (0.56 – 0.85) 0.55 (0.39 – 0.71) 0.93 (0.87 – 1.00) Table 3 The best five classifiers: Area under the curve and correlation with BMD, age and BMI. Area under the ROC curve ( A z ) for each of the best 5 classifiers and the correlation with age R age , femoral neck BMD (R BMD ) and body mass index (R BMI ) and associated significance values ( P ). Analysis Profile ROI A z (95% CI) R BMD ( P ) R age ( P ) R BMI ( P ) PCA PerP LN 0.93 (0.87 – 1.00) 0.09 (0.55) 0.14 (0.34) -0.08 (0.58) PCA PerP UH 0.84 (0.73 – 0.95) 0.09 (0.52) -0.17 (0.24) -0.03 (0.86) PCA PerP CH 0.84 (0.72 – 0.95) 0.06 (0.70) 0.27 (0.055) -0.11 (0.46) PCA CircP UH 0.76 (0.63 – 0.89) -0.16 (0.28) -0.15 (0.29) 0.07 (0.62) Fractal ParP UN 0.75 (0.61 – 0.89) -0.30 (0.034) 0.25 (0.081) -0.04 (0.78) Figure 4 Comparison of ROC curves. Comparison of the ROC curves for the strongest classifier from the combination of (A) PCA analysis of the perpendicular profile (Lower neck region), (B) PCA analysis of the circular profile (Upper head region) and (C) Fractal analysis of any profile (Upper neck region). Table 3 also shows the correlations between the top five classifiers with age, BMI and Neck-BMD. No significant correlations were found between any of these classifiers and either age or BMI and, for the top three, there was also no significant correlation with Neck-BMD ( P > 0.05). The fifth placed classifier, fractal analysis of the parallel profile in the upper neck region, was the only one significantly associated with Neck-BMD ( P = 0.034). A three-way analysis of variance was used to examine differences in performance due to the region, profile or type of analysis used. It showed that overall PCA analysis performed significantly better than fractal analysis ( P = 0.019) and that analysis of both the perpendicular and circular profiles performed significantly better than the parallel profile ( P = 0.003 and 0.011 respectively). No significant differences were found between the different regions of the femoral neck ( P = 0.241) (despite the apparently large differences in A z ). The power of this test was 0.69, 0.97 and 0.15 for the investigation of differences due to the method of analysis, type of profile used and the region analysed respectively. Table 4 presents the mean A z for the slope from each of the spatial frequency bands for all regions of interest. This was assessed for each profile individually and also for all the profiles together. A one-way ANOVA was used to test for significant differences in Az between slopes A, B and C. In the individual profiles, slopeA performed significantly better than slopeC for the circular profile ( P = 0.008), however when all the profiles were considered, no significant differences were apparent ( P = 0.26). Table 4 Comparing slopeA , slopeB and slopeC . The average and standard deviation of the area under the ROC curve ( A z ) are presented for each of the slopes used in the fractal analysis for all regions of interest. A significant difference was found between slopeA and slopeC in the circular profile, however when all the profiles were compared, no significant differences were found. SlopeA SlopeB SlopeC P All profiles 0.601 (0.074) 0.598 (0.055) 0.565 (0.067) 0.260 Circular 0.670 (0.072) 0.611 (0.022) 0.531 (0. 026) 0.008 Parallel 0.544 (0.042) 0.620 (0.083) 0.563 (0.037) 0.140 Perpendicular 0.589 (0.047) 0.563 (0.032) 0.600 (0.104) 0.678 Discussion and conclusions In these short series, this study found that texture analysis of standard radiographs using the fast Fourier transform can yield variables that are significantly associated with fracture but not significantly correlated with age, body mass index or Neck-BMD. Both PCA and fractal analysis of the FFT data could be used to discriminate successfully between the groups, although overall PCA was significantly stronger than fractal dimension. The best results from this study were not significantly correlated with femoral neck-BMD, age or BMI, indicating their potential for use as an independent predictor of fracture. The radiographic appearance of bone is known to be affected by factors including the size of the patient. As there was no significant difference in the BMI of the fracture and control groups, it is unlikely that this has influenced the results, however it is an issue that will need addressing in future studies. The PCA method extends a method previously developed for analysis of histological sections [ 26 ]. The use of oriented profiles improved the performance of the analysis by selecting directions in which there was the most information about bone structure i.e. perpendicular to the preferred orientation of the trabeculae. PCA considerably reduces the number of variables required to characterise the image via its power spectrum. For example, in this study, we start with a 256 × 256 pixel ROI (65,536 pixels), the Fourier transform is performed and a profile of 128 spatial frequency values is generated. For each profile, PCA was able to describe over 70 % of the variance present in the data using only 5 components or fewer. Overall, the performance of principal components analysis was significantly stronger than that of fractal analysis ( P < 0.01). One advantage of PCA that may contribute to this finding is the ability to summarise the information present in the dataset with a small number of components via an economical mapping of the variance present in the data. In addition, the property of orthogonality between these components ensures that the variables generated are linearly independent (Fig. 5 ). Benefits can also be found by the use of a model built on the mathematical distributions present in the data, rather than expecting the data to meet a given mathematical property, such as fitting a fractal distribution. Figure 5 Plot of two principal components. Example of a scatterplot of two principal components. For FFT/PCA analysis of the upper head region, principal components 4 and 5 were selected by stepwise analysis and are shown here. They are plotted against each other with fracture and control subjects identified using separate markers. The lack of correlation between the components can be seen ( r = 0.040, P = 0.997). Previous studies using non-fractal analysis of the Fourier power spectrum have focussed on images of the spine or wrist, where the alignment of trabeculae is generally orthogonal [ 28 - 30 ]. In such images, analysis of trabecular orientation can be performed by examining the vertical and horizontal sectors as the trabeculae lie predominantly in these directions. The trabecular structure of the femur is more complicated as the trabeculae are aligned in arcs, so the preferred orientation changes throughout the proximal femur. Analysis parallel to the preferred orientation of the trabeculae was significantly poorer than analysis using either the perpendicular or circular profiles ( P < 0.05). Analysis in the perpendicular direction was strongest overall, although it was not significantly better than the circular profile. This accords with the increasingly anisotropic nature of trabecular bone with aging; bone loss is not evenly distributed but is lost primarily at angles perpendicular and oblique to the preferred orientation of the trabeculae [ 30 ]. This loss heightens the risk of fracture, especially if the impact is from the side, as expected from a typical fall from standing height, as there are fewer trabeculae orientated in this direction to absorb the force of impact. In summary, this paper presents a new method for analysing the structure of trabecular bone from standard radiographs. It demonstrates that the Fourier transform can be used to describe structural information in images which may be related to fracture, independently of BMD. This study is limited by the small size of the data set and further analysis is needed to validate these findings. This should be performed on a similar series of radiographs, consisting of fracture and control subjects scanned at the same resolution. The methods from this study could then be applied directly to this group (without recalculating the PCA) to evaluate whether they were generally applicable. However the success of both this and our previous study, using similar techniques to analyse histological sections, indicates that this may be an effective method with clinical utility for describing bone quality statistically in terms of structural parameters. Competing interests The authors declare that they have no competing interests. Authors' contributions Author JG helped design the study, performed the image and data analysis and drafted the manuscript Author AS collected the data/images used within this study and helped with writing of the paper. Author PU assisted with some of the practical approaches, and the writing of the paper Author DMR designed the initial case control study and assisted with interpretation of the results and writing the paper Author RMA helped with the design of the study, the interpretation of the results and the writing of the paper. All authors read and approved the final manuscript Pre-publication history The pre-publication history for this paper can be accessed here:

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545204

Retinoic Acid and Arsenic for Treating Acute Promyelocytic Leukemia

What were the critical steps in the development of ATRA and arsenic as treatments for APL? Researchers in Shanghai tell the story and look to the future

Acute promyelocytic leukemia (APL) was first identified as a distinct subtype of acute myeloid leukemia in 1957 by Leif Hillestad. It is called M3 in the French–American–British classification, with a variant type referred to as microgranular (M3v in the French–American–British nomenclature) [1] . APL is characterized by three features: (1) the presence of an accumulation of abnormal promyelocytes (see Glossary ) that do not differentiate into mature granulocytes, (2) the occurrence of fibrinogenopenia and disseminated intravascular coagulation that is often worsened by chemotherapy, and (3) the presence of the specific chromosomal translocation t(15;17)(q22;q21) ( Figure 1 ). Glossary Apoptosis: A genetically determined process of cell death in which the cell uses specialized cellular machinery to kill itself and is then eliminated by phagocytosis or by shedding. Caspase: A family of cysteine proteases with aspartate specificity that are essential intracellular death effectors. Disseminated intravascular coagulation: A hemorrhagic disorder that occurs following the uncontrolled activation of clotting factors and fibrinolytic enzymes throughout small blood vessels, resulting in depletion of clotting factors and tissue necrosis and bleeding. Fibrinogenopenia: A decrease in concentration of fibrinogen in the blood. Granulocyte: Terminally differentiated myelocyte or polymorphonuclear white blood cell (as a basophil, eosinophil, or neutrophil) with granule-containing cytoplasm. Ligand-inducible transcription factors: Transcription factors that structurally have domains associated with DNA binding and ligand (hormone) recognition. When binding to its specific ligand, the transcription factor initiates a series of conformational changes and interacts efficiently with its specific DNA response element to recruit components of the transcriptional machinery. Nuclear receptor superfamily: One of the most abundant classes of transcriptional regulators including receptors for steroid hormones (e.g., estrogens, glucocorticoids, and vitamin D3), RAs, thyroid hormones, and so on. These transcription factors regulate diverse functions such as homeostasis, reproduction, development, and metabolism in animals. Promyelocyte: Granule-containing cell in bone marrow that is in an intermediate stage of development between myeloblasts and myelocytes and that gives rise to a granulocyte. Proteasome: Proteolytic complex that degrades cytosolic and nuclear proteins. Sumoylation: Post-translational modification of proteins by the small ubiquitin-like modifier SUMO. Ubiquitin: A chiefly eukaryotic protein that when covalently bound to other cellular proteins marks them for proteolytic degradation. Figure 1 The Three Features of APL The three features of APL are (A) accumulation of abnormal promyelocytes, (B) fibrinogenopenia and disseminated intravascular coagulation, and (C) the chromosomal translocation t(15;17)(q22;q21), the resultant fusion transcripts, and variants. APL accounts for 10%–15% of all cases of acute myeloid leukemia, with several thousand new cases diagnosed worldwide each year. Before the advent of differentiation therapy, APL was treated with anthracycline-based chemotherapy with a complete remission rate of 60%–76% and a 5-year event-free survival rate of 23%–35% [ 1 , 2 ]. Differentiation Therapy: From Hypothesis to Practice Failure to differentiate terminally characterizes most, if not all, cancer cells of every origin. Whether the induction of differentiation could be a treatment strategy for cancers was hotly debated for decades before the advent of differentiation therapy. An important discovery of the early 1970s was that myeloid leukemic cells could be reprogrammed to resume normal differentiation and to become non-dividing mature granulocytes or macrophages as a result of stimulation by various cytokines [ 3 , 4 ]. Based on this discovery, Leo Sachs at the Weizmann Institute of Science, Rehovot, Israel, hypothesized in 1978 that treatment with agents that induce cancer cells to complete differentiation could be a potential therapeutic option for patients with cancer [5] . In the early 1980s, Breitman and colleagues showed that retinoic acid (RA; Figure 2 ), a derivative of vitamin A, could induce terminal differentiation of human promyelocytic leukemic cells in vitro [ 6 , 7 ]. But the first clinical reports of using RA showed conflicting results. Some case reports showed beneficial effects of 13- cis RA in people with refractory or relapsed APL [ 8 , 9 , 10 ], but other reports showed that 13- cis RA was ineffective in treating APL [11] . Figure 2 Isomers of RA Beginning in the early 1980s, the Shanghai Institute of Hematology conducted a series of experiments on differentiation therapy for APL. These experiments showed that all-trans RA (ATRA) could induce terminal differentiation of HL-60, a cell line with promyelocytic features, and fresh leukemic cells from patients with APL. These intriguing results were the impetus for a clinical trial. Twenty-four patients with APL were treated with ATRA (45 to 100 mg/m 2 /day). The result was dramatic: 23 patients (95.8%) went into complete remission (CR) without developing bone marrow hypoplasia or abnormalities of clotting. The remaining one patient achieved CR when chemotherapy was added [12] . Morphological maturation of bone marrow cells was found in all patients studied. These results were later confirmed by many randomized studies in centers around the world. Further trials showed improved rates of CR, a decrease in severe adverse effects, and lengthening of the duration of remission. Table 1 summarizes the CR rates obtained in most large series of patients. Currently, ATRA combined with anthracycline-based chemotherapy can achieve CR in 90%–95% of patients with APL and overall 5-year disease-free survival in up to 75% of patients [13] . Table 1 CR Rate in Patients with APL Treated with ATRA (in Series Including More Than 50 Cases) Chemo, chemotherapy Arsenic: “Ancient Remedy Performs New Tricks” Arsenic is a common, naturally occurring substance that exists in organic and inorganic forms in nature. The organic arsenicals consist of an arsenic atom in its trivalent or pentavalent state linked covalently to a carbon atom. There are three inorganic forms of arsenic: red arsenic (As 4 S 4 , also known as “realgar”), yellow arsenic (As 2 S 3 , also known as “orpiment”), and white arsenic, or arsenic trioxide (As 2 O 3 ) [14] . Arsenic was used to treat chronic myelogenous leukemia in the 18th and 19th centuries, but was discarded as a treatment in the early 20th century because of its toxicity and the advent of radiation and cytotoxic chemotherapy. In the early 1970s, a group from Harbin Medical University in China found that intravenous infusions of Ailing-1, a crude solution composed of 1% arsenic trioxide with a trace amount of mercury chloride, induced CR in two-thirds of patients with APL. There was an impressive 30% survival rate after 10 years [ 15 , 16 ]. Pure arsenic trioxide at 0.16 mg/kg/day for 28–54 days was shown to induce CR in 14 out of 15 (93.3%) patients with relapsed APL [17] . Tetra-arsenic tetra-sulfide was also reported to be effective in APL treatment [18] . Since 1996, a large number of reports have shown that arsenic compounds induce a CR in 85% to 90% of patients with both newly diagnosed and relapsed APL [13] . Furthermore, after CR is achieved by arsenic compounds, a molecular remission (i.e., negative for promyelocytic leukemia RA receptor a [PML-RARa] transcript detected by reverse transcriptase polymerase chain reaction) is obtainable either with arsenic compounds or with ATRA and chemotherapy as consolidation treatment. It seems likely that arsenic compounds appropriately used in post-remission therapy could prevent recurrence and achieve a longer survival time [ 13 , 18 ]. Studies have shown that arsenic trioxide exerts dose-dependent dual effects on APL cells—it induces apoptosis (programmed cell death) preferentially at relatively high concentrations (0.5 × 10 −6 to 2 × 10 −6 M) and induces partial differentiation at low concentrations (0.1 × 10 −6 to 0.5 × 10 −6 M). The rapid modulation and degradation of the PML-RARa oncoprotein by arsenic trioxide could contribute to these two effects [19] . How Do ATRA and Arsenic Work at the Molecular Level? To understand how ATRA and arsenic compounds act at the molecular level in treating APL, it is first important to understand the role of the PML-RARa fusion protein in the pathogenesis of APL. Retinoids that are crucial for normal myeloid differentiation act via RA receptors (RARs) and retinoid X receptors (RXRs). These belong to the steroid/thyroid/retinoid nuclear receptor superfamily of ligand-inducible transcription factors. Both RAR and RXR families consist of three subtypes: α, β, and γ [20] . RARα forms a heterodimer with RXR and binds to RA response element to control the expression of target genes in the presence of physiological concentrations (10 −9 –10 −8 M) of retinoids ( Figure 3A ). Figure 3 Leukemogenic Effects of PML-RARá and Mechanisms of ATRA/Arsenic Trioxide in the Treatment of APL (A) In the absence of RA, RARα/RXR heterodimers recruit the transcription corepressor (CoR), which mediates transcriptional silencing by mechanisms that include direct inhibition of the basal transcription machinery and recruitment of chromatin-modifying enzymes. Chromatin modification includes histone deacetylation, which leads to a compact chromatin structure that impairs the access of transcriptional activators. In the presence of physiological concentrations (10 −9 –10 −8 M) of RA, the transcription corepressor is released and the coactivator is recruited to the RARα/RXR heterodimer, resulting in histone acetylation (AC) and overcoming of the transcription blockage. (B) PML-RARα fusion protein binds to RARα target genes either on its own or with RXR and then recruits corepressors, leading to transcriptional repression and myeloid differentiation inhibition. PML-RARα oncoprotein sequesters the normal RXR and PML, inhibits the PML/P53 apoptotic pathway, and delocalizes PML and other proteins from the nuclear body. PML-RARα also may affect interferon (IFN) and other signal pathways. Abnormalities in protein tyrosine kinases (e.g., FLT3, c-fms) may collaborate with PML-RARα to cause APL. (C) In the presence of pharmacological doses of ATRA or arsenic trioxide, the PML-RARα fusion is degraded in ways that are dependent on caspases and proteasomes. The degradation of PML-RARα may lead to derepression of transcription suppression and restoration of PML nuclear body structure. The blockade of other signaling pathways is also released, and the anti-apoptotic effect of PML-RARα is lost. ATRA also induces cyclic AMP (cAMP), which reverses the silencing of RXR, induces the expression of RA-induced genes and cyclooxygenase 1 (Cox 1), inhibits angiogenesis, and downregulates tissue factor. Subsequently, ATRA induces terminal cell differentiation, while arsenic trioxide induces partial differentiation and/or apoptosis of APL cells. The effects of ATRA and arsenic trioxide are indicated with red and blue arrows, respectively. AF2, the ligand-dependent transcriptional activation domain contained within the C-terminal E domain of RARα; D522, aspartate at residue 522; K160, lysine at residue 160; K490, lysine at residue 490; RARE, retinoic acid response element; SUG-1, a component of proteasome 19S complex that can bind with the activated AF2 domain of RARα. More than 95% of patients with APL have the t(15;17)(q22;q21) translocation. This results in the fusion of the RARα gene on 17q21 and the promyelocytic leukemia (PML) gene on 15q22, which generates a PML-RARaα fusion transcript [ 21 , 22 ]. Variant translocations can also be detected in APL. The PML-RARα chimeric protein acts as a dominant negative mutant over wild-type RARα. The chimeric protein prevents activation of key target genes required for normal myeloid differentiation by sequestering RXR and other RARa cofactors and inhibiting normal RARα functions. The PML-RARα oncoprotein binds to RAR target genes either on its own or with RXR and then recruits histone deacetlyase complexes, which act as repressors of transcription. PML-RARa may affect transcription in other pathways including those in which the transcription factor AP1 and interferon-responsive factors are involved. PML-RARα also binds to promyelocytic leukemia zinc finger (PLZF) protein and potentially affects its functions (e.g., growth suppression and transcription repression; control of developmental programs and differentiation) [20] . In addition, PML-RARα prevents apoptosis through delocalization of PML and other proteins from the nuclear body. Finally, PML-RARα may cooperate with activated mutations in protein tyrosine kinases, such as FLT3 [23] , which confer proliferative and/or survival advantage to hematopoietic stem/progenitor cells. Whether PML-RARα affects the protein tyrosine kinase activity directly or indirectly is unclear. All these interactions of PML-RARα could be involved in the leukemogenesis of APL ( Figure 3B ). ATRA and arsenic trioxide degrade and cleave the PML-RARα oncoprotein. Although we now have a good understanding of the molecular mechanisms underlying ATRA in differentiation therapy for APL, these mechanisms were shown long after the identification of the efficacy of this drug in treating the disease. Now it is well established that pharmacological concentrations of ATRA (10 −7 –10 −6 M) exert their effects through targeting the PML-RARα oncoprotein, triggering both a change in configuration and degradation of the oncoprotein and the activation of transcription, leading to differentiation. Cleavage of the PML-RARα fusion protein by caspases at residue D522 has been shown in APL cells induced to differentiate by ATRA [24] . Further dissecting of the pathways involved in PML-RARα catabolism led to the discovery of ubiquitin/proteasome-mediated degradation of PML-RARα and RARα, which was dependent on the binding of SUG-1 in the AF2 transactivation domain of RARα with 19S proteasome [ 25 , 26 ]. In contrast to ATRA, which targets the RARα moiety of the fusion, arsenic targets the PML moiety of PML-RARα, through a still unclear mechanism, and causes PML to localize to the nuclear matrix and become sumoylated. Sumoylation at K160 is necessary for 11S proteasome recruitment and arsenic-trioxide-induced degradation, whereas sumoylation at K490 is needed for nuclear localization [ 27 , 28 ]. These results provide a striking similarity in the effect of these two otherwise unrelated agents ( Figure 3C ). The final result of treatment with ATRA and arsenic trioxide is the degradation of the PML-RARa oncoprotein, which results in restoration of normal retinoid signaling. RXR and PML sequestration is abrogated, and PML nuclear body is restored. The corepressor is released and the coactivator is recruited and bound with RARα; thus, the transcription of target genes is derepressed. ATRA also induces cyclic AMP, a differentiation enhancer that boosts transcriptional activation, reverses the silencing of the transactivating function of RXR, and restores ATRA-triggered differentiation in mutant ATRA-resistant APL cells [29] . Additionally, ATRA induces the expression of RA-induced genes [30] and cyclooxygenase 1 [31] , inhibits angiogenesis [32] , downregulates the expression of tissue factor [33] , and restores other signal pathways (e.g., the interferon pathway). Consequently, the abnormal promyelocytes differentiate and die through programmed cell death ( Figure 3C ). Combining ATRA and Arsenic: A Cure for APL? Since ATRA and arsenic trioxide degrade the PML-RARa oncoprotein via different pathways, and since studies in animal models have shown synergic effects of both drugs in prolonging survival or even eliminating the disease [ 34 , 35 ], the group at the Shanghai Institute of Hematology hypothesized that the combination of the two drugs might synergize in treating APL. To test this, 61 patients newly diagnosed with APL were randomized into three treatment groups: ATRA, arsenic trioxide, or a combination of the two drugs [36] . Although CR rates in all three groups were high (>90%), the time to achieve CR differed significantly—the time was shortest in patients treated with the combination. The disease burden (as reflected by fold change of PML-RARα transcripts at CR) decreased significantly more with combined therapy than with ATRA or arsenic trioxide monotherapy ( p < 0.01), and this difference persisted after consolidation therapy ( p < 0.05). Notably, all 20 patients in the combination group remained in CR whereas seven of 37 cases treated with monotherapy relapsed ( p < 0.05) after a follow-up of 8–30 months (median, 18 months). It seems that a combination of ATRA and arsenic trioxide for remission and maintenance treatment of APL produces better results than either of the two drugs used alone, in terms of the time required to achieve CR and the length of disease-free survival. We hope that the use of three treatments—ATRA, arsenic trioxide, and chemotherapy—will ultimately make APL a curable human acute myeloid leukemia [36] . Conclusion The story of ATRA in the treatment of APL shows that by targeting the molecules critical to the pathogenesis of certain diseases, cells can be induced to return to normal. Differentiation therapy is therefore a practical method of treating human cancer that has shown consistent effectiveness in trials. The clarification of the underlying molecular abnormalities of APL is an example of the benefits of a close collaboration between bench and bedside, and is necessary for our understanding of the mechanisms of ATRA in differentiation therapy. It is clearly important to elucidate the molecular and cellular basis of a particular cancer if we are to further develop mechanism-based target therapies. The sequencing of the human genome and ongoing functional genomic research are now accelerating the dissection of disease mechanisms and identification of therapeutic targets. This in turn may facilitate the screening of promising treatments. What we learn from developing curative treatment approaches to APL may help to conquer other types of leukemias and cancers.

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543463

Modulation of Statin-Activated Shedding of Alzheimer APP Ectodomain by ROCK

Background Statins are widely used cholesterol-lowering drugs that act by inhibiting HMGCoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. Recent evidence suggests that statin use may be associated with a decreased risk for Alzheimer disease, although the mechanisms underlying this apparent risk reduction are poorly understood. One popular hypothesis for statin action is related to the drugs' ability to activate α-secretase-type shedding of the α-secretase-cleaved soluble Alzheimer amyloid precursor protein ectodomain (sAPP α ). Statins also inhibit the isoprenoid pathway, thereby modulating the activities of the Rho family of small GTPases—Rho A, B, and C—as well as the activities of Rac and cdc42. Rho proteins, in turn, exert many of their effects via Rho-associated protein kinases (ROCKs). Several cell-surface molecules are substrates for activated α-secretase-type ectodomain shedding, and regulation of shedding typically occurs via activation of protein kinase C or extracellular-signal-regulated protein kinases, or via inactivation of protein phosphatase 1 or 2A. However, the possibility that these enzymes play a role in statin-stimulated shedding has been excluded, leading us to investigate whether the Rho/ROCK1 protein phosphorylation pathway might be involved. Methods and Findings We found that both atorvastatin and simvastatin stimulated sAPP α shedding from a neuroblastoma cell line via a subcellular mechanism apparently located upstream of endocytosis. A farnesyl transferase inhibitor also increased sAPP α shedding, as did a dominant negative form of ROCK1. Most conclusively, a constitutively active ROCK1 molecule inhibited statin-stimulated sAPP α shedding. Conclusion Together, these data suggest that statins exert their effects on shedding of sAPP α from cultured cells, at least in part, by modulation of the isoprenoid pathway and ROCK1.

Introduction Alzheimer disease is the leading cause of dementia among the elderly and is characterized by accumulation of extracellular and vascular amyloid in the brain [ 1 ]. Amyloid deposits are composed of the amyloid-β peptide (Aβ), a 4-kDa peptide released during “amyloidogenic” proteolytic processing of the Alzheimer Aβ precursor protein (APP) [ 2 ]. APP can also be cleaved by the nonamyloidogenic α-secretases, a disintegrin and metalloproteinase 10 (ADAM-10) and ADAM-17 [ 3 ], in a reaction that is believed to occur primarily on the plasma membrane [ 4 ] and is known as “ectodomain shedding.” α-Secretase-type ectodomain shedding divides the Aβ domain of APP, thereby generating α-secretase-cleaved soluble APP ectodomain (sAPP α ) [ 4 ]. This reaction can be stimulated by activation of protein kinase C (PKC) or extracellular-signal-regulated protein kinases (ERKs) [ 5 , 6 , 7 ] or by inactivation of protein phosphatase 1 or 2A [ 5 ]. Reports from retrospective analyses suggest that the statin class of cholesterol-lowering HMGCoA reductase inhibitors may lower the risk for Alzheimer disease by as much as 70% [ 8 , 9 , 10 , 11 ]. Studies in wild-type guinea pigs and in plaque-forming transgenic mice have demonstrated that chronic statin treatment can attenuate cerebral amyloidosis [ 12 , 13 ], suggesting that statins may exert their risk-reducing effects, at least in part, by modulating APP metabolism. In cell culture, lovastatin and simvastatin decrease the release of Aβ by rat hippocampal neurons [ 12 , 14 ] while activating α-secretase-type ectodomain shedding [ 15 , 16 ]. However, the molecular mechanisms by which statins modulate ectodomain shedding remain to be elucidated [ 17 , 18 ]. Statin effects on APP metabolism are, to some extent, attributable to cholesterol lowering, but statin actions on APP may also involve cholesterol-independent actions [ 19 ]. Reduction in synthesis of mevalonate leads to decreased generation of a number of isoprenoid lipid derivatives. Isoprenoids, such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate, are 15- or 20-carbon lipid moieties. Through the action of farnesyl transferases and type I geranylgeranyl transferases, isoprenoids are attached to the amino acid sequence Cys-Ala-Ala-Xaa (“CAAX”) at the C-terminus of the Rho family of GTPases [ 20 ]. These posttranslational lipid modifications are essential for attachment of the GTPases to the cytosolic face of intracellular vesicles and/or to the cytosolic leaflet of the plasma membrane, thereby specifying subcellular targets for GTPase action(s). Some members of the Rho GTPase family exert their actions through modulation of protein kinase activities. One of the best characterized is Rho-associated protein kinase 1 (ROCK1, also called ROKβ). ROCK1 is a serine/threonine kinase with an apparent mass of 160 kDa that can be activated by either RhoA or RhoB [ 21 , 22 , 23 ]. Structurally, the ROCK1 N-terminus contains the protein kinase domain, while the C-terminus has both a Rho-binding domain and a pleckstrin homology domain, either of which can modulate protein–protein interactions. In the inactive state, the Rho-binding domain and the pleckstrin homology domain form an autoinhibitory loop by binding and blocking the kinase domain at the N-terminus of the molecule. Activation of ROCK1 occurs when a Rho protein binds to the Rho-binding domain, causing a conformational change that opens the kinase domain for the phosphorylation of downstream effectors [ 23 ]. Once activated, ROCK1 phosphorylates several substrates, including myosin light chain phosphatase, LIM kinases (Lin11, Isl1, and Mec3), and ezrin-radixin-moesin proteins [ 24 , 25 , 26 , 27 ]. ROCK1 has recently been implicated in modifying the site of substrate cleavage by APP γ-secretase [ 28 ], perhaps acting via ROCK1-dependent phosphorylation of a component of the γ-secretase enzyme complex. In the current study, we demonstrate that activation of sAPP α shedding from cultured cells by atorvastatin or simvastatin involves isoprenoid-mediated protein phosphorylation. Treatment of cells with a farnesyl transferase inhibitor or expression of a dominant negative (DN) ROCK1 molecule led to enhanced sAPP α shedding, supporting the notion that shedding is modulated by the isoprenoid pathway. Transfection with the cDNA for a constitutively active (CA) ROCK1 molecule led to inhibition of statin-activated sAPP α shedding. These results raise the possibility that the apparent beneficial effect of statins in the prevention of Alzheimer disease could be, at least in part, mediated by isoprenoid modulation of APP metabolism. Methods Reagents The APP C-terminal specific polyclonal antibody 369 [ 29 ] was used to detect full-length APP and its C-terminal fragments. Monoclonal antibody 6E10 against residues 1–16 of human Aβ (Signet, Dedham, Massachusetts, United States) was used to detect human holoAPP or sAPP α . Anti-ROCK1 antibody was purchased from Chemicon (Temecula, California, United States). Streptavidin-antibody HRP-conjugated C-Myc antibody 9E10, mevalonic acid, arachidonic acid, and phenylarsine oxide were purchased from Sigma (St. Louis, Missouri, United States). Atorvastatin was obtained from Pfizer (Groton, Connecticut, United States), and simvastatin was obtained from LKT Labs (St. Paul, Minnesota, United States). N2 supplement was obtained from Gibco (Carlsbad, California, United States). Sulfo-NHS-LC-Biotin was purchased from Pierce (Rockford, Illinois, United States). CA and DN Myc-tagged ROCK1 vectors were generated as previously described [ 30 , 31 ] and were generous gifts from Liqun Luo (Stanford University). Fugene 6 was purchased from Roche (Basel, Switzerland). Farnesyl transferase inhibitor 1 (FTI-1) was obtained from Biomol (Plymouth Meeting, Pennsylvania, United States). Tumor necrosis factor α (TNFα) protease inhibitor 2 and Y-27632 were purchased from Calbiochem (San Diego, California, United States). Protein concentration assay kit was purchased from Biorad (Hercules, California, United States). LIVE/DEAD Viability/Cytotoxicity Assay Kits and Amplex Red Cholesterol Assay Kits were purchased from Molecular Probes (Eugene, Oregon, United States). Culture Methods and Sample Preparation N2a mouse neuroblastoma cells stably transfected with the Swedish mutant form of APP (SweAPP N2a cells; APP695, 595–596 KM/NL) (gift from G. Thinakaran and S. Sisodia, University of Chicago, Chicago, Illinois, United States) were maintained in DMEM, 10% FBS, and 200 μg/ml G418 in the presence of penicillin and streptomycin [ 32 ]. For the 24 h prior to pharmacological treatments, the culture media were changed to N2-supplemented lipid-free medium. In some studies, cells were transfected in N2-supplemented FBS-free medium 48 h before pharmacological treatments. Transfections were carried out using the Fugene reagent, according to the manufacturer's instructions. All treatments were performed in the presence of 1 μM mevalonic acid, unless otherwise specified. Cells were lysed in 1% Triton-X/PBS buffer containing 1 X complete proteinase inhibitor cocktail (Roche), sonicated twice for 30 s, and centrifuged at 5,000 g for 5 min. Protein concentration in the supernatant was determined using the Biorad Protein Assay kit, following the manufacturer's instructions. For the detection of holoAPP and sAPP α , samples were separated in 7.5% polyacrylamide gels, transferred to nitrocellulose, and the proteins detected with either 369 (1:3,000 for holoAPP and C-terminal fragments) or 6E10 (1:1,000 for holoAPP or sAPP α ), followed by incubation of the transfers with appropriate secondary anti-rabbit or anti-mouse antibodies. For the detection of transfected ROCK1 proteins, samples were immunoprecipitated with 2 μg of anti-Myc antibody, separated in 5% polyacrylamide gels, transferred, and the proteins detected with anti-ROCK1 antibody (1:1,000 dilution). Cell-Surface Biotinylation Cells were plated in a 100-mm dish at a concentration of 5 × 10 6 cells/dish. After treatment, media were harvested, and sAPP α levels were evaluated by immunoblotting as described above. Cells were washed twice in PBS and then incubated with Sulfo-NHS-LC-Biotin for 30 min at 4 o C. Biotinylation reactions were terminated by one wash in Tris followed by two washes in PBS. Cells were lysed in 1% Triton-X/PBS buffer containing protease inhibitor cocktail as indicated above. Lysates were immunoprecipitated with 3 μl of whole 369 antibody serum and 30 μl of protein A beads. After washing twice with 1% Triton/PBS, and then twice with PBS, samples were boiled in sample buffer for 3 min, separated in a 7.5% polyacrylamide gel, and transferred to nitrocellulose. The biotinylated proteins were detected using streptavidin HRP polymer (1:10,000 dilution). Viability/Cytotoxicity Assays Cells were plated in an eight-well slide at a concentration of 1 × 10 4 cells/well. After treatments as indicated, LIVE/DEAD assays were performed following the manufacturer's instructions (Molecular Probes). Cholesterol Assays Cholesterol levels in cell lysates were measured using Amplex Red following the manufacturer's instructions (Molecular Probes). We have previously demonstrated that standard doses of either simvastatin or atorvastatin reduce cholesterol levels in N2a cells by 65%–67% [ 16 ]. Quantification and Statistical Analysis Quantification of protein bands was performed using the UVP Bioimaging System, and statistical analysis was performed on paired observations using the Student's t test. Results Atorvastatin Activates sAPP α Shedding at a Subcellular Site Upstream of Endocytosis from the Plasma Membrane We confirmed our previous observation [ 16 ] that atorvastatin produces an increase in sAPP α shedding that is dose-dependent, reaching a maximum effect at 5 μM. The increase in sAPP α shedding is accompanied by a corresponding increase in levels of the nonamyloidogenic APP α-C-terminal fragment (C83; data not shown). In order to refine our localization of the subcellular target of statin action, we evaluated the steady-state levels of cell-surface APP (csAPP) in the absence or presence of statins. After drug treatments, cells were subjected to the surface biotinylation protocol. Cells and media were harvested, and levels of sAPP α , holoAPP, and csAPP were measured. Treatment with atorvastatin increased csAPP by approximately 1.6-fold, similar to the effect of the drug on holoAPP ( Figure 1 ), while sAPP α shedding was increased by approximately 7-fold ( p < 0.05). Since csAPP levels were only slightly raised in the same statin-treated cells in which sAPP α shedding was dramatically increased, we interpret this disparity to indicate that the effector of statin-stimulated shedding is probably intrinsic to the plasma membrane. In other studies, the plasma membrane has been proposed to be, or to contain, the statin target. For example, statins have been proposed to cause co-localization of α-secretase and APP within lipid rafts [ 15 , 33 ]; statins might also induce modification of the structure and activity of a protein in the plasma membrane α-secretase complex, perhaps in an action similar to how statins bind and “lock” cell-surface integrins [ 34 ]. Figure 1 Atorvastatin Activates sAPP α Shedding Out of Proportion to Its Effect on holoAPP or csAPP (A) SweAPP N2a cells were treated with atorvastatin (Atv) for 24 h and then surface biotinylation was performed as described in Methods. Evaluation of csAPP was performed by immunoprecipitation–immunoblot after surface biotinylation, while holoAPP and sAPP α were evaluated by immunoblot as described in Methods. C, control. (B) Graphic representation of data. Y-axis shows effect of treatment (in arbitrary units) divided by effect of untreated control (in arbitrary units); n = 3 independent experiments; *, p < 0.05; **, p < 0.01; Student's t test). A pulse-chase protocol was also used to study post-transcriptional regulation of APP metabolsm by statins (data not shown). This protocol avoids any confound that might arise because of altered APP transcription. Pulse-chase studies were performed using a 10-min pulse with [ 35 S]methionine followed by various chase times from 0 to 120 min. Typical maturation and half-life of mature cellular holoAPP were observed, as was subsequent release of sAPP α [ 5 , 29 ]. In the presence of either atorvastatin or simvastatin, the time course of maturation and release perfectly paralleled that observed in the absence of either drug, except that the fractional content of cellular mature holoAPP was approximately 2-fold greater in the presence of drug (i.e., at 15 or 75 min chase, mature APP in the presence of statin was approximately 310% of the level of immature APP at t = 0 versus a control [vehicle treatment] of 150% of the level of immature APP at t = 0; also, at t = 30 min, the relative percent values for drug versus vehicle were 380% and 200%, respectively). Fold increases in released sAPP α in the same experiments were approximately 3- to 4-fold (2.0 arbitrary units versus 5.5–8.0 arbitrary units at 120 min for atorvastatin and simvastatin, respectively). Secretory maturation toxicity is one possible mechanism for elevated levels of intracellular mature holoAPP and causes retarded conversion of mature holoAPP to sAPP α . This pattern was not observed following statin treatment, excluding maturation toxicity as a mechanism underlying the altered levels of mature cellular holoAPP. Instead, the pattern that we observed raises the possibility that statins, presumably via isoprenoids (given the reversibility with mevalonate), as discussed in the next section, may alter sorting of cellular holoAPP, diverting holoproteins away from terminal degradation in the endosomal/lysosomal pathway and into the constitutive secretory pathway that generates sAPP α . However, the fold effect on reduced intracellular turnover in the endosomal/lysosomal pathway (or sorting out of the endosomal/lysosomal pathway and into the constitutive secretory/shedding pathway) is apparently insufficient to explain the fold effect on sAPP α generation (2-fold for the former, vs 3- to 4-fold for the latter), indicating a contribution from a downstream site in the processing pathway. When these results are taken together with independent work on regulated shedding of transforming growth factor α (TGFα) [ 35 , 36 ], a parsimonious explanation is that an important target for activation of ectodomain shedding is probably located at the plasma membrane or downstream of APP residence at the plasma membrane. The identification of the regulatory components of the ectodomain shedding machinery have been long-sought in other studies employing phorbol esters to stimulate shedding of sAPP α or TGFα [ 4 , 35 , 36 , 37 ]. Munc-13 has recently been implicated as a phorbol target in regulated shedding [ 38 ]. In our opinion, this molecule is rather unlikely to play a major role in shedding regulation, given the specificity of Munc-13 effects for phorbols and the generalization of the regulated shedding phenomenon to include activation by protein phosphatase inhibitors and neurotransmitters. Neither of these would be predicted to act via the phorbol-binding C1 domain of Munc-13. Sisodia and colleagues [ 39 ] demonstrated that arrest of APP endocytosis from the plasma membrane by deletion of its NPXY clathrin-coated vesicle targeting sequence [ 40 , 41 ] can dramatically stimulate sAPP α shedding, presumably by extending the half-life of co-localized α-secretase and APP on the plasma membrane. In order to exclude the possible contribution of altered endocytosis to statin-stimulated shedding, we evaluated the effect of phenylarsine oxide (PO), an inhibitor of endocytosis, on statin-stimulated shedding ( Figure 2 ). Treatment with either atorvastatin, simvastatin, or PO alone increased sAPP α shedding, as expected. Co-treatment of cells with PO plus either atorvastatin or simvastatin caused stimulation of sAPP α shedding to levels greater than the maximal levels of shedding achievable with inhibition of endocytosis using PO alone or with maximal doses of either statin alone ( p < 0.05). The additivity of statin- and PO-stimulated shedding is consistent with the hypothesis that statins act at or near the plasma membrane, prior to internalization of csAPP. Under all circumstances, stimulated sAPP α shedding was completely blocked using TNFα protease inhibitor 2, a standard α-secretase/metalloproteinase inhibitor ( Figure 2 ). We interpret this as an indication that the statin-induced α-cleavage of APP is probably mediated by one of the molecules usually associated with the phenomenon, i.e., ADAM-10 or ADAM-17 [ 16 ]. Figure 2 Simultaneous Treatment of SweAPP N2a Cells with Statins and an Inhibitor of Endocytosis (PO) Yields More sAPP α Shedding Than Does Treatment with Either Statins or PO Alone (A) SweAPP N2a cells were treated for 24 h with atorvastatin (Atv) or simvastatin (Sim) as indicated. Media were then replaced and cells were treated for an additional 20 min with atorvastatin, simvastatin, TNFα protease inhibitor, PO, or combinations, as indicated. Evaluation of sAPP α and holoAPP was performed by Western blot as described in Methods. C, control. (B) Graphic representation of data. Y-axis shows effect of treatment (in arbitrary units) divided by effect of untreated control (in arbitrary units); n = 3 independent experiments; *, p < 0.05 versus control; **, p < 0.01 versus C; #, p < 0.05 versus atorvastatin alone; ##, p < 0.05 versus simvastatin alone; Student's t test). Compounds that Modulate Isoprenoid Levels Activate sAPP α Shedding As discussed above, there is an established relationship between statins and isoprenoid-modulated protein phosphorylation. We therefore tested the effects of FTI-1 on statin-stimulated sAPP α shedding. FTI-1 increased the shedding of sAPP α , but the combination of a statin plus FTI-1 increased sAPP α shedding to levels greater than those achievable by using either compound alone ( Figure 3 ; p < 0.05). In the same experiment, levels of holoAPP were modestly increased but, again, to an extent insufficient to account for the increase in shed sAPP α ( Figure 3 ). Figure 3 Simultaneous Treatment of SweAPP N2a Cells with a Statin and FTI-1 Causes Greater sAPP α Shedding Than Either Drug Alone (A) SweAPP N2a cells were treated for 24 h with atorvastatin (Atv, 5 μM), simvastatin (Sim, 1 μM), FTI-1 (5 μM), or a combination of FTI-1 plus a statin. Levels of sAPP α (top panel) or holoAPP (bottom panel) were evaluated as described in Methods. C, control. (B) Graphic representation of data. Y-axis shows effect of treatment (in arbitrary units) divided by effect of untreated control (in arbitrary units); n = 3 independent experiments; *, p < 0.05 versus control; **, p < 0.05 versus atorvastatin alone; ***, p < 0.05 versus simvastatin alone; Student's t test). To test whether statin-activated shedding might be attributable to a metabolite downstream of HMGCoA reductase, cells were treated with 1 μM simvastatin, 5 μM FTI-1, and a series of concentrations of mevalonic acid. Since FTI-1 acts downstream of HMGCoA reductase, the stimulatory effect of FTI-1 on sAPP α shedding would not be predicted to be modified by mevalonate supplementation. Low doses (<1 μM) of mevalonic acid did not affect statin-induced sAPP α shedding, but complete inhibition of statin-activated sAPP α shedding was achieved with higher doses of mevalonic acid (100 μM). As predicted, the shedding observed following treatment with FTI-1 was not inhibited by any of the concentrations of mevalonic acid tested ( Figure 4 ). These data are consistent with a role for isoprenoids in statin control of APP metabolism in cultured cells. Figure 4 Mevalonic Acid Reverses Statin-Induced, but Not FTI-1-Induced, sAPP α Shedding SweAPP N2a cells were treated for 24 h with simvastatin (Sim, 1 μM), FTI-1 (5 μM), mevalonic acid (0–100 μM), or combinations as indicated. Levels of sAPP α were evaluated by western blot as described in Methods. This figure is representative of the results of two independent experiments. C, control. Expression of ROCK-Related Molecules Modulates sAPP α Shedding in a Bidirectional Manner Since many isoprenoid-mediated Rho effects converge on ROCKs, we next transfected N2a cells with cDNAs encoding either green fluorescent protein (GFP) (control), CA ROCK1, or DN ROCK1 ( Figure 5 ). Simvastatin caused a typical activation of sAPP α shedding from GFP-transfected cells. When CA ROCK1 was introduced, however, shedding of sAPP α from both untreated and simvastatin-treated cells was diminished ( Figure 5 ; p < 0.05 versus GFP control). Conversely, DN ROCK1 alone activated shedding of sAPP α . Cellular levels of holoAPP were not affected by transfection ( Figure 5 ; p < 0.05 versus GFP control). In studies aimed at independent confirmation of the involvement of ROCK activation in sAPP α shedding, we treated SweAPP N2a cells with arachidonic acid, an activator of ROCK. As shown in Figure 6 , arachidonic acid reduced the shedding of sAPP α without altering levels of holoAPP. Based on this series of results, we concluded that both basal and activated sAPP α shedding from cultured cells are controlled by ROCK activity. Figure 5 Structure and Expression of ROCK cDNAs, and Their Effect on Basal and Statin-Stimulated sAPP α Shedding (A) Graphic representation of the ROCK1 constructs. Myc, Myc tag; KD, kinase domain; PH domain, pleckstrin homology domain; RBD, Rho-binding domain. (B) SweAPP N2a cells were transfected with GFP, CA ROCK1, or DN ROCK1 for 48 h. Cells were lysed and levels of expressed ROCK1 protein evaluated by immunoprecipitation–immunoblot as described in Methods. (C) Model for ROCK activity modulation by Rho. (D) SweAPP N2a cells were transfected for 48 h with control (GFP), CA ROCK1, or DN ROCK1 cDNAs. At the end of this incubation, cells were treated for an additional 24 h with simvastatin (Sim, 1 μM). sAPP α and holoAPP were evaluated by immunoblot as described in Methods. (E) Graphic representation of data. Y-axis shows effect of treatment (in arbitrary units) divided by effect of untreated control (in arbitrary units); n = 3 independent experiments; *, p < 0.05 versus GFP; Student's t test). C, control. Figure 6 Arachidonic Acid Inhibits Basal sAPP α Shedding but Has No Effect on holoAPP Levels (A) SweAPP cells were treated for 24 h with arachidonic acid (5 or 50 μM, represented by AA5 and AA50, respectively). Levels of sAPP α were evaluated by immunoblot as described in Methods. C, control. (B) Graphic representation of data. Y-axis shows effect of treatment (in arbitrary units) divided by effect of untreated control (in arbitrary units); n = 6 independent experiments; *, p < 0.05 versus control; Student's t test. In some experiments, cells were treated with Y-27632 (10 nM to 50 μM), a compound that can inhibit ROCKs. Y-27632 showed no effect on basal sAPP α release and blocked statin-activated sAPP α shedding (data not shown). This result was unexpected in light of the effects of DN ROCK1. Given the internally consistent actions of DN ROCK1 and CA ROCK1, as well as the results employing either FTI-1 or arachidonate, we concluded that the Y-27632 result might be due to inhibition by Y-27632 of protein kinases other than ROCK1 [ 23 ]. The possibility was also considered that cytotoxicity of Y-27632 for the central vacuolar pathway might explain the disparity between the effects of DN ROCK1 and those of Y-27632, but neither impairment of intracellular APP maturation nor increased apoptosis as measured by LIVE/DEAD assay were apparent following Y-27632 treatment (data not shown). Ultimately, we were unable to document any explanation for the disparate results of Y-27632 and DN ROCK1. Discussion The isoprenoid pathway involves lipid modification of various members of the Rho family of small GTPases by the addition of either farnesyl or geranylgeranyl moieties [ 20 , 42 ]. Isoprenylation serves to target the GTPases to the proper organelle membrane, where their actions often relate to cytoskeletal dynamics and/or vesicle trafficking [ 20 , 42 ]. ROCKs are important downstream targets of Rho ( Figure 7 ), catalyzing the phosphorylation of effector phosphoprotein substrates [ 23 ]. The foregoing data indicate that statin-induced activation of APP shedding in cultured cells involves the Rho/ROCK pathway. More specifically, the data indicate that ROCK1 activation blocks the effects of statins on APP ectodomain shedding, while ROCK1 blockade alone can mimic the effect of statins on APP shedding. By extension, these data predict that application of statins to neurons might directly or indirectly inhibit ROCK1 activity. Evaluation of this possibility will be the subject of future investigation. Figure 7 Isoprenoid Pathway and Sites of Action of Compounds Used in This Study FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate. The first evidence that APP might be a substrate for ectodomain shedding was provided by Weidemann et al. [ 43 ] who identified sAPP α in the cerebrospinal fluid and blood. This aspect of APP metabolism bears resemblance to the proteolytic signal transduction pathways involved in processing pro-TGFα [ 35 , 36 ] and Notch [ 44 ]. In the case of Notch, the process is set in motion by the binding of a ligand to the Notch ectodomain, triggering its release (shedding). For APP, intracellular signal transduction appears to be more important [ 2 , 29 , 45 ]. In early studies, the existence of the shed ectodomain of APP was used to deduce the existence of the proteolytic activity designated α-secretase, which has the unusual specificity of cleaving its substrates at a proscribed distance from the extracellular leaflet of the plasma membrane [ 4 , 39 ]. Ultimately, the integral cell-surface metalloproteinases ADAM-10 and ADAM-17/TACE were found to underlie α-secretase-type ectodomain shedding [ 3 , 46 ]. Why is APP a substrate for ectodomain shedding? To answer this question requires contemplation of the physiological function of APP. APP is a type 1 integral protein that is subjected to a host of post-translational processing events, including N- and O-glycosylation, tyrosyl sulfation, phosphorylation, and proteolysis [ 39 , 43 , 47 , 48 ]. Most (60%–80%) newly synthesized APP is subjected to terminal intracellular degradation that generates no discrete fragments [ 5 ]. A smaller fraction of APP molecules (approximately 20% in PC12 cells under basal conditions [ 5 ]) undergoes ectodomain shedding catalyzed by either the α-secretase (nonamyloidogenic) or β-secretase (potentially amyloiodgenic) pathway. When PKC is activated, the stoichiometry of shed sAPP α rises from 2 mol shed per 10 mol synthesized to 4 mol shed per mole synthesized. Most of this shedding is catalyzed by the α-secretase pathway, but a trace amount (<5% [ 49 ]) is catalyzed by β-secretase/β-site APP cleaving enzyme [ 50 ]. sAPP α and sAPP β differ by the inclusion in sAPP α of the first 16 residues of Aβ. Unlike sAPP α , which is generated at the plasma membrane, most sAPP β is probably generated by cleavage within the trans -Golgi network and endocytic pathway vesicles. HoloAPP levels are likely limiting at one or more sorting steps in the late secretory pathway, since activated sAPP α shedding is apparently accompanied by diminished generation of sAPP β [ 51 ]. What is the function of shed sAPP α ? Again, from other molecules, we know that shedding can serve important cellular functions by releasing diffusible ligands from their membrane-bound precursors (e.g., TGFα and TNFα) or by terminating intercellular signaling (e.g., Notch). A popular model holds that sAPP α may function as a neurotrophic and/or neuroprotective factor, and may promote neurite outgrowth [ 52 ]. More recent evidence suggests that released APP derivatives modulate efficacy of neurotransmission at the synapse [ 53 ]. Targeted deletion of APP has not revealed a striking phenotype [ 54 ], presumably because of functional redundancy supplied by APP-like proteins [ 55 ]. Mice with double and triple null mutations in various combinations of APP, APP-like protein 1, and APP-like protein 2 are now being created, in search of evidence for a definitive function for APP. Cao and Sudhof [ 56 ] have recently discovered that the APP C-terminal fragment generated by α- or β-secretase is itself cleaved to release Aβ and an APP intracellular domain (AICD) that diffuses into the nucleus, possibly acting there as a transcription factor. The pathway leading to AICD must be initiated by ectodomain shedding: holoAPP cannot directly give rise to AICD. Therefore, one important function for α- and/or β-secretase processing of APP may be the eventual generation of AICD. Our results suggest that Rho/ROCK signaling provides modulation of basal and stimulated α-secretase activity. It will now be important to dissect pathways upstream of Rho/ROCK signaling in order to identify the intracellular and intercellular events that participate in Rho/ROCK regulation of α-secretase under physiological and pathological conditions. The potential role of cholesterol in α-secretase-mediated shedding was discovered by Bodovitz and Klein [ 57 ] who used β-cyclodextrin to lower cellular cholesterol. Kojro et al. [ 15 ] confirmed this observation, using not only β-cyclodextrin but also lovastatin to lower cellular cholesterol. These investigators proposed that elevated ADAM-10 activity and protein levels contributed to these effects. These basic observations dovetailed with emerging epidemiological evidence that administration of statins might lead to a diminished incidence of Alzheimer disease [ 8 , 9 , 10 , 11 ]. Despite this, however, the association of statins and cholesterol levels with activated α-secretase-mediated shedding of the APP ectodomain was unexpected and not readily explicable by existing knowledge regarding regulation of α-secretase activity. The best characterized regulation of α-secretase processing typically involves protein phosphorylation via PKC [ 5 , 29 ] or ERKs [ 7 ] or protein dephosphorylation by protein phosphatase 1 or 2A [ 29 ]. We recently excluded the possibility that either PKC or ERK plays a role in statin-activated shedding [ 16 ], raising the possibility that other protein phosphorylation signaling pathways might link statins and/or cholesterol to α-secretase activation. Maillet et al. [ 58 ] implicated the Rho pathway in modulation of α-secretase activity while dissecting the activated shedding process that accompanies serotonergic signal transduction. These investigators discovered that Rap1 acts through Rac to modulate α-secretase processing of APP. Soon thereafter, ROCK1 was discovered by Zhou et al. [ 28 ] to modulate a downstream processing step in APP metabolism that involves presenilin/γ-secretase-mediated proteolysis of APP C-terminal fragments C99 and C83. These investigators discovered that activation of ROCK1 may account for how nonsteroidal anti-inflammatory drugs specify the scissile bond within the APP transmembrane domain that is cleaved by presenilin/γ-secretase to generate the C-terminus of Aβ. Based on these reports, we asked whether the Rho/ROCK pathway might play a role in controlling shedding of sAPP α following statin application. CA ROCK1 and DN ROCK1 molecules yielded direct and complementary evidence that ROCK1 was indeed a candidate for modulation of statin-activated α-secretase action. Further, we were able to demonstrate that α-secretase activity could be modulated by molecules further upstream in the isoprenoid pathway (see Figure 7 ). FTI-1, an inhibitor of farnesyl transferase also known as L-744,832 [ 59 ], mimicked and potentiated statin-activated shedding, presumably by blocking transfer of isoprenoid moieties to a Rho protein by farnesyl transferase, and thereby decreasing Rho activity. However, FTI-1 treatment can also increase the level of geranylgeranylated isoforms of certain Rho proteins, e.g., the inhibitory geranylgeranylated RhoB protein [ 60 ]. In further support of a role for isoprenoids, we were able to demonstrate that supplementation of cells with mevalonate abolished statin-activated shedding (see Figure 4 ). Statins block HMGCoA reductase generation of mevalonate from 3-hydroxy-methyl-glutarate (see Figure 7 ). Therefore, the addition of mevalonate would be predicted to antagonize statin action via the isoprenoid pathway, by relieving statin-induced mevalonate deficiency. As predicted by this model, we observed that statin-activated shedding was abolished by adding mevalonate. Taken together, these results suggest the existence of a reciprocal relationship between isoprenoid-mediated Rho/ROCK signaling and sAPP α shedding, i.e., activation of ROCK1 blocks basal and stimulated shedding while ROCK1 inhibition apparently relieves a tonic negative influence exerted on shedding by ROCK1 activity. As in PKC- and ERK-activated shedding, the ROCK1 substrate effector molecule or molecules that regulate proteolysis by ADAMs remain to be identified. The cytoplasmic domains of both APP and ADAM-17 have been evaluated as candidates for important targets of protein phosphorylation during the regulated shedding process, but neither “substrate activation” nor “enzyme activation” appears to explain the phenomenon, i.e., phosphorylation of neither APP nor ADAM-17 dramatically increases the efficiency of α-secretion [ 61 , 62 ], indicating that activation is more indirect. Our data using statins and PO localize the mediator of statin-activated shedding to the plasma membrane, upstream of endocytosis, as appears to be the case for PKC-activated shedding [ 36 , 63 , 64 , 65 ]. Similar conclusions were drawn by Bosenberg and colleagues [ 36 ] who used streptolysin-porated cells and N-ethylmaleimide to demonstrate that reconstitution of activated shedding of TGFα from CHO cells does not require membrane trafficking and apparently occurs on the plasma membrane. These results suggest that a tightly membrane-associated regulatory subunit of the α-secretase complex is likely to be the key phosphoprotein that mediates α-secretase activity as a function of its state of phosphorylation by PKC and perhaps also ERK and ROCK1. The molecular identity of this phosphoprotein remains unknown. α-Secretase activation is a potential therapeutic strategy for modifying cerebral amyloidosis in Alzheimer disease [ 66 ]. This proposal is supported by recent evidence that either genetic modification of ADAM-10/α-secretase activity [ 67 ] or administration of bryostatin, a PKC activator [ 68 ], can modulate levels of brain Aβ in plaque-forming transgenic mice. α-Secretase activation may explain how statins lower the risk for Alzheimer disease [ 69 ], since atorvastatin diminishes Aβ burden in plaque-forming transgenic mice [ 13 ]. If α-secretase stimulation is to be truly viable as a human clinical intervention, it will be essential to assess the possibility that enhanced APP ectodomain shedding might incur mechanism-based toxicity (analogous to the concerns currently surrounding γ-secretase inhibitors). Along this line, extension of this work to other shed proteins will be important to determine the impact of enhanced shedding via ADAM proteinases on other substrates of those proteinases, including Notch, pro-TGFα, pro-TNFα, and CD44 [ 3 ]. Preliminary results from a pilot proof-of-concept using atorvastatin in a human clinical treatment trial are consistent with the proposed beneficial effects of this class of compounds [ 70 ]. Since atorvastatin has low blood–brain barrier permeability [ 71 ], this beneficial effect, if attributable to Aβ lowering, must be due to altered Aβ metabolism in the periphery. Reduction in levels of free Aβ in the circulation has been demonstrated to lead to diminution in brain plaque burden following active or passive immunization [ 72 ]. It is conceivable that, if statins lower circulating Aβ, this effect could secondarily cause diffusion of central nervous system interstitial Aβ down its concentration gradient and into the cerebrospinal fluid and circulation, from which it is cleared. To date, however, this mechanism is not supported by data from human clinical trials, where statin administration has shown no consistent effect on levels of circulating or cerebrospinal fluid Aβ [ 73 ]. The results reported here point to several areas for additional investigation. As described above, the key substrate or substrates linking cytoplasmic protein phosphorylation to intralumenal or cell-surface protelysis remain to be identified. Nonetheless, α-secretase activation has been validated as a viable therapeutic strategy for modulating cerebral amyloidosis [ 67 ]. Identification of the role of the Rho/ROCK pathway in regulating α-secretase provides a new avenue for its therapeutic activation, even though the potential relevance of atorvastatin-mediated ROCK1 inhibition in neurons may not explain the apparent clinical benefits of the drug. Still, if the reported disease-modifying effect of atorvastatin is confirmed in the National Institute on Aging's large, multi-center trial of simvastatin, one or more compounds of this class may be among the first disease-modifying compounds approved by the Food and Drug Administration for slowing the progression of Alzheimer disease. Supporting Information Accession Numbers The GenBank ( http://www.ncbi.nlm.nih.gov/Genbank/ ) accession numbers for the proteins discussed in this paper are ROCK1 (NP_005397) and APP695, 595–596 KM/NL (NP_958817). Patient Summary Background Large-scale studies have found a link between taking cholesterol-lowering drugs called statins and a decreased risk of developing Alzheimer disease. But it is not clear why statins might protect people from getting the disease. The brains of people who have died from Alzheimer disease show remnants of damaged cells called “tangles” as well as “amyloid plaques” in the spaces between the cells. These plaques are mostly made up of collections of a protein called amyloid-beta. It is the buildup of this protein that is thought to cause the brain damage and dementia associated with Alzheimer disease. The protein itself is formed when another, larger protein called APP (Alzheimer amyloid-beta peptide precursor protein) is broken down (or cleaved). There are two ways in which APP can be broken down. “Bad cleavage” releases the toxic amyloid-beta, whereas “good cleavage” destroys it. When researchers gave statins to animals over a long period of time, they found that statins could slow down the formation of amyloid plaques. From the animal experiments, it seemed that statins somehow caused more good cleavage to occur. Why Was This Study Done? This study examined how statins can affect APP cleavage. What Did the Researchers Do? They studied cells to see which of the players known to be involved in APP cleavage were affected by statin. What Did They Find? Statin's ability to promote “good cleavage” of APP involves a molecular pathway called the Rho/ROCK1 pathway. It seems that when ROCK1 is active, less good cleavage takes place. But in the presence of statins, ROCK1 is less active, shifting the balance toward good cleavage. Consistent with this, when the scientists blocked the Rho/ROCK1 pathway, they saw the good cleavage pattern even without statin. What Does This Mean for Patients? Inhibition of the Rho/ROCK1 pathway could explain some of the beneficial effects of statins against Alzheimer disease. And the pathway itself seems worth more research to see whether it might be a good target for new ways to prevent and treat Alzheimer disease. What Are the Limitations of the Study? Statins are likely to influence the risk for Alzheimer disease by several different pathways, and future studies will need to show how important this particular pathway is in the overall picture. Moreover, studies like this one are by necessity done in cells under carefully controlled laboratory conditions and still a long way from the development of safe and effective drugs. More Information Online Factsheet on statins from the Alzheimer's Association: http://www.alz.org/Resources/TopicIndex/statins.asp General information at the Alzheimer's Disease Education and Referral Center at the United States National Institute of Aging: http://www.alzheimers.org/index.html Homepage of Alzheimer's Disease International, an umbrella organization of Alzheimer disease associations around the world: http://www.alz.co.uk/

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529453

Enhanced cell-permeant Cre protein for site-specific recombination in cultured cells

Background Cell-permeant Cre DNA site-specific recombinases provide an easily controlled means to regulate gene structure and function in living cells. Since recombination provides a stable and unambiguous record of protein uptake, the enzyme may also be used for quantitative studies of cis - and trans -acting factors that influence the delivery of proteins into cells. Results In the present study, 11 recombinant fusion proteins were analyzed to characterize sequences and conditions that affect protein uptake and/or activity and to develop more active cell-permeant enzymes. We report that the native enzyme has a low, but intrinsic ability to enter cells. The most active Cre proteins tested contained either an N-terminal 6xHis tag and a nuclear localization sequence from SV40 large T antigen (HNC) or the HIV Tat transduction sequence and a C-terminal 6xHis tag (TCH 6 ). The NLS and 6xHis elements separately enhanced the delivery of the HNC protein into cells; moreover, transduction sequences from fibroblast growth factor 4, HIV Tat or consisting of the (KFF) 3 K sequence were not required for efficient protein transduction and adversely affected enzyme solubility. Transduction of the HNC protein required 10 to 15 min for half-maximum uptake, was greatly decreased at 4°C and was inhibited by serum. Efficient recombination was observed in all cell types tested (a T-cell line, NIH3T3, Cos7, murine ES cells, and primary splenocytes), and did not require localization of the enzyme to the nucleus. Conclusions The effects of different sequences on the delivery and/or activity of Cre in cultured cells could not be predicted in advance. Consequently, the process of developing more active cell-permeant recombinases was largely empirical. The HNC protein, with an excellent combination of activity, solubility and yield, will enhance the use of cell-permeant Cre proteins to regulate gene structure and function in living cells.

Background The Cre recombinase from bacteriophage P1 has been widely used to induce DNA sequence-specific recombination in mammalian cells [ 1 ]. The enzyme, which catalyzes recombination between 34 nucleotide LoxP sequences during P1 genome replication, has been used in a variety of genetic applications to regulate gene structure and function. These include conditional mutagenesis, gene replacement, chromosome engineering, and regulated gene expression [ 2 - 4 ]. However, the use of site-specific recombination in genetic studies is frequently hampered by difficulties expressing the recombinase in cells at the desired time and place [ 5 ]. Moreover, the use of Cre expression vectors is constrained by the fact that prolonged exposure to the enzyme can be lethal to cells [ 4 , 6 , 7 ]. To address these problems, we [ 8 ] and others [ 9 - 12 ] have developed membrane-permeable Cre recombinase proteins that are capable of entering cells by a process of protein transduction. Protein transduction exploits properties of specific protein sequences [termed protein transduction domains (PTDs)] that enhance the delivery of macromolecules – including peptides, proteins, and DNA fragments – into living cells [ 13 - 15 ]. Cell-permeant Cre proteins provide an effective means to regulate gene structure and function in living cells, and Cre-mediated recombination provides a potentially useful reporter system with which to study the process of protein transduction itself. In particular, recombination provides a stable and quantitative record of protein uptake that circumvents problems of distinguishing between internalized and cell-associated proteins [ 16 ]. In our previous report, recombinant fusion proteins bearing the 12 amino acid membrane translocation sequence (MTS) from fibroblast growth factor 4 (FGF-4) were used to deliver enzymatically active Cre proteins directly into mammalian cells. Of the four recombinant proteins tested, an enzyme containing an N-terminal 6xHis affinity tag, a nuclear localization sequence (NLS) from SV40 large T antigen, and the FGF-4 MTS (HNCM), displayed the best combination of yield, solubility, nuclear localization and enzymatic activity within cells. Recombination was observed in greater than 70% of cells treated with 10 μM HNCM for 2 hours. Widespread recombination was also observed in mice following intraperitoneal administration of HNCM, indicating that a wide variety of terminally differentiated cell types can internalize cell-permeant Cre and are competent to undergo site-specific recombination. In the present study, eleven recombinant Cre proteins were prepared in order to evaluate sequences affecting the uptake and/or activity of the enzyme in cultured cells and if possible to develop more active recombinases. Several constructs were designed to compare the activities of different PTDs, including the FGF-4 MTS [ 17 ], sequences from HIV TAT [ 18 ] and a (KFF) 3 K sequence that was previously used to deliver peptide nucleic acid (PNA) conjugates into cells [ 19 ]. Only the Tat sequence promoted the delivery of active Cre into cells, while all three PTD sequences adversely affected the solubility of recombinant proteins containing polyhistidine tags. The contribution of the SV40 large T antigen NLS [ 20 ] was also examined to understand apparent differences in the behaviour of cell-permeant Cre and proteins expressed following gene transfer. Thus, the activity of cell-permeant Cre was enhanced by the SV40 large T antigen NLS [ 8 , 10 ], whereas, the native Cre protein appears to possesses a functional NLS, whose activity was not augmented by the T antigen NLS [ 21 ]. We report that polyhistidine tags (6xHis) frequently used for protein affinity purification [ 22 ] and the large T antigen NLS each separately enhance cellular uptake of enzymatically active Cre, and we describe the development of a cell-permeant Cre recombinase with an excellent combination of activity, solubility and ease of purification. Results Recombinant Cre fusion proteins Eleven recombinant Cre proteins were prepared in order to evaluate sequences affecting the uptake and/or activity of cell-permeant enzymes (Figure 1A ). Native Cre (Cre) corresponds to the protein encoded by the P1 phage genome [ 23 ]. HC and H 6 C have amino-terminal hexahistidine tags consisting of MGSSHHHHHHSSLVPRGSH and MHHHHHH, respectively, while CH 6 is similar to H 6 C except the His tag is on the C-terminus. His-NLS-Cre (HNC) is similar to HC except a nuclear localization sequence (PKKKRKV) from SV40 large T antigen [ 20 ] is positioned between His and Cre. HT 7 N'C is similar to HNC except the His tag contains 11 additional amino acids (MASMTGGQQMG) from the pET28a(+) polylinker and the arginine of the NLS sequence was converted to a lysine. This change, which resulted from an altered PCR primer, is unlikely to affect nuclear localization activity. HNCM, described previously [ 8 ], contains a membrane translocation sequence (MTS) from the leader sequence of FGF-4 positioned at the C-terminus of HNC. Finally, four different sequences, each reported to have protein transduction activity [ 17 , 19 , 24 ], were placed on the amino terminus of CH 6 . These consisted of the FGF-4 MTS (MCH 6 ), an SV40 large T antigen nuclear localization sequence (NCH 6 ), the HIV Tat sequence (TCH 6 ), and a (KFF) 3 K sequence (KCH 6 ). Cre proteins were expressed from pET28a(+) plasmids in E. coli and, except for native Cre, were purified by Ni 2+ affinity chromatography under non-denaturing conditions [ 8 ]. The native enzyme was purified by a combination of hydroxyapatite column chromatography and Sephacryl S-100 HR FPLC. All proteins were expressed at high levels, with yields of purified proteins ranging from 5 to 41 mg/L of E. coli culture (Figure 1 ). All of the enzymes except TCH 6 and KCH 6 could be dialyzed against DMEM or RPMI media and stored at -20°C until use. However, TCH 6 precipitated under these conditions and was dialyzed instead against PBS supplemented with 0.3 M NaCl (0.45 M total NaCl) and 8% glycerol. The pH of the buffer (ranging from 7.5–8.5) had no obvious effect on protein solubility. The KCH 6 protein was insoluble over a range of pH values and salt concentrations (up to 0.8 M), and the protein was not evaluated further. The remaining proteins could be prepared at final concentrations above 1 mg/ml except HNCM, which precipitated out of solution at protein concentrations above 500 μg/ml. The specific activities of the tagged fusion proteins were similar, ranging from 4.3–8.5 × 10 4 U/mg protein, corresponding to 47–92% of the activity of the native enzyme (Figure 1 ). Native Cre recombinase has protein transduction activity that is enhanced by polyhistidine and NLS sequences The uptake and enzymatic activity of Cre proteins was monitored in Tex.loxP.EG cells [ 8 ]. These cells contain a single integrated retrovirus (Fig 2A ) in which the expression of an enhanced green fluorescent protein (EGFP) gene is prevented by a "stop cassette" consisting of multiple polyadenylation sites positioned between two loxP sites. Deletion of the stop cassette by Cre mediated-recombination activates the expression of the EGFP reporter gene. Cells were exposed to Cre, HC, HNC, HT 7 N'C, HNCM, H 6 C, CH 6 , NCH 6 , and TCH 6 for 2 hrs at concentrations ranging from 0 to 8 μM. The cells were then washed extensively with PBS, were cultured for 24 hrs to provide time for EGFP expression, and the percentage of EGFP-expressing cells was determined by flow cytometry. Recombination was also monitored by Southern blot hybridization (Fig 2C ), thus confirming that expression of the EGFP reporter accurately reflected the extent of template recombination. As shown in Figure 2B , all of the proteins tested induced recombination in a concentration-dependent manner. The native enzyme had the lowest activity, inducing recombination in up to 17% of the cells. Uptake and/or activity was increased by polyhistidine tags positioned on either the amino- (HC and H 6 C) or carboxy-terminal (CH 6 ) ends of the enzyme. HC, which contains the 6xHis tag from pET28a(+), and H 6 C and CH 6 , which contain simple 6xHis sequences, induced similar levels of recombination, ranging between 45 and 60% of cells. Activity was further increased by the addition of an SV40 large T antigen nuclear localization signal (HNC, HT 7 N'C and NCH). At low concentrations, TCH 6 was the most active protein tested, but higher levels of the protein were toxic to cells, presumably because of the concentration of NaCl required to maintain protein solubility during preparation of the enzyme. Finally, presence of the FGF-4 MTS sequence proved inhibitory for recombination in cultured cells. HNC, which lacks the MTS sequence, was approximately 8 times more active than HNCM as determined by the concentration of enzyme required to induce recombination in 50% of the cells. Thus, while the FGF-4 MTS has been used to transduce a variety of proteins and peptides into mammalian cells, the sequence suppressed the activity of cell-permeant Cre. This confirms the results of a earlier study, reported while this work was in progress [ 10 ]. Temperature-dependent protein transduction Transduction of cargoes containing the FGF-4 MTS [ 17 ], including the HNCM protein used in this study [ 25 ], is greatly decreased at 4°C as compared to 37°C. By contrast, there have been conflicting reports with regard to the transduction of proteins containing the Tat and Antennapedia PTDs [ 26 - 33 ]. Cre fusion proteins with (HNCM) and without (HNC, HT 7 N'C and HC) the FGF-4 MTS were therefore tested for their ability to enter cells at 4°C (Fig 3A ). Tex.loxP.EG cells were incubated with varying concentrations of the proteins for one hour at either 37°C or 4°C; were washed extensively and were cultured at 37°C to allow time for EGFP expression. In each case, the uptake of the enzyme was inhibited at 4°C, indicating that the inhibitory effects of low temperature are not limited to cargoes containing the FGF-4 MTS. Low levels of recombination observed in cells treated with higher concentrations probably results from cell-associated enzyme that the washing steps fail to remove and that gains entry when cells are later cultured at 37°C. Serum and cell density effects on protein transduction Serum has been reported to inhibit the transduction of cargoes containing the FGF-4 MTS; whereas, transduction of proteins containing the Tat and Antennapedia PTDs appears to be unaffected by serum. The effects of serum on protein transduction were assessed by treating TexloxP.EG cells with either HC (5 μM) or HNC (2 μM) for 1 hour in the presence of varying concentrations of either fetal bovine serum (Figure 3B , FBS) or normal mouse serum (Figure 3B , MS). Recombination induced by both proteins was inhibited by up to 60% and 80% in media containing 10% FBS and MS, respectively. Serum appeared to inhibit protein transduction specifically, since it had no discernable effect on either the stability or activity of the proteins in vitro (data not shown). These results indicate that the inhibition of protein transduction by serum is not limited to cargoes containing the FGF-4 MTS. To assess the effects of cell density on protein transduction, TexloxP.EG cells were seeded at different concentrations in 2 cm 2 culture dishes and were treated with HNC (2 μM) for 1 hour (Figure 3C ). Recombination efficiencies increased by about 40% as the number of number of cells was increased from 10 4 to 6 × 10 4 cells/cm 2 , and then declined sharply at concentrations above 2 × 10 5 cells/cm 2 . Tex.loxP.EG is a T-cell line and is non-adherent; however, the cells settle to the bottom of the culture dish. The optimum density for recombination was similar to the number of cells (.75 × 10 4 cells/cm 2 ) required to cover the culture dish. Uptake and localization of cell-permeant Cre proteins The kinetics of cell-permeant Cre uptake in cultured cells was monitored by examining cells for recombination after exposure to cell-permeant Cre for different lengths of time (Figure 4 ). Cells were treated with HC (5 μM), HNC (3 μM) and HNCM (10 μM) for 5 to 120 mins, were washed extensively and the extent of recombination was monitored 24 hours later by flow cytometry. Thus, the assay measures the time required for extracellular enzyme to become committed to cell entry. The uptake of HC, HNC and HNCM increased with time, reaching half-maximum levels after 15–20 min. Recombination was induced in cells following exposure to HNC and HNCM proteins for less time than to HC, suggesting a direct role for the NLS in promoting cell binding and/or entry. Note that the observed differences (apparent within minutes after exposing cells to Cre) cannot reflect potential differences in nuclear trafficking since a delay in nuclear import of a few minutes would not affect the extent of recombination measured 24 hours later. The uptake and localization of cell-permeant Cre in cultured cells was also monitored in living cells by using proteins labeled with the fluorescent dye, Alexa 488 (Figures 5 and 6 ). The uptake of tagged HC, HNC and HNCM increased with the time as measured by flow cytometry (Figure 5 ). Again, HNC and HNCM appeared to enter cells more rapidly than HC, which lacks an NLS. Note that the magnitude of the fluorescence is less important than the rate of increase (slope) since the proteins were not labelled to the same extent with Alexa 488. The localization of Alexa 488-tagged HNC and HC proteins was monitored in living cells by fluorescence microscopy. The HNC protein was localized to the nuclei of Cos7 cells, but was predominately cytoplasmic in NIH3T3 cells (Fig 6B ). Cre protein was also localized to the nuclei of Tex.loxP.EGcells (data not shown). Recombination in different cell types Several mammalian cell types were analyzed for their ability to undergo Cre-mediated recombination. Clones of Cos7 and NIH3T3 cells containing a single pBABE.lox.stp.EGFP provirus were treated with HNC for two hour and analyzed by Southern blot hybridization (Figure 7 ). Although the concentration of enzyme necessary to achieve nearly complete recombination was approximately 10 times higher in Cos7 cells than in NIH3T3 cells (10 versus 1 μM, Figure 7 ), the recombination efficiency did not correlate with localization of the enzyme to the nucleus (Figure 6 ). Extensive recombination was also observed in murine embryonic stem cells containing a floxed IKK γ gene (Figure 7 ) and in primary splenocytes explanted from ROSA26R mice (Figure 8 ). Efficient recombination was therefore observed in all mammalian cell types examined. Discussion Cell permeant Cre proteins have generated considerable interest as genetic tools to regulate gene structure and function in mammalian cells [ 5 ]. In addition, Cre mediated recombination provides a quantitative reporter for studies on the protein transduction process itself. In the present study, 11 recombinant fusion proteins were analyzed to characterize sequences and conditions that affect protein uptake and/or activity and to develop more active cell-permeant enzymes. We report that the native enzyme has a low, but intrinsic ability to enter cells. Uptake and was enhanced by the addition of a 6xhistidine-tag on either the amino or carboxyl terminal ends of the protein and was enhanced further by the addition of a nuclear localization sequence from SV40 large T antigen or the transduction sequence from the HIV Tat protein. Finally, the hydrophobic membrane translocation sequence (MTS) from fibroblast growth factor 4 (FGF-4) had a net deleterious effect on Cre-mediated recombination in cultured cells. We had hoped that the native or 6xHis-tagged enzymes would lack transduction activity so that the effects of additional sequences on cell entry, nuclear transport and recombination could be compared. However, we found that the native Cre protein has an intrinsic ability to enter cells, thus confirming observations by Will, et al. [ 11 ]. The mechanism by which Cre gains entry into cells remains to be determined. The enzyme may possess a protein transduction domain analogous to those described for HIV Tat, Antennapedia and a growing list of proteins that can enter cells directly, without requiring specific receptor and transporter systems [ 34 ]. The fact that Cre is a basic protein [ 23 ] is potentially significant considering that many basic peptides are capable of entering cells [ 24 , 35 - 38 ]. The transduction activity of the native enzyme hinders quantitative studies of sequences incorporated into recombinant Cre proteins, since structural changes associated with each modification may have varying effects on the intrinsic ability of Cre to enter cells. Even so, the 6xHis sequence appeared to facilitate cell entry, since two different 6xHis sequences enhanced Cre-mediated recombination while having little effect on the activity of the enzyme in vitro . Moreover, 6xHis sequences were active when positioned on either the amino- or carboxyl-terminal ends of the enzyme. L-histidine heptamers have been shown to enter cells, although much less efficiently than arginine homopolymers [ 37 , 38 ]. Positively charged histidine sequences also bind cell surface heparin sulfate proteoglycans [ 39 , 40 ], and thus may enhance uptake as has been suggested for the HIV Tat transduction sequence [ 41 ]. The transduction activity of the 6xHis sequences is potentially significant given their widespread use as affinity tags to purify recombinant fusion proteins [ 22 ]. We [ 8 ] and others [ 10 ] have shown that the activity of cell-permeant Cre fusion proteins in cultured cells can be enhanced by the addition of an SV40 large T antigen nuclear localization sequence (NLS). The NLS has been shown to enhance the activity of Cre expression vectors [ 42 ], presumably by targeting the protein to the nucleus. However, in the present study the NLS enhanced the delivery of Cre fusion proteins into cultured cells as assessed either by cell-based recombination or by uptake of fluorescent Cre proteins. Moreover, nuclear localization did not appear to contribute to cell type-specific differences in the activity of the HNC protein. These results are consistent with the observation that the T antigen NLS, like a number of other basic sequences, has protein transduction activity [ 24 ]. HNC, which consisted of a 6xHis tag and T antigen NLS attached to the amino-terminus of the enzyme, was highly active despite the absence of a canonical protein transduction sequence. The 6xHis tag and NLS sequences separately contributed to the transduction of the enzyme, which was 5–8 times more active in cultured cells than the HisNLSCreMTS (HNCM) protein described in our earlier study [ 8 ]. By virtue of their positive charge, these elements share similarities with basic protein transduction domains such as HIV Tat and Antennapedia. Early studies suggested that the basic PTDs entered cells through an energy-independent process that was relatively unaffected by low (4°C) temperature [ 26 - 30 ]. However, later studies suggest that the positively charged PTDs promote cell uptake by endocytosis possibly by binding negatively charged proteins on the cell surface [ 12 , 31 - 33 , 43 , 44 ]. Similarly, the uptake of Cre fusion proteins is consistent with an endocytic mechanism. In particular, uptake (time required for commitment to entry) was relatively rapid (10 to 15 min for half maximum uptake), was greatly decreased at 4°C, and was inhibited by up to 80% by serum. The inhibition by serum appeared to involve cell binding and/or entry specifically, since serum had no discernable effect on the stability or activity of Cre fusion proteins in vitro (data not shown). Recombination was markedly suppressed at higher cell densities, possibility because binding sites in or on cells sequester the enzyme, thus lowering the effective protein concentration. Alternatively, cell density may suppress protein transduction by reducing cell size and/or available surface area [ 25 ]. In either event, similar observations have been reported for a TatCre protein that lacks a 6xHis tag [ 9 ], illustrating the need to control for cell density when comparing the effects or cell-permeant proteins in different cell populations. Conclusions The effects of different sequences on Cre uptake or activity were difficult to predict in advance, and consequently, the process of developing a more active cell-permeant recombinase was largely empirical. Proteins containing amino-terminal Tat and (KFF) 3 K sequences (TCH 6 and KCH 6 ) were poorly soluble when dialyzed against isotonic media, and the FGF-4 MTS interfered with the activity of the enzyme in cultured cells. Since Cre is probably not unique in this regard, investigators seeking to develop other cell-permeant proteins would be advised to test a variety of sequences positioned at different places on the protein. In the present case, the HNC protein possesses an outstanding combination of activity, solubility and yield that will enhance the use of cell-permeant Cre to regulate gene structure and function in living cells. Methods Cre expression vectors Native Cre and Cre fusion proteins were expressed in E. coli from pET28a (+)-based plasmids (Novagen). A plasmid expressing native Cre (Cre) was constructed by using the Cre #1 and Cre-stop-HindIII primers to amplify Cre coding sequences from MBP-NLS-Cre-MTS [ 8 ] which were cloned between the Nco I and Hind III sites of pET28a (+). HC, His 6 C, HNC, and HT 7 C were similarly constructed by PCR amplification, using primers Cre #2, Cre #3, Cre #4 and Cre #5, respectively, together with Cre-stop-HindIII primer. The amplified DNA fragments were cloned into Nhe I and Hind III sites of pET28a (+). CHis 6 , NCHis 6 , MCHis 6 , TATCHis 6 , and (KFF) 3 KCHis 6 were constructed by using primers Cre #6, Cre #7, Cre #8, Cre #9 and Cre #10, respectively, together with the Cre-XhoI primer, and then cloned into the Nco I and Xho I sites of pET28b(+). Cre #1: AGAGAGCCATGGGCTCCAATTTACTGACCGTACACCAA Cre #2: GTACATGCTAGCTCCAATTTACTGACCGTACACCAA Cre#3: AGAGAGCCATGGGCCATCATCATCATCATCACAGCTCCAATTTACTGACCGTACACCAA Cre#4: GTACATGCTAGCCCAAGAAGAAGAGGAAGGTGCTCCAATTTACTGACCGTACACCAA Cre#5: GTACATGAATTCTCCAATTTACTGACCGTACACCAA Cre#6: AGAGAGCCATGGGCTCCAATTTACTGACCGTACACCAA Cre#7: AGAGAGCCATGGGCCCCAAGAAGAAGAGGAAGGTGTCCAATTTACTGACCGTACACCAA Cre#8: AGAGAGCCATGGGCGCAGCCGTTCTTCTCCCTGTTCTTCTTGCCGCACCCTCCAATTTACTGACCGTACACCAA Cre#9: AGAGAGCCATGGGCGGTCGCAAGAAACGTCGCCAACGTCGCCGTTCCAATTTACTGACCGTACACCAA Cre #10: AGAGAGCCATGGGCAAATTCTTTAAGTTCTTTAAGTTCTTTAAGTCCAATTTACTGACCGTACACCAA Cre-stop-HindIII: GATACGAAGCTTCTACTAATCGCCATCTTCCAGCAGGCGC Cre – XhoI: AGAGAGCTCGAGATCGCCATCTTCCAGCAGGCGCACCATTGCCCCTGT Protein purification Polyhistidine-tagged Cre fusion proteins were expressed in E. coli strain BL21 (DE3) and purified by Ni 2+ affinity chromatography as described previously [ 8 ]. Bacterial cultures (2L) were grown to an A 600 of 0.6–1.0, were induced with 0.4 mM IPTG, and after harvesting the cells were lysed in 40 ml 50 mM Tris (pH 8.0), 50 mM sodium phosphate and 300 mM NaCl. After affinity chromatography, recombinant proteins were dialyzed against DMEM or RPMI media, except TCH 6 and KCH 6 , which precipitated under these conditions. TCH 6 was dialyzed instead against PBS supplemented with 0.3 M NaCl (0.45 M NaCl final) and 8% glycerol. The native recombinase was similarly expressed; however, lysates were applied to a 50 ml hydroxyapatite column and step-eluted with sodium phosphate buffers (pH 8.0) of increasing concentration (50, 100, 200, 300, and 500 μM). The peak fraction (200 μM) was dialyzed against PBS, was concentrated by centrifugal ultra filtration to about 20 mg/ml and was fractionated by Sephacryl S-100 HR gel filtration FPLC. Peak fractions identified by SDS PAGE, were concentrated and dialyzed against PRMI-1640 medium containing 1% streptomycin/penicillin and 2% glycerol. In vitro assays of Cre enzyme activity measured the release of a circular plasmid inserted into a λ phage (Novagen, Madison, WI) by transformation of E. coli [ 8 ]. One unit (U) of enzyme produces 10 4 colonies (equivalent to 2 × 10 6 circular molecules) in a 30 minute reaction containing 200 ng DNA substrate in 50 mM Tris HCl, pH 7.5, 33 mM NaCl and 10 mM MgCl 2 in a total volume of 15 μl. All Cre proteins were stable for at least 6 months at -80°C without significant loss of enzymatic activity. Cell culture and protein transduction Tex.loxp.EG, 3T3.loxp.EG and Cos7.loxp.EG cells were derived from Tex (a murine thymoma line derived from p53-deficient mice), NIH3T3 and Cos7 cells, respectively, following infection with the pBABE.lox.stp.EGFP retrovirus [ 8 ]. Cells were incubated with serum-free RPMI 1640 (Tex.loxp.EG) or DMEM (3T3.loxp.EG and Cos7.loxp.EG) containing Cre fusion proteins for 2 hours, then washed with PBS twice and cultured in normal growth medium at 37°C incubator for 24 hours. Cre mediated recombination, which induces the expression of an enhanced green fluorescence protein (EGFP) in Tex.loxp.EG cells, was measured by flow cytometry using a FACSort instrument (Becton Dickinson). Alternatively, recombination was monitored by Southern blot hybridization [ 8 ]. For experiments involving low temperature protein transduction, the cells and all solutions were maintained at 4°C until after washing with cold PBS, after which the cells were returned to normal medium at 37°C. Preparation of fluorescent HNC, HC, and HNCM proteins Fluorescent HNC, HC, and HNCM proteins were prepared by using the Alexa 488 protein labeling kit (Molecular Probes, catalog number A-10235), according to the manufacturer's instructions. The proteins were dialyzed against PBS, were labeled at a concentration of 2 mg/ml and purified by gel filtration through a Sephadex G50 column. Cells were treated with the fluorescent proteins at a final concentration of 1 μM. The localization of fluorescent Cre proteins was monitored in living NIH3T3 and Cos7 cells by fluorescence microscopy. The cells were cultured to 50–80% confluence in a slide chamber (NUNC), washed with PBS and then incubated with fluorescent Cre proteins at a final concentration of 1 μM for 1.5 hours. The cells were washed three times with PBS, counterstained with 1 μg/ml 4',6-diamidino-2-phenylindole (DAPI) in PBS for 20 minutes, washed three times with PBS and mounted in anti-fade fluorescence mounting medium. The stained cells were photographed with an Olympus BX60 fluorescence microscope using green and blue filters. Ex vivo recombination by HNC recombinase in primary cells Splenocytes from ROSA-26R mice were cultured for one day in RPMI 1640 medium containing 10% FBS and were treated with HNC recombinase in serum free media for 2 hours. The cells were washed, cultured for 24 hours, and stained with a fluorescent β-galactosidase substrate (ImaGene green™, C 12 FDG) according to instructions provided by the manufacturer (Molecular Probes, Inc.) Recombination, which activates the expression of a floxed lacZ gene, was assessed by flow cytometry. Authors' contributions QL developed and characterized most of the recombinant Cre proteins described in this study. DJ provided the HNCM protein and assisted with in vitro assays for Cre activity. KDGA. investigated inhibition of Cre activity by serum. ER supervised the project and drafted the final manuscript. All authors approved the final manuscript.

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514711

A faster way to make GFP-based biosensors: Two new transposons for creating multicolored libraries of fluorescent fusion proteins

Background There are now several ways to generate fluorescent fusion proteins by randomly inserting DNA encoding the Green Fluorescent Protein (GFP) into another protein's coding sequence. These approaches can be used to map regions in a protein that are permissive for GFP insertion or to create novel biosensors. While remarkably useful, the current insertional strategies have two major limitations: (1) they only produce one kind, or color, of fluorescent fusion protein and (2) one half of all GFP insertions within the target coding sequence are in the wrong orientation. Results We have overcome these limitations by incorporating two different fluorescent proteins coding sequences in a single transposon, either in tandem or antiparallel. Our initial tests targeted two mammalian integral membrane proteins: the voltage sensitive motor, Prestin, and an ER ligand gated Ca 2+ channel (IP 3 R). Conclusions These new designs increase the efficiency of random fusion protein generation in one of two ways: (1) by creating two different fusion proteins from each insertion or (2) by being independent of orientation.

Background Biosensors based on GFP-fusion proteins are powerful tools for observing real-time events within living cells. Insertion of GFP within another protein has produced biosensors capable of signaling intracellular events through intrinsic fluorescence changes [ 1 , 2 ], fluorescence resonance energy transfer (FRET) [ 3 , 4 ], and changes in sub-cellular localization [ 5 ]. The difficult task of finding the right insertion site to produce a biosensor can be accelerated by screening libraries of random GFP insertions [ 6 - 8 ]. The insertional strategies described to date, however, are limited in two ways. First, each insertion produces only one kind, or color, of fluorescent fusion protein. Creating the multicolored libraries necessary for co-expression or FRET analyses requires either separate rounds of insertion and screening for each fluorescent protein or additional subcloning to exchange fluorophores. Second, the efficiency of any random approach is limited to a maximum of 1:6 because a fusion protein can only be produced if the GFP coding sequence lands in the correct orientation and reading frame with respect to the target coding sequence. We reasoned that it might be possible to overcome these limitations by placing two different fluorescent protein coding sequences in a single transposon, either in tandem or antiparallel. Here we present the results of our initial tests with these designs. Results and Discussion The mosaic ends (MEs) that define the hyperactive Tn 5 transposon [ 9 ] have two possible open reading frames (ORFs) through them. We used one of these reading frames to construct the Either-Or transposon (<EYOR>, Figure 1A ). <EYOR> carries the sequence encoding the yellow fluorescent protein (YFP) at its 5' end, flanked by two 8 bp restriction sites ( Asc I – 5' and Srf I – 3'). An identical cassette encoding cyan fluorescent protein (CFP) flanked with Asc I and Srf I sites, is positioned in the same orientation at the 3' end of the transposon. Tn 5 transposition, in vitro , is only ~1% efficient [ 10 ], so the kanamycin resistance gene ( Kan R ) was incorporated between the two fluorescent protein cassettes. Since the YFP sequence has no start codon, it should only be translated if it inserts within another protein coding sequence in the correct orientation and reading frame. Plasmids with transposon insertions that are in-frame with respect to the target coding sequence can be rapidly identified by screening for YFP fluorescence in transiently transfected mammalian cells. Each of these clones produces a truncated fusion protein with YFP at the C-terminus. A stop codon is positioned downstream of the Srf I site to prevent translation beyond the YFP coding sequence. These truncated fusion proteins may provide additional information about which parts of the primary sequence contain trafficking signals. Full-length YFP and CFP fusion proteins are then generated in parallel from each clone by digestion with Srf I or Asc I and re-ligation. By producing identical full-length YFP and CFP fusion proteins from each in-frame insertion <EYOR> should double the efficiency with which multicolored fusion protein libraries can be generated. To test the <EYOR> transposon we targeted Prestin, an integral membrane protein expressed in outer hair cells of the cochlea and believed to be the motor responsible for their rapid changes in length in response to fluctuations in membrane voltage [ 11 ]. The 2.2 kb cDNA encoding Prestin was expressed in a 4.8 kb Ampicillin resistant ( Amp R ) CMV expression plasmid, pBNJ12.5. Transposon insertions that disrupt the plasmid origin or Amp R (together ~1.5 kb) are not recovered [ 8 ], so the predicted number of in-frame insertions in Prestin, is ~7%. After transposition, plasmids conferring Amp R and Kan R were isolated by standard mini-prep procedures and transiently expressed in HEK-293 cells in a 96-well format. In a random sample of 192 transposed clones, 32 produced detectable fluorescence. Eighteen of these (~9%) were clearly localized to intracellular membranes. (Figure 2A ). Of the remaining fluorescent proteins 11 appeared to be YFP alone, with evenly distributed fluorescence throughout the entire cell, and 3 were too dim to determine any sub-cellular localization. Sequencing revealed that the 18 proteins targeted to intracellular membranes were truncated Prestin-YFP fusion proteins resulting from in-frame insertions at 12 unique sites (Figure 2D ). One of the YFP-like clones was an in-frame insertion in the intracellular N-terminus of Prestin (at amino acids 28–30) upstream of the first predicted transmembrane domain. The remaining 13 fluorescent proteins resulted from <EYOR> insertions outside the Prestin coding sequence, most of them being clustered just downstream of the CMV promoter. We could not identify an in-frame start codon (AUG) in any of these clones. There is however, an in-frame CUG codon at the 5' end of the Tn 5 ME sequence that may present an alternate translation initiation site in the presence of the strong CMV promoter [ 12 ]. To verify that <EYOR> could be used to generate full-length fusions with either YFP or CFP unique in-frame clones were digested in parallel with Srf I or Asc I and re-ligated. The resulting fusion constructs were transiently expressed in HEK-293 cells and screened for YFP and CFP fluorescence (Figure 2B,2C ). All 13 unique insertion sites produced fluorescent full-length fusions with both CFP and YFP and all were localized to intracellular membranes. The <EYOR> design could be expanded for a wide range of protein tagging applications by replacing the secondary CFP cassette with another open reading frame. With such a transposon, YFP fluorescence would be used as a reporter to rapidly identify random in-frame insertions. Subsequent digestion with Asc I and re-ligation could then generate fusion proteins that might otherwise be difficult to screen for such as epitope tags, protease cleavage sites, or even a new N-terminus complete with a secretory signal peptide. Several groups have reported similar strategies based on multi-domain transposons for the random insertion of small peptide tags [reviewed in:[ 13 ]]. Like <EYOR>, these transposons utilize a primary reporter domain to identify in-frame insertions. Subsequent excision of the reporter domain (and selectable marker) then restores the full-length target coding sequence with an inserted peptide tag. The <EYOR> design is unique, however, in that its overlapping pairs of Asc I and Srf I restriction sites, allow the user to create identical full-length fusion proteins from both the reporter domain and the secondary coding sequence. The second transposon design, the Double-Barrel transposon (<DBT>, Figure 1B ), encodes green and red fluorescent proteins (GFP and DsRed) in opposite orientations. This means that any <DBT> insertion within another protein coding sequence has a 1:3 chance of being in-frame regardless of its orientation. Therefore, <DBT> should double the efficiency of random fusion protein generation, by producing equal numbers of GFP and DsRed fusions. In addition to their antiparallel orientation, the GFP and DsRed coding sequences in <DBT> each use a different relative reading frame through the Tn 5 MEs. As in <EYOR>, GFP fusion proteins are created by insertions after the third nucleotide of a target codon. The DsRed coding sequence, however, has been shifted by 1 nucleotide relative to the Tn 5 MEs. Therefore, DsRed fusion proteins are generated by transposon insertions between the second and third nucleotides. Using different reading frames for GFP and DsRed doubles the total number of insertion sites in the target coding sequence from which fusion proteins could potentially be made. While this does not alter the frequency of in-frame insertions, it does reduce the screening cost of saturating a target clone by increasing the probability of recovering unique in-frame insertions. To test the efficiency of fusion protein generation with the <DBT> transposon, we targeted pCMVI-9, a CMV expression plasmid carrying cDNA encoding the type 1 IP 3 receptor (IP 3 R) [ 14 ]. The IP 3 R is a ligand gated Ca 2+ channel, composed of 4 homomeric subunits, expressed within the membranes of the endoplasmic reticulum. Each IP 3 R subunit is over 2700 amino acids, and creating a full-length fluorescent IP 3 R fusion protein with such a large cDNA presents a formidable challenge for traditional molecular biological techniques. The high ratio of coding sequence to vector makes it an excellent target for transposition, however, with a predicted frequency of in-frame insertions of ~11% per fluorophore for <DBT>. At 24 hrs post-transfection, visual screening for fluorescence of 288 Amp R + Kan R clones in HEK-293 cells identified 44 clones that produced green fluorescent proteins. Of these, 35 displayed a uniform cytoplasmic distribution and exclusion from the nucleus (Figure 3A ), 3 showed fluorescence throughout the entire cell and 6 were too dim to determine any sub-cellular localization. Screening at several time points between 2 and 4 days post-transfection identified 7 clones producing red fluorescent proteins, 2 of which were clearly excluded from the nucleus (Figure 3B ). The remaining 5 red proteins were too dim to determine any sub-cellular localization. Sequencing out of the transposon confirmed that 41 of the clones encoding green proteins (~14%) and 2 clones encoding red proteins represented in-frame insertions. Consistent with the results of the Prestin transposition, all of the proteins with sub-cellular localization different from that seen with GFP alone were the product of in-frame insertions. After digestion and religation, only 20 of the full-length fusion proteins retained detectable levels of fluorescence (18 green, 2 red). These full-length proteins displayed a dramatic shift in their distribution, with clear ER localization (Figure 3C ). We chose GFP and DsRed to build the <DBT> transposon because their coding sequences are so dissimilar. Our concern was that if we chose two similar sequences, CFP and YFP for example, the antiparallel orientation of these coding sequences could produce extensive mRNA hybridization and secondary structure that would inhibit protein translation. It appears however, that DsRed is not well suited for insertion within other proteins. Indeed, DsRed has not been reported as a fusion protein in the middle of another protein, and even N- and C-terminal fusions with DsRed can be problematic [ 15 ], perhaps due to its being an obligate multimer [ 16 ]. Despite the low yield of DsRed fusions, these results demonstrate that <DBT> can be used to simultaneously generate full-length fusion proteins in two different reading frames. As novel fluorescent proteins are isolated from new species, or old ones are altered, this type of bi-directional transposon could potentially double the output of the screening process. Conclusions The transposons described here should greatly accelerate the creation of multicolored libraries of fluorescent fusion proteins. By creating identical full-length YFP and CFP fusion proteins from each in-frame insertion, the <EYOR> transposon not only facilitates the generation potential FRET pairs, it enables the direct comparison of different fluorophores in otherwise identical fusion proteins. The <DBT> design, on the other hand, has the capacity to double both the throughput of fusion protein generation by virtue of its bi-directionality as well as the total output of novel fusion proteins through the simultaneous use of multiple reading frames. Ultimately, the ability to generate large numbers of novel fusions proteins in days rather than months, should shift the limiting rate at which novel fluorescent protein biosensors are identified to functional screening rather than protein design and construction. Methods Plasmids PCR and standard subcloning procedures were used to create the plasmids encoding <EYOR> (pBNJ55b.1), <DBT> (pBNJ38.5) and Prestin (pBNJ12.5) (full sequences in supplementary material). The fluorescent protein coding sequences used were Venus (YFP) [ 17 ], ECFP-N164H (CFP), EGFP (GFP) and DsRed2 (Clontech). The Kan R gene was obtained from pUniV5-His-TOPO (Invitrogen). The construction of the IP3 receptor expression plasmid, pCMVI-9, was previously described [ 14 ]. Tn5 transposition and plasmid isolation Transposons were amplified from their host plasmids via PCR with a single primer complementary to the 19 bp Tn 5 ME (5'-CTGTCTCTTATACACATCT-3') and purified as previously described [ 8 ]. Purified transposon and target concentrations were each quantified against an independent DNA standard using a DynaQuant 200 fluorimeter. The transposition reaction was performed according to manufacturer's recommendations (Epicentre Technologies, Madison, WI) with 200 ng of target DNA and a molar equivalent of purified transposon. Electrocompetent XL-10 Gold E. coli (Stratagene, La Jolla, CA) were transformed with 0.5 μL of the transposition reaction and plated on LB agar with Ampicillin (100 μg/mL) and Kanamycin (50 μg/mL). Parallel plating of the transformation on LB agar with Ampicillin alone was used to establish the transposition efficiency. Transposed plasmids were isolated in a 96-well format from 1.25 mL LB cultures with Eppendorf PerfectPREP-96 Vac Direct Bind miniprep kits on a PerkinElmer MultiPROBE II HT liquid handling robot and eluted in 70 μL of ddH 2 O. Visual screening HEK-293 cells (American Type Culture Collection CRL-1573) were plated 24 hr prior to transfection, in 96-well glass bottom tissue culture plates (NalgeNUNC) at 6 × 10 4 cells in 100 μL of MEM-E with 10% fetal bovine serum. Transfections were performed with ~300 ng of plasmid DNA and 0.3 μL of Lipofectamine 2000™ (Gibco BRL) in a total volume of 50 μL of Opti-MEMI (Gibco BRL) per well. The cells were screened for fluorescence 24 hr after transfection with a 20× objective on a Zeiss inverted microscope with excitation and emission filter sets optimized for CFP/YFP or GFP/DsRed imaging (Omega, Brattleboro, VT). Sequencing and generation of full-length fusion proteins Exact insertion sites were identified for all fluorescent transposed clones by sequencing 5' out of the transposon with a primer complimentary to the <EYOR> YFP coding region (5'-CTGCAGGCCGTAGCC-3') or <DBT> GFP coding region (5'-TGGCCGTTTACGTCGCCGTCCA-3'). To generate full-length fusion proteins, plasmids with unique in-frame insertions were digested and re-ligated. After restriction digestion (100 ng of plasmid DNA and 0.5 U of Asc I or Srf I in 10 μL total volume), 1 μL of the digest reaction (~20 ng DNA) was re-ligated with Fast-Link™ ligase (Epicentre Technologies) for 15 min at room temperature (0.5 mM ATP, 1X Fast-Link™ buffer, 1 U ligase, 7.5 μL total volume). After heat inactivation (70°C for 15 min.), XL-10 Gold E. coli were transformed with 0.5 μL of the ligation reaction and plated on LB agar with Ampicillin. The following day, colonies were co-inoculated in LB with Ampicillin and Ampicillin + Kanamycin to verify loss of the Kan R prior to plasmid isolation. Authors' contributions D.S. conceived of the transposon designs and carried out their construction, performed the transposition and screening for fluorescent fusion proteins, and drafted the manuscript. T.H. participated in the study design, coordination, and analysis. All authors have read and approved the final manuscript. Supplementary Material Additional File 1 Supplementary Material-hughes This is a PDF file containing both plasmid maps and full sequence data for the plasmids used in this study. Click here for file

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Dissection and Design of Yeast Prions

Many proteins can misfold into β-sheet-rich, self-seeding polymers (amyloids). Prions are exceptional among such aggregates in that they are also infectious. In fungi, prions are not pathogenic but rather act as epigenetic regulators of cell physiology, providing a powerful model for studying the mechanism of prion replication. We used prion-forming domains from two budding yeast proteins (Sup35p and New1p) to examine the requirements for prion formation and inheritance. In both proteins, a glutamine/asparagine-rich (Q/N-rich) tract mediates sequence-specific aggregation, while an adjacent motif, the oligopeptide repeat, is required for the replication and stable inheritance of these aggregates. Our findings help to explain why although Q/N-rich proteins are relatively common, few form heritable aggregates: prion inheritance requires both an aggregation sequence responsible for self-seeded growth and an element that permits chaperone-dependent replication of the aggregate. Using this knowledge, we have designed novel artificial prions by fusing the replication element of Sup35p to aggregation-prone sequences from other proteins, including pathogenically expanded polyglutamine.

Introduction The aggregation of misfolded proteins underlies a diverse range of human diseases, including sporadic amyloidoses such as Alzheimer's disease and hereditary neuropathies such as Huntington's disease ( Dobson 1999 ). Prions are a special class of protein aggregates that replicate their conformation and spread infectiously ( Prusiner 1998 ). After the discovery that prion aggregates are responsible for the mammalian transmissible spongiform encephalopathies, several epigenetically heritable traits in fungi were also found to depend on a prion mechanism ( Wickner 1994 ; Uptain and Lindquist 2002 ; Osherovich and Weissman 2004 ). In Saccharomyces cerevisiae and Podospora anserina, prions are transmitted from cell to cell through mating and cell division, resulting in readily assayed phenotypes with a non-Mendelian pattern of inheritance ( Liebman and Derkatch 1999 ). The yeast non-Mendelian factors [ PSI + ] ( Cox 1965 ) and [ URE3 ] ( Lacroute 1971 ), which are prion forms of the translation termination factor Sup35p and the transcriptional activator Ure2p, respectively, have served as useful models for the formation and replication of heritable protein aggregates. Prion forms of Sup35p and Ure2p lead to defects in their respective biochemical activities (translation termination and nitrogen catabolite repression). Mutational analysis has shown the glutamine/asparagine-rich (Q/N-rich) amino-terminal (N) domains of these proteins to be critical for prion behavior ( Ter-Avanesyan et al. 1993 ; Masison and Wickner 1995 ; Patino et al. 1996 ; Paushkin et al. 1996 ; DePace et al. 1998 ). In vitro, these Q/N-rich domains form self-seeding, β-sheet-rich amyloid fibrils similar to those associated with Alzheimer's and Huntington's diseases ( Glover et al. 1997 ; King et al. 1997 ; Taylor et al. 1999 ). The autocatalytic aggregation of yeast prion proteins often shows a high specificity for like molecules; for example, Sup35p N domains from different yeast species form prion aggregates that preferentially interact with molecules of their own kind ( Santoso et al. 2000 ; Chernoff et al. 2000 ; Kushnirov et al. 2000 ; Zadorskii et al. 2000 ; Nakayashiki et al. 2001 ). [ PSI + ] and [ URE3 ] can be eliminated by transient growth in the presence of guanidine hydrochloride (GuHCl), which “cures” cells of prions by inhibiting Hsp104p, a molecular chaperone needed for prion replication ( Chernoff et al. 1995 ; Jung et al. 2002 ; Ness et al. 2002 ). A surprisingly large number of proteins in S. cerevisiae and other eukaryotes have lengthy Q/N-rich tracts ostensibly similar to those found in the prion-forming domains of Sup35p and Ure2p ( Michelitsch and Weissman 2000 ). From among these, we and another group identified two novel proteins, New1p and Rnq1p, with prion-forming domains resembling those of Sup35p and Ure2p ( Santoso et al. 2000 ; Sondheimer and Lindquist 2000 ). When these Q/N-rich domains were fused to green fluorescent protein (GFP) and overexpressed, they formed visible aggregates resembling those of GFP-labeled Sup35p in [ PSI + ] cells. Fusion proteins in which these domains were introduced in place of the Sup35p prion domain could support distinct, self-specific prion states that recapitulated the translation termination defect associated with [ PSI + ]. Rnq1p was later shown to underlie a naturally occurring prion called [ PIN + ], which promotes the aggregation of Q/N-rich proteins such as Sup35p; overexpressed Sup35p forms aggregates and stimulates the appearance of [ PSI + ] only in [ PIN + ] strains ( Derkatch et al. 1997 ; Derkatch et al. 2001 ). Aggregates of the New1p prion domain, whether resulting from overexpression or from a constitutive prion form (termed [ NU + ]), also promoted the aggregation of other Q/N-rich proteins, emulating the effect of [ PIN + ] ( Osherovich and Weissman 2001 ). Many sequences with Q/N content as high as that of Sup35p and Ure2p, including human polyglutamine expansion disease proteins, form visible aggregates when overexpressed in yeast as GFP fusions ( Krobitsch and Lindquist 2000 ; Osherovich and Weissman 2001 ; Meriin et al. 2002 ). However, only a limited number of Q/N-rich sequences are bone fide prion domains capable of propagating these aggregates over multiple cell generations even when expressed at low levels (J. Hood and J.S.W, unpublished data). To understand what distinguishes generic Q/N-rich aggregates from heritable prions, we conducted a detailed dissection of the prion-forming regions of Sup35p and New1p. We found that the prion properties of Sup35p and New1p require the presence of two independent and portable sequence elements within their prion domains. One element mediates the growth of prion aggregates by incorporation of soluble monomers. The second promotes the inheritance of aggregates, generating new heritable “seeds” which can be partitioned between mother and daughter cells during cell division. Results Distinct Regions of the New1p Prion Domain Mediate Prion Growth and Division Sup35p can alternate between a biochemically active, soluble form ([ psi – ]) and an aggregated prion state ([ PSI + ]) with diminished translation termination activity, which can be monitored by nonsense suppression of the mutant ade1–14 allele ( Liebman and Derkatch 1999 ). Whereas [ psi – ] strains form red colonies on yeast extract-peptone-dextrose (YEPD) medium and cannot grow in the absence of adenine, [ PSI + ] strains suppress the premature stop codon in ade1-14 , and thus appear pink or white on YEPD medium and grow on adenine-free medium (a phenotype termed adenine prototrophy, Ade+). The N or prion domain of Sup35p (residues 1-112) is required for [ PSI + ] formation but is dispensable for the translation termination activity of the carboxy-terminal C domain ( Ter-Avanesyan et al. 1993 ). The charged middle domain (M) is not required for prion behavior, but modulates the efficiency of chaperone-dependent prion transmission ( Liu et al. 2002 ; L.Z.O., unpublished data) ( Figure 1 ). Two distinct regions in the N domain have previously been implicated in Sup35p aggregation: a Q/N-rich tract (residues 1–39) ( DePace et al. 1998 ) and an oligopeptide repeat (residues 40–112) that consists of five and a half degenerate repeats of the consensus sequence P/QQGGYQQ/SYN ( Liu and Lindquist 1999 ; Parham et al. 2001 ; Crist et al. 2003 ). Figure 1 Schematic Diagram of Sup35p and New1p Prion domains of both proteins are enlarged in the center, highlighting the Q/N-rich tract of Sup35p (blue), the NYN tripeptide repeat of New1p (purple), and the oligopeptide repeat sequences (orange) found in both proteins. The sequence of the NEW1 oligopetide repeat (residues 50–70) is QQQRNWKQGGNYQQGGYQSYN, while that of the adjacent tripeptide repeat region (residues 71–100) is SNYNNYNNYNNYNNYNNYNNYNKYNGQGYQ. We had earlier identified New1p as an uncharacterized protein with a Sup35p-like N-terminal domain; when fused to the M and C domains of Sup35p, the first 153 residues of New1p (New1 1–153 ) supported a [ PSI + ]-like prion state termed [ NU + ] ( Santoso et al. 2000 ). Sup35p and New1p have regions of clear similarity beyond their high Q/N content ( Figure 1 ). The prion domains of both have Q/N-rich tracts and oligopeptide repeat regions, although their order is reversed. The C-terminal domains of New1p and Sup35p also appear to be related, based on modest homology and the similarity of the translation termination defects in sup35 ( Song and Liebman 1985 ) and new1 mutants (L.Z.O., unpublished data). To understand the sequence requirements for the prion behavior of New1p, we constructed a series of truncated prion domains ( Figure 2 A) and examined their participation in several critical steps of the prion replication cycle. We first asked whether these truncated prion domains could form visible foci when fused to GFP (aggregation). Next, we examined whether such aggregates could convert New1 1–153 into a [ NU + ] prion state (induction). Finally, we fused these constructs to the M and C domains of Sup35p (–M-C), introduced them in place of endogenous SUP35, and assessed whether these proteins could adopt stable prion states (maintenance). Figure 2 Dissection of the New1p Prion Domain Reveals Distinct Regions Responsible for Aggregation and Prion Inheritance (A) Indicated fragments of New1p (left) were expressed as GFP fusions (inducers) in a [ nu – ] [ pin – ] strain, examined by microscopy for GFP aggregation, then plated on SD-ade medium to assess induction of [ NU + ]. The symbol “+” indicates induction frequencies of at least 5%; the symbol “–” indicates no induction. Maintenance was assessed by the ability of an episomal maintainer version of the indicated fragment to support an Ade+ state after overexpression of New1 1–153 -GFP (see Materials and Methods ). The aggregation of New1-GFP fusions (second column) has been previously reported ( Osherovich and Weissman 2001 ). (B) The NYN repeat of New1p induces [ NU + ] and [ NU + ] mini . New1 70–100 -GFP was overexpressed in [ nu – ] and [ nu – ] mini strains ([ pin – ] and [ PIN + ] derivatives of each), along with vector only or New1 1–153 -GFP controls. Averages of three independent trials, representing 600–2000 colonies, are shown for most induction experiments; inductions using New1 70–100 -GFP were conducted twice. Error bars show minimal and maximal observed induction efficiencies. (C) Reversibility of [ NU + ] mini . The [ pin – ] Ade+ convertants obtained in (B) were colony purified on SD-ade medium and confirmed to have lost the inducer plasmid. A stable [ NU + ] mini isolate is shown before and after induction, as well as after GuHCl treatment, along with [ nu – ] and [ NU + ] reference strains. We found that distinct regions within the New1p prion domain are necessary for the induction and maintenance of [ NU + ] ( Figure 2 A). The asparagine-tyrosine-asparagine (NYN) repeat (residues 70–100), which we had earlier shown to be sufficient for aggregation ( Osherovich and Weissman 2001 ), also proved sufficient for induction of [ NU + ]. As with the full-length New1p prion domain, overexpression of the NYN repeat efficiently stimulated the appearance of Ade+ in [ nu – ] cells ( Figure 2 B, left). However, stable prion maintenance required both the NYN repeat and the adjacent oligopeptide repeat. In a strain with this minimized New1p prion domain (residues 50–100), overexpression of the full prion domain or of the NYN repeat alone promoted the appearance of Ade+ colonies ( Figure 2 B, right). The resulting convertants remained Ade+ after loss of the inducer plasmid but reverted to Ade- after transient GuHCl treatment ( Figure 2 C). We conclude that the oligopeptide repeat and the NYN repeat of New1p together are sufficient to support a prion state, termed [ NU + ] mini , which recapitulates the characteristics of [ NU + ]. Dissection of the Sup35p Prion Domain In light of the similarity between New1p and Sup35p prion domains, we asked whether separate regions of Sup35p were involved in the induction and maintenance of [ PSI + ] aggregates ( Figure 3 ). We constructed a series of truncated Sup35p N domains and analyzed their behavior in the aggregation, induction, and maintenance assays described above for [ NU + ]. Additionally, we examined the ability of truncated N domains to decorate preexisting Sup35p aggregates in [ PSI + ] strains. Figure 3 Dissection of the Sup35p Prion Domain At top are schematic diagrams of these experiments; positive outcomes are shown below the arrows. In some cases, similar experiments have been reported by Parham et al. (2001 ) (indicated by “a”) and are repeated here as controls. Aggregation: Plasmid-borne M-GFP fusions of the indicated Sup35p N domain fragments (green) were overexpressed in a [ psi – ] [ PIN + ] strain and examined for fluorescent focus formation. The symbol “+” indicates that 10% or more of cells displayed aggregates. Sup35 1–57 -M-GFP displayed a lower frequency of aggregation (approximately 1%). Induction: Strains from the aggregation experiment were plated onto SD-ade medium and scored for growth to test whether aggregates of truncated protein (green) convert chromosomally encoded protein (blue) to [ PSI + ]. The symbol “+” indicates approximately 5–10% conversion frequency. Consistent with the aggregation experiment, Sup35 1–57 -M-GFP displayed a lower frequency of [ PSI + ] induction (approximately 1%). Decoration: Indicated proteins were expressed as –M-GFP fusions in [ PSI + ] [ PIN + ] cells, which were examined to determine whether GFP-labeled truncations (green) decorate preexisting aggregates of full-length Sup35p (blue). Curiously, Sup35 1–49 -M-GFP in [ PSI + ] cells formed abnormally large “ribbon” aggregates of the kind typically observed during de novo [ PSI + ] induction; furthermore, approximately 10% of the cells reverted to [ psi – ] (indicated by “*”). Thus, this truncation was a potent dominant PNM mutant. Maintenance: A SUP35-deleted [ PSI + ] [ PIN + ] bearing wild-type SUP35 maintainer (blue) was transformed with maintainer plasmids containing the indicated truncation (purple). The wild-type maintainer was lost by counterselection, and the resulting strain was tested for [ PSI + ] by color and growth on SD-ade medium. The Sup35 1–93 mutant displayed an intermediate pink color and grew poorly on SD-ade medium, as previously reported ( Parham et al. 2001 ). Note: King (2001 ) reports that Sup35 1–61 -GFP fusion could decorate [ PSI + ] aggregates in certain strains and could induce [ PSI + ] de novo when overexpressed. We found that the Q/N-rich tract and a small portion of the adjacent oligopeptide repeat are responsible for Sup35p aggregation and de novo [ PSI + ] induction. Deletions within the Q/N-rich tract or of oligopeptide repeat 1 abolished these properties, whereas a construct containing only the Q/N-rich region and the first two oligopeptide repeats (residues 1–64) aggregated and induced [ PSI + ] at levels comparable to the full prion domain, in agreement with King (2001 ). A construct (residues 1–57) with a partial deletion of oligopeptide repeat 2 could still aggregate and induce [ PSI + ], albeit with decreased efficiency. Although a construct lacking oligopeptide repeat 2 entirely (residues 1–49) did not induce [ PSI + ] de novo, this GFP fusion could nonetheless decorate preexisting Sup35p aggregates. Thus, while oligopeptide repeat 2 contributes to the aggregation of Sup35p, the primary determinants of prion induction reside in the amino-terminal Q/N-rich region and oligopeptide repeat 1. In contrast, the rest of the oligopeptide repeat region is needed for stable inheritance of [ PSI + ] aggregates. Constructs that did not form fluorescent foci could not retain [ PSI + ], suggesting that aggregation is a prerequisite for prion maintenance. However, aggregation is not sufficient for prion inheritance, as Sup35p constructs with deletions spanning oligopeptide repeats 3–5 could not support a prion state despite their ability to form aggregates and efficiently induce [ PSI + ]. Only the sixth (incomplete) oligopeptide repeat proved dispensable for [ PSI + ] maintenance, consistent with an earlier report ( Parham et al. 2001 ). The PNM2-1 Mutation in Oligopeptide Repeat 2 Specifically Compromises the Inheritance of [ PSI + ] Our deletion analysis suggested that oligopeptide repeat 2 participated in both the formation and inheritance of Sup35p aggregates. We made use of a point mutation within oligopeptide repeat 2 known as PNM2-1 (G58D) to distinguish between these two functions. PNM2-1 ( P SI N o M ore) shows strong interference with [ PSI + ] in certain strain backgrounds through a poorly understood mechanism ( McCready et al. 1977 ; Doel et al. 1994 ; Kochneva-Pervukhova et al. 1998 ; Derkatch et al. 1999 ). Using both in vivo and in vitro assays, we established that PNM2-1 does not have a defect in aggregation or [ PSI + ] induction. Earlier work indicated that PNM2-1 is capable of seeding [ PSI + ] in vivo ( Kochneva-Pervukhova et al. 1998 ; Derkatch et al. 1999 ; King 2001 ). Consistent with these reports, we found that overexpression of a PNM2-1-GFP fusion in [ psi – ] [ PIN + ] cells with a wild-type SUP35 locus led to both focus formation and [ PSI + ] induction ( Figure 4 A). A previous study of Sup35p polymerization in extracts had suggested that PNM2-1 might interfere with [ PSI + ] through a defect in seeding ( Kochneva-Pervukhova et al. 1998 ). We tested this by examining the rate of seeded polymerization of recombinant PNM2-1 protein. Like wild-type Sup35p, purified PNM2-1 spontaneously formed amyloid fibrils in vitro; this was accelerated by the addition of preformed Sup35p polymer seeds (data not shown). We measured the initial rates of polymerization of wild-type and PNM2-1 protein seeded by preformed wild-type polymers ( Figure 4 B) and by PNM2-1 polymers ( Figure 4 C) using a thioflavin-T–binding assay. We observed that wild-type and PNM2-1 monomers were seeded by wild-type polymers with similar kinetics; likewise, PNM2-1 polymers seeded both wild-type and PNM2-1 monomers equivalently. Thus, the PNM2-1 mutation does not affect polymerization or seeding. Figure 4 PNM2–1 (G58D) Prevents Inheritance But Not Aggregation of Sup35p Prions (A) PNM2-1 protein can seed [ PSI + ]. A Sup35p inducer containing the PNM2-1 (G58D) mutation was overexpressed in [ psi – ] [ PIN + ] cells; shown are cells (inset) with representative fluorescent foci, which were the same in frequency and appearance as cells with a wild-type inducer. Cells overexpressing inducer versions of wild-type Sup35p (SUP), an aggregation-defective N-terminal truncation (Δ1–38), and PNM2-1 were plated and scored for Ade+. Approximately 1000 colonies were counted. (B) PNM2-1 protein polymerization is similar to that of wild-type protein. (C) Preformed PNM2-1 polymers seed wild-type and PNM2-1 monomers with comparable efficiency. Endpoint PNM2-1 polymers were used to seed fresh reactions. (D) PNM2-1 displays a partially dominant, incompletely penetrant defect in [ PSI + ] maintenance. [ psi – ] (1) and [ PSI + ] (2) SUP35::TRP1 pSUP35 controls are shown. [ PSI + ] [ PIN + ] SUP35::TRP1 pSUP35 was transformed with a second maintainer expressing PNM2-1 (3). The wild-type maintainer (pSUP35) was then lost through counterselection (4). Red sectors from (4) were isolated, retransformed with the wild-type maintainer, and allowed to lose the PNM2-1 maintainer (5). (E) Mitotic instability of [ PSI + ] in the PNM2-1 strain. A pink (Ade+) [ PSI + ] [ PIN + ] PNM2-1 isolate was grown to log phase in SD-ade liquid then shifted into nonselective (YEPD) medium. At indicated time points, aliquots were plated onto SD-ade and YEPD media to determine the fraction of [ PSI + ] cells (minimum of 200 colonies counted per time point). Whereas a wild-type control remained [ PSI + ] through the experiment, the PNM2-1 strain rapidly lost [ PSI + ] during logarithmic growth; during stationary phase (18 h and beyond), the percentage of [ PSI + ] PNM2-1 strains remained unchanged (approximately 5%). (F) Propagon count of PNM2-1 vs. wild-type [ PSI + ] strains. The majority of PNM2-1 cells had no [ PSI + ] propagons (i.e., were [ psi – ]). In both strains, a small number of “jackpot” cells contained over 200 propagons; see Cox et al. (2003 ). Instead, the PNM2-1 strain shows a marked defect in the inheritance of [ PSI + ]. When the wild-type SUP35 gene of a [ PSI + ] strain was replaced with PNM2-1, the strain retained the prion on synthetic defined (SD) yeast medium that selected for [ PSI + ] (SD-ade medium) but reverted to [ psi – ] at a high frequency in nonselective YEPD medium, resulting in sectored colonies ( Figure 4 D). We measured the rate of [ PSI + ] loss in a PNM2-1 strain by growing it in YEPD medium and, at various time points, plating aliquots of the culture onto SD-ade medium to determine the fraction of cells that had retained [ PSI + ] ( Figure 4 E). A wild-type strain retained [ PSI + ] in all of the cells throughout the experiment. By contrast, in the PNM2-1 strain the fraction of [ PSI + ] cells decreased rapidly while the cells grew logarithmically, but remained at a constant level when the cells entered stationary phase. These findings indicate that PNM2-1 acts to eliminate [ PSI + ] in dividing cells, consistent with a defect in prion replication. We next used a recently described assay to measure the number of heritable prion seeds (propagons) in a PNM2-1 strain. Here, prion replication is inhibited by GuHCl treatment. As the cells divide, preexisting propagons are diluted but not destroyed. The number of propagons present in a colony arising from a single cell is then evaluated by removing the GuHCl prion replication block after a large number (10 or more) of cell divisions and counting the total number of [ PSI + ] cells in that colony ( Cox et al. 2003 ). Whereas a wild-type strain had a median of 92 ( n = 24) propagons per cell, the PNM2-1 strain had dramatically fewer: 41 of 50 cells had no [ PSI + ] propagons at all (i.e., were [ psi – ]), and among the remaining nine [ PSI + ] cells, the median propagon number was six ( Figure 4 F). Thus, although a PNM2-1 strain can harbor [ PSI + ] prions, a defect in propagon replication causes mitotic instability, demonstrating the importance of oligopeptide repeat 2 in prion replication or segregation. Design of Novel Prion Domains Our data suggested that the formation and inheritance of prions involve distinct regions of Sup35p and New1p prion domains. To assess the interchangeability of these prion domain components, we constructed a chimeric prion domain, termed F, in which the aggregation-determining NYN repeat of New1p was fused to the oligopeptide repeats of Sup35p ( Figure 5 A). While initially soluble and active, a fusion of F and the Sup35p M and C domains (F-M-C) could be converted into an aggregated state, termed [ F + ], after transient overexpression of F-M-GFP. As with [ NU + ], [ F + ] induction did not require [ PIN + ] (data not shown). [ F + ] could be eliminated by GuHCl treatment ( Figure 5 B) and was inherited in a dominant, non-Mendelian manner ( Figure 5 C). As with Sup35p in a [ PSI + ] strain, F-M-C protein in [ F + ] but not in [ f – ] extracts sedimented entirely to the pellet fraction following high-speed centrifugation ( Figure 5 D). Thus, [ F + ] results from a prion state of F-M-C. Figure 5 F, A New1p–Sup35p Chimera, Shows Prion Characteristics of New1p (A) Schematic diagram illustrating the construction of chimera F. (B) Chimera F forms a prion, [ F + ]. The SUP35 gene in a [ psi – ] [ pin – ] strain was replaced with the F-M-C fusion; after transient overexpression of F-M-GFP, approximately 10% of these cells converted from an Ade- ([ f – ]) to an Ade+ ([ F + ]) state. Shown are examples of[ f – ] and [ F + ] strains, before and after GuHCl treatment, along with [ psi – ] and [ PSI + ] controls. (C) Non-Mendelian inheritance of [ F + ]. A diploid made by mating a [ F + ] MAT a strain against an [ f – ] MAT α displayed a [ F + ] phenotype and, when sporulated, produced four [ F + ] meiotic progeny. All 11 tetrads examined showed this 4:0 pattern of inheritance. (D) Sedimentation analysis of F-M-C. Extracts of [ f – ] and [ F + ] strains, along with [ psi – ] and [ PSI + ] controls, were subjected to 50K × g ultracentrifugation for 15 min. Total, supernatant, and pellet fractions were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-SUP35NM serum. As with Sup35p, the prion form of F-M-C sediments primarily to the pellet but remains in the supernatant in [ f – ]. (E) F-M-GFP overexpression induces [ NU + ] but not [ PSI + ]. Indicated inducers and maintainers were used in an induction experiment. The symbol “+” indicates approximately 5–10% conversion to Ade+. F induced [ NU + ] at a comparable efficiency to New1 1–153 ; although New1 1–153 overexpression promoted the appearance of Ade+ colonies in the F-M-C strain, these were fewer in number (less than 5%) and reverted to Ade- after restreaking. (F) [ F + ] and [ NU + ] prion proteins interact with each other but not with [ PSI + ]. Episomal “second maintainers” were introduced into the indicated strains, along with an empty vector control. Antisuppression (red) indicates that the second maintainer is soluble, while white/pink indicates coaggregation of the endogenous and episomal maintainers. We next explored the specificity of [ F + ] prion seeding. Overexpression of the Sup35p prion domain did not induce [ F + ]; conversely, F-M-GFP overexpression did not induce [ PSI + ] ( Figure 5 E). However, F-M-GFP readily induced [ NU + ], indicating that mismatched sequences outside of the aggregating region did not prevent cross-interactions between heterologous proteins. Interestingly, overexpression of New1 1–53 -GFP induced Ade+ colonies in the [ f – ] strain, but this adenine prototrophy proved unstable. We also examined the ability of preexisting prion aggregates to recruit different prion-forming proteins using an antisuppression assay ( Santoso et al. 2000 ) ( Figure 5 F). [ PSI + ], [ F + ], and [ NU + ] strains were transformed with Sup35p–, F-M-C– or New1 1–153 -M-C–encoding plasmids; the color of the resulting colonies indicates whether the second maintainer protein is soluble (red) or aggregates as a result of the resident prion (pink/white). Consistent with the induction data, F-M-C and New1 1–153 -M-C were not incorporated into [ PSI + ] aggregates; likewise, Sup35p did not interact with [ F + ] or [ NU + ] aggregates. However, [ F + ] prions recruited New1 1–153 -M-C and, to a lesser extent, [ NU + ] recruited F-M-C. Thus, F and New1p prion domains can cross-interact during de novo induction and at normal levels of expression, indicating that the NYN repeat is sufficient to specify homotypic interaction between two otherwise distinct prion domains. Can a simple aggregation-prone sequence such as polyglu-tamine ( Zoghbi and Orr 2000 ) be turned into a heritable prion by fusion to an oligopeptide repeat? We designed artificial prion domains containing short (Q22) and pathogenically expanded (Q62) polyglutamine tracts, either alone or adjacent to the Sup35p oligopeptide repeat ( Figure 6 A). These domains were fused to –M-GFP and –M-C to create polyglutamine inducers and maintainers, respectively. Q22 inducers did not aggregate upon overexpression, but Q62 inducers (with and without oligopeptide repeats) formed visible foci in [ psi – ] [ PIN + ] cells ( Figure 6 B). Transient overexpression of Q62 inducers had no effect on the Q22 maintainer with the oligopeptide repeat or on the Q62 maintainer lacking the oligopeptide repeat. However, the Q62 maintainer with an oligopeptide repeat supported prion inheritance, converting to a stable Ade+ state following overexpression of the cognate inducer ( Figure 6 C). Several tests confirmed the prion nature of this state, termed [ Q + ]. Like [ PSI + ], [ Q + ] did not require the presence of the inducer plasmid after transient overexpression, was sensitive to GuHCl treatment ( Figure 6 D), and displayed a dominant, non-Mendelian pattern of inheritance ( Figure 6 E). We further tested the specificity of the [ Q + ] state by introducing a plasmid encoding a noncognate second maintainer into a [ Q + ] strain ( Figure 6 F). The Q62 maintainer failed to be incorporated into [ PSI + ] aggregates, causing antisuppression (red); conversely, Sup35p did not enter [ Q + ] aggregates. Figure 6 [ Q + ], a Prion Form of Pathogenically Expanded Polyglutamine (A) Schematic illustrating the construction of polyglutamine-derived prion domains. (Op) indicates the presence of the Sup35p oligopeptide repeats (residues 40–124). (B) Fluorescence micrographs of [ psi – ] [ PIN + ] strains expressing indicated polyglutamine inducers. (C) Polyglutamine-based prion inheritance. Strains with indicated inducers and maintainers were plated onto SD-ade and YEPD media to determine the fraction of Ade+ after a standard induction experiment. Interestingly, Q62 inducer forms aggregates but does not promote Ade+ in the Q62(Op) maintainer strain. Note that Q62(Op) shows a high rate of spontaneous appearance of Ade+. (D) GuHCl sensitivity of the [ Q + ] state. An Ade+ convertant obtained in (C) was restreaked to lose the inducer plasmid, then grown on GuHCl. Shown are plates before and after GuHCl treatment, along with [ psi – ] and [ PSI + ] controls. (E) Dominance and non-Mendelian inheritance of [ Q + ]. See Figure 5 C. (F) [ Q + ] does not interact with Sup35p and vice versa. [ Q + ] and [ PSI + ] strains were transformed with indicated maintainers; mismatches between the maintainer and the chromosomally encoded allele result in antisuppression (red). Discussion A number of epigenetic traits in fungi result from the stable inheritance of self-propagating, infectious protein aggregrates (prions) ( Uptain and Lindquist 2002 ). Prion inheritance requires three sequential events that must keep pace with cell division to preserve the number of heritable prion units, or propagons, per cell ( Osherovich and Weissman 2004 ). First, prion aggregates must grow in size by incorporating newly synthesized protein. Next, these enlarged aggregates must be divided into smaller ones through the action of cellular chaperones ( Kushnirov and Ter-Avanesyan 1998 ; Borchsenius et al. 2001 ; Ness et al. 2002 ; Kryndushkin et al. 2003 ). Finally, these regenerated propagons must be distributed to mother and daughter cells ( Cox et al. 2003 ); for small, cytoplasmic aggregates, this distribution may occur passively by diffusion during cytokinesis. In the present study, we have dissected the prion-forming domains of Sup35p and New1p to discover the sequence elements involved in these steps. We have found that these domains consist largely of modular, interchangeable elements that serve distinct functions of prion growth and division or transmission. Aggregation underlies the growth phase of the prion replication cycle ( Figure 7 A) and occurs through the templated addition of conformationally compatible monomers onto preexisting seeds. Like other amyloids, yeast prions display a high specificity for homotypic aggregation ( Santoso et al. 2000 ; Chernoff et al. 2000 ; Kushnirov et al. 2000 ; Zadorskii et al. 2000 ; Nakayashiki et al. 2001 ). This discrimination arises from differences in the amino acid sequence and the conformation ( Chien and Weissman 2001 ) of the aggregation-promoting Q/N-rich elements found in each yeast prion protein. Aggregation and specificity are dictated by the NYN repeat (residues 70–100) of New1p and by the Q/N-rich amino terminal region (residues 1–57) of Sup35p. Figure 7 Model for Prion Growth and Division (A) During prion growth, polymers seed the incorporation of monomers through interactions between Q/N-rich aggregation sequences (blue). Proteins with noncognate aggregation sequences (red) are excluded. (B) The division phase of prion replication requires the oligopeptide repeats (orange), which may facilitate the action of chaperones such as Hsp104p (scimitar) in breaking the polymer into smaller, heritable units. In contrast, the conserved oligopeptide repeat sequence mediates the division and/or segregation phase of prion replication ( Figure 7 B). In New1p, the NYN repeat alone can aggregate and induce [ NU + ] but requires an adjacent oligopeptide repeat to form a minimal heritable New1p prion, [ NU + ] mini . Similarly, in Sup35p, the Q/N-rich amino terminal region mediates aggregation whereas most of the oligopeptide repeats are needed for the inheritance of [ PSI + ] propagons. Oligopeptide repeats 1 and 2 appear to contribute to both growth and inheritance, consistent with earlier reports that expansion and deletion within this region modulate in vitro polymerization of Sup35p and the appearance of [ PSI + ] in vivo ( Liu and Lindquist 1999 ). However, the two functions can be distinguished by a point mutant in oligopeptide repeat 2 (PNM2-1), which displays a specific defect in [ PSI + ] inheritance despite normal aggregation. Certain [ PSI + ] variants are resistant to the dominant negative effect of PNM2-1 ( Derkatch et al. 1999 ; King 2001 ); this suggests that although oligopeptide repeat 2 is critical for the replication of the [ PSI + ] variant used in our studies, it may be less important for the replication of other Sup35p prion conformations. Many studies have established that prion inheritance requires the action of cellular chaperones such as Hsp104p and Hsp70 proteins (reviewed in Osherovich and Weissman 2002 ), although how these proteins contribute is poorly understood. We propose that oligopeptide repeats turn nonheritable aggregates into prions by facilitating chaperone-mediated division. Oligopeptide repeats may allow the division of aggregates by providing direct binding sites for chaperones or by altering the conformation of the amyloid core to allow chaperone access. An earlier study established that deletion of residues 22–69 of Sup35p (which include parts of both the Q/N tract and the oligopeptide repeat) interferes with both [ PSI + ] induction and chaperone-mediated prion disaggregation ( Borchsenius et al. 2001 ). Unlike the Δ22–69 mutant, the prion replication defect in PNM2-1 could not be corrected by increasing Hsp104p levels (data not shown), arguing that the mitotic instability of PNM2-1 [ PSI + ] is not due solely to inadequate Hsp104p binding. Our findings help to explain why, among many Q/N-rich proteins in yeast, only a small subset form heritable prions. While many Q/N-rich proteins can aggregate when overexpressed ( Sondheimer and Lindquist 2000 ; Derkatch et al. 2001 ; Osherovich and Weissman 2001 ), prion inheritance of such aggregates requires that they be divided and passed on to the next generation. Although the inheritance of Sup35p and New1p prions is mediated by oligopeptide repeats, other sequences could also serve this purpose. Ure2p lacks an oligopeptide repeat; interestingly, many isolates of [ URE3 ] are mitotically unstable in the absence of selection ( Schlumpberger et al. 2001 ). Rnq1p, which underlies [ PIN + ], also lacks a strict oligopeptide repeat, but a region (residues 218–405) within its prion domain has an amino acid content reminiscent of the oligopeptide repeat sequence (i.e., numerous Q, N, S, Y, and G residues) ( Resende et al. 2003 ). Only two other yeast proteins, YDR210W and YBR016W, have clearly recognizable oligopeptide repeats; both proteins also have Q/N-rich regions. YBR016W forms aggregates when overexpressed ( Sondheimer and Lindquist 2000 ), but it is not known whether either protein can maintain a heritable aggregated state. Although the mammalian prion protein PrP contains a sequence resembling the oligopeptide repeat that can functionally replace one of the Sup35p repeats ( Parham et al. 2001 ), it is unclear whether this sequence is important in the replication of the PrP Sc state. The interchangeable nature of prion domain components allowed us to design novel artificial prions. The F chimera, consisting of the aggregation sequence of New1p and the oligopeptide repeat of Sup35p, demonstrates that the growth and specificity of prions is largely determined by the Q/N-rich tract, not by the oligopeptide repeat. Despite a sequence derived primarily from Sup35p, the F chimera behaved like New1p rather than like Sup35p. The [ F + ] prion cross-interacted with New1p but not Sup35p. Like [ NU + ], [ F + ] could be induced in the absence of a prion-promoting (PIN) factor. Finally, [ F + ] could itself act as a PIN factor, as does [ NU + ] (data not shown). Notably, the NYN repeat of New1p functions as an aggregation module apparently without regard to its position within a protein; this sequence induced prions when overexpressed by itself or with oligopeptide repeats at its N-terminal (in New1 1–153 and New1 50–100 ) or C-terminal regions (in the F chimera). These results suggest that aggregation sequences are portable and functionally separable from the oligopeptide repeat, perhaps constituting a structurally discrete amyloid core. Indeed, a peptide derived from the amino-terminal region of Sup35p forms a self-seeding amyloid in vitro ( Balbirnie et al. 2001 ). A simple aggregation-prone sequence, pathogenically expanded glutamine, also supports prion inheritance when adjacent to the oligopeptide repeat, suggesting that prion domains can consist of little more than a generic, aggregating core sequence and an inheritance-promoting element. In addition to illuminating the principles of yeast prion domain architecture, artificial prions with distinct specificity may be useful as controllable epigenetic regulators of protein activity. Such prion “switches” can be turned on and off by transient overexpression and genetic repression; for example, the Q prion domain could be fused to other proteins in order to conditionally and reversibly inactivate them independently of [ PSI + ]. It may also be possible to design additional artificial yeast prion domains whose aggregation is driven by non-Q/N-rich amyloidogenic proteins such as the Aβ peptide that accumulates in Alzheimer's disease ( Koo et al. 1999 ) or the mammalian prion protein PrP ( Cohen and Prusiner 1998 ). Such artificial prions could serve as models for aggregate–chaperone interactions in metazoans and could provide a genetic system for the high-throughput screening of modulators of human aggregation diseases. Materials and Methods Yeast strains and methods Derivatives of W303 ( Osherovich and Weissman 2001 ), with the initial genotypes ade1-14, his3-11,15, leu2-3, trp1-1, and ura3-1, were used throughout unless otherwise noted; all strains were [ PIN + ]. Strain numbers, with indicated genotypic differences, are as follows: YJW 584 [ psi – ] MAT a , YJW 508 [ PSI + ] MAT α , YJW 716 [ nu – ] MAT α sup35 ::TRP1 pRS315SpNew1 1–153 -M-C, YJW 717 [ NU + ] MAT α sup35 ::TRP1 pRS315SpNew1 1–153 -M-C, YJW 844 [ f – ] MAT α sup35 ::F-M-C C.g. HIS3, YJW 881 [ F + ] MAT a sup35 ::F-M-C C.g. HIS3, YJW 867 [ q – ] MAT α sup35 ::Q-M-C C.g. HIS3, YJW 868 [ Q + ] MAT a sup35 ::Q-M-C C.g. HIS3. Maintainer plasmids used in Figure 3 (see plasmid and gene replacement construction, below) were introduced by plasmid shuffling into YJW 716 or YJW 753 ([ PSI + ] MAT a sup35 ::TRP1 pRS316SpSUP35), followed by loss of the maintainer spontaneously or through 5-FOA counterselection. The PNM2-1 strain in Figure 4 was generated in this manner and was subsequently restreaked on SD-ade to select for [ PSI + ]. HIS3-marked oligopeptide repeat truncations and PNM2-1 maintainers were from Parham et al. (2001 ); all other Sup35p and New1p maintainers were marked with LEU2. The [ f – ] strain was generated by “gamma” chromosomal integration of pRS306 F-M-C into the SUP35 locus of YJW 584; excision of the wild-type gene was confirmed by PCR of Ade- colonies arising from subsequent growth on 5-FOA. The [ q – ] strain was made by “omega” chromosomal gene replacement ( Kitada et al. 1995 ) of SUP35 with a C.glabrata HIS3-marked –M-C variant (with or without oligopeptide repeats) into the SUP35 locus of a diploid [ PSI + ] [ PIN + ] strain. After sporulation, gene replacement was confirmed by PCR and by loss of [ PSI + ] in half of the haploid progeny. Yeast culture methods were according to standard procedures ( Sherman 1991 ), but YEPD-medium plates contained 1/4 of the standard amount of yeast extract to accentuate color phenotypes. For prion curing, strains were grown on YEPD medium plus 3 mM GuHCl, then restreaked onto YEPD medium. Plasmid and gene replacement construction The modular SUP35 cloning system described in previous reports was used throughout ( Santoso et al. 2000 ; Osherovich and Weissman 2001 ). All plasmids are derived from Sikorski and Hieter (1989 ); sequence files of all constructs are available as a web supplement ( Data S1 ). Maintainer plasmids are low-copy CEN/ARS (pRS31x series) with the native SUP35 promoter (Sp) driving the expression of the indicated prion domain followed by the M and C domains of Sup35p. Inducer plasmids are high-copy 2μ (pRS42x series) with the inducible CUP1 promoter (Cp) driving the expression of the indicated prion domain followed by the Sup35p M domain and GFP. New1p inducers did not include the Sup35p M domain. For polyglutamine constructs, polyglutamine tracts (22 and 62) were amplified out of the MJDtr constructs used in an earlier study ( Osherovich and Weissman 2001 ). To permit amplification, primers contained sequences homologous to several codons adjacent to the 5′ and 3′ ends of the polyglutamine tracts plus an initiator ATG codon. Thus, the polyglutamine sequences read MAYFEK(Q22/62)DLSG. The resulting PCR fragments were cloned into maintainer and inducer plasmids, which were used as templates for gene replacement PCR (see yeast strains and methods, above). In vivo prion assays For aggregation, inducers were overexpressed by growth of cells in selective medium with 50 μM CuSO 4 until the culture reached stationary phase; cells were then examined by fluorescent microscopy (Zeiss Axiovert, Zeiss, Oberkochen, Germany; Metamorph imaging software, Universal Imaging Corporation, Downingtown, Pennsylvania, United States). Unless otherwise noted, cultures displaying 10% or more cells with foci were scored as positive. For induction, dilutions of the above cultures were plated onto SD-ade and YEPD media to determine percentage of Ade+. In qualitative assessments, strains were scored as positive if 5% or more of plated cells grew on SD-ade medium after 5 d. In [ NU + ] maintenance experiments, strains with indicated maintainers were tested for the ability to support an Ade+ state following New1 1–153 -GFP overexpression. In [ PSI + ] maintenance experiments, strains that began as [ PSI + ] were tested for Ade+ after plasmid shuffle gene replacement with the indicated maintainer. For decoration, a [ PSI + ] [ PIN + ] strain was transformed with the indicated inducers, grown in selective medium with 50 μM CuSO 4 , and examined by fluorescence microscopy during midlogarithmic phase. Propagon counts were performed as described in Cox et al. (2003 ). For the antisuppression assay, indicated strains were transformed with a second, differently marked maintainer plasmid, and color phenotypes were assayed on medium selective for both plasmids. In vitro prion assays Centrifugation was performed as described in Ness et al. (2002 ). Immunoblots were visualized with MT130 anti-Sup35p N-M domain serum.For the polymerization of PNM2-1, the PNM2-1 N and M domains were cloned as 7-histidine fusions into pAED4 and expressed and purified as described in DePace et al. (1998 ). Thioflavin-T binding was conducted as in Chien et al. (2003 ). The slope of early (0–6 min) dye binding was obtained from seeded polymerization reactions conducted in triplicate. To correct for a difference in dye binding between wild-type and PNM2-1 protein, these values were normalized to the end point (90 min) maximum signal for each protein. Monomer concentrations were 2.5μM. Supporting Information Data S1 DNA Sequences of Constructs (30 KB ZIP). Click here for additional data file. Accession Numbers The GenBank accession numbers for the proteins discussed in this paper are Hsp104p (NP_013074), New1p (NP_015098), Rnq1p (NP_09902), Sup35p (NP_010457), Ure2p (NC_014170), YDR210W (NP_010496), and YBR016W (NP_010319).

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514705

Is drug-induced toxicity a good predictor of response to neo-adjuvant chemotherapy in patients with breast cancer? -A prospective clinical study

Background Neo-adjuvant chemotherapy is an integral part of multi-modality approach in the management of locally advanced breast cancer and it is vital to predict the response in order to tailor the regime for a patient. The common final pathway in the tumor cell death is believed to be apoptosis or programmed cell death and chemotherapeutic drugs like other DNA-damaging agents act on rapidly multiplying cells including both the tumor and the normal cells by following the same common final pathway. This could account for both the toxic effects and the response. Absence or decreased apoptosis has been found to be associated with chemo resistance. The change in expression of apoptotic markers (Bcl-2 and Bax proteins) brought about by various chemotherapeutic regimens is being used to identify drug resistance in the tumor cells. A prospective clinical study was conducted to assess whether chemotherapy induced toxic effects could serve as reliable predictors of apoptosis or response to neo-adjuvant chemotherapy in patients with locally advanced breast cancer. Methods 50 cases of locally advanced breast cancer after complete routine and metastatic work up were subjected to trucut biopsy and the tissue evaluated immunohistochemically for apoptotic markers (bcl-2/bax ratio). Three cycles of Neoadjuvant Chemotherapy using FAC regime (5-fluorouracil, adriamycin, cyclophosphamide) were given at three weekly intervals and patients assessed for clinical response as well as toxicity after each cycle. Modified radical mastectomy was performed in all patients three weeks after the last cycle and the specimen were re-evaluated for any change in the bcl-2/bax ratio. The clinical response, immunohistochemical response and the drug-induced toxicity were correlated and compared. Descriptive studies were performed with SPSS version 10 and the significance of response was assessed using paired t-test. Significance of correlation between various variables was assessed using chi-square test and coefficient of correlation calculated by Pearson correlation coefficient. Results There was a statistically significant correlation observed between clinical, immunohistochemical response (bcl-2/bax ratio) and the drug-induced toxicity. Conclusion Responders also had significant toxicity while non-responders did not show significant toxicity following neoadjuvant chemotherapy. The chemotherapy-induced toxicity was observed to be a cost effective and reliable predictor of response to neo-adjuvant chemotherapy.

Background Neo-adjuvant chemotherapy (NACT) is an integral part of multi-modality approach in the management of locally advanced breast cancer (LABC). It is required both for the local control (to ensure microscopically free margins during surgery) and distant or systemic control [ 1 - 5 ]. In the past few years, considerable research has been done on the molecular aspects of breast cancer. The recognition that tumor growth rate is a product of the proliferative activity and the rate of cell death has lead to a reappraisal of traditional views of tumor response and resistance to cytotoxic Drugs [ 2 ]. Apoptosis is a closely regulated form of active cell death defined by characteristic biochemical and morphological criteria. A large number of anti-cancer agents with widely differing modes of action have been demonstrated to induce apoptosis in vitro, suggesting this as a significant final common pathway for exerting their clinical effects. Mechanisms that suppress apoptosis may be important in the development of intrinsic and acquired resistance to cytotoxic drugs [ 3 ]. It was suggested more than 20 years ago that apoptosis might account for much of the spontaneous cell loss (known from kinetic studies) to occur in many tumors and its extent often is enhanced by well-established modalities such as chemotherapy, irradiation and hormone ablation. However, during the past few years, advances in the understanding of the control of apoptosis at the molecular level has extended its potential oncologic significance far beyond the mere provision of a mechanistic explanation for tumor cell deletion. In particular, the discovery that the products of certain proto-oncogenes can regulate apoptosis has opened up exciting avenues for future research [ 4 ]. The protean effects of various neoplastic agents on synthesis of DNA, RNA, and inhibition of synthesis which may or may not lead to cell death, requires only that some critical concentrations of active drug or metabolite be present in a cell. Proliferating normal cells may therefore be subject to the same detrimental effects of chemotherapy experienced by neoplastic tissue and successful chemotherapy dictates that recuperative abilities of normal tissues are greater than those of cancer. The two tissues generally most adversely affected by antineoplastics are the hemopoetic cells of the bone marrow and the epithelium of the aero digestive tract as a result of high growth fractions and short cycling times of the cells. The ability of the cancer patients to perform normal activities and function is also recognized both as a determinant of how well the patient may respond to chemotherapy and an index of the general toxicities of the anti cancer agents. A variety of anti-cancer drugs have been shown to induce extensive apoptosis in rapidly proliferating "normal" cell population and the tumors alike. Thus enhanced apoptosis is also likely to be responsible for many of the adverse effects observed following chemotherapy [ 5 ]. Apoptosis being the common final pathway both for tumoricidal effect and also for the toxic side effects, a significant correlation should therefore exist between the effects and the toxicity produced. Toxic effects could thus serve as reliable indicators of apoptosis Apoptosis is a regulated phenomenon capable of being inhibited and activated. Indeed there is evidence that stimulation of some cells by trophic cytokines or increase in their levels of expression of Bcl-2 proto-ontogeny can greatly increase their resistance to the apoptosis-inducing effects of anticancer drugs. Thus Bcl-2 proto-ontogeny expression may be implicated in the development of resistance of tumors to therapeutic agents and may contribute to tumor growth and perhaps to ontogenesis by allowing the inappropriate survival of cells with DNA abnormalities [ 6 ]. Deregulated expression of the Bcl-2 protein represents the best-known example of a potent blocker of apoptosis. Over expression of Bcl-2 has now been shown to protect a wide variety of cell types from induction of apoptosis by many different anticancer agents. Several homologues of Bcl-2 protein have also been shown to act as inhibitors of apoptosis, including Bcl-Xl and others as apoptotic proteins such as Bax. In vitro data suggest that it is the relative ratios of anti-apoptotic and pro-apoptotic proteins that determine the likelihood of cells to undergo apoptosis in response to chemotherapeutic drugs [ 2 , 7 ] The increasing use of pre-operative chemotherapy (PCT) in breast cancer offers an in vivo test bed to further confirm the clinical relevance of these observations. The clinical response or the absence of response along with the toxic effects observed could well help predict the outcome with a particular chemotherapeutic regime and facilitate planning of an alternate regime for better response [ 5 - 8 ]. It is vital to assess the response to NACT in order to tailor the regime for a particular patient to predict the intrinsic or acquired chemo resistance. DNA-damaging agents such as chemotherapeutic drugs can induce apoptosis and increased resistance to chemotherapeutic agents, which has been found to be associated with decreased capacity to undergo apoptosis [ 5 - 9 ]. Central to this are proteins that modulate apoptosis, including bcl-2 and bax products. The change in expression of apoptotic markers brought about by various chemotherapeutic regimens is being used to identify drug resistance in the tumor cells. Various other biological markers like p-glycoprotein expression have also been used to predict the response to neoadjuvant chemotherapy [ 9 - 11 ]. The clinical response along with complete pathological response (CPR) is still considered a surrogate marker of response against which all other predictive markers are compared. The need to have a reliable and inexpensive predictor of response in a third world scenario can not be over emphasized since majority of patients present relatively late and the resources are limited and scarce [ 2 , 3 ]. Since both the response and drug related toxicity due to chemotherapeutic agents are the result of the same common final pathway of apoptosis the toxicity due to chemotherapy along with clinical response may be utilized as a cost-effective and reliable indicator (predictor) of response to neo-adjuvant chemotherapy. Against this background a prospective study was contemplated with the following aims and objectives: 1. To assess the clinical and immunohistochemical response to NACT in patients with LABC. 2. To correlate immuno-histochemical (apoptotic markers i.e. Bcl-2/Bax ratio) and clinical response to the drug induced toxicity. 3. To ascertain whether the drug induced toxicity could be utilized as a reliable indicator and predictor of response to neoadjuvant chemotherapy. Methods 50 FNAC proven cases of locally advanced breast carcinoma according to AJCC (American Joint Committee On Cancer) classification were included in the study A thorough clinical and ultrasonographic examination (USG) of all the patients including the opposite breast was performed to stage the disease accurately. A core biopsy using a tru-cut needle was performed for immuno-histochemical estimation of the apoptotic markers i.e. base-line Bcl-2/Bax ratio before initiating the chemotherapy. Routine and metastatic work up was done including complete blood examination (total blood count, platelet count), chest radiograph, ECG (Echocardiography when ECG had a positive finding), liver function tests, Bone Scan, USG abdomen, KFT (Kidney function tests). Patients were subjected to three cycles of FAC regime (cyclophosphamide 600 mg/m 2 , adriamycin -50 mg/m2, 5-fluorouracil-600 mg/m2) at an interval of three weeks. Before each cycle the patient was clinically and sonologically examined for the breast tumor size, axillary lymph node status & appearance of systemic metastasis. All patients were given the same antitoxicity treatment according to a standardized unit protocol including adequate hydration and inject able antiemetics before initiating the chemotherapy. The patients were observed for three main toxicities of the FAC regime i.e. vomiting, alopecia and leucopenia. Vomiting with minimum of four episodes on day one and two after chemotherapy was taken as significant. Total alopecia was considered significant. Leucopenia was assessed in terms of the WHO grading of hematological toxicity. All patients were subjected to Patey's modified radical mastectomy three weeks after the last cycle and the specimen were again subjected to immuno-histochemistry to evaluate for any change in the Bcl-2/Bax ratio and for the histological tumor size, margins. Objective clinical response was defined as >50% reduction in the tumor size after completion of three cycles of NACT, as assessed clinically, sonologically and histologically. Immunohistochemical response was taken as decrease in the Bcl-2/Bax ratio. Any increase or no change in this ratio was considered as no response. Immuno-histochemical methods Biopsy specimen was preserved in buffered formalin solution. Five-micron sections were prepared from paraffin embedded blocks on poly-l-lysine coated glass slides. Sections were deparaffinized in xylene for 15 min. and hydrated in alcohol for 15 minutes. Further, incubation was done in 0.3% hydrogen peroxide in methanol solution for 45 min. The slides were washed with citrate buffer and kept in a water bath at 90–95°C for 45 min. for antigen retrieval. Sections were washed with Tris buffer saline (TBS) solution and incubated with blocking antibodies (DAKO monoclonal mouse antihuman Bcl-2 oncoprotein for Bcl-2 expression and polyclonal rabbit antihuman for Bax expression) at 37°C. Sections were washed with TBS solution. Incubation with avidin-biotin complex (ABC) was done at 37°C for one hour and washed with TBS solution. 3,3 Diaminobenzidine tetra hydrochloride solution applied for 3–5 min. Counter-staining with haemotoxylin solution done for 3–5 min. Sections were washed with distilled water, air dried and mounted using DPX mountant. For Bax, positive controls were taken as germinal centers of the lymphoid follicles and normal breast tissue and negative control was taken as the test slide without primary antibody. For Bcl-2, positive controls were the mantle zone of lymphoid follicles and the negative controls were the test slides without primary antibody. The pattern of positive staining for bcl-2 and bax was cytoplasmic, granular. The primary antibodies for bcl-2 and bax were procured from DAKO. Bax-Rabbit Anti-Human code no. A 3533. Bcl-2 Monoclonal Mouse Anti-Human code no. M 0887. Dilution for both was 1: 40. The results were interpreted on the basis of two criteria: (1) Percentage of cells showing immune bodies; <5%: score = 0, 5–25%: score = 1, 25–75%: score = 2, >75%: score = 3 (2) Intensity of staining; mild: score = 1, moderate: score = 2, intense: score = 3. " Since there was a strong correlation between the intensity of staining and percentage of cells showing immune bodies, the percentage of cells showing immune antibodies alone was considered for calculating the bcl-2/bax ratio ". Statistics Descriptive studies were performed with SPSS version 10. The significance of response assessed using paired t-test. Significance of correlation between various variables assessed using chi-square test and coefficient of correlation was calculated by Pearson correlation coefficient. Results 50 cases of locally advanced breast cancer were included in this study. Mean age of the patients was 45.5 years (range: 28 to 71 years) and 53.3% patients were pre-menopausal. Size of the tumor was measured clinically as well as by ultrasound and the patients were subdivided into four groups: <5 cm(0%), 5–8 cm(56.6%), 8–10 cm(26.6%), >10 cm(16.6%). According to the axillary lymph node status the patients were divided into three groups: N0 (0%), N1 (33%), N2 (67%). Objective clinical response was defined as more than 50% reduction in the tumor size after three cycles of neoadjuvant chemotherapy. Immuno-histochemical response was defined as decrease in the Bcl-2/Bax ratio. Clinical response including the reduction in the tumor size and axillary lymph node status was observed in 70% of patients and was found to be statistically significant (p < 0.0001). There were no patients in the No group and 29.4%of the N1 patients were down staged to N0 while70.6% remained N1. In patients with N2 disease 7.7% were down staged to N0 status while 46.2% were downstaged to N1 status and 46.2% did not show any response. Immuno-histochemical response was observed in 60% and was also found to be statistically significant (p = 0.008). Correlation between immuno-histochemical and clinical response was also found to be statistically significant (p < 0.0001) [Table 1 ]. Acute vomiting was observed in 63.3% patients. 81% clinical responders had vomiting (p = 0.002) and 78% immunohistochemical responders also had vomiting which was statistically significant (p = 0.04). Alopecia was observed in 86% clinical responders (p = 0.000) and 94% immuno-histochemical responders (p = 0.000), which was also significant. Leucopoenia was observed in only 14% and 17% of clinical and immuno-histochemical responders respectively and was found to be an insignificant predictor of response in the present study. When multiple toxicities were correlated with the clinical and immuno-histochemical response, 46.7% of patients had both acute vomiting and alopecia. 67% clinical responders (p = 0.001) had both vomiting and alopecia.72% immunohistochemical responders (p = 0.001) had both vomiting and alopecia. A significant positive correlation was observed between the presence of vomiting (r = +0.558), alopecia(r = +0.802) and response to neoadjuvant chemotherapy. A significant negative correlation was observed between the absence of side effects and poor response to neoadjuvant chemotherapy (Table 2 ). Discussion Carcinoma of the breast is the leading cause of cancer in women all over the world and the second most common malignancy in India after carcinoma of the uterine cervix [ 1 ]. No other common epithelial cancer has been so carefully studied and so extensively characterized biologically [ 1 , 2 ]. In developing countries like India rate of locally advanced breast cancer at first diagnosis is estimated to be as high as 25%–30%[ 2 , 5 ]. The treatment of locally advanced breast carcinoma (LABC) has also evolved from primarily local modalities to treatment regimens that combine both systemic and local therapy. The realization that patients with LABC are likely to have undetectable micro metastases at diagnosis has lead to systemic treatment assuming major focus of the multi-modality approach as the studies have confirmed that surgery alone is an inadequate treatment in the management of these patients. Even aggressive surgical techniques have been observed to have a higher incidence of local recurrence in these patients [ 10 , 11 ]. Most importantly surgery does not change the pattern of distant failure in patients who probably have micrometastatic disease at the time of diagnosis [ 10 - 13 ]. Multi-modality therapy that included surgery, radiation therapy, chemotherapy, hormonal therapy has had the greatest impact on survival in patients with LABC [ 10 - 13 ]. Neoadjuvant chemotherapy (NACT) A new approach in the form of neoadjuvant chemotherapy was first reported in the 1970s and was initially utilized to convert unresectable tumors to smaller tumors making them more amenable to local control with either surgery or radiotherapy. An added advantage of this approach was the ability to assess patient's response to treatment both clinically after a defined number of courses of chemotherapy and pathologically after surgical resection. Perez and colleagues reported their results of a pilot study by the South-Eastern Cancer Study Group in 1979 that the primary tumor showed partial regression (>50%)in 65% of patients after two courses of FAC [ 16 ]. NACT has also shown benefits in the operable breast cancers by increasing the chances of breast conservation by up to 90% in some trials [ 10 - 13 ]. The other important advantage of NACT is that it represents an in vivo chemo sensitivity test for assessment of tumor response from which prognostic information can be obtained. It provides an early treatment of the micrometastatic disease, counteracting the increased growth rate possibly determined by the shrinkage of the tumor. The down staging converts an inoperable case amenable to curative resection [ 10 - 13 ]. Apoptosis Introduced by Kerr et al (1972) to describe characteristic morphological changes seen during programmed cell death [ 3 ]. It is defined as a closely regulated form of active cell death defined by characteristic biochemical and morphological criteria [ 3 , 14 , 15 ]. A wide range of anticancer drugs with widely differing modes of action have been demonstrated to induce apoptosis in vitro, suggesting this as a significant common final pathway through which they exert their clinical effect. Further more the mechanisms that suppress apoptosis may be important in the development of acquired resistance to cytotoxic drugs. Apoptosis or programmed cell death plays an important role in the regulation of tissue development, differentiation and homeostasis. It is therefore possible that deregulation of apoptosis contributes to the pathogenesis of cancer [ 3 , 13 - 15 ]. Apoptosis can be differentiated biochemically and morphologically from necrosis by the following criteria [ 16 ]: (1) Chromophin condensation (2) Membrane blebbing (3) Appearance of apoptotic bodies (4) Fragmentation of genomic DNA Certain biochemical and genetic events have been identified that are associated with multiple cell types including mammary epithelium. These include the DNA fragmentation via end nuclease activation and cleavage of intracellular proteins, expression of bcl-2 family members, tumor suppressor gene p-53 directed events, proto-oncogene activation and activation of transmembrane receptor signaling pathways such as tumour necrosis factor [ 4 , 17 - 22 ]. Although little is known about the mechanisms, which regulate apoptosis in epithelial cells, it is conceivable that defects in apoptosis related genes are involved in the pathogenesis of human cancers. The hypothesis is supported by the fact that the tumor suppressor gene product p-53, which is frequently mutated or deleted in breast cancer, is involved in regulating apoptosis [ 23 ]. The heterogeneous nature of breast cancer has resulted in overwhelming interest in search for prognostic markers to identify patients who might benefit most from the therapeutic modalities available. Assessment of apoptosis and individual components of apoptotic pathway are therefore relevant in determining prognosis in a particular patient [ 24 ]. DNA damaging agents such as ionizing radiations and chemotherapeutic drugs also induce apoptosis. Sakakura et al have shown an association between increased resistance to chemotherapeutic agents and decreased capacity to undergo apoptosis [ 25 ]. Central to this response are proteins that modulate apoptosis, including bcl-2 and bax gene products. Bcl-2 is anti-apoptotic in function, whereas bax is proapoptotic and it is the interaction between the two that determines the likelihood of a tumor to undergo cytotoxic drug mediated regression. Therefore any increase in bcl-2 or decrease in bax will push the balance towards chemo resistance and an increase in bax or decrease in bcl-2 will result in increased apoptosis [ 26 - 30 ]. It was observed in a study conducted by Kymionis et al [ 15 ] that increase in the ratio of anti apoptotic protein bcl-2 to pro-apoptotic protein i.e. bax results in markedly enhanced resistance of tumor cell lines to the cytotoxic effects of essentially all currently available chemotherapeutic drugs. In the present study the clinical response in terms of reduction of tumor size and immuno-histochemical response in terms of change of bcl-2/bax ratio correlated significantly with the drug induced toxicity following NACT. Toxicity related to chemotherapeutic agents [ 19 - 23 ] The time course of various toxic manipulations depends on the drug, its dose and frequency of administration, intrinsic characteristics of the tissue of interest and any local circumstances (e.g. radiation therapy, infection, trauma). There are few general rules however like mucosal toxicities of pain, erythema, ulceration etc. occur 3–10 days after the administration of most offending drugs. Bone marrow effects can be manifestated a few days later averaging 7–14 days. The recovery of normal functioning tissues in both cases is well under way 4–5 days after the zenith of the toxic effects. Alopecia may involve the scalp or the whole body can present within 2 weeks of the drug dose or be a progressive cumulative event. Other cumulative toxicities seen only after administration of a certain quantity of drug over some length of time include cardiomyopathy from anthracyclins. Cyclophosphamide An alkylating agent belonging to the nitrogen mustard subgroup. It is inactive as such produces few acute effects and is not locally damaging. It is transformed in to active metabolites like aldophosphamide and phospharimide mustard in the liver, which then produces the wide variety of antitumour action. The prominent side effects are alopecia and cystitis, which are caused by another metabolite acrolein. 5-Fluorouracil It is a pyrimidine antagonist and is converted in the body to the corresponding nucleotide, 5-fluro-2-deoxy-uridine monophosphate, which inhibits thymidine synthetase and blocks the conversion of deoxyuridilic acid to deoxythymidilic acid. Selective failure of DNA synthesis occurs due to non-availability of thymidylate. Flourouracil may itself get incorporated in to nucleic acids and this may contribute to its toxicity. Even resting cells are affected, though multiplying cells are more susceptible like the cells in the gastrointestinal tract (GIT) and bone marrow. Doxorubicin It is an anti tumor antibiotic and is capable of causing breaks in the DNA strands by activating topoisomerase II and generating quinone type free radicals. They have mutagenic potential. Maximum action is exerted at S phase, but toxicity is usually exerted in G2 phase. Cardiotoxicity is a unique side effect. Rapidly multiplying cells are more susceptible therefore it also acts on cells of GIT, bone marrow in addition to the tumor cells. Leucopenia has not been observed to be a frequently encountered chemotherapy induced toxicity using commonly used regimen in most of the studies [ 19 - 24 ]. This was observed in the present study also. Conclusions The rapidly proliferating normal and tumor cells are more susceptible to the action of chemotherapeutic agents, which could explain the significant correlation observed between the effects and the toxicity in the present study. There was a strong correlation observed between the immunohistochemical response (bcl-2/bax ratio), clinical response and drug toxicity. This indirectly indicates a correlation between chemotherapy induced apoptosis and the toxicity and therefore like apoptotic markers, chemotherapy induced toxic effects along with objective clinical response could serve as reliable and cost-effective indicators or predictors of response to NACT in patients with LABC. While many biological markers are in use and many are under trial to tailor the chemotherapy for a particular patient, most of these markers including apoptotic markers or p-glycoprotein etc. are not very frequently available and are expensive for a third world cancer set up. Thus the chemotherapy-induced toxicity along with clinical response may be utilized as a cost effective and reliable predictor of response to NACT in patients with LABC. This would also serve as an intermediate end point in determining drug sensitivity for adjuvant treatment, especially when adjuvant therapy is planned with the same regimen as induction chemotherapy. This can also help in planning an alternative regime in non-responders. Competing interests None declared. Authors' contribution • CM , the principal and the corresponding author was the Supervisor and the Chief surgeon who performed and standardized surgery on the patients and designed the study. • VS participated in the designing of the study, performed the statistical analysis and was the first surgical assistant and Senior Postgraduate in charge of the cases in the study. • JP , Postgraduate surgical resident was the second assistant in charge of the cases and participated in the sequence alignment. • AL, Resident surgery participated in the data processing and statistical analysis. • SS was in charge of the molecular genetic studies at the Tumor biology lab ICMR. • AB participated in the genetic studies and data processing. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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549589

A Person-Centred Approach to Communicating Risk

Standard approaches to communicating risk to patients do not appear to be very effective, argues Alaszewski. We need a new approach that takes patients' own perceptions into account

Doctors and other health professionals play a key role in communicating risk information. They are advisers to patients, especially when patients have to make fateful decisions that can irrevocably change their lives. There is a developing body of literature on the ways in which risk information can be effectively communicated [ 1 , 2 ]. However, much of this literature focuses on the nature of risk information and ways in which the transfer of this information can be improved. It does not fully take into account the complexity of the real world of clinical practice, nor the importance of considering patients as active partners in communication. The Rational Model of Risk Communication Much of the discussion of risk communication is grounded in the rational model of risk communication [ 3 , 4 ]. This model emphasises the role and position of experts such as doctors who have the ability to identify relevant risk knowledge. In the context of medical decision-making this is knowledge about the probable consequences of different courses of action based on scientific research. The role of the doctor is to make such knowledge available so that the patient can then use it to make an informed decision. With the rational model, when there is evidence that patients have not used risk knowledge effectively, then the response of the professional is to consider ways in which risk communication can be improved, such as by improving its presentation or mode of communication. When patients appear to be making irrational or harmful decisions, for example, continuing to smoke, choosing not to vaccinate a child against measles, mumps, and rubella, or not complying with medication, the professional's response is to work harder to convey the risks. Patients actively seek information on risks from many different sources (Illustration: Margaret Shear, Public Library of Science) But the rational model contains two key flaws. One relates to the nature of risk knowledge and the second to the nature of communication [ 5 ]. Within the rational model, risk knowledge is treated as a relatively simple and straightforward matter—in other words, there is a single uncontested source of knowledge that is relatively easy to access. In reality, risk knowledge is often a complex matter. While such knowledge may be produced by scientific research, it can and often is contested. There may be a scientific consensus, for example, that eating beef or having your child vaccinated against measles, mumps, and rubella is relatively safe, but there are often alternative scientific views, sometimes represented by high-profile media “mavericks” who emphasise the potential hazards [ 6 ]. Risk knowledge cannot actually be used directly by patients to inform their decision-making. Scientific research such as in epidemiology generates knowledge about the probability of harmful events occurring within populations . Individual patients need information on their own personal risks. Expert assessments of risk tend to focus on the knowable and measurable components of risk, that is, the extent to which future events are the same as and predictable by the knowledge of past events. Such assessments by definition exclude uncertainty—those aspects that cannot be assessed and measured. Given the speed of social and technological change, it is not clear that the past is an effective guide to the future. As such, there is an increasing awareness of the uncertainty of risk assessment, for example, in relationship to new diseases such as HIV/AIDS or new technologies such as mobile phones or genetically modified foods. The Need for a Person-Centred Approach Within the rational model of risk communication, the emphasis is on the flow of knowledge from the knowledgeable doctor to the uninformed patient. However, communication is a two-way process, and increasingly there is awareness of the active role of patients and the public [ 7 ]. Patients actively seek information, especially when they are aware that they are facing a crucial decision. While they can use traditional sources such as friends and relatives, if they have the skills and resources they can, through media such as the Internet, access highly sophisticated risk knowledge. For example, via the Cochrane Collaboration Web site ( www.cochrane.org ) they can find the latest evidence-based assessments of medical treatments and technologies, or via the Dr. Foster Web site ( www.drfoster.co.uk ) they can find the risks associated with different treatment facilities in the United Kingdom. Many patients access a variety of different sources, so they can clearly compare and evaluate the information provided by each. Patients do give particular credibility to sources that they know, which may include family and friends but also medical advisers with whom they have developed a relationship. They are particularly concerned about the trustworthiness of particular sources. While individuals can use their personal experience to evaluate the trustworthiness of personal sources, such as a particular relative or doctor, they often use contextual information to judge the trustworthiness of impersonal sources [ 8 ]. For example, information provided by a source that has an identifiable commercial interest, such as a company marketing a food product, will be considered as less trustworthy than a source without such an interest, for example, an expert committee of scientists. Patients will actively interpret risk information. If the information is timely and relevant it will tend to be accepted. Patients tend to define relevance in terms of the way they view or frame a situation, and there may be considerable differences between the ways that experts and patients view the same situation. As Zinn notes, the ways in which individuals frame and perceive risk will be highly influenced by their social situation, especially their personal biography [ 9 ]. Individuals may identify and respond to the same risks in very different ways. For example, Ziegeler has shown how the background and social context of individuals who have been diagnosed as having multiple sclerosis influence the ways in which they identify and manage their risks and opportunities [ 9 ]. Features of a Person-Centred Approach Standard approaches to risk communication, whether targeted at groups or individuals, do not appear to be very effective. For example Ruston and Clayton have shown the ways in which women disregard information and conceptually distance themselves from the risk of coronary heart disease—this applies even to those admitted to hospital with the disease [ 10 ]. Coleman has documented the failure of strategies that focus on providing information about the risks of teenage pregnancy to have any marked effect [ 11 ]. If doctors want to communicate effectively, then they need to develop a person-centred approach to risk communication, one that recognises that communication forms part of a relationship and builds upon it. Communication should be a dialogue that develops as the relationship develops, and those involved should have complementary and linked roles. Thus, the initial stage of communication could involve identifying the key issues, that is, those that cause concern for the patient. In this phase the emphasis might be on the patient talking and the doctor listening. If there is a major difference in the ways in which patient and doctor are framing the risk issues, then there might need to be an exchange or negotiation in which both parties adjust their mutual expectations and seek a mutually acceptable definition of what the problem is. If such an exchange does not take place, and if the patient's definitions are disregarded and not acknowledged, there is the danger that the patient will passively acquiesce but treat much of the information provided by the doctor as irrelevant and disregard it. If and when there is agreement, then there is the possibility of discussing the future and the likely consequences of taking different actions (risk communication in its traditional form). During this part of the exchange the emphasis might shift towards the doctor talking more and the patient listening more. There is a transfer of information, but it is a two-way process. The doctor should learn something about the patient's situation, including the risks that the patient is concerned about and the patient's beliefs about the nature of such risks. The patient should learn about the doctor's views of the nature of the risks that the patient is facing and the options for managing such risks. Underpinning the development of an effective relationship is the development of mutual trust. While trust usually takes time to develop, it is possible even during a short but positive exchange, in which mutual respect is shown, for a form of “swift” trust to be developed [ 12 ]. While I have focussed on communication in face-to-face relationship, the same issues and processes can be identified in more impersonal communication, such as the provision of risk information in health promotion campaigns. In such campaigns special mechanisms need to be created for dialogue. For example, Jones has described a project that engages young drug users in Hong Kong by helping them make videos about drug use, and has shown how such techniques can be used to evaluate and improve current health promotion adverts [ 13 ]. Conclusion There are no quick technical fixes for communicating risk information. If health professionals are serious about communicating risk information so that patients and others can make informed choices, they need to recognise that communication is a two-way process, and they need to take time to access patients' accounts and perceptions. Such investment should pay off both in an improved relationship and also in improved concordance with treatments.

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555759

Silhouette scores for assessment of SNP genotype clusters

Background High-throughput genotyping of single nucleotide polymorphisms (SNPs) generates large amounts of data. In many SNP genotyping assays, the genotype assignment is based on scatter plots of signals corresponding to the two SNP alleles. In a robust assay the three clusters that define the genotypes are well separated and the distances between the data points within a cluster are short. "Silhouettes" is a graphical aid for interpretation and validation of data clusters that provides a measure of how well a data point was classified when it was assigned to a cluster. Thus "Silhouettes" can potentially be used as a quality measure for SNP genotyping results and for objective comparison of the performance of SNP assays at different circumstances. Results We created a program (ClusterA) for calculating "Silhouette scores", and applied it to assess the quality of SNP genotype clusters obtained by single nucleotide primer extension ("minisequencing") in the Tag-microarray format. A Silhouette score condenses the quality of the genotype assignment for each SNP assay into a single numeric value, which ranges from 1.0, when the genotype assignment is unequivocal, down to -1.0, when the genotype assignment has been arbitrary. In the present study we applied Silhouette scores to compare the performance of four DNA polymerases in our minisequencing system by analyzing 26 SNPs in both DNA polarities in 16 DNA samples. We found Silhouettes to provide a relevant measure for the quality of SNP assays at different reaction conditions, illustrated by the four DNA polymerases here. According to our result, the genotypes can be unequivocally assigned without manual inspection when the Silhouette score for a SNP assay is > 0.65. All four DNA polymerases performed satisfactorily in our Tag-array minisequencing system. Conclusion "Silhouette scores" for assessing the quality of SNP genotyping clusters is convenient for evaluating the quality of SNP genotype assignment, and provides an objective, numeric measure for comparing the performance of SNP assays. The program we created for calculating Silhouette scores is freely available, and can be used for quality assessment of the results from all genotyping systems, where the genotypes are assigned by cluster analysis using scatter plots.

Background High-throughput single nucleotide polymorphism (SNP) genotyping assays generate large amounts of data, which usually is presented as scatter plots of signals corresponding to the two SNP alleles. A robust SNP genotyping assay is characterized by large distances between the three clusters that define the genotypes and small distances between the data points within each cluster. Numeric quality measures for the scatter plots would allow objective and automatic assessment of the success of a SNP assay. "Silhouettes" were introduced in 1987 as a general graphical aid for interpretation and validation of cluster analysis [ 1 ]. In a Silhouettes calculation, the distance from each data point in a cluster to all other data points within the same cluster and to all data points in the closest cluster are determined. Thus Silhouettes provides a measure of how well a data point was classified when it was assigned to a cluster by according to both the tightness of the clusters and the separation between them. This feature renders Silhouettes potentially well suited for assessing cluster quality in SNP genotyping methods. In high-throughput SNP genotyping, Silhouettes could be used for assessing the quality of automatic genotype assignment by alerting the operator if the quality of the genotype clusters fall below a certain limit. During assay development and optimization, Silhouettes could be used to compare the performance of a genotyping assay at different reaction conditions. It could also be applied for comparing the robustness of different SNP genotyping technologies. In this study we created a program (ClusterA) to calculate numeric Silhouettes for assessing the quality of genotype clusters obtained in SNP genotyping assays. We show the utility of Silhouettes and the program by applying it to our "in-house" developed four-color fluorescence minisequencing system for SNP genotyping in a microarray format [ 2 ]. Single nucleotide primer extension ("minisequencing") is the reaction principle underlying several of the commonly used systems for genotyping single nucleotide polymorphisms (SNPs) [ 3 - 8 ]. In minisequencing a DNA polymerase is employed to specifically extend a detection primer designed to anneal directly adjacent to the SNP position in the complementary DNA strand with a single labelled nucleotide analogue. The DNA polymerase is the most important factor that determines the efficiency and specificity of the primer extension reaction, irrespectively of the assay format. We used Silhouettes to compare the performance of three new commercially available DNA polymerases to the ThermoSequenase DNA polymerase, which is routinely used in minisequencing assays in many laboratories, including our own. We found Silhouettes to provide a relevant measure, in addition to signal-to-noise ratios and genotyping success, for selecting the most favourable enzyme for our assay. Results and Discussion We created a program, denoted ClusterA, for calculating numeric "Silhouettes" for clustered data, such as for example the three clusters of signal ratios commonly obtained in SNP genotyping assays. Figure 1 illustrates the Silhouette calculation for one data point in a typical scatter plot obtained in a SNP genotyping assays. A Silhouette close to 1.0 is obtained when the average distance from a data point to the other data points within its own cluster is smaller than the average distances to all data points in the closest cluster. A Silhouette close to zero indicates that the data-point could equally well have been assigned to the neighbouring cluster. A negative Silhouette is obtained when the cluster assignment has been arbitrary, and the data point is actually closer to the neighbouring cluster than to the other data points within its own cluster [ 1 ]. The mean value from the Silhouette calculations for all data points in each cluster yield an "average Silhouette width" for the cluster. Figure 1 Principle for Silhouette scores. Principle for quality assessment of genotyping clusters using Silhouette scores, illustrated for one data point (i). The SNP genotypes have been assigned based on cluster formation in scatter plots with the signal intensity fraction on the x-axis and the logarithm of the signals from both alleles on the y-axis. For each data point (i) in the scatter plot, the Silhouette s(i) is calculated by the formula in the figure, where a(i) is the average distance from i to all data points in the same genotype cluster (green lines), and b(i) is the average distances from i to all data points in the cluster closest to the data point, either b 1 (i) (blue lines) or b 2 (i) (red lines) [1]. Max and min in the formula denote the largest or smallest of the measures in the brackets. The "average silhouette width" is calculated by calculating the mean of all s(i) for each genotype cluster and the "Silhouette score" for the whole scatter plot (SNP assay) is obtained by taking the mean of the average silhouette width for all clusters. Here, we applied ClusterA to calculate "Silhouettes" for comparing the quality of the genotype clusters obtained in our "in-house" Tag-array minisequencing system. For each scatter plot, the mean of the average silhouette widths for the three genotype clusters were used to define a "Silhouette score" for each SNP assay. Thus the Silhouette score condenses the cluster quality for each SNP assay into a single measure that ranges from 1.0 to -1.0. When calculating the Silhouette score, the distance between data points can be measured either in one dimension, for example on the x-axis, or in two dimension using vectors, as illustrated in Figure 1 . In our Tag-array minisequencing system we used distances measured only in one dimension, along the x-axis, where the signal fraction (Signal Allele2 / (Signal Allele1 +Signal Allele2 ) is plotted, since this is the major determinant for genotype assignment in our system. The logarithm of the sum of the signals from both alleles (Signal Allele1 +Signal Allele2 ) plotted on the y-axis is only used to set the cut-off values for failed genotype calls. Figure 2 shows nine examples of SNP genotype clusters that yielded different Silhouette scores. Negative controls and assays with signals below signal cut-off level are not shown in Figure 2 since they are not included in the Silhouette score calculations. Figure 2 Examples of Silhouette scores. Examples of genotype clusters from nine SNP assays, each with the results from 16 samples genotyped in duplicate using Tag-array minisequencing with the calculated Silhouette scores shown in the right hand upper corner of each panel. The blue circles represent homozygotes for allele 2, the red triangles are heterozygotes and the green squares are homozygotes for allele 1. The SNPs are denoted by their dbSNP identification number, and the DNA polarities analyzed are indicated by "cod" or "nc". The examples in panels E, F and G of Figure 2 illustrate how different clustering patterns can yield similar Silhouette scores. Based on the results from the scatter plots used to assign genotypes in this study, our recommendation is to accept the results from SNP assays with Silhouette scores >0.65 and to fail the whole assays if the Silhouette scores is <0.25. Individual genotype calls for assays where the Silhouette score falls between 0.25–0.65 may be accepted or failed after visual inspection. Excluding some of the outliers will then increase the Silhouette score. Our recommendations is in line with Liu et al., who have included silhouette calculations in the complex algorithm used to interpret the data from the Affymetrix 10K HuSNP hybridization microarray [ 9 ]. Here we exemplify the use of Silhouette scores by comparing the performance of the TERMIPol, Therminator, KlenThermase and ThermoSequenase DNA polymerases in the Tag-array minisequencing system [ 2 ]. Twenty-six SNPs were analyzed in both polarities in 16 DNA samples in two independent experiments. As our Tag-array genotyping system utilizes an "array of arrays" format [ 10 ] with 80 subarrays on each microscope slide, we were able to test all four enzymes in all samples on the same slide at exactly the same conditions, to facilitate a fair comparison between the enzymes. Figure 3 shows the distributions of Silhouette scores in these SNP assays. For all enzymes, 75% of the scatter plots (indicated by light blue rectangles in Figure 3 ) yielded silhouette scores above or close to our recommended limit of 0.65. Results from a total of 79 scatter plots/SNP assays are included in Figure 3 and Table 1 . If a SNP assay failed for all samples with one enzyme, the results from this assay were excluded from the whole enzyme comparison. It should also be noted that a non-stringent genotype calling strategy was applied to reveal possible differences between the enzymes both in clustering properties and genotyping results. This is the reason for the very low Silhouette scores for some SNP assays, which normally would be considered as failed. Using 0.65 as cut-off, 70–76% of the SNP assays would have been successful in this study. Figure 3 Distribution of Silhouette scores from minisequencing assays using four DNA polymerases. The Silhouette score is given on the y-axis. Each black diamond represents the Silhouette score for one SNP assay. The light blue rectangular boxes indicate those 75% of the scatter plots that yielded the highest silhouette scores for each enzyme. Quartiles are indicated by the black horizontal lines. Table 1 Silhouette scores, signal to noise ratios and genotyping performance for four DNA polymerases in Tag-array minisequencing 1 Silhouette score 2 S/N 3 Genotype calls 4 Average Median Highest Average Highest Correct Errors n % n % n % n % TERMIPol 0.72 0.78 20 25.3 4.3 11 13.9 2337 98.9 18 0.8 Therminator 0.69 0.79 15 19.0 3.6 7 8.9 2323 98.3 32 1.4 KlenThermase 0.74 0.79 22 27.8 8.0 21 26.6 2346 99.3 10 0.4 ThermoSequenase 0.71 0.82 22 27.8 8.9 40 50.6 2324 98.3 34 1.4 1 Duplicate experiments, each with duplicate SNP assays in both DNA polarities, were performed and the results are composite values from both experiments. 2 The Silhouette scores were calculated as described in Figure 1. The average and the median score for all SNPs are given for each enzyme together with the number of SNP assays (n) and frequency (%) where an enzyme yielded the highest Silhouette score. 3 Signal to noise ratios (S/N) were calculated from each spot by dividing the fluorescence intensity values from the fluorescently labelled ddNTP/ddNTPs corresponding to a true genotype (signal) by the fluorescent intensity value from the other ddNTPs (noise). The average S/N ratios are given together with the number of SNP assays (n) and frequency (%) where an enzyme yielded the highest S/N. 4 Number of genotype calls (n) and call rate (%). The genotype obtained from the majority of the assays was considered to be the correct one. The percentages of the samples not accounted for in the table failed to give genotypes. In the comparison between the enzymes, KlenThermase displayed the highest average Silhouette score, ThermoSequenase had the highest median Silhouette score and also obtained the highest Silhouette score most frequently (Table 1 ). In addition to the Silhouettes scores, that represent a measure of the robustness of a SNP assay, the signal to noise ratios (S/N) and the genotyping success was assessed (Table 1 ). All four enzymes performed satisfactorily in our minisequencing assay taking into account the non-stringent genotyping criteria used. However, performance varied between the evaluated features with high error rates for Therminator and ThermoSequenase. KlenThermase showed the best results over all and, also taking into account the cost, would be the enzyme of choice based on the results from this study. Conclusion We conclude that "Silhouette scores" for assessing the cluster quality is well suited for comparing the performance of SNP assays. Here we used a one-dimensional calculation of the Silhouette scores, by measuring the distances between the data-points along the x-axis only. A two-dimensional Silhouette calculation using vectors should be applied when genotypes are assigned by scatter plots with the fluorescence signals corresponding to the two alleles on the y- and x-axis. Both options are available in the ClusterA program that also calculates mean, variance and F-statistic for the input data set. The program is freely available through our website . We believe that the ClusterA program for calculating Silhouette scores created in the present study is a useful and general tool for any genotyping system, where the genotypes are called by cluster analysis with the aid of scatter plots. Methods DNA samples Genomic DNA was extracted from blood samples from 16 volunteer blood donors using the Wizard genomic DNA purification kit (Promega, Madison, WI). Genotyping procedure Twenty-six SNPs, selected to be located in unique PCR amplicons, were included in the test panel. For information on the single nucleotide polymorphisms and oligonucleotides used, see the Additional file 1 : SNPinformation.pdf. PCR primers were designed and combined in multiplex PCR reactions. Minisequencing primers with 20 bp 5'-Tag sequences were designed for both DNA polarities. The experimental details of the genotyping procedure have been described in detail previously [ 11 ]. In short it included the following steps: The regions containing the sequence variations were amplified in six optimized multiplex PCRs. For each sample the PCR products were pooled and divided into four aliquots, one for each enzyme. The remaining dNTPs and primers from the PCR reaction mixture were removed by treatment with Exonuclease I and shrimp alkaline phosphatase. The cyclic minisequencing reactions were performed in solution as described below, and the extended minisequencing primers were hybridized to microarrays carrying immobilized covalently coupled oligonucleotides (cTags) complementary to the Tag-sequences of the minisequencing primers. The cTags had been immobilized to CodeLinkTM Activated Slides (Amersham Biosciences, Uppsala, Sweden) via their 3'-end NH 2 -groups to form 80 subarrays per slide, each with 60 cTags as duplicate spots. Finally the microarray slides were scanned, and the fluorescent signals were measured. Minisequencing reaction Cyclic minisequencing reactions were performed in solution with 10 nM of each of the 52 tagged minisequencing primers using 0.1 μM ddATP-Texas Red, ddCTP-Tamra and ddGTP-R110 and 0.15 μM ddUTP-Cy5 (Perkin-Elmer Life Sciences, Boston, MA), and 0.064 U/μl of one of the four DNA polymerases in 15μl of 0.02% Triton-X, 4.1 mM MgCl2 and 33.6 mM Tris-HCl pH 9.5. The cyclic extension reactions were performed on a Thermal Cycler PTC-225 (MJ Research, Watertown, MA) with an initial 96°C for 3 min followed by 55 cycles of 95°C and 55°C for 20 s each. The DNA polymerases were; TERMIPol (Solis BioDyne, Tartu, Estonia), Therminator (New England BioLabs Inc., Beverly, MA, USA), KlenThermase (Gene Craft, Lüdinghausen, Germany), or ThermoSequenase (Amersham Biosciences, Uppsala, Sweden). A custom made reaction rack holding the arrayed slides with a silicon grid to give 80 separate reaction chambers was used during capture of the minisequencing reaction products on the Tag-arrays. Data analysis and genotype assignment The fluorescence signals were measured from the microarray slides using a ScanArray Express ® instrument (Perkin-Elmer Life Sciences, Boston, MA). The excitation lasers were: Blue Argon 488 nm for R110; Green HeNe 543.8 nm for Tamra; Yellow HeNe 594 nm for Texas Red and Red HeNe 632.8 nm for Cy5. The fluorescence signal intensities were determined using the QuantArray ® analysis 3.1 software (Perkin-Elmer Life Sciences, Boston, MA). The QuantArray file was exported to the SNPSnapper v4.0 software ) for genotype assignment. Raw data as fluorescence signals and signal ratios are provided as supplementary material, see Additional file 2 : Rawdata.txt. Genotypes were assigned based on scatter plots with the logarithm of the sum of both fluorescence signals (Signal Allele1 +Signal Allele2 ) plotted on the y-axis, and the fluorescence signal fraction, obtained by dividing the fluorescence signals from one allele by the sum of the fluorescence signal from both SNP alleles (Signal Allele2 / (Signal Allele1 +Signal Allele2 ), on the x-axis [ 11 ]. The result file with the assigned genotypes and the corresponding signal ratios were exported as a text file and used to calculate Silhouettes scores using the ClusterA program. ClusterA is implemented in Microsoft Visual Basic 6.0, and can be run on PCs with the Microsoft Windows operating system. The ClusterA program also provides the mean, variance and F-statistic for the input data. Authors' contributions LL planned the experiments, guided the laboratory work and performed the analysis of results, interpreted the data and drafted the manuscript. AA carried out the laboratory work and part of the data analysis and provided input to the manuscript. MJ programmed the ClusterA program and took part in the interpretation of Silhouettes. ACS initiated the study, supervised it, and coordinated the manuscript writing process. All authors have read and approved the final manuscript. Supplementary Material Additional File 1 Lists the dbSNP identification numbers and the sequences of the PCR and minisequencing primers. Click here for file Additional File 2 Includes the raw fluorescence signals and the fluorescence signal intensity ratios for the two experiments as a tab delimited text file. Click here for file

/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC555759.xml

555942

Gene fusions and gene duplications: relevance to genomic annotation and functional analysis

Background Escherichia coli a model organism provides information for annotation of other genomes. Our analysis of its genome has shown that proteins encoded by fused genes need special attention. Such composite (multimodular) proteins consist of two or more components (modules) encoding distinct functions. Multimodular proteins have been found to complicate both annotation and generation of sequence similar groups. Previous work overstated the number of multimodular proteins in E. coli . This work corrects the identification of modules by including sequence information from proteins in 50 sequenced microbial genomes. Results Multimodular E. coli K-12 proteins were identified from sequence similarities between their component modules and non-fused proteins in 50 genomes and from the literature. We found 109 multimodular proteins in E. coli containing either two or three modules. Most modules had standalone sequence relatives in other genomes. The separated modules together with all the single (un-fused) proteins constitute the sum of all unimodular proteins of E. coli . Pairwise sequence relationships among all E. coli unimodular proteins generated 490 sequence similar, paralogous groups. Groups ranged in size from 92 to 2 members and had varying degrees of relatedness among their members. Some E. coli enzyme groups were compared to homologs in other bacterial genomes. Conclusion The deleterious effects of multimodular proteins on annotation and on the formation of groups of paralogs are emphasized. To improve annotation results, all multimodular proteins in an organism should be detected and when known each function should be connected with its location in the sequence of the protein. When transferring functions by sequence similarity, alignment locations must be noted, particularly when alignments cover only part of the sequences, in order to enable transfer of the correct function. Separating multimodular proteins into module units makes it possible to generate protein groups related by both sequence and function, avoiding mixing of unrelated sequences. Organisms differ in sizes of groups of sequence-related proteins. A sample comparison of orthologs to selected E. coli paralogous groups correlates with known physiological and taxonomic relationships between the organisms.

Background Eschericia coli remains a useful resource to the genomic community as it provides important knowledge which can be applied to the analysis of most microbial genomes. Its central role devolves from two facts; first, the accumulated results of seven decades of laboratory experimentation have identified the function(s) of over half of its gene products; second being a metabolic generalist, E. coli 's metabolic functions are widely shared among other organisms. Common practices of annotation rely, more than one might realize, on the accuracy of the annotation of E. coli 's genes. While searches for sequence matches to unknown genes usually yield a large number of matches, chances are high that firm functional information comes only from experimental studies on E. coli . Because annotations of genes do not always indicate that the assignments are derived, and because derived annotations are used serially for further annotation without experimental confirmation, many genes carry original E. coli annotations. It is therefore important to the entire genome-analyzing community that the data on E. coli gene products be as accurate as possible. Since the original GenBank deposit of E. coli K-12 (U00096), new and updated annotations are available at NCBI (U00096.2) and at more specialized databases including, ASAP [ 1 ], coliBASE [ 2 ], CyberCell [ 3 ], EchoBASE [ 4 ], EcoCyc [ 5 ], GenProtEC [ 6 ], and RegulonDB [ 7 ]. An effort is under way to coordinate the current E. coli annotations [ 8 ]. Over recent years, our work on the E. coli genome has led us to an appreciation of the pernicious role that gene fusions often play as troublemakers in function assignments and in relating groups of sequence similar proteins [ 9 ]. The fusion of two independently functioning genes results in the formation of a composite (multimodular) protein encoding for two independent functions located at separate parts of the protein. This type of fusion is not equivalent to the joining of protein domains, i.e. domains encoding binding sites for a cofactor or a substrate, which is seen in multidomain proteins. An example being the enzyme glyceraldehyde-3-phosphate dehydrogenase which according to the domain databases Pfam [ 10 ] and Superfamiliy [ 11 ] contains two domains, an NAD binding site and a dehydrogenase catalytic site. In our studies the entire protein including both domains represents one independent functional unit with one activity. Multidomain proteins are more prevalent and most often encode one overall function for the gene product [ 12 ]. Annotation involving transfer of function from composite proteins to sequence similar matches requires that the alignment regions be evaluated in order to determine whether all activities or only one of them should be assigned to the matching sequence. Currently fused proteins are themselves not always annotated to reflect that they encode more than one function, and rarely is the location of the separate functions indicated. Different combinations of fused genes are seen in the sequenced genomes, adding potential sources for annotation errors. Errors in functional assignments including those caused by fused genes have been noted years ago [ 13 ] and that such proteins may contribute to propagation of annotation errors in databases [ 14 ]. The fused proteins also interfere with the generation of sequence related protein groups as they link proteins based on their coexistence in a fused protein and not purely based on sequence similarity. Components of fused genes are often not sequence related, so generating protein groups without taking gene fusions into account may result in "mixed" groups of proteins with different sequence relatedness, functions and evolutionary histories. Previous work has been published where we identified fused E. coli proteins from partial alignments between proteins encoded in the E. coli genome [ 15 ]. This work resulted in the identification of 287 multimodular proteins. As our analysis continued and more genome sequences were incorporated in our studies we realized that most of these identified multimodular proteins actually contained multiple domains and had one overall function. We have therefore revised our method of detecting fused proteins. We are making use of sequence information from 50 genomes including E. coli to detect proteins which are fused in the E. coli genome and are present as individual components in one of the other genomes. We have also made use of published experimental data on E. coli gene fusions. As a result the number of fused proteins in E. coli has been reduced to 109. The number of groups of sequence related proteins was also reduced from 609 to 490 since some of the previously identified groups are made up of protein domains catalyzing only part of an overall reaction. This work represents a revision of the state of fused proteins in the E. coli genome their affect on genome analysis both within E. coli and across genomes. Results Multimodular vs. multifunctional proteins To prevent confusion, we define multimodular proteins as those seeming to result from gene fusion in which two independent proteins are connected. Multimodular proteins encode separate functions in different parts of the molecule. These functions might be the same if two like elements have fused, or as we see more often in E. coli , they differ in sequence and activity. Distinctly different, multifunctional proteins are defined as those that carry out more than one reaction or activity in the same part of the protein. Examples of such multifunctional proteins are encoded by the genes cob U, bir A, ubi G, fol D, cys G, tes A, and ndk (for details see gene products at GenProtEC [ 16 ]). A protein that illustrates both properties is the FadB protein of E. coli [ 17 , 18 ]. FadB is a multimodular protein with N-terminal and C-terminal modules. Its N-terminal module is multifunctional with three activities that are catalyzed at the same active site and cannot be spatially separated along the length of the protein. The three activities are 3-hydroxybutyryl-CoA epimerase, delta(3)-cis-delta(2)-trans-enoyl-CoA isomerase, and enoyl-CoA hydratase. The C-terminal module of FadB encodes a single function, 3-hydroxyacyl-CoA dehydrogenase. Adding the N-terminal and C-terminal modules, there are 4 activities for the FadB protein. Identifying multimodular proteins in E. coli In earlier work, before the genomic sequence of E. coli was completed, we saw that sequence similarity among its proteins was widespread [ 9 , 19 ]. After the entire sequence was available, we identified 287 E. coli proteins as being multimodular and encoded by fused genes [ 15 , 20 ]. The modularity of the proteins was inferred from the alignments among E. coli proteins. However, we have since found that many of these so-called multimodular proteins were proteins containing more than one domain and not more than one protein. Such multidomain proteins may appear to encode two functions but in reality encode two or more conserved motifs (i.e. DNA-binding and effector-binding domains of LysR type transcriptional regulators). By including sequence information from other genomes besides E. coli we were able to better distinguish fusions of complete proteins versus the more common fusions of protein domains. Of the 287 proteins previously identified as multimodulars only 70 remained as fused proteins in this study with the remaining representing domain fusions. In the present work, some of the fused proteins were identified by searching the literature for experimental data. Examples of E. coli proteins long known to contain multiple functions encoded at separate parts of the proteins include GlnE [ 21 ], MetL [ 22 ], ThrA [ 23 ], and TyrA [ 24 ]. We have collected such experimentally verified information over time [ 9 ], labeled as multimodular proteins with literature citations in our database GenProtEC [ 16 ]. Other multimodular proteins were identified by selected types of alignments between E. coli proteins and proteins encoded in 50 sequenced genomes. The component proteins of a multimodular protein may be unimodular and unfused in another genome. We looked for alignments between the larger potentially multimodular proteins in E. coli and smaller orthologous proteins that are homologous to only one of the modules (Figure 1a ). Not all gene fusions of E. coli will be detected by this method. For instance elements of a fused gene may have diverged to the point where the component modules no longer have detectable similarity to their homologous counterparts, or the independently existing modules may have been lost from the gene pool of the 50 genomes analyzed, or the 50 organisms may contain only the multimodular form. Figure 1 Identification and sequence similarity of multimodular E. coli proteins. (a) An E. coli protein (gi1787250) aligns with two smaller proteins from C. acetobutylicum , histidinol phosphatase (gi15026114) and imidazoleglycerol-phosphate dehydratatase (gi15023840). The E. coli protein represents a fused or multimodular protein encoding the two functions in separate parts of the protein as indicated by the two non-overlapping alignment regions. Based on the alignment regions, the E. coli protein is separated into two separate components, modules. The modules are identified with the extensions "_1" or "_2" to indicate their location in the gene product as N-terminal or C-terminal, respectively. (b) Sequence similarity between modules of the multimodular proteins is shown. No detectable similarity between the joined modules is indicated by a difference in the module patterns in the cartoon. Similarity is measured by Darwin and indicates that the proteins align at a distance of ≤ 200 PAM units over at least 83 amino acid residues or >45% of the length of the proteins. This level of similarity also reflects whether the modules belong to the same paralogous group. In total we identified 109 E. coli proteins to be multimodular, with 101 containing two modules and 8 containing three modules. The largest number of multimodular proteins joined modules of dissimilar sequence (illustrated in Figure 1b ). An abbreviated list of the modules and their functions is shown in Table 1 . A complete list of the multimodular E. coli proteins is made available: ' [see Additional file 1 ]'. The remaining proteins, 97.5 % of the total, were considered to be unimodular. The average length of the multimodular proteins was 637 residues compared to 309 for the remaining proteins in the chromosome (Figure 2 ). Individual modules from the multimodular proteins were on average 300 residues long, similar to the length of the unimodular proteins. However, the size alone of a protein does not reflect multimodularity as we found many large proteins to be unimodular. Table 1 Examples of multimodular E. coli proteins. Gene Module Start End Gty 1 Module Function thr A b0002_1 1 461 e aspartokinase I, threonine sensitive thr A b0002_2 464 820 e homoserine dehydrogenase I, threonine sensitive rib D b0414_1 1 143 e diaminohydroxyphosphoribosylaminopyrimidine deaminase rib D b0414_2 147 366 e 5-amino-6-(5-phosphoribosylamino) uracil reductase put A b1014_1 1 569 e bifunctional: transcriptional repressor (N-terminal); proline dehydrogenase, FAD-binding (C-terminal) put A b1014_2 618 1320 e pyrroline-5-carboxylate dehydrogenase adh E b1241_1 1 400 e acetaldehyde-CoA dehydrogenase adh E b1241_2 449 891 e iron-dependent alcohol dehydrogenase thi P b0067_1 1 274 t thiamin transport protein (ABC superfamily, membrane) thi P b0067_2 285 536 t thiamin transport protein (ABC superfamily, membrane) mdl A b0448_1 1 310 pt putative transport protein, multidrug resistance-like (ABC superfamily, membrane) mdl A b0448_2 314 590 pt putative transport protein, multidrug resistance-like (ABC superfamily, ATP_bind) mod F b0760_1 1 260 t molybdenum transport protein (ABC superfamily, ATP_bind) mod F b0760_2 261 490 t molybdenum transport protein (ABC superfamily, ATP_bind) hrs A b0731_1 1 178 t PTS family enzyme IIA, induction of ompC hrs A b0731_2 186 454 t PTS family enzyme IIB, induction of ompC hrs A b0731_3 456 628 t PTS family enzyme IIC, induction of ompC ato C b2220_1 1 125 r response regulator ato C b2220_2 145 461 r sigma54 interaction module of response regulator (EBP family) evgS b2370_1 1 935 e histidine kinase of hybrid sensory kinase evgS b2370_2 953 1197 r response regulator of hybrid sensory histidine kinase gln G b3868_1 1 120 r response regulator, two-component regulator with GlnL, nitrogen regulation gln G b3868_2 139 469 r sigma54 interaction module of response regulator (EBP family) kef A b0465_1 1 779 o unknown function module of mechanosensitive channel kef A b0465_2 780 1120 t mechanosensitive channel (MscS family) arg A b2818_1 1 293 o acetylglutamate kinase homolog (inactive) arg A b2818_2 298 442 e N-alpha-acetylglutamate synthase (amino acid acetyltransferase) ydc R b1439_1 1 117 pr putative transcriptional regulator (GntR family) ydc R b1439_2 118 468 pe putative amino transferase rnf C b1629_1 1 448 pc Fe-S binding module of electron transport protein rnf C b1629_2 450 740 o unknown function module of electron transport protein 1 Gene product type: e, enzyme; pe, putative enzyme; r, regulatory protein; pr, putative regulatory protein; t, transport protein; pt, putative transport protein; pc, putative carrier protein; o, unknown function. Figure 2 Size distribution for multimodular and single module proteins. The protein lengths in amino acid residues are shown for single module proteins (□) and for multimodular proteins (■). On average the multimodular proteins are longer than the unimodular proteins, 637 amino acids versus 314 amino acids. The length of a protein alone does not infer multimodularity and long single module proteins are seen. Characteristics of multimodular proteins of E. coli Table 2 shows some characteristics of the modules in the multimodular proteins. The majority of the E. coli modules, 90%, were found to have homologs existing as independent proteins in one of the 50 genomes analyzed. Independent unimodular homologs within E. coli were detected for only 57% of the modules (data not shown). A list of the major types of multimodular proteins is shown in Table 3 . Table 2 Features of multimodular E. coli proteins: No. Modules 109 multimodular proteins 226 101 bimodular proteins 202 8 trimodular proteins 24 with identity to unfused orthologs 203 without identity to unfused orthologs 23 known function 151 putative function 66 unknown function 9 type of protein 1 : enzyme 97 transport protein 85 regulatory protein 26 other 18 1 includes putative assignments Table 3 Types of multimodular proteins. Protein type 1 Protein names 2 Enzyme Aas, AdhE, AegA, ArgA, ArnA, CysG, Dfp, DgoA, DsbD, FadB, FadJ, FtsY, GlcE, GlmU, GlnE, Gsp, HisB, HisI, HldE, HmpA, MaeB, MetL, MrcA, MrcB 3 , NifJ 3 , PaaZ, PbpC, PheA, PolA, PurH, PutA, RbbA 3 , RibD, Rne 3 , ThrA, TrpC, TrpD, TyrA, YdiF, YfiQ, YgfN, YgfT, YjiR Transport protein AlsA, AraG, CydC, CydD, DhaH, Ego, FeoB, FhuB, FruA, FruB, FrvB, HrsA 3 , KefA, MacB, MalK, MalX, ManX, MdlA, MdlB, MglA, ModF, MsbA, MtlA, NagE 3 , PtsA, PtsG, PtsP, RbsA, ThiP, Uup, XylG, YbhF, YbiT, YddA, YejF, YheS, YjjK, YliA, YnjC, YojI, YpdD 3 , YphE Regulatory protein Ada, Aer, ArcB, AtoC, BarA, BglF, CheA, CheB, EvgS, GlnG, KdpD, MalT, RcsC, TorS, YdcR, YfhA, YieN, ZraR Other InfB, MukB 3 , RnfC, YegH, YfcK, YoaE 1 Gene type includes known and putative functions. 2 Protein names derived from gene names. 3 Genes encoding three modules. • Many of the multimodular enzymes function in the biosynthesis or degradation of compounds (amino acids, cofactors, peptidoglycan and fatty acids). • The majority of the multimodular transport proteins encode fusions of components of the ABC superfamily transporters (ATP-binding and membrane component). Also, fusions of the PTS proteins were detected in different combinations. Thirteen proteins contained two or more PTS components, including Hpr, enzymes I, IIA, IIB, or IIC. • Among the multimodular regulatory proteins, two-thirds were part of two-component regulatory systems and contained histidine kinases fused to response regulators. Seldom were known domain subdivisions within these modules detected by the rules we applied. While the fraction of enzymes (39%) is similar to the fraction of enzymes encoded in the genome as a whole (36%), the proportion of multimodular transport proteins (38%) and regulatory proteins (17%) were higher than their proportion genome wide (14% and 8% respectively). The over-representation in transporters and regulators is a reflection of the level of gene duplication seen for these proteins. Large paralogous groups are detected for some of the ABC transporter protein subunits and for components of the two-component regulators. Pairwise similarity of E. coli single modules All unimodular proteins, including the modules obtained from multimodular proteins, were tested pairwise for sequence similarity. Matching all single module E. coli proteins to each other using the AllAllDb algorithm of the Darwin package, we collected all aligned pairs with a similarity score of less than or equal to 200 PAM units, with an alignment of at least 83 residues. Altogether 9,626 unique pairs met these criteria (data available at GenProtEC [ 16 ]). Paralogous groups of E. coli protein modules We used the data on pairwise similarity to assemble groups of proteins of similar sequence that were unlike other proteins in the cell. Besides the PAM less than 200 and alignment length of at least 83 residues, two additional requirements were imposed; that more than 45% of each protein in each pair be aligned, and that a module could not belong to more than one group. A transitive clustering process was used to form the sequence-similar groups [ 9 ]. This grouping method requires only that each member of the group have sequence similarity to at least one other member of the group and does not require a detectable similarity among all the members of a group. Both closely related groups and groups with more divergent proteins were found. We identified 490 sequence-similar or paralogous groups in E. coli ' [see Additional file 2 for a complete list of the sequence-similar E. coli groups and their members]'. Altogether 1946 unimodular proteins belonged to one of the groups. Modules from 94 of the multimodular proteins were present in 61 of the groups. Table 4 shows the power law type of distribution of the number of members in the groups, smaller groups being more abundant than large ones. There were 279 groups of two proteins, and only 10 % of the groups had 7 or more members. As shown in Table 5 , the smaller groups tended to be tight groups in which the majority of sequences were related by our criteria to all or most others in the group. Larger groups were more divergent with a minority of members related to all others. At group size 8 and above, no members have the property of relating to all others. Table 4 Size distribution of paralogous groups. Group size No. Groups 2 279 3 91 4 32 5 31 6 6 7 18 8 7 9 2 10 2 11 3 12 1 13 2 14 2 18 2 20 1 21 1 22 2 24 1 30 2 40 1 43 1 46 1 51 1 92 1 Table 5 Sequence relationships within paralogous groups. Group size No. Groups All See All All See Some 3 92 56 36 4 32 21 11 5 31 7 24 6 6 0 6 7 18 2 16 The largest groups of paralogous enzymes, transport proteins and regulatory proteins are shown in Table 6 , 7 and 8 , respectively. While enzymes represent the largest gene product type in E. coli with known or predicted function, they tend to be present in smaller paralogous groups as compared to the transporters and regulators. Among the larger groups the oxidoreductases and the subunits of oxidoreductases are most common, making up 8 of the top 20 enzyme groups (Table 6 ). Table 6 Paralogous enzyme groups in E. coli . No. Members Group function 20 oxidoreductase, Fe-S-binding 18 oxidoreductase, NAD(P)-binding 18 oxidoreductase 1 , NAD(P)-binding 13 aldehyde oxidoreductase, NAD(P)-binding 13 oxidoreductase, FAD/NAD(P)-binding 11 sugar kinase 10 terminal oxidoreductase, subunit 9 aldo-keto oxidoreductase, NAD(P)-binding 8 phosphatase 8 nucleoside diphosphate (Nudix) hydrolase 8 acyl-CoA ligase 7 glutathione S-transferase 7 RNA helicase, ATP-binding 7 sugar epimerase/dehydratase, NAD(P)-binding 7 alcohol oxidoreductase 7 acyltransferase 7 aminotransferase, PLP-binding 7 decarboxylase, TPP-binding 7 crotonase 7 acyltransferase 1 Contains GroES-like structural domain (SCOP sf50129). Table 7 Paralogous transport protein groups in E. coli No. Members Group function 92 ABC superfamily transport protein, ATP-binding component 51 ABC superfamily transport protein, membrane component 40 MFS family transport protein 24 ABC superfamily transport protein, periplasmic binding component/ transcriptional regulator (GalI/LacR family)/ 22 APC family transport protein 12 ABC superfamily transport protein, membrane component 11 PTS family transport protein, enzyme IIA 9 ABC superfamily transport protein, periplasmic binding component 8 ABC superfamily transport protein, periplasmic binding component 7 GntP family transport protein 7 RND family transport protein 7 ABC superfamily transport protein, membrane component 5 HAAP family transport protein 5 PTS family transport protein, enzyme IIB 5 PTS family transport protein, enzyme I 5 GPH family transport protein 5 NCS2 family transport protein 5 HAAP family transport protein 5 transport protein 5 PTS family enzyme IIC 5 RhtB family transport protein 5 outer membrane porin Table 8 Paralogous regulatory protein groups in E. coli. No. Members Group function 46 LuxR/UhpA or OmpR family transcriptional response regulator of two-component regulatory system 43 LysR family transcriptional regulator 30 GntR or DeoR family transcriptional regulator 22 sensory histidine kinase in two-component regulatory system 14 sigma54 activator protein, enhancer binding protein 14 AraC/XylS family transcriptional regulator 7 ROK family transcriptional regulator/sugar kinase 7 IclR family transcriptional regulator 5 methyl-accepting chemotaxis protein 5 MerR family transcriptional regulator 4 DNA-binding regulatory protein 3 AraC/XylS family transcriptional regulator 3 MarR family transcriptional reguator 3 AsnC family transcriptional regulator ATP-binding components of the ABC superfamily of transport proteins are highly conserved and make up the overall largest paralogous group in E. coli (Table 7 ). The other two components of the ABC superfamily transporters are less conserved with membrane components in groups of 52 or less and periplasmic binding components in groups of 9 or less. Components of the PTS system; enzyme IIA, IIB, IIC and I also formed sequence similar groups. One of the groups classified as a group of transporter proteins actually contains both transport proteins (periplasmic binding components of the ABC superfamily) and regulatory proteins (transcriptional regulators of the GalR/LacI family). These two functional types are sequence related, and all of the proteins contain a common structural domain (SCOP sf53822) for the binding of small molecules [ 25 , 26 ]. The difference lies in the presence or absence of a DNA-binding domain. Response regulators of two-component regulatory systems make up the largest group of regulatory proteins in E. coli (Table 8 ). Sensory histidine kinases of two-component regulatory systems and the sigma54 activating proteins also constitute paralogous groups. A group almost equal in size to the response regulators is the LysR-family of transcriptional regulators. Other large groups of transcriptional regulators are also present. Cross genome comparisons of paralogous groups In addition to using paralogous groups for intra-genomic analyses, the groups were also used in cross genome comparisons (see Table 9 ). The sizes of selected sequence related groups are shown for three bacteria, the closely related enterics E. coli and Salmonella enterica serovar Typhimurium and the more distantly related organism Bacillus subtilis . The sizes of the groups in the closely related bacteria are similar, whereas there are differences in relation to B. subtilis , a gram positive soil organism. For instance, the largest E. coli enzyme group containing Fe-S-binding oxidoreductases was represented by only one homolog in the B. subtilis genome. However, B. subtilis encodes for 31 oxidoreductases homologous to the group of 18 NAD(P)-binding oxidoreductases of E. coli . The number of homologous sugar kinases, respiratory reductase subunits, and nucleoside diphosphate (Nudix) hydrolases appeared overall to be lower in B. subtilis . Table 9 Cross genome comparisons of enzyme groups. Ec 1 So 2 Bs 3 Group function 20 18 1 oxidoreductase, Fe-S-binding 18 14 31 oxidoreductase, NAD(P)-binding 18 13 10 oxidoreductase 4 , NAD(P)-binding 13 13 11 aldehyde dehydrogenase, NAD(P)-binding 13 11 13 oxidoreductase, FAD/NAD(P)-binding 11 16 6 sugar kinase 10 13 5 respiratory reductase, alpha subunit 9 8 8 aldo-keto reductase, NAD(P)-binding 8 7 5 phosphatase 8 8 2 nucleoside diphosphate (Nudix) hydrolase 1 No. proteins in Escherichia coli paralogous group 2 No. sequence matches for E. coli paralogous group in Salmonella typhimurium LT2 3 No. sequence matches for E. coli paralogous group in Bacillus subtilis 4 Contains GroES-like structural domain (SCOP sf50129). Discussion Protein modules vs. protein domains We have attempted to enumerate fused genes in E. coli in earlier work. Although we recognized the difference between independent proteins with complete function, called modules [ 9 ], as opposed to parts of proteins such as motifs and domains, we were not successful in our most recent effort in collecting only complete proteins to the exclusion of domains [ 15 , 27 ]. In earlier work we depended on size as a criterion to eliminate domains, but we know now some domains are large and overlap the lower range of sizes of independent proteins [ 28 ]. We also limited our previous studies to alignments between E. coli proteins. In this report we make use of information from 50 genomes to detect complete and independent protein homologs for the components of the fused E. coli proteins. The need to make use of additional genome sequences is supported by the fact that only 57% of the modules in fused E. coli proteins had unfused homologs within the E. coli genome while 90% had homologs among the 50 genomes. This result suggests that additional fused E. coli proteins might be detected in the future with more available genome sequences. The overall effect of changing the methodology has been to reduce the numbers of multimodular proteins identified in E. coli K-12. As a result of reducing the number of fused proteins, the number of paralogous protein groups was also reduced. The grouping process is based on similarity between the sequences hence many parts of the same proteins remained together in the new groups. The effects of multimodular proteins on annotation of genes For many years we have known that the E. coli contained fused genes and groups of sequence-similar proteins [ 19 ]. Today with the sequence of the entire genome and that of many other microbial genomes, we can quantify the gene fusions in E. coli and apply this information to generate paralogous groups. Even though we find that multimodular proteins are a minor fraction, 2.5%, of the proteins in E. coli K-12 MG1655, they significantly affect the annotation of related genes and the ability to define paralogous genes within a genome. Examples of the types of errors arising in the annotation of fused proteins are shown in Figure 3a . The multimodular protein ThrA (gi1786183) encodes an aspartokinase in the N-terminal module (aa 1–461) and a homoserine dehydrogenase in the C-terminal module (aa 464–820). A sequence similar protein from Lactococcus lactis , gi12723655, aligning only to the N-terminal module is erroneously annotated as having both aspartokinase and homoserine dehydrogenase activities. The correct annotation should be aspartokinase. In a second example, a protein from Bacillus halodurans , gi10174117, aligns to the aspartokinase module of ThrA but is described as homoserine dehydrogenase. The correct assignment should be aspartokinase. Figure 3 Annotation and composition of multimodular proteins. (a) Annotation is complicated by multimodular proteins. An E. coli protein (gi1786183) contains two modules, an N-terminal aspartokinase and a C-terminal homoserine dehydrogenase. Two single module proteins from L. lactis and B. halodurans (gi12723655 and gi10174117) align to the N-terminal aspartokinase module of the E. coli protein. Based on the sequence alignments, both of these proteins should be annotated as aspartokinases. However, errors are seen in the annotation of the L. lactis and B. halodurans proteins stemming from transfer of functions between multimodular proteins and partially aligned sequences without taking into account the alignment regions. (b) Different combinations of modules are seen in multimodular proteins of different organisms. While aspartokinase is fused to homoserine dehydrogenase in E. coli it is fused to DAP decarboxylase in X. fastidiosa . In both organisms the fusions are between enzymes of metabolic pathways, threonine biosynthesis for E. coli and lysine biosynthesis in X. fastidiosa . As shown in Figure 3b , different genes are sometimes fused to the same gene in different organisms. In E. coli an aspartokinase is fused to a homoserine dehydrogenase (gi1766183), while in Xylella fastidiosa , an aspartokinase is fused to a diaminopimelate decarboxylase (gi9106073). One needs to be alert to partial alignments. In this case, the annotation is correct for both activities of the Xylella protein, although the description does not follow the convention of stating the N-terminal activity first, raising the potential for misidentification of the activity of a partial homolog. Generality of gene fusions and remedies The details of gene duplication and divergence and of gene fusions have followed different courses in separate lines of descent of bacteria. The fusions of different gene partners to aspartokinase in E. coli and X. fastidiosa connected proteins acting in the same pathway. However, the pathways are different for the two organisms, threonine biosynthesis for E. coli and lysine biosynthesis in X. fastidiosa . Fusions of genes in a pathway have long been known and also the fusions of different genes in different organisms. In the tryptophan biosynthesis pathway of E. coli both the trp C gene (formerly trpC(F)) and the trp D gene (formerly trp G(D)) encode two enzymes as indicated in their former names. In contrast Rhizobium meliloti has a fusion between the trp E and trp G genes, trp E(G) [ 29 ]. Such differences not taken into account in annotation have generated errors in assignment of activities in some of the tryptophan synthesis proteins in a number of organisms. The variability in gene fusions among bacteria means that definition of multimodular proteins cannot be transferred from one organism to another, but must be worked out by analyzing the partial homology patterns with smaller independent proteins found in other organisms. To promote awareness of fused proteins, databases should list such proteins with their separate component activities and the approximate locations of these; either by start and end residues, or by module location (N-terminal, C-terminal, or Middle for proteins with >2 modules). Such a format has been implemented in GenProtEC [ 16 ]. When analyzing protein sequence alignments, one should make use of information on the alignment lengths and on the percent of each sequence that is involved in the alignment. Such information may hold clues to detecting fused proteins. Properties of paralogous groups of E. coli Groups of unimodular E. coli proteins similar in sequence vary in size from two (simple pairs) up to 92 members (Table 4 ). From pairs to groups of 8, the number of paralogous groups follows a power law. Above size 8, most sizes are represented by just one or two groups. For the smallest groups, two to four members, the degree of sequence similarity (PAM scores) tend to range widely (Figure 4 ). As the groups are larger, a clear distribution around PAM 150 emerges. Perhaps the larger groups are ones whose success is reflected in many duplication events over time with a retained function if the sequence drift is held to the range 100 to 200 PAM units. It appears that choosing 200 PAM as the upper ceiling has not eliminated an important number of groups with highly diverged members. Also, the broad range of degree of relatedness among members of paralogous groups (Table 5 , Figure 4 ) suggests that some types of proteins diverge further than others. The cluser around PAM 150 is populated by large successful paralogous groups, some of which are closely related in catalytic function while others have diverged to more distantly related activities. Figure 4 Sequence similarity of E. coli paralogous protein groups versus the group size. Protein sequences were aligned by the AllAllDb program of Darwin. Multimodular proteins were separated into modules (independent functional units) prior to the Darwin analysis. Alignments with similarities of ≤ 200 PAM units over 83 amino acids and where >45% of the length of both proteins in the pair were aligned were used to generate protein groups. The average PAM distances for the protein pairs in the smaller groups having 2–4 members (▲) and in the larger groups of ≥ 5 members (△) are shown. The smaller groups are more abundant and show a wide range of similarities. The larger groups appear to be more divergent with higher average PAM values clustering around PAM 150. The largest paralogous groups are transporters and regulators (Tables 7 & 8 ). Paralogous groups of enzymes tend to be smaller (Table 6 ). The largest enzyme classes tend to be oxidoreductases or subunits of oxidoreductases, and the relationships among members of these groups point in the direction of shared binding capacities accounting for the sequence relatedness, e.g. Fe-S clusters. In earlier work we found that some sequence related enzymes are alike in their ligand-binding characteristics, others are alike in mechanism of the catalytic action [ 30 ]. Both types of shared properties are seen in Table 6 . The ABC transporters have been a successful formula in bacterial evolution. The ATP-binding subunits maintain detectable sequence similarity. More divergent are the membrane subunits, and least similar are the periplasmic ligand-binding subunits, perhaps understandably divergent as their binding specificities for each transported compound will differ with the properties of the compounds [ 31 ]. One of the groups of periplasmic binding components also contains sequence related transcriptional regulators of the GalR/LacI family, agreeing with previous reports [ 25 , 26 ]. The major difference between these two functions is the presence or absence of a DNA-binding domain. According to Fukami-Kobayashi et al. [ 26 ], the regulators in this group are believed to have arisen by the fusion of a DNA binding domain to an ancestral periplasmic binding protein. The substrate specificity is thought to have evolved subsequently. Only a few of the transporters and regulators in this group bind the same substrates; galactose (MglB and GalR), ribose (RbsR and RbsB) and xylose (XylF and XylR). Among the regulator groups (Table 8 ), the class of two-component regulators is large. The two major activities of sensory histidine kinase and response regulators separate by the rules for grouping modules, but their known internal structures do not emerge. Many other groups are different kinds of transcriptional regulators. Another example of different functions related by sequence has been reported for a class of repressors and kinases, the ROK family [ 32 ]. In this case the two different functions are sequence related via their sugar-binding domains and differ in their DNA-binding or kinase activity. Cross genome comparisons Examining comparable paralogous groups among organisms may provide insight into functional and physiological differences among organisms. Illustration of the possibilities is shown in Table 9 where the sizes of comparable paralogous groups are shown for the closely related enteric bacteria E. coli and S. enterica serovar Typhimurium and the distant gram positive soil organism B. subtilis . Major difference is seen for one category of oxidoreductases. The largest enzyme group in E. coli contains 20 FeS-binding proteins whereas the B. subtilis genome has only one protein of this type. Members of the E. coli group include subunits of formate dehydrogenases, hydrogenases 3 and 4, DMSO reductase, and a NADH dehydrogenase. The presence of elements of the formate hydrogen lyase system and of the DMSO reductase in E. coli but not B. subtilis illustrates information on metabolic differences that emerges from such cross-genome comparisons. B. subtilis does not have the diverse anaerobic respiratory capability of E. coli and S. enterica . Duplication and divergence of this common ancestral gene seems to have taken a different course in the two bacterial lineages. In another example, B. subtilis has made use of one enzyme type to a greater extent than the two enteric organisms. The number of one of the types of NAD(P)-binding oxidoreductases is much larger in B. subtilis (31 proteins) than in the enterics (18 proteins). The B. subtilis enzymes in this group are fatty acid biosynthesis enzymes, agreeing with the known fact that this organism synthesizes a greater variety of fatty acids and has dedicated more of its proteome towards diversifying its fatty acid biosynthetic capabilities [ 33 , 34 ]. Thus sequence similar groups may be used in comparative analysis between genomes, highlighting areas where genetic resources have been expanded, pointing up metabolic differences between organisms. Conclusion • Proteins encoded by fused genes, multimodular proteins, require special attention in genome analysis. Such multimodular proteins contain two or more functional components that are located at separate parts of the protein and that may exist as independent proteins in other genomes. Annotation of the multimodular proteins should include the separate functions and their corresponding locations in the gene product. This will improve transfer of function between the fused proteins and sequences matching their entire length or only the length of one of their module components. Current annotation errors involving fused genes can be remedied by introducing this approach. • The identification of multimodular proteins in E. coli was improved by making use of sequence information from 50 genomes to detect alignments between the fused proteins and smaller, un-fused homologs corresponding to the component modules. The more common multidomain proteins, proteins containing fused sequence domains or motifs that together make up one overall function, were not detected as multimodular proteins by this approach. As a result the current number of fused E. coli proteins was reduced to 109 proteins with 8 containing three modules and 101 containing two modules. The multimodular E. coli proteins consist mainly of enzymes, regulators and transport proteins. Their component modules are often not related by sequence but many are related in that they function in a common pathway or cell role. Components of fused genes appear to vary from genome to genome hence complicating their detection and function assignment. • Multimodular proteins are different from multifunctional proteins in that the latter catalyze more than one reaction in the same region of the protein. • The generation of paralogous or sequence related groups is improved when the modules of multimodular proteins are separated and treated as independent proteins for the grouping process. 490 groups of sequence related E. coli proteins ranging in size from 2 to 92 were generated from the new module data. The smaller groups range widely in degree of relatedness while the larger groups have diverged from one another to about the same extent. Transport proteins and regulatory proteins were found in the larger groups while enzyme groups tended to have fewer members. • Over half of the E. coli proteins belong to paralogous groups, reflecting the prominent role of duplication and divergence in the evolution of the genome. The number and sizes of paralogous groups reflect the distinctiveness of the organisms and they can be used in cross genome comparisons. Methods Sequence sources Protein coding sequences were obtained from GenBank and included the following genomes: Aquifex aeolicus , (AE000657); Archaeoglobus fulgidus , (AE000782); Aeropyrum pernix , (BA000002); Agrobacterium tumefaciens , (AE007869/AE007870); Borrelia burgdorferi , (AE000783); Bacillus halodurans , (BA000004); Bacillus subtilis , (AL009126); Buchnera sp. APS, (BA000003); Campylobacter jejuni , (AL111168); Clostridium acetobutylicum , (AE001437); Chlamydia muridarum , (AE002160); Chlamydophila pneumoniae CWL029, (AE001363); Deinococcus radiodurans , (AE000513/AE001823); Escherichia coli K-12, (U00096); Escherichia coli O157:H7 EDL933, (AE005174); Escherichia coli O157:H7, (BA000007); Haemophilus influenzae , (L42023); Helicobacter pylori 26695, (AE000511); Halobacterium sp. NRC-1, (AE004437); Lactococcus lactis subsp. lactis , (AE005176); Mycobacterium leprae , (AL450380); Mycoplasma genitalium , (L43967); Mycobacterium tuberculosis H37Rv, (AL123456); Methanococcus jannaschii , (LL77117); Mesorhizobium loti , (BA000012); Mycoplasma pneumoniae , (U00089); Mycoplasma pulmonis , (AL445566); Methanobacterium thermoautotrophicum , (AE000666); Neisseria meningitidis MC58, (AE002098); Pseudomonas aeruginosa , (AE004091); Pyrococcus horikoshii , (BA000001); Pasteurella multocida , (AE004439); Pyrococcus abyssi , (AL096836); Rickettsia prowazekii , (AJ235269); Salmonella enterica subsp. enterica serovar Typhi, (NC_003198); Salmonella typhimurium LT2, (AE006468); Shewanella oneidensis MR-1, (NC004347); Sinorhizobium meliloti , (AL591688); Staphylococcus aureus subsp. aureus Mu50, (BA000017); Streptococcus pneumoniae TIGR4, (AE005672); Streptococcus pyogenes M1 GAS, (AE004092); Sulfolobus solfataricus , (AE006641); Synechocystis PCC6803, (AB001339); Thermoplasma volcanium , (BA000011); Thermotoga maritima , (AE000512); Treponema pallidum , (AE000520); Ureaplasma urealyticum , (AF222894); Vibrio cholerae , (AE003852/EC003853); Xylella fastidiosa 9a5c, (AE003849); Yersinia pestis , (AL590842). Analysis of protein sequence similarities Pairwise sequence alignments and scores were generated using the AllAllDb program of Darwin (Data Analysis and Retrieval With Indexed Nucleotide/peptide sequence package), version 2.0, developed at the ETHZ in Zurich [ 35 ]. Maximum likelihood alignments are generated with an initial global alignment by dynamic programming [ 36 - 38 ] followed by dynamic local alignments [ 39 ]. A single scoring matrix is used for these steps. After the initial alignment, the scoring matrix is adjusted to fit the approximate distance between each protein pair to produce the minimum PAM value. PAM units are defined as the numbers of point mutations per 100 residues [ 37 ]. The final report includes PAM distances and variances. For the work reported here, sequence pairs were collected that had alignment lengths of at least 83 amino acids and distances of 200 PAM units or less. We chose the length requirement of 83 residues as it improves the significance of the sequence alignments for the more distantly related protein pairs [ 40 ]. The requirement for at least 83 residues also avoids a class of commonly occurring protein domains smaller than 83 residues that appear widely in many otherwise unrelated proteins (such as small binding sites for a type of substrate, cofactor, or regulator). In addition for this study we removed proteins directly involved in horizontal gene transfer (IS proteins, transposases, and known prophage components) from the dataset. Identification of multimodular proteins Proteins encoded by fused genes were identified from the E. coli literature and from unequal sequence alignments. The literature was searched for E. coli proteins with more than one function encoded at separate parts of the protein. The locations of the alignment regions in the proteins were analyzed for orthologous and paralogous protein pairs. We identified proteins with two or more non-overlapping alignment regions where each region aligned separately to smaller homologs. Figure 1a illustrates the alignment of two unfused proteins with parts of a fused protein. Multimodular proteins so identified were separated into independent modules. Using the pairwise data, start and end positions of the modules were estimated from the many alignment regions and were set to cover as much of the sequence as possible, not only the most conserved regions of all the alignments. No overlap was allowed between any adjacent modules. Generation of internal sequence similar groups (paralogs) The sum of the separated modules from the multimodular proteins and the naturally occurring unimodular proteins of E. coli were aligned against themselves. Protein pairs aligning with >45% of the length of the peptides were used in a transitive grouping process as previously described [ 15 ]. The transitive nature of the process ensures sequence similarity to at least one member of the group and does not require all members of the group to have detectable similarity to one another. This type of clustering allows for more divergent sequences to be grouped. The restriction of PAM value to no more than 200 prevents groups from expanding beyond significant similarity. Authors' contributions MS designed the study, performed the sequence analysis, and participated in the data analysis and in writing the manuscript. MR participated in the data analysis and in writing the manuscript. Supplementary Material Additional File 1 Multimodular E. coli proteins. The table contains a complete list of the multimodular proteins in E. coli . Each module is described by its Gene name, Module Id, Module Start and End positions, Gene type, and Module Product. Click here for file Additional File 2 E. coli paralogous groups and their members. The table contains a complete list of the paralogous protein groups in E. coli . The members of the 409 paralogous groups are indicated by their Group Membership, Module Id, Module Start and End Position, Module Product. Click here for file

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Comparative promoter region analysis powered by CORG

Background Promoters are key players in gene regulation. They receive signals from various sources (e.g. cell surface receptors) and control the level of transcription initiation, which largely determines gene expression. In vertebrates, transcription start sites and surrounding regulatory elements are often poorly defined. To support promoter analysis, we present CORG , a framework for studying upstream regions including untranslated exons (5' UTR). Description The automated annotation of promoter regions integrates information of two kinds. First, statistically significant cross-species conservation within upstream regions of orthologous genes is detected. Pairwise as well as multiple sequence comparisons are computed. Second, binding site descriptions (position-weight matrices) are employed to predict conserved regulatory elements with a novel approach. Assembled EST sequences and verified transcription start sites are incorporated to distinguish exonic from other sequences. As of now, we have included 5 species in our analysis pipeline (man, mouse, rat, fugu and zebrafish). We characterized promoter regions of 16,127 groups of orthologous genes. All data are presented in an intuitive way via our web site. Users are free to export data for single genes or access larger data sets via our DAS server . The benefits of our framework are exemplarily shown in the context of phylogenetic profiling of transcription factor binding sites and detection of microRNAs close to transcription start sites of our gene set. Conclusion The CORG platform is a versatile tool to support analyses of gene regulation in vertebrate promoter regions. Applications for CORG cover a broad range from studying evolution of DNA binding sites and promoter constitution to the discovery of new regulatory sequence elements (e.g. microRNAs and binding sites).

Background Comparative sequence analysis has been a powerful tool in bioinformatics for addressing a variety of issues. Applications range from grouping of sequences (e.g. protein sequences into families) to de novo pattern discovery of functional signatures. Speaking of gene regulation, it has been known for a long time that there is considerable sequence conservation between species in non-coding regions of the genome. A comprehensive explanation of this observation is still elusive. However, sequence conservation within promoter regions of genes often stems from transcription factor binding sites that are under selective pressure (see [ 1 ] for a review and [ 2 ] for a systematic assessment of binding site conservation in man and mouse comparisons). Conserved sequence elements of other types have recently caught much attention. Not all non-coding conserved DNA in the vicinity of a gene's transcription start site necessarily functions at the level of transcriptional regulation. For example, most known methylation-guide snoRNAs are intronencoded and processed from transcripts of housekeeping genes [ 3 ]. A few microRNAs are apparently linked to protein coding genes, most notably mir-10 and mir-196 which are located in the (short) intergenic regions in the Hox gene clusters of vertebrates [ 4 - 7 ]. A second class of conserved sequence elements exert their function as regulatory motifs in the untranslated region (UTR) of the primary transcript or the mature mRNA. The UTRsite database [ 8 ], for example, lists about 30 distinct functional motifs including the Histone 3'UTR stem-loop structure (HSL3) [ 9 ], the Iron Responsive Element (IRE) [ 10 ], the Selenocysteine Insertion Sequences (SECIS) [ 11 ], and the Internal Ribosome Entry Sites (IRES) [ 12 ]. Most of these elements are contained in CORG since short intergenic regions or introns upstream of the translation start site are entirely covered by our definition of an upstream region. Phylogenetic footprinting The CORG framework aims at detecting and describing regulatory elements that are proximal to the transcription start site. In this context, the comparison of upstream regions of orthologous genes is particularly valuable. This concept is called "phylogenetic footprinting" and an overview of this approach can be found in [ 13 ]. Phylogenetic footprinting in a strict sense is carried out on orthologous promoter regions. Local sequence similarities can then be directly interpreted as related regions harboring conserved functional elements. We denote these similarities as Conserved Non-coding Blocks (CNBs). Multi-species sequence conservation Comparative approaches gain power from the inclusion of sequences from more than two species [ 14 ]. Multi-species comparisons help to increase specificity at the expense of intra-species sensitivity since supporting evidence (conservation) stems from many observations. To give an example, Man-mouse-rat comparisons enhance the detection of transcription factor binding sites since the rat genome is more divergent from the mouse genome than anticipated [ 15 ]. A nice property of vertebrate microRNAs is the high degree of sequence conservation which is found in alignments of man, mouse and fish microRNAs [ 16 ]. Both types of comparisons are available in CORG. In CORG, we consider cross-species conservation between promoter regions from 5 vertebrate genomes, namely Homo sapiens , Mus musculus , Rattus norvegicus , Danio rerio and Fugu rubripes . Multiple alignments are built from pairwise CNBs as described in the subsequent section. Construction and content Groups of orthologous genes In this work, we take a gene-centered view of phylogeny. Homology among proteins and thus genes is often concluded on the basis of sequence similarity. The EnsEMBL database [ 17 ] allows to distinguish orthologous from merely homologous genes by taking information on conserved synteny into account. We employed single linkage clustering on the graph of EnsEMBL orthologous gene pairs to define the CORG gene groups. Genomic mapping of validated promoter regions Various recent experimental efforts supply information about the position of transcriptional start sites in the human and mouse genome. Table 1 gives an overview on the resources that were employed in CORG. Table 1 Resources for validated transcription start sites Database name Features Eukaryotic promoter database (EPD) [44] The Eukaryotic promoter database is the smallest in size, but largely consists of manually curated entries. DataBase of Transcriptional Start Sites (DBTSS) [45] The DBTSS contains reliable information on the transcriptional start sites for man and mouse promoters. They exploit the oligo-capping technique to enrich their pool of clones for full-length 5'-to-3' cDNAs H-Invitational Database (H-InvDB) [46] H-InvDB is an international effort to integrate annotation of 41,118 full-length human cDNA clones that are currently available from six high throughput cDNA sequencing projects. FANTOM 2 (RIKEN) [47] The RIKEN consortium presented the FANTOM collection of RIKEN full-length cDNA clones. FANTOM stands for Functional Annotation of Mouse cDNA clones. The Reference Sequence project (RefSeq) [48] The Reference Sequence project aims to provide a comprehensive, integrated, non-redundant set of sequences, including full-length transcripts (mRNA) Some repositories offer genomic coordinates for their start site entries. Existing genomic mapping information was incorporated unless the underlying genome assembly build differed. The remaining data were projected onto the genome with SSAHA (Sequence Search and Alignment by Hashing Algorithm), a rapid near-exact alignment algorithm [ 18 ]. Sequence retrieval The notion of "promoter region" deserves some further explanation in the context of our approach. Typically, though not exclusively, we expect conserved regulatory regions to appear in the vicinity of the transcription start site of a gene. Since we do not know the precise location of the start of transcription for each and every gene, we chose to compare the sequence regions upstream of the start of translation from orthologous genes. If verified transcription start sites are known, we define a sequence window that is large enough to hold both, translation and transcription start sites, plus 5 kB upstream sequence. In case we lack this information, our observations on known transcription start sites indicate that most promoter regions should be captured in a sequence window of 10 kb size (Additional File 1 ). The size of a promoter region may be bounded by the size of the corresponding intergenic region. If an annotated gene happens to lie within the primary sequence window, the promoter region is shortened to exclude exonic sequence. Figure 1 Genomic context of human SRF. This image is displayed after the user selected a gene identifier on the search page. It provides the user with the genomic context of the selected gene. Known and predicted transcription start sites are shown as labelled red dots. Local similarities to homologous regions from other species are shown as connected purple boxes. Blue bars depict all upstream regions as contained in CORG. The structure of the corresponding EnsEMBL transcripts as well as the extent of RefSeq transcripts is show in the bottom track. Detection of pairwise local sequence similarities Significant local sequence similarities (phylogenetic footprints) in two sequences are computed with an implementation of the Waterman-Eggert algorithm. We have already given an account of the algorithm and statistics in [ 19 , 20 ]. The underlying alignment scoring scheme is the general reversible model [ 21 ]: where Q is the transition rate matrix. We left out the elements on the diagonal, which are constrained by the requirement that the sum of all elements in a row equals zero. The π i are the stationary nucleotide frequencies, their sum is constrained to be one. Although the two genomes under consideration are in general not in their stationary state with respect to the substitutional process we take the mean of the two observed nucleotide frequencies, , to be the best estimate of the stationary base composition. From other studies we have further knowledge about the relative rates between transversions, the transition A:T→G:C, and the transition G:C→A:T, which occur in roughly in the ratio 1:3:5 along vertebrate lineages [ 22 ]. These ratios of rates would generate sequences with 40% GC in their stationary state. To accommodate the observed nucleotide frequencies π i we have to allow for deviation from those ratios. We do this by choosing for example α ∝ ( R ( A → T )/ π T + R ( T → A )/ π A )/2, where R ( i → j ) is either 1, 3, or 5 depending on the process under consideration. At the end we scale the matrix Q , such that the PAM distance [ 23 ] of the substitution model equals the observed degree of divergence between the two species under comparison. Since we were mainly interested in highly conserved regulatory elements, we demanded an average similarity level at least as high as the average exon conservation between the species under comparison. The score for aligning two nucleotides i and j is then s ( i , j ) = log( P ( i , j )/( π i π j )) where P ( i , j ) is the probability of finding the pairing of i and j under the above substitution model [ 21 ]. Joining pairwise into multiple alignments All CNBs from pairwise sequence alignments are split up into groups as defined by gene homology. For each group a graph O = ( V , E ) with vertices V and edges E is constructed, which represents the species-internal overlap of CNBs on the genomic coordinate level. Each vertex a ∈ V represents a footprint, which is a pairwise local alignment between two species. An undirected edge is placed between two vertices if the corresponding CNBs have only one species in common and show an overlap of at least 10 bp on the sequence level. In our graph O , cliques of minimal size three are detected with an implementation of the Bron-Kerbosh algorithm [ 24 ]. Only those cliques are selected whose species count is equal to their size. This move prohibits the emergence of multiple alignments by similarity of multiple short CNBs to a single long CNB. Multiple alignments are then computed based on all cliques that meet the outlined criteria. We chose to employ the multiple alignment method of [ 25 ] who applies partial order graphs (POG) to the multiple alignment problem. Partial order graphs belong to the class of directed acyclic graphs (DAGs). A DAG is a graph consisting of a set of nodes N and edges E , which are one-way edges and form no cycles. The multiple alignment problem is then reduced to to subsequent alignment steps of individual sequences to a growing multiple alignment graph. If the sequences to be aligned share substantial sequence similarity, the number of bifurcation points within the POG stays low and allows rapid computation of the multiple alignment. Alignment results are subsequently trimmed to encompass the leftmost and rightmost ungapped block of at least 6 nucleotides. Annotation of promoter regions Exon detection with assembled EST clusters Promoter regions in CORG always extend upstream from the most downstream coding start (ATG). As a consequence, promoter regions may contain exons that are not translated. Our way of detecting such exons is a similarity search of man-mouse footprints versus GENENEST [ 26 ], a database of assembled EST clusters. Database searches are carried out for human and mouse footprints with the BLASTN program [ 27 ]. An E-value cut-off of 10 -4 is applied. Annotation with predicted binding sites The TRANSFAC database [ 28 ] is a repository of experimentally verified binding site sequences and representations thereof. These representations are used for querying the collection of man-mouse CNBs for known binding site patterns. Potential binding sites are detected with TRANSFAC weight matrices by the method of [ 29 ]. Here, the intuition is that there are two random models for a given sequence S : one is given by the signal profile F and the other one by the background model B . Under both models the distribution of weight matrix scores can conveniently be calculated by convolution, since the score is a sum of independent random variables. Probability mass distributions of P F (Score( S )) as well as P B (Score( S )) can be computed by dynamic programming if column scores are reasonably discretized. This allows a fine tuning of the proportions of false positives and negatives for each TRANSFAC weight matrix. Both error levels were set to be equal. All details are given in [ 29 ]. Utility and discussion We now present an overview of the web interface of the database and several example applications. Interface The CORG database is accessible via its home page and offers a redesigned web interface. From the search page one can quickly jump to gene loci via EnsEMBL or other standard identifers (e.g. HUGO symbol, LocusLink identifier, ...). The search query is processed according to the chosen reference source and a list of all matching database entries is returned to the user. This list serves as a springboard to a summary page where the genomic context of the selected gene and its similarities to other upstream regions is visualized as in Figure 1 . Pairwise as well as multiple comparisons are displayed on demand at this stage with a JAVA applet that complies with the JDK 1.1 standard. Alternatively, upstream region sequence and corresponding annotation can be exported in EMBL format (sequence data also in FASTA format). The JAVA applet should run on all JAVA-compatible web browsers. Detailed information about the conserved non-coding block structure are simultaneously shown for multiple upstream regions of different species. If available, annotation information on putative binding sites of transcription factors and EST matches are displayed for the query sequence. The applet facilitates zooming into sequence and annotation. In addition, web links are assigned to sequence features that relate external data sources to the corresponding annotation. CORG data may be also embedded into other viewers or programs via the distributed annotation system ( DAS , [ 30 ]). DAS facilitates the display of distributed data sources in a common framework with respect to a reference sequence. Our DAS server constitutes such an external data source. Position information on all conserved non-coding blocks and mapped promoters is accessible from this DAS server. Each DAS sequence feature provides a link to the corresponding CORG database entry. New DAS sources can be easily added to the ENSEMBL display. A small tutorial on installing external DAS data sources is available on our web page . Additionally, tools for on-site batch retrieval of CORG data will be added to the web portal in the near future. Phylogenetic profiling of binding sites One potential application of CORG is phylogenetic profiling of promoter regions. We define phylogenetic profiling in the context of gene regulation as comparative analysis of presence/absence patterns of binding sites in promoter regions. Here, we consider conserved predicted binding sites and contrast them with validated ones. Serum Response Factor (SRF) promoter SRF, a MADS-box transcription factor, regulates the expression of immediate-early genes, genes encoding several components of the actin cytoskeleton, and cell-type specific genes, e.g. smooth, cardiac and skeletal muscle or neuronal-specific genes [ 31 , 32 ]. Mouse embryos lacking SRF die before gastrulation and do not form any detectable mesoderm [ 33 , 34 ]. SRF mediates transcriptional activation by binding to CArG box sequences (Consensus pattern: CC(AT) 6 GG) in target gene promoters and by recruiting different co-factors. SRF regulates transcription downstream of MAPK signaling in association with ternary complex factors (TCFs) (for a review see [ 35 ]). TCFs bind to ets binding sites present adjacent to CArG boxes in many SRF target gene promoters. Figure 1 gives an overview of the genomic context of human SRF. As expected, the upstream region of SRF shows substantial conservation to its rodent orthologs. Additionally, significant alignments were found in comparisons with fish homologs (one from zebrafish and two from fugu). The same data is presented in the multiple alignment view of the JAVA applet in Figure 2 . This view gives a better idea on the location of alignments in the corresponding source sequences. Note, that the spacing between translation start and alignment is greater in fish than in mammals, which hints at different extension of the promoter region in the two subgroups. Figure 2 Graphical multiple alignment view (JAVA applet). Multiple alignment view of 6 homologous sequences from 5 species . All consistent local similarities in the upstream region of SRF homologs are placed relative to the species-specific translation start sites. The distance of the aligned segment to the translation start site is almost equal for all mammals and larger for the fish. The extent of each upstream region is shown as orange bar. Regions covered by flanking genes would be shown in red. We get a better idea on the cause of sequence conservation by browsing the multiple alignment. Textual information can be obtained by clicking on the alignment boxes. Then, the alignment appears in a pop-up window and may be copied to another destination. In Figure 3 , we used CLUSTAL X ([ 36 ]) to render the conservation structure on to the nucleotide level. Here, a striking observation is the conservation of the regulatory feedback loop of SRF to its own promoter in all species under consideration. So far, this feedback loop was experimentally verified in the mouse system [ 37 ] but could exist in all other species under comparison. Figure 3 Textual multiple alignment view . Multiple alignment as rendered by CLUSTAL X . The largest multiple alignment was retrieved from the JAVA applet by a cut and paste operation and rendered in CLUSTAL X [36]. Conserved binding sites are highlighted by red or blue boxes. Known sites as given in TRANSFAC are marked with a dollar sign [42]. Note that the validated Egr-1 site is only conserved in mammals. This site is bound by the serum-inducible Krox-24 zinc finger protein. Non-coding RNAs Non-coding RNA can be classified as transcribed regulatory elements. Non-coding RNAs are also accessible to the user via the CORG database. Since we were primarily interested in non-coding RNAs rather than small mRNA motifs we restricted our search here to long CNBs. A blast search of our multiple alignments with length L ≥ 50 against the Rfam database [ 38 ] and the microRNA Registry [ 39 ] identifies 21 alignments as 7 distinct microRNAs and a single snoRNA, Table 2 . Table 2 Rfam non-coding RNAs in CORG A + sign indicates that a sequence fragment from the corresponding species (hsa Homo sapiens , mmu Mus musculus , rno Rattus norvegicus , dre Danio rerio , tru Takifugu rubripes ) is contained in the CORG CNB; ∅ indicates that a blast search for an orhologous sequence in the Ensemble database was unsuccessful; n.d. mean no descriptive Ensemble gene annotation. The CNBs containing mir-196a-2 are shifted compared to the known microRNA sequences, preventing the detection of the correct stem-loop structure. The B columns marks whether a candidate was identified by a blast search against the Rfam or microRNA Registry, the A column shows whether a hairpin structure was identified by RNAalifold. p RNAz is the p -value for being an evolutionary conserved RNA secondary structure element returned by RNAz. CNB B A p RNAz ncRNA hsa mmu rno dre tru gene 119596 + + 0.995 mir-34c + + + + ∅ n.d. (BCT-4) 119607 + + 0.938 mir-34b in hsa 119658 + + 0.985 159914 + + 0.998 mir-138-2 + + + + ∅ SLC12A3, n.d. in teleosts 159932 + + 0.999 159939 + + 0.998 194777 + + 0.998 mir-196b + - + + + HOXA9, dre: HOXA9a and HOXA9b 194820 + + 0.999 194839 + + 0.999 194941 + + 0.999 226470 + + 0.999 mir-10a + + + + + HOXB4, dre: HOXB4a and HOXB4b 226514 + + 0.999 226555 + + 0.999 226677 + - 0.004 238163 + + 0.992 mir-10b + + + + + HOXD4, dre: HOXD4a, n.d in tru 238188 + + 0.984 238265 + + 0.994 391314 + - 0.125 mir-196a-2 + + - + + HOXC9, dre: HOXC9a 391315 + - 0.999 391318 + - 0.511 470004 + - 0.218 U93 + + + 0 + n.d. 110374 - + 0.995 IRES ? + + + + + DGCR8 146100 - + 0.891 + + + + 0 Ptf1a 393794 - + 0.999 IRE + + + + + SLCA1 The snoRNA U93 is an unusual mammalian pseudouridinylation guide RNA which accumulates in Cajal (coiled) bodies and it is predicted to function in pseudouridylation of the U2 spliceosomal snRNA [ 40 ]. It appears to be specific for mammals. The genomic copy of the human U93 RNA is located in an intron of a series of reported spliced expressed sequence tags (ESTs); furthermore, it has been verified experimentally that U93 is indeed spliced from an intron [ 40 ]. It was detectable in the CORG footprint dataset because of its location upstream of a conserved putative gene C14orf87 with unknown function. The known microRNAs belong to four different groups. The mir10 and the mir196 precursors are located at specific positions in the Hox gene clusters [ 4 - 7 ]. The mir-196 family regulates Hox8 and Hox7 genes, the function of mir10 is unknown. Substitution pattern of non-coding RNAs For a microRNA we expect a subsequence of about 20 nt that is almost absolutely conserved among vertebrates (the mature miRNA) and a well-conserved complementary sequence forming the other side of the stem from which the mature microRNA is excised. In contrast, the substitution rate should be much larger in the loop region of the hairpin [ 41 ]. mir10 is a good example of this typical substitution pattern, which gives rise to a hairpin structure. The pairwise correlation structure of nucleotides is depicted on top of the multiple alignment in Figure 4 . A different pattern is observed for the Iron Responsive element in the 5'UTR of SLCA1 , a member of the sodium transporter family. This time the substitution pattern does not meet the minimal length of the microRNA definition above. Nevertheless, it is conserved across all vertebrate species as shown in Figure 5 . Figure 4 Alignment and predicted RNA structure of mir-10b . The mir-10b CNB shows the typical pattern of substitutions in a microRNA precursor hairpin: There are two well-conserved arms, of which the mature microRNA is almost absolutely conserved, and a much more variable loop region. [43]. Figure 5 Alignment and predicted RNA structure of the Iron Response Element . The Iron Responsive Element (UTRdb [8] identifier: BB277285) shows a substitution pattern that is different from the hairpin structure in Figure 4. Additional orthologous sequences from the frog Xenopus tropicalis (xtr), the chicken Gallus gallus (gga) and the pufferfish Tetraodon nigroviridis are included. Conclusion We have improved and extended our framework of comparative analysis and annotation of vertebrate promoter regions over previous releases (see [ 20 ]). The following features have been added to the CORG framework: • Mapping of validated promoter regions and proper adjustment of the extent of upstream regions. • Multiple alignments from significant local pair wise alignments. • Novel approach to predict transcription factor binding sites. • Web site offers now a genomic context view (as in Figure 1 ) and an option to export sequence and annotation data. The CORG database is accessible via our web site. The user is guided step-by-step through the process of selecting and analyzing her promoter region of choice. CORG features an interactive viewer based on JAVA technology, which is tailored to detailed promoter analysis. Large-scale studies make direct use of our DAS service or the MySQL implementation of CORG in conjunction with an application interface (contact authors for details). We presented selected application examples from the realm of vertebrate gene regulation. Conserved regulatory elements of different kinds (binding sites, microRNAs and UTR elements) are readily accessible to CORG users. New genomes and annotation will be continuously added to CORG. Availability and requirements The database is freely accessible through the website . Programs, scripts and MySQL database dumps are available from the authors upon request. Authors' contributions Christoph Dieterich built the entire pipeline and some parts of the web interface. Steffen Grossmann annotated transcription factor binding sites and provided parts of the web interface. Andrea Tanzer analyzed known and novel RNA elements in the multiple alignments of the CORG database. Stefan Röpcke set up our database of binding site descriptions. Peter F. Arndt worked on an appropriate alignment scoring scheme. Peter F. Stadler and Martin Vingron initiated this work and provided all necessary infrastructure. Supplementary Material Additional File 1 Distribution of distance between start of transcription and translation . Histogram of observed genomic distances between start sites of transcription and translation in man for 1,700 entries from the EPD. The red and blue line indicates the 90% and 95% quantiles, respectively. Distances greater than 10 6 bp were exluded from the analysis as they mostly occur due to mismappings in the ENSEMBL database. Click here for file

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521184

Endangered Frogs Coexist with Fungus Once Thought Fatal

Amphibian declines have reached crisis proportions in various parts of the world. In many areas, habitat loss is the likely culprit. But when mass die-offs suddenly occurred in relatively undisturbed habitats, the cause was far less obvious. Fourteen species suffered either extinctions or major declines in the pristine rainforests of Queensland, Australia, between 1979 and 1993. It was suggested in 1996 that some unknown disease had spread through the populations, but no pathogen was discovered until 1998, when the fungus Batrachochytrium dendrobatidis was identified from sick and dead frogs. Since then, several lines of evidence suggest that B. dendrobatidis may be involved in frog declines: the fungus has been found on frogs in afflicted areas; lab studies show that it's highly pathogenic to some frog species; and pathological evidence links it to host mortality. But with little information about the prevalence of this fungal infection in wild frogs, or how the disease impacts frogs in the wild, the causal role of this chytrid fungus remains unclear. To evaluate the effects of B. dendrobatidis on frogs in their natural habitat, Richard Retallick et al. focused on six species living in the high-elevation rainforest streams of Eungella National Park in Queensland, Australia, where frog losses were “particularly catastrophic.” Two species vanished between 1985 and 1986: the Eungella Gastric-Brooding Frog ( Rheobatrachus vitellinus ), which is now thought extinct, and the Eungella Torrent Frog ( Taudactylus eungellensis ), which later reappeared in a few small populations. Other local frog species escaped this period relatively unscathed. Taudactylus eungellensis (Photo: Richard Retallick) Retallick captured frogs from six sites from 1994 to 1998, clipped one or two toe tips from each frog to age and identify them, and then released the frogs back into the wild. At the time, B. dendrobatidis had yet to be identified, but Retallick retained the toe tips, and the authors tested the toes for disease in 2002–2003. Fungal infections were found in two species— T. eungellensis and Litoria wilcoxii / jungguy (the latter consists of two species that are indistinguishable without genetic analysis); the other four species were infection-free. L. wilcoxii / jungguy did not decline to any great extent during the 1985–1986 die-off. The proportion of infected T. eungellensis frogs was greatest at three particular sites, which showed peak infections during cooler months. Prevalence of infection was highest during winter and spring, but did not vary from year to year, suggesting that the infection is now endemic. Fungal infections were found in 27.7% of L. wilcoxii / jungguy frogs, with no evidence that prevalence differed among sites, seasons, or individuals (males, females, or subadults). The probability of recapture was significantly lower for frogs that were already infected when first captured. While this might suggest a correlation between infection and death, it's impossible to distinguish death from simple failure to recapture the animal. On further analysis, McCallum and colleagues found no evidence that survival differed between infected and uninfected frogs, suggesting that this potentially devastating amphibian disease now coexists with the frogs, with little effect on their populations. These results, the authors conclude, “show unequivocally” that remaining populations of T. eungellensis , a rainforest frog listed as endangered, “now persist with stable infections of B. dendrobatidis .” While these findings do not exonerate the fungus as the agent of mass declines, they do rule out the possibility that the fungus caused the decline, then vanished from the area, allowing frog populations to recover. The authors allow that it's possible that B. dendrobatidis did not cause the initial T. eungellensis declines. Or alternately, the fungus could have emerged as a novel pathogen in the ecosystem, causing massive casualties before some form of evolutionary response took hold. Surviving frog populations may have evolved resistance to the pathogen, for example, or less virulent strains of the fungus may have evolved. If it turns out that frog populations can develop resistance to the chytrid fungus, the researchers point out, then a conservation program of captive breeding and selecting for resistance could potentially thwart the extinction of these, and other, critically endangered frogs. A critical next step, then, is to determine whether frogs and fungus do coevolve.

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212701

Supersensitive Worms Reveal New Gene Functions

The past ten years saw great progress in the field of molecular genetics, as new tools gave scientists the ability to investigate entire genomes instead of just one or two genes at a time. In this paper, Ronald Plasterk and colleagues developed a systemic approach using Caenorhabditis elegans , a tiny nematode and the first animal to have its genome sequenced, to gather functional information on nearly 400 genes. Many of the systemic approaches to discovering gene function involve either measuring or deleting messenger RNA (mRNA), the molecule that helps translate genes into proteins. The method used here, called RNA interference, or RNAi, follows the deletion approach by taking advantage of a cellular process bearing the same name. In nature, RNAi is thought to be an important part of the innate defense machinery in plants and animals, protecting them from invaders like viruses by interrupting the manufacture of viral proteins. To do this, short double-stranded RNA molecules with complementary sequences to the target gene inhibit the gene's function by disabling mRNA, which effectively shuts down the gene. By mimicking this natural process to turn off selected genes, scientists can find clues to how those genes might normally function by watching what happens when they are taken out of the picture. With the fully sequenced worm genome, it is possible to create interfering RNAs for all of its 20,000 or so genes. And because worms eat bacteria—which can themselves be used to deliver interfering RNAs—worms are the perfect RNAi model organism. The researchers fed the worms RNAi-producing bacteria, then observed the effects on the worm or its offspring to infer the function of the targeted gene. As previously reported, repeating this experiment for every gene in the worm genome, yields about 10% of the worms displaying abnormalities ranging from embryonic death to uncoordinated movement, suggesting defects in genes controlling development or muscle control, respectively. Having previously identified an RNAi-hypersensitive mutant worm strain, Plasterk and his colleagues repeated the experiment in the mutants and report proposed functions for 393 previously unknown genes. The types of abnormalities observed in the short-lived mutations induced by RNAi, they say, resemble the more stable mutations seen in the collection of worm mutations cataloged by worm researchers over the years. Though the DNA alterations for many of these mutations are not yet known, researchers know roughly where they occur in the genome. And the researchers show here that they can use their RNAi experimental results along with what is known about the mutants to identify several of the sequence alterations. They also performed what is believed to be the first analysis in which independently generated large-scale RNAi results were systematically compared to see how variable such RNAi results are, and the results have implications for similar approaches not just in worms but in plants and other animals. Caenorhabditis elegans worms

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539046

Human Carrying Capacity and Human Health

The issue of overpopulation has fallen out of favor among most contemporary demographers, economists, and epidemiologists. Discussing population control has become taboo. This taboo could be hazardous to public health

The issue of human overpopulation has fallen out of favor among most contemporary demographers, economists, and epidemiologists. Discussing population control has become a taboo topic. Yet, this taboo has major implications for public health. The silence around overpopulation prevents the global health community from making the necessary link between the planet's limited ability to support its people (its carrying capacity—see sidebar on following page) and health and development crises. In this article, I describe how popular thinking on population control has been shaped over the last 200 years, and how our failure to address the population explosion may be one cause of recent epidemics and social unrest. Overpopulation Concerns Peak, Then Decline The question of human overpopulation and its relationship to human carrying capacity has been controversial for over two centuries. In 1798 the Reverend Thomas Malthus put forward the hypothesis that population growth would exceed the growth of resources, leading to the periodic reduction of human numbers by either “positive checks”, such as disease, famine, and war, or “preventive checks”, by which (in the absence of contraception) Malthus meant restrictions on marriage. This “Malthusian view” was rapidly accepted by most politicians, demographers, and the general public, and remained popular until fairly recently. Malthus's worst fears were not borne out through the century following his death in 1834—food production largely kept pace with the slowly growing global population. However, soon after 1934, the global population began to rise steeply as antibiotics, vaccines, and technology increased life expectancy. By the 1960s, concerns of a mismatch between global population and global food supply peaked—expressed in books such as Paul Ehrlich's 1968 The Population Bomb [1] . This book predicted a future scarred by increasing famine, epidemic, and war—the three main Malthusian positive checks. In 1966, United States President Lyndon Johnson shipped wheat to India to avert a famine on the condition that the country accelerate its already vigorous family planning campaign [2] . Johnson was part of an unbroken series of US presidents concerned with the harmful effects of rapid population growth in developing countries. This line extended (at least) from John F. Kennedy to Jimmy Carter. George H. W. Bush was also sympathetic to this view, prior to becoming vice president in 1981. But the 1970s surprised population watchers. Instead of being a period shadowed by calamitous famine, the new crop strains introduced by the “Green Revolution” (especially grains such as rice, wheat, and maize) caused a dramatic increase in the global production of cereals, the main source of energy in the global diet. Among the development community, despair turned into cautious optimism. By the end of the decade, the public health community felt sufficiently empowered to proclaim “Health for All by the Year 2000”. Average life expectancy continued to zoom upwards almost everywhere—even in sub-Saharan Africa. The introduction of safe contraception contributed to a rapid fertility decline in many countries. But while the rate of global population growth declined from its peak in the late 1960s, the absolute increment of increase in annual global population continued to grow. Most population-related scientists, including food scientists and demographers, as well as US President Jimmy Carter, continued to be very concerned about global overpopulation. In 1970, the father of the Green Revolution, the agricultural scientist Norman Borlaug, was awarded the Nobel Peace Prize. In his Nobel lecture, Borlaug warned that the success of the Green Revolution would buy a breathing space for humankind of three decades, unless equivalent action was taken to reduce fertility rates [3] . China tightened its fertility policy in this decade, introducing its one-child policy in 1979. We are failing to confront the population explosion (Illustration: Sapna Khandwala) Concern for the Third World Fades With hindsight, the 1970s can be seen as the decade when widespread concern about overpopulation started to fade. The social and economic milieu of many developed countries, especially in the US, started to change. US foreign aid, as a percentage of the gross national product, declined from the late 1960s, perhaps in part because of the competing needs of the Vietnam War but also perhaps because of the apparent success of development in the Third World. The economic policies known as Keynesianism, which had been dominant since the end of World War II in many developed nations, came under sustained attack. These policies had placed a high value on full employment and social security. Keynesian policies restrained domestic inequality through high taxation and the promotion of social norms that censured conspicuous consumption (such as company executives exercising restraint in their personal salaries and people buying small houses). Shortly before his death, J. M. Keynes had also been crucially involved in the establishment of the World Bank. Keynes appears to have been personally committed to the advance of global justice, and to the reduction of inequality both within and between nations [4] . The world oil shock in 1973 contributed both to “stagflation”—a combination of rising unemployment with higher prices—and to increased economic power for the oil-producing countries of the Third World. Indeed, the term “Third World” came to be considered pejorative and was replaced by the “South”. Stagflation was interpreted as a failure of Keynesian policy. The demise of Keynesianism was accompanied by a further decline in concern for Third World development among elite economists and the general public. It is unlikely that the issue of global population policy figured into the election that put US President Ronald Reagan into office in 1980. Nevertheless, Reagan's policies were to cement a new orthodoxy about global overpopulation and development strategies. Unlike his republican predecessor, Richard Nixon, Reagan considered concerns about global population size to be “vastly exaggerated” [5] . In the same year, the US surprised the family planning world by abdicating its previous leadership in the effort to promote global family planning, at the International Conference on Population, held in Mexico City in 1984. The US took this position against the strenuous opposition of the Population Association of America, which represented many US demographers [5] . As foreign aid budgets fell, the “Health for All” targets began to slip from reach. Instead, international agencies promoted structural adjustment programs, health charges for patients (“user fees”), and the “trickle down” effect as the best ways to promote development. It is plausible that a fraction of the public who remained concerned about Third World development thought that these new economic policies deserved a chance. Less charitably, the new economic policies also appeared to allow people already financially comfortable to abdicate concern for Third World development because the new orthodoxy asserted that market deregulation, rather than aid, was the royal road to development. The increased domestic inequality of recent decades in developed countries [6] probably also contributed to a reduction in concern for the Third World, as working people have had to struggle harder to keep their position in their own society. It is now clear that market deregulation and generally high birth rates have proven disastrous in many Third World countries. “Health for All”, if recalled at all, is now seen as absurdly optimistic. The failure of development is most obvious in many sub-Saharan countries, where life expectancy has fallen substantially. But life expectancy has also fallen in Haiti, Russia, North Korea, and a handful of other nations [7] . The causes for this decline in life expectancy are multiple and complex. Causes that are usually listed include HIV/AIDS (Zimbabwe and Haiti) [ 8 , 9 ], ethnic hatred (Rwanda) [10] , crop failure (North Korea) [11] , poor governance and poverty (several parts of Africa) [12] , and alcoholism (Russia) [13] . Causal theory is complex. Every cause has a cause, and, increasingly, causes are being considered as a part of causal chains, causal webs, and causal snowballs. Some theorists distinguish between identifiable “proximal” causes and deeper, underlying, or “distal”, causes [14] . Yet, among the multitude of causes that can be identified for declines in either total population or life expectancy, overpopulation is hardly considered, except by dissident public health workers such as Maurice King [15] . Demography, the discipline that would appear to be the most likely holder of the Malthusian baton, is now almost entirely silent about overpopulation in developing countries [16] . Instead, most mainstream demographers appear to consider population ageing and European underpopulation as the most important demographic issues for this century. On the other hand, the role of the rapid demographic transition in China (from large to small families, with an average of two or fewer children) is rarely credited as central to the Chinese economic miracle. Overpopulation: A Cause of Crises in Africa? Often, the carrying capacity of one region at one point in time is boosted by the appropriation of the carrying capacity from other people and even other generations. Such resources include oil, deep sea fish, and the stability of the global climate and ecological systems. But in Rwanda, the most densely populated country in Africa, the importation of such resources has long been limited. Unlike other densely populated countries such as Hong Kong and Holland, Rwanda's economy at the time of its most infamous genocide, in 1994, depended almost exclusively on its primary production [17] . The country had little industry, few exports, and little tourism. The price of its most important export, coffee, had declined steeply just before the genocide [18] . Unlike many Asian countries, Rwanda also received few remittances from Rwandans working as guest workers abroad [17] . Among the many different explanations for the horrific 1994 Rwandan genocide, the possibility of a Malthusian check (also called “demographic entrapment”) is scarcely mentioned [ 17 , 19 ]. A Malthusian check in Rwanda was plausible not only because the total population was too large, but perhaps more importantly because the rate of population growth in Rwanda was faster than the capacity of Rwandan society to process the additional people. As a result, many indicators of development went backwards. The limited agricultural capacity forced many young men into Kigali, causing a concentration of young men with few prospects other than what they might gain from violence. There is even less scientific discussion that entertains the possibility that the sub-Saharan epidemic of HIV/ AIDS may also be a Malthusian check [19] . This is plausible if one applies a conceptual framework that combines the erosion of human carrying capacity through the same rapid population growth seen in Rwanda, with a consequent decline in per capita income and food supply. Furthermore, slowly operating feedbacks occurring as a result of the epidemic further undermined development, including the loss of human capital as teachers died [20] , the loss of agricultural expertise as farmers died [21] , and a deepening debt and loss of productivity from the countless funerals. And leaders in the developed world and many within Africa itself failed to devote the resources and provide the leadership required to quell the epidemic. Conclusion Maurice King refers to the silence on overpopulation as the “Hardinian Taboo”, named after the American ecologist Garett Hardin, who described the taboos that humans use to avoid confronting the need for population control [22] . Daniel Orenstein, at the Center for Environmental Studies at Brown University, has argued that powerful social norms inhibit debate about overpopulation in one of the world's most intractable trouble spots, Israel and Palestine [23] . Whatever the cause of the scarcity of modern academic analysis, the related issues of human carrying capacity and overpopulation deserve fresh consideration. The entrapment model has an explanatory power that is lacking in more superficial causal explanations. Of course, solving entrapment is very difficult, but as with most medical problems, a proper diagnosis will help identify the proper treatment. Human Carrying Capacity Human carrying capacity is the maximum population that can be supported at a given living standard by the interaction of any given human-ecological system. This apparently simple concept has many nuances and is rarely used by population scientists. However, in rejecting this term, purists risk making a terrible conceptual flaw, that of thinking that environmental and human resources are largely irrelevant to human population size. It is irrefutable that human ingenuity and cooperation can increase human carrying capacity [24] . But even so, human welfare will continue to depend on the external world, including for resources such as food and water. Humans are neither computer ciphers nor caged mice. That is to say, while a given area might tolerate a theoretically higher density of human population than it does, the reality of human evolution in distinct groups, separated by culture, religion, and language, means that this theoretical maximum will rarely be attained. A degree of underused carrying capacity can be viewed as a desirable buffer around disparate groups, vital for reducing tension and preventing conflict. Even culturally homogenous groups can outgrow their carrying capacity, as in the case of the Great Hunger in Ireland in the 1840s, when the population crashed because of famine, disease, and emigration. Indeed, Malthusian theory was used, in part, to justify the scanty aid provided to the Irish from Britain, a country that did not identify closely with the Irish.

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515369

Genetic Response to Climatic Change: Insights from Ancient DNA and Phylochronology

Understanding how climatic change impacts biological diversity is critical to conservation. Yet despite demonstrated effects of climatic perturbation on geographic ranges and population persistence, surprisingly little is known of the genetic response of species. Even less is known over ecologically long time scales pertinent to understanding the interplay between microevolution and environmental change. Here, we present a study of population variation by directly tracking genetic change and population size in two geographically widespread mammal species (Microtus montanus and Thomomys talpoides) during late-Holocene climatic change. We use ancient DNA to compare two independent estimates of population size (ecological and genetic) and corroborate our results with gene diversity and serial coalescent simulations. Our data and analyses indicate that, with population size decreasing at times of climatic change, some species will exhibit declining gene diversity as expected from simple population genetic models, whereas others will not. While our results could be consistent with selection, independent lines of evidence implicate differences in gene flow, which depends on the life history strategy of species.

Introduction Phylogeography has advanced our understanding of the spatial distribution of genetic diversity within and between species ( Avise 2000 ). However, empirical evidence of temporal change in genetic diversity in a single locality over time has not yet been placed in a population genetic or phylogeographic framework over ecologically long periods of time. In this paper we attempt to determine variation in genetic diversity experienced by populations of two mammalian species in situ and to place that diversity in the context of a changing environment through time. We view this approach as “phylochronology,” or the study of populations in space and time using phylogenetic and population genetic methods. Similar studies have not used such a long temporal record ( Pergams et al. 2003 ), have not considered gene flow ( Lambert et al. 2002 ; Pergams et al. 2003 ), or have used a spatially averaged sample as a proxy for a single locality ( Leonard et al. 2000 ). Our study takes advantage of a continuous, well-sampled mammalian fossil sequence spanning the last 3,000 years (Lamar Cave, Yellowstone National Park, Wyoming, United States). Lamar Cave has an extraordinarily complete representation of the local species in the vicinity, with over 10,000 identified mammalian specimens representing over 80% of the mammal species in the local habitat ( Hadly 1999 ). Late-Holocene climatic change, including the Medieval Warm Period (1,150 to 650 years before present [ybp]) and Little Ice Age (650 to 50 ybp) ( Soon and Baliunas 2003 ), affected the local abundances of common small mammals in a manner consistent with their habitat preferences ( Hadly 1996 ). We focus on two mesic habitat specialists, Microtus montanus (montane vole) and Thomomys talpoides (northern pocket gopher), species that presently are widespread in mountain habitats of western North America. Due to their preferences for wetter habitats, both responded demographically by increasing in relative abundance during wetter climates and declining during warmer climates. M. montanus showed an increase in abundance relative to other common rodents during periods of wet, cool climate in Yellowstone ( Hadly 1996 ). A 40% decline in M. montanus abundance occurred from 2,525 ybp to about 470 ybp during the Medieval Warm Period. Because T. talpoides also demonstrates a preference for mesic montane conditions, shifts in their relative abundance mimic the response seen in Microtus, decreasing by 50% between 2,525 and 470 ybp. In addition, T. talpoides showed a significant reduction in body size during this time ( Hadly 1997 ). These data highlight the influence of climatic change on the population dynamics and phenotypic response of these species, especially during warming events. Although the population responses of T. talpoides and M. montanus are similar, the ways in which they respond to climatic change at the genetic level are predicted to diverge because of differences in dispersal ability and population substructure. Advancement of ancient DNA (aDNA) techniques allows us to investigate directly the impacts of these environmental perturbations on neutral genetic diversity concurrent with these species population responses. We obtained ancient and modern mitochondrial DNA sequences from M. montanus and used previously published data for T. talpoides. Although these two species are broadly similar in body size ( M. montanus, 50–100 g; T. talpoides, 75–150 g) and are principally herbivorous, they differ in their natural history. T. talpoides is characterized by low population densities (1–62 gophers/hectare [ha]), a fossorial mode of life, maximum dispersal distances of a few hundred meters, and fiercely territorial behavior ( Verts and Carraway 1999 ). Populations of T. talpoides from Lamar Cave exhibit very little genetic variation through time but considerable genetic differences between present localities ( Hadly et al. 1998 ). This spatiotemporal pattern suggests that late-Holocene gene flow did not influence modern genetic variation of T. talpoides within localities over relatively short time scales (hundreds to thousands of years) despite the absence of obvious migration barriers. M. montanus achieves higher average population densities (60–186 voles/ha) ( Sullivan et al. 2003 ) than T. talpoides. In addition, genetic studies of closely related species and other arvicoline species (Microtus pennsylvanicus, Microtus longicaudus, and Microtus agrestis) have found little evidence for population subdivision over the scale of hundreds of kilometers ( Plante et al. 1989 ; Conroy and Cook 2000 ; Bjørnstadt and Grenfell 2001 ; Jaarola and Searle 2002 ), as expected from the ability and proclivity of voles to disperse hundreds to thousands of meters, resulting in migration between populations on generational time scales ( Jenkins 1948 ; Lidicker 1985 ). Such demography, however, implies that historical gene flow may be difficult to detect in M. montanus if only genetic data from the modern animals are used. This is contrary to the pattern expected in T. talpoides because this species has high genetic differentiation between extant populations, and past movement between such populations would be relatively easy to ascertain by the historic presence of unique, divergent haplotypes ( Hadly et al. 1998 ). The primary advantage of a phylochronologic approach as opposed to a single time slice for understanding mammalian response is the ability to reveal changes in genetic variation through time. This is in contrast to modern genetic studies that seek to reconstruct demographic history based on inferences from past climate or geologic records (e.g., Storz and Beaumont 2002 ; Lessa et al. 2003 ). Our study separates demographic and genetic response explicitly, allowing us to understand the microevolutionary forces responsible for the differences in species response over a time scale relevant to evolution within species. This approach is particularly powerful when coupled with environmental data so that perturbations may be linked to organismal response. In order to reveal these microevolutionary forces from our serial ancient data, it was necessary to explore the influence of sampling from the fossil record and to determine how variation in stochastic evolutionary forces (gene flow, drift, and mutation rate) might influence the record of gene diversity over time. Thus, we combined four methods of estimating population size, determining statistical significance, and assessing gene diversity through time. (1) We derived independent ecological estimates of population size through time from abundances of M. montanus and T. talpoides fossil specimens and modern population densities. (2) We calculated gene diversity over this 3,000-year period to determine the impact of environmental perturbations on genetic effective population size and gene diversity in M. montanus and T. talpoides. Unlike previous aDNA work ( Consuegra et al. 2002 ; Hofreiter et al. 2002 ; Lambert et al. 2002 ; Orlando et al. 2002 ; Paxinos et al. 2002 ), we used mitochondrial DNA sequences for samples taken through time from a single locality. (3) While there have been advances in the use of nuclear markers for ancient genetic analyses, we also confine our analyses to more easily derived mitochondrial DNA (mtDNA) data, thus limiting our analyses to a single locus, usually seen as a neutral marker within mammalian species ( Moritz et al. 1987 ). Our approach also constrains us to the fossil sample sizes from this locality, which are extremely large for ancient DNA studies, but limited relative to population genetic studies. Thus, we assessed the statistical power of a single locus for our empirical data using a neutral population model. (4) We used a neutral population model and serial coalescent simulations to determine whether our observed genetic data reflect our ecological estimates of population size and to evaluate statistical significance in changes of gene diversity through time. Despite similar population-level responses to climatic change of the late Holocene, we expected differences in gene diversity change for the two species. For T. talpoides, we predicted that changes in genetic variation through time would be dominated by drift, as suggested by the modern life history characteristics of small effective population size, low dispersal, and high amounts of population substructure. Therefore, as the ecological effective population size of T. talpoides declined with warmer climates, we expected genetic variation to decline. For M. montanus, we predicted that changes in genetic variation through time may be influenced more by migration, as suggested by large effective population sizes, high rates of dispersal, and low amounts of population substructure. As a result, past declines in ecological estimates of population size of M. montanus would not necessarily have resulted in a decrease in genetic variation. Results Fossil Abundance Our assessment of population response to climatic change ( Figure 1 ) depends on reconstruction of population size. Fossil relative abundances give a hint of the census size through time while genetic data (gene diversity, Figure 1 ) should yield independent assessments of the effective population size. The relationship between these measures varies, although most studies suggest that the estimate of effective size derived from ecological data is higher than that derived from genetic data (N e_ecol >> N e_gen ) ( Frankham 1996 ; Kalinowski and Waples 2002 ). However, we can convert census size estimates at any point in time into effective size estimates and vice versa. This allows us to compare explicitly ecological and genetic measures of population size. Figure 2 shows N e_ecol estimates based on low-, high-, and moderate-density estimates for T. talpoides ( Figure 2 A) and M. montanus ( Figure 2 B). For M. montanus they range from 218,652 to 436,981 for low-density estimates and from 677,825 to 1,354,650 for high-density estimates. For T. talpoides low-density estimates range from 2,219 to 5,015 and high-density estimates range from 4,586 to 10,488 individuals in the 7-km radius around Lamar Cave. Figure 1 Proportional Population Size and Gene Diversity of M. montanus and T. talpoides (A) Proportional population size (relative abundance) of M. montanus and T. talpoides ( n = 8,589 fossils) by years before present. (B) Gene diversity (H) of M. montanus and T. talpoides by years before present; 95% confidence intervals are shown. Squares indicate M. montanus; triangles indicate T. talpoides. Figure 2 Estimates of N e_gen and N e_ecol (A) T. talpoides and (B) M. montanus through time. Circles and dashed lines show N e_ecol estimates based on low-, high-, and moderate-density estimates. N e_gen estimates ([A] triangles and [B] rectangles) are based on θ S estimates from Arlequin. Standard errors for N e_gen are represented. Genetic Data: M. montanus The genetic evidence we have assembled from M. montanus suggests that the sequences we obtained for this study are target mtDNA. Of the 312 bp we sequenced for M. montanus, 96.5% of all the mutations were third-position codon changes, with first- and second-position mutations accounting for 3.5% and 0%, respectively. These ratios of variation are concordant with expectations for within-species variation and small overall sequence divergences ( Yang and Yoder 1999 ). Nucleotide base composition is similar to that of other Microtus species ( Conroy and Cook 2000 ; Jaarola and Searle 2002 ), with an excess of adenine (31.2%) and a deficit of guanine (15.7%) (χ 2 ; α = 0.71). Most of the mutations are synonymous (97.7%); the transition-to-transversion ratio of the entire data set was 4.1 to 1, which is consistent with expectations for mammalian cytochrome b and evolution in other Microtus species ( Conroy and Cook 2000 ; Jaarola and Searle 2002 ). Fossil and modern transition-to-transversion ratios are similar (3.1 and 4.2, respectively). All M. montanus sequences are reciprocally monophyletic (including M. pennsylvanicus as outgroup taxon) and translated successfully. Together with the frequency distribution of our pairwise differences, the prevalence of silent and third-position codon changes, and the standard of obtaining both forward and reverse fragments of overlapping sequence regions, these data permit us to conclude that the genetic diversity we have sampled represents authentic mitochondrial population variation and is unlikely to be from nuclear copies or pseudogenes. A total of 282 experiments included 47 fossil extractions and 1,644 PCRs. Eighty-eight percent of our aDNA specimens yielded readable sequence data, with no relationship found between success rate and age of the specimen ( R 2 = 0.004, not significant). All but one (out of 121) of the extraction controls were negative. When sequenced, this extraction blank BLASTed similar to Montanus townsendii, a taxon we had never worked on in the facility; this sequence has not since been amplified in the lab, and that extraction was not used further. Out of 87 successfully amplified samples and one sequence obtained from GenBank (AF119280), we identified 17 haplotypes within four haplogroups (A–D) of M. montanus ( Figure 3 A). The distribution of haplotypes within haplogroups suggests that our groups are defined appropriately. Each haplogroup was defined by at least 3% sequence divergence (≥10 bp) from other haplogroups in the 312-bp cytochrome b fragment. The majority of individuals (98.8%) fall within haplogroups A and D, with 84% of the samples within one substitution of the locally ancestral haplotype A ( Figure 3 A). Figure 3 Haplotype Networks for M. montanus and T. talpoides Haplotype networks ( Clement et al. 2000 ) for (A) M. montanus and (B) T. talpoides from Lamar Cave fossils and from modern specimens collected within a 400-km radius of Lamar Cave. Haplogroups for both species are indicated as A–D. Each haplogroup within a species is defined by at least 3% sequence divergence within the cytochrome b fragment. M. montanus haplogroup B is taken from GenBank. Haplogroup C is a sample from outside our 400-km radius (NK5897, Mono County, California; Museum of Southwestern Biology #53376). Light shading shows modern samples; dark shading shows fossil samples; bars indicate substitutions; cytochrome b sequence positions are indicated by number above base designation. Numbers within parentheses indicate sample sizes for each haplotype. The maximum uncorrected sequence divergence for our complete spatial and temporal data set was 4.5%, demonstrated between haplogroups A and B. Given that the highest average rodent divergence rate for cytochrome b is 6% to 10% per million years ( Irwin et al. 1991 ), these haplogroups have been evolving separately for at least 450,000 years. A similar age (422,000 years) is found when using a rate of 2.3% per million years for third-position transversions ( Conroy and Cook 1999 ). The maximum uncorrected sequence divergence of M. montanus from throughout the Lamar Cave temporal sequence was 4.2%. The maximum sequence divergence of 19 modern individuals from populations of this species within Yellowstone National Park and surroundings was 3.8%. Genetic Data: T. talpoides Protocols for T. talpoides are found in Hadly et al. (1998) . A haplotype network of this species shows three haplogroups and a total of eight haplotypes from 76 specimens ( Figure 3 B). For T. talpoides, 98.0% of all the mutations were third-position codon changes, with first- and second-position mutations accounting for 0% and 2.0%, respectively. Nucleotide base composition shows an excess of thymine (34.7%) and a deficit of guanine (11.4%). All mutations were synonymous; the transition-to-transversion ratio of the entire data set was 4.0 to 1. Gene Diversity through Time Estimates of gene diversity, nucleotide diversity, and number of segregating sites differed between M. montanus and T. talpoides ( Table 1 ). The estimates for M. montanus were higher than those for T. talpoides, as predicted by life history traits including higher ecological effective population size and higher dispersal between populations. Table 1 Summary Statistics, Sample Sizes, Sequence Length, and Number of Haplotypes for the Ancient DNA Samples for T. talpoides and M. montanus from Lamar Cave, Wyoming Gene diversity, number of segregating sites, and nucleotide diversity for each time interval were calculated with Arlequin v. 2.000 ( Jaarola and Tegelström 1996 ; Schneider et al. 2000 ) Our raw data on these species show similar relative abundance patterns but disparate trends in gene diversity (see Figure 1 ). These patterns cannot be linked directly to relative abundance because gene diversity estimates depend on true population size as well as sampling. We investigate how both of these parameters impact the observed trend in the following sections. Comparing Ecological and Genetic Estimates of Effective Size Ecological estimates of population size for T. talpoides (N e-tt_ecol ) exhibit the same trend as the genetic estimates (N e-tt_gen ), namely a population size decline of >50% after 1,500 ybp (see Figure 2 A). For T. talpoides, N e-tt_gen is consistently higher than N e-tt_ecol . When compared to those for M. montanus (see Figure 2 B), the estimates of total ecological and genetic effective population size are much lower. Additionally, unlike with M. montanus, the ecological and genetic effective sizes follow similar trends through the entire time period sampled, indicating that T. talpoides is acting as a closed population. For M. montanus, the estimates of effective size derived from the ecological data (N e-mm_ecol ) are lower than those derived from genetic data (N e-mm_gen ) for all time points (see Figure 2 B). While the estimates are not expected to be identical, comparison of their trends is instructive. Both genetic and ecological estimates follow similar trends between 2,525 and 845 ybp, after which the two estimates follow opposite trajectories. While the ecological size decreases by 50%, the genetic estimates show an initial decline of 30%, followed by an increase in population size equivalent to the pre–1,438-ybp level. This demonstrates that although the population is not recovering ecologically, it does recover genetically from population decline between 1,438 and 845 ybp. Effects of Sampling For M. montanus, our observed data were within the 95% confidence intervals for both sets of simulations ( n sample and n = 100; mutation rate = 4% per million years; moderate density values used to calculate abundance) for all time points except for 2,525 ybp, where observed gene diversity was significantly lower than could have been calculated given our sample size ( Figure 4 ). Since the observed gene diversity is lower than expected, we repeated simulations for five additional combinations of mutation rate and abundance (low mutation rate, high abundance; low mutation rate, moderate abundance; low mutation rate, low abundance; moderate mutation rate, low abundance; high mutation rate, low abundance). Results revealed that the observed gene diversity at 2,525 ybp was within the lower fifth percentile of the predicted distribution for two of the five combinations (when both mutation rate and abundance were low and for low mutation rate, moderate abundance). The overlap between the 95% confidence intervals for both sets of simulations suggests that sampling bias does not significantly impact the observed patterns of gene diversity except at 1,438 ybp ( n = 4), suggesting that we do not have sufficient power to detect processes at this time period. Figure 4 Expected and Observed Gene Diversity of M. montanus Boxes represent the 95th, 50th, and fifth percentiles for expected gene diversity of M. montanus given estimates of N e-mm_ecol at 2,525, 1,438, 845, 470, and 166 ybp and the associated sample sizes ( n = 7, 7, 18, 4, and 6) based on the Ewens sampling distribution (assumed mutation rate = 4% per million years per bp for a 312-bp fragment). Bars represent the 95th and fifth percentiles for a sample size of 100 at the same points in time. Diamonds represent observed gene diversity from empirical genetic data. The empirical data for each time unit fall within the expected ranges of gene diversity, except those for 2,525 ybp, which are much too low for the seven samples to detect, suggesting that observed gene diversities are not limited by sample size. Although the gene diversity for T. talpoides is not different given expectations from a closed population, we attempted to determine the statistical limitations of these data. Investigation of the effects of sampling for T. talpoides revealed that given the smaller number of base pairs (64 bp), we do not have enough statistical power to reject the null hypothesis. For every sampling time point, the Ewens distribution predicted that only one haplotype would be present in the genetic samples (unpublished data). As a result, the predicted gene diversity was zero for all time points. Because the observed gene diversity for T. talpoides was higher than predicted we also simulated five combinations of mutation rate and abundance, which could result in a higher predicted diversity (high mutation rate, high abundance; high mutation rate, moderate abundance; high mutation rate, low abundance; moderate mutation rate, high abundance; low mutation rate, high abundance) for 166 ybp ( n = 34). Results for all five combinations predicted presence of a single haplotype. Since we do not have adequate statistical power given the genetic data for T. talpoides, we did not conduct significance tests for this species. However, the observed values of gene diversity are not unexpected from dynamics within a closed population. Significance of Changes in Gene Diversity in M. montanus For eight of the nine combinations of mutation rate and effective size, we could reject the null hypothesis (closed population, no selection, changes in abundance inferred through fossil abundance) based on the observed change in M. montanus gene diversity throughout the entire time series (2,525 to 166 ybp) ( Table 2 ). The expected distribution of change in gene diversity given the null hypothesis and based on moderate M. montanus densities and moderate mutation rate is shown in Figure 5 ( Table 2 shows all combinations), along with the observed change. However, given the observed change in gene diversity specifically between 2,525 and 845 ybp, we were able to reject the null hypothesis for all nine combinations of mutation rate and effective size. These results suggest that M. montanus was not acting as a closed population during this period of time. Because the serial coalescent model presented here does not discriminate between selection and migration, either of these processes could have caused the observed change in gene diversity. Figure 5 Distribution of Change in Gene Diversity for M. montanus between 2,525 and 166 ybp, Based on Serial Coalescent Simulations Sampling is modeled at two points in time. N e-mm_ecol estimates from Figure 2 are used to specify demographic history. Eight of the nine combinations of mutation rate and density allow us to reject the null hypothesis for a closed population ( Table 2 ). This figure illustrates simulation results for moderate density and moderate mutation rate (4% per million years per bp). The probability of the observed change (shown by dashed arrow) is significant ( p = 0.015). Table 2 The Average (over 1,000 Simulations) Expected Change in Gene Diversity for Nine Combinations of Density and Mutation Rate for M. montanus between 2,525 and 166 ybp Significance values for observed change are in parentheses. All combinations other than moderate mutation rate and low density allow rejection of the null hypothesis of a closed population Our results may depend on our assumptions of equilibrium population size prior to 2,525 ybp. We investigated sensitivity to this assumption by modeling a population bottleneck in M. montanus prior to 2,525 ybp. We modeled population reduction to 104,577 (0.75 × N e-mm_ecol2525ybp ), 69,718 (0.5 × N e-mm_ecol2525ybp ), and 34,859 (0.25 × N e-mm_ecol2525ybp ) prior to 2,525 ybp. For a 75% bottleneck prior to 2,525 ybp, we were no longer able to reject the null hypothesis of a closed population. These results indicate that an extreme bottleneck where population size was reduced to 75% or more might result in the observed change in gene diversity. Our simulations reveal that unless an extreme bottleneck happened prior to 2,525 ybp, we can be confident that the observed change in gene diversity is not likely to be from events that occurred immediately prior to our historic data, and thus is due to migration or selection. Coalescent simulations used to investigate the significance of the observed gene diversity value for the modern samples demonstrated that the null hypothesis of past population size change could be rejected at only two of the nine mutation rate and density combinations. These results reveal that given data from only the modern samples, it was not possible to reject the null hypothesis of past population size change in M. montanus. Historic genetic data allow us to discriminate between population processes over millennia much better than do modern data alone. Other Evidence for Migration Independent lines of genetic and demographic evidence also point to the influence of gene flow in M. montanus populations. Estimates of β -diversity (used here to measure haplotypic turnover) from haplotypic data reveal that turnover was highest between 2,525 and 845 ybp ( β 2525–845ybp = 3; β 845–166ybp = 1.5; and β 2525–166ybp = 1.5). Closer examination of the haplotypic distributions demonstrates that five novel haplotypes appeared by 845 ybp, three of which are ≥3.2% different from the most common haplogroup (A) ( Figure 3 A) at 2,525 ybp, further implicating gene flow between 2,525 and 845 ybp. Discussion Our results demonstrate different genetic responses by two species of small mammals to changes in population size driven by climatic change. Fossil abundance data reveal population decline for both T. talpoides and M. montanus between 1,438 and 470 ybp, a period spanning the Medieval Warm Period ( Hadly 1996 ). For T. talpoides, the genetic response is directly related to changes in population size: Decrease in population size results in lowered gene diversity. M. montanus demonstrates the opposite relationship: A decrease in population size (between 1,438 and 166 ybp) results in an increase in gene diversity. We attempted to statistically validate our results by the use of serial coalescent simulations to demonstrate that the change in gene diversity of M. montanus between 2,525 and 845 ybp is significantly different from that expected based on the decrease in ecological estimates of population size. Taken together, these results indicate a departure from conditions of equilibrium (closed population without selection) for M. montanus. Our results have the following possible explanations: (1) the sampling area for fossils changed, (2) the local population size expanded, (3) selection occurred, and/or (4) gene flow occurred. Selection versus Gene Flow Results from all three of our analyses suggest that gene flow could be responsible for the patterns in gene diversity observed in our empirical data. Additionally, recent results of experimental studies of density dispersal dynamics in the root vole, Microtus oeconomus, indicate that migration occurs most frequently in and between low-density patches ( Andreassen and Ims 2001 ). These results indicate that density and dispersal in voles may be inversely related, a finding that is consistent with our results. An alternative explanation is that selection is governing the observed gene diversity patterns. While cytochrome b may not be under intense selection ( Irwin et al. 1991 ), it is linked to other portions of the mitochondrial genome that may be selectively advantageous in particular environments. Using cytochrome b as a marker for the accumulations of adaptations elsewhere on the genome may yield information about the effects of selection on local populations through time. Further exploration is necessary to investigate and identify the presence of locally adapted mtDNA and the rates of evolutionary change necessary to produce the variation in gene diversity we have observed (e.g., Pergams et al. 2003 ). Conclusions Here we demonstrate, using a phylochronologic approach, that it is possible to distinguish the dynamic processes that govern gene diversity over relatively short time scales (hundreds to thousands of years). We have documented environmental change, population response, genetic diversity change, and the correlations between the three. Without serial data, we would capture just a single record of these historic processes: modern genetic diversity. Although it is possible to hypothesize about historic events using modern data, phylochronology affords a unique look into the past and the potential ability to separate cause from effect. In particular, we show that M. montanus has a history recording responses both within populations (fluctuations in population size, possible selection) and between populations (gene flow). Discrepancy between the ecological and genetic estimates of population size and significant changes in haplotypic diversity prior to the Medieval Warm Period implicate increased gene flow into the Lamar Cave M. montanus population. Additionally, the observed haplotypic turnover in the Yellowstone population during this period suggests that as abundance of M. montanus declined through the last 845 years, relatively more individuals carried newly introduced haplotypes. Our results indicate that the presently observed widespread genetic variation across the geographic range in this species arose not because gene flow was equivalent through all populations through time, but because during particular time periods, certain local populations (and/or genotypes) declined while others expanded. Our data show that even with a prolonged ecological population size decline, the genetic diversity of M. montanus was maintained. In contrast, gene flow has not played a significant role in the recent genetic history of T. talpoides. This species, instead, responded more as a closed population over this time. The disparate nature of population response to climatic change of these two species is likely due to differences in demographic dispersal patterns between their populations. Such differences in species demography have resulted in differential genetic response to climatic change, even when ecological response is similar. Thus, genetic response to environmental change can be viewed as “individualistic,” similar to unique adjustments of species ranges ( Root et al. 2003 ). Life history traits such as dispersal ability contribute to the overall gene diversity of species in both space and time. If life history has such a large impact for common species, such differences will be particularly important in understanding how entire communities are affected by global change. Ultimately, knowledge from such analyses will lead to distinct, and perhaps predictable, patterns of species persistence through climatic changes, insights that will prove invaluable to future conservation of biodiversity. Materials and Methods Fossil locality Lamar Cave contains well-stratified, thoroughly radiocarbon-dated deposits, which display high fidelity to the local mammalian community ( Hadly 1996 , 1999 ). The most common animals from Lamar Cave are also the most common in the sagebrush grassland ecosystem in which Lamar Cave is located. Relative abundances are based on the entire data set of 10,597 specimens (except for those in Figure 1 A, which uses the five most common small mammals [ n = 8,589]) and are concordant with expectations of taxonomic diversity in montane mammal communities of western North America ( Hadly and Maurer 2001 ). The cumulative number of bones in Lamar Cave is correlated with time, demonstrating a constant “rain” of bones from the past to the present environment ( R 2 = 0.86; unpublished data). Age assignment The historical Microtus samples encompass 15 of 16 radiocarbon-dated stratigraphic levels from Lamar Cave ( Hadly 1996 ), with a maximum radiocarbon age of 2,860 ± 70 ybp (CAMS-20356). Data from the stratigraphic levels were pooled into five discrete intervals for Lamar Cave representing the last 3,000 years ( Hadly 1996 ). Interval boundaries were based on the stratigraphic pattern of deposition observed during excavation as well as a detailed radiocarbon chronology. Thus each interval represents a biologically significant packet of specimens. Age for each interval was assigned as the midpoint of the span of the calibrated radiocarbon ages ( Hadly 1996 ). Ecological estimates of population size Absolute population sizes in both ancient and modern communities are difficult to estimate. However, relative abundance changes of the small mammals are consistent with the climatically caused changes in habitats and the habitat preferences documented by modern trapping data proximate to the fossil site ( Hadly 1999 , Figure 1 A). By calculating the area preferred by M. montanus and T. talpoides using the geographical information system, we were able to standardize the relationship between taphonomy and population size in these species. This was possible because the collection radius of the fossils from Lamar Cave has been documented to be less than 7 km by using strontium isotopes ( Porder et al. 2003 ). We then estimated ecological effective population size from relative abundance through time, current population density, and the current area of preferred habitat in the collection radius. The current area of M. montanus habitat within the 7-km radius totals 1,992 ha; for T. talpoides it totals 971 ha. High, moderate, and low densities for T. talpoides ( Verts and Carraway 1999 ) and M. montanus ( Sullivan et al. 2003 ) were used in association with the corresponding areas of occupied habitat to estimate current census size. Because the rate of accumulation of bones through time is constant, the current census size was indexed against the percentage of Microtus bones from the uppermost level of Lamar Cave and used to calculate historic census sizes through time. In order to compare ecological estimates of effective population size to genetic estimates, we estimated mitochondrial effective population size assuming that N e /N census = 0.5 ( Storz et al. 2002 ; estimates from small mammals), and given that mitochondrial effective size is N e /4. Our estimates of N e were based entirely on relative fossil abundance and current ecological data and assume a mitochondrial effective size; thus we labelled them N e-tt_ecol (T. talpoides) and N e-mm_ecol (M. montanus). Genetic data from M. montanus From the fossil material, we used the upper first molar (from only one side of the jaw per level) to avoid sampling the same individual multiple times. Some Microtus species are cryptic with respect to these teeth. Genetic diagnosis indicated that these 78 fossil samples are derived from multiple arvicoline species, with a total of 47 M. montanus specimens (see Table 1 ). Fossil samples ( Microtus molariform teeth) ranged from 3.5 to 15.3 mg (average, 8.8 mg). We used two previously described extraction methods on the fossil teeth ( Hadly et al. 2003 ). We obtained a 312-bp fragment of mitochondrial cytochrome b from ancient samples ( n = 47) using the following primers: forward primers (5′–3′), CLETH 37 TAY AAY ATA ATY GAA ACH TGA A (5′ end of cyt b 319 equals Mus musculus 14458), CLETH 37L AYG GMT CTT AYA ACA TAA TCG AAA CAT G (cyt b 311, M. musculus 14450), MMONT 1 CAG TAA TTA CAA AYC TWC TAT CA (cyt b 452, M. musculus 14591), and MMONT 3 AGT GAA TCT GAG GGG GCT TCT CAG TAG A (cyt b 485, M. musculus 14621); reverse primers (5′–3′), ARVIC 08 CAG ATY CAY TCY ACT AGT GTT G (cyt b 473, M. musculus 14612), ARVIC 08L CTC AGA TTC ACT CTA CTA GTG TTG TG (cyt b 471, M. musculus 14610), MMONT 4 TTR TTT GAT CCT GTT TCG TGT AGG AAT A (cyt b 595, M. musculus 14631), and MMONT 2L TTG ACT GTG TAG TAA GGG TGA AAT GGG A (cyt b 653, M. musculus 14792). Attempts were made to amplify the region in two overlapping fragments using CLETH37/ARVIC 08, CLETH37L/ARVIC 08L, and MMONT 1/MMONT 2L. However, the low rate of success for MMONT 1/MMONT 2L (40%) in the fossil samples necessitated the breaking of the second fragment into two overlapping fragments using MMONT 1/MMONT 4 and MMONT 3/MMONT 2L. Ancient DNA samples were run on an ABI PRISM 310 Genetic Analyzer in the post-PCR lab, and modern DNA samples were run on an ABI PRISM 377 Sequencer in a separate sequencing facility (Protein and Nucleotide Facility, Beckman Center, Palo Alto, California, United States). Fragments were sequenced in both directions, primer regions were overlapped, and sequences with any ambiguous sites were rerun until completely resolved in order to provide additional corroboration and eliminate ambiguity. We adhered to strict extraction and amplification protocols ( Hadly et al. 2003 ). The protocol further included (1) independent sequence corroboration of two samples (J. Mountain lab, Anthropological Sciences, Stanford University), (2) processing of modern samples using personnel and reagents in another lab (D. Petrov lab, Biological Sciences, Stanford University) all physically separate from the aDNA facility, (3) monitoring contamination with several extraction and PCR controls, (4) primer design specific to arvicoline species, and (5) no prior or concurrent history of working with murid species in any of the DNA facilities involved. Modern (spatial) genetic sampling was obtained from a variety of sources including modern skins, modern liver tissue, museum skins, and teeth derived from modern raptor pellets. DNA was successfully extracted from 16 modern skins (n = 13 M. montanus; n = 3 other Microtus) and 12 teeth (n = 5 M. montanus; n = 7 other Microtus) from the vicinity (within 10 km) of the fossil site. In addition, DNA was successfully extracted from 51 modern specimens (liver tissue and museum skins) collected within a 400-km radius of Lamar Cave. Of these 51 samples, 22 were derived from museum skins (n = 21 M. montanus; n = 1 M. longicaudus) and 29 from liver tissue (n = 9 M. montanus; n = 18 other Microtus) of specimens trapped in the field. We followed the animal tissue protocol using the Qiagen Dneasy Tissue Kit (Qiagen, Valencia, California, United States) on 6.5 mg of liver samples and 7.5 mg of tooth samples ( n = 3). For museum samples, DNA was extracted from the ventral skin incision (0.5 to 10.3 mg; average, 2.7 mg). We amplified the entire cytochrome b gene (1,143 bp) for some modern skins ( n = 13) and liver tissue ( n = 29) with the following primers: forward primers (5′–3′), MVZ 05 CGA AGC TTG ATA TGA AAA ACC ATC GTT (cyt b −51, M. musculus 14088) and ARVIC 07 AAA GCC ACC CTC ACA CGA TT (cyt b 514, M. musculus 14653); reverse primers (5′–3′) MICRO 06 GGA TTA TTT GAT CCT GTT TCG T (cyt b 602, M. musculus 14741) and VOLE 14 TTT CAT TAC TGG TTT ACA AGA C (cyt b 1170, M. musculus 15309). DNA was extracted from a total of 81 modern Microtus samples, and 79 of those yielded successful amplification. Of these 79 samples, 48 were positively identified as M. montanus . Of the M. montanus samples, 41 specimens were successfully haplotyped. Sequences have been deposited in GenBank ( see Supporting Information ). Genetic data from T. talpoides We have built upon the previously published T. talpoides data set ( Hadly et al. 1998 ) with additional temporal sampling ( n = 3) (see Table 1 ). T. talpoides from Lamar Cave demonstrated remarkable continuity in gene diversity through time, with only three haplotypes present, all of which differ from each other by one synonymous third-position mutation. The majority of the fossil T. talpoides specimens are from haplotype A (83%), which is not found elsewhere in a 400-km radius around Lamar Cave. This constancy in the genetic lineage of T. talpoides within a single locality persists in spite of climatic changes and concurrent significant population and body size changes ( Hadly et al. 1998 ). Data analysis Arlequin v. 2.000 ( Jaarola and Tegelström 1996 ; Schneider et al. 2000 ) was used to calculate gene diversity, number of segregating sites, and nucleotide diversity for each time interval (see Figure 1 B and Table 1 ). Additionally, genetic data were used to estimate the mitochondrial effective population size (N e ) for all points in the past. Assuming a neutral model of molecular evolution, θ S (where S is the number of segregating sites; θ S = 2 N e μ , where μ is the mutation rate for the complete sequence per generation) was used to estimate N e . θ S was preferred over other estimators of θ since θ H is biased for single locus estimates, θ k does not incorporate sequence information, and θ π has higher variance. These estimates are determined entirely from genetic data; thus we label them N e-tt_gen (T. talpoides) and N e-mm_gen (M. montanus). We assumed a range of μ (2% [low], 4% [moderate], or 10% [high] per million years per bp) ( Table 2 ). Additionally, to estimate haplotype turnover we calculated β -diversity (used traditionally in ecology to measure species turnover) using Cody's index ( Cody 1968 ). For β -diversity each haplotype was treated as a “species.” Effects of sampling Given the general limitations of obtaining aDNA sequence data, sample sizes will always present challenges to ancient population genetic studies. To investigate whether our samples are adequate for addressing temporal gene diversity in both species, we used a neutral population model to evaluate expected diversity. Ewens (1972) derived expressions for the sampling distribution characterizing a closed population of size N, a gene with mutation rate μ, and an infinite alleles model given a sample size n. Here we use the Ewens distribution to ascertain the ability of our data to detect variation in values of gene diversity. In addition, this distribution allows us to (1) predict the distribution of expected gene diversity at each point in time (independently) given estimates of N e_ecol , a moderate mutation rate of 4% per million years, typed sequence length (312 bp for M. montanus and 63 bp for T. talpoides ), and sample size ( M. montanus: n 166ybp = 7, n 470ybp = 7, n 845ybp = 18, n 1438ybp = 4, and n 2525ybp = 6; T. talpoides: n 166ybp = 34, n 470ybp = 5, n 845ybp = 29, n 1438ybp = 4, and n 2525ybp = 11) and (2) predict change in the expected gene diversity distribution for a large sample size of 100. A modified version of MONTE CARLO ( Slatkin 1994 , 1996 ) was used to generate possible allele configurations for a given set of parameters (θ, K, and n), and gene diversity was calculated for each configuration. Simulations were repeated 1,000 times for each sampling time point, resulting in the 95% confidence intervals for the distribution of predicted gene diversities given a closed, selectively neutral population through time. Serial coalescent simulations We assessed the significance of the observed changes in gene diversity between time points using simulations. The serial coalescent was used as a framework to model M. montanus evolution during the past 2,525 years. Simulations were repeated 1,000 times to generate genetic data and changes in gene diversity for the null hypothesis. The significance of the observed changes was then inferred by comparing it to the generated null values (we thus used a Monte Carlo significance test). Estimates of N e_ecol were used to set up a null hypothesis specifying population size change through time in a closed population. We assumed that past changes in population size between intervals were due to exponential growth or decline. The effective population size at two time points and the time between the points was used to calculate a growth rate. The estimated growth rates were r 1438–2525ybp = −0.000178, r 845–1438ybp = 0.000732, r 470–845ybp = 0.0004385, and r 166–470ybp = 0.0003212. The null hypothesis thus corresponds to effective population sizes at particular points in the past and exponential growth or decline between those intervals, and represents an ecologically realistic description of the past 2,525 years. We assumed a constant population size prior to 2,525 ybp, as we have no data before this point in time. The coalescent program SIMCOAL ( Excoffier et al. 2000 ) was modified to incorporate temporal sampling (also known as heterochronous sampling): n 1 samples modeled back in time, with n 2 samples added to the genealogy at a time point t 1 generations in the past. The serial coalescent ( Rodrigo and Felsenstein 1999 ; Drummond et al. 2003 ) has been used to estimate parameters such as μ for HIV and ancient mtDNA (most recently using an MCMC approach; Drummond et al. 2002 ; Lambert et al. 2002 ). In this paper, we present what we believe to be its first application as a simulation tool used to predict change in gene diversity for the null model of population size change described above. Running the model 1,000 times provides the expected distribution for change in gene diversity. Using N e-mm_ecol estimates based on high-, moderate-, and low-density estimates for M. montanus (186, 126, and 60 voles/ha; Sullivan et al. 2003 ) and a high, moderate, and low mutation rate ( μ = 10%, 4%, and 2% per million years per bp; sequence length = 312 bp; finite sites mutation model, no rate heterogeneity), we investigated the significance of observed changes in gene diversity at all nine parameter combinations over the entire time span (2,525 to 166 ybp) and between 2,525 and 845 ybp (spanning the Medieval Warm Period). Additionally, we also investigated the relevance of temporal data to our ability to reject the null hypothesis. Significance tests were repeated assuming the observed data were from only the most recent genetic samples. Again, simulations were repeated for all nine combinations of mutation rate and density estimates. Supporting Information Accession Numbers Sequences for the successfully haplotyped M. montanus specimens described in Materials and Methods have been deposited in GenBank under accession numbers AY660606 to AY660629.

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535898

Verbal autopsy of 80,000 adult deaths in Tamilnadu, South India

Background Registration of the fact of death is almost complete in the city of Chennai and not so in the rural Villupuram district in Tamilnadu, India. The cause of death is often inadequately recorded on the death certificate in developing countries like India. A special verbal autopsy (VA) study of 48 000 adult (aged ≥ 25 yrs) deaths in the city of Chennai (urban) during 1995–97 and 32 000 in rural Villupuram during 1997–98 was conducted to arrive at the probable underlying cause of death to estimate cause specific mortality. Methods A ten day training on writing verbal autopsy (VA) report for adult deaths was given to non-medical graduates with at least 15 years of formal education. They interviewed surviving spouse/close associates of the deceased to write a verbal autopsy report in local language (Tamil) on the complaints, symptoms, signs, duration and treatment details of illness prior to death. Each report was reviewed centrally by two physicians independently. Random re-interviewing of 5% of the VA reports was done to check the reliability and reproducibility of the VA report. The validity of VA diagnosis was assessed only for cancer deaths. Results Verbal autopsy reduced the proportion of deaths attributed to unspecified and unknown causes from 54% to 23% (p < 0.0001) in urban and from 41% to 26% (p < 0.0001) in rural areas in Tamilnadu for adult deaths (≥ 25). The sensitivity of VA to identify cancer was 95% in the age group 25–69. Conclusion A ten day training programme to write verbal autopsy report with adequate feed back sessions and random sampling of 5% of the verbal autopsy reports for re-interview worked very well in Tamilnadu, to arrive at the probable underlying cause of death reliably for deaths in early adult life or middle age (25–69 years) and less reliably for older ages (70+). Thus VA is practicable for deaths in early adult life or middle age and is of more limited value in old age.

Background In developed countries, data on disease-specific mortality by age are readily available from national vital registration. In developing countries, where 80% of the world's deaths occur, estimation of cause of death is more difficult because the levels of coverage of vital registration and reliability of cause of death stated on the death certificate are generally low (especially in rural areas). A reliable assessment of disease-specific mortality rates is not yet possible in many parts of India, either because the underlying cause of the terminal illness was never known or because the relevant information was not recorded. For legal purposes death records do usually subdivide the causes of death into medical and non-medical (external) causes. But once non-medical causes have been excluded, specification of the underlying cause of a death from disease may be inaccurate, misclassified or missing for about 50% of adult deaths. For example, in Chennai, Tamilnadu, (south India) about half of those who died at home soon after the diagnosis of cancer (and whose deaths were therefore, in almost all cases, likely to have been caused by their cancer) do not have cancer mentioned on their death certificate [ 1 ], and for other diseases the problems might well be even worse. Elsewhere, 'Verbal Autopsy' i.e., 'systematic retrospective inquiry of family members about the symptoms and signs of illness prior to death' has been used to help determine the underlying medical cause of death, particularly in childhood [ 2 , 3 ]. For childhood deaths in populations that are not covered by adequate medical services such "verbal autopsies" are now of established value in helping to classify the broad patterns of mortality. Verbal autopsies have also been used to assess the medical causes of maternal deaths [ 4 - 7 ]. Although in India there are about as many deaths in middle age as in childhood, there is less experience with verbal autopsies of adult deaths. A special study on 'verbal autopsy' of adult deaths was conducted in urban and rural areas in the state of Tamil Nadu, south India during 1998–2000. The aims of the study were (a) to develop a verbal autopsy instrument and test its utility to determine the underlying cause of death and (b) to estimate cause specific mortality using underlying cause of death arrived based on verbal autopsy reports. Now we report the type of training given to the field interviewers to interview and write the verbal autopsy report for adult deaths, the procedure followed to arrive at the probable underlying cause of death, the accuracy of the instrument developed and change in the proportions of cause of death based on the underlying cause of death arrived by reviewing 80 000 verbal autopsy reports. Methods A: Training of field interviewers to write a 'Verbal autopsy' report Male non-medical graduates with at least 15 years of formal education were selected. A ten days training was given on verbal autopsy interview techniques and writing verbal autopsy reports. There are four steps in training; 1). introduction to anatomy, signs and symptoms of various diseases, 2). mock interviews, 3). hands-on-training on writing verbal autopsy reports and 4). feed back session. Step 1 This consisted of a basic three day introduction to anatomy, collecting data on history of past illness (refer Appendix I in Additional file 1 ), using symptoms/signs checklist of various diseases (refer Appendix II in Additional file 1 ) and to interview the surviving spouse/close associates or relatives of the deceased, the other members of the community such as neighbours to get data on train of events or circumstances preceding the death. Reports are to include complaints, symptoms, signs, duration of illness and treatment details of the illness prior to death. The following data are to be ascertained (for all deaths due to medical causes) from the respondent to write the verbal autopsy narrative report: • onset of illness prior to death: sudden or gradual • major symptom(s) and associated symptom(s) – in chronological order • If a symptom was present it was used as a filter to define what questions to be asked. For example, the filter symptom for heart attack was chest pain and the associated symptoms were breathlessness, sweating, vomiting and pain in the retrosternal area radiating to hand, shoulder, back etc. Cough for more than 4 weeks was a filter for lung cancer and tuberculosis. For each symptom, the duration should be recorded. Details of additional symptoms are built into the narrative in chronological order, by prompting, if necessary. • progress of the illness • any treatment received : Yes/No • If yes, type of treatment received • details of hospitalization prior to death: ○ name of the hospital (e.g. tuberculosis hospital, cancer hospital, coronary care unit etc), ○ duration of hospitalization, ○ whether discharged from the hospital against medical advice or not. ○ Status at the time of discharge from the hospital: alive/dead • history of similar episodes and treatment(s) given • abstract information related to the illness prior to death from the investigation reports done for any illness close to the time of death (within 6 months prior to the death) / hospital discharge summary etc, if available • If a death certificate is available, copy the cause of death given on the death certificate (In the Tamilnadu study death certificates were available for only 20% of total deaths). • While recording history of adults with long standing illness, the description should include details that occurred in the month preceding the death, with other information recorded in the past history section (Appendix I in Additional file 1 ). • For deaths that occur during pregnancy, delivery, or within six weeks of delivery: use Appendix II (A and B) in Additional file 1 If the respondent is able to give the major symptoms and circumstances leading to death, then additional probing questions are asked about the associated symptoms using the symptoms/signs checklist (Appendix II(A & B) in Additional file 1 ) If the respondent is not able to give sufficient information on the symptoms of the illness prior to death or have difficulty in remembering any major symptom, then get necessary information to rule out non-medical causes of death. When the interviewer is sure that the death was not due to unnatural cause, the following procedure is used to collect necessary data on the symptom. ○ read out the filter symptom/sign of each module in the symptom/sign checklist ○ check responses to each, and note down positive responses ○ Where there is a positive response, additional details on that symptom and associated symptoms, if any, should be obtained. Thus, the methodology of collecting data in the open format using 'symptoms/signs checklist' is an interactive process, with the respondent taking the lead in providing the information, and the interviewer prompting where necessary for more details. The Field Interviewer gathers as much information as possible on the underlying cause of death from the respondent. It is imperative to get a logical and complete history of symptoms, signs, events, investigations and treatment, so that the medical reviewer gets sufficient information to assign a probable specific underlying cause of death. Step 2 In the following two days, mock interviews were organized to illustrate techniques of probing a respondent to get the required information on cause of death as well as how to write the verbal autopsy report in local language (Tamil) in Appendix I in Additional file 1 as stated by the respondent. Step 3 The third component of training included three days of hands on verbal autopsy training in the field. To limit distress over the terminal event, the field visit was carried out at least six months after death. Name of the deceased, father's name (if the deceased was a male) or spouse name (if the deceased was a female), age, gender, informant's name and address of the deceased at the time of death were given to field interviewers to locate the house of the deceased. The Field Interviewers carry Appendix I and II (symptoms/signs checklist) in Additional file 1 to the field. They were blind to the cause of death stated on the death certificate. The Field Interviewer located the house of the deceased based on the data given to him. He introduced himself to the respondent and began the interview. Each one completed twenty reports which were reviewed and feedback was provided two days after completion of field work to maximize quality of writing the verbal autopsy report. Step 4 The final component of training was feed back session for 2 days. This session involved teaching them how to include essential information in report writing. The feedback session mainly focused discussion on reports which did not have a specified underlying cause of death and reports with minimal information to arrive at the probable underlying cause of death; for example, a report may say that a person had a stroke ten days ago but did not specify the type of onset (sudden or gradual, whether the person was conscious or unconscious, had difficulty in speaking or not, which parts of the body may have been affected etc.) or a report may say that the deceased had fever for ten days and died. It did not give details about the fever and other associated symptoms if any. B: Verbal autopsy of 80 000 adult deaths (≥ 25 years at the time of death) in Tamilnadu, South India This special verbal autopsy study was carried out in two areas in Tamilnadu. The Chennai city (urban) with a population of 4.2 million, and the Villupuram district (rural), with a population of 2.5 million were chosen for this study. We have successfully traced 48 000 adult deaths (≥ 25 years at the time of death) in urban area and 32 000 adult deaths in rural Villupuram district and reviewed 80 000 verbal autopsy reports to arrive at the probable underlying cause of death. Mortality data in urban area (Chennai) Information on deaths that occur in Chennai has been maintained manually by trained staff in Chennai Vital Statistics Department (VSD). The following data on deaths that occurred in Chennai during 1995 to 1997 were collected from the death registers in the Vital Statistics Department: deceased name, age, gender, marital status, father/spouse name, informant's name, occupation, place of death, address at the time of death, date of death and recorded cause of death (immediate, underlying and/or contributory). 72,000 deaths occurred during the study period of 1995–97. Of 72 000 found, 5000 deaths were attributed to external causes (unintentional injuries, suicide or homicide) in the death certificate, and were excluded. Of the remaining 67 000 deaths attributed in the VSD to medical causes, 48 000 of the households were successfully visited during 1998–99 to try to assign cause of death by verbal autopsy. Mortality data in rural area (Villupuram district) All formal and informal village records were to be sought to identify all deaths at any age during 1997–98. 41,000 such records were identified and 39 000 of the households were successfully visited during 1999–2000 to try to assign cause of death by verbal autopsy. Of 39 000 deaths, 7000 were before age 25. Feed back sessions and re-interview Feed back sessions were organized regularly throughout the study period to improve the quality of the verbal autopsy reports and 5% of the field visit reports were validated by re-interview one week after completion of the main interview, and blind to its results. This re-interviewing was done by one or other of two special interviewers because knowledge that a resurvey might well take place would ensure reliably motivated fieldwork at the initial survey, and also to check whether there were any systematic defects in the technique of any of the field workers: none were found. The underlying cause of death arrived based on re-interview data was not substantially different from the one arrived based on main interview data. Arriving at underlying cause of death All verbal autopsy reports were centrally reviewed by two physicians independently in order to arrive at "probable underlying cause of death". Each made a diagnosis based on signs, symptoms and sequence of events prior to death given in the verbal autopsy report, which were then coded according to the 9 th International Classification of Diseases, Injuries and Causes of Death [ 8 ]. The same 2 physicians reviewed all the 80,000 verbal autopsy reports. The discrepancies in the underlying causes of death were noted in 5% of verbal autopsy reports. These were discussed and resolved. The disagreement between 2 physicians in arriving at underlying cause of death was noted before classifying causes of death into broad groups. For example, 'Pneumonia' and 'Lower respiratory infection' were grouped under 'Infection'. According to one physician the underlying cause of death was pneumonia and for another physician it was lower respiratory infection. Results Urban study In Chennai city, the study was done in 1998–99 and the verbal autopsy reports were reviewed for 27 726 male deaths and 20 631 female deaths. Table 1 shows about 1100 (M:683, F:456) deaths due to medical causes were reassigned to external causes based on verbal autopsy reports. Deaths from unspecified medical causes and unknown causes decreased from 54% to 23% (p < 0.0001). Table 1 Cause of death from Vital Statistics Department* and based on Verbal Autopsy of 48 000 adult deaths (aged ≥ 25) in Chennai (urban), south India: 1995–97 Causes of death (ICD9 codes) Cause of death in VSD Cause of death based on Verbal Autopsy M (%) F (%) M (%) F (%) Vascular disease (390–415, 418–459) 8319 (30) 5168 (25) 11056 (41) 7435 (37) Respiratory tuberculosis (TB) (011, 012, 018) 1399 (5) 372 (2) 2231 (8) 575 (3) Other respiratory diseases (416, 417, 460–519) 1088 (4) 596 (3) 1597 (6) 855 (4) Neoplasm (140–239) 1163 (4) 1002 (5) 2344 (9) 1999 (10) Infection except respiratory & TB (rest of 1–139, 279.8 [HIV], 320-6, 590, 680-6) 584 (2) 303 (2) 1034 (4) 618 (3) Unspecified medical causes (780-9, 797-9) 12291 (44) 115 11 (56) 4367 (16) 5889 (29) Other specified medical causes 1899 (7) 1045 (5) 4414 (16) 2804 (14) No cause given in VSD (hence probably medical) 983 (4) 634 (3) Nil Nil Total deaths – medical 27 726 20 631 27 043 20 175 Re-assigned by VA to external causes *Excluded from the study 683 456 Total deaths (medical causes+external causes) 27 726 20 631 27 726 20 631 *Deaths(M: 3644; F:1644) that were assigned by the Vital Statistics Department(VSD) to non-medical causes were excluded from the study Rural study In Villupuram district, verbal autopsy report was written for all deaths i.e., deaths due to medical and external causes. So verbal autopsy reports of 19 294 male deaths and 12 494 female deaths were reviewed. Deaths from unspecified medical causes and unknown causes decreased from 41% to 26% (p < 0.0001) (Table 2 ). Table 2 Cause of death from various local records in Villupuram district and based on Verbal Autopsy of 32 000 adult deaths (aged ≥ 25) in Villupuram (rural), south India: 1997–98 Causes of death (ICD9 codes) Cause of death in local records Cause of death based on Verbal Autopsy M (%) F (%) M (%) F (%) Vascular disease (390–415, 418–459) 3351 (20.3) 1614 (14.4) 3928 (24.6) 2404 (22.0) Respiratory tuberculosis (TB) (011, 012, 018) 1659 (10.1) 686 (6.1) 1841 (11.5) 671 (6.1) Other respiratory diseases (416, 417, 460–519) 717 (4.4) 471 (4.2) 1044 (6.5) 728 (6.6) Neoplasm (140–239) 415 (2.5) 594 (5.3) 488 (3.1) 664 (6.1) Infection except respiratory & TB (rest of 1–139, 279.8 [HIV], 320-6, 590, 680-6) 1818 (11.0) 1584 (14.1) 1954 (12.2) 1411 (12.9) Unspecified medical causes (780-9, 797-9) 5829 (35.4) 4565 (40.7) 4173 (26.1) 2737 (25.0) Other specified medical causes 2237 (13.6) 1346 (12.0) 2570 (16.1) 2334 (21.3) No cause given (hence probably medical) 451 (2.7) 343 (3.1) Nil Nil Total deaths – medical 16 477 11 203 15 998 10 949 External causes 2817 1291 3296 1545 Total deaths (medical causes+external causes) 19 294 12 494 19 294 12 494 Validity of verbal autopsy tool The cause of death stated on the death certificate is often inaccurate. Studies, which have been undertaken around the world, show substantial difference (10–40%) between the clinical diagnosis or clinical cause of death and postmortem findings [ 9 - 12 ] and many of the completed death certificates failed to provide relevant information to allow adequate ICD-10 coding [ 13 ]. In India, individuals whose deaths might have been due to external causes are often subjected to postmortem examination, but others are not. So it is not possible to compare (clinical diagnosis of) medical causes of death against postmortem findings in India. Gajalakshmi et al [ 1 ] had done a study in Chennai to determine the sensitivity of the death certificate to identify cancer by comparing the cause of death stated on the death certificate with the morbidity date base of Chennai population-based cancer registry. It was found that the sensitivity of the death certificate to identify cancer as the underlying cause of death was 57%. In Chennai, about 75–80% of cancer patients attend health care facilities at late stage of the disease; about half of those who died at home soon after the diagnosis of cancer (and whose deaths were therefore, in almost all cases, likely to have been caused by their cancer) did not have cancer mentioned on their death certificates. Hence verbal autopsy tool was developed to determine specific cause of death, to compute cause specific death rates. Where a cause recorded on the death certificate in the VSD differed from the underlying cause assigned by the VA, there was often no absolute way of knowing which was correct (where the assigned cause by a medical doctor on the death certificate lacked detail, the VA may well be more reliable, and vice-versa) except for cancer deaths which could be verified with the Chennai cancer registry records. Hence, the validity of VA diagnosis was assessed only for cancer deaths(ICD 9:140–208) by comparing with the stated cause of death in the VSD records and verifying with the Chennai cancer registry records and hospital medical records (only for cancer diagnosis). Chennai Population-Based Cancer Registry is a demographic registry in the network of the Indian Council of Medical Research, Govt. of India and has been functioning since 1982 at the Cancer Institute(WIA), Chennai. Cancer is not a notifiable disease in India. Hence registration has been done by active method. Cancer patients attending the Govt. hospitals are interviewed to collect data on age, sex, address, duration of stay in Chennai city, marital status, mother tongue and educational level. Interviews are done at the houses for those who have been missed by the registry staff during their (Govt.) hospital visit. The clinical data, such as date of cancer diagnosis, method of diagnosis, site of cancer, any spread of the disease, histology, treatment details and status (alive or dead, if dead at the hospital, then, date and cause of death) for all registered patients are abstracted from the hospital medical records. All data on cancer patients attending the private hospitals are abstracted from the hospital records. The mortality data available at the Vital Statistics Department are linked with the Chennai Cancer Registry data base. Therefore the cause of death arrived based on verbal autopsy report was compared with the hospital data on cancer patients available in the Chennai Cancer Registry data base. Table 3 shows that 3053 deaths were identified as being due to cancer by VA. Review of VSD records revealed that 1435 of 3053 deaths as being due to non-cancer causes (majority of deaths were attributed to ill-defined/unknown followed by vascular causes) and 1618 (of 3053 deaths) as being due to cancer. Since the sensitivity of death certificate to identify cancer as underlying cause of death is only 57% in Chennai [ 1 ], all deaths at ages 25–69 included in the present study were verified with Chennai population-based cancer registry records. Table 4 shows that out of 3053 deaths identified by VA as cancer underlying cause of death, 2765 deaths matched with Chennai population-based cancer registry data base and 288 deaths did not. The cancer deaths identified by VSD records and not by VA (n = 107) (Table 3 ) matched with Chennai population-based cancer registry data base. Thus 288 cancer deaths, identified by VA, were not registered in the Chennai population-based cancer registry [ 14 , 15 ]. These were missed by the Chennai population-based cancer registry, both in the routine morbidity and mortality data registration process. We were successful in identifying all 288 cancer deaths, not available in the Chennai population-based cancer registry, in the medical records of the hospitals located in Chennai city. Thus all 3053 cancer deaths identified by VA were confirmed by linking with Chennai Cancer Registry records and hospital medical records. So there were no false positive cancer deaths recorded by VA. The sensitivity of VA to identify cancer was 94% (1618/1725) compared to VSD records and 96% (2765/2872) compared to Chennai population-based cancer registry in the age group 25–69 [ 15 , 16 ] and the Chennai population-based cancer registry missed 9% (288/3160) of total cancer deaths in the early adult life and middle age during the study period of three years. Table 3 Cancer (ICD 9: 140–208) deaths at ages 25–69 by Verbal autopsy (VA) and in Vital Statistics Department records (VSD) in Chennai (urban), South India VSD VA Cancer Noncancer Total Cancer 1618 1435 3053 Noncancer 107 21941 22048 Total 1725 23376 25101 Table 4 Cancer (ICD 9: 140–208) deaths at ages 25–69 by Verbal autopsy (VA) and in Chennai population-based cancer registry in Chennai (urban), South India Cancer Registry VA Registered Not registered Total Cancer 2765 288 3053 Noncancer 107 21941 22048 Total 2872 22229 25101 Discussion The Tamilnadu study on verbal autopsy [ 15 , 16 ] used university graduates since it is very expensive to send professionally trained individuals to field visits, to write verbal autopsy reports. We have found it very difficult to get female graduates willing to do field work. Hence only males were recruited for the field work. Responders of female deaths were usually males who did not hesitate to reveal the circumstances/ symptoms etc prior to death. The participation rate was 100%. The informants were given full information about the objectives of the study and the participation in this study was entirely voluntary basis. The verbal autopsy tool for adult deaths is an open narrative format uses the check list of symptoms and signs with filters to get more information on train of events or circumstances preceding the death. The sensitivity of this tool is 95% (94% compared to VSD records and 96% compared to Chennai population-based cancer registry) in the age group 25–69 during the study period. The validity of this verbal autopsy tool is influenced by the training given to the interviewers, on the immediate random checking of the 5% of interview data and reviewing of the field reports centrally by 2 physicians to arrive at the probable underlying cause of death which is better than that arrived by opinion-based algorithm [ 17 ]. There is little information or literature about validity of cause of death for adults by verbal autopsy in India. As a result of using verbal autopsy method, adult deaths (≥ 25) from unspecified and unknown causes decreased from 54% to 23% (p < 0.0001) in urban and from 41% to 26% (p < 0.0001) in rural areas in Tamilnadu. Ten day training to write verbal autopsy reports followed by constant monitoring of the submitted reports resulted in arriving at the probable underlying cause of death for most of the deaths and to compute broad classification of the underlying causes of about 90% of deaths in early adult life or middle age: in old age, however, the proportion classifiable is substantially lower. The specific causes of death arrived based on verbal autopsy reports were used to estimate death rates for Chennai city [ 16 ] and to estimate the risk of death associated with smoking for broad groups of causes of death[ 18 ]. This methodology of arriving cause of death for adult deaths by verbal autopsy is now being adopted by the Registrar General of India, Govt. of India, for nationwide use in the Sample Registration System(SRS) that consists of 6671 units (4436 rural and 2235 urban) spread across the country covering 1.1 million households and about 6 million population. The SRS is a large demographic survey of vital events occurring in a national random sample of urban and rural areas in India by the Registrar General of India (RGI) to provide annual estimates of age-specific birth and death rates at the national and state levels. This exercise on verbal autopsy for adult deaths along with the verbal autopsy questionnaire developed by the SRS collaborators for deaths that occur at ages less than 15 years and for maternal deaths is expected to yield reliable cause-specific death rates for India. Conclusion A ten day training programme to write verbal autopsy report with adequate feed back sessions and random sampling of 5% of the verbal autopsy reports for re-interview worked very well in Tamilnadu, to arrive at the probable underlying cause of death reliably for deaths in early adult life or middle age (25–69 years) and less reliably for older ages (70+). Our experience shows that the open narrative, if well written, provides adequate information for assigning probable underlying cause of death for adult deaths. Competing interests The authors declare that they have no competing interests. Authors' contributions VG and RP participated in designing the study, analysis and preparation of the report. Development of verbal autopsy tool, training on verbal autopsy method, co-ordination of field work and data management were done by VG. Verbal autopsy review by VG and TS Kanaka, physician. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 Appendix I – used to write the history of past illness and verbal autopsy report for adult deaths (25 years or older) and in Appendix II – Symptoms/signs checklist for adult deaths (≥ 25 years) is given Click here for file

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544898

Case-based medical informatics

Background The "applied" nature distinguishes applied sciences from theoretical sciences. To emphasize this distinction, we begin with a general, meta-level overview of the scientific endeavor. We introduce the knowledge spectrum and four interconnected modalities of knowledge. In addition to the traditional differentiation between implicit and explicit knowledge we outline the concepts of general and individual knowledge . We connect general knowledge with the "frame problem," a fundamental issue of artificial intelligence, and individual knowledge with another important paradigm of artificial intelligence, case-based reasoning , a method of individual knowledge processing that aims at solving new problems based on the solutions to similar past problems. We outline the fundamental differences between Medical Informatics and theoretical sciences and propose that Medical Informatics research should advance individual knowledge processing (case-based reasoning) and that natural language processing research is an important step towards this goal that may have ethical implications for patient-centered health medicine. Discussion We focus on fundamental aspects of decision-making, which connect human expertise with individual knowledge processing. We continue with a knowledge spectrum perspective on biomedical knowledge and conclude that case-based reasoning is the paradigm that can advance towards personalized healthcare and that can enable the education of patients and providers. We center the discussion on formal methods of knowledge representation around the frame problem. We propose a context-dependent view on the notion of "meaning" and advocate the need for case-based reasoning research and natural language processing. In the context of memory based knowledge processing, pattern recognition, comparison and analogy-making, we conclude that while humans seem to naturally support the case-based reasoning paradigm (memory of past experiences of problem-solving and powerful case matching mechanisms), technical solutions are challenging. Finally, we discuss the major challenges for a technical solution: case record comprehensiveness, organization of information on similarity principles, development of pattern recognition and solving ethical issues. Summary Medical Informatics is an applied science that should be committed to advancing patient-centered medicine through individual knowledge processing. Case-based reasoning is the technical solution that enables a continuous individual knowledge processing and could be applied providing that challenges and ethical issues arising are addressed appropriately.

Background A meta-level view of science Our aim is to place Medical Informatics in the context of other sciences and to bring coherence in its formal education [ 1 ]. This will necessarily place the discussion at a meta-level view of science, which traditionally was the concern of philosophers. From such a general perspective, science could be defined as "the business of eliciting theories from observations in a certain context , with the hope that those theories will help to understand, predict and solve problems." Also revolving around the "business of creating theories," R. Solomonoff's ideas [ 2 ], summarized in [ 3 ], contribute to the basis of Algorithmic Information Theory (AIT) [ 4 ], a relatively new area of research initiated by A. Kolmogorov, R. Solomonoff and G. Chaitin, and regarded as the unification of Computer Science and Information Theory. According to Solomonoff's view, a scientist's theories are compressions of her observations (i.e., her experimental data). These compressions are used to explain, communicate and manage observations efficiently and, if valid, to help solving problems, understanding and predicting. Intuitively, the higher the compression achieved by the theory, the more "elegant" that theory and the higher its chances of acceptance. This very general perspective of the scientific endeavor also makes science to appear twofold: it comprises the creation of theories (i.e., theory elicitation) as well as their subsequent use in understanding, predicting and solving problems (i.e., theory application). Therefore, science seems to be driven by two opposite forces: that of creating theories, and that of applying those theories to practical applications. The four-dimensional space-time continuum we live in (i.e., our universe) forms the reality (i.e., the context) of all scientific observations. The compression of the immense complexity and dynamicity of this reality in concise "theories of everything" was already demonstrated by Zuse [ 5 , 6 ] and recently Schmidhuber [ 7 ]. These results of theoretical computer science demonstrate the power of human theory elicitation and provide important answers to old questions of science and philosophy. However, their unfeasibility when applied to practical problems, which would be equal to building computing devices capable of running precise simulations of our reality, also widens the gap between theoretical research and practical sciences. For the time being, humanity still needs to divide science and define human knowledge as a collection of individual theories elicited from scientific observations. The immense number of theories that comprise the collective human knowledge about every possible subject, as well as its extraordinary dynamics, have forced us to divide it into what we commonly refer to as knowledge domains , thereby reducing the contexts of our observations to smaller space-time continuums. The attempts to process with computers the knowledge in a domain have taught us that we need to recognize the reality of the "knowledge acquisition bottleneck" [ 8 ] and to not underestimate the importance of common-sense knowledge (see [ 9 ] and [ 10 - 13 ]). The particularities with regard to the context retention, acquisition, representation, transferability and applicability of domain knowledge, causes us to distinguish between different modalities of domain knowledge, and place them on what we refer to as the knowledge spectrum . The knowledge spectrum The knowledge spectrum (Figure 1 ) spans from a complex reality (the source of experimental data and information gathered from observations and measurements) to high-level abstractions (e.g., theories, hypotheses, beliefs, concepts, formulae etc). Therefore, it comprises increasingly lean modalities of knowledge and knowledge representations media and the relative boundaries and relationships between them. Two forces manifest on the knowledge spectrum: that of creating abstractions and that of instantiating abstractions for practical applications. The former is the theory elicitation and is synonymous to processes of context reduction and knowledge decomposition. The latter, theory application, equates to context increase and knowledge composition processes. The engines behind the two knowledge spectrum forces are the knowledge processors, natural or artificial entities able to create abstractions from data and to instantiate abstractions in order to fit reality. Knowledge is traditionally categorized into implicit and explicit (Table 1 ) and ranges from rich representations grounded in a reality, to highly abstracted, symbolic representations of that reality. The classical distinction between data, meta-data, information, knowledge and meta-knowledge is simplified by our subscription to the unified view of Algorithmic Information Theory (AIT) [ 4 ] which recasts all knowledge modalities and their processing into a general framework requiring a Universal Turing Machine, its programs and data represented as finite binary sequences. From this perspective a precise distinction between these modalities becomes unimportant. Implicit knowledge (U, from unobvious, unapparent) is the rich, experiential, sensorial kind of knowledge that a knowledge processor acquires when immersed into an environment (i.e., grounded in an environment), or presented with detailed representations of that environment (e.g., images, models, recordings, simulations). It is very well applicable to specific instances of problems and relies on processing mechanisms such as feature selection, pattern recognition and associative memory. Explicit knowledge (E) is the abstract, symbolic type of knowledge present explicitly in documentations of knowledge such as textbooks or guidelines. It requires a representation language and the capability of a knowledge processor to construe the meaning of concepts of that language. It is applicable to both specific and generic problems and relies on explicit reasoning mechanisms. The distinction between implicit and explicit knowledge are useful to characterize the nature of human expertise, but become problematic when one wants to describe fundamental differences between theoretical and applied sciences: many applied sciences, especially knowledge intensive ones, in addition to general theories of problem solving, also make use of explicit knowledge in order to describe, with various degrees of precision, particular instances of problem solving and theory application. This represents the rationale for further dividing the knowledge spectrum into general and individual knowledge (Table 2 ). General knowledge General knowledge (G) is the explicit, abstract, propositional type of knowledge (e.g., guidelines), well applicable to context-independent, generic problems. However, it is more difficult to use in specific contexts because of the gap between the general knowledge itself and a particular application context. This knowledge gap translates into uncertainty when a general knowledge fact is instantiated to a specific situation. For example, knowing generally that a certain drug may give allergic reactions but being uncertain whether a particular patient may or may not develop any, is an example of what we consider the uncertainty associated with general knowledge. The creation of general knowledge (i.e., abstraction, generalization, context reduction, theory elicitation) is a relevance-driven process done by "stripping away irrelevancies" [ 9 ]. This causes general knowledge to have a lower complexity and be more manageable: "generalization is saying less and less about more and more" [ 9 ]. Formal representations of explicit knowledge have been common in early artificial intelligence (AI) applications in the context of expert system development. They operated under the "closed world assumption" and were meant to make the representation of knowledge manageable, reproducible and clear. However this assumption also rendered the expert systems "brittle" or completely unusable when applied to real world problems [ 14 ]. The completeness necessary for automatic reasoning using explicit reasoning mechanisms can be illustrated with the following formal definition of the concept of "a brick" in a limited, hypothetical world, containing only simple geometric objects such as bricks and pyramids (Figure 2 ) (adapted from [ 15 ]): "being a brick implies three things: 1. first, that the brick is on something that is not a pyramid; 2. second, that there is nothing that the brick is on and that is on the brick as well; and 3. third, that there is nothing that is not a brick and the same thing as the brick." This definition could have the predicate calculus representation: This representation shows that an intelligent agent who has no implicit knowledge of the hypothetical physical world and no capacity of generalization or analogy making, must be explicitly provided with all knowledge necessary to reason about "bricks" in that limited reality. Such approaches are known to suffer from a fundamental shortcoming, the "frame problem." The frame problem Daniel Dennett was the first philosopher of science who clearly articulated the "frame problem" and promoted it as one of the central problems of artificial intelligence [ 16 ] (also see [ 17 ]). Janlert [ 18 ] identifies the frame problem with "the problem of representing change." In [ 14 ] the frame problem is defined as "the problem of representing and reasoning about the side effects and implicit changes in a world description." In order to articulate and circumvent the abstract nature of its definition, Dennett has invented a little story involving three generations of increasingly sophisticated robots. These fictitious robots are products of early artificial intelligence (AI) technology that use automated reasoning based on formal representations similar to the brick example. These particular robots are specifically designed to solve a problem consisting of the retrieval of their life-essential batteries from a room, under the threat of a ticking bomb set to go off soon. Although increasingly sophisticated in their reasoning, all three successive versions of the robot fail: • The first robot fails by missing a highly relevant side effect of pulling the wagon with the batteries out of the room: the ticking bomb sitting on the same wagon was also retrieved, together with the batteries. • The second robot did not finish its extensive, irrelevant side-effect reasoning procedures before the bomb goes off. As Dennett ironically puts it, the robot "had just finished deducing that pulling the wagon out of the room would not change the color of the room's walls and was embarking on a proof of the further implication that pulling the wagon out would cause its wheels to turn more revolutions than there were wheels on the wagon – when the bomb exploded." • The third robot failed because it was " busily (i.e., explicitly) ignoring some thousands of implications it has determined to be irrelevant " and its batteries were therefore lost in the inevitable explosion. The frame problem can therefore be recast as a problem of relevance [ 17 ] (see preface), which is compounded by time constraints. It demonstrates that relevance judgment mechanisms based on general knowledge are time consuming and cause the failure to solve time-constrained decision problems. It is a problem only because in the real world we do have time constraints . Individual knowledge Individual knowledge (I) or instance specific knowledge, on the other hand, is a knowledge modality very well applicable to real problems, because it identifies uniquely and matches precisely an application context. The knowledge gap and uncertainty are reduced but still exist because of our changing reality (time dimension) which may render individual knowledge about a patient collected in the past (e.g., value of blood pressure from a month ago), less applicable in the present or future. Because it preserves context (i.e., it is more grounded), individual knowledge has a higher complexity than general knowledge and hence is more difficult to manage (i.e., has high memory requirements). For example, knowing the drugs and the precise description (e.g., numeric, textual, visual) of the allergic reactions that they caused in a certain person, as well as many other particular knowledge facts about individual, is what we call individual knowledge. The uncertainty and knowledge gap related to the application of such knowledge to future instances of decision making involving that individual are reduced: individual knowledge is supposed to fit very well the application context where it was originally captured. Case-based reasoning Individual knowledge captured from a very specific context (e.g., diagnosing a particular patient with a particular disease) can be extrapolated to similar contexts. The higher the similarity between contexts, the smaller the knowledge gap and instantiation uncertainty and the higher the chances for a successful solution to a new problem. For this reason, individual knowledge processing has become increasingly important for artificial intelligence applications and is defined as the approach to solving new problems based on the solutions of similar past problems [ 14 , 19 - 21 ]. It has several flavors (e.g., exemplar-based, instance-based, memory-based, analogy-based) [ 21 ] which we will refer to in this paper interchangeably, through the generic term of "case-based reasoning" (CBR). There are four steps (the four "RE") that a case-based reasoner must perform [ 14 , 20 , 21 ]: 1. RETRIEVE: the retrieval from memory of the cases which are appropriate for the problem at hand; this task involves processes of analogy-making or case pattern matching; 2. REUSE: the decomposition of the retrieved cases in order to make them applicable to the problem at hand; 3. REVISE: the compositional adaptation and application of the knowledge encoded in the retrieved cases to the new problem; and 4. RETAIN: the addition of the current problem together with its resolution to the case base, for future use. CBR entails that an expert system has a rich collection of past problem-solving cases stored together with their resolutions. CBR also hinges on a proper management of the case base and on appropriate mechanisms for the matching, retrieval and adaptation of the knowledge stored in the cases relevant to a new problem. Ideally, the individual knowledge in a case-base will progress asymptotically towards an exhaustive knowledge base, which represents the "holy grail" of knowledge engineers. From a learning systems point of view, similarly to artificial neural networks [ 22 , 23 ] and inductive inference systems [ 24 ] that learn from training examples, a CBR system acquires new knowledge, stores it in a case base and makes use of it in new problem solving situations. The absolute positions and shapes of boundaries between the four knowledge modalities, although admittedly not as precise as drawn on the knowledge spectrum in Figure 1 , are not of importance for this discussion. However, the relative relationships between knowledge modalities are, and can be represented formally as a Venn diagram (Figure 3 ), which implies that: • Individual knowledge has a higher complexity than the explicit knowledge elicited from the same context. This is equivalent to stating that, for example, the picture of a person encodes more knowledge than the textual description of that person's appearance. • Implicit knowledge is a subset of the individual knowledge. • General knowledge is a subset of the explicit knowledge. • The set of individual knowledge represented explicitly formed by the intersection of individual knowledge with explicit knowledge is a nonempty set. This is equivalent to stating that it is possible, for example, for an explicit textual description to identify a context uniquely (e.g., the complete name and address of a person at a specified moment in time). A meta-level view of Medical Informatics The meta-level overview of sciences and the definitions and properties of the knowledge spectrum and knowledge modalities enable us to draw some fundamental differences between theoretical sciences and applied sciences such as Medicine [ 25 ] and Medical Informatics. From this perspective, theoretical sciences (e.g., theoretical computer science): • Make use of observations which are highly abstract symbolisms and create far more limited contexts of application of their theories, when compared to the complexity of the human body or of any social or biological system, • Have as a primary purpose the creation of general knowledge comprising valid, powerful theories which explain precisely and completely the observations, and therefore, • Include a relatively limited number of precise theories which are evaluated primarily by their power of explaining experimental observations, elegance, generality, and • Are less concerned with the acquisition of the individual knowledge required by the practical implementation and by the application of results to real world problems. Applied sciences such as Medicine and Medical Informatics, on the other hand: • Gather extensively data and observations ( individual knowledge ) from very complex systems [ 9 , 26 ] (e.g., human body), which are characterized by high individual variation and randomness; • Have as a primary purpose not only distilling data and observations into general knowledge , but are also concerned with the implementation details and with the application of theories to individual problem solving (e.g., diagnosis and treatment of real patients), • May lack the incentive to refine existing theories which are objectively wrong as long as practical success is achieved [ 25 ], • Contain very few simple, "elegant" theories ( general knowledge ) that can solve individual problems completely or explain and predict accurately [ 27 ] because of the complexity of the human body and its individual variation and, therefore, • May pursue the application of a multitude of mutually contradictory, poorly grounded, general theories (e.g., the general theory of medical reasoning and the concepts of "diagnosis" and "symptom") [ 1 , 25 ], • Abound in general theories (e.g., guidelines) which are "lossy" (i.e., ignore individual context variation) and which are evaluated statistically by their practical success relative to existing ones (e.g., cancer therapy), • Attempt to make up for the knowledge gap between general knowledge and the reality where knowledge is applied, by employing experienced clinicians who require extensive training and information technology (e.g., decision support), and, in addition, • Are compounded by time-constrained circumstances and largely unsolved ethical issues (e.g., privacy and confidentiality, genomics research). Given the special circumstances of our applied science in the context of other sciences and the increasing recognition of the importance of knowledge processing to Medical Informatics [ 28 ], we propose, as part of the thesis of this paper, that Medical Informatics should complement the traditional quest for general biomedical knowledge with the advance of acquisition, storage, communication and use of individual knowledge . By doing so, Medical Informatics will provide a solution to the problems that arise during the use of general knowledge and, in the same time, will enable clinical research as well as advanced decision support and education of both healthcare providers and patients . Individual knowledge processing equates to a case-based reasoning (CBR) approach that employs collections of patient cases. Currently, such collections are the focus of research on Electronic Health Records (EHR). Envisioned as "womb to tomb" collections of patient-specific data, EHR contain a wealth of data that could be used to support case-based decisions. If EHR are to be used in a CBR context, the issues pertinent to the design of case-bases automatically become pertinent to the EHR design, and the CBR paradigm becomes important to Medical Informatics . The overall knowledge processing capacity of healthcare systems can be thought to be distributed between two sources: human resources (i.e., healthcare professionals) and information technology (Medical Informatics). An ideal CBR approach would increase this knowledge processing capacity by allowing for the automatic processing (acquisition, representation, storage, retrieval and use) of individual knowledge present in increasingly rich knowledge media such as natural language artifacts, images, videos and computer simulations of reality (Figure 4 ). The storage and communication of knowledge are well advanced by current information technology. However, most of the acquisition, retrieval and knowledge use are, and will continue to be the task of professionals until advanced processing (e.g., real-time computer vision, scene understanding and synthesis, image understanding, robotics, natural language understanding) are applicable. Given the widespread use of natural languages as knowledge representation and communication media, it follows that natural language processing (NLP) research is a very important component of Medical Informatics, required to advance the organization and processing of individual knowledge in reusable case-bases. Further, the goal to advance processing of increasingly complex knowledge representations (e.g., natural language, sounds, images, simulations) and create intelligent machines that can hear, see, think, adapt and make decisions, brings Informatics even closer to what traditionally was the concern of Artificial Intelligence (AI) . Finally, because the knowledge processing capacity of human resources tends to remain relatively constant, moving towards the ideal of individual knowledge processing, no matter how slowly, may also have ethical implications because it proves that medical informaticians are trying to do everything they can in order to serve the interest of the individual . Discussion In order to support our thesis, the following discussion will focus initially on fundamental aspects of medical decision-making and biomedical knowledge creation from the standpoint of the knowledge spectrum. This will lead to a discussion of fundamental knowledge representation and processing principles and the proposal of a CBR perspective on EHR, including challenges and potential solutions. Human and computer knowledge processing Decision making in medicine Medicine is a knowledge intensive domain where time-constrained decisions based on uncertain observations are commonplace. In order to successfully cope with such situations, health professionals go through a tedious learning process in which they gain the necessary domain knowledge to evolve from novices to experts. As experts, health professionals have attained, among other things, two important, highly interrelated abilities: • To be able to reduce knowledge complexity by determining efficiently what is relevant for solving a problem in a particular situation, and, • To be able to reduce the knowledge gap between knowledge facts and reality which translates into being able to reduce the uncertainty of knowledge instantiation to a particular context. For example, both the presence and the absence of a past appendectomy are relevant and contribute (potentially unequally) to reducing the uncertainty of instantiation of the biomedical knowledge of an expert to a particular context of a patient with right lower abdominal pain. Fundamental to decision making, relevance judgments and uncertainty reduction seem both closely connected with the quality and quantity of knowledge available for solving a problem as well as with the nature of knowledge processing mechanisms. Studies of expert-novice differences in medicine [ 29 ] have shown that the key difference between novices and experts is the highly organized knowledge structures of the latter, and not the explicit strategies or algorithms they use to solve a problem. This is supported by expert system development experiences which showed that a system's power lies in the domain knowledge rather than in the sophistication of the reasoning strategies [ 14 ]. Studies of predictive measures of students' performance indicate that tests which measure the acquisition of domain knowledge are the best predictors [ 30 ]. The work on naturalistic decision-making (NDM) and the development of psychological models of "recognitional decision-making" such as the Recognition-Primed Decision (RPD) [ 31 - 33 ], suggest the heavy dependence of decision makers on their previous experience of problem-solving and also on their ability to perform mental simulations. The discussion around the amount of problem solving experience of a decision maker becomes critical in time-constrained decision circumstances. The exhaustiveness of the knowledge base and the efficiency of retrieval mechanisms now become paramount to the decision speed. Empirical evidence that shows the existence of "systematic changes of cognitive processes" related to time stress, comes from the studies on the psychology of decision-making under time constraints [ 34 ]. Although most of these studies attest the overall negative effect of time stress on the "effectiveness of decision-making processes" [ 35 ], others [ 31 , 33 ] argue that even extremely time-constrained situations could be handled successfully by human subjects, given enough expertise (i.e., enough problem solving experience). Since humans are able to make sound relevance judgments and reduce instantiation uncertainty of knowledge most of the times, the following questions arise: What is their strategy for increasing the exhaustiveness of their knowledge base while managing its exponential complexity? How do they represent and organize their knowledge and how do they manage time-constrained situations? At least some of these questions have been under intense scrutiny that has resulted in important empirical work on naturalistic decision-making [ 32 , 33 , 36 , 37 ]. Important insights have been gained at the individual but also at the organizational and social levels. Coherent with the importance of the social aspects of decision-making, Armstrong [ 38 ] builds an interesting argument about the Darwinian evolution, social networking and the drive for knowledge discovery of the humanity as being some of the reasons that contribute to the human decision making potential. From the perspective of the knowledge spectrum, it seems reasonable to associate expert decision makers with individual knowledge and novices with the more abstract general knowledge about a subject, available in explicit knowledge artifacts (e.g., textbooks, guidelines). It is also conceivable that mental models of experts span a great length of the knowledge spectrum, causing them to efficiently perform implicit processing (feature selection, pattern recognition, associative recall) and also just-in-time explicit reasoning (Figure 5 ). The ability to move freely across the knowledge spectrum causes experts to efficiently reduce data to abstractions and to create hypotheses and micro-theories through sound relevance judgments. The powerful mental simulations that experts can perform allow them to construe appropriate meanings of concepts and to verify their hypotheses against contexts of reality. Novices, on the other hand, have limited mental models of reality situated towards the abstract region of the spectrum. This causes them to have difficulties with construing appropriate meanings of concepts due to the increased knowledge gaps between their mental models and reality. Novices are therefore unable to make sound relevance judgments and limited in their ability of interpreting data and creating abstractions. They are also usually overwhelmed by the explicit, general knowledge present in textbooks and guidelines and unable to fully construe the meanings of concepts present in such knowledge artifacts. In conclusion, in information and knowledge intensive domains such as medicine, explicit reasoning is important but individual knowledge acquisition (i.e., experience) and processing (i.e., CBR) are crucial for decision-making. Because the nature of expertise seems largely connected with individual knowledge processing, it follows that the evolution of novices into experts is unattainable only by the provision of extensive general knowledge. In addition, not only the individual learning but also the collective sharing of experiences (e.g., case records, personal stories, etc.) between individuals and between generations, contribute to the way humans deal with decision problems . Patient-centered vs. population-centered healthcare The major driving force of science is universally applicable knowledge (i.e., general knowledge). While creating and communicating new knowledge, scientists move across the knowledge spectrum from the data that captures the reality of their experiments and observations towards abstract representations that allow them to communicate their theories. In biomedical research, such an example is the randomized controlled trial (RCT), currently regarded as the gold standard for knowledge creation. The correct design of an RCT is crucial for the validity of the medical evidence obtained. A correct randomization process in RCTs will limit the bias and increase the chance for applicability of the evidence obtained, to a specifically selected group of patients (e.g., "women aged 40–49 without family history of breast cancer"). However, at the same time, the randomization process removes the circumstances of individual cases and creates a knowledge gap between the RCT evidence and future application instances. As with any statistical approach, the RCT-based evidence is best applicable at the population level rather than at the individual level. This depersonalization of medical knowledge and evidence was also noted by others [ 39 , 40 ] and could also be illustrated by the observation that most patients feel relieved when told that the chances of being successfully treated for a certain condition are 99%, for example. Although this is psychologically very positive, the patients should not necessarily be relieved, as they could very well happen to fall among the 1%, for whom things could go wrong and for whom, usually, the RCT-based evidence does not provide additional information. An experienced physician and, from a CBR perspective, a highly efficient case-based reasoner, is most of the times able to individualize the medical decision for a particular patient for whom things are likely to go wrong and fill in the knowledge gap between the RCT evidence and the medical problem at hand. This could lead to avoiding a therapeutic procedure recommended by the medical evidence. The individual knowledge that this decision is based on is usually not provided by the RCT, but is acquired through a tedious process of training. This decision is often so complex that it cannot be easily explained as it becomes heuristic in nature and is motivated by the individual knowledge that a decision maker possesses. Others [ 41 ] have also pointed out that when physicians manage their cases (e.g., diagnosis and treatment), their previous experience allows them to make informed decisions based on heuristics rather than on a sound, complete and reproducible reasoning, such as logical inference based on a predicate calculus representation of a problem. In addition, human experts often disregard probabilistic, RCT-type of evidence and consistently detach themselves from the normative models of classical decision theory (e.g. probability theory, Bayes theory) in favor of heuristics-based approaches. Although prone to occasional failures, heuristics-based decisions are much more efficient in time-constrained and uncertain situations [ 33 ]. From the perspective of the knowledge spectrum, the driving forces of Health Informatics and RCT methodology seem to have opposite directions: while Informatics aims towards individual knowledge and personalized health care, the general knowledge gained through populational studies (e.g., RCTs) targets the ideal of universal applicability (Figure 6 ). The value of a single bit of data (e.g., a Yes/No answer to a specific question such as a past appendectomy) can be very relevant in a decision-making context if it reduces the overall uncertainty of knowledge. However, such individual bits of data are inevitably lost during the creation of general knowledge. Rigorously and expensively collected, general, populational level knowledge is useful only in situations where individual knowledge lacks (e.g., new drugs), providing the decision makers have access to it and are able to apply it to specific situations. However, general knowledge is unlikely to be used as such in many naturalistic decision-making processes, because it does not support the way expert decision makers think. The knowledge gap and inherent instantiation uncertainty manifested in the application of general knowledge does not fully enable the education of providers and patients which would require additional knowledge about individual contexts of successful or unsuccessful application instances. Informatics, on the other hand, by advancing individual knowledge processing, provides an alternative solution to the problems that arise from the use of general knowledge that targets universal applicability. An integral part of individual knowledge, genomic data is already recognized [ 39 , 40 ] as being of extreme importance for a solution to the problems of general knowledge . Knowledge representation by formal methods The application of formal knowledge representations to real problems suffers from a fundamental shortcoming: the frame problem. As explained above (see "The frame problem"), the frame problem can be recast as a problem of relevance. Given the capability of relatively effortless human relevance judgments, the frame problem seems a rather "artificial" creation, difficult to grasp and which usually goes unnoticed. In order to circumvent its abstract nature, Dennett uses a story-telling approach. However, the frame problem also applies to and could be illustrated from the perspective of humans, who in their first years of life, learn and can easily and efficiently reason about the side effects and the implicit changes of the complex four-dimensional spatio-temporal physical world in which they live. As this learning gradually becomes common sense knowledge, it causes us to efficiently determine the relevant implicit changes while ignoring the non-relevant ones for a given situation. For example, such facts as that the clothes we are wearing are moving with us while walking or traveling are most of the times irrelevant given the context of a planned trip. However, if the trip involves some rapid movement through the air such as riding a motorbike, suddenly wearing a sombrero becomes a relevant fact. As experts at managing our physical world, we are able, through an effortless but powerful mental simulation, to determine the relevance of such a particular fact. The recall of our personal experiences of moving fast through the air and of the dragging force of the air becomes paramount. Therefore, intelligent agent must be endowed with efficient mechanisms for determining the relevance of particular facts for a decision . We suggest that what made the robots vulnerable was their creators' choice for knowledge representation and reasoning: the robots did not have quick access to implicit knowledge about the relevance of particular facts (i.e., records of problem solving instances) but only to explicit facts in frames which had to be employed in time-consuming, immense number of explicit relevance judgments about the effects of particular actions. Although they were supposed to be experts at their task, the robots were behaving like novices. The frame problem is not a problem of the knowledge representation per se, but a problem of the choice for representation of knowledge needed to solve time-constrained decisions. In other words, formal representations and logic reasoning work, but not in time constrained, complex situations. From the perspective of our knowledge spectrum, explicit, formal representations sit on the abstract side of the spectrum (Figure 7 ). The retrieval of explicit knowledge representation is currently the subject of the increasingly important field of research of information retrieval (IR). It is commonly accepted that IR is strongly coupled with the notion of intended meaning of concepts: a retrieved document is considered to be relevant to a query if the intended meanings of the authors of a document are relevant to the intended meaning of that query. We propose that "meaning," a property that characterizes all concepts present in explicit knowledge, is intimately connected (if not identical) with the notion of context . According to this rather paradoxical view, meaning, a property which characterizes the abstract side of the knowledge spectrum, is strongly coupled with context which, by definition, is a feature of the reality side of the knowledge spectrum. Therefore, in order to construe meaning appropriately one needs to be able to efficiently move from abstractions towards richer representations of reality. This movement on the knowledge spectrum is necessary in order to fill the knowledge gap between abstract concepts and the richer mental representations required for construing their meaning. Explicit, formal representations attempt to capture general truth and generally applicable problem solving strategies, but become too abstract in nature. Through the abstraction process, which is essentially a reduction driven by the relevance judgments of knowledge creators, the context of a problem is lost. Losing context creates difficulties with construing meaning (which is context-dependent by definition) and widens the knowledge gap between the representation itself and the reality of a future problem-solving instance. The knowledge gap translates into the instantiation uncertainty that characterizes the application of general knowledge to specific problems. Making up for the knowledge gap through explicit relevance reasoning becomes time consuming and consequently takes its toll on the applicability of the representation. In sensitive applications such as medical decision-making and health research, general knowledge may potentially be harmful (e.g., prescribing an highly recommended drug to which a patient has a undocumented allergy). In addition, abstractions and general methods and theories of problem solving and decision making (e.g., guidelines) do not fully enable the education of individuals and the learning from successes and mistakes. Knowledge representation approaches must therefore preserve to the extent possible, the context of a problem-solving instance. By efficiently recalling similar past instances of problem solving and their contexts, intelligent agents are immediately provided with implicit knowledge about relevance, encoded in the retrieved contexts and, in the same time, with more possibilities to reduce the instantiation uncertainty of general knowledge when applied to specific problems. To enable this, informatics research must advance the processing of rich representations of the knowledge encoded in past problem solving cases: this is the definition of CBR research . Knowledge representation by natural language Similar to formal specifications (e.g., predicate calculus) natural language uses abstractions, i.e., concepts. Its richness and power of expression place it in the knowledge spectrum to the left side of formal specifications but to the right side of rich descriptions consisting of images, sounds, video-clips and simulations of reality (Figure 4 ). Natural language has power of expression but loose semantics and inherent ambiguity. However, despite its abstract nature, it remains the indispensable, main knowledge representation and transfer medium between humans. In order to illustrate our point about ambiguity we direct the reader to the previous, natural language definition of the concept of "a brick." Although the definition may look unequivocal, there are subtle ambiguities that make a difference in the predicate calculus representation. The first condition of an object to be "a brick" (i.e., "the brick is on something that is not a pyramid," highlighted in the equations 2 and 3) is an ambiguous natural language construction and could have slightly different formal representations: In (2) this condition has been interpreted as: "the brick being on something IMPLIES that that something is not a pyramid" and was therefore represented as "for all Y, if X is on Y, this implies that Y is not a pyramid." In (3), which is identical to (1) but is repeated to the benefit of the reader, this condition was interpreted as "the brick MUST BE (or is always) on something that is not a pyramid" and that was represented as "there exists Y such as X is on Y and Y is not a pyramid." The first definition is therefore more "relaxed" as it allows the possibility that a brick sits on nothing. The second definition is more restrictive, because it requires the brick to be on something that is "not a pyramid" or otherwise X is not a brick anymore. Therefore, the first definition is more general and defines the concept of "a brick" in such a way that the definition would be true even in a world with no gravity (i.e., the brick is on nothing). In addition, definition (3) does not reject the possibility that an object sits on both another brick and a pyramid, at the same time (Figure 8 ). The point is that, most often, humans receive and transmit knowledge without the deep understanding and completeness required by an exact mathematical representation of the knowledge to be transmitted. This shallowness has also been recognized by others [ 42 ] who are trying to draw natural language processing researchers' attention to the fact that humans are rather superficial in their knowledge acquisition and processing and often make use of "underspecified" representations. Although, since the early days of science, scientists have fallen in love with the pure reasoning approaches, as they were reproducible, unambiguous means to express new knowledge, the problems with the use of classical predicate calculus as a knowledge representation method and of the classical logic inference as a reasoning strategy are discouraging. This is due to the requirements of complete, unequivocal representations, which prevents them from dealing with the messiness of the real world problems. If possessing the necessary knowledge, humans are able to effortlessly fill the knowledge gaps between natural language representations and their richer representations of reality (i.e., mental models), and to easily construe the appropriate meaning of potentially ambiguous concepts. Although current technology allows for its storage, knowledge present in richer media (e.g., images, videos, simulations) is currently very difficult to process (e.g., real-time computer vision, scene understanding and synthesis, image understanding) using today's technology. Because natural languages are used by people universally and allow rich representations that no other language specification can attain, natural language processing (NLP) research is a first step that Informatics should take in order to advance the organization and processing of individual knowledge in case-bases that can be reused. The insights gained will advance knowledge processing towards richer knowledge representation media, will reduce the knowledge processing gap and consequently increase the knowledge processing capacity currently supported largely by human knowledge processors . Memory-based knowledge processing One of the main features of information processing systems is their memory. It is accepted that storage and manipulation of information are necessary for complex cognitive activities in humans [ 43 ]. Memory is also considered crucial for both the "situation recognition" and mental modeling processes which are part of naturalistic decision models [ 33 ]. From a computational point of view, one could easily argue that without a random access memory structure there can be no effective processing. In the context of "the computational architecture of creativity," this argument is clearly outlined in [ 44 ]. It is based on the examination of the classes of computational devices, in the ascending order of their computational power, ranging from finite-state machines to pushdown automata and linear automata. These are paralleled by their corresponding grammars, arranged similarly in the Chomsky hierarchy, consisting of regular grammars, context-free grammars, context-sensitive grammars and of the unrestricted transformational grammars for machines with random access memory [ 44 ]. Recent natural language processing (NLP) research stresses the importance of memorization of individual natural language examples [ 45 ]. The importance of memory is also emphasized in earlier [ 46 ] and more recent models of language processing in humans [ 47 - 49 ]. These converge on the idea that natural language processing, regardless of the processor, is memory-based (i.e., case-based). Additional evidence comes from the fact that most language constructs (e.g. words, phrases) have very low frequencies. In fact, the very low frequency of most words in the English language (i.e., Zipf's law) is known from the 1940s since Zipf's famous book "Human Behavior and the Principle of Least Effort" [ 50 ] which is discussed in [ 51 ]. The main implication of "Zipf's law" is that purely statistical approaches or language processing algorithms that do not memorize training examples will either lose important information or may need extensive data (potentially impossible to collect) in order to be able to retain important features which have extremely low frequencies [ 52 ] and which may be crucial for construing the appropriate meanings of a language's concepts. The tradeoff between learning effort and communication efficiency seems to be biased naturally towards memorization rather than towards logical reasoning. The processing complexity of natural language might therefore not be an intrinsic quality of the algorithms, but rather a function of the memorization capabilities of the language processor, given the sparseness of natural language pattern space. By analogy, the advanced knowledge processing in humans might not be the result of very sophisticated reasoning strategies, but rather the utilization of a limited reasoning apparatus on a huge knowledge base, consisting of rich representations of one's experience. The limitations in reasoning are balanced by complex spatio-temporal pattern recognition capabilities operating on a case base built from years of experience. This case base includes common-sense knowledge. Furthermore, people and computers memorize information differently. Both have a short term, working memory and long-term memory for storing data and information. However, the memory access is carried out in different ways. Computers can reliably store large streams of data, which most of the times have a very well defined spatial and temporal structure (e.g., a movie clip). In contrast, people can only store information and knowledge rather than data and their storage is unreliable, temporally fragmented and spatially incomplete. Computers have very reliable memories capable of error checking at the bit level while the human memory supports only a high-level semantic consistency check. Finally, computers access their memory in a random seek fashion, being able to position their "reading heads" at any position in the data streams in order to extract a certain block of data. People can access their memory by content, by being provided with an incomplete description of a potentially complex, spatio-temporal pattern serving as a retrieval key. Therefore, one of the main differences between computers and humans is that computers have address-based random access memories, while humans possess content-addressable memories. In conclusion, from a case-based reasoning perspective, humans seem to be naturally endowed with the necessary structures for efficient case base acquisition, organization and retrieval while computers do not directly support this way of processing information and knowledge . Pattern recognition, comparison and analogy-making Pattern recognition is an undisputed feature of human cognitive abilities and a research area in its own right. However, it does not seem to be as pervasive as it should, in the information processing systems in current use. Natural language, as a product of human cognition, offers compelling evidence that people are naturally inclined toward processing information using pattern recognition and similarity principles. This evidence is supported by the widespread use of language devices such as the simile and the metaphor. These are examples of comparison and analogy making that humans perform without effort, in contrast to the difficulty of implementing them in the artificial information processing systems [ 53 ]. Analogy making is essential to generating new knowledge and new artifact designs [ 54 - 56 ], as well as to problem solving and inductive reasoning [ 57 , 58 ]. In a case-based reasoning context, the essential tasks of case matching and retrieval rely on pattern recognition, comparison and analogy making. In a decision making process, these mechanisms provide the immediate, implicit access to information about relevance stored in the contexts of similar instances of problems solving. The patterns and analogies that humans are able to handle are often represented by complex spatio-temporal events with a potentially multi-sensorial impact. For example, while humans have no difficulty in understanding a metaphor like "the computer swallowed the disk," an artificial information processing system that has no visual input sensors and which lacks the capability of image understanding, would probably never be able to perceive this particular analogy with the same speed, because of the extensive reasoning and amount of explicit knowledge needed to bring the swallowing process, as it occurs in living things, close to the action of inserting a disk into a computer's disk drive. In addition to operating on high dimensional, spatio-temporal complex patterns, analogy making in humans may also possess a dynamic component that could yield different relevance judgment outcomes, depending of context. A very illustrative example is given by French and Labiouse in [ 59 ], using the concept of a "claw hammer." According to its designed purpose, the "claw hammer" is semantically close to other concepts like "nail," "hit" and "pound." However, it may be dynamically "relocated" or reassigned in the semantic space, through a complex spatio-temporal mental simulation and analogy-making process, to the dynamically created class of "back-scratching devices," in the semantic neighborhood of the "itch," "scratch" and "claw" concepts. Similarly, one could think about the concept of a wooden decoy duck, which inherits properties from at least the "wooden object", "animal duck", "toy" and "hunting gear" classes. This concept may also be dynamically relocated into the semantic neighborhood of any of the classes, depending on the context of use that may be focused on themes such as "combustibles" or "hunting" for example. In the medical domain, the contextual dependence of relevance judgments, classifications and analogies is even more important, as these are often based on uncertain information and may be dynamically reevaluated in the light of new information about the patients or about their diseases. Polyhierarchy and multiple inheritance are indisputable desiderata of terminology systems [ 60 ]. However, building multiple inheritance mechanism using current technology seems very difficult, simply because the number of possible alternative classifications increases exponentially with the number of concepts. It is also very unlikely that this kind of taxonomic dynamicity (e.g., the claw hammer circumstantially classified as a back-scratching-device) of the human semantic space could work on such fixed conceptual structures which are constructed beforehand through learning, in human semantic memory. A more plausible hypothesis is that such ad-hoc classifications are circumstantially created using mechanisms that are closer to a distance calculation between high dimensional, distributed, vector representation of concepts. This is in agreement with neurolinguistic evidence from functional brain imaging studies of the human semantic memory. These studies suggest the existence of distributed feature networks for the representation of object concepts [ 61 ] and help the case for less structured approaches to capturing and representing semantics such as compositional terminology schemes (e.g. as in GALEN-GRAIL [ 62 ] and SNOMED-RT [ 63 ]), latent semantic indexing (LSA) [ 64 - 70 ] and connectionist models [ 49 , 71 , 72 ]. These approaches allow for a multidimensional semantic space where concept features can vary in importance, evolve or change dynamically, accounting for many possible classifications and subtle variations of concept meaning, including the new and the less plausible ones. This contrasts with the fixed or highly structured semantic representation schemas (e.g. fixed knowledge frames, semantic networks, ontologies), which fail to capture concept semantics in a way that provides richness, dynamicity and reusability. The dynamicity of concept meanings and relevance judgments may offer at least one of the reasons why fixed classification schemes, controlled terminology systems or open domain ontologies have not turned out satisfactory. It may also explain why existing lexical databases based on carefully handcrafted knowledge such as WordNet [ 73 ] often contain either too fine-grained or too coarse-grained, "static" semantic information [ 64 ]. In information intensive domains like medicine, concept dynamicity may account for why the development of a universal (i.e., one size fits all) clinical terminology system is so difficult [ 74 ]. From a case-based reasoning perspective, humans are naturally equipped with powerful pattern matching and classification capabilities which allow them to cope with complex, time-constrained relevance judgments, to easily construe meaning of concepts and to tolerate the ambiguity of natural language . Only relatively recently have computers come close to this functionality with the introduction of data mining and machine learning techniques such as self organizing maps and clustering algorithms based on similarity metrics [ 75 ]. In such machine learning approaches, the important problem of feature selection equates to a problems of relevance. CBR enabled EHR – Proposals, Challenges and Solutions Iatrogenic causes are said to be important causes of death in the US [ 76 ]. The reported incidence of adverse effects among patients in Canadian acute care hospitals is 7.5% [ 77 ]. A proposed means to counteract such medical errors is information technology, through the education and decision support offered to health care professionals. One very effective form of medical education is the retrospective analysis of case records where health professionals, both experienced or novices, learn from their own and from others' successes and failures [ 78 ]. Providing that legal and ethical implications such as provider and patient protection are dealt with appropriately, the efficacy of this teaching method can be improved if case records are continuously created, enriched, accumulated and organized on similarity principles. This is possible through a CBR approach of the EHR which, from this perspective, could serve as a comprehensive case base of managed patients that will evolve asymptotically towards an exhaustive knowledge base. Medical errors are also connected with the complex human cognitive task of planning [ 79 ]. CBR approaches, devised originally as a solution to automated planning tasks [ 80 ], have been since used in various applications including healthcare, legal and military (e.g., battle planning) [ 21 ]. This demonstrates a particularly good fit of a medical decision support based on CBR with its human users, the healthcare professionals. Providing that the privacy and confidentiality issues, which are even more stringent in this case, are dealt with appropriately, opening EHR to patients could benefit them [ 81 ]. It is perfectly conceivable that patients could learn from the history of other cases similar to theirs, which could be presented in an anonymized, story-telling format and organized on case similarity principles. It is also possible that patients may be willing to directly provide some of their own case information in order to be matched with previously managed cases, for example in the context of online chronic disease support groups. These principles are already realized in form of bulletin boards, mailing lists and forums, where actual patients interact with each other and occasionally with health professionals and exchange information regarding health related problems ([ 82 ] and [ 83 ]). The unstructured, textual exchange of information in such resources would ideally be moderated by knowledgeable individuals (e.g., providers). Although the automatic processing of text still is not readily available, case matching is possible so far and is performed by the very individuals who are able to offer useful information and knowledge to others, based on the similarity of their own experiences (i.e., their own story). Medicine has always and will always be a case oriented profession. Medical Informatics has recognized this early through the works of various researchers who pioneered the area of decision support systems [ 84 ]. Relevant to CBR work are also the attempts to enhance early decision support systems with domain knowledge from simulated patient cases [ 85 ]. Currently, the exploration of CBR in medical contexts is increasing [ 86 - 94 ]. Regardless of the problem nature, the most important components of a CBR expert system are • The case base, the memory of past problem-solving instances • The case matching or pattern matching procedure which retrieves the relevant cases for a certain problem While humans seem to possess a natural support for these two components, there is still work to be done in order to make the computer support this kind of knowledge acquisition and processing. We envision four important challenges in advancing towards CBR enabled EHRs: 1. Case record comprehensiveness 2. Organization on similarity and associative principles (associative memory) and development of advance data visualization techniques 3. Development of pattern recognition and similarity measures between heterogeneous records 4. Solving ethical issues and provision of privacy and confidentiality measures 1) Case record comprehensiveness EHR comprehensiveness is required because the exhaustiveness of a case base is not only a function of the number of records but also of the richness of each case record. Current knowledge processing technology limits the acquisition and especially the processing of comprehensive EHR records which incorporate structured data, images, video-clips, bio-signals, genomic data, unstructured textual data covering clinical findings, detailed patient history, etc. However, as knowledge processing technology advances and knowledge acquisition bottlenecks are overcome, it might be possible to overcome the heterogeneity and sparseness of EHR and allow the creation of representative case-bases and the organization of knowledge on principles that facilitate similarity based retrieval. Temporal knowledge is also a good example of a heterogeneously represented type of knowledge in the form of potentially non-interoperable standards for dates and times and temporal knowledge of various degrees of precision, embedded in knowledge facts such as "soon after receiving the drug, the patient developed a rash." Currently, for many people, the problem may seems to boil down to devising yet another standard which encompasses all the different temporal representations of dates, times and temporal concepts into a unified, common representation. From a knowledge engineering standpoint, and again currently for many researchers, this may equate to the creation of a comprehensive ontology of temporal knowledge. However, the problem of representing time starts to look like a somewhat limited version of another burning problem of Medical Informatics: that of medical terminologies. The fact that all these issues remain largely unsolved, can only help the case for CBR and for adaptive, empirical methods and approaches to knowledge processing. We believe that such approaches have the potential to cope and overcome the problems with redundant, possibly ambiguous representations, which have arbitrary degrees of precision. Thereby we are specifying a goal towards which the development of EHR should proceed. 2) Organization on similarity and associative principles (associative memory) and development of advanced data visualization techniques Similarity based retrieval is difficult with current database technology. For example, queries to retrieve cases which are similar to a textual description of a given case are difficult to answer. The comprehensiveness of EHR must be complemented with the possibility of indexing its records on similarity principles. Conceptually, the functionality of EHR will be that of an associative memory of cases that will enable the CBR paradigm. The organization of a case-base must be complemented by the development of advanced data visualization techniques that comply with the principles of organization of information by similarity. One example of such data visualization techniques are self-organizing maps [ 75 ]. These models are able to perform cluster analyses on high dimensional data sets and provide a visual display which can help with the navigation through and retrieval of similar cases. For instance, the self-organizing map obtained from the analysis of the Wisconsin Breast Cancer Dataset [ 95 ] used to cluster and classify cases based on their similarity in [ 96 ], could also be used for data visualization and navigation purposes, in a CBR context (Figure 9 ). It also demonstrates how high level abstractions (i.e., benign tumors forming the green cluster on the map) can be derived through an entirely automatic, data driven approach. 3) Development of pattern recognition and similarity measures between heterogeneous records CBR relies on the proper management of the case base and on appropriate mechanisms for matching and retrieval of these case records. All similarity retrieval mechanisms are based on some sort of distance calculation between the problem at hand and the records in the case base, followed by the retrieval of the most relevant ones. Clinical narratives and other EHR components containing unrestricted text represent a particularly difficult challenge for semantic similarity measures. The development of terminology systems based on less structured (e.g., latent semantic indexing, connectionist models) and data-driven approaches will provide the semantic richness, dynamicity and reusability needed for such complex tasks. A concrete example for the potential feasibility of such approaches, is the automated knowledge induction based on contextual similarity modeling ranging from morphological to sentential context [ 97 ] (Figure 10 ). An experimental knowledge processing system can induce automatically the new knowledge fact that Ayercillin , an item unknown to the system and hence not appearing in Figure 10 , is most likely to be a drug, precisely a penicillin. The decision is based on morphological (e.g., "-cillin"), semantic (e.g., six of the similar items are known to be drugs, precisely, penicillins) and pragmatic (e.g., the six, semantically similar items are consistent with the use in a medical context) similarities that help in filtering out the non-relevant information (e.g., book of common prayer). On the same basis, the system can also induce that surgical procedures ending in "-tomy" (e.g., perineotomy, valvulotomy, myringotomy, strabotomy) are usually incisions while those ending in "-ectomy" (e.g., myringectomy, tonsillectomy, splenectomy, nephrectomy) are usually removals, that concepts containing the morpheme "leuco" (e.g., leucocyte, leucothoe, platalea leucorodia) are usually associated with color white while those containing "eryth" (e.g., erythroblast, erythema, erythrina) with color red. However, despite such proof-of-concept applications and other progress in data mining and knowledge extraction from heterogeneous databases, case matching remains largely an open research question. 4) Solving ethical issues, provision of privacy and confidentiality measures We discuss this challenge last, not because it is less important but, on the contrary, because of its potential to become the most important obstacle to individual knowledge processing. The very fact that individual knowledge has the potential to contribute to solving future problems instances, raises the important ethical issue whether such knowledge should be made available to decision makers and researchers. Because the definition of individual knowledge implies the possibility to match it in time and space with an application context, i.e., with a patient, sharing individual knowledge is counterbalanced by the need for privacy and confidentiality. In addition, to further complicate matters, it may turn out that some of the most useful records for future instances of decision making are instances of medical errors or other unexpected events that are unique in their course of events and therefore easily identifiable together with their contexts of development (i.e., patients, providers, family members). The high complexity of individual knowledge renders explicit, manually controlled access to individual knowledge cases and their components unfeasible. The only solution to this problem seems to be of technological nature. Current privacy and confidentiality measures which include de-identification, de-nominalization and scrambling of the unique personal identifiers automated or semi-automated seem insufficient to counteract the potential to identify patients from unique, individual knowledge patterns. As a general approach, we propose that the accurate measuring of similarity of individual knowledge could form the basis of a confidentiality risk assessment. This could be intuitively understood by considering that: • very similar individual knowledge patterns which are in great numbers are a very low threat to the privacy and confidentiality infringement, and, at the other extreme, • stand-alone patterns which possess unique features or combinations of features, are at high risk of privacy and confidentiality breaches. In addition, the provision of privacy and confidentiality could be regarded as a special case of knowledge processing, which involves knowledge about the proper use (e.g., access, modification, transfer) of individual knowledge . This potentially complex, particular case of meta-knowledge processing could be implemented and managed using the principles of CBR paradigm itself, by building case-bases with examples of both proper and improper (simulated, not necessarily real) individual knowledge accesses and that can be compared with future access instances. Overcoming this very important challenge hinges on the possibility to effectively measure the similarity between heterogeneous records and on the advancement of knowledge processing on CBR principles. If successful, CBR research might therefore fulfill a longstanding need for intelligent information processing and advance informatics towards the ideal of individual knowledge processing. This calls for further investigation of information processing models that are, similarly to human experts, capable to efficiently move across the knowledge spectrum. One class of such models is represented by artificial neural networks [ 14 , 23 ], which are highly adaptive information processing models able to create high-level abstractions from raw data, completely automatically [ 75 ] and "learn by themselves" new information processing functions from data. From this perspective, Informatics aligns closely to the goals of AI to create intelligent machines that can hear, see, speak, think, adapt and make decisions. Conclusions CBR provides potential solutions to important problems that, among other, stymy the usefulness of EHR. The natural integration of learning with reasoning and the CBR resemblance to the cognitive models of human decision-making hold the promise to overcome the "brittleness" and "knowledge acquisition bottleneck" of classical expert systems. The CBR applications to the medical field have the potential to offering the training and decision support needed by health professionals and the means towards a true patient-centered healthcare. With a CBR theoretical foundation still in its infancy and with limited medical applications in existence, more research is needed for providing proofs of the feasibility of practical CBR-EHR applications. Challenges in the way to accomplish this include the increasing complexity, ethical issues as well as the paradigm shift that our current computing devices must undergo in order to support the CBR principles of knowledge processing. Summary 1. Science is twofold and is driven by two opposite forces: that of creating theories (theoretical sciences), and that of applying theories to practical applications (applied sciences). Medical Informatics is fundamentally an applied science that should be committed to advancing patient-centred medicine through individual knowledge processing. 2. Case-based reasoning is the technical solution that enables a continuous individual knowledge processing that could be integrated with the Electronic Health Records. 3. Medicine is an information and knowledge intensive domain where time-constrained decision problems can only be solved effectively based on the recollection of similar problems and their solutions (i.e., a case-based reasoning strategy). The collective sharing of experiences is important for making future decisions as well as for learning how to make decisions. 4. Unlike computers, human decision makers possess the components necessary to perform case-based reasoning naturally (i.e., a content addressable memory to organize a case base efficiently by similarity principles, as well as the capability to perform pattern recognition, comparison, and analogy-making). 5. Applying the CBR approach to EHR might be a way to overcome the important obstacles of EHR acceptance and use, providing that technical challenges and ethical issues arising are addressed appropriately. List of abbreviations AI Artificial intelligence AIT Algorithmic Information Theory CBR Case Based Reasoning E Explicit knowledge EHR Electronic Health Records G General knowledge LSA Latent Semantic Indexing I Individual knowledge NDM Naturalistic Decision Making RCT Randomized Controlled Trial RPD Recognition-Primed Decision U Implicit (Unobvious) knowledge Competing interests The author(s) declare that they have no competing interests. Authors' contributions Before the reviews SP researched the paper and provided a first draft. JA, JM critically revised the manuscript three times each and provided their own additions to the initial draft. JA provided more feedback on the cognitive aspects and decision-making as well as writing style and missing references. JM additions were with regard to the writing style, clarity, missing references and the overall organization of the paper. After the reviews SP and JM worked on the responses to reviewers' comments. SP wrote a first revision of the paper. JM provided extensive feedback as well as new references and suggested a major revision that includes recent ideas. JA also commented and made suggestions on the knowledge spectrum model and on the meta-level view on Medical Informatics. SP overhauled the entire paper. JM revised the new version and provided feedback. SP operated the changes and proposed new modifications. JM revised the second draft. JA provided feedback on the second draft of the paper with regard to fundamental aspects of knowledge modalities. SP and JM incorporated the minor changes suggested by the last review. All authors read and approved the final version of the paper. Pre-publication history The pre-publication history for this paper can be accessed here:

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The diagnostic value of endoscopy and Helicobacter pylori tests for peptic ulcer patients in late post-treatment setting

Background Guidelines for management of peptic ulcer patients after the treatment are largely directed to detection of H. pylori infection using only non-invasive tests. We compared the diagnostic value of non-invasive and endoscopy based H. pylori tests in a late post-treatment setting. Methods Altogether 34 patients with dyspeptic complaints were referred for gastroscopy 5 years after the treatment of peptic ulcer using a one-week triple therapy scheme. The endoscopic and histologic findings were evaluated according to the Sydney classification. Bacteriological, PCR and cytological investigations and 13 C-UBT tests were performed. Results Seventeen patients were defined H. pylori positive by 13 C-UBT test, PCR and histological examination. On endoscopy, peptic ulcer persisted in 4 H. pylori positive cases. Among the 6 cases with erosions of the gastric mucosa, only two patients were H. pylori positive. Mucosal atrophy and intestinal metaplasia were revealed both in the H. pylori positive and H. pylori negative cases. Bacteriological examination revealed three clarithromycin resistant H. pylori strains. Cytology failed to prove validity for diagnosing H. pylori in a post-treatment setting. Conclusions In a late post-treatment setting, patients with dyspepsia should not be monitored only by non-invasive investigation methods; it is also justified to use the classical histological evaluation of H. pylori colonisation, PCR and bacteriology as they have shown good concordance with 13 C-UBT . Moreover, endoscopy and histological investigation of a gastric biopsy have proved to be the methods with an additional diagnostic value, providing the physician with information about inflammatory, atrophic and metaplastic lesions of the stomach in dyspeptic H. pylori positive and negative patients. Bacteriological methods are suggested for detecting the putative antimicrobial resistance of H. pylori , aimed at successful eradication of infection in persistent peptic ulcer cases.

Background Treatment of peptic ulcer in accordance with relevant guidelines is becoming a common task for general practitioners [ 1 - 6 ]. In a post-treatment setting, in accordance with guidelines, prompt check-up of treatment results is recommended only in gastric ulcer cases with the use of 13 C-urea breath test ( 13 C-UBT ) [ 2 - 5 ]. In a situation where patients have clinical symptoms after H. pylori eradication therapy, endoscopy is favoured in all peptic ulcer cases [ 6 ]. The aim of endoscopy is to establish the reason for clinical symptoms and to prove presence of peptic ulcer or malignancies, but also to support physicians and patients in the understanding of complaints [ 7 ]. Moreover, endoscopy allows determination of persistent H. pylori infection using endoscopy-based tests. Endoscopic biopsies alone are not considered adequate for confirming eradication of bacteria, although they might provide additional information about gastritis and dysplasia [ 8 ]. Use of more than one method in testing gastric specimens definitely enhances the diagnostic value when assessing the post-treatment H. pylori status [ 9 ]. Our aim was to assess the diagnostic value of different non-invasive ( 13 C-UBT ) and endoscopy-based diagnostic methods (visual endoscopy, classical cytological and histological examination of mucosal specimens, PCR and bacteriological methods) for monitoring patients after eradication therapy in a late post-treatment setting. Methods Patients The study group was formed of 134 consecutive peptic ulcer outpatients who had been treated by 7-day triple therapy with metronidazole, amoxicillin and omeprazole in 1996. The group was observed at the outpatient department of Tartu University Hospital at 4 weeks, at 1 year (1997) and at 5 (2001) years after treatment [ 10 ]. Five years after treatment, 108 patients (81% of the initial group) were available for the follow-up of the clinical course of peptic ulcer. During the 5-year follow-up period only 11 (10 %) patients had relapses of peptic ulcer. For comparison of the diagnostic value of different diagnostic methods in a post-treatment setting, 34 patients were recruited. The inclusion criteria for this study group were resistant upper abdominal pain as the predominant complaint and compliance with all investigations (clinical symptoms, 13 C-UBT , endoscopy, biopsy, bacteriology, PCR and cytology). The studied patients were not NSAID users. Methods The patients passed the Gastrointestinal Symptoms Rating Scale (GSRS) test [ 11 ] in a validated Estonian translation. Dyspeptic syndrome (abdominal pain, heartburn, acid regurgitation, sucking sensation, nausea and vomiting) was registered on the 7-grade Likert scale for assessing severity of symptoms. The mean score of dyspeptic syndrome was calculated for each patient. 13 C-UBT The subjects passed 13 C-UBT drinking 100 mg 13 C -urea; the test meal was citric acid and the time of specimen collection was 30 min. The test was provided, according to a standard protocol, from the Helsinki Keskuskatu Laboratory, Finland. The ratio of 13 CO 2 to 12 CO 2 in expired breath was measured by mass spectrometry and expressed in ml/mmol/kg (δ). An automated breath 13 C analyser (ABCA) with chromatographic purification and a single inlet isotope ratio mass spectrometer (IRMS) were used. A difference of 5‰ in the content (δ 13 C ) was considered positive for H. pylori infection. Endoscopy of the upper gastrointestinal tract The procedure was performed with the gastroscope Olympus-GIF 21. All mucosal defects were registered according to the Sydney classification for endoscopic evaluation [ 12 ]. Gastric ulcer was diagnosed if the ulcer was located at the angulus or above it. Duodenal ulcer was diagnosed if the ulcer was found in the duodenal bulb area. Gastrobiopsy and histological examination Five specimens from the antrum mucosa and five from the corpus mucosa were taken with medium-sized forceps. Two specimens were embedded in paraffin and the paraffin sections were stained using haematoxylin-eosin and Giemsa methods. The mucosal specimens were evaluated histologically according to the Sydney classification: presence of neutrophil infiltration, chronic lymphocytic inflammation, surface epithelial damage, atrophy, intestinal metaplasia, lymphoid follicles and H. pylori colonisation were evaluated on a three-grade scale both for the antrum and the corpus [ 12 - 14 ]. Bacteriological examination One specimen from the antrum and one from the corpus were placed in the Stuart Transport Medium (Oxoid) and taken to the laboratory within two hours for bacteriological examination. The biopsy samples were homogenised with sterile glass powder and under a stream of CO 2 and diluted in the Brucella broth (Oxoid). H. pylori was isolated on the Columbia Agar Base supplemented with 7% horse blood and 1% Vitox (Oxoid) or Isovitalex (BBL). The plates were incubated for 3–7 days at 37°C under microaerobic conditions (CampyBak, BBL or CampyGen, Oxoid). H. pylori was identified by Gram staining and by oxidase, catalase and urease reactions [ 15 ]. The sensitivity of the isolated H. pylori strains to clarithromycin was estimated by E-test. The antibiotic cut-off points employed for the E-test were 1.0 mg/l (NCCLS, 2002). Cytological examination One specimen was used for imprinting the cytology slides from the antrum and corpus mucosa, fixed with 96% ethanol and stained by Acridine Orange (Difco, BBL) [ 16 ]. The cytological specimens were studied under a fluorescence microscope (AXI Phot 2) where the morphotypes and the density of bacterial colonisation were evaluated [ 17 ]. A positive cytological diagnosis was based on the presence of typical helical H. pylori cells on the gastric mucosa and in the mucus layer. PCR For DNA extraction of H. pylori from a frozen gastric biopsy specimen, a previously described procedure was used [ 18 ]. The presence of the glmM gene in each strain was established by PCR using primers, the reaction mixture, and thermal cycling [ 19 , 20 ]. DNA from H. pylori NCTC 11637 (National Collection of Type Cultures, Central Public Health Laboratory, Colindale Ave., London NW9 5HT, England, United Kingdom) and the DNA-free reaction mixture were assayed in separate tubes in each PCR and were run as the positive and negative controls of the reaction, respectively. The PCR products were identified by electrophoresis on 2% agarose gels. Criteria for evaluation H. pylori was assessed positive if at least two tests were positive according to golden standard [ 21 ]. Statistical analysis The data were analysed by Fisher's exact tests using the Jandel SigmaStat 2.0 program. Measurements from the GSRS were expressed as the mean values for dyspeptic syndrome. Ethics The study was carried out in accordance with the Helsinki Declaration and was approved by the Ethics Committee of the University of Tartu. Results Dyspeptic syndrome was found in all 34 cases. The mean GSRS score for the patients varied from 1.2 to 4.3. The applied non-invasive test revealed H. pylori infection in half of the investigated patients: positive 13 C-UBT was found in 17 out of 34 cases. There was no difference between the mean GSRS score values for the H. pylori positive and negative cases (2.8 ± 1.8 vs. 2.9 ± 1.7, p > 0.05). On endoscopy, among the 34 patients, no ulcer or other mucosal defects were observed in 24 cases; erosions in the duodenal bulb were revealed in 6 cases and peptic ulcer was found in 4 cases (2 duodenal ulcers and 2 gastric ulcers). The data of H. pylori status and of the endoscopic finding are presented in Table 1 . Table 1 Comparison of the findings in H. pylori positive and negative cases in a late post-treatment setting Patients (n = 34) Non-invasive method 13 C-UBT (+) n = 17 13 C-UBT (-) n = 17 Invasive methods Endoscopy: Normal 11 13 Duodenal ulcer 2 0 Gastric ulcer 2 0 Erosions 2 4 Cytology: H. pylori (+) 4* Diverse forms of bacteria Histology: H. pylori (+) 17 0 Bacteriology: H. pylori (+) 16 1 PCR: H. pylori (+) 17 0 * typical morphology of H. pylori (the other cases showing diverse forms of bacteria) A poor concordance was found between the visual examination of the gastric and duodenal mucosa on endoscopy and the applied non-invasive and invasive tests of H. pylori (accepting 13 C-UBT , histological examination and PCR as the reference tests). The gastric and duodenal mucosa was visually normal in 11 H. pylori positive cases out of 17. On the contrary, only in 4 H. pylori positive cases did the endoscopic examination reveal the above mentioned peptic ulcers. Among the 6 cases with erosions of the duodenal mucosa, only two patients were H. pylori positive. Comparison of the different diagnostic methods used for the detection of H. pylori is shown in Table 1 . The results of 13 C-UBT and PCR were consistent with the data of histological examination both in 17 H. pylori positive and 17 negative cases. On bacteriological examination, only one case, which was H. pylori positive both by PCR and the histological tests, was H. pylori negative. In contrast, cytological examination assessed typical H. pylori bacterial cells in only 4 of the 17 H. pylori positive cases (24%), while all other cases (both positive and negative for H. pylori by the other methods) displayed abundant bacteria of different morphotypes. The data of the histological findings are presented in Table 2 . Colonisation of the gastric mucosa by H. pylori was detected in 17 patients out of 34. Neutrophil infiltration, chronic inflammation, and surface epithelial damage both in the antrum and corpus mucosa were significantly expressed in the H. pylori positive cases (p < 0.001). Glandular atrophy and intestinal metaplasia were rarely observed both in the antrum and corpus mucosa of the H. pylori negative cases in comparison with the H. pylori positive cases, but the difference was not statistically significant (p > 0.05). Lymphoid follicles were more frequent in the antrum colonised with H. pylori (p < 0.05). Table 2 Gastric mucosal findings (by the Sydney system) in H. pylori positive and negative cases Gastric mucosal findings (Sydney system) H. pylori (+) n = 17 H. pylori (-) n = 17 p values Activity of neutrophil polymorphs Antrum 11/17 0/17 <0.001 Corpus 7/16 0/17 <0.05 Chronic inflammation Antrum 16/17 1/17 <0.001 Corpus 13/16 0/17 <0.001 Surface epithelial damage Antrum 13/17 0/17 <0.001 Corpus 8/16 0/17 <0.001 Glandular atrophy Antrum 7/17 2/17 NS* Corpus 4/16 3/17 NS Intestinal metaplasia Antrum 1/17 2/17 NS Corpus 0/16 2/17 NS Lymphoid follicles Antrum 6/17 0/17 <0.05 Corpus 5/16 2/17 NS * NS, not significant (p > 0.05). Bacteriological investigation revealed H. pylori in 16 biopsy samples of the antral mucosa, while highly (> 256 mg/l) clarithromycin resistant H. pylori strains were found in 3 cases. Discussion Proper diagnostic and therapeutic management of patients with dyspeptic syndrome after H. pylori eradication therapy is of utmost importance for physicians as well for patients [ 7 ]. Several studies [ 22 , 23 ] have demonstrated the reliability of H. pylori tests used before treatment, while post-treatment testing is not yet adequately studied. However, in the case of long-lasting recurrent dyspepsia after H. pylori eradication therapy, endoscopy has been strongly recommended [ 4 ]. Our study shows that endoscopy gives useful information for the general practitioner both in the cases where peptic ulcer is found and in the cases where it is not found. In the case of a normal endoscopic finding, further management depends on the histological finding and on H. pylori status. Since persistent H. pylori positivity is always associated with possible peptic ulcer recurrence, the second line treatment according to bacterial susceptibility should be recommended. In the remaining cases where H. pylori is absent, the gastric mucosa is normal and no ulcer is detected, management of such patients should be aimed at establishment of other possible reasons for their complaints. Usually, a normal endoscopic finding reassures both the doctor and the patient [ 7 ]. A recent study of Ohkusa et al. [ 24 ] showed that even simple careful visual evaluation of the mucosa and the diagnoses of erythema and oedema correlated well with H. pylori infection. On the contrary, our results demonstrate that although all patients with recurrent peptic ulcer were H. pylori positive, the minor visual findings in the other cases were not in concordance with H. pylori colonisation. Usually, the mucosa was visually normal even when H. pylori was found, and, on the contrary, most duodenal erosions occurred in H. pylori negative patients. The clinical data of our patients did not suggest earlier use of NSAID, which would have been one of the main reasons for H. pylori negative erosions. Therefore, after treatment, in presence of complaints, it is important to obtain samples for the investigation of gastric mucosa specimens to enhance the value of endoscopic examination. We completely agree with the opinion that the value of using mucosal specimens for histological evaluation of late post-treatment H. pylori eradication is sometimes underestimated [ 9 ]. The non-invasive H. pylori test alone cannot solve the clinical problem of these patients. In our study, H. pylori negative patients had dyspeptic syndrome as well as gastric mucosal erosions, glandular atrophy and intestinal metaplasia. The last two lesions can presumably be associated with previous H. pylori infection and the follow-up of severe mucosal changes is recommended [ 25 ]. Hence it is evident that follow-up strategy should be considered also in H. pylori negative cases in accordance with endoscopic and histological findings. Our study demonstrates that evaluation of the gastric mucosa with a focus on neutrophil and lymphocyte infiltration and epithelial damage is specific and sensitive for diagnosing H. pylori infection even after treatment, and that the diagnostic value of a histology-based decision is high. Today, the value of mucosal specimens for the post-treatment histological diagnosis of H. pylori is considered low assuming that H. pylori colonisation may be patchy, or coccoid forms are difficult to detect [ 25 ]. We have excluded patchy damage by using 13 C-UBT in parallel with histological investigation. Next, for detecting the coccoid forms of the bacteria, we used additionally PCR method. Our results show that the histological finding of H. pylori completely correlates with the results of 13 C-UBT and PCR both in H. pylori positive and negative cases. This confirms the validity of the histological evaluation of mucosal specimens in the case of recurrent peptic ulcer or erosions. Moreover, in countries with a high rate of H. pylori infection and gastric cancer, it is especially important to follow up patients for detecting dysplasia and malignancies [ 26 - 29 ]. Surprisingly, brush cytology from the mucosa failed to detect H. pylori in cases where it was found by other methods. Cytology is highly evaluated for detection of H. pylori infection, as its agreement with histology is considered to be 100% [ 30 ]. Our results show that when patients had been treated with antibacterial drugs and still had dyspeptic complaints, cytological examination was not suitable for H. pylori detection, as different forms of the bacteria were found. The morphology of the helicobacters could have been modified for coccoid or otherwise non-typical forms. It is possible that some other bacteria might have colonised the mucosa due to reduced colonisation resistance after antibacterial treatment, failure of some intestinal functions or usage of medicines administered to relieve the feeling of discomfort [ 31 - 33 ]. Bacteriological investigation enabled to find a few clarithromycin resistant H. pylori strains, which may result in the failure of repeat triple therapy. As the macrolide clarithromycin is chemically stable and well tolerated [ 34 ], physicians often choose it for treatment of different infections. Therefore, if the physician plans to use macrolides, endoscopy and histological testing should be accompanied by bacteriological investigation. Regarding PCR, its main value, obtaining of fast results, is evidently not so important in post-treatment settings. Conclusions In a late post-treatment setting, patients with dyspepsia should not be monitored only by non-invasive investigation methods; it is also justified to use the classical histological evaluation of H. pylori colonisation, PCR and bacteriology as they have shown good concordance with 13 C-UBT . Moreover, endoscopy and histological investigation of a gastric biopsy have proved to be the methods with an additional diagnostic value, providing the physician with information about inflammatory, atrophic and metaplastic lesions of the stomach in dyspeptic H. pylori positive and negative patients. Bacteriological methods are suggested for detecting the putative antimicrobial resistance of H. pylori , aimed at successful eradication of infection in persistent peptic ulcer cases. Competing interests The author(s) declare that they have no competing interests. Authors' contribution HIM, IK and KLa carried out GSRS, endoscopy and gastrobiopsy. HK recruited patients, collected 13 C-urea breath tests, and performed GSRS. KLõ carried out bacteriological examination. PH performed cytological examination. HA carried out molecular analysis and participated in the writing of the manuscript. HIM performed histological examination and statistical analysis, and participated in the design of the study and in the writing of the manuscript. MM coordinated the study and participated in the completion of the manuscript. All authors have read and approved the final version of the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC529255.xml

529269

Detecting imbalanced expression of SNP alleles by minisequencing on microarrays

Background Each of the human genes or transcriptional units is likely to contain single nucleotide polymorphisms that may give rise to sequence variation between individuals and tissues on the level of RNA. Based on recent studies, differential expression of the two alleles of heterozygous coding single nucleotide polymorphisms (SNPs) may be frequent for human genes. Methods with high accuracy to be used in a high throughput setting are needed for systematic surveys of expressed sequence variation. In this study we evaluated two formats of multiplexed, microarray based minisequencing for quantitative detection of imbalanced expression of SNP alleles. We used a panel of ten SNPs located in five genes known to be expressed in two endothelial cell lines as our model system. Results The accuracy and sensitivity of quantitative detection of allelic imbalance was assessed for each SNP by constructing regression lines using a dilution series of mixed samples from individuals of different genotype. Accurate quantification of SNP alleles by both assay formats was evidenced for by R 2 values > 0.95 for the majority of the regression lines. According to a two sample t-test, we were able to distinguish 1–9% of a minority SNP allele from a homozygous genotype, with larger variation between SNPs than between assay formats. Six of the SNPs, heterozygous in either of the two cell lines, were genotyped in RNA extracted from the endothelial cells. The coefficient of variation between the fluorescent signals from five parallel reactions was similar for cDNA and genomic DNA. The fluorescence signal intensity ratios measured in the cDNA samples were compared to those in genomic DNA to determine the relative expression levels of the two alleles of each SNP. Four of the six SNPs tested displayed a higher than 1.4-fold difference in allelic ratios between cDNA and genomic DNA. The results were verified by allele-specific oligonucleotide hybridisation and minisequencing in a microtiter plate format. Conclusions We conclude that microarray based minisequencing is an accurate and accessible tool for multiplexed screening for imbalanced allelic expression in multiple samples and tissues in parallel.

Background Single nucleotide polymorphisms (SNPs) are highly abundant in the human genome, appearing on average at 0.1% of the nucleotide positions [ 1 ]. Thus, each gene or transcriptional unit will contain multiple SNPs that potentially give rise to sequence variation between individuals and tissues on the level of RNA. Recent studies indicate that differences in the expression levels of the alleles of heterozygous SNPs may occur frequently for human genes [ 2 - 6 ]. Imbalanced allelic expression was detected in foetal liver or kidney tissues for more than half of 602 genes analysed, and one third of the genes displayed more than four-fold differences in allelic expression [ 3 ]. Another study detected lower levels of allelic imbalance for one fifth of 129 genes analysed in lymphoblastoid cell lines [ 4 ]. Non-synonymous SNPs in coding regions of genes may be functional by altering an amino acid, which in turn may affect the structure and function of the encoded protein, while synonymous SNPs may have functional consequences by affecting the stability or folding of mRNA transcripts. Intronic SNPs may give rise to alternatively spliced mRNAs, while SNPs in 5'- or 3'-untranslated mRNA regions may affect the stability or processing of the RNA. Moreover, SNPs in non-protein coding regions of genes that affect binding of regulatory factors may cause imbalanced expression of SNP alleles. This form of genetic variation has been suggested as a common cause of both normal and disease-related inter-individual variation in complex phenotypes [ 7 ]. Clearly, methods with high accuracy that can be used in a high throughput setting are needed for systematic surveys of expressed sequence variation and its molecular causes. Owing to the high sequence specificity of nucleotide incorporation by DNA-polymerases, single nucleotide primer extension has proven to allow quantitative determination of SNPs in genomic DNA in several studies and assay formats (for a review, see Syvänen 2001 [ 8 ]). A frequently used quantitative application of the method is to determine SNP allele frequencies in pooled DNA samples [ 9 - 13 ]. The rationale for detecting imbalanced expression of the two alleles of a heterozygous SNP by minisequencing is to measure the ratio between the amounts of labelled nucleotides incorporated in the minisequencing reactions for the two SNP alleles in RNA (cDNA) samples from the tissue of interest. These ratios are then compared to the corresponding ratio measured in genomic DNA, where the two alleles are present in an equimolar ratio [ 2 , 4 , 14 - 16 ]. Imbalanced expression of the alleles of a SNP is revealed by a difference in the ratios measured in the RNA and DNA samples. We are currently using microarray based minisequencing for multiplex genotyping of SNPs. Our custom-made microarrays permit the genotyping of up to 100 SNPs in 80 samples per standard microscope slide, either using immobilised minisequencing primers [ 17 , 18 ] or using a "tag-array" format [ 19 , 20 ] of the method [ 13 ]. The purpose of this study was to evaluate the performance of these two microarray formats in quantitative determination of SNP alleles on the RNA level as alternatives with higher multiplexing capacity than previously used primer extension methods in which the SNPs are analysed in individual reactions. Using these systems, we were able to detect significant differences in the amounts of the two alleles of heterozygous SNPs on the RNA level. Results We used a panel of ten coding SNPs in five genes to choose the optimal microarray based minisequencing strategy for multiplex, quantitative genotyping of SNPs in DNA and RNA samples. The selected SNPs were located in genes shown by reverse transcriptase PCR analysis to be expressed in one or both of two endothelial cells lines, HUVEC (human umbilical vein endothelial cells) and HAEC (human aortic endothelial cells) that served as our model cell lines in this study (data not shown). We evaluated two formats of microarray based minisequencing by performing five parallel assays with each method for each sample in the evaluation. The SNPs were analysed in both DNA polarities and the evaluation of the methods was based on the DNA polarity yielding the highest signal-to-noise ratio. In Method I, immobilised minisequencing primers are extended with fluorescently labelled ddNTPs in reactions performed on the microarray surface after annealing of the multiplex PCR products to the primers [ 18 , 21 ]. In Method II, cyclic primer extension reactions are performed in solution in the presence of 5'-tagged minisequencing primers, PCR products and fluorescent ddNTPs [ 22 , 23 ]. After the cyclic reactions the extended primers are captured on a microarray surface carrying immobilised oligonucleotides complementary to the 5'-tag sequences on the minisequencing primers. Both these systems are performed in an "array-of arrays" format developed previously in our laboratory [ 24 ]. We analysed a dilution series with mixtures of DNA from two individuals with different genotypes for the panel of ten SNPs in both DNA polarities. The genotyping results from these mixtures of known amounts of the two SNP alleles are expressed as the signal ratio between the fluorescence signals corresponding to the two alleles of each SNP. The quantitative analysis of these ten SNPs is illustrated in Figure 1 by regression lines, in which the mean signal intensity ratios are plotted as a function of the known allelic ratios in the mixed samples. The coefficient of determination (R 2 ), which describes how well the regression line fits the data points, was used to assess the accuracy of quantification of the SNP alleles by Methods I and II. As can be seen in Table 2 , the R 2 values are close to one for most of the SNPs analysed, demonstrating little scatter of the data points around the regression line. For Method I, six of the ten SNPs analysed have R 2 values ≥ 0.95, while for Method II the R 2 values are ≥ 0.95 for eight of the SNPs. Thus, accurate quantification of SNP alleles is possible by both methods. The slopes of the regression lines vary between the ten SNPs as well as between the two methods (Figure 1 ). A regression line with a steep slope usually corresponds to a high R 2 value, as observed for the SNP rs5930 LDLR analysed by Method I and SNP rs5331 EDNRB analysed by Method II. A flat slope does not necessarily imply less accurate quantification, as exemplified by the SNP rs4331 ACE, where Method II yielded a flat slope with a higher R 2 value than Method I. We also determined the sensitivity of the methods for detection of a minority allele. The detection limit was defined as the percentage of the minority allele in the mixed sample, for which the signal ratio differed from the signal ratio in the corresponding homozygous sample with a p-value < 0.05 in a two sample t-test. Depending on the genotype of the DNA samples used for the dilution series, determination of the lower limit of detection was possible for seven of the ten SNPs with allele ranges 0–50% or 0–100% in the mixed samples (Table 1 ). For the remaining three SNPs with the allele range 50–100%, the smallest percentage of an allele that could be distinguished from a heterozygous genotype was identified by the same approach. Using Method I, we were able to detect less than 5% of the minority allele for two SNPs (rs1042713 ADRB2 and rs5925 LDLR) and less than 9% for rs4331 ACE, rs1042719 ADRB2, rs5351 EDNRB and rs5930 LDLR (Table 2 ). Method II allowed more sensitive detection of minority alleles than Method I. Less than 2% was detectable for the SNPs rs1042713 ADRB2, rs1042719 ADRB2 and rs5351 EDNRB, and less than 9% was detectable for the SNPs rs4331 ACE, rs5925 LDLR, rs5930 LDLR and rs1433099 LDLR (Table 2 ). For the SNPs rs1042714 ADRB2, rs1042718 ADRB2 and rs1799983 NOS3, we were able to measure 4–14% deviations from the heterozygous genotype (Table 2 ). These results show that the amount of SNP alleles can be accurately determined on the DNA level by Methods I and II using reference samples with the two SNP alleles present in known ratios. Next, the performance of the two methods in quantitative analysis on the RNA level was assessed. The ten SNPs were first genotyped in genomic DNA (gDNA) from the HUVEC and HAEC cells to identify those SNPs that were heterozygous in either or both cell lines. Three SNPs in the low density lipoprotein receptor gene (LDLR; rs5925, rs5930 and rs1433099) were heterozygous in the HAEC cell line, and one SNP in each of the genes encoding angiotensin I converting enzyme (ACE rs4331), β 2 -adrenergic receptor (ADRB2 rs1042719) and endothelin receptor type B (EDNRB rs5351) were heterozygous in the HUVEC cell line. These SNPs were genotyped in cDNA produced from total RNA extracted from the cells with the corresponding gDNA as reference samples using both methods. Table 3 presents the mean fluorescence signals with coefficients of variation (CV) obtained in five parallel reactions for the six SNPs in cDNA and gDNA from the HUVEC and HAEC cells. For the heterozygous SNPs the largest difference in the variability between parallel reactions was observed between SNPs, with the lowest CV values (3.6 – 8.6 %) for the rs1042719 ADRB2 SNP, and the highest CV values (13 – 41%) for the rs1433099 LDLR SNP. No systematic differences in the variability of parallel reactions were observed between Method I and Method II, or between cDNA and gDNA. Table 4 shows the differences in mean signal intensity ratios between the cDNA and gDNA assays for the six SNPs that were heterozygous in HUVEC or HAEC cells, respectively, together with the corresponding normalized cDNA/gDNA ratios. The SNPs in the ACE, ADRB2 and EDNRB genes displayed significant imbalanced expression in the HUVEC cells using both methods. For the SNP rs4331 ACE, the signal intensity ratio based on the raw data obtained by Methods I and II differed from each other, but despite this large difference, both methods yielded similar levels of allelic imbalance for this SNP after normalisation against the signal ratio in gDNA (Table 4 ). Only for one of the three LDLR SNPs (rs5930), the difference in fluorescence intensity ratios between cDNA and gDNA from HAEC cells reached statistical significance by both methods. Allelic imbalance of the LDLR gene was detected for the LDLR SNP rs5925 using Method II only. To test that the results on imbalanced allelic expression detected by the multiplexed microarray based methods represents the true biological situation in the cells, we analysed the heterozygous SNPs in five replicate RNA samples prepared from HUVEC or HAEC harvested at different time points from different cell culture flasks. We also analysed the three LDLR SNPs in five replicate reverse transcription reactions from the same RNA sample prepared from HAEC cells. For this analysis we used our first generation solid-phase minisequencing assay for individual SNPs in a microtiter plate format. The concordant cDNA/gDNA ratios from these control experiments from independent cell and RNA samples presented in Table 5 show that the detected allelic imbalance was not caused by the procedures for RNA extraction or cDNA synthesis. Finally, we verified the results obtained by microarray-based minisequencing for three of the SNPs by real-time PCR with allele specific hybridization probes (TaqMan). Table 4 shows these results together with the corresponding results by solid-phase minisequencing in a microtiter plate format. Allelic imbalance was detected with statistical significance for the SNP rs1042719 ADRB2 and the SNP rs1433099 LDLR by both methods. Particularly for the SNP rs1042719 ADRB2, the cDNA/gDNA ratios obtained by the two reference methods were highly similar to the results from the microarray-based methods presented in Table 4 , as well as with each other. As for the microarray-based Method II, the difference in signal ratios between cDNA and gDNA measured by the TaqMan assay for the SNP rs5925 LDLR did not reach statistical significance due to large variation between parallel assays. Analysis of the SNP rs1433099 LDLR by the reference methods confirms the imbalanced expression of the LDLR receptor alleles. Discussion The purpose of our study was to evaluate microarray based minisequencing for multiplexed detection and quantification of imbalanced expression of SNP alleles, as a prelude to further large scale screening for allelic imbalance. We found no significant differences in the performance of our two "in house" methods, minisequencing with primers directly immobilised on the microarrays (Method I)[ 18 ] and the "tag-array" format, based on cyclic minisequencing followed by capture on microarrays using immobilised complementary "tag" probes (Method II) [ 23 ]. Both methods showed a linear relationship between SNP allele ratios and the signal intensity measured in the four-colour fluorescence minisequencing assay for all SNPs. With respect to accuracy assessed by coefficients of variation (CV) between five parallel assays both methods performed equally well, and the CV values between parallel assays were indistinguishable between genomic DNA and reverse transcribed cDNA samples. The sensitivity of detecting a SNP allele present as a minority in a sample was defined as the percentage for which the signal ratio differed from the signal ratio in the corresponding homozygous sample with a p-value < 0.05 in a two sample t-test. The sensitivity differed between SNPs, and range from 1% to 9%, with a trend to be slightly better using the "tag-array" system (Method II). In several cases the p-values were lower than 0.05 (Table 2 ), which indicates that in practice the sensitivity of detection would be lower than the stringent limit set here. The sensitivity of our multiplex microarray based minisequencing methods compares well with the sensitivity of other single nucleotide primer extension assays performed for individual SNPs in recent studies [ 4 , 25 - 27 ]. It is notable that the largest differences in accuracy and sensitivity were observed between SNPs. Some of the SNP-to-SNP differences are likely due to differences is the accuracy and efficiency of incorporation of the four different fluorescently labelled nucleotide analogues by the DNA polymerase [ 13 , 26 ] as well as to other sequence context dependent factors. The large variation between parallel assays for the SNP rs1433099 LDLR prevented detection of the allelic imbalance for the LDLR gene, while imbalance was detected by the SNP rs5930 LDLR using both methods. This result demonstrates that it is preferable to analyse more than a single SNP in each gene in systematic screening for allelic imbalance in gene expression. As more data from primer extension assays accumulate, it may be possible to improve the accuracy of the system by improving the SNP selection and assay design further with the aid of algorithms developed based on this data [ 28 , 29 ]. Comparison of the relative amounts of the alleles of six SNPs on the RNA (cDNA) level to heterozygote SNPs in genomic DNA revealed four SNPs with imbalanced expression of the two alleles. A three-fold increase in the expression of the T-allele for the SNP rs4331 ACE was the most pronounced difference observed. In our study, 1.4–1.5-fold differences in allelic expression levels were detectable. The sensitivity of detecting a minority allele in our system would allow the distinction between 10-fold reduction in the expression of an allele and monoallelic expression, for example as a result of imprinting. Owing to its potential for high throughput screening of large numbers of samples, we have also performed a preliminary evaluation of the commercial SNPstream genotyping system (GenomeLab, Beckman Coulter) that also utilises the "tag-array" primer extension strategy in a semi-automated 384-well microtiter plate format for detection of imbalanced allelic expression [ 30 ]. The same trend of imbalanced allelic expression was observed for each of the SNPs, which is encouraging for future studies of imbalanced allelic expression in a high throughput semi-automated way. Other studies that have used fluorescent single base primer extension assays report that 1.2 – fold to 1.5 – fold differences in allelic expression are detectable [ 2 , 4 , 5 ]. Primer extension methods based on direct measurement of fluorescent signals, including the microarray-based methods evaluated here, are likely to provide better accuracy and sensitivity for allele quantification than homogeneous primer extension based on fluorescence polarisation [ 31 , 32 ], in which the allele quantification relies on measurement of small differences between large polarization signals. It is also reassuring for future large scale detection of imbalanced allelic expression that the accuracy of our methods seemed to be similar for cDNA and genomic DNA. Analysis of replicate RNA samples from different batches of both cell lines using a microtiter plate format of the minisequencing method evidenced for the biological authenticity of the allelic imbalance detected using minisequencing in the microarray format. The data obtained from independent cell samples also indicate an acceptable reproducibility of RNA extraction, RNA storage and cDNA synthesis. Another important factor besides sample to sample variation that may affect the accuracy of the relative allele quantification is the amount of mRNA subjected to the analysis. At a low copy number of mRNA, the stochastic distribution of the RNA templates may be a major source of variation [ 33 ]. The reason for the large variation between parallel assays for the LDLR receptor gene observed with all four methods used in our study may reflect a low expression level of the LDLR gene in the HAEC cells. Moreover, the amount of gene specific transcript in each RNA sample may vary which makes it difficult to perform balanced multiplex RT-PCRs to screen for allelic imbalances in several genes in one reaction. A similar minisequencing strategy as the one used for determination of imbalanced expression between SNP alleles can also be used for determination of the relative expression levels of highly homologous genes [ 15 ] and for determination of alternatively spliced transcripts [ 34 ], a resolution that is beyond the capacity of traditional microarray based RNA expression profiling. Conclusions Here we demonstrated the applicability of two formats of microarray based minisequencing for detecting imbalanced expression of SNP alleles. The accuracy and sensitivity of both systems allow detection of 1.4- to 10-fold differences in the expression levels of the two alleles of heterozygous SNPs. The microarray-based minisequencing systems utilise widely available reagents and equipment, and can thus easily be established "in-house". Moreover, the system is flexible with respect to number of SNPs and samples to be analyzed. Systematic quantitative screening of genetic diversity on the RNA level in multiple individuals and tissues will be a future approach in the elucidation of the molecular mechanisms that regulate gene expression. Methods DNA and RNA samples DNA samples from 30 volunteer donors were genotyped by Methods I and II to identify individuals of different genotypes for the panel of ten SNPs analysed. The SNPs are described in the section "SNPs and primers" below. DNA (10 ng/μl) from one individual was serially diluted 2:1 into DNA (10 ng/μl) from a second individual, to yield a series of DNA samples with different ratios between the SNP alleles. These mixed DNA samples were used for construction of quantification standard curves. Depending on the genotype of each SNP in the two individuals whose DNA was mixed, dilution series of samples with different allelic ranges were obtained for the ten SNPs, as specified in Table 1 . Human Umbilical Vein Endothelial Cells (HUVEC) and Human Aortic Endothelial Cells (HAEC) (Cascade Biologics, Inc., Portland, OR, USA) were grown in Medium 200 with Low Serum Growth Supplement (LSGS Kit, Cascade Biologics, Inc., Portland, OR, USA) at 37°C in a humidified atmosphere of 5% CO 2 . Cells from the cultures were harvested at 80% confluence according to the manufacturer's instructions. Total RNA was isolated from the cells using the TRIZOL ® Reagent (GIBCO BRL, Paisley, Scotland) and the RNA samples were stored at -70°C until use. High quality RNA with A 260 /A 280 ratio over 1.9 and intact ribosomal 28S and 18S RNA were used for cDNA synthesis. The RNA samples were treated with 1 U RQ1 RNase-free DNase (Promega, Madison, WI, USA) per μg RNA. Two to 2.5 μg total RNA was subjected to first strand cDNA synthesis using SuperScript™ II (RNase H - Reverse Transcriptase, Invitrogen, Carlsbad, CA, USA) reagents in a 20 μl volume. DNA was extracted from the cells using GenElute™ Mammalian Genomic DNA Kit (Sigma, St Louis, MO, USA) and stored at -20°C until use. PCR The fragments comprising the SNPs were PCR-amplified in individual reactions using 10–15 ng genomic DNA or one tenth of the cDNA products, 0.2 mM dNTPs, 1U AmpliTaq ® Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA), 1.5 mM MgCl 2 , and 0.2–0.3 μM of primers in 50 μl of 10 mM Tris-HCl pH 8.3 and 50 mM KCl. The PCR conditions were initial activation of the enzyme at 95°C for 10 min followed by 35 cycles of 95°C for 1 min, 56°C for 1 min and 72°C for 1 min and a final extension at 72°C for 7 min in a Thermal Cycler PTC225 (MJ Research, Watertown, MA, USA). The amplified fragments were combined and concentrated to 60 μl using Microcon ® YM-30 Centrifugal Filter Devices (Millipore Corporation, Bedford, MA, USA). SNPs and primers Ten SNPs located in coding regions of genes known to be expressed in HUVEC and HAEC cells were analysed. Information on the SNPs, including dbSNP [ 35 ] ID number and nucleotide variation is given [see Additional file 1 ] together with the sequences of the minisequencing primers. The primers for PCR and minisequencing were designed using the Oligo Primer Analysis software v6.65 (Molecular Biology Insights Inc., Cascade, CO, USA). Preparation of microarrays The minisequencing primers or the complementary tag-oligonucleotides were covalently immobilised on CodeLink™ Activated Slides (Amersham Biosciences, Uppsala, Sweden) by the mediation of a NH 2 -group in their 5'- or 3'-end, respectively. The oligonucleotides were applied in duplicates to the slides at a concentration of 25 μM in 150 mM sodium phosphate pH 8.5 using a ProSys 5510A instrument (Cartesian Technologies Inc, Irvine. CA, USA) equipped with one Stealth Micro Spotting pin (SMP3B, TeleChem International Inc., Sunnyvale, CA, USA) to minimise the variation between spots in different "subarrays". The oligonucleotides were spotted in an "array-of-arrays" configuration that facilitates analysis of 80 individual samples in parallel on each microscope slide [ 24 ]. In each "subarray" a fluorophore-labelled oligonucleotide was included as a control for the immobilisation process. A reference oligonucleotide, complementary to a synthetic template included in the minisequencing reaction mixtures to monitor the difference in incorporation efficiency of the four nucleotides by the DNA polymerase, was also included in each "subarray". Finally, an oligonucleotide designed not to hybridise to any of the oligonucleotides present in the reaction mixture was included in each "sub-array" to be used for background corrections. After printing, the slides were incubated in a humid chamber for at least 24 hours, followed by treatment with ethanolamine according to the manufacturer's instruction. The slides were then stored desiccated in the dark until use. Minisequencing using immobilised primers (Method I) Aliquots of 7.5 μl of the concentrated PCR products were analysed in five parallel "subarrays" for each sample, essentially as described previously [ 18 ]. The PCR products were allowed to anneal to the immobilised oligonucleotides. After washing, the extension reactions were performed with 0.75 U of Thermo Sequenase™ DNA polymerase (Amersham Biosciences, Uppsala, Sweden) and 0.35 μM Texas Red-ddATP, Tamra-ddCTP, R110-ddGTP and Cy5-ddUTP (Perkin Elmer Life Sciences, Boston, MA, USA) in Thermo Sequenase™ reaction buffer in a total volume of 15 μl, followed by washing of the slide. Minisequencing using "tag-arrays" (Method II) Five parallel reactions with a 4.5 μl aliquot of the concentrated PCR products were analysed for each sample, as described in detail in [ 23 ]. Excess of PCR primers and dNTPs were removed by treatment with 5 U of exonuclease I and 1 U of shrimp alkaline phosphatase (USB Corporation, Cleveland, OH, USA). The cyclic minisequencing reactions were performed in the presence of the 20 tagged primers at 10 nM concentration, 0.1 μM Texas Red-ddATP, Tamra-ddCTP and R110-ddGTP, 0.2 μM Cy5-ddUTP (Perkin Elmer Life Sciences, Boston, MA, USA) and 1 U of Thermo Sequenase™ DNA polymerase (Amersham Biosciences, Uppsala, Sweden) for 55 cycles of 95°C and 55°C for 20 s each. The extension products were allowed to anneal to the immobilised complementary tag oligonucleotides at 42°C for 2.5 hours followed by washing of the slide. Solid-phase minisequencing in a microtiter plate format PCR was run with one of the primers biotinylated. The biotinylated PCR products were immobilised in a microtiter plate coated with streptavidin (Combiplate 8, Labsystems, Helsinki, Finland) and the unbiotinylated strand was removed with alkali treatment [ 9 , 15 ]. The minisequencing mixture, containing the appropriate tritium labelled dNTP (Amersham Biosciences, Uppsala, Sweden), AmpliTaq ® DNA polymerase (Applied Biosystems, Foster City, CA, USA) and the minisequencing primer was added. The extension reaction was allowed to proceed for 10 min at 50°C. The extended primers were released with alkali and the amount of incorporated tritium labelled nucleotide was measured. Hybridisation with allele-specific TaqMan probes Primers and probes for the TaqMan assays were designed by Applied Biosystems as Assay-by-Design (rs1042719 ADRB2 and rs5925 LDLR) or Assay-on-Demand (rs1433099 LDLR) service. The probes for the two alleles were labelled with the reporter dyes FAM and VIC respectively. The sequences of the primers and probes for the SNPs rs5925 LDLR and rs 1042719 ADRB2 are found in [ Additional file 1 ]. The primer and probe sequences for the SNP rs1433099 LDLR were not made available to us by ABI since this SNP is included in their Assay-on-Demand program. Real time quantitative PCR was run in 25 μl TaqMan Universal PCR Master Mix (Applied Biosystems) with 200 nM of both labelled TaqMan probes, 900 nM PCR-primers and 10 ng genomic DNA or one tenth of the cDNA products. The PCR conditions were initial activation of the enzyme at 95°C for 10 min followed by 60 cycles of 95°C for 15 sec and 60°C for 1 min in a ABI7000 instrument (Applied Biosystems, Foster City, CA, USA). The signal intensity ratios were calculated based on normalised ΔRn fluorescence values obtained from the assay during the exponential phase of PCR. The ΔRn values were retrieved from cycle 38 for the SNP rs1042719 ADRB2, cycle 42 for the SNP rs5925 LDLR and cycle 43 for the SNP rs1433099 LDLR. Imbalanced expression of the SNP alleles was determined by a t-test as described below. Signal detection and data analysis In Methods I and II fluorescence was measured using a ScanArray ® Express instrument (Perkin Elmer Life Sciences, Boston, MA, USA) with the excitation lasers Blue Argon 488 nm, Green HeNe 543.8 nm, Yellow HeNe 594 nm and Red HeNe 632.8 nm with the laser power set to 80% and the photomultiplier tube gain adjusted to obtain equal signal intensities from reaction control spots for all four spectra. The fluorescence signals were extracted using the QuantArray ® analysis 3.1 software (Perkin Elmer Life Sciences, Boston, MA, USA). The mean of the fluorescence signals for the duplicate spots was corrected for the average background in each "sub-array" separately. The data was handled and interpreted using the Microsoft ® Excel program. The genotype for each individual SNP was assigned by calculating a ratio between the fluorescence signals for the two alleles. Coefficients of determination (R 2 ) were assigned by linear regression analysis of the relationship between the signal intensity ratios determined from the minisequencing assay and the known allelic ratios in the mixed samples for the quantification standard curves. Two-sample t-tests with two-tailed significance levels assuming unequal variance were performed to determine the lowest level of detection of a specific allele for the quantification standard curves and to evaluate the imbalanced expression of the two alleles of the SNPs in the cell lines. Authors' contributions UL participated in the design of the study and in RNA and DNA extraction, and performed all the laboratory work involving "in-house" minisequencing methods, performed the statistical calculations and drafted the manuscript. MF cultured the cells, performed RNA and DNA extraction, performed the assays with the reference method, and provided input to the manuscript. AD performed the assays using the SNPstream system. A-CS conceived the study, participated in its design, coordination and in preparation of the manuscript. All authors read and approved the final manuscript. Supplementary Material Additional File 1 Additional file1 is a pdf-file with information on the SNPs, including dbSNP ID number, nucleotide variation and the sequences of the primers and probes used in the microarray based minisequencing and TaqMan assays respectively. Click here for file

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539052

Sensitive, Noninvasive Detection of Lymph Node Metastases

Background Many primary malignancies spread via lymphatic dissemination, and accurate staging therefore still relies on surgical exploration. The purpose of this study was to explore the possibility of semiautomated noninvasive nodal cancer staging using a nanoparticle-enhanced lymphotropic magnetic resonance imaging (LMRI) technique. Methods and Findings We measured magnetic tissue parameters of cancer metastases and normal unmatched lymph nodes by noninvasive LMRI using a learning dataset consisting of 97 histologically proven nodes. We then prospectively tested the accuracy of these parameters against 216 histologically validated lymph nodes from 34 patients with primary cancers, in semiautomated fashion. We found unique magnetic tissue parameters that accurately distinguished metastatic from normal nodes with an overall sensitivity of 98% and specificity of 92%. The parameters could be applied to datasets in a semiautomated fashion and be used for three-dimensional reconstruction of complete nodal anatomy for different primary cancers. Conclusion These results suggest for the first time the feasibility of semiautomated nodal cancer staging by noninvasive imaging.

Introduction Most primary malignancies spread systemically via lymphatic dissemination [ 1 ]. For example, the finding of axillary nodal metastases predicts a much shorter disease-free survival in breast cancer [ 2 ]. The total nodal tumor burden (number of affected nodes and metastatic tumor volume) affects prognosis even more severely [ 3 ]. Accurate lymph node staging also remains a cornerstone in choosing the most appropriate therapy for a given stage. Therapeutic intervention of metastatic lymph nodes [ 4 ], prophylactic radiation of frequently affected drainage routes [ 5 ], and systemic therapies [ 6 ] all have been shown to improve survival. Genetic profiles identifying metastatic tumors [ 7 ], serum biomarkers, and proteomic profiles are currently being developed to identify patients at risk [ 8 , 9 ]. No direct genetic profile, however, has been demonstrated to date to accurately predict the presence of human nodal metastases in a given patient. Rather, surgical approaches, such as sentinel lymph node biopsy or lymph node dissection, are still commonly used. Careful histological analysis includes mapping, bisectioning, and rapid staining in the frozen tissue laboratory. Higher diagnostic accuracies can be achieved by serial sectioning (50 μm) and by immunohistochemical staining [ 10 , 11 ]. Noninvasive imaging studies are commonly used during the workup of primary malignancies. Typically, lymph nodes are diagnosed by tomographic techniques (computed tomography [CT], magnetic resonance imaging [MRI]) as malignant when their short axis is >10 mm in size [ 12 ]. Such size criteria, however, have been shown to be unreliable [ 13 ]. Similarly, the detection of cancer in nonenlarged (occult) nodes is often quite low by positron-emission tomography (PET) and single photon emission computed tomography imaging. For example, small nodal metastases (< 5 mm) are often missed by PET imaging in patients with breast cancer [ 14 ]. More recently, it has become possible to image anatomic regions at submillimeter resolutions by MRI, with excellent spatial coverage and reduced motion artifacts. The development [ 15 , 16 ] and clinical introduction of lymphotropic magnetic nanoparticles has been shown to significantly improve diagnostic accuracies of MRI for nodal staging (LMRI) in prostate cancer [ 17 ]. These nanoparticles serve as probes for lymphatic anatomy and function and enhance tumor detection through abnormal distribution patterns in malignant nodes [ 17 , 18 ]. Despite the advances of LMRI for cancer staging, image analysis has been challenging and occasionally controversial. Traditional analysis has been based on a reader's identification of certain structural abnormalities that can be variable, given differences in acquisition parameters and interpretation criteria [ 19 , 20 , 21 ]. Furthermore, it has been challenging to quickly and accurately analyze large datasets generated by LMRI. The goal of the current study was to develop and test technologies that would vastly improve the accuracy of current LMRI nodal staging. Specifically we set out to (a) determine whether unique magnetic parameters existed and could be used for semiautomated image analysis and (b) whether the technique could be applied to different primary cancers. Here we provide the first comprehensive analysis of tissue parameters validated against histopathology as an end point. Methods Study Design The Institutional Review Board approved the current study and all patients signed informed consent. The study was divided into a learning ( n = 97 lymph nodes with known histopathology) and a test dataset ( n = 216 lymph nodes with known histopathology; Table 1 ). Assignment into datasets was done in temporal fashion. The learning dataset represented retrospective cases at outset of the study, and the test dataset represented prospective cases collected during a 1-y interval. In the learning set, 55% of the nodes were benign, and 45% of the nodes were malignant. The learning dataset was obtained from 36 patients (24 male, 12 female, age 28–85 y, mean 59.7 y) with histologically proven primary genitourinary malignancies (prostate, 21; bladder, 9; testes, 5; ureter, 1). All patients completed the MRI study and then underwent surgical resection ( n = 26) and/or nodal biopsy ( n = 10). The investigated nodes had a mean short axis diameter of 10.5 mm (range 3–39 mm). Table 1 Overview of Patient Datasets The test dataset was obtained from 34 patients (25 male, nine female, age 30–82 y, mean 58.9 y) with histologically proven malignancies from different primaries ( Table 1 ), including prostate ( n = 18), breast ( n = 7), penile ( n = 4), bladder ( n = 2), testes ( n = 2), and colon ( n = 1). Seventy-nine percent of the nodes were benign and 21% of the nodes were malignant. The nodes in the test dataset had a mean short axis diameter of 10.0 mm (range 3–39 mm). Both datasets included the full spectrum of normal nodes to completely replaced nodes. MRI MRI was performed at 1.5 T (System 9X, General Electric Medical Systems, Milwaukee, Wisconsin, United States) using phased-array coils. All images were archived on DICOM PACS servers (MIPortal, CMIR and Siemens Medical Systems, Erlangen, Germany; and Impax RS 3000, AGFA Technical Imaging Systems, Richfield Park, New Jersey, United States) for subsequent analysis. Images of the pelvis ( n = 56) extended from the pubic symphysis to just above the level of aortic bifurcation. In patients with primary testicular cancers ( n = 7) imaging was extended superiorly to include the renal hilum and retroperitoneum. In patients with breast cancer ( n = 7) we obtained MR images of the bilateral axillae, including the internal mammary and supraclavicular regions. All patients were imaged with identical pulse sequences and timing parameters. Imaging was performed before and 24 h after intravenous ferumoxtran-10 administration (Combidex, Advanced Magnetics, Cambridge, Massachusetts, United States; 2.6 mg Fe/kg diluted in normal saline and infused over a 20-min period using a 5-μm filter). The acquired pulse sequences included (a) axial T2-weighted fast spin-echo (TR/TE, 4500/80; flip angle, 90°; field of view, 24–28 cm; slice thickness, 3 mm; matrix, 256 × 256; number of excitations, 2; in-plane resolution, 1.2 mm); (b) a T1-weighted two-dimensional gradient-echo sequence obtained in different anatomical planes (TR/TE 175/1.8; flip angle, 80°; field of view, 22–30 cm; slice thickness, 4 mm; matrix, 128 × 256; in-plane resolution, 2.0 mm); (c) an axial T2-weighted dual TE gradient-echo (TR/TE 2100/14–24; flip angle, 70°; field of view, 26–28 cm; slice thickness, 3 mm; matrix, 160 × 256; in-plane resolution, 1.7 mm); and (d) a three-dimensional (3D) T1-weighted gradient echo sequence; TR/TE 4.5–5.5/1.4; flip angle, 15°; field of view, 24–28 cm; slice thickness, 1.4 mm; matrix, 256 × 256; in-plane resolution, 1.0 mm). The above listed imaging sequences and parameters had previously been optimized to reduce motion artifacts, maximize signal-to-noise ratio (SNR), and provide diagnostically useful images of the pelvis, abdomen, and chest within clinically acceptable time limits. The T2-weighted fast spin-echo sequence, in (a) above, was primarily used for qualitative nodal detection, and hence a square pixel with more than one acquisition was obtained. The two-dimensional axial T1-weighted gradient-echo sequence, in (b) above, was chosen to achieve adequate anatomical coverage within a short imaging time. The axial dual-echo gradient-echo sequence, in (c) above, was developed specifically for this project to provide artifact-free datasets for quantitative image analysis. A matrix size of 160 × 256 was chosen for this sequence to achieve a balance between the upper limits for imaging time while reducing image noise. Finally, a 3D T1-weighted sequence was obtained, in (d) above to provide a dataset for vascular maximum intensity projection (MIP) reconstructions. Quantitative Image Analysis All image analysis was performed on archived DICOM images using different software packages (e.g., custom-built software such as CMIR-Image, MGH, Boston, Massachusetts, United States; Syngo, Siemens Medical Systems; Advantage Windows, General Electric Medical Systems). Lymph nodes were identified by readers who manually placed kernels onto each node for automated boundary detection and calculation of nodal dimensions and volumes. The thus identified regions of interest (ROIs) encompassed the entire lymph node (not only portions of it) and were used for quantitative signal-intensity (SI) measurements (see Table 2 ). Serial measurements of nodal dimensions on different pulse sequences or time points varied less than 2%. Table 2 Frequency of Imaging Parameters in Learning Dataset A number of quantitative tissue parameters were calculated either as differences between pre- and postcontrast scans (δ) or as single-value analysis on postcontrast scans (see Table 2 ). The lymph node/muscle (LNM) ratio was calculated by dividing signal intensities of an entire lymph node by that of adjacent muscle using a similar-sized ROI, drawn manually. The nodal SI change was calculated by obtaining SI before and after contrast administration. The nodal SNR was calculated by obtaining SD/SD noise . The T2* was calculated in nodal ROIs on dual TE images using CMIR-Image. T2* maps were constructed by performing fits of a standard exponential relaxation model (S = Ke –TE/T2* ) to the data on a pixel-by-pixel basis. Only pixels with intensity greater than a threshold level (2X of noise) were considered during the fitting process. Pixel variance was obtained from post-MR images. Comparative visual analysis included short axis measurements, and identification of heterogeneity, large focal defects, and central hyperintensity, according to criteria previously established [ 12 , 17 ]. To determine the diagnostic accuracy of the different tissue parameters in the learning dataset, we determined sensitivity, specificity, and predictive values for each parameter alone and in combination ( Table 3 ). The most discriminatory parameters were then applied to the test dataset ( Table 4 ). Table 3 Discriminatory Power of Imaging Parameters in Learning Dataset PPV, positive predictive value; NPV, negative predictive value Table 4 Application of Quantitative Parameters to Test Dataset ( n = 216) a Includes short axis > 10 mm or round > 8 mm PPV, positive predictive value; NPV, negative predictive value In the final set of semiautomated image analysis, 3D reconstructions were obtained for nodal mapping onto vascular anatomy using MIP projections. While the MIP projections do not aid in the differentiation between malignant and benign lymph nodes, they are invaluable in providing anatomic content to the dozens of lymph nodes identified. In particular, MIP images were generated interactively from postcontrast, fat-saturated, volumetric interpolated breath-hold images to outline vascular anatomy. The evaluated lymph nodes characterized as benign or malignant (by T2*/variance analysis) were then superimposed on the volumetric 3D images, using customized software (Advantage Windows, General Electric Medical Systems). Statistical Analysis Data were expressed as mean ± standard deviations (SD) and medians. All statistical testing was performed using GraphPad Prism (GraphPad Software, San Diego, California, United States). The significance between two individual groups was determined using the nonpaired Student's t -test (e.g., benign and malignant datasets in Figure 1 ). For the more discriminatory datasets alternative-free-response receiver operating characteristic curves were plotted. Ratios for cut-off single-value parameters were defined to yield highest sensitivity and specificity. Accuracy for a given parameter was expressed as the area under the curve (A z ), and values are summarized in Table 4 . Figure 1 Tissue Parameters in Learning Dataset Nodal tissue parameters for benign and malignant nodes are shown before (A and B) and after (C–E) intravenous administration of magnetic nanoparticles. Note the insensitivity of conventional MRI (A and B), better separation using single-value analysis (C and D) and excellent separation using two-value analysis (E). Histology All lymph nodes were sampled histologically within 2 wk of the MRI (mean: 6 d; range: 2–14 d). The analysis was done in surgically resected lymph nodes ( n = 55; both benign and malignant nodes) or in fine needle aspirates and core biopsies ( n = 15; malignant nodes only), implementing careful mapping procedures to correlate nodes. Surgically excised nodes were sectioned at 10–20 μm intervals after bihalving and were stained with hematoxylin-eosin. Results Learning Dataset The learning dataset consisted of 97 histologically validated lymph nodes from 36 patients with different primary malignancies (see Table 1 ). The mean short axis diameter was 10.5 mm (range 3–39 mm) with 56 of the 97 nodes (58.3%) measuring less than 10 mm, that is, below the traditional imaging cutoff for malignancy (“occult nodes”). Table 2 summarizes the incidence of different visual, comparative (before and after contrast administration), and semiautomated (postcontrast administration only) parameters in the two different groups. Figure 1 is a graphical representation of overlaps between malignant and benign groups for different parameters listed in Table 2 . Table 3 summarizes sensitivities, specificities, and predictive values for the different quantitative imaging parameters. Sensitivities of metastasis detection by visual image analysis ranged from 50%–94%, however, often with lower specificities. Volumetric measurements, in particular, were insensitive markers of malignancy in nonenlarged nodes (see Table 3 ). In contradistinction, image analysis of pre- and postcontrast image sequences resulted in higher specificities and sensitivities (see Table 3 ). Comparative differences between benign and malignant nodal groups were highest for T2* and pixel variance measurements (see Table 3 ). Of all the semiautomated parameters tested alone, T2* measurements showed the highest sensitivity (93%; 95% confidence interval: 82%–98%) and specificity (94%; 95% confidence interval: 84%–99%) in the learning dataset (see Figure 1 and Table 3 ). Of all the semiautomated parameters tested in combination, T2* measurements combined with pixel variance analyses postcontrast showed the highest sensitivity (98%; 95% confidence interval: 88%–99%) and specificity (94%; 95% confidence interval: 82%–98%) in the learning dataset ( Figure 1 E). Using the dual-value analysis, there was one malignant outlier in the benign dataset (the lymph node was 3 mm in overall size, with few malignant cells seen on histology, and probably too small for analysis) and two benign outliers in the malignant dataset (both these nodes showed hyalinosis replacing more than 50% of the nodal architecture). Test Dataset To determine whether feature extraction would be accurate for prospective nodal staging, we utilized the above criteria against a larger test dataset encompassing 216 validated lymph nodes from 34 patients, including different primaries (see Table 1 ). The sensitivity, specificity, and predictive values of the most discriminatory parameters of this prospective analysis are summarized in Table 4 . We primarily focused on semiautomated image analysis of postcontrast scans because of the high sensitivity and specificity determined in the learning dataset. T2* measurements showed a sensitivity of (93%; 95% confidence interval: 82%–99%) and a specificity of (91%; 95% confidence interval: 85%–96%). Combined T2* and pixel variance analysis achieved a sensitivity of 98% (95% confidence interval: 88%–99%) and a specificity of 92% (95% confidence interval: 87%–96%) comparable to that of the learning set and much superior to currently used size criteria. Using the dual-value analysis, there were two malignant outliers in the benign dataset (both of these nodes were less than 3 mm in overall size and probably too small for analysis—similar to the learning dataset) and three benign outliers in the malignant dataset (two of these nodes had hyalinosis replacing more than 50% of the nodal architecture and one had macrocalcifications). More important, all the misclassified nodes occurred in individual patients rather than in the same patient and, hence, did not affect the overall nodal staging on a patient-by-patient basis in this dataset. Image Reconstruction Video 1 Automated 3D Reconstruction of Pelvic Nodal Anatomy Utilizing semiautomated feature extraction to identify lymph nodes and image analysis (based on T2* and pixel variance), we subsequently proceeded to map individual lymph nodes onto vascular anatomy in the different anatomic drainage patterns. Figure 2 summarizes the different steps in image analysis. Figure 3 and Video 1 shows an example of a 45-y-old patient with colorectal cancer undergoing semiautomated nodal staging. In this particular patient, MRI identified six positive lymph nodes (< 10 mm each), reconstructed as a 3D dataset, whereas all positive lymph nodes were missed by PET scans. Figure 4 and Video 2 show reconstructions and analyses from a patient with a breast cancer primary with bilateral nodal metastases. Note the high spatial resolution allowing the detection of a 3-mm nodal metastasis. Figure 2 Steps in Semiautomated Image Analysis Semiautomated image analysis involves recognition and automated segmentation of each lymph node (A), quantitation of magnetic tissue parameters (T2*, variance of pixel values; [B]), comparison of extracted tissue parameter to a database (C), and 3D reconstruction of nodal anatomy onto vascular anatomy (D). Figure 3 Pelvic Nodal Staging Nodal staging in patient with colorectal cancer. A PET scan using 18 FDG as a tracer (A) and a CT scan (B) were interpreted as negative for nodal metastases. LMRI identified six small pelvic lymph nodes ([C] and [D]; red arrowheads), which had magnetic parameters of malignancy. Semiautomated reconstruction (E) identifies multisegmental metastases, subsequently proven histologically (F). For 3D reconstruction of pelvic nodal anatomy see Video 1 . Figure 4 Breast Cancer Mapping Patient with breast cancer prior to sentinel lymph node biopsy. (A) Conventional axillary MRI shows nonenlarged lymph nodes that do not meet the size criteria of malignancy (white bar = 5 mm). (B) Following intravenous administration of nanoparticles, a single 3-mm intranodal metastasis was correctly identified. (C) Ex vivo MRI of sentinel node specimen confirms metastasis. (D) Semiautomated nodal analysis and reconstruction correctly juxtaposed solitary lymph node metastases adjacent to two normal lymph nodes. (E) Correlative histopathology confirms the diagnosis. For 3D reconstruction of axillary nodal anatomy see Video 2 . Video 2 Automated 3D Reconstruction of Axillary Nodal Anatomy Discussion We show that it is feasible to extract various quantitative tissue parameters to predict the likelihood of nodal metastases in vivo. These results are highly relevant in cancer staging because they provide evidence that (a) quantitative tissue parameters enable diagnosis of lymph node metastases while reducing interobserver variability and (b) that semiautomated reconstructions allow spatially more extensive mapping than is currently possible. Metastases to lymph nodes occur during growth of most primary malignancies, and their presence mandates the need for more extensive and systemic therapy. Nodal cancer staging currently relies on invasive procedures (surgical lymph node dissection, sentinel lymph node resection, biopsy) with significant morbidity and cost [ 22 , 23 ], or insensitive tomographic imaging methods [ 24 ]. For example, detection sensitivities using size criteria with state-of-the-art multislice CT are as low as 50%, whereas PET imaging of nonenlarged nodes has equally low sensitivities [ 14 ]. Based on the observation that nanoparticulate solutions accumulate in nodal macrophages upon systemic injections [ 25 , 26 ], lymphotropic superparamagnetic preparations have been developed [ 16 ]. In earlier clinical trials (using lower spatial resolution sequences), metastases of 1–2 mm have been detected [ 17 ], whereas as few as 1,000 tumor cells have been detected in nodes in experimental mouse models [ 18 ]. Despite these advances, it has been difficult to acquire images of sufficiently high resolution and to derive parameters to automate diagnosis. The data presented here indicate that unique magnetic parameters allow identification of nodal metastases and accurate 3D reconstructions, including surgically inaccessible lymph nodes. The significance of the above findings is 3-fold. First, the ability to directly and noninvasively monitor nodal tumor involvement represents a powerful diagnostic tool for cancer. Accurate staging represents the cornerstone for triaging patients to either localized or to more aggressive and systemic therapies. Second, the method described here was sensitive for the limited subsets of primary cancers tested. It is reasonable to hypothesize that such analysis could be applied to staging of other common primaries. In particular, lung, colorectal, genitourinary, and head and neck cancers could benefit from this staging procedure. In addition to nodal staging, the nanoparticle-enhanced MRI can also be used to measure microvascularity in primary tumors [ 27 ] and to improve the detection of liver metastases [ 28 ]. Third, our results are significant because the semiautomated staging method is highly accurate and reduces variability in visual image analyses between different observers. The LMRI staging technique is believed to be clinically relevant in several key areas. First, LMRI may play a significant role in avoiding unnecessary surgeries, that is, those in node-positive patients. Second, since LMRI can detect lymph nodes outside traditional surgical fields, this information may influence surgical approaches. In colorectal cancer, LMRI may provide a “sentinel-node-like” guide to staging. Third, it is likely that LMRI would be useful to identify appropriate patients to receive neoadjuvant chemotherapy prior to surgery. Currently, neoadjuvant therapy is often reserved for postoperative patients, once the nodal status has been determined. Fourth, LMRI may be particularly useful to guide radiation therapy by mapping the complete nodal status onto bony and vascular landmarks. Finally, LMRI could be used to avoid invasive diagnostic procedures, which are not part of therapy. For example, LMRI could replace lymphangiography, mediastinoscopy, or endoscopic ultrasound for nodal staging. Our findings have a number of direct implications for technology development and in clinical care. Accurate measurements of T2* relies on motion artifact-free multiecho pulse sequences that are not routinely available on clinical scanners at spatial resolutions required for nodal staging. Such sequences will have to be implemented and combined with postprocessing tools to simplify and semiautomate analysis. Similar software approaches are already used routinely in lung nodule characterization [ 29 ] or screening for breast cancers [ 30 ]. We predict that in the case of LMRI, such automation routines will be highly specific, given the unique mechanism of image contrast. As a proof-of-principle, we implemented approaches to identify, segment, analyze, and display nodal information. While the current technology is already highly accurate, we anticipate further improvements with hardware and software advances. We hope that this will ultimately translate into clinical practice and replace unnecessary intervention. Patient Summary Background When deciding on treatment for patients with cancer, it is very important to assess whether the cancer has spread to lymph nodes—both to help decide what treatment a patient should have and what the eventual outcome might be. Previous ways of finding involved lymph nodes included taking out the nodes by surgery, ultrasound, and CT and MRI scans. What Does This Study Show? A solution of magnetic nanoparticles that tend to go to lymphoid organs was injected and then tracked by MRI. The pattern of the particles was abnormal when there was metastasis in the nodes, and it was possible to train a computer to recognize this abnormality. The authors developed the program in one group of patients and then tested it in another group, in which they were able to correctly predict whether the nodes were involved in about nine of ten nodes. In addition, they could use the information to display a virtual picture of the involved nodes. What Does This Study Mean for Patients? The technique will need to be validated in a larger group of patients, and by other investigators. However, it means that it is potentially possible to work out much more precisely, and with less chance of error, whether lymph nodes are involved in cancer. Hence, treatment can be better planned, and if surgery is needed to remove nodes for analysis, then this technique could ensure that the surgery is as minimal as possible. Where Can I Get More Information? RadiologyInfo, a public information site developed by the American College of Radiology and the Radiological Society of North America: http://www.radiologyinfo.org/ Medline Plus, which has health information from the National Library of Medicine: http://www.nlm.nih.gov/medlineplus/cancer.html

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523234

Controlling the Timing of Gene Expression during Organ Development

For more than 2,000 years, from the time of Aristotle onwards, it was thought that the complete body plan of human beings (and that of other animals) was present in the fertilized egg. During pregnancy, a preformed miniature human being, or homunculus, grew bigger and bigger; development was simply a process of growth. Then, in the mid-18th century, Carl Friedrich Wolff described how the chick gut, basically a tube, forms from an initially flat sheet of cells, overthrowing at a stroke the preformation theory of embryology. We now know that development is a complex series of coordinated processes that transforms the amorphous ball of cells produced from the fertilized egg by cell division into an intricate body containing numerous specialized tissues and organs. And we are beginning to understand how a wide array of transcription factors—proteins that bind to regulatory sequences within genes to control their expression—guide the sequential stages involved in development. It seems that these factors form regulatory networks that control the temporal and spatial waves of gene expression that underlie and are required for organized body building. Susan Mango and her colleagues are studying the role of transcription factors in controlling organ development. The organ they are studying—the pharynx of the nematode worm—is relatively simple. This muscular tube, which passes bacteria (the food of this small soil-dwelling organism) from the mouth to the midgut, contains fewer than 100 cells of only seven different types. To get an overall picture of the regulatory sequences within genes that are involved in the temporal control of pharyngeal development, the researchers identified 339 candidate pharyngeal genes by comparing gene expression profiles in mutant worm embryos that had excess pharyngeal cells with those in mutant embryos lacking pharyngeal cells. Then, by referring to a database that details gene expression patterns in nematode worms and embryos, the researchers classified 37 of their candidate genes as having early-onset expression and 34 as having late-onset expression. Next, the scientists carefully examined the DNA sequence of each gene for candidate regulatory regions that might contribute to its temporal regulation. Of nine candidate motifs revealed by this search, six functioned as regulatory sites in in vivo assays. The researchers estimated that these six elements, together with sites that bind PHA-4—a member of a family of transcription factors that are important in digestive tract development in many animals—account for the timing of onset of expression of about half of the nematode's pharyngeal genes. Finally, the researchers used combinations of the newly discovered temporal regulation sites and PHA-4 sites in a genome-wide search that predicted pharyngeal genes and their time of onset of expression with greater than 85% accuracy. Fluorescent reporter genes expressed in the developing C. elegans foregut From these results and those of previous studies, Mango and her colleagues propose a model to explain how the temporal control of pharyngeal gene expression needed for pharynx development is achieved. The earliest time for pharyngeal gene expression, they suggest, is determined by how well PHA-4 sticks to a particular gene's binding site. However, gene expression only occurs if other factors that bind to the regulatory sites are also present, and the exact combination of these factors determines which gene is active at any given time. The identity of these factors remains to be discovered. Nevertheless, at least for this simple organ, we now have a much better idea of how the complex process of organ formation is controlled at a molecular level, and it is likely that similar regulatory networks will underlie the formation of other organs as well.

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548132

Expression of Bcl-2 and p53 at the fetal-maternal interface of rhesus monkey

To study the apoptosis and its mechanism at the fetal-maternal interface of early gestation, localization of apoptotic cells in the implantation sites of the rhesus monkey on day 17, 19, 28 and 34 of pregnancy were first examine by using the TUNEL technique. The expression of Ki67, a molecular marker of proliferating cells, and two apoptotic proteins, B cell lymphoma/leukaemia-2 (Bcl-2) and P53, were then studied by immunohistochemistry. Apoptotic nuclei were observed mainly in the syncytiotrophoblast. Ki67 was confined almost exclusively to cytotrophoblasts. The localization of Bcl-2 protein follows that of the apoptotic nuclei and its expression level increased as the development of the placenta progressed on. P53 was detected to some extent in cytotrophoblasts and syncytiotrophoblast covering the basal feet of the anchoring villi during the late stage of placentation. Based on these observations, it might be suggested that Bcl-2 could be possible to play an interesting role in limiting degree of nuclear degradation and sustaining cell suvival in the multi-nucleated syncytiotrophoblast cells during early pregnancy, and P53 could also be essential in regulating the trophoblastic homeostasis by controlling its proliferation or apoptosis.

Introduction Apoptosis plays important roles in placentation and embryonic development [ 1 ]. The cells undergoing apoptosis have characteristic structural changes in the nucleus and cytoplasm. The nuclear disintegration involves DNA cleavage into oligonucleosomal length DNA fragments [ 2 - 4 ], and the DNA fragments can be detected by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end-labelling (TUNEL) technique. Expressions of apoptotic regulatory molecules, such as Fas, Fas ligand, P53, and the proteins of Bcl-2 family, have been reported in human placenta [ 5 - 8 ]. Bcl-2 and P53 are two of the key players in the apoptotic signaling cascades. Bcl-2, a proto-oncogene first discovered in human follicular lymphoma [ 9 ], is involved in the inhibition of apoptosis and the survival of a variety of cell types [ 10 ]. Bcl-2 protein is located in the membranes of endoplasmic reticulum, nuclear envelope, and mitochondria. Over-expression of Bcl-2 suppresses apoptosis by preventing the activation of caspases that carry out the process. P53 is well known as a tumor suppressor. It is a transcription factor that induces apoptosis mainly through inducing the expression of a batch of redox-related genes [ 11 ] and the down-regulating Bcl-2 [ 12 ]. The expression of Bcl-2 and P53 human placenta has been studied [ 1 , 13 ]. However, their cellular distribution in the implantation site at early stage of pregnancy has not been reported. Because the monkey and the human share a very similar implantation process in terms of timing, morphological changes, and cell types involved [ 14 ], we aimed, in the present study, to investigate the expression, localization of Bcl-2 and P53 in the implantation site of the rhesus monkey, in order to gain some insights to the mechanism of time-dependent apoptosis occurring at the fetal-maternal interface. Materials and methods Animals Healthy adult male and female rhesus monkeys ( Macaca mulatta ) were purchased from the monkey colony of the Primate Research Center (PRC), Kunming Institute of Zoology (KIZ), Chinese Academy of Sciences (CAS). All experimental procedures were approved by the Animal Ethics Committees of both the Institute of Zoology and PRC. The animals were caged individually and were evaluated daily by visual examination of the perineum for menses, with the onset of menses defined as Day 1 of the menstrual cycle. Adult female monkeys with regular menstrual cycles of approximately 28 days were chosen for this study. Female monkeys on Day 11 of their menstrual cycle were caged with a male monkey of proven fertility from previous mating for 3 days. Vaginal smears were examined the next morning for the presence of sperm. The day when the smear was detected as positive for sperms was designated as Day 1 of pregnancy (D1). The presence of a conceptus was confirmed by ultrasound examination. The monkeys were anesthetized by pentobarbital sodium (3 animals each group), and the uteri were removed surgically from early villous to villous placenta stages: on D17, D19, D28 and D34 of pregnancy respectively and cut into pieces, the specimens were quickly washed in cold phosphate-buffered saline (PBS) to remove adherent blood, then placed in cold 4% paraformaldehyde fixative for 16 h at 4°C and further processed through graded dehydration, clearing and embedding in paraffin for immunohistochemistry and TUNEL assay. Part of the specimen was cryopreserved at -70°C for Western blot analysis. Reagents Primary antibodies including rabbit anti-human P53 (SC-6243) and mouse anti-human Bcl-2 (SC-7382) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Their quality and specificity were confirmed by the result of the Western blot analysis of D34 placental proteins (Figure 1 ). Rabbit anti-human cytokeratin (ZA0070), mouse anti-human actin (ZA0001), and mouse anti-Ki67 (ZM0166) were purchased from Zymed laboratories (San Francisco, CA, USA). Biotin labeled secondary antibodies, alkaline phosphatase (AP) conjugated avidin, horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG, HRP-conjugated horse anti-mouse IgG, and AP substrates "Vector-red" were from Vector Laboratories (Burlingame, CA, USA). Digoxigenindideoxy (DIG)-11-dUTP, TdT, blocking reagent, AP-conjugated anti-DIG antibody and 5-bromo-4-chloro-3-indoxyl phosphate/nitro-blue tetrazolium chloride (BCIP/NBT) were purchased from Roche-Boehringer-Mannheim (Mannheim, Germany). Proteinase K was purchased from Merck-Schuchardt (Darmstadt, Germany). Levamisole were purchased from Sigma-Aldrich (St Louis, MO, USA). SuperSignal ® West Pico substrate was from PIERCE Biotechnology (Rockford, IL, USA). Figure 1 Western blot analysis of Bcl-2 and P53 for monkey tissue obtained on D34 of pregnancy . Specific signals of Bcl-2 and P53 proteins were detected. No band was found in the control when antibodies were replaced with normal IgG of the same concentration and origin. Western blot Western blot was done as previously described [ 15 ] with slight modifications to verify the cross-reactive specificity of the antibodies with the monkey tissue. The tissue of implantation sites on D34 of pregnancy was homogenized and the supernatant (50 μg) from centrifugation was run on a 10% SDS-PAGE gel under reduced conditions. After being transferred to the polyvinylidene difluoride membrane, individual lanes were cut and blocked with 5% nonfat milk/PBS for 1 h, followed by incubation at 20°C for 1 h with the primary antibodies (IgG, 0.2 μg/ml) in 5% milk/PBS. The membranes were washed three times, 5 min for each, in 5% milk/PBS and incubated with HRP-conjugated horse anti-mouse IgG (0.2 μg/ml, for Bcl-2) or HRP-conjugated goat anti-rabbit IgG (0.04 μg/ml, for P53) in 5% milk/PBS for 1 h respectively. The membranes were washed in PBS three times 5 min for each, followed by 10 min of incubation with SuperSignal ® West Pico substrate, then exposed on x-ray film. For negative controls, primary antibodies were replaced with normal IgG of the same concentration and origin. TUNEL Apoptotic cells were identified by using the TUNEL technique [ 1 , 16 ]. The procedure was slightly modified based on Gao et al. [ 17 ] as the following. Deparaffinized and hydrated 4 μm sections were first treated with 10 μg/ml proteinase K at 37°C for 20 min, and then subjected to 3'-end-labelling of the DNA with 1 μM DIG-11-dUTP and 1 U/μl TdT at 37°C for 1 h. The sections were washes three times in Tris buffer, and incubated with blocking buffer (100 mM Tris, 150 mM NaCl, pH 7.5, and 1% blocking reagent) for 30 min at room temperature. Next, sections were incubated with the primary AP-conjugated anti-DIG antibody (1:500 in 1% blocking reagent, 100 mM Tris, and 150 mM NaCl, pH 7.5) at room temperature for 2 h, and then washed with Tris buffer. Staining was developed using the standard substrates NBT (337.5 μg/ml) and BCIP (175 μg/ml). Negative controls were similarly processed with the omission of TdT. Immunohistochemistry Serial 4 μm sections of tissue were deparaffinized and rehydrated through degraded ethanol. Antigen retrieval was performed by incubating the sections in 0.01 M citrate buffer (pH 6.0) at 98°C for 20 min followed by cooling at room temperature for 20 min. Non-specific binding was blocked with 5% (v/v) normal goat serum in PBS for 1 h. The sections were incubated with primary antibodies specific for P53 (1 μg/ml), Bcl-2 (2 μg/ml) or Ki67 (2 μg/ml) respectively in 2% goat serum overnight at 4°C. Sections were then washed three times with PBS (10 min each) and incubated with biotinylated secondary antibody (2 μg/ml) at RT for 30 min. 3 × 10 min successive washes were followed by incubation with avidin-AP complex (1:200, RT, 20 min). Sections were developed with standard substrates (337.5 μg/ml NBT and 175 μg/ml BCIP) or Vector Red AP substrates according to the manufacturer's protocol after another three washes. Endogenous AP activity was inhibited by supplement of 1 mM levamisole into substrate. The sections stained with Vector Red substrates were counter-stained using haematoxylin. Sections incubated with normal IgG instead of primary antibody served as negative controls. A double immunostaining technique using the antibodies to cytokeratin and actin was performed to localize the extravillous endovascular trophoblast cells. De-paraffinized sections were incubated with 3% H 2 O 2 in methanol for 10 min at room temperature to quench endogenous peroxidase after antigen retrieval treatment as described above. To detect the cytokeratin signal, the sections were washed (3 × 10 min in PBS), blocked for nonspecific signals, incubated sequentially with primary anti-human cytokeratin anbibody (1 μg/ml, RT, 1 h), secondary biotinylated goat anti-rabbit IgG (2 μg/ml, RT, 30 min), and avidin-peroxidase complex (1:200, RT, 20 min), and developed with DAB substrate solution in a similar way as described above. To detect actin signal, the procedure was repeated one more time with anti-human actin antibody (1 μg/ml, RT, 2 h) as primary antibody, AP conjugated horse anti-mouse IgG (1 μg/ml, RT, 40 min) as secondary antibody, and Vector Red developing AP substrate. As a result, the trophoblast cells were labeled brown and the blood vessel wall red. Microscopic assessment Placental samples from three individual monkeys for each group were analyzed. Experiments were repeated at least three times, and one representative from at least three similar results was presented. The mounted sections were examined using a Nikon microscope. For Ki67, the percentages of immunoreactive cells were assessed on at least 2000 cells in each tissue section; For TUNEL, the percentages of positive nuclei were assessed out of at least 2000 nuclei in each tissue section; For assessment of Bcl-2 staining intensity in cells of different compartments, semi-quantitative subjective scoring was performed by three blinded investigators using a 4-scale system with "-"= nil; "+/-"= weak; "+" = moderate; and "++" = strong as described by Yue et al. [ 18 ]. Results Apoptosis in implantation site of early pregnancy The TUNEL technique was used to identify cell types that underwent apoptosis in the implantation site of rhesus monkey on D17, D19, D28 and D34 of pregnancy. On D17 and D19, apoptotic nuclei were observed in the syncytiotrophoblast layer covering the basal feet of the anchoring villi (Figure 2 A, B , arrowhead) and in the villous stromal cells (Figure 2 A , arrow), but not in the cytotrophoblasts. The positive nuclei in the syncytiotrophoblast was only about 0.06%. On D28 and D34, the apoptotic nuclei were present in the syncytiotrophoblast covering the villi (Figure 2 C, D ), in the villous stromal cells (Figure 2 C, D , arrow), in the syncytiotrophoblast layer covering the basal feet of the anchoring villi (Figure 2 E ), and in the cytotrophoblasts within the cell columns (Figure 2 F ). On D28, the percentage of TUNEL-positive nuclei in the syncytiotrophoblast was 0.21%. As pregnancy progresses, the percentage increased to 0.34% on D34. In maternal compartment, a lot of apoptotic nuclei were detected in the stromal cells (Figure 2 G ) and glandular epithelium (Figure 2 H ). Figure 2 Apoptosis detected by TUNEL at the implantation sites of the rhesus monkey on D17 (A), D19 (B), D28 (C, G) and D34 (D, E, F, H) of gestation. Apoptotic nuclei were stained dark. Arrowhead and arrow in panel A – D indicated the nuclei of syncytiotrophoblast and villous stromal cells respectively. The insets in C and D showed the positive nuclei under a higher magnification. Note the syncytiotrophoblast layer covering the basal feet of the anchoring villi in E and the cell columns in F. G and H represent the stromal cells and glandular epithelial cells respectively in the endometrium. I was the negative control. St, syncytiotrophoblast; CT, cytotrophoblast; Vi, placental villi. Scale bars represent 50 μm. Proliferative activity in implantation site at early pregnancy Ki67 is a protein expressed in cycling cells from G1 to M phases and is widely used as a roliferative marker (19, 20). As shown in Figure 3 , Ki67 was expressed in the cytotrophoblasts and the villous stromal cells, but not in the syncytiotrophoblasts. As pregnancy progresses, the percentage of Ki67-positive cytotrophoblast cells lining the villi decreased from more than 85% on D17 to less than 25% on D34 (Panel A-D, and E, F for a higher magnification). However, the cytotrophoblasts at the proximal tip of cell columns remained highly proliferative (more than 70%) at all stages (Panel G and H). Figure 3 The proliferating activity revealed by Ki-67 immunostaining at implantation sites of the rhesus monkey on D17 (A, E, G), D19 (B), D28 (C) and D34 (D, F, H) of gestation. Panels A-D were under a lower magnification. Ki-67 protein was stained red, and nuclei blue. E and F were the placental villi under a higher magnification. G and H were the anchoring villi under a higher magnification. Vi, placental villi. ST, syncytiotrophoblast. CT, cytotrophoblast. Sc, stromal cell. En, endometrium. Scale bars represent 100 μm. Bcl-2 expression in implantation site at early pregnancy In order to study the mechanisms of the apoptosis observed at the fetal-maternal interface, the expression of Bcl-2 was investigated by using immunohistochemistry. At the early stages of placentation (D17, D19), Bcl-2 was only detected in the syncytiotrophoblast covering the cell columns (Figure 4, A and 4B ) and the extravillous cytotrophoblast (Figure 4C , arrow). At the later stages (D28, D34) it was detected in all the syncytiotrophoblast (Figure 4,D and 4E ), the villous stromal cells (Figure 4F , arrow), and the extravillous endovascular trophoblast cells (Figure 4G ), the fetal origin of these cells were indicated by the anti-cytokeratin antibody staining (Figure 4 G inset, brown), and the vascular wall was stained by anti-actin antibody (red). The pattern of Bcl-2 expression in the syncytiotrophoblast was similar to that of the apoptotic nuclei distribution (Figure 2 ). In the maternal compartment, Bcl-2 could be detected in some stromal cells (Figure 4H ). Notably, the cytotrophoblasts lining the villi, within the cell columns, and the glandular epithelia were negative for Bcl-2 staining. The semi-quantitative expression level of Bcl-2 in different cell types at the various stages was summarized in Table 1 . A gradual increase of Bcl-2 staining was observed in the syncytiotrophoblast as gestation advances. Figure 4 Immunohistochemical staining for Bcl-2 at implantation sites of the rhesus monkey. Bcl-2 staining is red, and nuclear counterstain blue. A, villous plancenta on D17. B, villous plancenta on D19. C, extravillous trophoblast cells in the basal plate of D17. D, villous plancenta on D28. E, villous plancenta on D34. F, villous plancenta on D34 under a higher magnification. G, the extravillous endovascular trophoblast cells; in the inset, the fetal origin of these cells was confirmed by anti-cytokeratin antibody (brown), and their position within the vascular wall was confirmed by anti-actin antibody staining (red). H, decidua. I, negative control. Vi, placental villi. ST, syncytiotrophoblast. CT, cytotrophoblast. Sc, stromal cells. Ge, glandular epithelium. Evc, extravillous cytotrophoblast. Scale bars represent 50 μm. Table 1 Semi-quantitative assessment of the immunohistochemical staining of Bcl-2 in the placenta of rhesus monkey. D17 D19 D28 D34 Syncytiotrophoblast lining the villi +/- +/- ++ ++ Syncytiotrophoblast covering the cell column + + ++ ++ cytotrophoblast lining the villi - - - - extravillous cytotrophoblast + + + + P53 expression in implantation site at early pregnancy The expression profile of P53 was also acquired by using the immunohistochemistry. On D17 and D19, the expression of P53 was only confined to a small number of nuclei in the syncytiotrophoblast (Figure 5,A,B ). On D28 and D34, its expression was observed not only in the syncytiotrophoblast (Figure 5C,D ) but also in the nuclei of cytotrophoblasts lining the villi (Figure 5E ) and within proximal tip of cell columns (Figure 5F ) where a proliferative activity was high as indicated by Ki67 staining (Figure 3 ). Clustered P53-positive nuclei were seen in the syncytiotrophoblast covering the basal feet of the anchoring villi (Figure 5G ), coincident well with the strong apoptosis detected by TUNEL (Figure 2E ). P53 was also expressed in some stromal cells (Figure 5H ) of the uterine endometrium. Figure 5 Immunohistochemical staining for P53 at implantation sites of the rhesus monkey on D17 (A), D19 (B), D28 (C, H), and D34 (D, E, F, G) of gestation. P53 was stained dark in nuclei. A-D were villous placenta under a lower magnification. The inset of panel A shows the staining in the syncytiotrophoblast covering the basal feet of the anchoring villi under a higher magnification. E, staining in villous placenta under a higher magnification. F, staining in cell columns. G, syncytiotrophoblast covering the basal feet of the anchoring villi under a higher magnification. H, the endometrium with arrows indicating stromal cells. ST, syncytiotrophoblast. CT, cytotrophoblast. Scale bars represent 50 μm. Discussion For the first time in present study, we investigated the expression of Bcl-2 and P53 in relation to apoptosis at the fetal-maternal interface of rhesus monkey at the very early stages (D17-D34) of gestation. Villous trophoblasts consist of cytotrophoblasts and syncytiotrophoblast. While cytotrophoblasts possess a brisk mitotic activity during the first trimester of gestation in human, the syncytiotrophoblast is incapable of cell division despite of a metabolic activity [ 1 ]. This fact implies that cell proliferation is differently regulated in these two cell types. The reports on the type of trophoblast cells undergoing apoptosis in the first trimester are controversial [ 1 , 21 , 22 ]. Our results further cleared that the apoptotic nuclei were distributed mainly in the syncytiotrophoblast at the early stages and in the cytotrophoblasts within the cell columns at later stages of pregnancy. In our previous study, Bax expression was found at the Fetal-Maternal Interface of Rhesus Monkey [ 17 ]. Bax is a Bcl-2 family member that promotes cell death susceptibility, possibly by countering the effect of Bcl-2 on cell survival through heterodimer interaction. Bax to Bcl-2 "rheostat" may be a critical factor in regulating apoptosis in multiplicate tissues. As shown in Figure 6 , Bax was found expressed in the placenta and glandular epithelium of endometrium and all kinds of cells in placental villi, and no obvious change was observed between different time points from D17 to D34 in placental villi. Therefore, we speculated that Bcl-2 may play a more important role on controlling the apoptosis in placental villi. The diffusive expression of Bcl-2 in syncytiotrophoblast obtained from the first trimester human placenta has been reported recently [ 7 , 23 - 25 ]. Our observation on the Bcl-2 expression in syncytiotrophoblast at later stages (D28-D34) agreed well with these data. As shown in this study, although Bcl-2 was expressed, apoptotic nuclei still exsisted in the same region. This phenomenon implies that the expression of Bcl-2 is not sufficient to completely inhibit the apoptosis in the syncytiotrophoblast. Therefore, the role of Bcl-2 here becomes an interesting question. Multiple nuclei sharing the same cytoplasm is a morphological characteristic of syncytiotrophoblast. In such cells, the apoptotic signal may be transmitted from one nuclear to another, and cause a spontaneous abortion. Therefore, the number of nuclei undergoing apoptosis in the syncytiotrophoblast should be limited by some mechanism in order to ensure normal embryo development in normal pregnancy [ 1 ]. We speculate that Bcl-2 may be included in this mechanism. The major role of apoptosis-associated Bcl-2 expression in the syncytiotrophoblast might be to limit the nuclear degradation to a special area and inhibit the spread of cell apoptosis signals to the other nuclei sharing the same cytoplasm, thus sustain cell survival in these multi-nucleated cells. Toki et al has also suggested that Bcl-2 might play a major role in avoiding the possible excessive nuclear degradation in syncytiotrophoblast [ 26 ]. Further studies, however, are needed to prove this speculation. The immunostaining for Bcl-2 was also detected in part of the extravillous and endovascular cytotrophoblast in our study. These subtypes of cytotrophoblast lost the capacity of proliferation (Ki-67-negative), but they did not undergo apoptosis (negative in TUNEL assay). Therefore, we hypothesize that Bcl-2 may also participate in regulation of the extravillous trophoblast apoptosis by stimulating the cellular survival. Figure 6 Immunohistochemical staining for Bax at implantation sites of the rhesus monkey on D28. Bax staining is brown, and nuclear counterstain blue. A, villous plancenta, positive staining was found in all the cells. B, endometrium, glandular epithelium was positive for Bax staining. Vi, placental villi. Ge, glandular epithelium. P53 was partly identified in some nuclei of the syncytiotrophoblast with the same position of apoptotic nuclei, in the basal feet of the anchoring villi in particular, but it is not clear whether the P53 was co-localized with the apoptotic signals. Activation of P53 in some cell types leads to either the cessation of cell growth or apoptosis [ 27 ]. Therefore, P53 protein might be related to cell cycle arrest or apoptosis in syncytiotrophoblast during early stage of placentation. Low level of P53 staining was detected in the cytotrophoblasts during the earlier stages of gestation (D17 and D19). However, at the later stages (D28 and D34), the expression was observed predominantly in the nuclei of cytotrophoblasts. The presence of P53 in cytotrophoblast in the primate was consistent with that observed in the human first trimester placenta [ 8 ]. Indeed, the TUNEL staining showed that the apoptosis seldom happened in the cytotrophoblast, with the exception of cytotrophoblast at proximal tip of cell columns during later stages of placentation (D28, D34) where a high proliferative activity and P53 expression were detected. This finding supports the hypothesis that a physiological upregulation of the P53 tumour suppressor gene might be a mechanism for controlling excessive trophoblastic proliferation in normal placentation [ 26 , 28 ]. It is known that early pregnancy is unique in its methods of cell proliferation control, the existing data suggest that some growth factors and transcription factors from the embryo and endometrium, such as CSF-1, VEGF, and transcription factors of the helix-loop-helix family, provide at least part of this control [ 29 ]. In addition, other studies found maternal age and some diseases, such as diabetes can also influence the apoptotic and proliferative activities in trophoblast cells [ 30 , 31 ]. Further investigations are required to uncover which endocrine event regulates the expression of Bcl-2 and P53.

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548668

Maps of the Sri Lanka malaria situation preceding the tsunami and key aspects to be considered in the emergency phase and beyond

Background Following the tsunami, a detailed overview of the area specific transmission levels is essential in assessing the risk of malaria in Sri Lanka. Recent information on vector insecticide resistance, parasite drug resistance, and insights into the national policy for malaria diagnosis and treatment are important in assisting national and international agencies in their control efforts. Methods Monthly records over the period January 1995 – October 2004 of confirmed malaria cases were used to perform an analysis of malaria distribution at district spatial resolution. Also, a focused review of published reports and routinely collected information was performed. Results The incidence of malaria was only 1 case per thousand population in the 10 months leading up to the disaster, in the districts with the highest transmission. Conclusion Although relocated people may be more exposed to mosquito bites, and their capacity to handle diseases affected, the environmental changes caused by the tsunami are unlikely to enhance breeding of the principal vector, and, given the present low parasite reservoir, the likelihood of a malaria outbreak is low. However, close monitoring of the situation is necessary, especially as December – February is normally the peak transmission season. Despite some losses, the Sri Lanka public health system is capable of dealing with the possible threat of a malaria outbreak after the tsunami. The influx of foreign medical assistance, drugs, and insecticides may interfere with malaria surveillance, and the long term malaria control strategy of Sri Lanka, if not in accordance with government policy.

Background After the tsunami hit Sri Lanka on 26 December 2004, news reports and public health agencies warned against the possibilities of an increase of vector borne diseases, in particular malaria and dengue. Immediately after the disaster, an estimated 860,000 people were displaced and more than 820 emergency camps established throughout the affected areas [ 1 ]. By 14 January, approximately 440,000 people were still sheltered in approximately 460 emergency camps [ 2 ]. Maps of the tsunami affected area, are presented elsewhere [ 3 ]. Malaria in Sri Lanka is of a highly unstable nature and has historically fluctuated greatly over the years and with significant seasonal differences. Sixty-five to eighty percent of the malaria cases are caused by Plasmodium vivax and the remainder by Plasmodium falciparum [ 4 ]. Recently, an overview of the spatial and temporal distribution of malaria in Sri Lanka over the period 1995 – 2002 was published in this journal [ 5 ]. The present publication aims at providing an update on the recent malaria situation, to October 2004 inclusive, and to discuss factors of relevance which may help in assessing the potential of the tsunami and ensuing events for exacerbating the malaria situation. Methods Malaria maps were based on monthly records over the period January 2004 – October 2004 (the most recent month for which data recording was complete at the time of writing) of microscopically confirmed malaria parasite positive blood smear readings, at district spatial resolution. These were collected by the Anti Malaria Campaign (AMC) Directorate of the Ministry of Health from aggregated disease records reported by governmental hospitals and mobile clinics. Additionally, in the temporal analysis, monthly data by district for the period 2001 – 2002, and data by sub district for 1995 – 2000 as described by Briët et al . [ 5 ] were used. The quality of routinely collected information on malaria is described elsewhere [ 5 ]. As denominator for the incidence calculations, population estimates for 2001 and beyond were made by exponential interpolation (and extrapolation to December 2004) (Figure 1 ) as follows. For the districts Mannar, Vavuniya, Trincomalee and Batticaloa, that were not or incompletely enumerated in the 2001 census because of limited access of the government to these conflict affected areas, the 2001 mid-year population was taken from data posted by the North East Provincial Council [ 6 ]. For all other districts, the 2001 mid-year population was taken from data posted by the Department of Census and Statistics [ 7 ]. The natural annual (mid-2001 to mid-2002 and mid-2002 to mid-2003) population growth rates for Jaffna, Kilinochchi, Mullaitivu, Mannar, Vavuniya, Trincomalee and Batticaloa were taken as the average annual growth rates of all the other districts, calculated from mid year population statistics estimated by the Department of Census and Statistics. For all other districts, these growth rates were calculated for each district separately. For mid 2003 to mid 2004 and beyond, the growth rates for mid-2002 to mid-2003 were used. Further, the number of internally displaced persons (IDPs) was taken into account [ 8 ]. For each month and for each district, the net number of immigrants was calculated as the total number of IDPs moved to or within a district since 2001, minus the number of IDPs moved from or within that district. This net number of immigrants was then distributed over the months proportionately to the monthly statistics of IDPs moved to or within a district. Additionally, the number of monthly immigrants from India was taken into account. Figure 1 Population. Map of population by divisional secretariat division in Sri Lanka estimated for mid December 2004. One dot represents 1,000 people. Sources: Department of Census and Statistics , North East Provincial Council and UNCHR . A focused review of literature has been performed, identifying crucial information for the outbreak preparedness and control during the emergency phase. The intent was not to present a complete review of malaria in Sri Lanka but to provide information useful for an assessment of the current situation. A general review of malaria in Sri Lanka can be found in Konradsen, Amerasinghe et al . [ 4 ]. Results and discussion Present malaria situation and parasite reservoir The country-wide malaria incidence increased from January 1996 to January 2000, with the typical seasonality of high peaks around January and lower peaks around June – July, but it has decreased dramatically since January 2000 (Figure 2 ). Figure 3 shows that the recent decrease in the overall malaria incidence in the country is predominantly due to a decrease in incidence in the districts of Vavuniya and Kilinochchi in the north. The decrease was least in the district of Ampara, making it the most malarious district during January to October 2004 (Figures 4 and 5 ). Although districts on the east coast which were badly affected by the tsunami had been relatively malarious in 2004 as compared to the rest of the country, the maximum of around 1 case per 1000 people over a 10 month period in these districts is remarkably low. The total number of malaria cases in 2003 was 10,510, the lowest since the resurgence of malaria in 1968 when the eradication campaign failed [ 9 ]. The year 2004 promises to be three times lower with only 3,037 cases recorded up to October, as opposed to 9,682 cases recorded during January – October 2003. The low incidence is not related to a decline in collection effort, which has decreased only marginally (Figure 2 ). At the time of writing, malaria incidence information for the months of November and December was still incomplete. In November 2004, without the figures for the non endemic districts Gampaha and Kalutara, and data from a few medical institutions in Mannar and Mullaitivu missing, thus far only 230 cases were recorded. In the malaria endemic districts, December, January and February are normally the months with the highest malaria incidence [ 5 ], so a rise in case numbers may normally be expected. However, neither the district authorities nor the Epidemiology Unit of the Ministry of Health have reported any malaria cases from the affected areas for 30 December 2004 – 13 January 2005, based on the spot checks performed and the review of available health information [ 10 ]. Asymptomatic infections of P. falciparum and P. vivax and dormant stages of P. vivax normally provide the parasite reservoir for bridging periods of low seasonal transmission (with unsuitable conditions for mosquito vectors). Under the present policy of administering primaquine in addition to chloroquine (see section on diagnosis and treatment), the reservoir of dormant stages of P. vivax will be low and this will delay a possible outbreak. It must be emphasized that the low level of malaria transmission in the recent past does not guarantee that localized or even island wide epidemics will not occur. In the past, even after periods of very low levels of malaria transmission, outbreaks have occurred, often due to constraints placed on the public health system, by unusual rainfall patterns or by yet unexplained factors. Figure 2 Monthly parasite and blood smear examination incidence patterns. Monthly parasite incidence patterns of P. falciparum and P. vivax malaria combined per 1000 population (red line on logarithmic scale), blood smears examined per 1000 population (black line on logarithmic scale), and percentage of blood smears positive for malaria (blue line) from January 1995 to October 2004 in Sri Lanka. Figure 3 Trends of parasite incidence. Trends of parasite incidence of P. falciparum (red bars) and P. vivax (blue bars) malaria over the years November 1995 – October 1996 (bar on far left) to November 2003 – October 2004 (bar on far right), at district resolution. The height of the bars in the legend represents an annual parasite incidence of 10 cases per 1000 persons. Figure 4 Parasite incidence of Plasmodium vivax. Map of the districts of Sri Lanka with P. vivax malaria cases per 1000 population over the period January – October 2004. Figure 5 Parasite incidence of Plasmodium falciparum. Map of the districts of Sri Lanka with P. falciparum malaria cases and mixed infections of both P. vivax and P. falciparum per 1000 population over the period January – October 2004. Capacity of health care services and disease surveillance An important factor to consider in the current situation is the capacity of the existing health care service. Following the tsunami the Sri Lanka Ministry of Health reported 22 hospitals and nine administrative buildings damaged or completely destroyed, mostly in Ampara and Trincomalee districts [ 11 ]. It has been reported that at least 40 doctors and hundreds of other medical staff have died as a consequence of the tsunami and a much higher number injured or in other ways affected by the disaster [ 12 ]. However, both the central government departments and organizations in the field report sufficient medical staff. Even in the conflict affected areas in the north and east, the AMC has been able to monitor malaria and react timely with control measures to outbreaks since the peace process started in 2002. Also, the AMC has long standing experience with mobile clinics for malaria detection and treatment in remote areas. Lack of co-ordination among the many government departments, international aid agencies, non-governmental organizations and private individuals involved in the first phase of the emergency continues to be an important issue weeks into the disaster. According to the Ministry of Health media reports, more than 600 foreign doctors are now working in the affected areas, but few, if any, are registered with the Sri Lanka Medical Council or other relevant authorities [ 13 ]. With doctors from many countries, language barriers are also a perceived problem. In some places, central stocks of medical supplies were destroyed, including the Regional Medical Supply Division in the Ampara District. However, sufficient drugs have been imported during the days and weeks following the disaster. The World Health Organization has drawn up plans for antimalarials, insecticides and spray equipment to be made available on request. Although the increased capacity at the district and provincial levels has improved the co-ordination, a risk remains that local needs for health care are not adequately covered in spite of the availability of significant resources. In some parts of the island, especially areas in the east, affected both by the destruction caused by the tsunami and by exceptionally heavy rainfall in the weeks following, distribution of drugs has been problematic and this has left certain communities vulnerable. Whereas the overall capacity to provide treatment and routine malaria control activities, in general, has not been severely hampered, the routine health information system will have been constrained by the large number of autonomous health camps set up, and their lack of integration with the established surveillance system. It is essential to establish a system for monitoring malaria in the affected areas. Many people are moving back to their old place of residence trying to rebuild livelihoods and it will be essential for the public health authorities to keep contact with these communities to prevent an increase in malaria going unnoticed. Diagnosis, treatment and drug resistance In Sri Lanka, microscopy on blood smears or use of rapid diagnostic test kits have been the standard diagnostic procedure, and precedes the prescription of drugs to the patient. In the current situation, with the many small health clinics established within emergency camps, it is likely that the use of rapid diagnostic kits would be the more feasible means of confirmation. The first line drugs recommended for malaria treatment in Sri Lanka is still a chloroquine and primaquine (PQ) combination for cases of P. vivax as well as P. falciparum infection. Primaquine is not administered to children below one year, and those with known G-6PD enzyme deficiency, and for pregnant mothers. So far, there have been no reports of chloroquine-resistant P. vivax infections in Sri Lanka. The first chloroquine-resistant P. falciparum case was reported in 1984 [ 14 ]. Up to 62% in vivo chloroquine resistance has been recorded in malarious areas [ 5 , 15 - 17 ]. For chloroquine resistant cases of P. falciparum the government recommended drug is sulphadoxine-pyrimethamine (SP). However, SP is not recommended for the last trimester of pregnancy, first six weeks of lactation and for children below two months of age. The first SP-resistant case of P. falciparum was reported in 1992 in Polonnaruwa district. Up to 1999, five to six cases have been reported by the AMC. More recently (January – June 2002), SP resistant P. falciparum has been documented in the Northern Province [ 17 ]. For SP resistant cases quinine is recommended, but only as an in-patient treatment. In the current emergency situation, with many (foreign) doctors working autonomously, the diagnosis and treatment practices may depart from the established government guidelines and new antimalarials are also likely to be brought in. Moreover, the current practice of restricting SP to government hospitals will be difficult to enforce. Similarly, introduction of low quality and obsolete drugs will be difficult to counter at community level at the current stage of supervisory capacity and co-ordination level. Drugs have been reported stolen from warehouses, allegedly finding their way to private trade establishments [ 18 ]. Overall, it is crucial that the development of drug resistance is monitored closely and inappropriate drugs are actively phased out of the market to avoid later complications in case management. Environmental changes and vector breeding The seawater brought inland by the tsunami has mixed with monsoon rainwater to form puddles of varying salinity. Also, thousands of muddy surface water puddles have been created as a result of destruction and rehabilitation activities that are already underway. The brackish puddles are expected to favour the breeding of Anopheles subpictus sibling species B, which is a well-known coastal breeding species in Sri Lanka. However, it has not been directly incriminated as a field vector in Sri Lanka, despite its susceptibility to P. falciparum [ 19 ]. Nevertheless, Abhayawardana et al. [ 20 ] found peak malaria transmission in coastal areas of Puttalam in the presence of An. subpictus sibling species B and the complete absence of Anopheles culicifacies (the main malaria vector in Sri Lanka), and suggested that this An. subpictus sibling may have a role in transmission. It is noteworthy, that freshwater An. subpictus (which is now known to consist of a mixture of species A, C and D), which breeds in muddy rain fed puddles, has been consistently incriminated in malaria transmission in many inland areas of Sri Lanka [ 4 ]. Another species that is likely to breed prolifically in muddy rain-fed pools is Anopheles vagus . This species has been linked as a vector responsible for a malaria outbreak in southern Sri Lanka [ 4 , 21 ]. On present evidence, neither An. subpictus nor An. vagus , are likely to cause major malaria epidemics but could, at high density, be responsible for focal outbreaks that need quick action. Thus, it is important that an entomological monitoring programme be set up in the period leading up to and during the south west monsoon that is expected during May to June 2005 in the tsunami affected western and southern Sri Lanka. It should be noted that the infamous Asian brackish water breeding malaria vector Anopheles sundaicus , which is a threat in the tsunami-affected areas in Indonesia, Myanmar, and the Andaman and Nicobar islands [ 22 ], does not occur in Sri Lanka. The main vector in Sri Lanka is An. culicifacies type E [ 23 , 24 ], which breeds mainly in pools formed in river and stream beds, and therefore, its density is mostly dependent on temporal and spatial variations in rainfall and river flow. Anopheles culicifacies also breeds in abandoned gem mining pits, agricultural wells and to a lesser extent in pools in agricultural water reservoirs [ 4 ]. It is unlikely that the rubble constituting a major part of the landscape in the affected areas creates breeding opportunities for An. culicifacies , unless it blocks waterways and creates pooling. Post-tsunami development activities may revive banned sand mining practices in rivers. If this happens, clear water pools created by these sand mining activities may serve as breeding sites for An. culicifacies [ 4 ]. Overall, it is very unlikely that the principal vector of malaria in Sri Lanka will breed prolifically in brackish water habitats or other habitats that may be created during the post tsunami reconstruction phase. Similarly, the principal dengue vector in Sri Lanka, Aedes aegypti , does not breed in saline water [ 25 ]. However, it may find plenty of rainwater-filled containers amidst the rubble created by the disaster for it to breed. Vector control strategies and insecticide resistance The Colombo based Head Office of the AMC gives the overall guidelines for island wide vector control, while each province works out a plan for control activities based on the distribution and level of malaria transmission. Several malaria vector control interventions are currently employed within the country. In all districts, residual insecticide spray activities are focused on areas where malaria transmission has been established by confirmed malaria cases. The control of anopheline larvae using mostly chemicals focuses on sites close to human habitation. Small-scale application of larvivorous fish and environmental modifications are also carried out. Since 1997, mosquito nets, which are biannually treated with insecticide, are distributed free of charge in malarious areas. During the last two years, the main control effort has been through these nets. Since January 2004, 80,000 nets with long lasting insecticide have been distributed. Also, nets are available for purchase from outlets in most parts of the country. Studies in Sri Lanka over the 1990s on An. culicifacies and a range of potential secondary vectors such as An. subpictus and An. vagus have shown high level of resistance to either organochlorines, organophosphates or to both groups of insecticides [ 4 , 26 - 28 ]. DDT and Malathion are no longer recommended since An. culicifacies and An. subpictus has been found resistant. Currently, synthetic pyrethroids such as Cyfluthrin, Deltamethrin, Etofenprox, and Lambda-cyhalothrin are being used in the country. At present, Fenitrothion is the only organophosphate used for vector control. A study conducted by Abhayawardana from 1990 to 1992 on An. subpictus found 68% and 54% susceptibility to Malathion and Fenitrothion, respectively, for inland species (sibling species A), whereas for coastal species (primarily sibling species B) it was 100%. However, the latter was found resistant to permethrin [ 20 ]. From several districts it was reported that, as a result of the tsunami, organisations have brought in insecticides not normally used or no longer recommended for vector control in Sri Lanka (P. Amerasinghe, personal communication). Vector resistance, in the light of the introduction of new insecticides, needs to be monitored and if necessary action should be taken. Exposure of the affected community The majority of the people initially affected by the disaster are still living in emergency camps or in other places close to the coast. At the time of writing, to the best of our knowledge, relatively few people have moved from areas of low or no malaria transmission to areas of high transmission. However, during the next phases, when people may be resettled in semi-permanent and later in permanent housing, communities may be relocated from areas where they have had no malaria experience to malarious areas. In these situations, the communities' capacity to cope with malaria infection will be low. Despite distribution of nets to many camps, and intensified vector control in some areas, people in the emergency camps (schools, temples, mosques, etc.) and those returning to damaged houses are more exposed to mosquito bites than in pre-disaster housing, due to the open nature of the shelter. Additionally, most families will have lost mosquito nets or other means to protect against mosquito bites. It is more difficult to assess the protective effect of tents that have been set up in most of the semi-permanent camps established. The location of semi permanent and permanent settlements may have a significant effect on the risk of infection. Epidemiological studies from other parts of Sri Lanka have shown that people living within 750 m of a stream with An. culicifacies breeding, were at significantly higher risk for malaria than people living further away [ 29 ]. Conclusions This paper provides maps of both P. vivax and P. falciparum malaria incidence distribution on the island of Sri Lanka at district resolution in the 10 months preceding the tsunami, and an analysis of monthly malaria incidence in the country since January 1995. The malaria incidence was historically low, which implies a limited parasite reservoir in the human population. In spite of the fact that the months of December - February are normally the peak period for transmission, given the transmission level in the months leading up to the disaster, the risk of a large-scale outbreak seems to be limited. However, the low transmission levels over the past years may also have made people less alert to possible outbreaks, and the population would have less protective immunity towards the disease. The environmental changes resulting from the tsunami do not create particular opportunities for breeding of the principal malaria vector An. culicifacies but potential does exist for less important species such as An. subpictus and An. vagus . People living in emergency camps or returning to pre-disaster areas of residence are at higher risk of mosquito bites than normal. In spite of the emergency, the capacity of the public health authorities to perform malaria preventive and curative interventions remains high and essential supplies and staff capacity is not a problem. However, co-ordination of assistance and maintaining a strong surveillance system remain significant areas of concern. Increased attention to the establishment of a monitoring system including both parasitological and entomological parameters is recommended. Likewise, the large inflow of donated drugs and insecticides outside government control will potentially have long term implications on malaria control and case management, and especially the quality of administered drugs and the development of drug resistance requires careful monitoring. Authors' contributions GNLG collected the malaria data. OJTB checked the data, calculated incidence, made the maps. FK, FPA and PHA performed the focused literature review. All authors helped write, read and approved the final manuscript.

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493285

Transient spleen enlargement in peripheral blood progenitor cell donors given G-CSF

The administration of granulocyte colony-stimulating factor (G-CSF) to peripheral blood progenitor cell (PBPC) donors causes spleen length to increase, but the duration of enlargement is not known. Eighteen healthy subjects were given 10 μg/kg of G-CSF for 5 days and a PBSC concentrate was collected by apheresis. Ultrasound scans were used to assess craniocaudal spleen length before and after G-CSF administration. Mean spleen length increased from a baseline length of 10.7 ± 1.3 cm to 12.1 ± 1.2 cm on the apheresis day (p < 0.001). Ten days after apheresis, spleen length fell to 10.5 ± 1.2 cm and did not differ from baseline levels (p = 0.57), but in 3 subjects remained 0.5 cm greater than baseline length. Increases in spleen length in PBPC donors are transient and reversible.

Background Peripheral blood progenitor cell (PBPC) concentrates donors are routinely given granulocyte colony-stimulating factor (G-CSF) to increase the concentration of circulating PBPCs and hence the number of progenitors that can be collected by apheresis. Typically 10 to 16 μg/kg of G-CSF are given subcutaneously daily for 4 to 6 days prior to the collection [ 1 - 3 ]. The administration of G-CSF to healthy PBPC concentrates donors is very safe, but there have been four reports of spontaneous rupture of the spleen and splenectomy in healthy allogeneic PBPC donors given G-CSF [ 4 - 7 ]. While spontaneous rupture of the spleen in PBSC donors given G-CSF is rare, the administration of G-CSF for five days causes spleen length to increase in almost all healthy donors [ 8 , 9 ]. The increase in length is highly variable, but the mean increase is approximately 13%. Spleen length begins to return to baseline levels quickly, but it is not known how long it takes to return to baseline. In a previous study of 20 PBPC donors given 10 μg/kg of G-CSF for 5 days, we found that spleen length measured four days after the last dose of G-CSF was less than the length on the day of apheresis but greater than baseline values [ 8 ]. Since allogeneic PBPC donors may be at risk for splenic rupture while the spleen is enlarged, it is important to determine when spleen size returns to baseline levels. The purpose of this study was to determine if spleen length returns to baseline 10 days after G-CSF-mobilized PBPC concentrates are collected by apheresis from healthy subjects. Methods Study design All of the subjects were in good health and were donating G-CSF-mobilized PBPC concentrates for laboratory investigations. The donors were given 10 μg/kg of G-CSF (Filgrastim, Amgen, Thousand Oaks, CA) daily for 5 days, and a PBSC concentrate was collected approximately 2 hours after the last G-CSF dose was given. PBPC concentrates were collected with a CS3000 blood cell separator (Baxter Health Care Corporation, Round Lake, IL). Spleen length was evaluated by ultrasound examination three times: prior to the administration of the first dose of G-CSF, on the day of apheresis, and 10 or 11 days after apheresis. This study was approved by the Institutional Review Board of the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland. Spleen length assessment Craniocaudal spleen length was assessed using ultrasound (Acuson Aspen Advanced, Siemens Medical Solutions Ultrasound Division, Mountain View, CA) with a sector transducer (Acuson, 4V1, 4.0 mHz frequency, Siemens Medical Solutions). The intra-observer error for measuring spleen length using ultrasound is 4.9 mm when healthy subjects are evaluated at separate settings [ 10 ]. Blood counts and chemistries Complete blood counts were performed with an automated cell counter (Cell Dyne 4000, Abbott Diagnostics, Santa Clara, CA). CD34+ cell counts were performed using a flow cytometer (Beckman Coulter, Miami, FL). Statistical analysis Spleen lengths measured before and after the G-CSF course were compared using 2-tailed paired t-tests. Spleen length changes were also compared among males and females and Caucasians and non-Caucasians using 2-tailed t-tests. The percent change in spleen length was compared with blood counts, CD34+ cell counts, and donor age using linear regression. Results and Discussion The median age of the 18 healthy subjects was 34 years old and ranged from 22 to 55 years of age. Eight of the subjects were male, 13 of the donors were Caucasian, 3 were African American, and 2 Asian. Apheresis day spleen length increased above baseline length in 17 of 18 donors (Figure 1 ). The mean spleen length increased from a baseline level of 10.7 ± 1.3 cm to 12.1 ± 1.2 cm on the day of apheresis (p < 0.001). The mean increase in length was 1.4 ± 0.8 cm or 13.1 ± 8.9%. Figure 1 Spleen length changes in healthy subjects donating G-CSF-mobilized PBPC concentrates. Eighteen subjects were given 10 μg/kg of G-CSF for 5 days and a PBPC concentrate was collected approximately 2 hours after the last dose of G-CSF. Spleen length was measured by ultrasound before G-CSF was given (day 1), immediately after the PBPC concentrate collection (day 5), and approximately 10 or 11 days after the collection (day 15 or 16). Spleen length was measured 10 or 11 days after apheresis in all 18 subjects. The mean spleen length 10 days after apheresis fell to 10.5 ± 1.2 cm and was less than the apheresis day spleen length (p < 0.001). There was no difference between the 10-day post-apheresis and pre-G-CSF spleen length (p = 0.57). The spleen length 10 days after apheresis was less than the apheresis day length in all 17 donors whose spleen length increased. However, the spleen length 10 days after apheresis remained more than 0.5 cm greater than baseline spleen length in 3 subjects (Table 1 ). All 3 were Caucasian and 2 were female. Their spleen length remained 6.7%, 11.0%, and 10.5% greater than baseline levels. Two of these subjects had relatively large increases in spleen length of 18.3% and 26.3%, but their spleen length fell considerably 10 days after apheresis. It is likely that the spleen returned to baseline length in these 2 subjects shortly after the third ultrasound was preformed. The other subject's spleen increased only 12.0% in length and 10 days after apheresis had changed little. It is not certain when this subject's spleen returned to baseline length. Table 1 Peripheral Blood Stem Cell Donors Whose Spleen Length 10 Days After Apheresis Remained More than 0.5 cm Greater than Baseline Length Spleen Length (cm) Donor Age (Yrs) Gender Race Baseline Apheresis Day 10 Days Post-Apheresis Enlargement 10 Days Post-Apheresis 7 23 Female Cauc 10.4 12.3 11.1 0.7 13 22 Female Cauc 10.0 11.2 11.1 1.1 14 54 Male Cauc 9.5 12.0 10.5 1.0 Cauc = Caucasian PBPC donors with the largest increase in spleen size may be at the greatest risk for spontaneous splenic rupture. In order to determine if subject age, gender, race, or post-G-CSF blood counts affected the magnitude of spleen enlargement, we assessed the relationship between these factors and percent change in spleen length in the 18 subjects in this study and 20 subjects studied previously [14]. There was no difference in the spleen length increase between males and females (12.3 ± 9.7% versus 14.8 ± 8.0%, p = 0.40) or between Caucasians and non-Caucasians (13.5 ± 8.4% versus 13.6 ± 10.4%; p = 0.98). Spleen length increase was not related to donor age (r = 0.13). In addition, spleen length increase was not related to preapheresis CD34+ (r = 0.04), WBC (r = 0.05), neutrophil (r = 0.07), lymphocyte (r = -0.14), monocyte (r = -0.04), and platelet counts (r = 0.19) or hemoglobin level (r = -0.04). Conclusions Healthy PBPC concentrate donors given G-CSF should be warned that their spleens will be enlarged for a brief time and that they may be at risk of splenic rupture. Most donors are likely at risk for splenic rupture only during the time of G-CSF administration and for about 10 days after the completion of the G-CSF course. Since splenic enlargement may persist for longer periods in some donor, until more data are available it may be worthwhile to counsel PBPC donors to avoid activities that could lead to abdominal and splenic trauma for 2 to 3 weeks after the last dose of G-CSF.

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539278

Effects of the group I metabotropic glutamate receptor agonist, DHPG, and injection stress on striatal cell signaling in food-restricted and ad libitum fed rats

Background Chronic food restriction augments the rewarding effect of centrally administered psychostimulant drugs and this effect may involve a previously documented upregulation of D-1 dopamine receptor-mediated MAP kinase signaling in nucleus accumbens (NAc) and caudate-putamen (CPu). Psychostimulants are known to induce striatal glutamate release, and group I metabotropic glutamate receptors (mGluR) have been implicated in the cellular and behavioral responses to amphetamine. The purpose of the present study was to evaluate whether chronic food restriction increases striatal MAP kinase signaling in response to the group I mGluR agonist, DHPG. Results Western immunoblotting was used to demonstrate that intracerebroventricular (i.c.v.) injection of DHPG (500 nmol) produces greater activation of ERK1/2 and CREB in CPu and NAc of food-restricted as compared to ad libitum fed rats. Fos-immunostaining induced by DHPG was also stronger in CPu and NAc core of food-restricted relative to ad libitum fed rats. However, i.c.v. injection of saline-vehicle produced greater activation of ERK1/2 and CREB in CPu and NAc of food-restricted relative to ad libitum fed rats, and this difference was not seen when subjects received no i.c.v. injection prior to sacrifice. In addition, although DHPG activated Akt, there was no difference in Akt activation between feeding groups. To probe whether the augmented ERK1/2 and CREB activation in vehicle-injected food-restricted rats are mediated by one or more GluR types, effects of an NMDA antagonist (MK-801, 100 nmol), AMPA antagonist (DNQX, 10 nmol), and group I mGluR antagonist (AIDA, 100 nmol) were compared to saline-vehicle. Antagonist injections did not diminish activation of ERK1/2 or CREB. Conclusions These results indicate that a group I mGluR agonist induces phosphorylation of Akt, ERK1/2 and CREB in both CPu and NAc. However, group I mGluR-mediated signaling may not be upregulated in food-restricted rats. Rather, a physiological response to "i.c.v. injection stress" is augmented by food restriction and appears to summate with effects of the group I mGluR agonist in activating ERK1/2 and CREB. While the augmented cellular response of food-restricted rats to i.c.v. injection treatment represents additional evidence of enhanced CNS responsiveness in these subjects, the functional significance and underlying mechanism(s) of this effect remain to be elucidated.

Background Chronic food restriction increases central sensitivity to rewarding and motor-activating effects of psychostimulants and direct dopamine receptor agonists [ 1 ]. Corresponding adaptive changes at the cellular level include increased psychostimulant-induced DA release in nucleus accumbens core [ 2 ], and upregulation of D-1 DA receptor-mediated MAP kinase signaling in dorsal and ventral striatum, with consequent increased activation of CREB and the immediate early gene, c-fos [ 3 , 4 ]. Changes in striatal glutamate receptor function have not been investigated but are of interest in light of findings that (i) psychostimulants induce striatal glutamate release [ 5 - 7 ], (ii) amphetamine-induced activation of striatal MAP kinase, CREB, and c-fos is attenuated by a group I metabotropic glutamate receptor antagonist [ 8 ], and (iii) the augmented effects of striatal D-1 receptor stimulation in food-restricted rats include hyperphosphorylation of the NMDA receptor NR1 subunit [ 9 ]. The group I mGluRs (mGluR1/5) are of particular interest. Group I mGluRs are densely expressed in striatal medium spiny neurons [ 10 ] and activate phospholipase C, resulting in hydrolysis of phosphoinositides and activation of Ca 2+ -dependent signaling cascades [ 11 ]. The group I mGluR agonist, DHPG, increases the phosphorylation of extracellular signal-regulated kinase (ERK) and the transcription factor CREB when infused into dorsal striatum [ 12 ], as do cocaine injected systemically and the D-1 agonist SKF-82958 injected into the lateral ventricle [ 4 , 13 ]. DHPG also elicits hyperlocomotion resembling that induced by DA receptor agonists, and the effect is not attenuated by the D-1 DA receptor antagonist, SCH23390 [ 10 ]. It is therefore possible that glutamate and group I mGluR function contribute to the changes in psychostimulant-induced behavioral responses and striatal cell signaling in food-restricted subjects. The purpose of the first experiment of this study was to compare dorsal and ventral striatal cell signaling in response to intracerebroventricular (i.c.v.) administration of the group I mGluR agonist, DHPG, in ad libitum fed and food-restricted rats. A dose of 500 nmol was used based on pilot work and published reports indicating that this dose is sufficient to exert behavioral effects [ 14 ] and stimulate PI hydrolysis [ 11 ] while being below threshold for producing convulsive behavior [ 15 ]. A second experiment was based on results of the first experiment and sought to elucidate the observed increase in striatal ERK1/2 and CREB phosphorylation in food-restricted rats injected i.c.v. with saline-vehicle. Results Experiment 1 In both the caudate-putamen and nucleus accumbens ERK1/2 phosphorylation was increased by food restriction (Cpu: F 1,18 = 10.8, p < .005; NAc: F 1,18 = 8.2, p < .01) and by DHPG injection (Cpu: F 1,18 = 6.6, p < .02; NAc: F 1,18 = 10.6, p < .005). However, there was no interaction between feeding condition and drug treatment in either brain region (Cpu: F 1,18 = 0.6; NAc: F 1,18 = 0.3). This analysis supports the impression (see Figures 1 & 2 , top panel) that food restriction did not actually increase the group I mGluR-mediated response to DHPG; rather, increased signaling in the food-restricted group, irrespective of i.c.v. injection treatment, appears to have summated with DHPG to produce a greater net effect in the food-restricted than ad libitum fed group. An identical pattern of results was obtained for CREB phosphorylation (Figures 1 & 2 center panel; Cpu: F diet; 1,18 = 6.2, p < .025; F drug; 1,18 = 3.9, p = .06; F diet × drug; 1,18 = 1.2; NAc: F diet; 1,18 = 12.9, p < .0025; F drug; 1,18 = 15.3, p < .001; F diet × drug; 1,18 = 2.7, p > .10). Interestingly, Akt phosphorylation was induced by DHPG but did not differ between food-restricted and ad libitum fed groups in either the Cpu (F drug; 1,18 = 4.5, p < .05; F diet; 1,18 = 0.4; F diet × drug; 1,18 = 0.4) or NAc (F drug; 1,18 = 12.7, p < .0025; F diet; 1,18 = 0.4; F diet × drug; 1,18 = 2.4, p > .10; Figures 1 & 2 bottom panel). In previous studies, striatal Fos-immunostaining was greater in food-restricted relative to ad libitum fed rats injected i.c.v. with d-amphetamine or the D-1 DA receptor agonist SKF-82958, but not saline-vehicle [ 3 , 16 ]. Therefore, in a small sample of ad libitum fed and food-restricted subjects, brains were processed for Fos-immunostaining and revealed stronger DHPG-induced staining in CPu (t(7) = 3.6, p < .01) and NAc core (t(7) = 2.8 p < .025) but not NAc shell (t(7) = 1.1) of food-restricted, relative to ad libitum fed, subjects (see Figure 3 ). During the period between DHPG injection and sacrifice, rats, which remained in their home cages, did not display any obvious behavioral responses to the drug. Experiment 2 In rats without lateral ventricular cannulas that received no injection treatment prior to sacrifice, ERK1/2 phosphorylation did not differ between feeding groups (Figure 4 ; Cpu: t(8) = 1.2; NAc: t(8) = 1.0), nor did CREB phosphorylation (Cpu: t(8) = 1.2; NAc t(8) = 0.1). In food-restricted rats injected with GluR antagonists prior to sacrifice, neither ERK1/2 nor CREB phosphorylation differed from that observed in food-restricted rats injected with saline-vehicle (Figure 5 ). Discussion Adding to the results of Choe and Wang who demonstrated DHPG-induced activation of ERK1/2 and CREB in dorsal striatum [ 12 ], the present study has demonstrated DHPG-induced activation of ERK/12 and CREB in both CPu and NAc following lateral ventricular infusion. While i.c.v. injection allows for the possibility that these effects were not directly mediated by striatal group I mGluRs, the proximity of striatum to the lateral ventrical and the high density of mGluRs in these structures support the likelihood of direct effects. Direct effects are also supported by the observed activation of Akt which mediates group I mGluR signal transduction [ 17 ]. As previously seen in rats challenged with the D-1 dopamine receptor agonist, SKF-82958, DHPG-induced activation of ERK1/2, CREB and c-Fos were greater in food-restricted than ad libitum fed rats. However, the pattern of results in this study – i.e. augmented ERK1/2 and CREB activation in food-restricted rats injected with vehicle, and no difference in Akt activation between feeding groups – suggests that group I mGluR signaling was not enhanced. Rather, a cellular-activating effect of the injection procedure was enhanced by food restriction and summated with the effect of DHPG. This response of vehicle-injected subjects was clearly a response to some aspect of the i.c.v. injection procedure because similarly food-restricted rats that were not challenged in any way prior to sacrifice displayed levels of pERK1/2 and pCREB that were essentially identical to those of ad libitum fed rats. Furthermore, the augmented activation of ERK1/2 and CREB in vehicle-injected food-restricted rats was not mediated by Akt because Akt, though activated by DHPG, did not differ between food-restricted and ad libitum fed rats injected with vehicle or DHPG. Attribution of the augmented DHPG-induced ERK1/2 and CREB phosphorylation to a "nonspecific" response to i.c.v. injection distinguishes the group I mGluR from the D-1 DA receptor. Although a modestly enhanced ERK response to i.c.v. vehicle infusion was seen in the NAc of food-restricted rats in the prior study, the dramatically increased activation of ERK1/2 and CREB by SKF-82958 in CPu and NAc was dissociable from any response to the injection procedure [ 4 ]. Even mild stressors are known to stimulate DA [ 18 ] and Glu [ 19 , 20 ] release in striatum, and it is possible that food-restriction augments this physiological response or the cell signaling induced by it. Because the ionotropic (AMPA and NMDA) as well as group I mGluRs are abundant in striatum and all three receptor types mediate MAP kinase signaling [ 12 , 21 , 22 ], corresponding antagonists were injected i.c.v. and compared to vehicle. It was reasoned that if the physiological response to the injection procedure involves one of these GluRs, ERK1/2 and CREB phosphorylation would be decreased, relative to vehicle, in the group(s) receiving the corresponding antagonist. The MK-801 treatment was also a potential probe for mediation by D-1 DA receptors because D-1 DA agonist-induced activation of ERK1/2 and CREB are dependent on the NMDA receptor in food-restricted subjects [ 9 ]. Results of this test did not provide support for mediation by one of the GluR types. Because only one dose of each antagonist was used, these results must be considered preliminary. However, considering the proximity of striatum to the lateral ventricle, and the fact that the doses used have exerted measurable cellular, physiological or behavioral effects in other studies [ 23 - 26 ] there is some doubt about involvement of GluRs in the effect of "injection stress". An interesting possibility to consider is mediation of the response by brain-derived neurotrophic factor (BDNF). Food-restricted rats have elevated striatal BDNF levels which appear to be involved in an enhanced neuroprotective response to diverse insults [ 27 ]. BDNF activates striatal ERK1/2 which, unlike glutamate-induced activation of Akt, ERK1/2 and CREB is not blocked by a PI 3-kinase inhibitor [ 21 ]. It is therefore possible that an enhanced BDNF-mediated activation of ERK1/2 and CREB in food-restricted rats represents a neuroprotective response to intraventricular infusion. Conclusions The present results indicate that a group I mGluR agonist activates Akt, ERK1/2 and CREB in both the CPu and NAc. Further, activation of ERK1/2, CREB, and c-Fos are stronger in food-restricted than in ad libitum fed rats. The augmented response is not attributed to increased Group I mGluR function but, instead, to an augmented response of food-restricted rats to the i.c.v. injection procedure. Some evidence casting doubt on attribution of the latter response to GluRs was obtained. It will be of interest to evaluate whether striatal MAPK signaling in food-restricted rats is generally augmented in response to stressors or whether this response is peculiar to i.c.v. infusion. In addition, it will be of interest to evaluate whether this physiological response is mediated by BDNF and its TrkB receptor. Methods Subjects and surgery All experimental procedures were approved by the New York University School of Medicine Institutional Animal Care and Use Committee and were performed in accordance with the "Principles of Laboratory Animal Care" (NIH publication number 85-23, revised 1996). Subjects were male Sprague-Dawley rats (375–425 g) housed individually in plastic cages with free access to food and water except when food restriction conditions applied. Animals were maintained on a 12-h light/dark cycle, with lights on at 07:00 h. Rats were anesthetized with ketamine (100 mg/kg; i.p.) and xylazine (10 mg/kg; i.p.) and stereotaxically implanted with a 26-gauge guide cannula (Plastics One, Roanoke, VA USA) in the right lateral ventricle. The cannula was permanently affixed to the skull by flowing dental acrylic around it and four surrounding mounting screws. Patency of the guide cannula was maintained with an occlusion stylet. Several days after surgery, cannula placements were confirmed by demonstration of a vigorous and short latency (i.e. <60 s) drinking response to 50 ng of angiotensin II. Food restriction and habituation Seven days following surgery, half the subjects were put on a food restriction regimen whereby a single 10 g meal of Purina (Gray Summit, MO USA) rat chow was delivered at approximately 17:00 h each day. Rats continued to have ad libitum access to water. Once body weight had declined by 20–25% (approximately 15 days) daily food allotments were titrated for an additional week to maintain stable body weight. During the 3 weeks required for food-restricted rats to attain and stabilize at target body weights, all rats were habituated, on five occasions, to the handling and injection procedures to be employed on the terminal day of the experiment. Drug treatment In Experiment 1, six food-restricted and six ad libitum fed rats received i.c.v injections of sterile 0.9% saline (5 μl). Six food-restricted and six ad libitum fed rats received i.c.v injections of the group I metabotropic glutamate receptor agonist, DHPG (500 nmol in 5 μl; Tocris Cookson, Ellisville, MO, USA). An additional five food-restricted and four ad libitum fed rats received i.c.v. injections of DHPG and brains were processed for Fos-immunostaining. In Experiment 2, six unoperated food-restricted rats and six unoperated ad libitum fed rats were prepared and habituated as above and received no unusual handling or drug injection on the terminal day of the experiment. In the second part of Experiment 2, twenty four food-restricted rats with lateral ventricular cannulas were prepared and habituated as in Experiment 1. On the terminal day of the experiment, six were injected i.c.v. with saline vehicle (5.0 μl), six were injected with the AMPA glutamate receptor antagonist, DNQX (10 nmol; Sigma-Aldrich, St. Louis, MO), six were injected with the NMDA glutamate receptor antagonist, MK-801 (100 nmol; Sigma-Aldrich), and six were injected with the group I mGluR antagonist, AIDA (100 nmol; Tocris Cookson). Antagonist doses were chosen on the basis of prior findings [e.g. [ 23 , 26 , 28 ]] and/or being just below threshold for producing noticeable motoric abnormalities. For i.c.v injection, solutions were loaded into a 30 cm length of PE-50 tubing attached at one end to a 250-μl Hamilton syringe filled with distilled water and at the other end to a 33-gauge injector cannula which extended 1.0 mm beyond the implanted guide. The 5.0 μl injection volume was delivered over a period of 95 s. One minute following injection, internal cannulas were removed, stylets replaced, and animals were returned to home cages. Lysate preparation Prior studies have indicated that MAP kinase activation is transient and the optimal time-point to study phosphorylation of ERK 1/2 after physiological or pharmacological treatment is 15–20 min [ 29 ]. Therefore, all rats, except the twelve unoperated/uninjected rats, were killed 20 min after injection by brief exposure to CO 2 followed by decapitation. The twelve unoperated/uninjected rats were simply removed from home cages and exposed to CO 2 followed by decapitation. Brains were rapidly removed and immediately frozen in powdered dry ice. Five hundred-micrometer sections were cut using an IEC Minotome cryostat, and CPu and NAc were micropunched, under an Olympus dissecting microscope, from a series of 8 consecutive frozen sections. The tissue was then homogenized in 10 volumes of 50 mM Tris-HCl, pH 7.5 containing 50 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1 mM Na 3 VO 4 , 40 mM β-glycerophosphate, 50 mM NaF and 5 mM Na 4 P 2 O 7 , 1% Tx-100, 0.5 μM okadaic acid, 0.5% sodium deoxycholate and 0.1% SDS, followed by centrifugation and protein determination using BCA reagent kit as described by the manufacturer (Pierce) Supernatants were mixed with 5 × SDS-PAGE sample buffer, boiled for 5 min, cooled on ice and kept at -80°C until future use. Western blotting Protein (10–30 μg per lane) was separated by electrophoresis on precast 10% polyacrylamide gels (Cambrex, East Rutherford, NJ, USA). Precision Plus protein standard molecular weight markers (Bio-Rad, Hercules, CA, USA) were also loaded to assure complete electrophoretic transfer and to estimate the size of bands of interest. The gels were transferred to nitrocellulose membrane (Osmonics) for 2 h, with a constant voltage of 100 volts. Membranes were blocked for 1 hr at room temperature with blocking buffer, 5% non fat dry milk in 50 mM Tris-HCl, pH 7.5 containing 150 mM NaCl and 0.1% Tween 20 (TBS-T), then probed overnight at 4°C using primary monoclonal antibodies for phospho-(Thr202/Tyr204)-p44/42 ERK1/2 (mouse monoclonal, 1:2000; Cell Signaling, Beverly, MA, USA), or polyclonal antibodies for phospho-Akt (Ser 473) (rabbit polyclonal, 1:1000 dilutuion, Cell Signaling), and phospho (Ser 133) CREB (rabbit polyclonal, 1:2000; Upstate Biotechnology, Lake Placid, NY, USA). Total levels of ERK1/2, Akt and CREB were detected on the same blots using anti-rabbit p42/44 ERK1/2 antibody 1:2000, (Cell Signaling), anti-rabbit total Akt (1:2000 dilution, Cell Signaling), or anti-rabbit CREB antibody (1:2000 dilution, Calbiochem). After detection of phosphorylated ERK1/2, phospho-Akt and CREB blots were stripped with 25 mM Glycine, pH 2.0 containing 1% SDS for 30 min at room temperature, washed six times in TBS-T buffer, blocked in blocking buffer for 1 h and then incubated overnight at 4°C in total ERK1/2, total Akt or CREB antibody. After probing with primary antibodies and washing with TBS-T buffer (3 × 5 min), membranes were incubated with 1:2000 dilution horseradish peroxidase conjugated anti-mouse or 1: 2000 dilution anti-rabbit IgG (Cell Signaling). Proteins were visualized using a chemiluminescense ECL kit (Pierce). Densitometric analysis of the bands was performed using NIH image software. Phospho-p42/44 MAPK, phospho Akt and phospho CREB values were normalized to total p42/44 MAPK, Akt and CREB values respectively. Immunohistochemistry Ninety min after DHPG injection rats were anesthetized with sodium pentobarbital (50.0 mg/kg,i.p.) and transcardially perfused with isotonic phosphate buffered saline (PBS) followed by 4% paraformaldehyde in PBS. Brains were then removed and maintained in 20% sucrose at 4°C for 48 h. Forty μm sections were cut on an IEC Minotome cryostat and collected in a cryoprotective solution. Fos immunostaining was carried out using a rabbit polyclonal c-Fos antiserum (Oncogene Science-Calbiochem, La Jolla, CA) and the avidin-biotin peroxidase complex (ABC; Vector laboratories). Sections were washed in 1% sodium borohydride followed by PBS and incubated for 2 hrs in 4% normal goat serum plus 1% BSA in PBS containing 0.2% Triton X-100 (Sigma-Aldrich) to block nonspecific binding. This was followed by incubation, overnight, with rabbit polyclonal c-fos antiserum (1:5000 dilution). Following several PBS washes, sections were incubated with a secondary antiserum (Vector, Burlingame, CA) for 60 min and subsequently reacted with avidin-biotin complex (ABC) (Vector). The peroxidase reaction was visualized with a chromogen solution containing 100 mM nickel sulfate, 125 mM sodium acetate, 10 mM imidazole, 0.03% diaminobenzidine (DAB), and 0.01% hydrogen peroxide at pH 6.5. Sections were then mounted on chrome-alum coated slides, dehydrated, and coverslipped. Objective counting of c-Fos positive cells in CPu at coronal levels +1.7 and -0.3 mm, and NAc core and shell at coronal level +1.5 mm in relation to bregma [ 30 ] was accomplished with a light microscope (Olympus, CK2) equipped with a Sony XC-77 video camera module coupled to an MCID image analysis system (Imaging Research Inc., St. Catherines, Canada). For each region, bilateral grain counts from three to five consecutive sections were measured to arrive at an average bilateral value per rat. Because results obtained from anterior and posterior levels of CPu were essentially the same, results from the two levels were combined and averaged for each rat. Data analysis For each Western blot, film exposure time was set as needed to visualize distinct bands in the control samples of each experiment. Immunoblots were analyzed using NIH imaging software. For each blot, relative phospho-protein levels were calculated from the ratio of optical density of the phosphorylated protein/total protein to correct for small differences in protein loading. In addition, tubulin levels were analyzed in several representative gels and no differences were observed between treatment groups. Results were expressed by comparison to the normalized control, which in Experiment 1 was defined as the ad libitum fed group injected with vehicle. In the first part of Experiment 2, unoperated/uninjected ad libitum fed rats served as control. In the second part of Experiment 2, food-restricted rats injected with i.c.v. saline vehicle served as control. Differences between treatment conditions in Experiment 1 were analyzed by two-way analysis of variance (ANOVA; feeding condition × injection treatment). Differences between treatment conditions in the first and second parts of Experiment 2 were analyzed by student's t-test and one-way ANOVA, respectively. Authors' contributions YP conducted the majority of immunoblotting experiments plus the immunohistochemistry experiment, contributed to experimental design and assisted in manuscript preparation. YB provided technical supervision of immunoblotting experiments and assisted in manuscript preparation. KC contributed to design of the study, assisted in all experiments, and wrote the final draft of the manuscript.

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514498

Mapping the distribution of Loa loa in Cameroon in support of the African Programme for Onchocerciasis Control

Background Loa loa has recently emerged as a filarial worm of significant public health importance as a consequence of its impact on the African Programme for Onchocerciasis Control (APOC). Severe, sometimes fatal, encephalopathic reactions to ivermectin (the drug of choice for onchocerciasis control) have occurred in some individuals with high Loa loa microfilarial counts. Since high density of Loa loa microfilariae is known to be associated with high prevalence rates, a distribution map of the latter may determine areas where severe reactions might occur. The aim of the study was to identify variables which were significantly associated with the presence of a Loa microfilaraemia in the subjects examined, and to develop a spatial model predicting the prevalence of the Loa microfilaraemia. Methods Epidemiological data were collected from 14,225 individuals living in 94 villages in Cameroon, and analysed in conjunction with environmental data. A series of logistic regression models (multivariate analysis) was developed to describe variation in the prevalence of Loa loa microfilaraemia using individual level co-variates (age, sex, μl of blood taken for examination) and village level environmental co-variates (including altitude and satellite-derived vegetation indices). Results A spatial model of Loa loa prevalence was created within a geographical information system. The model was then validated using an independent data set on Loa loa distribution. When considering both data sets as a whole, and a prevalence threshold of 20%, the sensitivity and the specificity of the model were 81.7 and 69.4%, respectively. Conclusions The model developed has proven very useful in defining the areas at risk of post-ivermectin Loa -related severe adverse events. It is now routinely used by APOC when projects of community-directed treatment with ivermectin are examined.

Background Knowledge of the spatial distribution of Loa loa is important in countries involved in the African Programme for Onchocerciasis Control (APOC) and is also now a significant issue for the Global Programme to Eliminate Lymphatic Filariasis (GPELF). Both programmes rely on the wide-scale distribution of anti-helminthic drugs to poor communities using community-directed drug distribution schemes. A problem was first observed in Cameroon where a series of reports of severe and sometimes fatal encephalopathic reactions to ivermectin (Mectizan ® ) in individuals with high Loa loa microfilarial counts was made [ 1 - 3 ]. Similar problems may occur when albendazole (used to control lymphatic filariasis) is distributed in Loa loa endemic areas although evidence for this is contradictory [ 4 - 7 ]. Loa loa is associated with tropical "eye worm" (migration of adult worms across the sub-conjunctiva), Calabar swelling, oedemas and prurities but is not considered as pathogenic as other filarial worms, and is consequently less well studied. It is transmitted by species of horse-flies ( Chrysops spp.), most commonly Chrysops dimidiata and C. silacea which inhabit the forest areas of West and Central Africa, extending to the Ethiopian border. Severe and fatal reactions to ivermectin have been associated with individuals with high Loa loa microfilarial loads (more than 30,000 microfilariae (mfs) per ml of blood) and those with more than 50,000 mfs/ml are considered at very high risk [ 1 , 8 - 10 ]. Changes in the protocols for drug administration and post-treatment surveillance in areas considered to be at-risk of severe reactions to ivermectin have been implemented [ 11 , 12 ], but the detailed geographic distribution of Loa loa remains unclear [ 13 ]. Such information is essential in terms of developing safe treatment, and above all surveillance strategies across the region and, given the vast area which may be affected, it is recognized that rapid assessment methods must be developed to evaluate the risk of severe reactions in communities co-endemic for loiasis and onchocerciasis. Recent studies have shown that there is a close relationship between intensity of microfilariae infection and prevalence rates of Loa loa [ 14 , 15 ] suggesting that a distribution map based on prevalence of infection alone (and not intensity, which would require time-consuming counting of mfs) would provide sufficient information to delineate areas of high risk of severe reactions. The aim of our study was to develop a map indicating the areas where Loa loa infection may be high enough (i.e. with a prevalence of Loa microfilaraemia in adults exceeding 20%) that poses an operational problem for drug distribution by the Community-Directed Treatment with Ivermectin (CDTI) strategy [ 16 ]. As a preliminary step we created a risk model for Loa loa in West and Central Africa based on the relationship of crude Loa loa prevalence data (obtained from a literature search) to a wide range of environmental variables. Initial results suggested that land/forest cover derived from NOAA-AVHRR satellite data (i.e. collected by the Advanced Very High Resolution Radiometer on board the satellite series operated by the National Ocean and Atmospheric Administration of the USA), and soil type (from the FAO digitised soil map of Africa), are significant predictors and a preliminary risk map was produced [ 17 ]. However, when this first model was tested against field data from Cameroon it was found to poorly represent areas of high risk of infection in certain districts [ 18 ]. Possible reasons for this include: (a) the low spatial resolution of the satellite and environmental data used (1 km) which was unable to identify narrow gallery forests in savannah areas and/or (b) the use of prevalence data from various sources which had not been standardised by age or sex. In order to improve the quality of epidemiological data being used to develop the model, we created a new prevalence database from a series of surveys conducted by the team of the Institut de Recherche pour le Développement (IRD) at the Centre Pasteur du Cameroun (CPC). A second independent data set, collected as part of a project supported by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) to calibrate a rapid clinical diagnosis of Loa loa (RAPLOA) [ 15 ], was used to validate the results of the model. Methods Epidemiological data Epidemiological data used to create the model was obtained from a series of field studies undertaken by the IRD-CPC laboratory in Cameroon during the period 1991–2001 in which the presence of Loa loa parasites in the blood of individuals was assessed. The data come from five regions: (a) the forest-savannah mosaic area of the Mbam and Kim division (Central province); (b) the degraded forest area of the Lekie division (Central province); (c) the dense forest area of the southern part of the Central province; (d) the highland savannah area of the Western Province; and (e) the savannah areas of the districts of Banyo and Bankim, in the Adamaoua province. The Ministry of Public Health of Cameroon provided ethical approval for these surveys. The total number of subjects included in the analysis was 14,225 individuals for 94 villages. Study participants consisted of individuals over the age of 5 years who gave their consent or for whom consent was obtained from the parent or guardian. Individuals who had had filaricidal drugs, such as ivermectin or diethylcarbamazine, in the previous five years were not included in the study. The arrival of the research team in the villages was announced to the population one week before the event through the local authorities and teachers. The objective of the examinations were clearly explained, and it was stated that each individuals results would be returned. On the given day, the team settled at a central place of the village (usually at the chief's home, or in a school), and all the volunteers were examined between 10.00 and 16.00 hours. A standardized quantity of capillary blood was obtained with a non-heparinized micro-capillary tube. Prior to 1994, the amount taken was 30 μl, and thereafter, 50 μl. After Giemsa staining, the slides were examined under a microscope and the presence of Loa mfs was recorded. The latitude and longitude of all study villages were obtained from either the ordinance survey map or a global positioning system. Thus the data set included village name, longitude and latitude, alongside data on individuals examined (age, sex, standard size of blood sample taken and presence/absence of Loa loa infection). A summary of the epidemiological data used is presented in Table 1 . Table 1 Epidemiological data sets used in development of the spatial model for predicting the prevalence of Loa microfilaraemia Region Sex* Blood sample (μl) Prevalence of Loa loa Age range No. subjects No. villages Banyo-Bankim M 50.00 22.21 5–98 1783 16 Banyo-Bankim F 50.00 20.00 5–90 1815 South of Central Province M 30.00 32.81 5–80 381 5 South of Central Province F 30.00 27.37 5–80 453 South of Central Province M 50.00 24.76 5–86 941 17 South of Central Province F 50.00 18.49 5–90 1206 Lekie Department M 30.00 26.33 5–91 1052 14 Lekie Department F 30.00 19.10 5–92 1340 Lekie Department M 50.00 23.96 5–80 359 3 Lekie Department F 50.00 21.41 5–90 369 Mbam et Kim M 30.00 12.37 5–99 1293 24 Mbam et Kim F 30.00 4.53 5–95 1281 Western Province M 50.00 6.22 15–99 916 15 Western Province F 50.00 3.76 15–99 1036 * M = male and F = female General principles of the analysis The aim of the study was to identify variables which were significantly associated with the presence of a Loa microfilaraemia in the subjects examined. Besides data on individuals (age, sex, size of blood sample), we investigated whether some variables describing the environment of their place of residence (see list of variables below) were significantly related to the microfilaraemia. For these environmental variables, distance operatives indicating whether or not villages were within 5 km of potential at-risk variables were created. Distance operatives were based on the normal dispersal range of Chrysops which has been shown to be within 5 km of their breeding sites [ 19 ]. In order to design a valid modelling structure, univariate analysis was first undertaken of the relationship between, on the one side, individual and environmental variables, and on the other side the variable of interest, i.e. the presence/absence of Loa loa mfs in the individual. Then we developed a series of logistic regression models (multivariate analysis) to describe variation in the prevalence of Loa loa microfilaraemia using individual level co-variates (age, sex, μl of blood taken for examination) and village level environmental co-variates. Logistic regression is useful to predict the presence or absence of a characteristic or outcome based on values of a set of predictor variables. It is similar to a linear regression model but is suited to models where the dependent variable is dichotomous. Logistic regression coefficients can be used to estimate odds ratios for each of the independent variables in the model. In our example the dichotomous variable is the presence or absence of Loa loa infection in each individual in the sample. Those variables which did not have a significant relationship with age and sex adjusted prevalence were excluded from further analysis. All variables were analysed at individual level. Environmental variables and data sets included in the analysis The distribution of the human population in sub-Saharan Africa is relatively well described from population census data obtained at the national and sub-national level. The population data for 1990 used in this analysis was obtained from the Global Resource Information Database of the United Nations Environment Programme (UNEP-GRID), Eros Data Centre [ 20 ]. These population density surfaces (with 1 km resolution) have been derived from a model including population estimation for countries, population of major urban centres and transportation network and accessibility. They have been used in numerous studies involving population distribution in Africa, including studies on estimating the burden of malaria [ 21 ]. The USGS (United States Geological Survey) hydrologic digital dataset provides detailed descriptions of the topography of the area, including elevation (Digital Elevation Model, DEM), slope, aspect (direction of maximum rate of change in elevation between each cell and its eight neighbours and representing direction of slope), flow accumulation (defining amount of upstream area draining into each cell) [ 22 ]. Satellite data indicating the 'greenness' of the environment (Normalised Difference Vegetation Index, NDVI) was obtained from the VEGETATION sensor launched in 1998 onboard the SPOT 4 satellite system. NDVI images have been specifically tailored to monitor global land surfaces' parameters on a daily basis with a medium spatial resolution of one km [ 23 ]. They permit monitoring of seasonal and inter-annual variation in vegetation status and have been shown elsewhere to be important correlates of the spatial and temporal changes in the distribution of insect vectors of disease [ 24 ]. Free access is given to 10 daily synthesized maximum value composite products 3 months after insertion in the VEGETATION archive [ 25 ]. Thirty-six decadal NDVI images were obtained from SPOT VEGETATION satellite sensor data archive for 1999. The VEGETATION data used consists of 10 daily NDVI products, compiled from daily synthesis over the previous ten days, for the entire African continent, to which both radiometric corrections and geometric corrections have been applied to enhance product quality. The true value of NDVI was calculated from the 8 bit decadal images (1–255) using ((digital number*0.004)-0.1). From the true values images, mean, minimum, maximum, median, standard deviation were calculated for the year 1999. In order to take into account the flight distance of the vector, 5 km buffers were created around each study village and the mean value of the NDVI variable was obtained and used in the development of the logistic regression model. Validation of the model Epidemiological data used to validate the model was obtained from a multicentric study supported by TDR which was designed to assess the relationship between clinical and parasitological indicators of Loa loa endemicity [ 15 , 26 ]. The surveys were conducted by three research teams (comprising epidemiologists, parasitologists, social scientists and clinicians) based in Buea and Yaoundé, both in Cameroon, and in Calabar, Nigeria. Only data from Cameroon are used in our validation process. The Buea study sites were in South-West and North-West Provinces of Cameroon; in these areas, a total of 4532 individuals over the age of 15 years, living in 42 villages, participated in the survey. The study sites for the Yaoundé team were located in the East Province of Cameroon, where 3181 persons of the same age, living in 32 localities, were examined. A standardized questionnaire was administered to participants from whom finger-prick blood samples were collected and examined for Loa loa mfs. Model validation was undertaken through correlating model outputs with the independent data set. Results Univariate analysis While Loa loa prevalence when regressed against population density was not found to be significantly correlated, a strong and significant linear relationship ( r 2 = 0.7699; P < 0.001) was found with age below 40 years (Figure 1 ). In our analysis we used this linear relationship for all individuals below 40 but then treated all those above 40 as though they remained at this age since any subsequent change with age was deemed non-significant. This broken stick approach was considered to be a reasonable strategy and much simpler than fitting a non-linear model. Figure 1 Relationship between prevalence of Loa loa microfilaraemia and age. Prevalence rates were significantly ( P < 0.001) higher in males than in females (19.8% n = 6725 and 15.2% n = 7500 respectively). Sex was therefore entered (as a categorical variable) in the logistic regression model. As one would expect, prevalence rates were significantly higher ( P < 0.001) in individuals from whom the blood smear was prepared using a volume of 50 μl as compared with those from whom 30 μl of blood were taken. Blood sample size was therefore entered (as a categorical variable) in the logistic regression model. After investigating the value of the DEM and its associated files (slope, aspect, flow accumulation) in predicting microfilaraemia prevalence using univariate and multivariate regression statistics, the DEM alone was chosen as a predictive variable for the development of a logistic regression model. Because the relationship between Loa loa prevalence and the DEM was found to be non-linear (Figure 2 ) the DEM data was divided up into 250 m interval classes and treated as a categorical variable. Figure 2 Relationship between prevalence of Loa loa microfilaraemia and elevation. Since univariate analysis of the SPOT VEGETATION NDVI data against prevalence indicated a non-linear relationship [Figure 3a and 3b ], the satellite data were entered into the model as a series of numeric variables (the original, the square and the cube) for the mean, the minimum, the maximum and the standard deviation. Standard deviation and maximum NDVI were most strongly correlated with prevalence using univariate analysis. Figure 3 Relationship between prevalence of Loa loa microfilaraemia and standard deviation of NDVI (a); and between prevalence of Loa loa microfilaraemia and maximum annual NDVI (b). Multivariate analysis Separate and combined models were created which included all individual level co-variates and altitude for both buffered and non-buffered SPOT VEGETATION data variables. Final model choice was based on (a) the simplicity and biological validity of the model (b) the predictive capacity of the model when assessed using bootstrap methodology (where half of the data was extracted randomly from the data set and tested against model results developed from the remaining half) and (c) the ability of the model to be extended over the large geographic area involved in the APOC programme. Thus, besides the three individual-level covariates (sex, age and blood sample size), only three village level environmental co-variates were kept in our model: maximum annual NDVI, standard deviation of the NDVI, and elevation. In the final model, the probability of a female individual being infected with Loa loa is represented by: 1/(1+e - z ), where (for our example a female aged ≥ 40 years, and a blood sample of 50 μl): [Z] = ([Sex]*0.221) + ([Age] * 0.049) - ([Blood sample size]*0.542) + ([Maximum NDVI 2 ] * 74.38) - ([Maximum NDVI 3 ] * 58.816) + ([Standard deviation NDVI] * 11.788) + (([Elevation] < 250) * 1.133) + (([Elevation] = 250-500) * 0.865) + (([Elevation] = 500-750) * 0.981) + (([Elevation] = 750-1000) * 1.171) - (([Elevation] = 1000-1250) * 0.623) - (([Elevation] = 1250-1500) * 1.516) - (([Elevation] = 1500-1750) * 1.342) - (([Elevation] = 1750-2000) * 0.669) - 22.883 (constant), where [Sex] = 0 for females, and 1 for males; and [Blood sample size] = 0 for 30 μl, and 1 for 50 μl. Thus for our example, age, sex and blood sample size variables are presented for a fixed value because of their non-spatial nature. The probability of infection increases (+) with increasing value for sex (0 versus 1), with increasing age, and with increasing standard deviation of NDVI and Maximum NDVI 2 ; it decreases (-) with Maximum NDVI 3 (indicating that the relationship to maximum NDVI is non linear), and increases for categories of elevation between 0 and 1000 metres after which it decreases. The probability of infection decreases with increasing value of blood sample size (0 versus 1) even if larger blood sample should normally allow better detection of the infection. This could be due to the fact that larger samples have been taken often in places where infection rates are low, in the highlands, and this is presumably a result of interacting with the other variables. A similar model can be made for males and individuals at any other age. The model was developed using 50% of the data (randomly selected) and then tested on the remaining 50% of the data. When the model results were plotted against the observed data an r 2 of 0.6033 was obtained (Figure 4 ). In addition, when one takes the threshold of 20% as the Loa microfilaraemia above which there is a risk of post-ivermectin encephalopathy, the sensitivity and specificity of the model (respectively: the proportion of villages with a measured prevalence ≥ 20%, and which were correctly predicted as such by the model; and the proportion of villages with a measured prevalence <20%, and correctly identified as such by the model) were found to be 77.1 and 80.0%, respectively (Table 2 ). Figure 4 Relationship between observed prevalence of Loa loa microfilaraemia and predicted prevalence. Table 2 Distribution of the villages examined, according to their observed and predicted prevalence of Loa microfilaraemia Observed prevalence <20% ≥ 20% Predicted prevalence First dataset <20% 48 8 (collected by IRD-CPC) ≥ 20% 12 27 Dataset used for validation <20% 20 5 (collected by TDR) ≥ 20% 18 31 Mapping the model results: an Environmental Risk Map for Loa loa In order to standardize the variables relating to individuals (which cannot be mapped) we chose values that best represent the average adult population (above the age of 15) i.e. 50:50 male – female ratio, age 40 and a blood sample of 50 μl. Age 32 was the approximate average age of individuals in the data sets used to develop the model. Using age 40 (the average age of individuals >15 years of age) in the model was therefore likely to slightly over-represent infection in these data sets but the final model would be directly comparable with current policy to exclude individuals below the age of 15 from rapid epidemiological surveys for loiasis [ 14 , 15 ]. In order to create a model, which included both males and females, two separate models were created, one for each sex and the mean of the two model results taken. Validation of the model Since the chosen model was based on environmental variables, which could be mapped across the entire country, it was possible to extrapolate the model results to the whole of Cameroon (Figure 5 ) and compare them with the results of the independent TDR survey. Correspondence between observed prevalence from the TDR verification data set and the model results was found to be very close: the sensitivity and the specificity of the model, when using the prevalence threshold of 20%, were 86.1 and 52.6%, respectively. Five villages were observed to have high prevalence rates (>20%) despite being model predictions for prevalence below 20% (Table 2 ). Two villages, Nguri and Ngu, located in the North-West province, were classified as extremes (Figure 6 ). Inaccuracies in the georeferencing of the village location or the spatial data used to create the model may account for this, as could the possibility that the local population regularly visit the Chrysops infested area nearby. The three other villages (Ntem, Boum and Baktala) are all villages from the Eastern Province of Cameroon which were sited on very localised areas where model predictions for <20% prevalence occurred. These villages were all within 1 km of areas where model results predicted >20% prevalence. Figure 5 Predictive model of Loa loa prevalence for Cameroon overlaid with the observed prevalence data. Figure 6 Inset of verification villages in North-West province of Cameroon (note the position of Nguri and Ngu). When one considers both datasets as a whole, and the prevalence threshold of 20%, the sensitivity and the specificity of the model were 81.7 and 69.4%, respectively. Discussion Detailed information on distribution of disease and levels of endemicity is a prerequisite for effective planning of control programmes. This has been an important element in the planning of the African Programme for Onchocerciasis Control (APOC) where community-directed treatment with ivermectin (Mectizan ® ) has been targeted at areas of hyper- and meso-endemicity, identified by Rapid Epidemiological Mapping [ 27 ]. The expansion of the APOC programme in Cameroon has been delayed due to severe (sometimes fatal) adverse reactions in patients receiving ivermectin who were co-infected with Loa loa. This has led to caution in defining new areas for the expansion of the programme and a requirement for identifying areas of high risk of Loa loa using a potentially rapid and extensive methodology. Thomson et al . [ 17 ] developed a preliminary model using satellite mapping based on existing knowledge of distribution and prevalence, and recently a method of rapid assessment using a simple questionnaire, called RAPLOA, has been developed [ 15 ]. This paper further develops the satellite-derived risk map of Loa loa by using improved satellite data sets and detailed information on infection rates in villages in various ecological zones in Cameroon. The resulting model is able to predict prevalence risk throughout southern Cameroon with a greater accuracy than hitherto available. The final model has been chosen because of the good fit between observed and predicted prevalence and is based on elevation and 1 km SPOT VEGETATION data. In order to understand the value of the risk map it is important to note that (a) while the 1 km resolution of the environmental data cannot reveal the small and localised muddy breeding sites of the Chrysops vectors, their general habitat is well captured by the model and it is recognised that the 1 km resolution is much less than the dispersal capacities of the adult flies; and (b) the model is not dependant on the seasonal or inter-annual variability of the vector density, since this is not reflected in fluctuations in Loa microfilaraemias, because the adult worm life-span is much longer (4–17 years). Among the areas in which the prevalences of Loa loa are particularly high, according to the Environmental Risk Map (ERM), two should be pointed out, because they are also those where most of the cases of serious adverse events (SAEs) have been recorded so far. The first one is the Lekie Department (Central Province), in the Sanaga valley, where 53 of the 63 probable or possible Loa loa encephalopathy cases recorded between 1989 and 2001 in Cameroon have occurred [ 3 ]. Besides the high level of endemicity, this cluster of cases may be related to the high population density there, as well as the proximity of the area with the capital, Yaoundé, which probably led to a high reporting rate; however, other hypothesis should be considered, such as specific susceptibility of the local human populations, or special pathogenicity of the local Loa "strain" [ 28 , 29 ]. The latter possibility is currently investigated in Cameroon, and in the Mayumbe forest (Democratic Republic of Congo), another area where some 15 cases of fatal SAEs were reported in December 2003. The second area of interest shown by the ERM in Cameroon is the Tikar plain, near Bankim, between the Western, North-West and Adamaoua provinces, where several cases have been recently reported, though the area's vegetation is of shrub savanna type. The ERM also shows those areas where CDTI projects against onchocerciasis may be implemented in a near future, and where the risk of SAEs is probably high: the Eastern and Southern Provinces, and the north-western part of the Littoral Province. While the use of satellite-derived environmental data for evaluation of disease risk has been developed for several vector borne diseases (malaria, Rift Valley fever, visceral leishmaniasis, tick-borne encephalitis) [ 24 ], limited use of such data in public health programmes for the control of infectious diseases has been achieved. This paper defines a model which identifies areas of potential high risk of severe adverse reactions to ivermectin, and will contribute to APOC's programme development by enabling resources to be effectively targeted to areas deemed at risk. The Technical Consultative Committee of APOC, and the Mectizan Expert Committee have developed a series of recommendations aimed at "facilitating effective detection and management of SAEs following treatment with Mectizan in known and suspected Loa loa endemic areas". Three types of mass treatment strategies have been defined, according to the levels of endemicity of onchocerciasis and of loiasis, the latter being defined by the prevalence of Loa microfilariae (<20 % versus ≥ 20%) or of history of eye worm passage (<40 % versus ≥ 40%). In those areas where onchocerciasis is meso- or hyperendemic, and the prevalence of Loa mfs exceeds 20%, a number of detailed measures should be taken, regarding training of community distributors and medical personnel, availability of medical supplies, duration of distribution, surveillance of the treated populations, and management of the patients who develop a SAE. Though lower, the risk that SAE occur in areas where the prevalence of Loa microfilaraemia is lower than 20% is not nil, and thus guidelines have also been developed regarding the strategy to apply in such situations [ 30 ]. The model developed is by no means a definitive product but provides a basis for decision making in terms of where rapid epidemiological surveys for loiasis should now be targeted. Modelling in this way permits an iterative process between field epidemiologist and modeller which not only means that decisions are made on the best available data but that such data is updated rapidly as new survey results are entered in the database and the model refined. Models are valuable in so far as they reflect reality. Given the complexity of disease transmission processes where human, parasite, vector and environment interact, it is impossible to think that all relevant factors can be incorporated into a general model which can be applied on a regional scale. What is significant here is the fact that important decisions need to be made now with regard to the likely spatial extent of the distribution of Loa loa . As it stands, the model presented here uses environmental features known to be associated with the biology of the vector, is robust for the area of data collection and predicts areas within Cameroon where Loa loa has been found in the past. It provides a rapid assessment methodology of areas where adverse reactions to ivermectin may occur and will be further developed in conjunction with the RAPLOA procedure which APOC intends to apply to CDTI areas potentially endemic for Loa loa [ 15 ]. The next step in the process will be to update the model with new data (e.g. the verification data set) from all the countries where loiasis is endemic, and to explicitly represent uncertainty in the model outputs so that decision makers will be able to assess for themselves the quality of the model results for their area of interest. List of abbreviations APOC African Programme for Onchocerciasis Control CDTI Community-Directed Treatment with Ivermectin CPC Centre Pasteur du Cameroun DEM Digital Elevation Model ERM Environmental Risk Map for Loa loa GIS Geographic Information System GPELF Global Programme to Eliminate Lymphatic Filariasis IRD Institut de Recherche pour le Développement Mfs Microfilariae NDVI Normalised Difference Vegetation Index NOAA-AVHRR National Ocean and Atmospheric Administration – Advanced Very High Resolution Radiometer RAPLOA Rapid Assessment of the Prevalence and Intensity of Loa infection SAE Serious Adverse Event SPOT Satellite Pour l'Observation de la Terre TDR UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases UNEP-GRID United Nations Environment Programme-Global Resource Information Database USGS United States Geological Survey Competing interests None declared. Authors' contribution MCT designed the study, planned the analysis, supervised the modelling, interpreted the results, and wrote the paper. VO prepared the data, developed the model, prepared the maps, and wrote the paper. JK and JG collected the field data used for the development of the model. SW, IT and PE collected the field data used for the validation of the model. JHR supervised the study thanks to which the validation data were collected. DHM proposed the study, contributed to its design and interpretation of the results. MB designed the study, planned the analysis, collected the field data, interpreted the results, and wrote the paper.

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539250

BAG-1 haplo-insufficiency impairs lung tumorigenesis

Background BAG-1 is a multifunctional co-chaperone of heat shock proteins (Hsc70/Hsp70) that is expressed in most cells. It interacts with Bcl-2 and Raf indicating that it might connect protein folding with other signaling pathways. Evidence that BAG-1 expression is frequently altered in human cancers, in particular in breast cancer, relative to normal cells has been put forward but the notion that overexpression of BAG-1 contributes to poor prognosis in tumorigenesis remains controversial. Methods We have evaluated the effect of BAG-1 heterozygosity in mice in a model of non-small-cell lung tumorigenesis with histological and molecular methods. We have generated mice heterozygous for BAG-1, carrying a BAG-1 null allele, that in addition express oncogenic, constitutively active C-Raf kinase (SP-C C-Raf BxB) in type II pneumocytes. SP-C C-Raf BxB mice develop multifocal adenomas early in adulthood. Results We show that BAG-1 heterozygosity in mice impairs C-Raf oncogene-induced lung adenoma growth. Lung tumor initiation was reduced by half in BAG-1 heterozygous SP-C C-Raf BxB mice compared to their littermates. Tumor area was reduced by 75% in 4 month lungs of BAG-1 haploinsufficient mice compared to mice with two BAG-1 copies. Whereas BAG-1 heterozygosity did not affect the rate of cell proliferation or signaling through the mitogenic cascade in adenoma cells, it increased the rate of apoptosis. Conclusion Reduced BAG-1 expression specifically targets tumor cells to apoptosis and impairs tumorigenesis. Our data implicate BAG-1 as a key player in oncogenic transformation by Raf and identify it as a potential molecular target for cancer treatment.

Background BAG-1 is a multifunctional protein that is expressed in most cells. Originally identified as a Bcl-2 binding protein [ 1 ], other interaction partners of BAG-1 were described, including the serine threonine kinase C-Raf [ 2 ]. The C-terminal "BAG domain" of BAG-1 mediates the interaction with the Hsc70 and Hsp70 heat shock proteins [ 3 ], molecular chaperones that bind proteins in non-native states assisting them to reach a functional active conformation [ 4 ]. BAG-1 acts as a nucleotide exchange factor in this activation cycle [ 3 ]. The above findings indicated that BAG-1 might connect protein folding with other signaling pathways. Signaling networks promoting cell growth and proliferation are frequently deregulated in cancer [ 5 ]. The classical mitogenic cascade transmits stimuli from growth factor receptors via Ras, Raf, MEK and ERK to the cell nucleus [ 6 ]. C-Raf, like A- and B-Raf kinases also act at the outer membrane of mitochondria to augment cell survival [ 7 , 8 ]. Previously we had observed the stimulation of C-Raf kinase activity by BAG-1 in vitro [ 2 ]. Ras and B-Raf mutations have been found in various human cancers [ 9 , 10 ]. Evidence that BAG-1 expression is frequently altered in human cancers, in particular in breast cancer, relative to normal cells has been put forward but the notion that overexpression of BAG-1 contributes to poor prognosis in tumorigenesis remains controversial [ 11 ]. Methods Animals Mice used in these studies were generated and maintained according to protocols approved by the animal care and use committee at University of Würzburg. To inactivate the BAG-1 gene, we constructed a vector where exons 1 and 2 are replaced with a neomycin resistance gene. A phage clone with a 15-kb genomic insert from mouse strain 129/Sv spanning all seven exons of BAG-1 was identified and characterised using standard methods. The targeting construct contained 1,1-kb from the BAG-1 locus upstream of the neomycin resistance gene of plasmid pPNT [ 12 ] and 6-kb downstream. The upstream arm of 1,1 kb is located 5' to the start codon in the first exon of BAG-1 and the 3' arm of 6 kb is located downstream of exon 2. The mutation was introduced into embryonic stem cells by homologous recombination. Positive clones were identified by Southern blot analysis. Germline transmitting chimeras were obtained and bred to C57BL/6 mice. Further details will be described elsewhere. Heterozygous BAG-1 mice were genotyped by a PCR assay. The targeted BAG-1 allele was detected with primers P1 (5'-GAG TCT CCC GAT CCC TTT TCC), located upstream of exon 1, and P2 (5'-GAT TCG CAG CGC ATC GCC TT), located in the neomycin resistance gene, yielding a product of 600 base pairs. BAG-1 heterozygous mice were backcrossed at least three times onto C57BL/6 background before crossing with SP-C C-Raf BxB mice. Lung tumour mice expressing oncogenic C-Raf BxB were backcrossed at least six times onto C57BL/6 background. Western blot For the analysis of BAG-1 expression, lung lysates of the indicated genotypes were separated on 12,5% polyacrylamide-SDS (sodium dodecyl sulphate) gel, transferred to nitrocellulose Protran BA83 membrane (Schleicher&Schüll) and probed with rabbit anti-BAG-1 (FL-274) antibody (1:250, Santa Cruz Biotechnology). Amounts of protein were determined by Bradford protein assay to ensure equal protein loading for the analysis. Blots were developed using the appropriate horseradish peroxidase coupled secondary antibody and the ECL system (Amersham Pharmacia Biotech). Subsequently, the membrane was stripped and reprobed with rabbit antibody to glyceraldehyde 3 phosphate dehydrogense (1:2000, ab9485, Abcam Ltd.). Histopathology and immunohistochemistry Animals were sacrificed and lungs were fixed under 25 cm water pressure with 4% paraformaldehyde and embedded in paraffin. 5 μm sections were stained with hematoxylin and eosin and analysed. Pictures were taken using a Leica DMLA microscope and a Hitachi HV-C20A colour camera. Immunohistochemical staining to detect activated caspase-3, phospho-ERK (extracellular signal-regulated kinase), PCNA (proliferating cell nuclear antigen) have been described elsewhere [ 13 ]. Apoptotic, PCNA and p-ERK indices were determined by evaluating randomly chosen adenomas or fields of normal lung in 3–4 sections and determining the percentage of positive cells per 2000 cells at ×400. Results and discussion BAG-1 heterozygosity impairs C-Raf driven tumorigenesis In order to assess the functional role of BAG-1 on tumorigenesis, we have generated a null allele of BAG-1. To inactivate the BAG-1 gene, exons 1 and 2 were replaced with a neomycin resistance gene. This strategy was chosen to disrupt the expression of all known isoforms of BAG-1 which are generated by alternate translation initiation of a single mRNA; the start codons are present in exons 1 and 2. Western blot analysis of liver protein extracts of BAG-1 deficient embryos showed the complete loss of all BAG-1 protein isoforms. Embryos homozygous for this allele died at midgestation at around E13,5, but the heterozygous animals (BAG-1 +/- ) are normal. A comprehensive description of the BAG-1 -/- phenotype is subject of another manuscript. Previously, we had generated a lung cancer mouse model by targeting constitutively active C-Raf kinase (SP-C C-Raf BxB) to the lung [ 14 ]. These mice develop multifocal adenomas early in adulthood. Based on the observation, that BAG-1 can activate C-Raf [ 2 ], we asked whether heterozygosity for BAG-1 would affect C-Raf BxB driven adenoma growth. We observed that lung tumour initiation was reduced by half in 1, 2 and 4 months old BAG-1 +/- mice transgenic for SP-C C-Raf BxB compared to their BAG-1 +/+ littermates. Tumour area was reduced by 75% in 4 month lungs of BAG-1 haploinsufficient mice compared to mice with two BAG-1 copies, see Figure 1 . The histological picture emphasises the difference in adenoma formation between a representative SP-C C-Raf BxB/BAG-1 +/+ and SP-C C-Raf BxB/BAG-1 +/- lung. The difference in the staining intensity of the two lung sections derives mainly from the observation that the adenoma cells have a tendency to bind more intensively hematoxylin and eosin compared to normal lung cells. Thus, reduction of the BAG-1 gene dosage impairs the oncogenic activity of C-Raf in vivo. Reduced BAG-1 expression in BAG-1 heterozygous lungs Quantitative immunoblots demonstrated that the specific BAG-1 protein concentration in the lungs of BAG-1 +/- mice was half the amount of BAG-1 +/+ littermates, see Figure 2a . Moreover, immunohistochemical staining showed that BAG-1 was expressed in adenoma cells, see Figure 2b . There was no obvious difference in the BAG-1 immunohistochemistry of SP-C C-RafBxB/BAG-1 +/+ and SP-C C-RafBxB/BAG-1 +/- lungs. Tumour cells of BAG-1 heterozygous mice show increased apoptosis Concerning the molecular mechanism how a reduction of the BAG-1 protein expression in the heterozygous mice would impair tumorigenesis, we determined the fraction of apoptotic cells. Staining for activated caspase-3 revealed indistinguishable apoptosis in healthy regions of the lung of 1 month old SP-C C-Raf BxB mice with either one or two BAG-1 alleles, in line with the unaltered, normal lung structure of BAG-1 +/- mice. In the adenomas, however, we observed a significant increase of apoptotic cells in BAG-1 +/- SP-C C-Raf BxB mice compared with their SP-C C-Raf BxB/BAG-1 +/+ littermates, see Figure 3a . This mechanism of action of BAG-1 on the regulation of cell survival is compatible with the phenotype of embryonic day 12,5 BAG-1 null embryos. Immunohistochemical staining for activated caspase-3 and trypan blue staining of dissociated cells showed hypocellularity and elevated levels of apoptosis in the livers of BAG-1 -/- embryos (unpublished observations). Proliferation and p-ERK signalling are unaffected in BAG-1 heterozygous mice To exclude the alternative mechanism that the decreased level of BAG-1 expression in heterozygous animals would cause reduced cell proliferation in the adenomas, we performed proliferating cellular antigen (PCNA) staining. No significant differences were observed in the fraction of proliferating adenoma cells between SP-C C-Raf BxB animals heterozygous or wild type for BAG-1, see Figure 3b . Also, the percentages of adenoma cells positive for Ki-67, another proliferation marker and Bmi-1, a chromatin-associated protein expressed in stem cells, were not affected by the BAG-1 heterozygosity (not shown). Furthermore, staining of lung sections for phosphorylated ERK revealed no quantitative differences in the adenomas of SP-C C-Raf BxB animals heterozygous or wild type for BAG-1, see Figure 3c . Thus, signalling through the mitogenic cascade was not affected by the BAG-1 heterozygosity in the adenoma cells. Conclusions Tumours often are highly dependent on signalling pathways promoting cell growth or survival and may become hypersensitive to downregulation of key components within these signalling cascades. This study identifies BAG-1 as a protein specifically required at wild type expression levels for the survival of tumour cells and reveals it as potential anticancer target. Since many key components of survival pathways are regulated by interaction with (co-)chaperones [ 15 ], our finding is not without precedent but novel insofar as we have uncovered that reduced BAG-1 expression specifically targets tumour cells to apoptosis and impairs tumorigenesis. Whether this effect on adenoma cell survival requires that BAG-1 interacts with C-Raf or Hsc70/Hsp70 or with both partners requires additional studies. Questions concerning specific roles of the different BAG-1 isoforms were not addressed with this BAG-1 deficient mouse as both isoforms of BAG-1, p50 and p32 are absent in protein extracts of knock-out embryos. Another setting where BAG-1 has a physiological role is the heart, where up-regulation of BAG-1 after ischemia rescues cells from apoptosis [ 16 ]. A possible model combining the findings of this report and other data indicates that BAG-1 functions as an activator of C-Raf at the outer mitochondrial membrane where enzymatically activated C-Raf finds apoptosis-related targets such as BAD [ 17 ], see Figure 4 . We can purify overexpressed C-Raf either in an enzymatically inactive form in a complex with Hsp70 or in an enzymatically active form in a complex with Hsp90/50 (unpublished observations), and BAG-1 is proposed to regulate this activation with ATP generated in the mitochondria. Experiments dealing with this questions are currently ongoing. Therefore, the therapeutic efficacy of a standard chemotherapeutic agent [ 13 ] should be increased dramatically by co-application with a BAG-1 inhibitor, since it would target the adaptability of cancer cells to environmental stress and overcome their genetic plasticity. One way to reduce BAG-1 expression is through use of RNA interference-based gene silencing, in particular as BAG-1 overexpression has been observed in human tumours [ 11 ]. Drugs that bind to the ATP binding site of Hsc70/Hsp70 might also be expected to be effective as they would inhibit the interaction of BAG-1 with the ATPase domain of heat shock proteins. Such new specific BAG-1 inhibitors may be identified, aided by the known three-dimensional structure of the BAG domain [ 18 , 19 ]. Competing interests The author(s) declare that they have no competing interests. Authors' contributions RG caried out the molecular and histological studies and participated in the design and co-ordination of the study. BWK carried out the histological and immunohisto-chemical studies. GC participated in the histological and immunohistochemical experiments. URR participated in the design and co-ordination of the study. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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549210

An evaluation of the metabolic syndrome in the HyperGEN study

Background In 2001 the National Cholesterol Education Program (NCEP) provided a categorical definition for metabolic syndrome (c-MetS). We studied the extent to which two ethnic groups, Blacks and Whites were affected by c-MetS. The groups were members of the Hypertension Genetic Epidemiology Network (HyperGEN), a part of the Family Blood Pressure Program, supported by the NHLBI. Although the c-MetS definition is of special interest in particular to the clinicians, the quantitative latent traits of the metabolic syndrome (MetS) are also important in order to gain further understanding of its etiology. In this study, quantitative evaluation of the MetS latent traits (q-MetS) was based on the statistical multivariate method factor analysis (FA). Results The prevalence of the c-MetS was 34% in Blacks and 39% in Whites. c-MetS showed predominance of obesity, hypertension, and dyslipidemia. Three and four factor domains were identified through FA, classified as "Obesity," "Blood pressure," "Lipids," and "Central obesity." They explained approximately 60% of the variance in the 11 original variables. Two factors classified as "Obesity" and "Central Obesity" overlapped when FA was performed without rotation. All four factors in FA with Varimax rotation were consistent between Blacks and Whites, between genders and also after excluding type 2 diabetes (T2D) participants. Fasting insulin (INS) associated mainly with obesity and lipids factors. Conclusions MetS in the HyperGEN study has a compound phenotype with separate domains for obesity, blood pressure, and lipids. Obesity and its relationship to lipids and insulin is clearly the dominant factor in MetS. Linkage analysis on factor scores for components of MetS, in familial studies such as HyperGEN, can assist in understanding the genetic pathways for MetS and their interactions with the environment, as a first step in identifying the underlying pathophysiological causes of this syndrome.

Background Metabolic and physiologic disorders for cardiovascular disease (CVD) and type 2 diabetes (T2D), including abdominal obesity, insulin resistance, hyperglycemia, dyslipidemia, and hypertension often cluster. This cluster is frequently identified as the "metabolic syndrome" (MetS). Reaven [ 1 ] related MetS to the presence of resistance to insulin-mediated glucose disposal, glucose intolerance, hyperinsulinemia, increased triglycerides, decreased high-density lipoprotein cholesterol, and hypertension. Later, the definition of MetS was extended to include obesity, inflammation, microalbuminuria, and abnormalities of fibrinolysis and of coagulation [ 2 - 4 ]. Clearly, insulin resistance is not considered equivalent to MetS [ 5 , 6 ]. Grundy et al. [ 7 ], at a recent National Heart, Lung, and Blood Institute (NHLBI) /American Heart Association (AHA) National Conference, concluded that abdominal obesity is strongly associated with MetS. Sonnenberg et al. [ 8 ] have hypothesized that increased adipose tissue mass contributes to the development of MetS by triggering an increase in proinflammatory adipokines, especially the tumor necrosis factor-α, which may play a role in the pathogenesis of dyslipidemia, insulin resistance, hypertension, endothelial dysfunction, and atherogenesis. Although several studies have targeted MetS, its genetic determination and its pathophysiology remain unclear [ 9 ]. Different definitions and multivariate statistical approaches have been applied to characterize the increasing high-risk MetS premorbid state. Recently, special attention has received the categorical definition of metabolic syndrome (c-MetS) of the National Cholesterol Education Program Adult Treatment Panel III (NCEP) [ 2 ]. The NCEP definition (see Material and Methods) has especially two components, its usefulness in the clinical diagnosis of MetS and its association with recommendations for its therapeutic treatment. Based on the NCEP c-MetS definition, it is reported that 20 to 25 percent of the U.S. adult population has MetS. This represents a high prevalence of the syndrome in the general population [ 10 , 11 ]. In addition, employing the multivariate statistical method factor analysis (FA) different studies in different sampled populations have documented the underlying latent traits of MetS [ 4 , 12 - 17 ]. Meigs [ 3 ] has reported that FA in different studies has yielded on average 2 to 4 latent traits (factors) of MetS. Different studies have found different numbers of latent factors, depending on the type and number of the original risk factors analyzed, sampled population(s), methods utilized, including the statistical rotation method, and decisions about how many factors appeared statistically meaningful. The objective of this study was to exemplify important facets of the MetS in the HyperGEN study. Two MetS aspects were assessed: a. The trait characterized as the categorical MetS (c-MetS) was studied according to the NCEP definition; b. The underlying (latent) traits or clusters of MetS (q-MetS) were evaluated by performing FA with and without Varimax rotation on 11 risk factors. All data were grouped by ethnicity and gender. Subgroups were created by excluding T2D participants, under the assumptions that T2D individuals may have a different pattern of glucose and insulin levels. Finally, our goal was to compare the expression of c-MetS and q-MetS in the Hypertension Genetic Epidemiology Network (HyperGEN) study. Results Sample size and relationships among original risk factors For c-MetS the sample sizes varied from 2,025 observations for fasting triglycerides (TG) to 2,300 for high density lipoprotein (HDL) cholesterol in Blacks, and from 2,171 observations for TG to 2,471 for HDL in Whites. In the HyperGEN study, a high percentage of individuals have body waist (WAIST) and systolic blood pressure (SBP) / diastolic blood pressure (DBP) above the NCEP thresholds (Figure 1 ). Whites tended to have greater percentages of participants with TG and HDL beyond the NCEP thresholds than Blacks. The prevalence of c-MetS was 34 and 39 percent in Blacks and Whites, respectively. Figure 1 Categorical MetS (c-MetS) in the HyperGEN Study For q-MetS, the sample sizes and variables studied are summarized in Table 1 (the statistics were similar when T2D subjects were excluded, results not shown). After participants with missing data for any of the 11 original variables were excluded, this resulted in a sample of 1,422 Blacks and 1,470 Whites with complete data. The samples reduced to 1,173 Blacks and 1,322 Whites when T2D participants were excluded. Table 1 Original Data Included in Factor Analysis Blacks (N = 1422) Whites (N = 1470) Variable Units Kurtosis Skewness Mean Std Dev Kurtosis Skewness Mean Std Dev BMI kg/m 2 0.51† 0.72 32.04‡ 7.54 0.67 0.77 28.86 5.57 GLUC mg/dl 0.68 0.70 107.37 44.06 1.43 -0.55 100.80 26.38 INS μU/ml 0.34 0.06 10.55 9.24 0.42 0.23 7.45 5.90 LDL mg/dl 0.31 0.33 118.89 36.63 0.36 0.26 116.41 31.56 HDL mg/dl 0.46 0.22 53.58 15.17 0.17 0.18 48.70 14.09 TG mg/dl 0.41 0.35 101.32 56.03 0.17 0.20 144.77 75.24 SBP mm Hg 0.46 0.68 128.45 21.78 0.44 0.62 120.50 18.33 DBP mm Hg 0.85 0.60 74.08 11.59 0.42 0.44 68.96 9.94 WAIST cm 0.51 0.64 102.05 17.78 0.74 0.71 99.22 15.48 WHR ratio 0.11 0.10 0.90 0.08 0.17 0.05 0.92 0.09 %BF % 0.44 0.17 40.00 12.06 0.33 0.57 33.41 9.29 †Kurtosis and skewness are reported after the data were transformed (where necessary) and adjusted (see Material and Methods); ‡number of observations, mean, and standard deviations represent measures from the final sample in factor analysis In terms of the participants with complete data, the age at clinic visit had a mean of 46 and a standard deviation of 13 years in Blacks, and a mean of 51 and a standard deviation of 14 years in Whites. Overall, when compared to Whites, Blacks tended to have a higher body mass index (BMI), fasting plasma glucose (GLUC), fasting insulin (INS), HDL, SBP, DBP, WAIST, and percent body fat (%BF), similar low density lipoprotein (LDL) cholesterol and waist to hip ratio (WHR), and lower TG. Kurtosis after adjustments varied from 0.11 for WHR to 0.85 for DBP in Blacks, and 0.17 for WHR to 1.43 for GLUC in Whites, which demonstrates normal distributions for the traits in study and also for the factors created by performing FA (see Material and Methods). Pearson correlations among the 11 adjusted and normally distributed variables are presented in Table 2 . The lower triangle correlations correspond to all data, whereas the upper triangle refers to the data excluding T2D participants. In both ethnicities, strong correlations were observed among BMI, WAIST, %BF, INS, and WHR. GLUC correlated significantly with INS (inversely, because GLUC was inversely squared transformed). HDL cholesterol had a significantly negative correlation with TG and INS. SBP was significantly correlated with DBP. Interestingly, WAIST correlated higher with BMI (about 0.9) than with WHR (about 0.7) in all groups. These inter-correlations determine the structure of the factors. Table 2 Correlation Matrix of the Variables Included in Factor Analysis Variables ** Blacks: Upper Triangle: All Data Excluding T2D (N = 1422 / 1173) BMI INS GLUC LDL HDL TG SBP DBP WAIST WHR %BF Lower Triangle:All Data BMI 0.56‡ - 0.33‡ 0.15‡ - 0.22‡ 0.15‡ 0.18‡ - 0.04 0.91‡ 0.45‡ 0.78‡ INS 0.51‡ - 0.50‡ 0.16‡ - 0.37‡ 0.32‡ 0.04 - 0.08† 0.57‡ 0.44‡ 0.49‡ GLUC - 0.29‡ - 0.40‡ - 0.16‡ 0.25‡ - 0.26‡ - 0.06 0.03 - 0.33‡ - 0.26‡ - 0.28‡ LDL 0.13‡ 0.11‡ - 0.11‡ - 0.18‡ 0.23‡ - 0.00 - 0.05 0.14‡ 0.11† 0.17‡ HDL - 0.20‡ - 0.35‡ 0.23‡ - 0.16‡ - 0.41‡ 0.02 0.06 - 0.25‡ - 0.25‡ - 0.20‡ TG 0.17‡ 0.32‡ - 0.31‡ 0.20‡ - 0.41‡ 0.05 - 0.01 0.20‡ 0.28‡ 0.16‡ SBP 0.17‡ 0.02 - 0.07* 0.02 0.05 0.06 0.76‡ 0.16‡ 0.12† 0.06 DBP - 0.04 - 0.08† 0.04 - 0.02 0.08† - 0.00 0.74‡ - 0.03 0.03 - 0.06 WAIST 0.90‡ 0.52‡ - 0.32‡ 0.14‡ - 0.23‡ 0.22‡ 0.15‡ - 0.03 0.70‡ 0.75‡ WHR 0.44‡ 0.42‡ - 0.32‡ 0.10† - 0.25‡ 0.30‡ 0.11‡ 0.02 0.69‡ 0.407‡ %BF 0.76‡ 0.44‡ - 0.24‡ 0.16‡ - 0.18‡ 0.16‡ 0.03 - 0.06* 0.74‡ 0.37‡ Variables Whites: Upper Triangle: All Data Excluding T2D (N = 1470 / 1322) BMI INS GLUC LDL HDL TG SBP DBP WAIST WHR %BF Lower Triangle:All Data BMI 0.55‡ - 0.30‡ 0.01 - 0.21‡ 0.20‡ 0.19‡ 0.07* 0.89‡ 0.46‡ 0.77‡ INS 0.52‡ - 0.30‡ - 0.01 - 0.35‡ 0.34‡ 0.22‡ 0.15‡ 0.52‡ 0.36‡ 0.45‡ GLUC - 0.32‡ - 0.31‡ - 0.08* 0.13‡ - 0.16‡ - 0.12† - 0.09* - 0.30‡ - 0.25‡ - 0.26‡ LDL 0.02 - 0.02 - 0.05 - 0.04 0.08* 0.05 0.07 0.04 0.08* 0.07* HDL - 0.22‡ - 0.37‡ 0.21‡ - 0.02 - 0.43‡ 0 0.01 - 0.20‡ - 0.21‡ - 0.12† TG 0.20‡ 0.33‡ - 0.21‡ 0.06* - 0.45‡ 0.13† 0.09† 0.22‡ 0.23‡ 0.20‡ SBP 0.17‡ 0.19‡ - 0.13‡ 0.04 0.00 0.10† 0.70‡ 0.15‡ 0.13‡ 0.09† DBP 0.03 0.09† 0.00 0.06 0.01 0.06 0.67‡ 0.06 0.09† 0.03 WAIST 0.89‡ 0.49‡ - 0.33‡ 0.04 - 0.21‡ 0.23‡ 0.14‡ 0.02* 0.70‡ 0.77‡ WHR 0.46‡ 0.35‡ - 0.25‡ 0.06 - 0.23‡ 0.25‡ 0.11† 0.07* 0.69‡ 0.44‡ %BF 0.76‡ 0.43‡ - 0.25‡ 0.08† - 0.12‡ 0.18‡ 0.11† 0.01 0.76‡ 0.43‡ *p < 0.05 † < 0.01 ‡ < 0.0001 **Variables were adjusted for age and center within ethnicity and gender (see Material and Methods) A negative correlation between GLUC and INS is result of the inverse squared transformation of the original GLUC FA with no rotation identified a factor (Factor 1) that was loaded mostly by central obesity, obesity risk factors and INS (Table 4, see Additional file 1 ). It explained 21 percent to 32 percent of the variance of the original risk factors. Three other factors, "Obesity," "Blood pressure," and "Lipids," were identified. In Blacks, the "Obesity" factor was primarily loaded by BMI, INS, WAIST, and %BF. In Whites, exclusion of T2D participants led to a similar pattern. However, in all data in Whites, the first factor represented a stronger mixture of central obesity, obesity and INS, leaving the fourth factor with mainly WHR loading. In order to distinguish the second factor from the first one, we labeled the first as "Central obesity" factor and the second as "Obesity" factor. In both ethnicities, blood pressure (BP) gave rise to a separate factor. Also the "Lipids" remained as a separate factor and was dominated mainly by HDL and TG. INS was associated mostly with the "Obesity" and "Lipids" factors. In the case of FA with Varimax rotation, again, 4 distinct factors explained about 60 percent of the variance in the original variables (Table 4, see Additional file 1 ). We are labeling them as "Obesity," "BP," "Lipids," and "Central obesity". Factor loadings less than 0.1 were not listed in Tables 3 and 4 (see Additional file 1 ). The first factor alone explained 23 percent to 25 percent of the variance in the original risk factors, while the fourth factor explained 7 percent to 9 percent of the variance. The "Obesity" factor (Factor 1) loaded mainly BMI, WAIST, WHR, %BF, and INS. SBP and DBP loaded separately. A distinct factor ("Lipids") loaded mainly HDL cholesterol, TG, INS and GLUC in Blacks and HDL cholesterol, TG and INS in Whites. The fourth factor contained a higher loading for WHR than for WAIST. Similar factor loadings were present in the samples when T2D participants were excluded (Table 4, see Additional file 1 ). Table 3 Factors, Loadings, and Sums of Squared Loadings in All Data (Males (M) and Females (F)), and by Gender (M, F) (Varimax Rotation) Factor 1 (Obesity) Sample BMI* %BF WHR WAIST INS GLUC SBP DBP LDL HDL TG SS Loadings Blacks M+F (1422) 0.95† 0.71 0.32 0.86 0.42 -0.20 0.13 2.49 M (483) 0.92 0.71 0.65 0.95 0.46 -0.15 0.11 0.11 -0.21 0.14 2.91 F (939) 0.96 0.71 0.23 0.84 0.40 -0.22 0.14 2.43 Whites M+F (1470) 0.94 0.77 0.40 0.86 0.46 -0.26 0.14 -0.11 0.11 2.69 M (721) 0.94 0.76 0.55 0.93 0.48 -0.26 -0.13 0.16 2.99 F (749) 0.94 0.81 0.31 0.84 0.46 -0.28 0.18 -0.11 0.11 2.69 Factor 2 (BP) Sample BMI %BF WHR WAIST INS GLUC SBP DBP LDL HDL TG SS Loadings Blacks M+F (1422) 0.99 0.76 1.58 M (483) 0.13 0.83 0.83 1.42 F (939) 0.99 0.77 1.59 Whites M+F (1470) 0.13 0.88 0.76 1.39 M (721) 0.14 0.87 0.80 1.41 F (749) 0.12 -0.14 0.98 0.65 0.11 1.45 Factor 3 (Lipids) Sample BMI %BF WHR WAIST INS GLUC SBP DBP LDL HDL TG SS Loadings Blacks M+F (1422) 0.22 0.19 0.31 0.24 0.52 -0.43 0.27 -0.57 0.64 1.49 M (483) 0.17 0.12 0.29 0.18 0.48 -0.19 -0.72 0.65 1.37 F (939) 0.18 0.11 0.29 0.21 0.49 -0.41 0.32 -0.55 0.59 1.32 Whites M+F (1470) 0.23 0.17 0.27 0.21 0.51 -0.27 -0.68 0.64 1.41 M (721) 0.14 0.41 -0.18 -0.18 -0.75 0.61 1.20 F (749) 0.25 0.19 0.24 0.23 0.5 -0.34 0.11 0.22 -0.70 0.60 1.44 Factor 4 (Central Obesity) Sample BMI %BF WHR WAIST INS GLUC SBP DBP LDL HDL TG SS Loadings Blacks M+F (1422) 0.90 0.40 0.15 -0.13 0.11 1.03 M (483) -0.19 0.37 0.11 0.2 F (939) 0.93 0.44 0.15 -0.16 0.11 1.13 Whites M+F (1470) 0.16 0.69 0.47 -0.10 0.76 M (721) 0.11 0.57 0.27 -0.11 0.13 0.45 F (749) 0.92 0.44 0.14 1.10 * Variables were adjusted for age and center within ethnicity and gender (see Material and Methods) GLUC negative loadings are result of inverse squared power transformation of the original GLUC;† Loadings ≥ 0.4 are in bold FA with Varimax rotation was performed also by gender (Table 3 ). Between genders, the factors loaded in a similar fashion in Blacks and Whites. WHR and WAIST reflected gender differences in their loadings in factors 1 and 4. Discussion The fact that MetS is more prevalent in the HyperGEN study as compared to the average of the US adult population [ 10 , 11 ], is probably due to selection bias arising from the hypertension selection criterion applied in HyperGEN (see Material and Methods). Although BP was an important contributor to the categorical definition of the MetS (c-MetS), other risk factors such as WAIST, HDL and TG were also important. The HyperGEN Whites had a higher prevalence of c-MetS than the Blacks. Blacks had a higher percentage of participants with WAIST, BP, and GLUC beyond the NCEP thresholds. In this study, FA of 11 potential risk factors for CVD and T2D yielded 4 latent factors, explaining about 60 percent of the variance in the original risk factors. Using the maximum likelihood estimation (MLE) method in S-PLUS (Insightful Corporation software), we found that the model p-values were significant, suggesting that additional factors may exist. However, although additional factors must exist to explain approximately 40 percent of the variance in the original variables, none of the remaining factors individually can explain more than about 5 percent of the variance. Therefore, we concluded that the quantitative structure of MetS can be described in terms of three to four factors when no rotation was performed, and four factors with Varimax rotation. One may even argue whether the fourth factor in the Varimax rotation is very meaningful. We chose to retain the "Central Obesity" factor particularly because it tends to reflect the well-known gender asymmetry (Table 3 ). FA without and with Varimax rotation can be useful in different settings. We believe that FA without any rotation makes more sense when investigating the pattern of risk factor clustering in the MetS. On the other hand, gene finding studies can be enhanced with Varimax rotation because, it is easier to find genes each of which influences a different (uncorrelated) factor. Therefore, depending on the goals of a study, rotation may or may not be used. We regard this flexibility as strength of the FA method. We tested the pattern of the factors between genders only for FA with Varimax rotation. This pattern was stable among ethnicities between genders (Table 3 ). WHR on Factors 1 and 4 and WAIST on Factor 4 had statistically different loadings in males and females. Another characteristic of the HyperGEN study was that at least two participants in each sibship had hypertension. A large proportion of the hypertensive participants have used anti-hypertensive and anti-cholesterol medications. Maison et al. [ 13 ] have compared medicated and unmedicated groups to see any implications of the medication use in FA. They applied FA to 9 risk factor changes over time, and separated data into groups of treated and untreated for hypertension and dyslipidemia. They found 3 and 4 factors respectively in males and females, the "BP", "Glucose," "Lipid," and "BMI, WHR, INS and TG" factors, which were similar between treated and untreated groups. It is a common belief that T2D participants may have a different expression of INS and GLUC, therefore it may influence also the factors pattern in the MetS analysis. In the present study, we found a consistency of the factors before and after removing type 2 diabetics. This finding is supported also by Hanson et al. [ 17 ] who studied two samples of Pima Indians classified as T2D and non-T2D. They identified consistently 4 latent factors out of 10 risk factors in the two samples, with a relative variation only on insulin loadings. They found that the "Insulinemia" factor explained 30 percent of the original variance, "Body size" 20 percent, "BP" 15 percent, and "Lipids" 14 percent. In our study, the INS variable loaded with BMI, WAIST, %BF, and also with lipids. INS was present mainly in 2 and sometimes in 3 factors with loadings mostly in the "Obesity" and "Lipids" factors. Other studies have provided similar results about the latent traits of MetS [ 4 , 16 , 17 ]. FA studies cited here, and other studies described by Meigs [ 3 ], have elements in common with our study: 2 to 4 factors were identified; BMI, INS, WHR and WAIST are major contributing risk factors; SBP and DBP load in a separate factor; INS was associated with more than one factor and mainly with obesity and lipids. Our study and several others have shown that FA is a useful method for studying the underlying traits of MetS. Nevertheless this methodology has not passed without been criticized. Lawlor et al. [ 18 ] reviewed 22 published studies of the MetS, all based on FA. None of the studies had clearly identified whether they used FA for exploratory analysis or for hypothesis testing purposes. Such ambiguous use was regarded by Lawlor et al [ 18 ] as a major misuse of FA in the study of the metabolic syndrome. In fact exploratory FA and the hypothesis-testing (confirmatory) FA are two distinct methods. Basically, the two analyses use different constraints and different approaches in the analytical software. The exploratory FA is driven by the data (example is our study). On the other hand, the hypothesis-testing FA is only performed with some prior knowledge of possible loadings for different risk factors. One applies confirmatory FA on the data to test if the factor structure of the hypothesized model specifying the interrelationships among the original variables and the latent factors included in the model is true or not. A detailed example of the confirmatory FA of the metabolic syndrome is provided by Shen et al. [ 16 ]. If c-MetS and q-MetS are explaining the same disorder in two different aspects in the HyperGEN study, can FA contribute to MetS gene finding? MetS is recognized as a precursor for cardiovascular disease and type 2 diabetes [ 19 ]. There are several studies that have used FA for understanding the complexity of the MetS. Our study brings in more evidence that FA provides not only insights about the latent factor traits for MetS, but it produces factor scores for each of the MetS domains at the same time. Can factor scores be used in genetic analysis, such as in linkage analysis? The concept of a latent factor has much (intellectual) parallel with the concept of a latent gene. Much like a latent gene might have pleiotropic effects on several correlated phenotypes (original risk factors), several correlated risk factors load onto a latent factor. This makes FA very attractive from a genetic analysis point of view since, unlike individual risk factors each of which may entail several genes, each factor is likely to involve only a few genes which simplify their discovery. We believe that FA is useful for complex disease gene finding. In a near future motivated from this analysis we plan in the HyperGEN study to perform linkage analysis on the trait established by c-MetS and also on factor scores created by q-MetS, for identifying essential MetS putative genes / QTLs. Conclusions These analyses demonstrated that obesity and hypertension were the most important factors contributing to the MetS in the HyperGEN Study. Three to four distinct factor domains were identified depending on the FA rotation applied and decisions made. Results support the hypothesis that MetS is a compound phenotype, where obesity and its relationship to lipids and insulin are clearly the driving force of MetS. Insulin may play a connecting role between obesity and lipid domains. In genetic analysis, it is well known that categorical data, especially a complex trait such as MetS, encounter reduced power as compared to quantitative variables. Therefore, we suggest that genetic analysis should be performed on specific combinations of traits that belong to a factor. It is possible that some common genes may exist in the pathways for the factors identified. Linkage analysis investigating putative quantitative trait loci for MetS factor domains can be a first step which may help discover the underlying mechanisms, or generate new hypotheses, in finding the causes of MetS. Material And Methods Data collection and MetS definition The sample represents data from the HyperGEN network, part of the Family Blood Pressure Program, supported by the NHLBI as described by Williams et al. [ 20 ] and Province et al. [ 21 ]. The ethnicity was recorded as a self-reported demographic category. In the HyperGEN study sibships were recruited, each with at least 2 members who were hypertensive before age 60. Also, parents and offspring of some of the hypertensive sibs, as well as random samples of unrelated Blacks and Whites, were recruited, totaling 4,781 participants. Insulin measurements are not available in a part of the sample and therefore the sample size was smaller for FA. Also, participants with missing values for any of the quantitative risk factors used in the definition of MetS were excluded. A detailed account is provided in the Results section. A participant was classified as having T2D if (s)he had a fasting plasma glucose value ≥ 126 mg/dl, or is a current user of hypoglycemic medication or insulin that was documented at examination in the clinic, or if diabetes was reported in the HyperGEN questionnaire. Also, an age at diagnosis ≥ 40 years was required for T2D individuals [ 22 ]. c-MetS according to the NCEP definition, was identified in participants by the simultaneous presence of 3 or more of the following conditions: WAIST > 102 cm in men, and > 88 cm in women; TG ≥ 150 mg/dl; high density lipoprotein (HDL) < 40 mg/dl in men, and < 50 mg/dl in women; systolic blood pressure (SBP) ≥ 130 mm Hg and/or diastolic blood pressure (DBP) ≥ 85 mm Hg or using antihypertensive medications; GLUC ≥ 110 mg/dl or on treatment for diabetes [ 2 ]. Factor analysis was founded on 11 variables: BMI expressed as the ratio of body weight divided by body height squared (in kg/m 2 ); WAIST measured at the level of the umbilicus in cm; WHR defined as waist circumference divided by hip circumference; GLUC in mg/dl; INS in μU/ml (where fasting time was defined as ≥ 12 hours before blood draw); LDL in mg/dl; HDL in mg/dl; TG in mg/dl; Sitting SBP in mm Hg; DBP in mm Hg (SBP and DBP were measured three times after the subject was asked to sit for five minutes, with the mean of the second and third measurements of each variable being used in the analysis); %BF derived from the bioelectric impedance measurements based on the Lukaski formula [ 23 ]. Statistical Analysis TG and INS had skewed distributions. A relatively skewed distribution was also present for HDL. Log transformation brought these variables distributions to approximately normal. GLUC and %BF were highly kurtotic. Using Box-Cox transformation, it was found that the inverse of the squared transformation of GLUC (1/GLUC 2 ) and the squared transformation of %BF (%BF 2 ) reduced the excess kurtosis considerably. The procedure transreg in SAS (version 9 for PC) was employed for finding power transformations. There were two field centers recruiting Blacks and four field centers recruiting Whites. Accordingly, dummy (0,1) field center variables, one for Blacks and three for Whites, were created. All 11 risk factors were adjusted within ethnicity and gender for age, age 2 , age 3 , and field center effects using stepwise regression analysis within ethnicity and gender by employing SAS (SAS version 8.2 for Linux). Any variables with outliers beyond ± 4 standard deviations (SD) were also adjusted for heteroscedasticity of the variance. After the adjustments for each variable, outliers beyond ± 4 SD were eliminated. Each final adjusted variable was standardized to a mean 0 and variance 1. Prevalence of c-MetS was estimated with the FREQ procedure of SAS. The multivariate method of factor analysis was employed for reducing a group of risk factors to a subset of latent factors. The primary goal of FA is to identify the interrelationships among a set of variables. In this study FA was used for exploratory analysis, because there was no a priori information about the structure underlying the variables. FA can be used also for a confirmatory analysis, when validation (or refutation) of a postulated structure is sought. In either case, FA seeks parsimony by summarizing a large group of interrelated variables (risk factors for a complex disease such as MetS) in terms of a small number of latent factors, thereby reducing the dimensionality. Theoretical statistical descriptions of FA can be found in the literature [ 24 - 26 ]. FA was performed with S-PLUS 6.0.1 software by using the factanal function, in which the MLE was employed. FA evaluated latent factors underlying the 11 original variables. FA was performed with and without the Varimax rotation. "No rotation" achieves the simplest latent factor structure, in the extreme case loading any variable in one of the factors and almost negligible loadings in the rest of the factors. That is the reason why some studies (extracting factors with no rotation) find a concentration of the major variables' loading on the first factor. This is also the reason why some investigators named the first factor in their studies as the "Metabolic syndrome" factor [ 3 , 27 ]. Conversely, when Varimax rotation is applied, the objective is to maximize the independence of the clusters for variables that load onto factors. This is achieved by loading in separate factors distinct combinations of the interrelated risk factors. A loading of 0.4 or larger was considered as a significant contribution of an original variable to a factor. List of abbreviations used MetS, metabolic syndrome; c-MetS, categorical MetS; q-MetS, latent traits of MetS; FA, Factor analysis; MLE, maximum likelihood estimate; T2D, type 2 diabetes; CVD, cardiovascular disease; BMI, body mass index; INS, fasting insulin; GLUC, fasting glucose; WHR, waist to hip ratio; SBP, systolic blood pressure; DBP, diastolic blood pressure; BP, blood pressure; TG, fasting triglycerides; LDL, low density lipoprotein cholesterol; HDL, high density lipoprotein cholesterol; %BF, percent body fat. Competing interests The author(s) declare that they have no competing interests. Authors' contributions All authors were equally involved in designing the MetS study, evaluating statistics, interpreting the data, writing the manuscript, and organizing the figure and tables. Supplementary Material Additional File 1 Table 4. This table contains information on factor loadings result of FA with and without rotation performed on 11 risk factors Click here for file

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Co-transcriptional folding is encoded within RNA genes

Background Most of the existing RNA structure prediction programs fold a completely synthesized RNA molecule. However, within the cell, RNA molecules emerge sequentially during the directed process of transcription. Dedicated experiments with individual RNA molecules have shown that RNA folds while it is being transcribed and that its correct folding can also depend on the proper speed of transcription. Methods The main aim of this work is to study if and how co-transcriptional folding is encoded within the primary and secondary structure of RNA genes. In order to achieve this, we study the known primary and secondary structures of a comprehensive data set of 361 RNA genes as well as a set of 48 RNA sequences that are known to differ from the originally transcribed sequence units. We detect co-transcriptional folding by defining two measures of directedness which quantify the extend of asymmetry between alternative helices that lie 5' and those that lie 3' of the known helices with which they compete. Results We show with statistical significance that co-transcriptional folding strongly influences RNA sequences in two ways: (1) alternative helices that would compete with the formation of the functional structure during co-transcriptional folding are suppressed and (2) the formation of transient structures which may serve as guidelines for the co-transcriptional folding pathway is encouraged. Conclusions These findings have a number of implications for RNA secondary structure prediction methods and the detection of RNA genes.

Background Most of the existing computational methods for RNA secondary structure prediction fold an already completely synthesized RNA molecule. This is done either by minimizing its free energy (e.g. done by MFOLD [ 1 - 3 ] and by the programs of the VIENNA package [ 4 - 8 ]) or by maximizing the probability under a model whose parameters can incorporate a variety of different sources of information, e.g. comparative information, free energy and evolutionary information (e.g. [ 9 ], TRNASCAN-SE [ 10 ], PFOLD [ 11 , 12 ] and QRNA [ 13 ]). All of these programs, including those that predict folding pathways by folding an already synthesized RNA sequence [ 14 , 15 ], therefore disregard the effects that co-transcriptional folding may have on the RNA's functional secondary structure. They essentially aim to predict the thermodynamic RNA structure , i.e. the secondary structure that minimizes the free energy of the molecule. However, theoretical studies of RNA molecules [ 16 ] indicate that the thermodynamic structure of even moderately long RNA molecules need not necessarily correspond to the functional structure which confers the desired functionality within the organism to the RNA molecule. RNA molecules are known to fold as they emerge during transcription [ 17 , 18 ]. Transcription is a directed process of variable speed, during which the 5' end of the RNA molecule is synthesized before its 3' end. Hydrogen-bonds at the 5' end of the RNA molecule can thus form earlier in time than hydrogen-bonds involving the 3' end of the molecule. The thus emerging secondary structure elements can be transient or not, depending on their stability, their formation times and the availability and stability of competing alternative pairing partners. The directedness and also the speed of transcription can influence both the folding pathway and the functional secondary structure of the RNA molecule. We call this phenomenon sequential or co-transcriptional folding and call the resulting secondary structure the kinetic structure of the RNA molecule. Co-transcriptional folding leads to the formation of temporary secondary structure elements [ 18 , 19 ]. The time that it takes to form and replace these transitory structure elements may successively narrow down the set of accessible folding pathways and may thereby guide the folding towards an ensemble of secondary structures which contains the desired functional secondary structure. However, these temporary secondary structure elements can also have distinct biological functions, e.g. in viroids [ 19 ] and as initial sites for protein anchoring during pre-mRNA transcription [ 20 ]. Based on experimental and theoretical investigations, Harlepp et. al. [ 21 ] and Isambert et. al. [ 22 ] found that temporary structures may form during transcription. All these results suggest that temporary secondary structure elements may play an important role in the correct folding of RNA sequences. The speed of transcription also has an effect on folding which can be investigated by varying the nucleoside triphosphate concentration [ 19 ] or by transcribing RNA genes with viral polymerase T7 which has faster elongation during transcription than bacterial polymerases [ 23 , 24 ]. Both decreasing and increasing the natural speed of transcription can yield inactive transcripts [ 23 , 24 ]. Recent in vitro investigations of the Tetrahymena ribozyme [ 25 ] show that its co-transcriptional folding in vitro is twice as fast as the refolding of the entire RNA molecule under the same conditions and that both lead to the same functional folding. Moreover, they find that the co-transcriptional folding in vitro is still much slower than in vivo. Among the multitude of biochemical processes which are known to occur transcriptionally [ 26 , 27 ], some processes act in order to prevent the mis-folding of RNA molecules. RNA chaperones are proteins which are believed to help refold mis-folded RNA structures by promoting intermolecular RNA-RNA annealing through non-specific interaction [ 28 ]. Without RNA chaperones, moderately long GC-rich helices have dissociation half-times of up to 100 years [ 29 ]. This time can be significantly reduced by RNA chaperones, which preferentially bind stretches of unfolded RNA and thereby decrease the kinetic barrier between the correct and incorrect secondary structure elements [ 28 ]. Specific RNA-binding proteins are also known to promote RNA folding by either guiding its folding or stabilizing its correct structure [ 30 , 31 ]. The hnRNP proteins non-specifically bind pre-messenger RNA and help in the splicing process [ 32 ]. RNA sequences can also promote the proper folding of other RNA sequences. It is known, for example, that the temporary interaction with highly conserved leader sequences of bacterial rRNA-operons is needed for the proper formation of 30S ribosomal subunits and the maturation of 16S rRNA [ 33 , 34 ]. All these experimental and the few theoretical findings suggest that co-transcriptional folding may play an important role in the correct folding of RNA molecules. They also show that the functional structure may only be a transient one which is available during a certain time span and that the functional structure need not correspond to the structure which would dominate the ensemble of structures after an infinite time span. Little is known whether co-transcriptional folding is mainly governed by the specific or non-specific binding of proteins (or other molecules) which target the emerging RNA or whether the primary structure of the RNA molecule itself conveys the desired properties to guide its own correct co-transcriptional folding. In this paper, we propose several statistics in order to detect, if and how co-transcriptional folding influences RNA sequences. Using these statistics, we show that the effects of co-transcriptional folding are widespread in RNA genes. Methods Theory We want to show that an RNA sequence is organized in such a way to help the formation of the functional secondary structure during transcription. We aim to support this hypothesis by detecting two different features: • Possible competitors of helices in the functional structure are suppressed. When the 3' end of a helix that is part of the final secondary structure emerges during transcription, the number of possible competitors for the 5' part of the helix should be as low as possible in order to promote the formation of the correct helix. • The folding pathway is engineered. During transcription, several temporary helices are formed which may guide the folding process. We investigate these features using several statistics which are based on the known primary and secondary structures of our RNA sequences. A crucial point in investigating these features is to define a set of statistics that have expectation of zero in the H 0 case, when we suppose no co-transcriptional folding. However, verifying that these statistics have an expectation value of zero in the H 0 case cannot simply be achieved by analyzing random sequences. Indeed, even generating random sequences is not trivial. First, it is hard to reliably predict the minimum free energy structure for the randomized sequences as most secondary structure prediction algorithms discard pseudo-knots and, even without pseudo-knots, predict only on average about 70 % of the base-pairs correctly. In addition, there is no guarantee that the secondary structure with the lowest free energy would correspond to the functional one. Second, even if the random sequences are generated by a shuffling algorithm which keeps the given secondary structure fixed, it cannot be guaranteed that the fixed structure remains the correct one for the new primary sequence. Generating random sequences therefore provides no straightforward solution for obtaining a H 0 statistics with expectation value zero. We circumvent this problem by studying pairs of statistics, where both statistics have the same, unknown expectation value in the H 0 case and where one statistics has a bias away from the H 0 expectation value in case of co-transcriptional folding, while the other statistics is not affected by co-transcriptional folding. By studying the difference of these two statistics, we thus gain a new statistics with expectation value zero in the case of no co-transcriptional folding and an expectation value larger or smaller than zero in the case of co-transcriptional folding. The statistics (which we will define in detail below) measure the presence of alternative helices which compete for at least one base-pair with the helices of the known secondary structure. These competing alternative helices are required to consists of at least min stem = 9 consecutive base-pairs of type {G - C, C - G, A - U, U - A, G - U, U - G} and are calculated by a dynamic programming procedure in which the known primary and secondary structure of the RNA is fixed, see Figure 1 for the definition of a competing, alternative helix. We checked that we obtain qualitatively similar results for smaller and larger min stem values (data not shown). While calculating all helices of at least min stem length, we test which of these helices constitute competing alternatives to helices of the known secondary structure and record each such competing case in one of our statistics. These alternative helices may be part of a pseudo-knotted structure and we do not discard them. As each of the two bases i and of a base-pair in a known helix can have a competing alternative base-pairing partner within an alternative helix and as this alternative partner can either be found 5' (before), 3' (behind) or between the two strands of the known helix, all cases can be classified into six different classes. Of these six, we discard the two classes where the alternative helix falls between the two strands of the known helix as this un-paired loop region is typically too short to accommodate an alternative helix of at least min stem length. The remaining four classes, see Figure 2 , can be sub-divided into two cis- and two trans- alternative classes, depending on whether the known base-pairing partners lie between the alternative base-pairing partners (trans) or not (cis). The four statistics 3' cis, 3'trans, 5'cis and 5'trans that we use correspond to these four classes. Figure 1 Definition of a competing, alternative helix. Pictorial definition of a competing, alternative helix. The known base-pair between sequence positions i and has to have at least two other directly adjacent base-pairs within the known secondary structure (right) and the competing, alternative helix has to contain an alternative base-pair between sequence positions i and c ( c is the competitor of ) which has to be contained within a helix of minimum stem length (left). Figure 2 Definition of the statistics. Pictorial definitions of the four configurations 3' cis , 3' trans , 5' cis and 5' trans which correspond to the four statistics used to measure the directedness of RNA folding. Sequence positions i and form a base-pair within the known secondary structure. Sequence position c is an alternative base-pairing partner for i (but according to the base-pairing rules therefore not for ) within a competing, alternative helix of a minimum length min stem . See the text for more explanation. It is important to note that even without co-transcriptional folding, the destabilizing effects of competing cis- and trans -alternative helices are not necessarily the same as the stacking energies are not symmetric with respect to the 5' → 3' direction of the RNA sequence [ 3 ]. In addition, alternative cis -pairing partners are closer to the known pairing partners than trans -pairing partners and may thus lead more easily to incorrect helices. We may therefore compare only cis -competitors with other cis -competitors and trans -competitors with other trans -competitors. This yields two possible comparisons: 3' trans versus 5' trans and 5' cis versus 3' cis, see Figure 2 , with which we can measure the effects of co-transcriptional folding. We proceed as follows to detect if co-transcriptional folding takes place: For every RNA sequence of the data set, we detect events of type 3' cis, 3'trans, 5'cis and 5'trans , where an alternative helix competes with a known helix. Each such event is given two different weights, see Table 1 for an overview of definitions: (1) a weight of 1/ ( d·log ( l )), where d is the distance between the two competing helices and l is the length of the sub-sequence 5' or 3' of the known helix on which the competing helix falls, or (2) a weight of | G | / ( d ·log( l )), where the former weight is multiplied by the absolute value of the free energy G of the competing, alternative helix. The factor 1/ d gives alternative helices that are far away from the known helix a smaller weight than closer ones. The factor 1 /log (l) accounts for the fact that log (l) is proportional to the expected sum of 1 /d statistics for a sub-sequence of length l (i.e. the integral ). The free energy factor G in the second type of weights gives stable alternative helices which have a larger impact on the folding pathway a greater weight than helices which are easily unfolded. Statistics derived from weights of type 1 /(d log( l )) are denoted by an index p (for plain) and those of type | G | / ( d ·log( l )) by an index g (for free energy). By summing the weighted counts for each of the four classes of events, we thus arrive at eight different scalar values which characterize each RNA sequence: 3'Trans x , 3'Cis x , 5'Trans x and 5'Cis x for x ∈ { p,g }. Table 1 Definitions of the different statistics. Definitions of the different statistics used. i and denote the sequence positions of a base-pair in the known structure, c is an alternative pairing partner for i (but according to the base-pairing rules therefore not for ), L is the length of the RNA sequence, N is the number of sequences in the data set and the index x indicates the type of weight used. Please refer to the text for a description of how alternative pairing partners are calculated. x p plain weights g free energy weights 3'cis x 1/(( c - i ) log( L - i )) | G ci |/(( c - i ) log( L - i )) 3'trans x 1/(( c - ) log( L - )) | G ci |/((c - ) log( L - )) 5' cis x 1/(( i - c ) log( i )) |G ic |/(( i - c ) log( i )) 5' trans x 1/(( - c )log( )) |G ic |/(( - c ) log( )) cis x 5' cis x - 3' cis x trans x 3' trans x - 5' trans x 3' Cis x Σ #3' cis 3' cis x 3' Trans x Σ #3' trans 3' trans x 5' Cis x Σ #5' cis 5' cis x 5' Trans x Σ #5' trans 5' trans x Cis x 5' Cis x - 3' Cis x Trans x 3' Trans x - 5' Trans x where x ∈ { p,g }, y ∈ {3' Cis , 3' Trans , 5' Cis , 5' Trans , Cis , Trans } We can now define the two statistics which are capable of measuring the two main types of asymmetry within each RNA sequence: Cis := 5' Cis - 3' Cis Trans := 3' Trans - 5' Trans which can calculate for both types of weights. Without co-transcriptional folding, the expectation value of these two statistics is zero. Co-transcriptional folding induces two types of asymmetries by suppressing the number of alternative helices which compete with the final helices (indicated by an increased number of configurations, see Figure 2 ) and by promoting the formation of transient helices which guide the correct folding (indicated by an increased number of configurations). Both types of effects are indicated by an expectation value larger than zero for the respective statistics. Without co-transcriptional folding, the introduced statistics have an expectation of zero, moreover, the distributions should be symmetric. The number of positive cases (pos) thus follows a binomial distribution with parameter p = 0.5 and the statistic where n is the number of all cases, approximately follows a standard normal distribution. If this value is sufficiently positive, we have to reject the hypothesis that co-transcriptional folding is not encoded within RNA genes. Data All 16S rRNA, 23S rRNA as well as Group I and Group II type intron sequences with completely known secondary structures were downloaded from the Comparative RNA Web (CRW) Site [ 35 , 36 ], resulting in 304 16S rRNA, 84 23S rRNA, 15 Group I intron and 6 Group II intron sequences from three main taxonomical units (Archea, Bacteria, Eukaryotes) and two organelles, see Table 2 . Table 2 Composition of the two data sets. Taxonomic unit all 16S rRNA 23S rRNA Group I Group II Data set A Archea 28 22 6 0 0 Bacteria 277 232 45 0 0 Eukaryotes 41 35 6 0 0 Chloroplasts 6 6 0 0 0 Mitochondria 9 9 0 0 0 Sum 361 304 57 0 0 Data set B Eukaryotes 15 0 0 15 0 Bacteria 5 0 5 0 0 Chloroplasts 5 0 5 0 0 Mitochondria 23 0 17 0 6 Sum 48 0 27 15 6 Organellar 23S rRNA sequences frequently contain Group I introns and recent research revealed that the 23S rRNA of several hyperthermophilic bacteria also have Group I intron [ 37 ]. Other species only rarely have introns in rRNA genes, however, some 16S rRNA introns are known [ 38 ]. rRNA genes in bacteria are encoded in the so-called rrn-operon (see for example [ 39 ]). The canonical order of rRNA genes in the rrn-operon is 16S-23S-5S, but some exceptions to this rule are known. In Vibrio harvey, the order is 23S-16S-5S [ 40 ], but not in Vibrio cholerae [ 41 ] and Vibrio parahaemolyticus [ 42 ], whose 16S rRNA sequences were downloaded from the Comparative RNA Web Site. We divided the gathered sequences into two sets: data set A which consists of all RNA sequences that are thought to correspond to the originally transcribed sequence units and data set B which contains all those RNA sequences that are known to differ from the originally transcribed sequence units. Data set B thus contains the Group I and II intron sequences, organellar and hyperthermophilic bacteria 23S RNA sequences. As we neither know the sequence nor the secondary structure of the original transcript units from which the sequences of data set B were derived, we are limited to detecting the effects of co-transcriptional folding within these shorter sequences. We expect this to be much more difficult than in sequences that correspond to the originally transcribed sequence units as co-transcriptional folding introduces long range effects which are harder to detect the shorter the investigated sub-sequence gets. See Table 2 for a detailed overview of the composition of each data set. Results We calculated the 3'Cis x , 3'Trans x , 5'Cis x and 5'Trans x values for both types of weights, i.e. x ∈ { p,g }, for each sequence in the two data sets. From these values we then derived each sequence's Cis x and Trans x values, again for both x types of weights. Their distributions are shown in Figure 3 . Averaging over the values of all sequences in each of the two data sets resulted in the final values shown in Table 3 . Figure 3 Distribution of Cis and Trans values. Distribution of Cis and Trans values for the sequences of data sets A and B and both types of weights (plain (p) or free energy based (g)). The area under each curve has been normalized to one to allow a direct comparison between the two data sets. Table 3 Average values for different statistics. Final values of the different statistics which were obtained by averaging the values of each sequence in the data set. The error shown is the standard deviation. dataset A 0.215 ± 0.009 0.461 ± 0.032 0.285 ± 0.009 0.382 ± 0.032 0.070 ± 0.004 0.079 ± 0.026 B 0.298 ± 0.040 0.562 ± 0.086 0.296 ± 0.043 0.521 ± 0.075 -0.003 ± 0.015 0.041 ± 0.082 dataset A 2.916 ± 0.106 6.236 ± 0.431 3.710 ± 0.111 5.134 ± 0.354 0.794 ± 0.061 1.102 ± 0.384 B 3.392 ± 0.406 7.033 ± 1.050 3.362 ± 0.456 6.380 ± 0.954 -0.030 ± 0.184 0.653 ± 1.253 The first thing to note in Figure 3 is that all distributions follow approximately a symmetric distribution, thus confirming our theoretical considerations, and that the distributions of data set B are always shifted towards lower values with respect to the corresponding distributions for data set A which are always centered around average values larger than zero. The mean values of Cis and Trans in Table 3 are positive for data set A for both types of weights, indicating the influence of co-transcriptional folding, whereas they are closer to zero or even negative in the case of data set B. A Cis value larger zero means that configurations of type outnumber those of type , see Figure 2 . The formation of potential transient helices involving base-pairs between c and i that can later yield to the final secondary structure element containing the base-pair between i and thus seems to be encouraged. However, these transient structure elements may not be too stable if they are to guide rather than impede the proper folding. The presence of transient helices could thus be further substantiated by showing that these transient helices are less stable than the final helix. In contrast to the configuration, the competing ic helices in the case are suppressed as they lie 3' of the final helix and thus emerge later in time during co-transcriptional folding. A Cis value larger than zero can therefore be explained by the presence of temporary helices which may guide the formation of the final, functional secondary structure during co-transcriptional folding. A Trans value larger than zero means that configurations are less frequent than configurations, see Figure 2 . In the configuration, both c and are competing pairing partners for i as they both emerge before i during transcription. This may lead to the formation of wrong ci helices, whereas the order of pairing partners in the configuration has a lower risk of mis-folding due the c emerging only after the and thus only after the helix could have already formed. In addition, 3'Trans > 3'Cis in Table 3 can be interpreted as a stabilization of the final, functional secondary structure. Imagine that the hydrogen bounds of the or helix temporarily break up. In the case of the 3'Trans configuration, the pairing partners come in the order along the RNA sequence, whereas they come in the order in the 3'Cis configuration. In the order, the c part is in vicinity to the i part, so the possibility of ending up with a wrong refolding due to a ic helix is larger than in the case. Overall, we can thus conclude from the average values in Table 3 , that the sequences of data set A are tailored towards co-transcriptional folding, whereas we cannot reliably detect the effects of co-transcriptional folding within data set B. We detected co-transcriptional folding in data set A by showing that the final secondary structure is actively stabilized (3'Trans > 3'Cis), that the formation of temporary helices may guide the structure formation and that these helices may thus be used to actively engineer a folding pathway (Cis > 0) and that secondary structure elements which may interfere with the formation of the final, functional secondary structure during co-transcriptional folding are suppressed (Trans > 0). In order to quantify the influence of co-transcriptional folding further, we calculated two statistics, a t-test for the hypothesis that the given statistics have an expectation value of zero as well as the p-value of the number of positive cases for our two co-transcriptional folding indicators, see Table 4 . The high p-values for data set B imply that the presence of co-transcriptional folding is not well supported in this data set. However, the corresponding indicators strongly support co-transcriptional folding within data set A. Table 4 Statistical significance of results. p-values of t-test for the hypothesis that the final values in Table 3 have an expectation value of zero as well as the p-values for the hypothesis that the number of positive cases follows a binomial distribution with parameter 0.5. dataset A B p-value for t-test p-value for pos p-value for t-test p-value for pos < 0.0001 < 0.0001 0.5733 0.6137 < 0.0001 < 0.0001 0.5650 0.6137 0.0012 < 0.0001 0.3093 0.8068 0.0021 < 0.0001 0.3011 0.5000 Discussion Recent experimental studies [ 23 , 24 , 19 ] have shown that the proper speed of transcription helps the correct folding of RNA molecules. In addition, theoretical studies [ 16 ] indicate that the functional structure of an RNA need not correspond to the minimum free energy structure, even for moderately long RNA molecules. These findings suggest that co-transcriptional folding may play a decisive role in the formation of functional RNA structures. Although our statistics are able to reveal two general effects of co-transcriptional folding within data set A, we cannot conclude that they would be powerful enough to serve as a reliable indicator of co-transcriptional folding for single RNA sequences, as some of the sequences in data set A may not correspond to the originally transcribed sequence units. In addition, all of our statistics consider only a first order effect of co-transcriptional folding by studying alternative helices for the known helices, but do not take higher order effects into account as e.g. alternative helices of alternative helices etc. Based on computer simulations, H. Isambert et. al. [ 43 ] conjecture that pseudo-knotted motifs are common in co-transcriptional folding. Pseudo-knotted structures are explicitly included in our statistics, as the corresponding calculations naturally allow for alternative helices which are part of a pseudo-knot and as we do not reject them. Conclusions To summarize, our findings show that co-transcriptional folding is a guiding principle in the formation of functional RNA structure and that it can influence both the primary and potential secondary structures of an RNA molecule. This has several implications. Current algorithms for RNA secondary structure prediction can probably be improved by adopting co-transcriptional folding as a guiding principle rather than only free energy minimization. This may hopefully provide the extra information needed to be able to reliably detect RNA genes [ 44 ]. Several groups have already come up with computer algorithms which attempt to fold an RNA sequence co-transcriptionally [ 45 - 48 , 22 ]. These findings also have implications for computational methods which infer the phylogeny of RNA sequences, as these consider only co-evolution within the base-pairs of the functional helices, but discard any information due to the conservation of folding pathways and may hence mis-estimate evolutionary times. Similar arguments hold for all comparative studies that aim to detect functional secondary structure elements, since co-evolution of nucleic acids does not necessarily imply that these nucleic acids are base-paired in the final functional secondary structure. As evolution probably not only selects for the correct functional secondary structure, but also for a suitable folding pathway, it should be possible to detect the effects of co-transcriptional folding also in a comparative way. Most importantly, co-transcriptional folding should lead to a better understanding of how RNA sequences fold. This should in turn enable us to also understand why some RNA sequences mis-fold and fail to function properly in the organism. Even though protein folding is known to differ in many respects from RNA folding, they also have some features in common [ 49 ]. One of the obvious similarities is that both proteins and RNA sequences are synthesized in a directional process. It would thus be interesting to investigate if protein folding is also influenced by co-translational folding. In this study, we neither attempted to study the effects that co-transcriptional folding may have on sequences that are transcribed together (e.g. genes in an operon) nor to study the influence that the binding by proteins or RNA sequences or RNA editing may have on the co-transcriptional folding pathway and the final, functional RNA structure. This will almost certainly require more refined investigation methods, but we hope that this study provides enough insight and motivation to start to tackle these exciting questions. Authors' contributions I.M.M. proposed this work and contributed the main idea for the statistics. I.M. selected the data and evaluated the statistical significance of the results. Both authors shared the programming tasks and the writing of the manuscript.

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Structural comparison of metabolic networks in selected single cell organisms

Background There has been tremendous interest in the study of biological network structure. An array of measurements has been conceived to assess the topological properties of these networks. In this study, we compared the metabolic network structures of eleven single cell organisms representing the three domains of life using these measurements, hoping to find out whether the intrinsic network design principle(s), reflected by these measurements, are different among species in the three domains of life. Results Three groups of topological properties were used in this study: network indices, degree distribution measures and motif profile measure. All of which are higher-level topological properties except for the marginal degree distribution. Metabolic networks in Archaeal species are found to be different from those in S. cerevisiae and the six Bacterial species in almost all measured higher-level topological properties. Our findings also indicate that the metabolic network in Archaeal species is similar to the exponential random network. Conclusion If these metabolic network properties of the organisms studied can be extended to other species in their respective domains (which is likely), then the design principle(s) of Archaea are fundamentally different from those of Bacteria and Eukaryote. Furthermore, the functional mechanisms of Archaeal metabolic networks revealed in this study differentiate significantly from those of Bacterial and Eukaryotic organisms, which warrant further investigation.

Background Classification of biological organisms is of fundamental importance to evolutionary studies. It is commonly believed that there are three domains of life: Archaea, Bacteria and Eukaryote. Currently, the most popular classification method is the so called "molecular approach", in which polymorphism information in DNA or protein sequence is exploited to assess the phylogenetic relationships among species [ 1 , 2 ]. To a large extent, this is a "local" approach since the choice of sequence for comparison greatly affects the final result, "lateral gene transfer" (LGT) and thus the resulting "genome chimerism" further complicates the situation [ 3 ]. A new "system" approach that takes "global" properties of each organism into consideration serves as a potential alternative to overcome this shortcoming. Indeed, recent advances in system biology and increasingly available genomic databases have made it possible to rebuild biological networks from genomic data and have offered opportunity for such a "system" approach [ 4 ]. Podani and co-workers [ 5 ] proposed classifying organisms based on two kinds of network indices: the Jaccard index, which measures proportions of common sets of nodes in two networks, and Goodman-Kruskal γ function, which measures the similarity between rankings of nodes in two networks. They studied metabolic and information network structures of 43 organisms using these two measures under the hypothesis that network structure and the network design principle(s) behind them contain phylogenetic information. Ma and Zeng [ 6 ] conducted a more extensive phylogenetic classification study on 82 fully sequenced organisms based on different cellular function systems (enzyme, reaction, and genes) at the genomic level. They constructed phylogenetic tree based on Jaccard index and Korbel's definition, and concluded that in general, the classification based on network indices are in good agreement with the one obtained by analyzing the 16S rRNA using molecular approach. These studies seem to support the notion that significant differences in the network design principle(s) exist among the three domains of life [ 7 ]. These differences may reflect on the different approaches that organisms take to organize their entire systems to serve their special needs in the environment they live during the evolutionary history. Motivated by these encouraging results, in this manuscript, we went on to conduct a thorough comparison of network structural properties which provide further and more compelling evidences that significant differences exist among the network design principle(s) in organisms from the three domains of life. Restricted by the theoretical network structural studies, there are not many deterministic and informative topological measurements available [ 8 - 11 ]. The established measurements can be roughly divided into two categories: higher-level (global) properties and low-level (local) properties. The difference between the two is that one needs to know the whole network in order to calculate the higher-level property measures (e.g. average path length) while the low-level properties can be worked out locally (e.g. marginal degree of individual node) [ 9 ]. We use three groups of topological measurements (both low and higher-level) that address different aspects of the network structure. The first group contains network indices such as average clustering coefficient, average path length [ 12 ]. The second group is composed of degree distributions (both marginal and bivariate joint degree distributions) [ 8 - 11 , 13 ]. The third group is composed of network motif profiles that are recently shown to represent the network design principle(s) and global statistical properties of the network when aggregating together [ 14 - 16 ]. These measurements have been well studied in the network literatures, and are able to capture most aspects of network degree information. Single cell model organisms such as E. coli and S. cerevisiae have been studied intensively in biochemistry, cell biology and genetics; hence the rebuilt networks in those organisms present the best chance to approximate the true underlying network. Moreover, single cell organisms are less likely to have experienced the Whole Genome Duplication (WGD), which might drastically change the network structure [ 17 , 18 ]. As a result, we selected eleven single cell organisms to study their network structural properties: one Eukaryote: S. cerevisiae ; six Bacteria: E. coli , V. cholerae , R. solanacearum , B. subtilis , L. lactis , S. coelicolor ; and four Archaea: S. solfataricus , S. tokodaii , M. acetivorans , T. acidophilum . There are three main types of intracellular networks: the protein-protein interaction network, the transcriptional regulation network and the metabolic network. The first two are rebuilt by using high throughput techniques such as yeast two-hybrid system, in vivo pull down assay or DNA microarray, which are subject to high uncertainties, and the resulting networks may not be good approximation to biological complexity [ 19 - 22 ]. On the other hand, the metabolic network is derived from metabolic pathways, many of which are inferred from biochemical experiment-defined stoichiometries of many reactions [ 23 ]. It is well known that central pathways contain "hub nodes" of the whole metabolic network [ 24 , 25 ] and are also main building blocks of the so-called Giant Strongly Connected Component (GSCC) and Giant Weakly Connected Components (GWCC) [ 26 ]. The former is defined as the largest cluster of nodes within which any pair of nodes is mutually reachable from each other, and the latter is defined as the largest cluster of nodes within which each pair of nodes is connected in the underlying undirected graph [ 10 ]. Therefore, our high confidence in the structure of GSCC and GWCC, based on experimentally verified pathways, guarantees high confidence in whole network structure. The long history of biochemical studies of enzymes ensures relatively low false positive and low false negative rates of connections. Therefore, we decided to use metabolic networks in single cell organisms to compare network topological properties in the three domains of life. Results In constructing metabolic networks, Ma and Zeng [ 28 ] argued that connections through "current metabolites", which is referred to as cofactors in biochemistry such as ATP, ADP, H 2 O, should be removed from metabolic networks. We followed their suggestions by removing such "current metabolites" before conducting the following analysis. Group I measures: network indices Before checking different types of network topological measurements, we visually compared different metabolic networks (Fig. 1 ). Metabolic networks in S. cerevisiae and the six Bacterial species appear much more heterogeneous than Archaeal metabolic networks. It is well known that the so-called exponential random network (marginal degree distribution follows a Poisson distribution, see Methods for details) appears homogeneous while scale-free network (marginal degree distribution follows a power-law distribution, see Methods for details) appears more heterogeneous and modular [ 9 ]. Calculations of the two classic network indices, average clustering coefficient and average betweenness (see Methods for definition) also indicate that the metabolic networks in S. cerevisiae andthe six Bacterial species are more clustered and modular than those in the four Archaeal species (Table 1 , Fig. 2 ). From Table 1 and Fig. 2 , it is evident that the Clustering Coefficient (C) and Betweenness (B) did a better job in separating Archaeal species from non-Archaeal species than Average Path Length (L) and Diameter (D). Note that since we removed connections through "current metabolites" when constructing metabolic networks, our average path lengths are much longer than those reported in Jeong et al. [ 25 ] but similar to those reported in Ma and Zeng [ 28 ]. To avoid the confounding effects stemming from different network sizes, we calculated the so-called concentrations (number of appearances of subgraphs divided by the number of nodes with edges or arcs (directed edges), see Methods for details) of three-node subgraphs and four-node subgraphs. The concentration of subgraphs is an objective measure of the extent of clustering and modularity of the network [ 8 , 9 ]. It is observed that the concentrations of subgraphs in S. cerevisiae and the six Bacterial metabolic networks are much higher than those in Archaeal metabolic networks (Fig. 3 ). Group II measures: degree distributions Marginal degree distributions Recently, a variety of real-life networks are found to share the "scale-free" property, i.e. the marginal degree distribution follows a power-law distribution [ 25 , 29 - 31 ]. Our analysis demonstrates that the outgoing and incoming marginal degree distributions in metabolic networks also follow the power-law distribution. A simple linear model fits the log-transformed data well (except for the incoming degree distributions for most of the Archaea) which indicates that in general, the power-law model is appropriate to capture the structure of degree data (Fig. 4 ). Parameters were estimated using the Least Square method. The results together with goodness of fit measure R 2 and 95% individual confidence intervals are summarized in Table 2 and Table 3 . The estimated power-law index γ is around -0.3 in all cases and the estimated log-transformed scaling parameter α ranges within 2.0 to 2.5. These indicate that marginal degree distribution, which is a low-level (local) topological property measure, although showed some distinction, is not enough to effectively differentiate networks from different domains. Overall, metabolic networks in most of the species we studied seem to follow the power-law distributions and thus are "scale-free". The fact that the incoming degree distributions of most Archaeal species we studied do not follow power-law well (Fig. 4B ) suggests that networks in Archaeal species tend to be less "scale-free" and more "random-like" compared to those of the non-Archaeal species. As we have shown, marginal degree distribution alone does not reveal the fundamental network structural differences between the Archaeal species and the non-Archaeal species. Simulation studies have shown that randomized networks preserving marginal degree distribution can be quite different in terms of global (higher level) topological properties such as average clustering coefficient [ 9 ]. In metabolic networks, we are unable to determine the preferred types of reactions based on just marginal substrate or product degree distributions. Since the metabolic network is rebuilt from chemical reactions, joint behavior of substrate and product in reactions should be more informative than disjoint behavior of metabolites. Therefore, we calculate the joint degree distributions hoping to gain more insight into the network organization. Joint degree distributions Joint degree distribution measures and describes correlation between connectivities of neighboring nodes. N ( K 0 , K 1 ) is defined as the number of edges connecting nodes of connectivity K 0 to those of connectivity K 1 . For metabolic networks, which are directed, N ( K out , K in ) is used to measure the number of arches where substrate (node) with out-connectivity K out transforms to product with in-connectivity K in . This quantity reflects intrinsic properties of the network and can be used to distinguish different types of networks. For instance, we can test whether N ( K out , K in ) of a particular network differs significantly from that of the random network. To be specific, we calculate , where ( K out , K in ) represents the mean of random variable N ( K out , K in ) in a large number (say, 1000) of random networks simulated by an edge-rewiring algorithm proposed by Maslov and Sneppen [ 13 ], ( K out , K in ) denotes the estimated standard deviation of N ( K out , K in ). The p -value can then be obtained by compare Z to a standard normal distribution. Comparing with "properly" randomized network ensembles allows us to concentrate on those statistically significant patterns of the complex network that are likely to reflect the design principle(s) [ 13 ]. We calculated statistically significant correlation profiles (Z-score profiles, see Methods for details) for the metabolic network in each organism (Fig. 5 ). The Z-score profiles of the four Archaeal species are similar to each other but quite different from those in S. cerevisiae and the six Bacterial species. Although the dark red regions of the Z-score profiles in Archaeal species are quite different in scale, they all seem to differ significantly from the random network preserving the corresponding marginal degree distribution in a similar way ( p -value < 0.1). Looking into the correlation profiles more carefully, we found that the number of statistically significant positive ( K out , K in ) increases in the order of S. cerevisiae , the six Bacterial species and the four Archaeal species. The significant Z-score of certain observation N ( K out , K in ) implies that the chemical reaction between substrates with out-degree K out and products with in-degree K in are statistically significant. We define substrates whose K out >= 2 or products whose K in >= 2 as versatile metabolites. Thus, the above trend implies that the preference to employ reactions involving versatile metabolites increases in the order of S. cerevisiae , the six Bacterial species and the four Archaeal species. Correspondingly, the variety of metabolites decreases in the above order and so does the number of distinct enzymes or variety of enzymes because of the high specific binding of metabolites and enzyme. This is consistent with the biological facts that S. cerevisiae (Eukaryote) encodes a greater variety of enzymes than Bacterial and Archaeal species. Group III measure: Network Motif The network motif is defined to be recurring and non-random building blocks of the network [ 14 , 15 ]. Just like sequence motif, which is an over-represented and biologically meaningful DNA or protein sub-sequence, network motif is an over-represented and biologically meaningful subgraph. Network motif has been shown to be informative of network design principle(s) and network structure. It was found that over 80% of the nodes in the E. coli transcription regulation network are covered by network motifs [ 14 ]. Dobrin et al. [ 16 ] recently discovered that in the E. coli transcriptional regulatory network, "individual motifs aggregate into homologous motif clusters and a supercluster forming the backbone of the network and play a central role in defining its global topological organization." More importantly, network motifs capture the information that is likely to be missed by the correlation profiles because motif actually describes the number of appearances of certain configurations of multiple nodes, and therefore nicely complement with the correlation profiles [ 9 ]. One might argue that there are certain amount of overlaps between the information they capture but the motif profile does not capture the degree information of the connecting nodes, which may be the most powerful feature of the correlation profiles. We searched for all of the 13 three-node subgraphs and all of the 199 four-node subgraphs in the metabolic networks of eleven species. The results showed that the three-node motif profiles found in S. cerevisiae and the six Bacterial species are identical while there is no three-node motif found in any of the four Archaeal networks (Fig. 6 ). Also there is no common four-node motif shared by Archaeal species and S. cerevisiae /Bacterial species while two four-node motifs (id4702, id4950) are shared by the latter ( Additional file 1 ). Among all the 13 possible three-node subgraphs, six of them have one pair of nodes not directly connected. Abundance of such subgraphs will lower the extent of clustering and modularity of the network. As expected, we found that all three-node motifs identified in S. cerevisiae and the six Bacterial species form triangles (Fig. 6 ). It may explain our main finding that metabolic networks in non-Archaeal species are more clustered and modular than those in Archaeal species. Discussion Based on our comparison of network structural properties beyond network indices, we were able to gain more insight into the structural differences across the three domains of life. Having shown that the metabolic network is "scale-free", we further showed that metabolic networks in the four Archaeal species are closer to "exponential random network" [9:Ch2, [ 11 ]] than those in S. cerevisiae and the six Bacterial species. The reasons are the following: First, the Archaeal metabolic networks are visually more homogeneous among themselves compared to their counterparts in the non-Archaeal species. In random networks, any pair of nodes is equally likely to be connected. The network topology should look homogeneous given that the size of network is large enough. The "scale-free" network, on the other hand, features a highly modular and heterogeneous topology since the marginal degree is power-law distributed [ 8 , 9 ]. Moreover, the marginal degree distributions of the metabolic networks in non-Archaeal species fit the power-law model better than Archaeal species (Table 2 and Table 3 ). Second, the average clustering coefficient and average betweenness of Archaeal metabolic networks are much smaller than those in S. cerevisiae and the six Bacterial species. The same is true for the concentrations of three-node and four-node subgraphs. As pointed out by Watts and Strogatz, real-life networks show strong clustering or network transitivity while exponential random network does not [ 12 ]. Third, there is no three-node motif and fewer four-node motifs found in Archaeal metabolic networks compared to non-Archaeal metabolic networks. In particular, the ubiquitous feed-forward loop (FFL) motif found in networks from biology (including metabolic networks in S. cerevisiae and the six Bacteria species in this study) to neurology and engineering fields was not found in any of the four Archaeal metabolic networks (Fig. 6 ). Since motifs are statistically significant subgraphs compared to "properly" randomized network ensembles, no motif or fewer than usual motifs found in a real-life network indicates that the network structure is closer to that of a random network. It has been shown by Milo et al. [ 15 ] that concentration of FFL motif is insensitive to the network size within E. coli transcription regulation network, but diminishes to zero in increasingly larger random networks. This also supports that Archaeal metabolic networks are closer to randomized network ensembles than other real-life networks. The metabolic networks in Archaea are both "random-like" and "scale-free", which might exert profound influences on their adaptability to the hostile environment. Archaeal species are typically restricted to marginal habitats such as hot springs or areas of low oxygen concentration and can assimilate different kinds of inorganic carbon and nitrogen sources. Indeed, the chemical structure and component of the macromolecules such as protein and lipid make significant contributions to the organism's adaptability to the environment. The seemingly ad hoc network organization (both "random-like" and "scale-free") in Archaeal species might also enabled them to survive in those extreme physiological conditions. Archaeal species might employ some biologically significant subgraphs (rather than statistically significant motifs) which can not be detected by current motif searching algorithm [ 15 ]. This makes the Archaeal metabolic networks appear random in statistical sense (not statistically significantly different from random networks) but not in biological sense. Our comparison results showed that many network structural properties measured in Archaeal species are different from those of non-Archaeal species. However, the hidden anthropomorphic factors might account for some of the differences observed. Specifically, the drastic differences of topological profiles between the metabolic networks of Archaeal species and non-Archaeal species may be partially explained by the fact that significantly less extensive metabolic pathway studies have been conducted in Archaeal species [ 32 ]. Robustness of topological profiles against random perturbations can alleviate the impact to a certain extent but is unable to eradicate it [ 9 ]. Conclusions Our network analysis results showed that in most of higher-level (global) topological properties measured, metabolic networks in the four Archaeal species are similar to each other but significantly different from those in S. cerevisiae and the six Bacterial species. This provides further evidence that the metabolic network structures and consequently the design principle(s) in the four Archaeal species are very different from those in S. cerevisiae (Eukaryote) and the six Bacterial species. Our finding that the metabolic networks in Archaeal species possess many properties of the exponential random network begs for better understanding of the design principle(s) in biological networks, which may be revealed by further systematic analyses. For example, locate and align conservative pathways such as glycosis between E. coli or S. cerevisiae and Archaeal species to understand the functional mechanisms of Archaeal metabolic networks. Methods Data source Chemical reaction data was obtained from metabolic database in Ma and Zeng [ 28 ], which consists of five related tables: reaction , enzyme , react , connect and organism . We compiled a new table from this database excluding any inconsistent or redundant connections between metabolites (details below). SQL was used to query the database. Identify and remove inconsistency Inconsistent connections refer to pairs of metabolites that have conflicting reversibility annotation. It is caused by the fact that a pair of metabolites can be in more than one reaction and the reversibility of these reactions can be different. For example, NAD + and Nicotinamide is a pair of metabolites in two reactions: 1) NAD + + L-Arginine = Nicotinamide + N 2 (ADP-D-ribosyl)-L-arginine 2) NAD + + H 2 O -> Nicotinamide + ADPribose. (Note that here the role of NAD + is NOT "current" metabolite, and hence connections established through it should NOT be removed). Reaction 1 is a reversible reaction while reaction 2 is not. We annotated an edge between the two metabolites as long as there was at least one reversible reaction that both of them were involved. For example, the type of connection between NAD + and Nicotinamide is edge (undirected connection). This step could be summarized as "edge ← edge + arc". Identify and remove redundancy There are also numerous redundant connections where the same pair of metabolites switch their roles between substrate and product in two or more different irreversible reactions. For example: 1) UDPglucose + N-Acylsphingosine = UDP + Glucosylceramide 2) Glucosylceramide + H 2 O = D-Glucose + N-Acylsphingosine . ( N-Acylsphingosine and Glucosylceramide is a pair of metabolites that switch their roles in two irreversible reactions). In case of redundancy, we annotated an edge between the pair of metabolites rather than the two arcs because they could be converted to each other through two reactions. This step could be summarized as "edge ← arc + arc". Definitions of some network topological measurements Clustering coefficient (C) We define two kinds of clustering coefficients for each node in the directed metabolic networks, i.e. C in and C out . C in measures the average clustering coefficient of the node representing the product that can be generated from its first-order "nearest neighbors" through chemical reactions. C out measures the average clustering coefficient of the node that generate its first-order "nearest neighbors" through chemical reactions. The larger the coefficients, the more clustered and modular the network appears to be. Betweenness (B) The betweenness for any node n i in the network is defined as , where g jk is the number of shortest paths between node j and node k . g jk ( n i ) is the number of shortest path between node j and node k containing node n i , g is the total number of nodes with edges/arcs. C B ( n i ) needs to be multiplied by two in the case of directed network [ 27 ]. The average betweenness is defined as: . Higher value of betweenness indicates the network is more clustered and modular. Average path length (L) Watts and Strogatz [ 12 ] defined the average path length as , where d ( j , k ) is the shortest path length between node j and node k (distance), V represents the set of all nodes with edges/arcs of the graph, and g is the number of nodes with edges/arcs. Diameter (D) The diameter of the directed graph G is the longest geodesic between any pairs of nodes. The geodesic is the shortest path between a pair of nodes. Pajek [ 33 ] was used to calculate the average betweenness, average path length and diameter. Concentration of subgraphs (S) Wasserman and Katherine [ 27 ] defined the subgraph as follows: A graph G s is a subgraph of G if the set of nodes of G s is a subset of the set of nodes of G , and the set of lines in G s is a subset of the lines in the graph G . Let M be the number of subgraphs, and N be the number of nodes with edges or arcs. Then the "concentration of subgraph" is defined as C = M/N . A high value of C indicates the network is more clustered and modular. Mfinder1.1 [ 15 ] was used to calculate both M and N . Marginal degree distribution calculations The marginal degree distribution of each network is calculated from the Boolean adjacency matrix A , a matrix of 0 or 1. Zero means there is no connection between nodes, and 1 the opposite. The outgoing degree of the node i , k out ( i ) is defined as , where . The incoming degree of the node i , k in ( i ) is defined as . Simple regression analyses of marginal degree distributions The power-law degree model was first log transformed into linear model, i.e. log P ( K i ) = γ log( K i ) + log( α ) + ε i ( i = 1,2,...,n ), γ and α are parameters, ε i is the residual. K i is the degree and P ( K i ) is the corresponding probability. Based on the fitted linear model, we made statistical inference including parameter estimation and individual confidence intervals on the estimates using the Least Square method. Correlation profile calculations Statistically significant correlation profiles were calculated using Matlab code downloaded from Dr. Maslov's website [ 34 ]. The adjacency matrix of the network is the input. Motif profiles calculations According to Milo et al.[ 15 ] , a subgraph is referred to as a motif if the following criteria are met: 1) Its empirical p -value is smaller than a pre-specified threshold, e.g. 0.01. 2) The number of appearances in real networks with distinct sets of nodes is larger than another pre-specified cut-off value, e.g. 4. 3) The number of appearances in real networks is significantly larger than that in randomized networks, i.e. . N real and N rand represent the number of certain subgraphs detected in real-life network and randomized networks, respectively. This is to avoid the situation where some common subgraphs are detected as motifs that have only slight differences in N real and N rand but have a narrow spread of distribution in randomized networks [ 14 , 15 ]. Motif profiles are generated using the Mfinder program. This program and the motif dictionary were downloaded from Dr. Uri Alon group's website [ 35 ]. Authors' contributions DZ and ZSQ conceived and designed the study; DZ wrote the computer code, analyzed the data and draft the manuscript. Both authors read and approved the final manuscript. Supplementary Material Additional File 1 Four-node motifs found in the metabolic networks in different species. The number of connecting nodes for each network is shown. For each motif, the numbers of appearances in real networks ( N real ) and in randomized networks ( N rand ± SD , all values rounded) are shown. The p -values of all motifs are less than 0.01, as determined by comparing to 1000 randomized networks. Each motif occurs at least four times in one network. Other restrictions apply. Motifs were detected and generated using program found in Milo et al. [ 15 ] and the motif dictionary therein. Click here for file

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300675

Structural Mechanism Shows How Transferrin Receptor Binds Multiple Ligands and Sheds Light on a Hereditary Iron Disease

Iron is an essential nutrient for sustaining life-forms as diverse as plankton and humans. But too much iron, or too little, can spell trouble. Mammalian cells maintain the proper balance partly with the help of a specialized cell surface protein called the transferrin receptor (TfR). TfRs bind to the iron-carrying transferrin protein (Fe-Tf) and escort their cargo to the cell's interior. (To learn more about iron metabolism, see the primer by Tracey A. Rouault in this issue [DOI: 10.3171/journal.pbio.0000079 ].) This receptor also binds the hereditary hemochromatosis protein (HFE), which is mutated in individuals who have the common iron-overload disorder hereditary hemochromatosis. While the molecular pathway that mediates cellular intake of iron through the TfR is known, it was not clear just how TfR assists in iron release to the cell and how it binds HFE and transferrin. By introducing multiple mutations in human TfRs, Pamela Bjorkman and colleagues identified functional binding sites for transferrin in both its iron-loaded and iron-free (apo-Tf) forms and for HFE. From these data, the researchers mapped out a scenario of the dynamic interactions between receptor and ligands (the bound molecule) and worked out a structure-based model for the mechanism of TfR-assisted iron release from Fe-Tf. Bjorkman's lab, which had previously solved the structures of both HFE and HFE bound to the TfR, used their structural information to investigate how the proteins interact, which amino acid residues are required for binding, whether the two ligands bind differently to the receptor, and how HFE binding affects transferrin binding. They found that Fe-Tf and HFE occupy the same or an overlapping site on the receptor, but since transferrin is much larger than the HFE protein, it appeared that transferrin could also interact with other parts of TfR. And it remained to be seen whether TfR discriminated between the iron-loaded and iron-free states of transferrin. In this study, Bjorkman and colleagues expanded their library of TfR mutants to clarify the transferrin binding signature on TfR and to see how the TfR mutations affect the way apo-Tf and Fe-Tf interact with the receptor. They characterized the binding affinities of 30 TfR mutants to HFE and Fe-Tf and to apo-Tf, and they report that mutations in 11 of the TfR residues interfere with either one or both forms of transferrin. Four of these residues are essential for transferrin binding and are conserved in all known TfR DNA sequences. Since residues that didn't have much impact are not conserved, the scientists say the results are likely to describe transferrin–TfR interactions for other species as well. As expected, the most critical residues required for transferrin binding fall within the receptor's helical domain and have significant physical overlap with residues required for HFE binding; though some residues that are required for apo-Tf binding do not affect Fe-Tf binding. Bjorkman et al. also identify additional residues in another domain on TfR (called the protease-like domain) that support Fe-Tf but not apo-Tf binding, confirming that the receptor binding footprints of the two metal-binding states of transferrin are indeed different. With a structural model showing where Fe-Tf and apo-Tf bind to the receptor, they could evaluate how they bind and thus explain how the receptor mediates iron release. By suggesting a mechanism through which TfR binding regulates iron release, this structural model of the transferrin–TfR complex will bolster efforts to elucidate the molecular details of this process. Confirmation that transferrin and HFE do indeed compete for docking privileges reveals a possible role for HFE in maintaining iron homeostasis and will provide valuable insights into the dysregulation that leads to the warehousing of iron and resulting tissue and organ damage associated with hemochromatosis. Ribbon diagram of transferrin receptor homodimer

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548683

FAM20: an evolutionarily conserved family of secreted proteins expressed in hematopoietic cells

Background Hematopoiesis is a complex developmental process controlled by a large number of factors that regulate stem cell renewal, lineage commitment and differentiation. Secreted proteins, including the hematopoietic growth factors, play critical roles in these processes and have important biological and clinical significance. We have employed representational difference analysis to identify genes that are differentially expressed during experimentally induced myeloid differentiation in the murine EML hematopoietic stem cell line. Results One identified clone encoded a previously unidentified protein of 541 amino acids that contains an amino terminal signal sequence but no other characterized domains. This protein is a member of family of related proteins that has been named family with sequence similarity 20 (FAM20) with three members (FAM20A, FAM20B and FAM20C) in mammals. Evolutionary comparisons revealed the existence of a single FAM20 gene in the simple vertebrate Ciona intestinalis and the invertebrate worm Caenorhabditis elegans and two genes in two insect species, Drosophila melanogaster and Anopheles gambiae . Six FAM20 family members were identified in the genome of the pufferfish, Fugu rubripes and five members in the zebrafish, Danio rerio . The mouse Fam20a protein was ectopically expressed in a mammalian cell line and found to be a bona fide secreted protein and efficient secretion was dependent on the integrity of the signal sequence. Expression analysis revealed that the Fam20a gene was indeed differentially expressed during hematopoietic differentiation and that the other two family members (Fam20b and Fam20c) were also expressed during hematcpoiesis but that their mRNA levels did not vary significantly. Likewise FAM20A was expressed in more limited set of human tissues than the other two family members. Conclusions The FAM20 family represents a new family of secreted proteins with potential functions in regulating differentiation and function of hematopoietic and other tissues. The Fam20a mRNA was only expressed during early stages of hematopoietic development and may play a role in lineage commitment or proliferation. The expansion in gene number in different species suggests that the family has evolved as a result of several gene duplication events that have occurred in both vertebrates and invertebrates.

Background Hematopoietic differentiation is a complex process whereby multiple functionally and morphologically distinct cell types arise from a population of pluripotent hematopoietic stem cells (PHSCs) [ 1 ]. The accurate and efficient regulation of hematopoietic development is controlled by a large number of regulatory proteins that have been identified over the past few decades. These regulatory molecules include the hematopoietic growth factors (HGFs), soluble proteins that recognize specific receptors on the surface of sub-populations of hematopoietic cells, thereby initiating signal transduction pathways that modulate the differentiation, proliferation, and/or survival of target cells [ 2 ]. The identification of regulators of hematopoiesis has been an ongoing effort for many years and has benefited from the existence of accessible cell line models as well as the characterization of genes affected by somatic mutations associated with specific human leukemias [ 3 ]. We have used a pair of factor-dependent murine cell lines to identify novel genes expressed within distinct hematopoietic lineages as an approach to the identification of novel candidate genes for development of diagnostic and therapeutic approaches to leukemia. The EML and MPRO cell lines were both established by infecting murine bone marrow cells with a retrovirus expressing a dominant negative retinoic acid receptor α (RARα) protein [ 4 , 5 ]. The infected cells were selected in the presence of either stem cell factor (SCF) or granulocyte/macrophage colony stimulating factor (GM-CSF). EML are SCF-dependent and resemble uncommitted hematopoietic progenitor cells. They can be induced to differentiate to the promyelocyte stage of granulopoiesis in the presence of interleukin-3 (IL-3) and high doses of all trans retinoic acid (atRA) [ 4 , 6 ]. Terminal neutrophil differentiation of EML cells can be induced by replacement of IL-3 and SCF with GM-CSF. MPRO cells are GM-CSF-dependent and can be induced to differentiate to neutrophils by adding high doses of atRA to the culture medium. The expression patterns of a number of genes expressed during hematopoiesis have been examined in EML and MPRO cells and generally agree with the patterns observed in other cell systems and in primary hematopoietic cells. Thus, EML and MPRO provide a powerful system for the identification and characterization of novel genes expressed within the hematopoietic lineage. We have employed the representation difference analysis technique [ 7 ] to identify cDNAs representing genes expressed at higher levels in EML cells 72 hours after induction of differentiation than in uninduced cells. We describe the identification of a clone derived from an uncharacterized putative secreted protein. We have performed a comparative genomics analysis and determined that this protein is the founding member of an extended family of highly related proteins. This family contains three members in mammalian species, one or two members in invertebrate or simple vertebrate species and five or six members in fish. We have determined that one family member is a secreted glycoprotein and describe the expression pattern of the human and mouse genes in tissues and during hematopoietic differentiation. Results Identification of differentially expressed genes by representational difference analysis (RDA) Total RNA was prepared from EML cells grown in the presence of SCF alone (0 hour) or in medium supplemented with IL-3 and atRA for 72 hours. The RNA was converted to cDNA and subjected to three rounds of RDA as previously described [ 6 ]. Six differentially-expressed clones were identified [ 6 , 8 ]. Clone number 1623 was chosen for further analysis and the differential expression of this gene was confirmed by Northern blot analysis. 1623 mRNA was essentially undetectable in the 0 hour sample but readily detectable in the 72 hour sample (data not shown and see figure 10 ). Sequence analysis of clone 1623 The initial cDNA isolated by RDA was a 273 bp fragment that appeared to contain the coding sequence of the C-terminus of a protein that was not present at that time in public databases. To identify the full open reading frame of this cDNA, we first performed rapid amplification of cDNA ends (RACE) in both the 5' and 3' directions. Extension in the 3' direction revealed the presence of a consensus polyadenylation signal located 154 nucleotides downstream of the putative translation stop codon. Extension in the 5' direction yielded an additional 443 bp of sequence containing a contiguous ORF. Comparison of this extended sequence to public databases identified a cDNA (NM_017565) that was identical to clone 1623 in the region of overlap. This cDNA was isolated from a mouse mammary tumor but no functional analysis had been performed. We designed PCR primers based on the published sequence and confirmed that the cDNA isolated from EML cells was identical to the published sequence. The full length ORF was 1623 bp in length and encoded a protein of 541 amino acids (Figure 1A ). The protein did not contain any recognizable motifs when examined using domain mapping software such as SMART or Profilescan (see Methods). However, a putative amino terminal signal sequence was identified using the SignalP analysis program. The cDNA mapped to an 11 exon gene located on mouse chromosome 11E1 (Figure 1B ). The gene spanned approximately 60,000 bp of genomic sequence and the relatively large size of the gene is primarily due to the fact that the first intron is greater than 44,000 bp in length (Figure 1C ). Figure 1 Characterization of the full length mouse 1623 (Fam20a) cDNA and genomic sequence. A. The full length cDNA derived from the original RDA clone was isolated using a combination of 5' and 3' rapid amplification of cDNA ends (RACE) procedures, comparisons to public databases, and amplification of putative full length clones by PCR. The full open reading frame was 1623 bp in length and encoded a 541 amino acid protein. The locations of regions conserved within the subsequently identified FAM20 family are indicated using underlines. Eight cysteine residues that are also conserved within the family are indicated in bold and four putative N-glycosylation sites are indicated in red type. B. The distribution of the 11 exons of the mouse Fam20a gene is shown with the exons indicated using numbers. A consensus polyadenylation signal is located downstream of the terminal exon. C. The sizes of the 11 exons and 10 introns of the Fam20a gene are shown. Identification of a family of related genes The full length cDNA and the encoded protein were compared to sequences in public databases. We reported previously a weak similarity to a protein named Fjx1, which is the mouse orthologue of a Drosophila protein named four-jointed [ 8 ]. However, the degree of sequence identity between these proteins was low (16%) and thus the search was extended to include uncharacterized proteins. We first identified two other mouse proteins that displayed significant similarity to, but were distinct from, the query sequence. One protein (Accession number NP_663388 aka Riken C530043G21) was 409 amino acids in length and displayed 27% identity to the query sequence while the second (NP_085042) was a truncated version of a 579 amino acid protein that displayed 40% identity to the query sequence. We have subsequently discovered that these proteins are members of a highly related family and this family has received the official name "family with sequence similarity 20" (FAM20) from the Human Genome Organization Gene Nomenclature Committee. The protein derived from our original 1623 cDNA is named Fam20a in mouse and the other two family members are named Fam20b and Fam20c, respectively. Continued searches of public databases revealed the existence of related proteins in several other species. Each mammalian genome contains genes encoding three members that are orthologous to the three mouse proteins mentioned above. The accession numbers for the relevant cDNAs in human and rat are listed in Table 1 and we have also identified the same number of related sequences in other mammalian genomes, including the pig, cow and dog (data not shown). However, most of these sequences are incomplete and will not be described in detail here. Table 1 Accession numbers for vertebrate FAM20 family members 1 Family Member Human Mouse Rat Fugu Zebrafish Ciona FAM20A NM_017565 NM_153782 XM_221067 (BK001521) SINFRUP00000146987 (BK001515) ND ND FAM20B NM_014864 NM_145413 XM_222770 SINFRUP00000138548 (BK001520) CAI11712 AK115425 FAM20C NM_020223 NM_030565 XM_221975 (BK001522) SINFRUP00000140879 (BK001516) SINFRUP00000141732 (BK001518) SINFRUP00000163431 (BK001517) BK001519 ENSDARP00000009272 ENSDARP00000005688 ENSDARP00000028589 ND 1. Accession numbers refer to nucleotide sequences in GenBank except in the Fugu and Zebrafish listing where the accession numbers refer either to predicted peptides in the Ensembl database (entries beginning SINFRUP or ENSDARP) or Genbank (entry beginning CAI). Entries beginning with BK refer to predicted sequences in the Third Party Annotation database arising from this study. ND: None detected. In order to gain further information concerning the origin of the FAM20 family, we searched for related sequences in several other invertebrate and vertebrate organisms. The ascidian Ciona intestinalis is a model for a basal chordate organism and has emerged as a powerful model for evolutionary and developmental studies [ 9 , 10 ]. In particular, many gene families or subfamilies are represented by single members in C. intestinalis and thus the identification of an orthologue in this organism can provide useful information for evaluating the evolutionary origin of the members of a gene family. Consistent with this concept, we identified a single cDNA and the corresponding genomic locus in C. intestinalis that displayed significant sequence similarity to the mammalian FAM20 genes and proteins (Table 1 ). Complete genome sequences are also available for several invertebrate species and two related sequences were identified in Drosophila melanogaster and Anopheles gambiae with one family member in Caenorhabditis elegans (Table 2 ). Finally, analysis of genomic cDNA and protein databases for the pufferfish ( Fugu rubripes ) and zebrafish ( Danio rerio ) revealed the presence of six and five family representatives, respectively (Table 1 ). The gene numbers in these various species are listed on an idealized evolutionary tree in Figure 2 and suggest that the FAM20 gene family has undergone a complex set of gene duplications in both the invertebrate and chordate lineages. Table 2 Accession numbers for invertebrate FAM20 family members 1 Family Member Drosophila Mosquito C. elegans FAM20A ND ND ND FAM20B NM_170079 NM_206490 EAA08010 EAA13434 NM_078126 Fam20C ND ND ND 1. Accession numbers refer to nucleotide sequences in GenBank. ND: None detected. Figure 2 Evolutionary distribution of FAM20 gene number. An idealized evolutionary tree (modified from [10]) is shown with the number of FAM20 genes identified in several genomes as described in the text. The gene numbers are supportive of a single gene duplication event occurring in invertebrates (at least in insects) and multiple gene duplication events occurring in higher vertebrates. Assignment of subfamily relationships To elucidate the nature of these putative gene duplications, we sought to assign the individual sequences from the various species into subfamilies based on protein sequence and gene structure, specifically using the number and size of exons in the latter case. Initially, the exon distribution of each of the Fam20 members in three mammalian species (human, mouse and rat) was compared and revealed obvious inter and intra orthologue similarities (figure 3A ). Each FAM20A gene contained 11 exons and exon sizes were identical in these three species. Likewise, each FAM20B gene contained 7 exons that were identical in size in human, mouse and rat. The FAM20C genes each contained 10 exons and only exon 1 displayed any variation in size amongst these three species. In intra-orthologue comparisons, the exons in FAM20B and FAM20C genes clearly aligned with exons in the FAM20A genes, with small variations (in multiples of three bases) in the size of the internal exons in FAM20B. FAM20B lacks exons corresponding to exons 2–4 of FAM20A while FAM20C lacks exon 3. In addition, exons 8 and 9 in FAM20A and FAM20C are represented by a single exon in FAM20B that is identical in size to the combined exons in the other two genes. Thus, the three mammalian genes are highly evolutionarily related and presumably are derived from a common ancestral gene. Figure 3 Assignment of FAM20 family members to subfamilies. A. Exon size and distribution of mammalian FAM20 members. The exons within each FAM20 gene in human, mouse and rat are indicated with the number of base pairs indicated within each exon. The sizes of exons that differ in size from the FAM20A genes are indicated. B. A dendrogram showing the relationships between FAM20 proteins from human (Hs), mouse (Mm), rat (Rn), Fugu rubripes (Fr), Danio rerio (Dr), D. melanogaster (Dm), A. gambiae (Ag), C. intestinalis (Ci) and C. elegans . The accession numbers of the cDNA sequences from which each protein sequence was derived are shown in parentheses except in the case of the mosquito family members where the accession number is used as the gene/protein name. Accession numbers for zebrafish peptide sequences are listed in Table 1. The FAM20 nomenclature has not been extended to the invertebrate sequences and the previous gene names have been used for Drosophila and C. elegans family members. The subfamily assignment of each family member is shown on the right. C. Exon number and size distribution within Fugu Fam20 members. The accession number of each sequence within the Third Party Annotation database is shown at left and family assignment based on dendrogram position and exon distribution is shown on the right. To assign the genes identified in other species to these three subfamilies, we performed a global comparison of the peptide sequences derived from 25 of the identified family members listed in Tables 1 and 2 . One zebrafish protein (from FAM20A) was omitted as its sequence is incomplete. A dendrogram showing the results of this comparison is presented in Figure 3B . As expected, the mammalian orthologues clustered together and thus defined the subfamilies. All of the invertebrate proteins and the single protein identified in C. intestinalis clustered with FAM20B proteins, suggesting that this represents the ancestral branch of the FAM20 family. A single protein from Fugu and zebrafish clustered with the FAM20A and FAM20B family members while two Fugu and two zebrafish proteins clustered with FAM20C members. However, two Fugu proteins and one zebrafish protein clustered on a separate branch between FAM20A and FAM20B. In order to determine the subfamily to which these proteins belonged, we made use of the high degree of conservation of exon size and number noted in the mammalian genes (Figure 3C ). The exon number and size of the Fugu and zebrafish genes encoding the two proteins assigned to FAM20A and FAM20B were consistent with their membership in these families. The only variations noted were a slightly larger exon 2 in the Fugu Fam20a gene and the division of exon 1 into two exons in the Fugu Fam20b gene. As in the mammalian family members, the sizes of the terminal exons varied more than the internal exons. The other four Fugu genes displayed exon distributions consistent with membership in FAM20C, despite the clustering of two of the encoded proteins between FAM20A and FAM20B. We have assigned each of these proteins to FAM20C with number suffixes (c1, c2, etc.) to designate individual genes and proteins. Each of these genes maps to distinct genomic loci and thus represents independent genes and not splicing variants of a smaller number of genes (data not shown). The gene structures of the three zebrafish family members were also consistent with this family assignment (data not shown). Comparisons of the derived protein sequences within each subfamily are shown in figures 4 , 5 , 6 . Figure 4 Sequence alignment of FAM20A protein sequences. The complete protein sequences of FAM20A members were compared using the AlignX component of the VectorNTI sequence analysis suite of programs. Identical amino acids are outlined in yellow, and similar residues are indicates in light blue. Conserved regions 1, 2 and 3 are underlined (see below). Gaps are indicated with dashes and the sequences are from human (H), mouse (M), rat (R) and puff erfish (F). Figure 5 Sequence alignment of FAM20B protein sequences. The complete protein sequences of FAM20B members are presented as described in figure 4. The sequences are from human (H), mouse (M), rat (R), pufferfish (F), zebrafish (D) and C. intestinalis (Ci). Figure 6 Sequence alignment of FAM20C protein sequences. The complete protein sequences of FAM20C members are presented as described in figure 4. The sequences are from human (H), mouse (M), rat (R), pufferfish (Fcl-4) and zebrafish (Dcl-3). Features of FAM20 proteins All of the identified FAM20 protein sequences contain putative signal sequences at their amino termini but no other functional domains were unambiguously detected using several different annotation search software programs. In order to search for potential functional domains, we compared the sequences of all family members. These comparisons revealed that the greatest similarity was located within the carboxy-terminal two thirds of each protein (Figure 7A ). We have named this region the conserved C-terminal domain (CCD) and it overlaps with a domain listed in the CDD database at NCBI as DUF1193. The CCD contains three distinct regions that are more highly conserved within all members of the family than the surrounding sequences (named conserved regions 1, 2 and 3 in figure 7A ) and the consensus sequences for each conserved region were derived (figure 7B ). Amino acids that are essentially invariant in all family members have been indicated in bold type and the heptapeptide DRHHYE in CR2 is the longest contiguous sequence that is conserved in all members of the family. A set of eight cysteine residues is also perfectly conserved within the CCD of each family member that may participate in inter-or intramolecular disulphide bond formation. Figure 7 Schematic representation of the structural features of FAM20 family members. A. Structural features of FAM20A showing domains and residues conserved within the entire family. Key: SS: signal sequence; CCD: conserved C-terminal domain; CR: conserved region; Cys: cysteine residues conserved within CCD (indicated with asterisk). B. Consensus sequences were derived for CR1, CR2 and CR3 using a global comparison of all the family members listed in Tables 1 and 2. Residues that are invariant or only differ in one sequence are indicated in bold. Non-conserved residues are indicated with an x and positions with more than one common residue are shown below the main sequence. Fam20a is a secreted protein As the putative signal sequence was the only known domain identified in all family members, we next tested whether this sequence is functional. Signal sequences are commonly found on proteins that are directed to the endoplasmic reticulum (ER) and either retained there or processed and transported into the Golgi apparatus and secreted from the cell. Many proteins are glycosylated during their transit through the ER and Golgi apparatus and the mouse Fam20a protein contains four potential sites for N-glycosylation (indicated in red type in figure 1 ). As Fam20a does not contain an ER retention signal, we predicted that it should be detected in the medium of expressing cells. A mammalian expression vector was constructed that contained the full length mouse Fam20a coding sequence fused to a C-terminal Myc epitope tag and a hexahistidine sequence to permit purification. The plasmid was transfected into monkey kidney COS-1 cells and total protein was isolated from both the cells and the cell medium. Proteins in the cell medium were first processed on a Nickel column to isolate and concentrate the recombinant Fam20a protein and both protein samples were analyzed by immunoblotting using an antiserum specific for the Myc epitope. The predicted molecular weights of the full length and processed forms of Fam20a are 61,500 and 57,500, respectively, and a recombinant form of the protein synthesized in rabbit reticulocyte lysates was run alongside as a molecular size marker. The recombinant protein migrated just below the 62,000 mol.wt. size marker (Figure 8A and 8B , lane 5); however, the proteins detected in both the cell medium and cell extract migrated slower (lane 3). To test whether this slower migrating band represented a glycosylated form of Fam20a, the protein samples were treated with the enzyme N-glycosidase F (PNGaseF). The protein detected after enzyme treatment migrated more rapidly than the untreated protein and comigrated with the recombinant form of the protein (compare lanes 3, 4 and 5). We noted a second band that migrated slightly more slowly than the recombinant protein in the PNGase F treated cell extracts that may represent an alternatively modified form of Fam20a (Figure 8B , lane 4). To confirm that Fam20a is a secreted protein, we also exposed Fam20a-expressing cells to Brefeldin A, a fungal metabolite that specifically blocks transport from the ER to the Golgi apparatus, and examined the effects on Fam20a secretion. Brefeldin A treatment resulted in a consistent decrease in the amount of Fam20a detected in the cell medium (Figure 8C , compare lanes 5 and 6). Thus, Fam20a is a secreted glycoprotein. Figure 8 Fam20a is a secreted protein. COS-1 cells were transfected with either an empty expression vector (-) or one encoding mouse Fam20a with a C-terminal myc epitope tag and proteins were isolated from either the medium (panel A) or the cells (panel B). The proteins were analyzed by immunoblotting using a Myc tag-specific antiserum. Samples in lanes 2 and 4 of each blot were pre-treated with protein N-glycosidase prior to analysis to remove glycosyl groups. A recombinant form of Fam20a synthesized in rabbit reticulocyte lysates (TnT) was included on each gel as a size marker. The position of glycosylated and deglycosylated Fam20a is indicated using arrowheads and cross reacting material detected in the medium is indicated using asterisks. The location of molecular size markers is shown on the left of each gel. C. Protein samples from the medium of transfected cells that were untreated or treated with Brefeldin A were analyzed by immunoblotting using the Myc tag-specific antiserum. As Brefeldin A was resuspended in DMSO, the untreated cells were exposed to DMSO alone as a vehicle control. The amount of Fam20a detected in the medium of Brefeldin A treated cells was consistently lower than that observed in untreated cells (indicated using an arrowhead). Fam20a secretion requires a functional signal sequence We next tested whether the integrity of the signal sequence was required for Fam20a secretion. Signal sequences typically contain a high proportion of hydrophobic amino acids and 19 of the first 34 amino acids of Fam20a are hydrophobic (Figure 9A ). Therefore, we expressed a Fam20a protein lacking the first 23 amino acids (FAM20a(Δ23)) in COS-1 cells and examined secreted and intracellular proteins by immunoblotting (Figure 9B ). Glycosylated FAM20a(Δ23) protein was not detected in the medium (compare lanes 2 and 3) and immunoreactivity that comigrated with the unglycosylated recombinant protein was detected in the cell extract (lane 6). We also compared the subcellular location of the FAM20a(Δ23) protein to the wild type protein using GFP fusion proteins (figure 9C ). The wild type Fam20a-GFP proteins displayed perinuclear and cytoplasmic staining consistent with ER localization. In contrast, the Fam20a (Δ23)-GFP protein was absent from the cytoplasm and appeared to be exclusively localized within the nucleus. To ensure that this effect was not a consequence of a gross change in protein structure due to the deletion of 23 amino acids, we also constructed an expression vector encoding a Fam20a protein with a two amino acid substitution within the putative signal sequence (Figure 9A ). These changes (Leu 14 –Leu 15 to Asp-Glu) were predicted to disrupt the signal sequence without grossly altering the protein structure. Again the mutant protein displayed nuclear staining and was absent from the ER (Figure 9D ). These results confirm that an intact signal sequence was necessary for secretion of Fam20a and that secretion was accompanied by prominent localization of the protein to the ER. Figure 9 Secretion of Fam20a requires an intact signal sequence. A. Schematic representation of the putative signal sequence of Fam20a. The predicted cleavage site is indicated with a red arrowhead. The two amino acid substitutions introduced in the SSmut construct and the sequence remaining in the Δ23 mutant construct are shown. B. Immunoblot analysis of Fam20a and Fam20a(Δ23) protein levels in transfected COS-1 cells. The position of the glycosylated form of Fam20a (which is absent in Fam20a(Δ23) transfected cells is indicated with an arrowhead. C. Fluorescence images of COS-1 cells expressing either Fam20a-GFP or Fam20a(Δ23)-GFP. The wild type protein was observed within the cytoplasm, predominantly in a structure that is likely to be the ER. The mutant protein was primarily localized to the nucleus. D. Immunofluorescence images of Fam20a and Fam20a (SSmut) proteins as detected by antiserum directed against the C-terminal Myc epitope. The wild type protein was again detected in the ER and the mutant protein primarily in the nucleus. The cells have been counterstained with DAPI to delineate the nucleus. Figure 10 RT-PCR analysis of mouse Fam20 mRNA levels during differentiation of EML and MPRO cells. Total RNAs were prepared from EML (panel A) or MPRO (panel B) cells at the indicated timepoints during myeloid and granulocytic differentiation. cDNAs prepared from each sample were amplified using primer pairs specific to each mouse family member. The PCR products were analyzed by agarose gel electrophoresis and stained using Gelstar SYBR Green DNA stain. GAPDH was used as a loading control. Expression patterns of FAM20 genes during myeloid differentiation We originally identified Fam20a as a differentially expressed mRNA in EML cells induced to differentiate along the myeloid lineage. To determine whether Fam20b and Fam20c are also expressed during hematopoiesis, we performed RT-PCR analysis of cDNAs prepared at various times during experimentally-induced differentiation of EML and MPRO cells using primers specific to each family member. Fam20a mRNA levels were low in uninduced EML cells maintained in the presence of SCF and increased during the subsequent 72 hours of incubation in atRA and IL-3 (Figure 10A ). EML cells mature to the promyelocyte stage of neutrophil differentiation under these conditions and can subsequently be differentiated into neutrophils by adding GM-CSF in place of SCF and IL-3. Fam20a mRNA levels decreased during terminal neutrophil differentiation in EML cells and also in MPRO cells induced to undergo the same differentiation process in the presence of atRA (Figure 10A and 10B ). Fam20b and Fam20c mRNAs were readily detected in both cell lines and their levels did not vary dramatically during the differentiation process in either cell line (Figure 10A and 10B ). Expression patterns Of FAM20 genes in human tissues Although we originally isolated Fam20a from a hematopoietic cell line, cDNAs and ESTs derived from each of the FAM20 family members have been isolated from non-hematopoietic tissues (data not shown). Therefore, we examined the expression patterns of the three genes in a panel of cDNAs derived from various human tissues. FAM20A displayed the most restricted expression pattern with high levels in lung and liver and intermediate levels in thymus and ovary (Figure 11 ). Low levels of FAM20A mRNA were detected in several other tissues. FAM20B and FAM20C were expressed in a wider variety of tissues and their expression patterns were very similar. Figure 11 RT-PCR analysis of human FAM20 mRNA levels in human tissues. A panel of commercially available human cDNAs prepared from the indicated tissues was analyzed by PCR using primer pairs specific for each of the human FAM20 family members. GAPDH was again used as a loading control although large variations were observed in the GAPDH signal in the different tissues. Discussion Several classes of secreted proteins, including the colony stimulating factors or hematopoietins, are important regulators of hematopoietic differentiation and function [ 11 ]. These molecules are of clinical significance due to their use in stimulating hematopoiesis in patients with neutropenias and other hematological disorders [ 11 ]. Consequently, the identification of novel secreted proteins that display specific spatiotemporal expression patterns in hematopoietic cells is of great interest. In this report, we describe the identification and initial characterization of a new family of secreted glycoproteins expressed within the hematopoietic lineage as well as several other cell types and tissues. The family has been named FAM20 to indicate the fact that the members are related by sequence similarity rather than a specific shared function and contains three members in mammals. We anticipate that the members will acquire new names as their specific functions are determined. The family contains three separate subfamilies which are referred to as FAM20A, FAM20B and FAM20C in humans. FAM20 proteins: features and potential functions At the present time, the specific function(s) of the FAM20 proteins is unknown. Analysis of the sequences of the proteins from various species failed to reveal obvious similarities to known functional domains except for an N-terminal signal sequence. Expression studies clearly demonstrated that the mouse Fam20a protein is a secreted protein and that disruption of the signal sequence prevented the detection of the glycosylated form of the protein in cell media. Surprisingly, disruption of the signal sequence resulted in redistribution of intracellular Fam20a from a cytoplasmic compartment that is likely to be the ER to the nucleus. It is unclear whether this redistribution is functionally significant and is presumably due to the presence of a cryptic nuclear localization signal (NLS) in the protein. NLSs are generally comprised of stretches of basic residues [ 12 ] and several candidate regions rich in basic amino acids are present in Fam20a that could direct the mislocalized protein to the nucleus. Overall, the FAM20 proteins vary in length between 400–670 amino acids with the FAM20B family members being the shortest and the FAM20C members generally being the longest. The variation in length is due to differences in the length of the less conserved N-terminal region. The FAM20 proteins are further characterized by a highly conserved C-terminal region of approximately 350 amino acids that we named the conserved C-terminal domain (CCD). We noted the presence of three regions that are more highly conserved amongst all family members, with the most invariant extended sequence being the DRHHYE heptapeptide within conserved region 2. The function of the peptide is as yet unknown but three possible functions can be proposed. First, the histidines within this sequence could be involved in the coordination of metal ions that may be required for FAM20 protein function. Second, FAM20 proteins may be enzymes and this sequence may be a component of the active site of the enzyme. Interestingly, a weak match was detected between this region of certain FAM20 family members and a conserved domain within the phosphatidylinositol 3- and 4-kinases [ 13 ]; however additional experiments must be performed to address the relevance of this similarity. Third, the highly charged nature of this peptide suggests that it may be located on the surface of FAM20 proteins, where it may participate in protein:protein interactions. Although we have been unable to identify any other proteins in public databases that contain the exact sequence, the sequence RHHYE is found between amino acids 41–45 in the N-terminal region of the viral infectivity protein (Vif) from human immunodeficiency virus 1 (Accession number AAQ09611) [ 14 ]. Vif enhances HIV-1 infectivity by blocking the antiviral activity of the nucleotide editing enzyme APOBEC3G [ 15 ]. Vif exerts its inhibitory effect by binding to and inducing the degradation of APOGEC3G and also by blocking its translation [ 16 , 17 ]. The APOBEC3G interacting domain is located within the N-terminal region that contains the FAM20-related pentapeptide although the specific involvement of this sequence has not yet been investigated [ 16 ]. Thus, this sequence within conserved region 2 may be a site for protein:protein interaction. However, APOBEC3G or related proteins are unlikely candidate binding partners as they are intracellular proteins. We also noted that one of the Drosophila FAM20 proteins (NM_170079; CG31145) was identified as a protein that interacted with the Dynein light chain protein Dlc90f [ 18 ]. Dyneins are motor proteins involved in intracellular transport [ 19 ]. It appears unusual that a secreted protein would directly interact with a motor protein; therefore, this may represent a specific interaction of this particular family member in fruitfly. Evolution of the FAM20 gene family Tremendous progress has been made over the past decade in the sequencing of genomes from species at different positions on the evolutionary tree [ 20 - 25 ]. This vast amount of information can be used for comparative studies to elucidate the evolutionary origin of members of gene families. We have reported here the identification of orthologues of the FAM20 members in two insect species ( D. melanogaster and A. gambiae ), a simple chordate ( C. intestinalis ), three mammals ( H. sapiens, M. musculus and R. norvegicus ) and two fish ( F. rubripes and D. rerio ). In each case, the identification of these genes as bona fide transcription units is supported either by direct experimental evidence for the human and mouse genes, or by the existence of multiple EST sequences in public databases. A single FAM20 gene was identified in C. intestinalis , which is considered to be a representative of a basal chordate related to the common ancestor of humans and other higher chordates [ 10 ]. The C. intestinalis genome encodes approximately 16,000 genes and generally contains single representatives of superfamilies in higher vertebrates, thereby permitting the elucidation of likely evolutionary origins of genes within these families [ 9 ]. The C. intestinalis FAM20 protein clustered with the FAM20B subfamily members, as did the family members identified in invertebrate species. Therefore, we propose that the FAM20B subfamily contains the direct descendents of the ancestral FAM20 gene and that the FAM20A and FAM20C subfamilies result from duplication and subsequent evolution of this ancestral gene [ 26 ]. This pattern is consistent with the 2R hypothesis of genome evolution proposed by Ohno in 1970 [ 27 ]. In the FAM20 case, the loss of one gene at some stage of higher vertebrate evolution would need to be hypothesized to explain the final paralogue number of three in mammals. A single round of gene duplication appears to have occurred subsequent to the divergence of the nematode and insect lineages, giving rise to the two paralogues in fruit fly and mosquito that both cluster within the FAM20B subfamily. A further round of gene duplication has been proposed to have occurred in fish [ 28 ] and the existence of five to six FAM20 genes in pufferfish and zebrafish is consistent with this hypothesis. However, it is interesting that the expansion appears to have occurred exclusively in the FAM20C subfamily. This pattern could be explained either by two successive rounds of gene duplication of a small genomic region that included the original FAM20C representative, or by two rounds of duplication of larger genomic segments followed by gene conversion of FAM20A and FAM20B descendents (or parents) to yield four FAM20C members. Analysis of the genomic regions surrounding each of the FAM20C genes in fish will be necessary to distinguish between these two possibilities. Nevertheless, the expansion of the FAM20C subfamily in fish suggests that these proteins have acquired specific functions required within these species. FAM20 expression patterns in mammalian cells and tissues Expression analysis of the three FAM20 family members in mammalian tissues and hematopoietic cells showed that FAM20A is expressed in a much more restricted pattern that the other two members. Importantly, Fam20a was also the only member to display obvious differential expression in hematopoietic cells undergoing myeloid differentiation. Fam20a mRNA levels were highest during intermediate stages of differentiation of EML cells, at a time when many cells are becoming committed to the myeloid lineages and undergoing extensive proliferation [ 4 , 8 , 29 ]. EML cell cultures also give rise to a small number of cells from B cell and erythroid lineages under these conditions but these cells do not survive after SCF and IL-3 is removed from the medium and replaced with GM-CSF. Thus, the decrease in Fam20a mRNA levels after the 72 hour timepoint could be interpreted in two ways. First, Fam20a may be expressed in one of the lineages that cannot survive in GM-CSF. Second, Fam20a may be expressed specifically in cells committed to the myeloid lineage and its expression may decrease during terminal granulocytic differentiation. The similarities in Fam20a expression patterns in EML cells after the 72 hour timepoint and in MPRO cells suggests that the second explanation is most likely. Therefore, we propose that Fam20a is primarily expressed in cells committed to the granulocytic lineage and presumably plays a role in either lineage commitment of cell proliferation. The hypothesis is currently under investigation using gene disruption techniques and through further characterization of the Fam20a protein. Methods Cell culture and Representational Difference Analysis (RDA) EML and MPRO cells were cultured as described previously [ 6 , 30 ]. Briefly, EML cells were cultured in Iscove's modified Dulbecco's medium containing 20% horse serum (Atlanta Biochemical, Norcross, GA) supplemented with 10% BHK-MKL conditioned medium (CM) as a source of stem cell factor (SCF). The cells were differentiated by adding 10% of WEHI-3 CM as a source of IL-3 and all trans retinoic acid (atRA, 1 × 10 -5 M, Sigma) for 72 hours. Terminal granulocytic differentiation was induced by removing IL-3 and SCF and adding 10% BHK-HM5 CM as a source of granulocyte/macrophage-colony stimulating factor (GM-CSF). MPRO cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT) and 10% BHK-HM5 CM. Terminal differentiation was induced by adding 1 – 10 -5 M atRA to the culture medium. For collection of RNA samples at different timepoints, 10 cm dishes were seeded with 150,000 cells and cultured under identical conditions. Cells were harvested from individual wells at 24 hour intervals for RNA preparation. Total RNA was prepared from isolated cells using a modified guanidium isothiocyanite/phenol extraction procedure as described previously [ 31 ]. Poly A + RNA prepared from EML cells at zero hours (stem cell stage) and 72 hours (promyelocyte stage) was converted to double stranded cDNA and three rounds of RDA was performed as described previously [ 6 ]. cDNA clones representing six putative differentially expressed genes were identified and named according to clone number. Initial characterization of these clones was described earlier [ 6 , 8 ] and clone number 1623 was examined further in this study. Cos-1 cells were maintained in Dulbecco's modified Eagle's medium (DMEM, BioWhittaker, Walkersville, MD) containing 10% FBS and Penicillin/Streptomycin (BioWhittaker) under standard cell culture conditions. For transfections, cells were plated at a density of 2.5 × 10 4 per well in six well plates and transfected using the Effectene Transfection Reagent (Qiagen, Valencia, CA) under conditions recommended by the manufacturer. Isolation of a full length 1623 cDNA Analysis of the sequence of the original 1623 cDNA isolated from the RDA experiments revealed that it contained the coding sequence from the C-terminus of an uncharacterized protein. 5' and 3' rapid amplification of cDNA ends (RACE) was performed using Marathon-Ready cDNA from mouse spleen using the Advantage cDNA PCR kit (both from Clontech) as described previously [ 30 ]. The RACE reactions were performed using gene specific primers designed based on the original 1623 sequence or on sequences identified in early RACE reactions and adaptor primers provided with the cDNA. The 5' RACE products were compared to sequences in public databases and identified a mouse cDNA (NM_017565; aka DKFZp434F2322) that was identical to the extended 1623 sequence. PCR primers were designed that overlapped the putative initiation and stop codons of the ORF encoded by NM_017565 and used to PCR amplify products from cDNAs prepared from undifferentiated MPRO cells. The product of this PCR reaction was subcloned and sequenced in its entirety. The sequence was identical to the original clone (which was isolated from mammary tissue) indicating that the same mRNA is expressed in both tissues. The full length clone (now named Fam20a) encoded a 541 amino acid protein. Sequence analysis All basic sequence manipulations were performed using the VectorNTI suite of sequence analysis programs (Informax/Invitrogen Corp., Carlsbad, CA). Identification of FAM20 members from different species was initially achieved using standard BLAST searches at NCBI . Genome specific searches were performed either through NCBI or Ensembl or through genome specific websites ( Ciona intestinalis : ; Fugu rubripes : ). Searches were performed either as nucleotide-nucleotide (blastn) searches or as protein-translated nucleotide (tblastn) searches. In general, these searches were sufficient to identify gene regions encoding the most highly conserved regions of the proteins, particularly in the fish and Ciona genomes. To assemble complete genes, genomic regions containing the identified regions of similarity were downloaded into VectorNTI and searches were performed for individual exons, initially derived from mammalian genes but subsequently, from potential orthologues in fish or lower vertebrates, depending on the sequence being examined. Putative exons were examined for the presence of consensus splice donor and acceptor sites and complete sets of exons were assembled into cDNAs for translation and further comparisons. These results were compared against genes assembled by two gene prediction programs, FGENESH: and GENSCAN: , however, these programs often made incorrect assignments that required manual assessment of the results. The protein sequences derived from each of the examined species were primarily used in the comparisons to define subfamily assignments and alignments were performed in the AlignX program in VectorNTI using the blosum62 scoring matrix. Protein domain searches were performed using the Simple Modular Architecture Research Tool (SMART) and/or Profilescan . Signal sequence searches were performed using the SignalP analysis program [ 32 ]. Sequences derived by annotation of genome sequences in this study have been submitted to the Third Party Annotation database at NCBI under accession numbers BK001515 to BK001522. The FAM20 family name has been approved by the Human Genome Organization nomenclature committee. Plasmid construction The Fam20a and Fam20aΔ23 were constructed in the pEGFP-N1 vector by PCR cloning. Xho I and Hind III restriction sites were engineered into the PCR primers (Fam20a: 5'GGCCTCGAGGCCATGCCCGGGCTGCGCAGG3', 5'GGCAAGCTTGCTCGTCAGATTAGCCTG3'; Fam20aΔ23: 5'GGCCTCGAGGCCATGTACTTCCACCTCTGGCCG3' and reverse primer was as above). Similar strategy was used to clone Fam20a and Fam20aΔ23 in the pcDNA3.1 vector (Forward primers were same as above; Fam20a and Fam20aΔ23 reverse primers: 5'GGCAAGCTTGGGCTCGTCAGATTAGCCTG3'). The Fam20a(SSmut) was generated using a PCR-based site-directed mutagenesis kit (Quickchange, Stratagene). All of the sequences were verified by sequencing using an ABI automated sequencer. Western blotting Whole cell extracts were prepared from transfected COS-1 cells by lysing directly in 2X Laemmli Sample Buffer as described previously [ 31 ]. Purified His-tagged proteins were prepared as described below and mixed with 2X Laemmli Sample Buffer (120 mM Tris-HCl (pH 6.8), 10% Glycerol, 3.3% SDS, 0.2 M dithiothreitol, 0.004% Bromophenol Blue). Equal amounts of total cellular and purified secreted proteins were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes (Micron Separations, Westborough, MA). Membranes were blocked in 5% non-fat milk in TBST (100 mM Tris-HCl (pH 7.5), 0.9% NaCl, 0.1% Tween-20) for 1 hour. Myc-tagged Fam20a proteins were detected using an anti-myc mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Horseradish peroxidase-conjugated donkey anti-mouse antibody (Promega, Madison, WI) was used for the secondary antibody. The immune complexes were detected using SuperSignal chemiluminescence detection kit (Pierce, Rockford, IL). In vitro transcribed and translated Fam20a was generated using the TnT T7 coupled reticulocyte lysate system (Promega) as described previously [ 33 ]. Purification of His-tagged proteins from cell media COS-1 cells were transfected as described above and media were collected after 48 hours and mixed with freshly made 10X binding buffer (500 mM NaH 2 PO 4 -H 2 O (pH 8.0), 150 mM NaCl, 10 mM Imidazole). His-tagged proteins were purified using Ni-NTA Magnetic Agarose Beads (Qiagen, Valencia, CA) according to manufacturer's recommendations. Following the purification step they were either used directly in western blot analysis or for deglycosylation experiments. Purified proteins were subjected to enzymatic deglycosylation using the GlycoPro deglycosylation kit (ProZyme, San Leandro, CA) following the manufacturer's protocol. Immunofluorescence COS-1 cells were plated on glass coverslips and transfected with the indicated constructs the following day. After 48 hours of incubation, the cells were fixed in 1% formaldehyde for 30 minutes at room temperature. Coverslips were incubated in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 (pH 7.3)) containing 1% Triton X-100 for 10 minutes to permeabilize the cell membranes. They were then transferred to PBST (PBS containing 0.1% Tween-20) and incubated for 30 minutes. Mouse monoclonal anti-Myc antiserum (Santa Cruz) was used at 1:2,000 dilution in PBST containing 1% bovine serum albumin (1 hour at room temperature). Coverslips were then washed with PBST three times and incubated with the secondary antibody (Alexa Fluor 488 [Molecular Probes, Eugene, OR]) for 1 hour. Coverslips were washed three times in PBST and once in water for 15 minutes each, and were mounted on glass slides. The cells were observed by epifluorescence microscopy using an Axiovert 135 TV microscope (Zeiss, Gottingen, Germany). Images were captured with a Kodak DC290 zoom camera and analyzed with MetaMorph 6.0 (Universal Imaging Corporation, Downingtown, PA). Reverse transcription-polymerase chain reaction Total RNA was purified as described above from EML and MPRO cells at 24 hour timepoints during the differentiation process of each cell line The primers were designed using the Vector NTI sequence analysis suite of programs (InforMax, Frederick, MD) and were follows: MmFam20a forward 5'-catagaggcccacggcgagcg-3' and reverse 5'-atggaatggggcaacag gggc-3'; Mmfam20b forward 5'-tggacaggattctgggtttc-3' and reverse 5'-ccagggatgtcgatgtttct-3'; MmFam20c forward 5'-agcagacgagagagcaggag-3' and reverse 5'-cggatctccttggtcatgtt-3'; HsFAM20a forward 5'-ctggcaggaaaagagtg-3' and 5'-cccgaacttggtgaacatct-3', HsFAM20b forward 5'-ccctgaagagacaccagaagagc-3' and reverse 5'-gaaacccagaatcctgtcca-3', HsFAM20c forward 5'-ggctcacgttccacattggt-3' and reverse 5'-aaagtcagggggtgtctcct-3'.; mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward 5'-aatggtgaaggtcggtg tgaac-3' and reverse 5'-gaagatggtgatgggcttcc-3'; human (GAPDH) forward 5'-tgaaggtcggagtcaacggatttggt-3' and reverse 5'-catgtgggccatgaggtccaccac-3'. GAPDH was used as control for cDNA integrity. Single stranded cDNA was reverse transcribed from 2 μg of total RNA using 400 Units of Superscript RNase H - Reverse Transcriptase (Invitrogen, Carlsbad, CA), 0.125 mM dNTPs, 10 mM DTT, and 1 μM oligo (dT) 15 in a total volume of 50 μl for 1 hour at 37°C. 2 ul of the RT reaction was then mixed with 1 ul of 10X PCR Buffer (500 mM Tris-HCl (pH8.3), 2.5 mg/ml crystalline BSA and MgCl 2 at 10, 20 or 30 mM (Idaho Technology Inc., Idaho Falls, ID), 0.2 mM each dNTP, 0.05 μM of each primer and 1.25 Units Taq DNA polymerase (Fisher Scientific, Pittsburgh, PA) in a total volume of 10 μl. Reactions were loaded into a capillary tube and PCR cycles were carried out using the Rapidcycler Thermal cycler (Idaho Technology). Annealing temperatures and Mg 2+ concentrations were initially optimized for each primer. A commercial set of tissue cDNAs, (Human Multiple Tissue cDNA (MTC) Panels, Clontech, Palo Alto, CA) was used for the RT-PCR analysis of human FAM20 family members. List of Abbreviations FAM20: family with sequence similarity 20; EML: Erythroid, myeloid, lymphoid cell line; MPRO: mouse promyelocyte cell line; GM-CSF: granulocyte/macrophage-colony stimulating factor; PHSC: pluripotent hematopoietic stem cell; HGF: hematopoietic growth factor; RAR: retinoic acid receptor; atRA: all trans retinoic acid; SCF: stem cell factor; IL-3: interleukin-3; RDA: representational difference analysis; RACE: rapid amplification of cDNA ends; CCD: conserved C-terminal domain; GFP: green fluorescent protein; CM: conditioned medium. Author Contributions DN isolated full length Fam20a cDNA, performed transfection experiments and drafted the manuscript. HY performed RT-PCR assays. IN assisted in construction of expression vectors. SS performed genomics analyses. EC and EGB provided reagents and assistance in performing RT-PCR assays. YD performed the original representational difference analysis and SCW directed the project and performed genomics analyses. SCW also wrote the final draft of the manuscript and all authors read and approved this version.

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546335

Opposing Fat Metabolism Pathways Triggered by a Single Gene

Regulating metabolism of fat is an important challenge for any animal, from nematodes to humans. Central players in the regulatory network are the nuclear hormone receptors (NHRs), which are transcription factors that turn on or off a set of target genes when bound by specific lipid molecules. NHR genes number 48 in mammals, and a surprising 248 in nematodes. Despite the difference in quantity, there are some structural similarities between NHRs in these two groups, in particular, between the nematode gene nhr-49 and the mammalian HNF4 . In this issue, Keith Yamamoto and colleagues show that nhr-49 controls two different aspects of fat metabolism, which interact to form a feedback system controlling the consumption and composition of fats in the nematode. Regulation of fat content and lifespan in C. elegans Using RNAi to suppress gene expression, the researchers discovered that when nhr-49 was absent, the lifespan of the nematode was reduced by more than 50%, and the animal displayed numerous gross abnormalities in the gut and gonad. This was accompanied by unusually high fat content in the larvae. By using quantitative PCR to measure output of fat and glucose metabolism genes, the researchers showed that deletion of nhr-49 changed expression of 13 of these genes, with the most dramatic effects occurring within two metabolic pathways: mitochondrial lipid oxidation and fatty acid desaturation. Oxidation degrades lipids to release energy, explaining the build-up of fat in nhr-49 -suppressed larvae. One of nhr-49 's normal functions is to increase expression of the mitochondrial acyl–Coenzyme A (CoA) synthetase gene acs-2 . A principal role for mitochondrial acyl-CoA synthetases is to “activate” free fatty acids for transport into mitochondria, where they are oxidized. This process involves attaching a CoA group to a free fatty acid, and often serves as a rate-limiting step in lipid oxidation. Indeed, the authors found that suppression of acs-2 alone was sufficient to reproduce the highfat phenotype, while overexpression of acs-2 rescued the phenotype even in the absence of nhr-49 . Fatty acid desaturation is the process of converting saturated fats into unsaturated ones, by forming one or more double bonds between adjacent carbons in the tail. This process is catalyzed by fatty acid desaturase enzymes. nhr-49 increases expression of several desaturases, most importantly fat-7 , which converts stearic acid to oleic acid; deletion of nhr-49 more than doubled the proportion of stearic acid compared to oleic acid. RNAi interference of fat-7 alone produced two interesting results. First, it shortened the nematode life span, suggesting this was the primary pathway through which nhr-49 suppression exerted that same effect. Second, it produced some effects that were opposite those of nhr-49 suppression: specifically, it reduced rather than increased fat content, and it increased rather than reduced expression of acs-2 . These results show that in its normal actions, nhr-49 sets in motion two opposing pathways: it increases acs-2 , which leads to reduction of fat content, and it increases fat-7 , which, by reducing acs-2 , increases fat content. Surprisingly, this behavior links nhr-49 most closely not to HNF4 , with which it shares the most structural similarity, but to another type of mammalian NHR, called peroxisome proliferator-activated receptors (PPARs). Further investigation of this link may lead to better understanding of the functions of PPARs, and provide opportunities for altering their function for treatment of fat metabolism disorders such as diabetes and obesity.

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549562

Surface expression, single-channel analysis and membrane topology of recombinant Chlamydia trachomatis Major Outer Membrane Protein

Background Chlamydial bacteria are obligate intracellular pathogens containing a cysteine-rich porin (Major Outer Membrane Protein, MOMP) with important structural and, in many species, immunity-related roles. MOMP forms extensive disulphide bonds with other chlamydial proteins, and is difficult to purify. Leaderless, recombinant MOMPs expressed in E. coli have yet to be refolded from inclusion bodies, and although leadered MOMP can be expressed in E. coli cells, it often misfolds and aggregates. We aimed to improve the surface expression of correctly folded MOMP to investigate the membrane topology of the protein, and provide a system to display native and modified MOMP epitopes. Results C. trachomatis MOMP was expressed on the surface of E. coli cells (including "porin knockout" cells) after optimizing leader sequence, temperature and medium composition, and the protein was functionally reconstituted at the single-channel level to confirm it was folded correctly. Recombinant MOMP formed oligomers even in the absence of its 9 cysteine residues, and the unmodified protein also formed inter- and intra-subunit disulphide bonds. Its topology was modeled as a (16-stranded) β-barrel, and specific structural predictions were tested by removing each of the four putative surface-exposed loops corresponding to highly immunogenic variable sequence (VS) domains, and one or two of the putative transmembrane strands. The deletion of predicted external loops did not prevent folding and incorporation of MOMP into the E. coli outer membrane, in contrast to the removal of predicted transmembrane strands. Conclusions C. trachomatis MOMP was functionally expressed on the surface of E. coli cells under newly optimized conditions. Tests of its predicted membrane topology were consistent with β-barrel oligomers in which major immunogenic regions are displayed on surface-exposed loops. Functional surface expression, coupled with improved understanding of MOMP's topology, could provide modified antigens for immunological studies and vaccination, including live subunit vaccines, and might be useful to co-express MOMP with other chlamydial membrane proteins.

Background Every Gram-negative bacterium in the order Chlamydiales is an obligate intracellular pathogen [ 1 ]. The organisms are dimorphic, and alternate between free-living, infectious "elementary bodies" (EBs) endocytosed by mucosal cells into vesicular inclusions, and metabolically active, intracellular "reticulate bodies" (RBs). RBs replicate and redifferentiate into EBs before being released to infect neighboring cells, and infections (including Chlamydia muridarum pneumonitis, an important animal model) are often complicated by a damaging immune response and chronic inflammation. Human genital C. trachomatis infections are associated with ectopic pregnancy and infertility, and serovars that target ocular membranes can lead to trachoma and blindness. Chlamydophila pneumoniae ( Ch. pneumoniae ) causes pneumonia in the elderly, and colonization of the placenta by Ch. abortus causes abortion in ewes (and, occasionally, in women). Uniquely among bacteria, the chlamydial outer membrane (OM) is reinforced by a network of disulphide bonds [ 2 ]. Treatment of EBs with Sarkosyl produces "chlamydial OM complexes" (COMCs) [ 3 ] containing three relatively detergent-resistant, cysteine-rich proteins: the Major Outer Membrane Protein (MOMP), encoded by ompA , and OmcB and OmcA, encoded by omp2 and omp3 , respectively. MOMP (~40 kDa) is expressed in both EBs and RBs [ 4 ]. It contains extensive β-sheet secondary structure and forms large pores [ 5 , 6 ], similar to β-barrel porins found in other outer bacterial membranes (e.g. E. coli OmpF). The MOMPs encoded by different C. trachomatis serovars share five well-conserved regions and four "variable sequence" (VS) domains [ 7 , 8 ]. C. trachomatis VS domains, and homologous regions in MOMPs from other species, could correspond to cysteine-rich surface-exposed loops in a porin β-barrel, and EB MOMP is oxidised and highly cross-linked, making the OM very stable. RBs in contrast are osmotically active with reduced, mainly monomeric, MOMP [ 9 ]. MOMP's pore-forming ability is enhanced by reduction [ 5 ], compatible with a link between reversible disulphide bond formation and the developmental stage of the bacteria. Supporting this idea, DTT-reduced EBs tend to resemble RBs [ 5 ], and native MOMP is monomeric when solubilised in SDS under reducing conditions, but forms monomers, dimers, trimers, tetramers and even larger complexes [e.g. [ 6 , 10 , 11 ]] under oxidising conditions. C. trachomatis MOMP is highly immunogenic. Antibodies to the protein neutralised EB infectivity [ 12 ], and triggered approaches to generate MOMP-based vaccines [e.g. [ 13 , 14 ]]. However, as implied earlier, the immunopathology of chlamydial infections is complicated [ 15 ], with T H1 type immune responses as well as specific antibodies (T H2 responses). MOMP is not equally immunogenic in all spp ., and it also stimulates T-cell division, including CD4+ and CD8+ T-cells, enhancing IFN-γ secretion [ 16 ]. C. trachomatis MOMP will probably need to be modified to form a safe and effective subunit vaccine, emphasizing the importance of understanding its structure in more detail. OmcA and OmcB (the other main components of the COMC) are present as approximately 1 OmcB:2 OmcA:5 MOMP [ 17 ]. Ch. psittaci (formerly known as C. psittaci ) OmcA is a 9 kDa lipid-anchored protein with 14 cysteine residues [ 18 ], while OmcB (60 kDa) contains 37 cysteines [ 19 ]. The Omc proteins may not be integral membrane proteins. Reduced OmcB is water-soluble, and although OmcA remains membrane-associated, it can be readily solubilised when reduced [ 20 ], and neither protein was detected on the surface of intact EBs by immunogold labeling [ 21 ]. Regardless of their membrane association, OmcB appears to be extensively cross-linked in the periplasm of EBs, forming disulphide bonds with both MOMP and OmcA. Appropriately, both Omc proteins are expressed late in the developmental cycle (from a bicistronic operon), as RBs are reorganized into EBs [ 22 ], consistent with the idea that RB MOMP is functional and exchanges nutrients and other factors (possibly including signaling molecules) with the host cell. Extensive disulphide cross-linking in EBs may inactivate the porin, and prevent expansion of the growing bacterial cell wall. Although MOMP is of major biological and clinical interest, chlamydia only grow in eukaryotic cells, and MOMP is difficult to isolate and purify because it can aggregate when oxidized, or interact with other cysteine-rich chlamydial proteins. As a result, many groups have expressed recombinant MOMP in E. coli using full-length ompA genes that include the signal sequence to target the translated protein to the OM. Although leadered MOMP can be expressed in a heterologous system [ 23 - 25 ], this approach has proved to be highly problematic, because the protein tends to misfold and aggregate. Koehler et al . [ 26 ] demonstrated surface-exposure, but with a dramatic reduction in cell viability, including OM disruption and substantial cell lysis (i.e. unincorporated, periplasmic MOMP may have been exposed). Jones et al . [ 27 ] co-reconstituted recombinant MOMP with endogenous E. coli porins, and showed altered solute permeabilities in liposome-swelling assays. Although attributed to novel porin activity, this could have reflected modification of endogenous porins. Wyllie et al . [ 28 ] pursued an alternative approach with truncated versions of Ch. abortus and Ch. pneumoniae MOMP, and obtained small amounts of folded proteins without prior denaturation and refolding, sufficient for incorporation into planar bilayers and single-channel recording. Other expression systems, pioneered because of their potential for vaccine delivery, include mammalian COS cells [ 29 ] and Vibrio cholerae [ 30 ]. PorB (37 K), a second putative porin, is also surface-exposed in chlamydia [ 31 ]. Recombinant PorB specifically transported dicarboxylates in liposome-swelling assays [ 32 ], although. it was used with a C-terminal His tag. The terminal residues of porins normally meet to complete a transmembrane β-strand, and may even be linked by a salt bridge. Being integral to the protein fold, additional terminal residues might affect the conformation and, therefore, the specific function of a porin. We expressed PorB as well as MOMP to help determine the factors affecting chlamydial porin expression, but because of these theoretical concerns concerning porin folding and function, we avoided tagged proteins in the present study, and built on previous work with leadered constructs. We developed improved conditions for the surface expression of MOMP in E. coli cells, and demonstrated unambiguously by single-channel recording that recombinant C. trachomatis MOMP folded and formed a functional protein in the absence of many endogenous porins. We showed that MOMP can insert into the outer membrane of E. coli cells and form SDS-sensitive oligomers in the absence of cysteine residues, and generated a "working model" of the topology of MOMP to provide structural hypotheses that could be tested by engineering the recombinant protein. Results Optimised MOMP expression in E. coli cells Our first objective was to obtain properly folded recombinant chlamydial porins in the outer membranes of E. coli cells. Building on previous work (e.g. [ 26 ]), BL21(DE3) cells were transformed with pET- ompA or pET- porB constructs, and expression was induced by 1 mM IPTG at 37°C after growth to an OD 600 of 0.6. Compared to the expression of non-leadered proteins (which accumulate in cytoplasmic inclusion bodies), cells expressing leadered porins must transport the immature full-length porin across the inner membrane, cleave the leader sequence in the periplasmic space, and fold and insert the mature protein into the OM. Expression of mature, leaderless C. trachomatis MOMP did not inhibit growth compared to non-transformed cells, in contrast to substantial inhibition with full length MOMP (Fig. 1A ). To investigate whether different leader sequences could improve processing, C. trachomatis MOMP was expressed with the OmpT leader rather than its native leader. Initial growth rates were comparable to those shown by non-transformed cells, and similar to cells expressing mature MOMP (i.e. MOMP without a leader sequence), although the cultures again showed a reduced final cell density. We next investigated the expression of MOMPs from other chlamydial spp . to determine whether the observed effects were specific to C. trachomatis MOMP, and we also expressed C. muridarum PorB to exclude a universal problem with the expression of all putative chlamydial porins in E. coli . The constructs had different effects on cell viability (Fig. 1B ). Bacteria expressing C. muridarum MOMP grew more slowly than bacteria expressing C. trachomatis MOMP, although the bacteria continued to grow slowly throughout the entire period of induction. The growth of bacteria expressing Ch. abortus MOMP or C. muridarum PorB was markedly reduced, and the density decreased after 30 min. The "recovery" at later stages reflected multiplication of non-expressing cells in the presence of β-lactamase released from dead or dying cells (growth ceased on fresh Ampicillin plates, data not shown). We then changed the leader sequences. The growth of cells expressing C. trachomatis MOMP and C. muridarum PorB was improved by replacing the native chlamydial leader with the E. coli OmpT leader, and the decrease in optical density occurred later in the induction and continued more slowly. In contrast, no significant improvement was seen when Ch. abortus MOMP was expressed with the OmpT leader (data not shown). We also expressed full-length constructs in E. coli BL21(DE3)omp8 cells lacking expression of the endogenous porins LamB, OmpA, OmpC and OmpF [ 34 ]. Toxicity was more pronounced than in unmodified BL21 cells, and after establishing conditions for detergent extraction of recombinant MOMP ( Additional Data File #1 ), expression conditions were further optimised to improve the yield of processed, recombinant protein. Native and OmpT-leadered C. trachomatis MOMP constructs were induced rapidly at 37°C with 1 mM IPTG or slowly at 16°C with 0.1 mM IPTG (Fig. 2 ). At 37°C both versions of MOMP were expressed, and by 4 hours about half the protein was processed, as shown by the doublet band of OM-associated MOMP with and without its signal sequence (Fig. 2 ). The ~2 kDa difference between the cleaved and non-cleaved protein bands (38 kDa and 40 kDa, respectively), is similar to the difference seen when leadered versions of E. coli OmpF are expressed). There was a slight decrease in total protein when MOMP was expressed with its native leader at 16°C, but the proportion of processed protein was unchanged. Although protein decreased following slow induction of MOMP containing the OmpT leader, most of the protein was processed. Based on these observations, slow induction of native-leadered MOMP was carried out in different growth media for prolonged periods. After growing for 6 hours, cultures in LB medium plateaued at an OD 600 ~0.85, after which the cells began to lyse. In contrast, cells cultured in more supportive SOC medium continued to grow steadily, and began to plateau about 12 hours after induction ( Additional Data File #2 ). Processing and surface expression of mutagenised and engineered MOMPs Given the known difficulties associated with protein misfolding and aggregation (e.g. [ 23 - 26 ]), a particular problem for chlamydial MOMPs compared to other bacterial porins, our next objective was to determine whether MOMP was actually inserted into the E. coli outer membrane. Although recombinant MOMP was associated with the OM fraction following subcellular fractionation, the observation that its leader sequence was not always cleaved (Fig. 2 ) suggested that some leadered protein co-fractionated with OMs, possibly as a peripheral membrane protein. This raised the possibility that even cleaved recombinant proteins might not be fully integrated into the OM. To determine whether processed MOMP was actually inserted into (and across) the OM, we carried out whole cell immunoblots to probe for the presence of MOMP epitopes on the surface of intact E. coli BL21 cells. Because of the importance of reduced temperature (Fig. 2 ), we carried out inductions for whole cell immunoblotting at 37°C, 16°C and an intermediate temperature of 25°C. MOMP was incorporated into the OM at both 25°C and 16°C, when induced in the presence of either 1 mM or 0.1 mM IPTG, respectively. Expression and processing were more rapid at 25°C, and because the presence of some unprocessed protein was irrelevant in this experiment, we induced the cells at 25°C for 2 hrs. Non-transformed BL21 cells, or cells transformed with an empty plasmid, and BL21 cells transformed with constructs encoding mature, leaderless C. trachomatis MOMP, or with OmpT-leadered MOMP and native leadered-MOMP, were applied to a nitrocellulose membrane (avoiding methanol-activated PVDF, and the risk of OM permeabilisation and exposure of periplasmic MOMP), and probed with anti-MOMP pAb (Fig. 3A ). The absence of a signal from control cells and cells expressing MOMP in its non-leadered, mature form confirmed the incubation and blotting conditions did not cause cell lysis and expose unincorporated protein. Both OmpT- and native-leadered MOMP were detected on the cell surface (Fig. 3A , whole cell blots), confirming they were inserted into the OM. Unfortunately, BL21omp8 cells were too fragile to survive the same blotting procedure. SDS-PAGE analysis of OG-solubilised OM fractions (Fig. 3A , middle panel) confirmed MOMP expression and processing, although parallel immunoblots (Fig. 3A , lower panel) showed faint additional bands of ~40 kDa for the leadered proteins, indicating that processing was incomplete, as expected. Parallel immunofluorescence data (Fig. 3B ) showed MOMP was confined to cytoplasmic inclusion bodies containing the mature protein when the appropriate cells were fixed and permeabilised before staining (Fig. 3B , panel b). As expected, staining was absent when the antibody was applied before permeabilisation (data not shown). However, OM staining was seen for MOMP expressed with both the OmpT leader and the native leader (panels c and e, respectively). When these cells were permeabilised before staining (panels d and f, respectively), immunoreactive protein was also noted internally, as expected (e.g. Fig. 3A , lower panel), although reduced or absent in BL21omp8 cells induced for 12 hrs at 16°C in more supportive SOC medium (Fig. 3B , inset in panel d). We concluded that MOMP constructs encoding appropriate leaders could be expressed in E. coli , cross the inner membrane, and be processed in the periplasm. Furthermore, under modified incubation and induction conditions (especially at reduced temperatures, and in the relatively supportive medium SOC), MOMP could be folded and incorporated into the outer membrane. Membrane topology of MOMP Having confirmed that C. trachomatis MOMP was inserted into the OM of E. coli cells, we set out to investigate how the protein was organized in the membrane. While noting that predictive algorithms must always be deployed with care, and with reference to established findings for a given protein, we first analyzed MOMP's primary sequence for membrane crossings using a neural network trained with OM proteins of known structure [ 36 ]. The analysis (Fig. 4A ) showed 16 membrane crossings. As expected, the VS domains of C. trachomatis MOMP generally corresponded to regions of the protein predicted to be extracellular. We then reanalyzed the sequence using two β-strand prediction programs (Fig. 4B ). The combined analysis revealed a total of 16 strands, corresponding numerically to the initial "membrane crossing" prediction (which does not on its own appear to be sufficient to identify the specific extramembrane domains). We discarded the strand coinciding with VS1 in B2TMPRED (see Methods) because VS domains are likely to be extracellular loops, and inserted an extra strand between G210 and S218 to bring the chain back across the membrane, so that all 4 VS domains remained external. Minor adjustments were made to accommodate known constraints on β-strand organization and porin structures [ 39 , 40 ]. The final working model (Fig. 5 ) provided testable hypotheses concerning the pattern of transmembrane folding. All the cysteine residues were predicted to be accessible for inter- or intrasubunit disulphide bond formation or cross-linking with other proteins. Most were predicted to be external, but two were periplasmic. Although one thiol group was in a predicted transmembrane domain, it faced the central water-filled pore rather than the lipid bilayer, where it could potentially interact with a cysteine thiol on a pore-confined loop. We designed four C. trachomatis MOMP constructs (with intact cysteines and native leaders, to correspond exactly in these respects to the "wild-type" protein) in which substantial regions of VS domains 1, 2, 3 or 4 (shown in Fig. 6A ) were deleted, to test the prediction that these domains are surface-exposed loops that can be shortened without compromising the main β-barrel fold and membrane insertion. The region removed from VS1 was G63 to Y87; from VS2, E141 to F156; from VS3, Y220 to G238; and from VS4, D278 to T318. Our strategy (see Methods) resulted in some mutations. Most were conservative changes (M62T in VS1, T239V in VS3 and A277V in VS4), apart from G219D in VS3. However, our topology prediction placed this residue in an external loop, where the additional charge was unlikely to be significant. We also generated another pair of constructs with deletions of either one or two of the predicted β-strands between VS domain 1 and VS domain 2 (summarized in Fig. 6B–C ), in an attempt to disrupt the formation of OM-inserting β-barrels. These constructs were designated: Δβ5, with removal of E95 to F111 (with no residue changes) and Δβ5,6, with removal of F97 to A129 (with 2 changes, E95D and M96V). Before expressing the cDNAs encoding putative loop or strand deletions, we re-examined the expression and OM insertion of full-length C. trachomatis MOMP using a construct in which all 9 cysteine residues (Fig. 4A , circles; Fig. 5 , shaded residues) were replaced by alanine. The results (Fig. 7A ) were similar to those for the non-mutagenised protein, showing that folding and membrane insertion could proceed without cysteine residues and without the controlled formation of disulphide bonds (as it may do in RBs). We then expressed each of the "loop-deleted" MOMP proteins in BL21 cells. All four were detected on the cell surface (Fig. 7B ), demonstrating incorporation into the OM. In contrast, recombinant proteins expressed from constructs with putative β-stand deletions were not detectable on the surface of E. coli cells (Fig. 7 ). We considered the unusual possibility that all the epitopes in the "strand-deleted" proteins might have been unreactive in the E. coli membrane, due to masking or oligomerisation, but suspension of the cells in Tris (rather than phosphate) buffer (100 mM NaCl, 50 mM Tris-HCl, pH 7.4), or the addition of 2 mM EDTA, failed to "unmask" any immunoreactivity ( Additional Data File #3 ). MOMP forms oligomers in the E. coli outer membrane Native MOMPs are difficult to purify free from other chlamydial proteins [ 6 ], precluding firm conclusions about native subunit structure, especially in the absence of protein (cysteine) oxidation. In preliminary investigations of the subunit organisation of recombinant MOMP, we noted that the recombinant protein did not form SDS-resistant oligomers ( Additional Data File #4 ). However, unlike trimeric E. coli porins [ 40 ], oligomers of isolated MOMP, away from their normal membrane environment [ 6 ], may be unstable in the presence of SDS, so we subjected detergent-solubilised OM extracts to large-scale non-denaturing GE chromatography in milder detergents. For these and all subsequent experiments, MOMP was expressed in BL21omp8 cells with the OmpT leader (in SOC medium, at 16°C), to exclude heterooligomers containing endogenous E. coli porins, and minimize uninserted periplasmic protein, respectively. We carried out GE chromatography in LDAO or Zwittergent 3–14 (having previously noted these to be cheaper but equally effective detergents to replace OG, Additional Data File #1 ), with excess (5 mM) DTT in the presence of MOMP cysteine residues (calibrating the column in the presence of detergent). Under these conditions, MOMP appeared to form oligomers containing 2–4 subunits, although some recombinant MOMP always formed higher-order oligomers (Fig. 8 ). Similar results were obtained after repeating each experiment at least twice. The apparent subunit stoichiometry of recombinant MOMP depended on the detergent, with putative dimers in LDAO, and trimers or tetramers in Zwittergent 3–14, depending on the presence or absence of cysteine residues, respectively. However, it should be emphasized that only the major quaternary species was identified in each case. The presence of oligomers in LDAO or Zwittergent 3–14 contrasted with the absence of SDS-resistant oligomers during SDS-PAGE, and oligomer formation even in the absence of cysteine residues argued against an essential role for disulphide bonds. We also investigated the subunit organization of MOMP by covalent cross-linking following expression and insertion into BL21omp8 OMs, by removing DTT to allow in situ cysteine oxidation by dissolved oxygen. OM proteins were then incubated in SDS sample buffer with or without reducing agent at room temperature for 10 mins, separated by SDS-PAGE, and detected by Western blotting (Fig. 9 ). Reduced MOMP appeared as a single band of ~38 kDa, but non-reduced MOMP occupied several distinct bands. SDS-denatured, monomeric MOMP appeared as a band of ~38 kDa (labeled "denatured monomer"), corresponding to the reduced sample. However, monomeric MOMP also formed a band of ~35 kDa, running "ahead" of its normal apparent molecular mass, as previously seen with "folded" porin monomers [ 41 , 42 ]. Additional, fainter bands at higher molecular masses corresponded to dimers, tetramers and possible trimers (~80 kDa, ~160 kDa and ~120 kDa, respectively), similar to the findings following GE chromatography, with an upper band of aggregated protein that failed to enter the gel. Surface-expressed MOMP is functional Fully processed and correctly folded MOMP should function as a porin-like ion channel [ 6 ]. We tested this crucial prediction by expressing "wild-type" full-length recombinant C. trachomatis MOMP in BL21omp8 cells which express only a small subset of native E. coli porins, and not OmpF or OmpC [ 34 ]. We then functionally reconstituted solubilised BL21omp8 OM protein GE fractions in voltage-clamped planar lipid bilayers. Fractions containing "oligomeric" MOMP complexes gave rise to large-conductance, porin-like ion channels (Fig. 10 ). Similar channels were recorded irrespective of whether the detergent was LDAO or Zwittergent 3–14 (using fractions corresponding to 195 ml or 180 ml, respectively). The channels were voltage-dependent, closing at relatively high holding potentials (e.g. + or - 100 mV), but remaining open around 0 mV. The single-channel conductance in symmetric 500 mM KCl was 480 ± 19 pS (mean ± SEM, n = 6 independent experiments), and the reversal potential in 500 mM vs 50 mM KCl ( cis vs trans ) was -31 ± 1.5 mV (mean ± SEM, n = 9 independent experiments). This corresponded to a relative cation vs anion selectivity of 3.8 under these specific ionic conditions. Control preparations (detailed under Methods), including membrane proteins from control BL21omp8 cells subjected to the same experimental conditions, where OM proteins were solubilised and subjected to GE chromatography in exactly the same way, did not give rise to similar channel activity (6 experiments). Discussion Functional reconstitution of recombinant C. trachomatis MOMP at the single-channel (single molecule) level from cells lacking many endogenous porins provides very strong evidence that MOMP adopted its native fold when expressed in E. coli under suitable conditions. Although a leadered version of recombinant chlamydial MOMP was expressed and functionally analysed previously [ 27 ], membranes containing the protein were co-reconstituted with endogenous E. coli porins for liposome-swelling studies. Although MOMP may have contributed additional porin-like activity, functional modification of endogenous porins could not be ruled out. Interestingly, the successful expression and processing of recombinant chlamydial porins in E. coli cells depends on the precise leader sequence, as well as on the specific protein. PorB is less "toxic" with its native leader, in contrast to MOMP, which is less "toxic" with the E. coli OmpT leader, and native-leadered C. muridarum MOMP is less deleterious to E. coli than Ch. abortus MOMP. Although a full investigation of the role of leader sequences could not be undertaken here, it is known that successful OM insertion, as well as prior transport across the inner membrane and processing, is also signal sequence-dependent. For example, a large proportion of E. coli LamB porins with signal sequence mutations remained "tethered" to the inner membrane (probably by their unprocessed signal sequence), even though the protein was also closely associated with the OM [ 43 ]. For C. trachomatis MOMP, use of the Omp-T leader and induction at 16°C (not induction at 37°C, as previously employed), in either "wild-type" cells or "porin knockout" cells in a supportive medium (SOC), provides improved processing and OM insertion, and there is also significant insertion at 25°C in "wild-type" E. coli . The single-channel properties of C. trachomatis MOMP are consistent with previous data on bacterial [ 40 ] and putative chlamydial [ 6 ] porins. In particular, the channels show "bell-shaped" voltage-dependent gating and are mainly open around ~0 mV, with very high conductances (close to the saturating conductances predicted for a large water-filled pore) and poor ionic selectivity, showing only a slight preference (~4:1) for cations over anions (using a Nernst-Plank analysis because relatively wide, water-filled porin channels are probably electroneutral [ 35 ], and poorly-described by electrodiffusion theory). The channels often appeared in groups of three, as might be expected for a trimeric "triple-barrelled" porin (e.g. Fig. 10 ). However, unless the channels were randomly incorporated into the bilayer (which is difficult to demonstrate), these complexes may represent a selected sub-population. Despite the lack of sequence similarity to known bacterial porins, a combination of different predictive approaches (none of which was entirely satisfactory in isolation), set in the context of elegant and pioneering work from many laboratories on the properties of VS domains, predicted that C. trachomatis MOMP, like putative porins in the intracellular pathogens Burkholderia thailandensis and B. pseudomallei [ 44 ], could be a 16-stranded β-barrel. Our working model pays due attention to the construction principles for β-barrels [ 39 , 40 ]. The N and C termini complete final strand 16, the periplasmic turns are short, and most external loops are long and include the immunogenic VS domains. The barrel surface in contact with the bilayer consists largely (though not exclusively) of hydrophobic side chains, and all 18 strand residues with charged side chains project into the pore to line the central water-filled central channel. 6 cysteines lie in extracellular loops, and 2 periplasmic cysteines lie on opposite sides of the barrel where they are unlikely to form an intrasubunit disulphide bond, although they could form intersubunit bonds, or bonds with other proteins. A single membrane thiol projects into the barrel pore, where it could be involved in disulphide bond formation if a loop (e.g. L1) were to fold into the barrel. Our working model for the membrane topology of C. trachomatis MOMP differs in some significant respects from the recent prediction for C. muridarum MOMP [ 45 ] (which was based partly on hydrophobicity plots). Although both studies predict that MOMPs are 16-stranded β-barrels with an average strand length of ~8 residues, periplasmic thiols are absent from the C. muridarum prediction. This would preclude the significant interactions with OmcB and OmcA, described in the Background. We also assigned L2, 4, 6 and 7 as C. trachomatis VS domains, not L2, 3, 5 and 6, the homologous regions in C. muridarum MOMP. Experimental tests of the predicted membrane topology of C. trachomatis MOMP are consistent with our model, because individual VS domains can be substantially truncated without preventing incorporation of the protein into the bacterial OM. If MOMP is a β-barrel porin, as suggested, and VS domains are confined to specific extracellular loops, it is conceivable that MOMP can continue to fold into a β-barrel in the absence of one of these domains. On the other hand, the removal of β-strands would disrupt folding. Removal of a single strand, bringing periplasmic residues into direct contact with external residues, is predicted to be particularly destructive to the global fold. Removal of more than one strand might be better tolerated, provided the β-barrel can form with a significantly reduced diameter. In practice, it appears that C. trachomatis MOMP cannot accommodate either type of strand modification. GE chromatography suggested that MOMP forms oligomers in the presence of Zwittergent or LDAO, and in line with these findings, in situ cysteine cross-linking of recombinant MOMP in E. coli OMs revealed oligomeric MOMP complexes, together with a species of folded or partially-folded MOMP monomers containing at least one intramolecular disulphide bond. This species contrasts with reduced, denatured MOMP monomers seen when chlamydial MOMP is solubilised directly from OMs (or native EBs [ 6 ]). However, the exact stoichiometry of MOMP oligomers in the E. coli OM remains uncertain because our size estimates for the oligomers, and thus their stoichiometries, may be too high because of uncorrected bound detergent. Also, it is clear that the stability of MOMP oligomers is detergent-dependent. Native Ch. abortus MOMP forms SDS-resistant oligomers of ~100 K [ 6 ], unlike the SDS-unstable MOMP oligomers isolated from E. coli OMs. We speculate that this may be because native MOMP oligomers are stabilised by interactions with other chlamydial components (e.g. co-purified Omp90 [ 6 ]), and possibly also by disulphide bonds. Disulphide bond formation (whether transient or permanent) does not appear to be essential during protein folding and OM insertion, because a cysteine-free mutant can be fully processed (Fig. 7A ) and can also form oligomers. Overall, our results show that the subunit stoichiometry of detergent-solubilised MOMPs expressed and processed in E. coli is detergent-dependent, that MOMP subunits can be cross-linked by disulphide bridges, and that folded monomers contain at least one intrasubunit disulphide bond (Fig. 8 ). Conclusions C. trachomatis MOMP, an immunodominant, cysteine-rich, chlamydial surface protein of crucial importance in the immune response to infection, is a major subunit vaccine target. However, unlike many other bacterial porins, it has been difficult to refold from inclusion bodies or to achieve and demonstrate functional surface expression. This study is the first to report unambiguous functional analysis, by single-channel recording, of recombinant chlamydial MOMP recovered from bacterial outer membranes. The modified expression system described in the present study provided a means to test specific hypotheses provided by a working model for the C. trachomatis protein. However, although our results are consistent with a working model of MOMP as a 16-stranded β-barrel, more mutations or other approaches are needed before a specific model can be accepted. The protein also formed oligomers, even in the complete absence of cysteine residues. The surface display of modified, functional MOMP in E. coli cells (potential vehicles for a live, subunit vaccine), together with a working topological model, could guide the removal of unwanted or harmful epitopes from engineered proteins, and it might also be possible to display external loops containing specific MOMP epitopes on other porin "scaffolds" in living cells. However, it is important to note that such approaches will be limited if essential disulphide bonds in the native chlamydial envelope, including bonds involving non-MOMP cysteines, stabilise the conformation of key immunogenic VS domains. Methods DNA manipulations C. trachomatis ompA (corresponding to X62918, from the Da serovar) and Ch. abortus ompA were cloned without their leader sequences into the Nde -I/ Nco -I sites of pET22b(+) (Novagen) after destroying an internal Nde -I site in C. trachomatis ompA by Quik-Change PCR mutagenesis (Stratagene). This did not alter the encoded protein. C. muridarum ompA and porB were amplified with and without their leaders from genomic DNA and cloned into the Nde -I/ Bam- HI sites and Nde -I/ Nco -I sites, respectively, of the same vector ( C. muridarum ompA also required null mutation removal of an internal Nde -I site). The E. coli OmpT protease leader sequence or the native C. trachomatis MOMP leader sequence was added to the 5' end of the leaderless C. trachomatis and Ch. abortus inserts by sequential gene extension PCR using three overlapping primers. A 5' Nde -I site was again used to provide the starting methionine codon in the final full-length construct. Quik-Change PCR was also used to create pairs of unique internal restriction sites in native-leadered C. trachomatis ompA to permit the deletion of specific domains by plasmid restriction and religation [ 33 ]. These sites were: for VS1, Age -I; for VS2, Bcl -I; and for VS3, VS4 and the predicted β-strands, Aat- II. Successful deletions were confirmed by hemi-nested single-colony PCR (using Taq polymerase) to identify clones that could be amplified by gene-spanning primers but not by primers complementary to regions that had been removed. We also generated C. trachomatis MOMP expression constructs containing inserts in which all 9 cysteine residues (C26, C29, C33, C102, C115, C182, C184, C207 and C335) were replaced by alanine using Quik-Change PCR. Most of the modifications were carried out in a pSTBlue-I/NovaBlue system, and the fidelity of each insert was confirmed by automated DNA sequencing (MWG Biotech). Protein expression and recovery E. coli BL21(DE3) or BL21(DE3)omp8 [ 34 ] cells were harvested from cultures of LB (Luria-Bertani) medium (10 g/l Bacto tryptone, 5 g/l yeast extract, 10 g/l NaCl, pH 7.0) or SOC medium (20 g/l Bacto tryptone, 5 g/l yeast extract, 0.5 g/l NaCl, 20 mM glucose, pH 7.0) by centrifugation at 6,000 × g for 5 mins after inductions as described in the Results section, and washed in 50 ml phosphate buffered saline (PBS). The cell pellet was resuspended in 5 ml TEN buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 100 mM NaCl) containing 1 mg lysozyme and incubated for 30 min. at room temperature. Following sonication (6 × 15 s, 6 μm amplitude, Sanyo Soniprep 150 sonicator) the cell lysate was incubated with 20 U/ml Benzonase (Novagen) for 15 min at room temperature. OM fragments were pelleted by centrifugation at 15,000 × g for 10 min, and washed twice in 20 ml TEN buffer. Membrane proteins were solubilised by resuspending the pellet in 6 ml solubilisation buffer containing 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 50 mM NaCl and 10 mM DTT with either 1% (w/v) octyl glucoside (OG, Anatrace), 1% (w/v) lauryl (dodecyl) dimethylamine oxide (L(D)DAO, Anatrace) or 1% (w/v) Zwittergent 3–14 (Anzergent 3–14, Anatrace), and incubating at 37°C for 1 hour. The solution was clarified by ultracentrifugation (Beckman TLA-100) for 20 mins at 100,000 rpm. Protein concentrations were determined after TCA precipitation. SDS-PAGE and Western blotting Unless otherwise indicated, SDS-PAGE was carried out under reducing conditions using 10–12% (w/v) gels. Molecular masses were estimated from plots of relative mobility vs the logarithm of the molecular mass of Precision Plus unstained protein markers (BioRad). For Western blotting, proteins were electrophoretically transferred to PVDF membranes under conditions compatible with the transfer of high-MW proteins including native MOMP oligomers [ 6 ]. The membranes were blocked in 5% (w/v) non-fat milk in PBS-T (0.005% (v/v) Tween-20 in PBS) then incubated in 1:5000 goat anti- C. trachomatis MOMP antibody (Fitzgerald International) for 1 hour at room temperature. Following 2 × 30 sec and 3 × 5 min washes in PBS-T, membranes were incubated in 1:10,000 HRP-conjugated anti-goat/sheep antibody (Sigma) for 1 hr at room temperature. After washing, immunoreactive proteins were detected by ECL. Whole cell immunoblotting and immunofluorescence 10 ml of LB medium was seeded 1:100 with cultures grown to saturation overnight, and incubated until the OD reached 0.6. The cells were pelleted by centrifugation (6,000 g × 10 mins) and resuspended in fresh medium. Following incubation at the selected temperature for 10 mins, 0.1–1 mM IPTG was added and incubation was continued for another 2–16 hrs. Intact cells were harvested by gentle centrifugation (4,500 g × 5 mins) and washed in 1 ml PBS. The pellets were resuspended in 200 μl PBS, and 10 μl was applied to a nitrocellulose membrane and allowed to dry. The membrane was blocked and probed with anti- C. trachomatis MOMP polyclonal antibody as described above. Immunofluorescence was carried out as described previously [ 26 ], with fixation and permeabilisation either before or after immunostaining, using 1:200 dilutions of the above primary antibody and fluorescein-conjugated anti-goat secondary antibody (Sigma). The cells were then observed by bright field, phase contrast and fluorescence microscopy using a Leica TCS-NT confocal microscope. Gel-exclusion chromatography Solublised OM proteins were separated by GE chromatography using a high resolution 26/60 HiLoad Superdex 200 prep grade column (Amersham Pharmacia Biotech) freshly equilibrated in 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 50 mM NaCl and 5 mM DTT (omitting the latter for the cysteine-less MOMP mutant). The buffer also contained either 0.05% (w/v) LDAO or 0.05% (w/v) Zwittergent 3–14. 2 ml aliquots of solubilised OM proteins (containing up to 10 mg protein, solubilised as described earlier under protein expression) were loaded, and the column was eluted with the same buffer for 800 min. at a flow rate of 0.5 ml/min. 5 ml fractions were collected and 10 μl of each protein-containing fraction was deposited onto a pre-prepared PVDF membrane and probed for MOMP as described earlier under Western blotting. The column ( V t 320 ml) was calibrated in the presence of detergent using standard proteins. V 0 (the void volume) was 115 ml, and K av was calculated as ( V e - V 0 )/( V t - V 0 ), where V e is the elution volume. Bilayer reconstitution and single-channel analysis Planar bilayers were cast from diphytanoyl phosphatidylcholine (Avanti) between two 0.5 ml chambers containing 50 mM KCl, 20 mM Tris-HCl (pH 8.0) and 1 mM DTT, designated cis and trans [ 6 ]. The cis chamber was voltage clamped with respect to the trans chamber using an Axon 200B amplifier or a Biologic RK300 amplifier. 1–5 μl aliquots of pre-diluted solubilised proteins (containing up to 10 ng protein and no more than 5 ng detergent) were added to the cis chamber, followed by aliquots of 5 M KCl to raise the salt concentration to 500 mM. Channel incorporation usually occurred within 30 min, accelerated by switching the holding potential between +/- 60 mV. Experimental protocols were programmed and the digitised data were low-pass filtered (1 kHz, 8-pole Bessel-type response) and recorded using pClamp8 software (Axon Instruments), and analysed offline. The bilayer potential was slowly and repeatedly ramped between -100 mV and +100 mV (each sweep taking 32 s) in the presence of an asymmetric (500 mM vs 50 mM, cis vs trans ) gradient of KCl, or with equimolar 500 mM or 1 M KCl. At least 3 voltage ramps were recorded and analysed for each experiment, and equilibrium recordings were obtained at defined holding potentials. Holding potentials refer to the cis chamber, and upgoing deflections represent net movement of cations from cis to trans or of anions from trans to cis . Relative ionic permeabilities were determined from the equilibrium solution of the Nernst-Planck flux equations [ 35 ]. When the cation and anion fluxes are equal: E r is the equilibrium (zero current, or reversal) potential in asymmetric KCl, and R, T and F have their usual significance. (the permeability ratio of K + to Cl - ) was calculated using appropriate activity coefficients ( a ) from standard tables. Control experiments were carried out using equivalent amounts of detergent and with equivalent amounts of solubilised OM proteins purified from non-transformed bacteria, selecting identical GE column fractions. Membrane topology prediction The number of membrane crossings was predicted using a neural network based-outer membrane protein topology prediction program trained with known porins [ 36 ]. We discounted a predicted membrane crossing very near the N-terminus that was only apparent after numerical rounding. β-strands were predicted independently by similar computational approaches using B2TMPRED [ 37 ] and TMBETA [ 38 ], respectively. The three predictions were combined and adjusted manually, taking account of the accessibility of the VS domains of C. trachomatis MOMP and the known characteristics of antiparallel, amphipathic β-barrel strands in porins. Authors' contributions HEF and HM carried out most of the experiments. HEF analysed and organised the data, and drafted the first version of the manuscript. RHA conceived the overall project, provided experimental guidance, carried out some of the experiments, and redrafted the manuscript. All the authors read and approved the final manuscript. Supplementary Material Additional File 1 Detergent extraction of recombinant MOMP. Immunoblot with ECL detection following SDS-PAGE of OM proteins (10 μg per lane) from BL21 cells expressing OmpT-leadered C. trachomatis MOMP, induced for 2 hrs at 37°C. OM proteins (see Methods) were solubilised in 1% (w/v or v/v) octylglucoside, Triton X-100, Zwittergent 3–14 or LDAO, as indicated. NB & B are non-boiled and boiled samples, respectively. NE = non-expressing (control) cells. Click here for file Additional File 2 Optimisation of C. trachomatis MOMP expression and processing in BL21omp8 cells. Growth curves of BL21omp8 cells expressing C. trachomatis MOMP with its native leader, in LB or SOC medium (means ± SEM, n = 4). Click here for file Additional File 3 MOMP epitopes are not unmasked by Tris buffer or EDTA in whole cell immunoblots. Control BL21 cells and cells expressing "strand-deleted" constructs were suspended in 100 mM NaCl containing 50 mM Tris-HCl (pH 7.4) with or without 2 mM EDTA, applied to nitrocellulose membranes, and probed with anti- C. trachomatis MOMP polyclonal antibody. Click here for file Additional File 4 Recombinant MOMP does not form SDS-resistant oligomers. SDS-PAGE and immunoblot analysis of C. trachomatis MOMP expressed with its native leader in BL21omp8 cells at 16°C (induced for 12 hrs in the presence of 0.1 mM IPTG). Lanes 1 & 2 contain 10 μg non-boiled and boiled OM proteins, respectively, solubilised in 1% (w/v) OG. Note successful transfer of high-MW proteins. Click here for file

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300885

A Truly Broad View of Gene Expression Spotlights Evolution and Diversity

Bioinformatics and microarrays have given scientists powerful new tools to investigate the structure and activity of genes on a global scale. Rather than studying just a few genes, scientists can analyze tens of thousands within and across species. Microarrays flag which genes are expressed under particular cellular conditions in an organism, while genome sequencing offers clues to gene function and regulation. By comparing the genomic properties of different species, scientists can spot patterns that help them identify functional and regulatory elements, learn about genome structure and organization, and gain a better understanding of the evolutionary forces that shape life on Earth. The potential of these technologies to reveal insights into the fundamental structure and function of biological systems continues to grow along with the wealth of gene sequence and expression data—but the ability to interpret and merge these datasets lags behind the ability to collect them. In an effort to overcome these limitations, Sven Bergmann, Jan Ihmels, and Naama Barkai developed a comparative model that integrates gene expression data with genomic sequence information. Because functionally related genes are expected to be coexpressed in different organisms and because the sequence of some of these functionally related genes may also be conserved between organisms, Bergmann and colleagues hypothesized that “conserved coexpression” could serve as an indicator of gene function on a genomic level. (Conserved genes are those that have changed little since they first evolved. Conserved coexpression describes functionally related genes that are activated together in different species.) But first they had to determine whether coexpression was conserved among species. Analyzing the gene expression profiles of six distantly related organisms—bacteria, yeast, plant, worm, fruitfly, and human—the researchers found that functionally related genes were indeed coexpressed in each species. The most strongly conserved sets of coexpressed genes are associated with core cellular processes or organelles. These results indicate that conserved coexpression can improve the interpretation of genome sequence data by providing another functional indicator for homologous sequences. Since functionally related genes are expressed together in different organisms, it would be reasonable to think their regulatory networks are also conserved. To explore this idea, the researchers grouped coexpressed genes and their regulatory elements into “transcription modules” for each organism. They found significant variation in the number, organization, and relative importance of these modular components. Which components contributed most to an organism's global transcription program, for example, depended on the organism. But they also found that the transcription networks are highly clustered—meaning that genes connected to a specific gene are also connected to each other. This finding indicates that gene expression programs, regardless of their size or individual components, are highly modular. Each transcriptome contains modules that have been conserved over time along with “add-on” modules that reflect the needs of a particular species. This modularity supports the notion that variation between and among species arises from the diversity of gene expression programs. Although the regulatory details of individual gene groups varied, the researchers found common ground in the overall landscape of the expression data. The transcription programs exhibit properties typical of dynamically evolving “real-world” networks that are designed to perform in uncertain environments and to maintain connections between elements independent of scale. These properties were originally identified in studies of social networks and the World Wide Web, but they aptly describe the real-world challenges of the cell. Studies of dynamically evolving networks show that nodes (i.e., genes and proteins) added at an early stage (much like highly conserved genes) are more likely to develop many connections, acting as a hub. Following these organizational principles, transcription networks would have a relatively small number of highly connected “hub genes”—though a much higher number than one would expect in a random network. And that is what the authors observed: the networks they constructed from the expression data had the expected number of highly connected hub genes, which tend to be essential and conserved among organisms. Since these highly connected genes are likely to have homologues in other organisms, they can serve as powerful and efficient tools for assigning function to the thousands of uncharacterized sequences found in sequence databases. This model presents a framework to explore the underlying properties that govern the design and function of the cell and provides important clues—in the form of conserved transcription modules—to the evolutionary building blocks that generate diversity. Regulatory relations among transcription modules

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526370

Inhibitory effects of proanthocyanidins from Ribes nigrum leaves on carrageenin acute inflammatory reactions induced in rats

Background The anti-inflammatory effects of proanthocyanidins (PACs), isolated from blackcurrant ( Ribes nigrum L.) leaves, were analysed using carrageenin-induced paw oedema and carrageenin-induced pleurisy in rats. Results Pretreatment of the animals with PACs (10, 30, 60 and 100 mg/kg, i.p.) reduced paw oedema induced by carrageenin in a dose and time-dependent manner. PACs also inhibited dose-dependently carrageenin-induced pleurisy in rats. They reduced (A) lung injury, (B) pleural exudate formation, (C) polymorphonuclear cell infiltration, (D) pleural exudate levels of TNF-α, IL-1β and CINC-1 but did not affect IL-6 and IL-10 levels. They reduced (E) pleural exudate levels of nitrite/nitrate (NOx). In indomethacin treated rats, the volume of pleural exudate was low, its content in leukocytes and its contents in TNF-α, IL-1β, IL-6 and IL-10 but not in NOx were reduced. These data suggest that the anti-inflammatory properties of PACs are achieved through a different pattern from those of indomethacin. Conclusion These results suggest that the main mechanism of the anti-inflammatory effect of PACs mainly lies in an interference with the migration of the leukocytes. Moreover, PACs inhibited in vivo nitric oxide release.

Background Proanthocyanidins are compounds, naturally occurring in various plants, with anti-inflammatory [ 1 , 2 ] and anti-arthritic activities [ 3 ]. They are reported to prevent skin aging and heart diseases, they scavenge oxygen free radicals and inhibit UV radiation-induced peroxidation [ 4 - 10 ]. We have isolated prodelphinidins and procyanidins, proanthocyanidins (PACs) from blackcurrant ( Ribes nigrum L., Grossulariaceae) leaves which are used in European traditional medicine for the treatment of inflammatory disorders such as rheumatic diseases [ 11 ]. Majority of these compounds are water soluble monomers and oligomers (2 to 3 units) consisting of flavan 3-ol monomer units linked together by mostly C-4 to C-8 (Figure 1 ) and to a lesser extent C-4 to C-6 bindings. Few tetramers are also found. Figure 1 Chemical structure of proanthocyanidins. Where R = H, it is a procyanidin: catechin (R 1 = H and R 2 = OH) and epicatechin (R 1 = OH and R 2 = H); Where R = OH, it is a prodelphinidin: gallocatechin (R 1 = H and R 2 = OH) and epigallocatechin (R 1 = OH and R 2 = H). Previously, we have observed that, in vitro, these compounds profoundly affect the metabolism of chondrocytes : they increase the secretion from these cells of type II collagen and proteoglycans while they decrease the secretion of prostaglandin E2 (PGE2) [ 12 ]. On the other hand, while these compounds inhibited purified cyclo-oxygenase-1 and cyclo-oxygenase-2, they did not reduce the release of thromboxane B2 and PGE2 from human in vitro stimulated platelets and neutrophils respectively [ 12 ]. Moreover, PACs might influence the contractile status of smooth muscles of blood vessels : intravenous and intraperitoneal injection of PACs induced a drop of the blood pressure without a significant bradycardia [ 13 ]. This effect counteracts the hypertensive activity of norepinephrine. The present studies were designed to evaluate the potential anti-inflammatory activities of these compounds, in vivo , on carrageenin-induced paw oedema and pleurisy in rats. This latter inflammatory reaction allowed us to examine the influence of PACs not only on the exudate volume and polymorphonuclear cell accumulation but also on the release of several cytokines, IL-1β, TNF-α, IL-6, IL-10, CINC-1 and of nitric oxide (NO). These cytokines and NO are among the more important mediators involved in inflammatory processes [ 14 - 16 ]. Results Influence of PACs on rat paw oedema Carrageenin-induced oedema was significantly inhibited by PACs dose-dependently (Figure 2 ). This inhibitory effect was efficient from 2 h after the carrageenin injection for the two upper doses of PACs and was significative 4 h after the carrageenin administration for all doses of PACs. The maximum inhibitory effect of PACs reached 63% at 4 h after carrageenin, time of the maximum development of the oedema. Figure 2 Time course of inflammatory reaction induced by injection of carrageenin 1% in rat hind paw and its antagonism by PACs (10, 30, 60 and 100 mg/kg -1 ). Inflammation is expressed as the increase of the rat paw volume (ml) from 0 to 4 h following injection of carrageenin. The volume of the paw was reduced by PACs at the four doses tested and the inhibition is time and dose-dependant. Each value is the mean ± s.e. mean of n = 6 experiments. *P < 0.05 versus carrageenin. Influence of PACs on the carrageenin-induced pleurisy In control rats, the volume of the exudate collected 4 h after carrageenin injection reached 0.87 ± 0.18 ml per rat (n = 12). This exudate contained a large number of cells, mostly (> 95%) polymorphonuclear leukocytes (PMNs). The total leukocytes number in the exudate was 119.71 ± 29.29 × 10 6 per rat (Figure 3A ). PACs significantly reduced the volume of the exudate in a dose-dependent relationship, showing a maximum inhibitory effect (48%) from the dose of 30 mg/kg which was not increased by the upper doses of PACs. As expected, the volume of the exudate was reduced in indomethacin-treated rats. On the other hand, PMNs infiltration (Figure 3B ) was significantly inhibited by PACs in a dose-dependent way and by indomethacin. Figure 3 Effect of indomethacin and PACs on carrageenin-induced pleurisy. At 4 h after carrageenin injection, the volume of the exudate (A) was reduced by PACs (10, 30, 60 and 100 mg/kg) and indomethacin (10 mg/kg) administration. The accumulation of polymorphonuclear cells (PMNs, B) in the pleural cavity was inhibited by all tested drugs. Each value is the mean ± s.e. mean of n = 6 experiments. °P < 0.05 versus sham. *P < 0.05 versus carrageenin. Effects of PACs on the release of cytokines High levels of TNF-α, IL-1β, IL-6, IL-10 and CINC-1 were found in pleural exudates induced by carrageenin (Figure 4 ). Indomethacin reduced the level of the five cytokines studied while PACs lowered significatively the levels of TNF-α (Figure 4A ), inhibited the release of IL-1β (Figure 4B ) but did not affect IL-6 levels (Figure 4C ) and IL-10 production (Figure 4D ). PACs also lowered significantly CINC-1 levels (Figure 4E ). Figure 4 Effect of indomethacin and PACs on cytokines release in pleural exudate. Pleural injection of carrageenin caused by 4 h an increase in the release of the cytokines, tumor necrosis factor alpha (TNF-α, A), interleukin-1β (IL-1β, B), interleukin-6 (IL-6, C), interleukin-10 (IL-10, D) and cytokine-induced neutrophil chemoattractant-1 (CINC-1, E). TNF-α, IL-1β and CINC-1 levels were reduced by PACs, but IL-6 and IL-10 levels were not modified. Indomethacin lowered the level of all these cytokines. Each value is the mean ± s.e. mean of n = 6 experiments. °P < 0.05 versus sham. *P < 0.05 versus carrageenin. Effect of PACs on nitrite/nitrate (NOx) levels in pleural exudate The pleural exudate of carrageenin-treated rats contained a large amount of NOx (716 ± 32 μM; n = 6) (Figure 5 ). The amount of NOx in pleural exudate of rats treated with 10 mg/kg indomethacin was similar to the content found in the control group. On the other hand, PACs, at 30 mg/kg, significantly decreased the amounts of NOx in pleural exudate from 51%. Figure 5 Effect of PACs and indomethacin on NOx formation in pleural exudate. Production of NOx release was not significantly affected by pretreatment of rats with indomethacin (10 mg/kg, intraperitoneally) while PACs caused an inhibition in NOx production. Each value is the mean ± s.e. mean of n = 6 experiments. °P < 0.05 versus sham. *P < 0.05 versus carrageenin. Histological examination of lung sections Histological examination of lung sections revealed significant tissue injury (Figure 6 ) when compared with lung sections taken from saline-treated rats (Figure 6A ). Lung withdrawn from rats treated with carrageenin showed oedema, tissue injury and an extensive infiltration of the tissue by PMNs (Figure 6B ). Pretreatment of rats with indomethacin (10 mg/kg, i.p.) or PACs (30 mg/kg, i.p.) showed a reduced lung injury as well as a decrease in the infiltration of PMNs (Figures 6C,6D ). Figure 6 Effect of PACs on lung injury. When compared to lung sections taken from control animals (A), lung sections from carrageenin-treated rats (B) demonstrated interstitial haemorrhage and polymorphonuclear leukocyte accumulation. Lung sections from a carrageenin-treated rat that had received PACs (30 mg/kg) (C) or indomethacin (10 mg/kg) (D) exhibited reduced interstitial haemorraghe and a lesser cellular infiltration. Original magnification: × 125. Discussion Proanthocyanidins (PACs) from Ribes nigrum leaves reduced the inflammatory reactions induced by carrageenin in rats : the extent of the paw oedema was halved, the volume of the pleural exudates and its content in TNF-α, IL-1β, CINC-1 and NOx were reduced, the infiltration of leukocytes into the lungs and the accumulation of leukocytes into the pleural cavity were largely diminished. PACs have been reported to be able to scavenge free radicals and NO [ 17 ]. This property could be an explanation of the reduction of NOx level in the pleural fluid after PACs treatment. According to Ialenti et al [ 18 ], during the development of carrageenin-induced pleurisy, the main role of NO is the inhibition of leukocytes migration to the inflammatory site. However, in rats pretreated with PACs, the level of NOx and of leukocytes are simultaneously reduced. This result suggests that PACs could more or less directly affect the transmigration of leukocytes. The development of carrageenin-induced inflammatory reactions in rats results from the activation of the kinin system, the accumulation of leukocytes and the release of several mediators such as prostanoids and cytokines [ 19 , 20 ]. Indeed, these inflammatory reactions are greatly reduced in kininogen-deficient rats, in animals pretreated with kinin-antagonists and in leucopenic rats [ 19 , 21 ]. Previous studies [ 22 ] have demonstrated that PACs can reduce other inflammatory reactions such as the oedemas induced in rats by nystatin and concanavalin-A in which the kinin system is not involved [ 19 ] but in which leukocytes play a major role [ 23 ]. The comparison of the major determinants of these three kinds of reactions, all inhibited by PACs, is another argument suggesting that the main target explaining the anti-inflammatory activity of PACs would be the involvement of leukocytes. Pro-inflammatory cytokines TNF-α, IL-1β and IL-6 are sequentially released in the pleural exudates induced by carrageenin in rat [ 14 ]. These cytokines cause chemotaxis to attract granulocytes and monocytes and then, migrating leukocytes produce, in turn, further cytokines, such as TNF-α and IL-1β, and other pro-inflammatory mediators [ 15 ]. IL-6 has been proposed as a crucial mediator for the development of carrageenin-induced pleurisy and for the accumulation of leukocytes in the inflammatory site. Indeed, in carrageenin-induced pleurisy in IL-6 knock-out mice, the degree of plasma exudation, leukocyte migration and the release of TNF-α and IL-1β were greatly reduced. Moreover, a positive feedback plays an important part in the development of the oedema as levels of TNF-α and IL-1β are attenuated in IL-6 knock-out mice [ 24 ]. PACs did not affect the level of IL-6 and of IL-10, an anti-inflammatory cytokine, but reduced the pleural content of TNF-α, IL-1β and leukocytes. This result indicates that the release of IL-6 does not depend on the presence of leukocytes, of TNF-α and IL-1β on one hand, and, on the other hand, suggest that the main target of PACs would be the accumulation of leukocytes and the associated release of inflammatory mediators. TNF-α plays an important role in promoting and amplifying lung inflammation through the release of chemotactic factors such as CINC-1 (rat IL-8), an important mediator that promotes the migration of neutrophils [ 25 ] and oesinophils [ 26 ]. CINC-1 can increase the expression of LFA-1 integrin on rat neutrophils [ 27 ] and because expression of leukocyte adhesion molecules such as E-selectin is dependent on CINC [ 28 ], the inhibition of CINC-1 levels in pleural exudates by PACs may exert both direct and indirect effects on neutrophil vascular adhesion and extravascular migration. PACs probably acts by disrupting TNF-α, IL-1β, CINC-1 and PMNs accumulation pathways. One of the mechanism for the anti-inflammatory effect of PACs may be attenuation of the migration of PMNs in the exudate, because CINC-1, a representative cytokine for PMNs migration in rats, is suppressed by PACs in parallel with PMNs number dose-related fashion. Although, clarification for the precise mechanism would remain in future study. Recently, grape seed proanthocyanidins have been demonstrated to reduce the expression of soluble adhesion molecules, ICAM-1, VCAM-1 and E-selectin in the plasma of systemic sclerosis patients [ 29 ]. The same compounds have been shown to inhibit TNF-α-induced V-CAM-1 expression in human umbilical vein endothelial cells cultures [ 30 ]. A possible mechanism of the anti-inflammatory effect of PACs would be an interference with the expression or the effect of adhesion molecules. This interference would result in a reduction of polymorphonuclear cell migration and subsequently in a reduction of the release of pro-inflammatory factors such as TNF-α and IL-1β. Injection of carrageenin into the pleural cavity induces the accumulation of leukocytes, a release of cytokines, the expression of inducible NO synthase and of cyclo-oxygenase-2, and thus the release of large amounts of nitric oxide and of prostanoids [ 16 ]. The inhibitory effect of PACs on the accumulation of leukocytes and on the release of TNF-α and IL-1β could have resulted in a decrease in the induction of inducible NO-synthase and of cyclo-oxygenase-2 and finally of plasma exudation. Comparatively, some animals have been treated with indomethacin. The inhibitory effect of this well-known non-steroidal anti-inflammatory drug is larger than that obtained with PACs. Indomethacin greatly reduced plasma exudation, nearly suppressed the accumulation of leukocytes and decreased the levels of the cytokines while, it did not modify the pleural content of NOx. Indomethacin is known to inhibit the cyclooxygenase-1 and -2 responsible of the release of PGE 2 production. The peak of cyclooxygenase-2 activity measured by prostanoid levels in carrageenin-induced pleural exudates spreads from 2 to 6 h after irritant injection [ 31 , 32 ]. Both IL-6 and IL-10 release are, in part, stimulated by PGE 2 [ 33 , 34 ]. An inhibition of PGE 2 production by high doses of indomethacin could result in a downregulation of IL-6 and IL-10 production [ 35 , 36 ]. Moreover, Cuzzocrea et al [ 24 ], using carrageenin-induced pleurisy in IL-6 knock out mice, showed that IL-1β and TNF-α production in the pleural exudates is, at least, partly IL-6 dependent. Our results showing a reduction in the levels of IL-1β, TNF-α, IL-6, IL-10 and CINC-1 by indomethacin four hours after the induction of the pleurisy, could be mainly explained through the inhibition of PGE 2 and IL-6 pathways. Conclusions In conclusion, we have shown that proanthocyanidins isolated from Ribes nigrum leaves interfere with the accumulation of circulating leukocytes, associated with a reduction of pro-inflammatory factors such as TNF-α, IL-1β and CINC-1, a decrease of NOx level and a decrease in plasma exudation. Methods Animals We used male Wistar rats, weighing 250 – 300 gm. The animals were maintained on a standard laboratory diet with free access to water. The experiments were conducted as approved by the Animal Ethics Committee of the University of Liège, Belgium. Paw oedema Rats were pretreated with an intraperitoneal administration of saline or PACs (10, 30, 60 and 100 mg/kg). Thirty minutes later, lambda carrageenin, (0.1 ml, 10 mg/ml) was injected into the plantar region of the right hind paw. Each experimental group contained six animals. Paw volume was measured using a water plethysmometer (Ugo Basile) before and 1 h, 2 h and 4 h after the injection of carrageenin. After 4 h, the animals were anaesthetized with a large dose of sodium pentobarbital (80 mg/kg). Carrageenin-induced pleurisy Rats were pretreated with an intraperitoneal injection of saline, PACs (10, 30, 60 or 100 mg/kg) or indomethacin (10 mg/kg) 30 min before the intrapleural injection of the irritant. They were then anaesthetized with ketamine HCl (75 mg/kg) and carrageenin (0.2 ml, 10 mg/ml) or saline (0.2 ml) was administered into the right pleural cavity. Each experimental group contained 6 animals. Four hours later, the animals were anaesthetized with sodium pentobarbital (80 mg/kg). The chest was carefully opened and the pleural cavity rinsed with 2.0 ml saline solution containing heparin (5 U/ml). Exudates and washing solutions were removed by aspiration and the total volume measured. Exudates with blood were rejected. Exudates were aliquoted and kept frozen at -32°C. After removal of the exudates, lungs were withdrawn and fixed for one week under 30 cm pressure with 10% formaldehyde aqueous solution containing 0.480 M Na 2 HPO 4 and 0.187 M KH 2 PO 4 (pH 7.2) at room temperature. They were then dehydrated by graded ethanol and embedded in Paraplast. Tissue sections (thickness 7 μm) were deparaffinized with UltraClear, stained with hematoxylin-eosine and examined using light microscopy. The volume of the exudates was calculated by subtracting the volume of the washing solution (2.0 ml) from the total volume recovered. A sample of each exudate was diluted in phosphate buffer and total leukocyte count was performed using a hemocytometer. The levels of IL-1β, TNF-α, IL-6 and IL-10 in the exudates were measured using a colorimetric commercial ELISA kit (Biosource, Nivelles, Belgium) with a lower detection limit of 4, 3, 8 and 5 pg/ml, respectively. The levels of CINC-1 in the exudates were measured using a colorimetric commercial ELISA kit (Amersham Biosciences, Freiburg, Germany) with a lower detection limit of 0.49 pg/ml. The amount of NOx (nitrite/nitrate) present in the exudates was determined using a microplate assay method (Calbiochem, Leuven, Belgium) based on Griess reaction after reduction of NO 3 - to NO 2 - with a lower detection limit of 1 μM. Extraction and purification of proanthocyanidins Proanthocyanidins from Ribes nigrum leaves were extracted and isolated according to a previously described method [ 37 ]. A voucher sample (RN 210590) has been deposited in the Pharmaceutical Institute of Liège, Belgium. Briefly, leaves were powdered separately and then extracted at room temperature with acetone (70 % v/v in water). The acetone was removed under vacuum at 40°C. The resulting aqueous solution was freeze-dried. Isolation was carried out by MPLC on reversed-phase RP8 with water-acetone (9:1) to obtain a total proanthocyanidin-enriched fraction (PACs). Materials We used ketamine-HCl from Pfizer (Bruxelles, Belgium), sodium pentobarbital from Ceva (Bruxelles, Belgium) and heparin from B. Braun Medicals (Diegem, Belgium). PACs and lambda carrageenin (Sigma, Bornem, Belgium) were dissolved in saline. Indomethacin (Merck, Sharp and Dohme, Leuven, Belgium) was dissolved in Tris-HCl (0.15 M, pH 7.4). Statistical evaluation Results are given as mean ± standard error of the mean (s.e. mean) of N observations. For the oedema paw studies, a Mixed Procedure SAS (normal distribution) was used to compare difference of least square means. For the pleurisy studies, data sets were examined by one-way analysis of variance (ANOVA) followed by a Scheffe post-hoc test. A P -value of less than 0.05 was considered significant. Authors' contributions NG carried out PACs isolation, animal experimentation, immunoassays, lung sections and statistical analysis. MT coordinated and participated to the PACs isolation. LA coordinated the PACs isolation. JD participated in animal experimentation, conceived of the study and participated in its design and coordination.

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